Contributors to V o l u m e X I X Article numbers are shown in parentheses followfiag the names of contributors. Ai~liations listed are current.
E. D. AvAMso~r (60, 61), Department o/
Biochemistry, University oJ Toronto, Toronto, Ontario, Canada BEI~JA~tII~ ALCXAND~a (72), The New York Blood Center, New York, New York S. Y. ALl (65), Department o/Pathology, Institute o] Orthopaedics (University o] London), Royal National Orthopaedics Hospital, Stanmore, Middlesex, England KBI AIilMA (30), Department o] Agricultural Chemistry, The University o/ Tokyo, Bunkyo-ku, Tokyo, Japan Rtrra AeNo~ (14), Department o/ Chemical Immunology, The Weizmann Institute o/ Science, Rehovot, Israel TAGv. ASa'RtrP (64), The James F. Mitchell Foundation, Institute lot Medical Research, Washington, D.C. EsT~.~E B~-ELI (24), Department o/ Biochemistry, Tel Aviv University, Tel A viv, Israel GSANT H. Bara.ow (48), Molecular Biology Department, Abbott Laboratories, North Chicago, Illinois D. Jo~. BAtrOHMA~ (9), Ortho Pharmaceutical Corporation, Raritan, New Jersey MAKONI~m~ BELEW (40), Ethiopian Nutrition Institute, Children's Nutrition Unit, Addis Ababa, Ethiopia MYRON L. BE~rD~.R (12), Department o/ Chemistry, Northwestern University, Evanston, Illinois A. BEEa~ (35), Department o] Biophysics, The Weizmann Institute o] Science, Rehovot, Israel Zw BOI-IAK (22), Department o/ Biophysics, The Weizmann Institute of Science, Rehovot, Israel
K. BROCKLEHURST (71), Department o]
Biochemistry and Chemistry, Medical College o] St. Bartholomew's Hospital, Charterhouse Square, London, England JoYcE BRUNER-LoRAND (59), Division o] Biochemistry, Department o] Chemistry, Northwestern University, Evanston, IUinois F. M. BuMPUS (51), Cleveland Clinic Foundation, Cleveland, Ohio PHILIP J. BtmcK (67), Biochemical Research, Eli Lilly and Company, Indianapolis, Indiana BE~EVlOr J. CAMPBELL (54), Department o] Biochemistry, University o] Missouri, Columbia, Missouri R. C~APuIs (37b), Laboratorium ]iir Molekular-Bialogie, Chemischer Richtung, Eidgeni~ssische Technische Hochschule, Z~2rich, Switzerland THv.ODOmS CI-IaSE,JR. (2), Department o] Biochemistry and Microbiology, College o/Agriculture and Environmental Science, Rutgers University, New Brunswick, New Jersey ICHIRO CHm^TA (58), Research Laboratory, Tanabe Seiyaku Company, Ltd., Higashiyodogawa-ku, Osaka, Japan G. E. COrCNELL (60, 61), Department o/ Biochemistry, University o] Toronto, Toronto, Canada E. M. CeooK (71), Department o] Biochemistry and Chemistry, Medical College o/St. Bartholomew's Hospital, Charterhouse Square, London, England SAM T. DOl~T^ (23), Department o/ Medicine, Boston University School o] Medicine, Boston, Massachusetts R. F. DooLrrrLE (38), Department o] Chemistry, University o] California, San Diego, La Jolla, Cali]ornia S. D. ELLIOTT (16), Department o] Pathxi
xii
CONTRIBUTORS TO VOLUME X I X
ology, Corpus Christi College, Cambridge, England ARACELIM. E~GEL (72), The New York Blood Center, New York, New York M. P. ESNOUF (53), Nuffield Department o] Clinical Biochemistry, Radcliffe Infirmary, Ox]ord, England L. FISHMAX (49), Department o] Biochemistry, College o] Dentistry, New York University, New York, New York J. E. FOLK (6, 32), Section on Enzyme Chemistry, Laboratory o] Biochemistry, National Institute o] Dental Researoh, National Institutes o] Health, Bethesda, Maryland B. FOLTMANN (28), University o] Copenhagen, Institute o] Genetics B, (Biochemical Genetics), Copenhagen, Denmark B~.RNARD FRI~.I)ENSON (17), Department o] Biochemistry Research, University o] Buffalo, Buffalo, New York KITTY L. A. GLaNVmLE (52), Department o] Pathology, Institute of Orthopaedics, Royal National Orthopaedic Hospital, Brockley Hill, Stanmore, Middlesex, England LEON GOLDSTEIN (70), Department o] Biophysics, The Welzmann Institute o] Science, Rehovot, Israel T. Goa'oH (59a), Division o] Biochemistry, Department o] Chemistry, Northwestern University, Evanston, Illinois EDWARD E. HALEY (55, 56), Veterans Administration Hospital, West Haven, Connecticut ELVI~r HARPER (44), Developmental Biology Laboratory, Department o] Medicine, Massachusetts General Hospital, Boston, Massachusetts WILLIAMF. I'IARRINGTON(45), McCoUum Pratt Institute, The Johns Hopkins University, Baltimore, Maryland S. RALPH HIMMELHOCH (33), Laboratory o] Biochemistry, National Cancer Institute, National Institutes o] Health, Department o] Health, Education and Wel]are, Bethesda, Maryland T. HOFMA~rN (25), Department o] Big-
chemistry, University o] Toronto, Toronto, Ontario, Canada JEaOME F. HRUSKA (13), University o] Minnesota, Minneapolis, Minnesota EIJI ICHmHIMA (26), Laboratory o] Microbiology and Enzymology, Tokyo Noko University, Fuchu, Tokyo, Japan TSUTOMU ISHIm~WA (58), Research Laboratories, Tanabe Seiyaku Company, Ltd., Higashiyodogawa-ku, Osaka, Japan SHINJmO IWASAKX (30), Meito Sangijo Company, Ltd., Chiao-ku, Tokyo, Japan RICHARD L. JACKSON (42), Experimental Therapeutic~ Branch, National Health Institute, National Institutes o] Health, Bethesda, Maryland E. T. KAISER (1), Departments o] Chemistry and Biochemistry, University o] Chicago, Chicago, Illinois BEAVaICE KAssr.ta, (21, 66, 66a, 66b, 66c, 66d, 66e, 66f, 66g), Department o] Bio-
chemistry, Marquette School o] Medicine, Milwaukee, Wisconsin F. J. K~zvY (1), Department o] Biochemistry, University o] Chicago, Chicago, Illinois PREBE~rK0K (64), The James F. Mitchell Foundation, Institute ]or Medical Research, Washington, D.C. DONALI) K. KU~IMITSU (15), Department o] Chemistry, Fresno State College, Fresno, Cali]ornia CHARLES H. LACK (52), Department o! Pathology, Institute o] Orthopaedics, Royal National Orthopaedic Hospital, 8tanmore, Middlesex, England At)EL LAJTHA (36), New York State Institute o] Neurochemistry and Drug Addiction, Ward's Island, New York JOH~ H. LAw (13), Department o] Biochemistry, University o] Chicago, Chicago, Illinois MILTON LEVY (49), Department o] Bigchemistry, College o] Dentistry, New York University, New York, New York IRWN E. LmNER (17), Department o] Biochemistry, College o] Biological
CONTRIBUTORS TO VOLUME XIX
xiii
Sciences, University ol Minnesota, St. MARY J. MVC~.K (19), New Jersey ColPaul, Minnesota lege o/ Medicine and Dentistry, TV.H-YuNo Ltv (16), Department o] BiNewark, New Jersey ology, Brookhaven National Labora- ROLF MYHRMAN (59), Division o] Biotory, Upton, New York chemistry, Department o~ Chemistry, L. LORANV (59a), Division o] BiochemisNorthwestern University, Evanston, Illinois try, Department ot Chemistry, Northwestern University, Evanston, Illinois YaSUSHI NAKAnAWA (41), The RockeJAMES McCoNN (39), Division o] Bio]eller University, New York, New York chemistry, Department ot Chemistry, YOSHIKO NARAHASHI (47), Polymer Northwestern University, Evanston, Chemistry Laboratory, Institute o] Illinois Physical and Chemical Research, YaSTXFFAN MAGNUSSON (9a), Institut liar mato-Machi, Saitama Pre]ecture, Molekylar Biologi, Aarhus Universitet, Japan Aarhus, Denmark MARTIN OTTwS~.N (11, 30a), Chemical NEVILLE MARKS (36), New York State Department, Carlsberg Laboratory, Institute o] Neurochemistry and Drug Copenhagen-Valby, Denmark Addiction, Ward's Island, New York PHILIP H. P~TRA (31), Departments o] Obstetrics, Gynecology, and BiochemisFRITZ MARKWAaDT (69), Pharmakologitry, University o] Washington, Seattle, sches Institut, Medizinische Akademie, Washington Er]urt, Germany ( D.D.R.) HmOSHI MATSmat.aA (46), The Central G. PFLEIDERER (34), Lehrstuhl /fir Biochemie, Abteilung ]fir Chemie, RuhrResearch Institute, Sunory Limited, Universitiit Bochum, Bochum, GerKita-ku, Osaka, Japan many GARy R. Mn~stmvA (42), Department o] Biochemistry and Biophysics, Uni- MANFRED PHILIPP (12), Department o] Chemistry, Northwestern University, versify o] Hawaii, Honolulu, Hawaii Evanston, Illinois PAVaICL~ A. MErrNEa (21), Department ol Biochemistry, Marquette School o] LASZLO POLGAR (12), Institute o] Biochemistry, Hungarian Academy o] Medicine, Milwaukee, Wisconsin Sciences, Budapest, Hungary TV.RENCE G. Mv.mm-r (24), The Radiochemical Centre, Amersham, Bucking- JERK~IR PORATH (40), Institute o] Biohamshire, England chemistry, Uppsala, Sweden KENT D. MILLER (8), Department o] ELINE S. PRADO (50), Departamento de Medicine, Division o] Laboratory Bioquimica e Farmacologia, Escola Medicine, University o] Miami School Paulista de Medicina, Sao Paulo, o] Medicine, Miami, Florida Brasil WILLIAM M. MITCHELL (45), Department W. RICKERT (30a), Department o] Stao] Microbiology, Vanderbilt University, tistics, University o] Waterloo, WaterNashville, Tennessee loo, Ontario, Canada FRANK C. MONKHOUSE (68), Depart- KENNETH C. ROBBINS (10), Biochemistry ment of Physiology, University o] Section, Michael Reese Blood Center, Toronto, Medical Sciences Building, Chicago, Illinois Toronto, Ontario, Canada G. RONC.~RI (37a), Laboratorium /iir Rosin E. MoNao (62), Instituto de Molekular-Biologie, Chemischer RichBiologia Celular, Centro de Investigatung, Eidgen6ssische Technische Hochciones Biologieas, Madrid, Spain sckule, Zi~rich, Switzerland TAKASHI MUaACHI (18), Department o] C. A. RYAN (66f), Department o] AgriBiochemistry, Nagoya City University, cultural Chemistry, Washington State School o[ Medicine, Nagoya, Japan University, Pullman, Washington
xiv
CONTRIBUTORS TO VOLUME XIX
A. P. RVLE (20), Department o1 Bigchemistry, University o] Edinburgh Medical School, Edinburgh, Scotland I. SCHV.NKEX~ (49), Irvington House Laboratories, Department o] Medicine, New York University School o] Medicine, New York, New York CHRISTIAN SCHWABE (57), Department o]
Biolooical Chemistry, Harvard School o] Dental Medicine, Boston, Massachusetts SAM Sv.IFTES (44), Department ot Biochemistry, Albert Einstein College O] Medicine, Yeshiva University, Bronx, New York ELLIOTT SHAW (2), Biology Department, Brookhaven National Laboratory, Upton, New York DAVID M. SHOTTO~r (7), Department o] Biochemistry, University o] Bristol, Bristol, England R. R. SMv.nY (51), Cleveland Clinic Foundation, Cleveland, Ohio J. ~ODV.K(25), Department o] Biochemistry, University o] Alberta, Edmonton, Alberta, Canada LOUIS SUMMARIA(10), Biochemistry Section, Michael Reese Research Foundation, Chicago, Illinois Is SV~.NVS~.N (11), Carlsberg Laboratory, Copenhagen, Denmark A. SZEWCZUK (61), Institute o] Immunology and Experimental Therapy, Wraclaw, Poland JOaDAN TANO (27), Neurascienees Section, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma FLETCHER B. TAYLOal JR. (63), Department o] Medicine, Allergy and Immunology Division, University of Pennsylvania School o] Medicine, Philadelphia, Pennsylvania RUSSELL H. TOMAR (63), Department o] Medicine, Allergy and Immunology Division, University o] Pennsylvania
School o] Medicine, Philadelphia, Pennsylvania TETSUYA TOSA (58), Research Laboratories, Tanabe Sciyaku Company, Ltd., Higashiyodogawa-ku, Osaka, Japan HELEN VAN VUNAKm (23), Graduate Department o] Biochemistry, Brandeis University, Waltham, Massachusetts K. A. WALEH (3, 4), Department o] Bigchemistry, University o] Washington, Seattle, Washington MARmN E. WEBSTEa (50), Experimental Therapeutics Branch, National Heart and Lun¢ Institute, National Institute of Health, Bethesda, Maryland C. W. WHARTON (71), Department o] Biochemistry, The University o] Birmingham, Birmingham, London, England D. R. WHITAXER (43), Biochemistry Laboratory, National Research Council o] Canada, Ottawa, Ontario, Canada JOHN R. WHITAKER (29), Department o]
Food Science and Technology, University o] Cali]ornia, Davis, California WILFRm F. WHITE (48), Biochemical Research Department, Abbott Laboratories, North Chicago, Illinois P. E. WXLCOX (3, 5), Department o] Chemistry, Harvard University, Cambridge, Massachusetts A. YAaON (35), Department o] Biophysics, The Weizmann Institute o] Science, Rehovot, Israel KERRY T. YASUNOSU (15, 39), Depart-
ment o] Biochemistry and Biophysics, University o] Hawaii, Honolulu, Hawaii JUHYUN YU (30), Department o] Food Technology, College o] Science and Engineering, Yonsei University, Seudaimun-ku, Seoul, Korea H. ZUB~R (37a, 37b), Laboratorium ]fir Molekular-Biologie, Chemischer Richtung, Eidoen6ssische Technisehe Hochschule, Ziirich, Switzerland
Preface The "Proteolytic Enzymes" like other volumes of "Methods in Enzymology" provides information on the purification and mode of assay of enzymes. It is meant to be of value to the graduate student as well as to the more advanced investigator. The authors have been asked to give an up-to-date synopsis of other knowledge relating to physical properties (molecular weight, electrophoretic behavior, optical rotatory dispersion) and chemical structure (amino acid composition and sequences) as well as of some details on the kinetics of the various reactions catalyzed by the enzymes discussed. We, thus, believe that this volume will fill some of the needs of a primary reference work in this area. Classification of enzymes is a difficult task, and proteolytic enzymes are no exception. Regardless of the criteria adopted for their grouping, one soon notices t h e arbitrary nature of imposed systematization, and contradictions become obvious. While it may be convenient to group a number of enzymes together on the basis of a catalytic functionality common to the enzyme proteins themselves (e.g., serine proteases, cf. Section II [3]) it is also of some advantage to have sections based on entirely different criteria such as the pH optimum of activity (acidic proteases) or substrate specificities (carboxypeptidases and aminopeptidases, etc.). Other difficulties are also obvious; nevertheless, we felt that the presentation of a volume of this sort would gain even by such ambiguous subdivisions into various sections. Similarity of the catalytic pathway for a number of estero-proteolytic enzymes provided the idea for including methods on enzyme titrations. Apart from being of immediate use for the standardization of the particular enzymes discussed in the special chapters of Section I, hopefully they will provide an impetus, in general, for designing appropriate methodology for a variety of other proteases as well. Eventually, these titration methods are bound to be of prime usefulness in enzyme standardization for a variety of purposes (e.g., World Health Organization). Although there are several enzymes which could be assayed by the rate of hydrolysis of given common substrates (be it casein, hemoglobin, or tosylarginine methyl ester), optimal conditions for the assay of each enzyme will vary appreciably and, in spite of possible duplications, these rate assays are better dealt with in connection with the descriptions of individual enzymes. In addition to proteases and peptidases, this volume includes a number of transpeptidating (transamidating) enzymes. Of course, several of these also catalyze hydrolytic reactions just as many of the enzymes conxv
xvi
PREFACE
ventionally regarded as proteases function in transpeptidating reactions. Special reasons could be offered for including a few other miscellaneous enzymes, too. Because water-insoluble enzymes hold great promise, detailed coverage was given to some solid-phase enzyme derivatives. The section on the naturally occurring activators and inhibitors of proteolytic enzymes will, no doubt, enhance the value of this book. Some duplications in a task of this nature are unavoidable. However, we are somewhat more unhappy about possible omissions, a few of which we are already aware {serum trypsin inhibitors; the complement system), and hope that the reader will not hesitate to draw our attention to these. The proteolytic enzyme field appears to be in a state of rapid expansion, and it is conceivable that some of the omissions could be included in a later volume. Although we did recommend certain guidelines for presentation, it was decided that we would not try to mold contributions of the different authors into a uniform format. It is doubtful that we could have accomplished this even if we attempted it. Such is the inherent diversity of the subject matter of proteolytic enzymes--and of those who write about them. Special thanks are due to Dr. Joyce Bruner-Lorand for valuable help.
METHODS IN ENZYMOLOGY EDITED BY
Sidney P. Colowick and Nathan O. Kaplan VANDERBILT UNIVERSITY
DEPARTMENT OF CHEMISTRY
SCHOOL OF MEDICINE
UNIVERSITY OF CALIFORNIA
NASHVILLE, TENNESSEE
AT SAN DIEGO LA JOLLA, CALIFORNIA
I. II. III. IV. V. VI.
Preparation and Assay of Enzymes Preparation and Assay of Enzymes Preparation and Assay of Substrates Special Techniques for the Enzymologist Preparation and Assay of Enzymes Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques VII. Cumulative Subject Index
xvii
METHODS IN ENZYMOLOGY EDITORS-IN.CHIEF
Sidney P. Colowick
Nathan O. Kaplan
VOLUME VIII. Complex Carbohydrates
Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate Metabolism Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation
Edited by RONALDW. ESTABROOKAND MAYNARDE. PULLMAN VOLUME X I . Enzyme Structure
Edited by C. H. W. Hms VOLUME XII. Nucleic Acids (Parts A and B) Edited by LAWRENCEGROSSMAN AND KIVIE MOLDAVE VOLUMEXIII. Citric Acid Cycle
Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpeneids Edited by RAYMONDB. CLAYTON VOLUMEXVI. Fast Reactions
Edited by KENNE'rH KUSTIN VOLUMEXVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited by HERBERTTABORAND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)
Edited by DONALDB. MCCORMICK AND LEMUEL D. WRIGHT VOLUME X I X . Proteolytic Enzymes Edited by GERTRUDE E. PERLMANN AND LASZLOLORAND xix
XX
VOLUMES I N SERIES
VOLUMEXX. Nucleic Acids and Protein Synthesis (Part C)
Edited by KIVlE MOLDAVEAND LAWRENCEGROSSMAN VOLUMEXXI. Nucleic Acids (Part D)
Edited by LAWRENCEGROSSMANAND KIVIE MOLl)AVE VOLUMEXXII. Enzyme Purification and Related Techniques Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis and Nitrogen Fixation (Part A)
Edited by ANTHONY SAN PIETRO
[1]
A c ~ v ~ . SITE TITRATION OF PROTEOLYTIC ENZ~fMES
3
[ 1 ] Principles of Active Site Titration of Proteolytic E n z y m e s I By F. J. K~ZDY and E. T. KAISER Introduction
The purpose of this chapter is to discuss methods which have been developed for the measurement of the active site molarities of enzyme solutions. Rather than presenting an exhaustive survey of the active site titration techniques now available, we will consider first the basic principles involved in such measurements, and then we will describe a few representative titration methods for ~-chymotrypsin. Any quantitative chemical study has to rely ultimately on the knowledge of the concentration of each reacting species in the reaction mixture. The measurement of concentrations the titration ideally would require a method which should be accurate, precise, and specific for each compound. The development and perfection of such methods is still one of the major preoccupations of analytical chemistry. In the field of enzymology, accurate titration methods only recently became a realistic goal, after the remarkable progress achieved in the past decades concerning the purification, structural analysis, and understanding of the mechanism of a multitude of enzymes. Conceptually, the process of establishing a method of titration could be divided into the following elementary steps: (1) definition of the function(s) or the molecular species to be measured; (2) selection of an experimentally measurable physical or chemical property which is common and proper to these compounds or functions; and (3) the development of theoretical equations permitting the translation of the experimental results into concentrations. The methods of titration of proteolytic enzymes are no exception to this rule although the concepts involved are further complicated by the complex chemical nature of the enzymes. Enzymes are proteins of well-defined chemical composition and theoretically the molecular species to be titrated could be defined as the protein molecules having amino acid sequences identical to an established standard. For the present, the lack of adequate criteria for protein purity renders impossible the attainment of this goal. Furthermore, the most important property of an enzyme is its catalytic activity, • Supported in part by grants from the National Institute of Arthritis and Metabolic Diseases and the National Institute of General Medical Sciences.
4
~rITP~rZON M~.THODS
[1]
which is determined not only by the primary sequence, but also depends critically on the secondary and tertiary structure of the protein. Finally, the catalysis occurs at a limited area of the protein surface the active site or active center of the enzyme--and the enzymatic activity is in many cases independent of slight chemical or structural changes undergone by other parts of the protein molecule. For these reasons it would be impractical to define the goal of enzyme titrations as the determination of the number of protein molecules of identical chemical structure and of identical catalytic activity. In the present discussion, therefore, we would like to limit ourselves to the titration of active sites, i.e., the determination of the concentration of catalytic centers possessing identical enzymatic properties. The selection of the method should take into account the fact that the enzyme to be titrated will be contaminated by inactive protein, most likely of identical or similar amino acid composition. Thus, even if the active center contains a unique chemical function--such as the sulfhydryl group of several proteolytie enzymes of plant origin--a simple determination of that particular function will not necessarily yield the concentration of active centers. The titrating agent should be chosen in such a way that its reaction would be dependent upon the integrity of the active center. The enzyme should either catalyze the reaction in some way, or at least the environment of the active site should modify the reactivity of the particular chemical function in order to distinguish it from a similar group on enzymatically inactive fragments. TM In other words, in designing titrating reagents, one should exploit fully the specificity of the enzyme. In general, the reagents suitable for titration will be small molecules of high purity possessing a pronounced chemical or physical affinity toward the active site. The reaction of the reagent with the enzyme should be accompanied by an easily and accurately measurable change in a physical property. The reaction should have a one-to-one stoiehiometry and optimally should consist of the irreversible formation of a covalent bond. Reagents which combine reversibly with the active site or which bind to it noncovalently are less satisfactory from the experimental point of view. Covalent bond-forming reagents can be further classified according to whether they react with groups involved in the catalytic process or whether they react with other "structural" groups of the active site. Several criteria will help to establish that the titration occurs at the active center: This modification of the reactivity might sometimes be obtained by adsorbing substrate or competitive inhibitor molecules to the active site.
[1]
ACTIVE SITE TITRATION OF PROTEOLYTIC ENZYMES
5
1. Comparison of the rate constant of the enzyme reaction with that of an appropriate model system will show the presence of catalysis. 2. The kinetics of the reaction should be simple first or second order--depending on the relative concentration of the reagent and the enzyme--or it should show a saturation phenomenon if the titrant binds to the active site before reaction can occur. Substrates and competitive inhibitors specific to the enzyme should normally inhibit the titration reaction. The presence of such an inhibition is a good indication--but not a rigorous proof that the reaction occurs at the active site. In the same vein, the absence of inhibition does not rule out the participation of the active center, especially when the group to be titrated is not one of the catalytic functions. ~. The combination of the titrant with catalytic groups should yield an inactive enzyme. Even if the reaction involves only "structural" groups, a considerable modification of the activity should result. As a corollary, reversing the reaction of titration should restore fully the enzymatic activity. 4. In the case of a titrating agent possessing an asymmetric center, a large difference in activity of the antipodes is again excellent evidence for the involvement of the active site in the reaction. This difference should be more pronounced when the structure of the titrating agent better approximates the structure of substrates specific for the enzyme. 5. Finally, the use of experimental conditions which lead to the suppression of enzymatic catalysis--such as extreme pH's, high temperature, or 8 M urea--should also result in the disappearance of the reaction of titration. The establishment of theoretical equations allowing one to convert the experimental data into concentrations of active centers is a relatively simple process only in the case of titrations accompanied by the irreversible formation of a covalent bond. The use of titrants based on the formation of reversible products and transient intermediates necessitates a more complicated mathematical treatment, the validity of which has to be ascertained by a detailed kinetic study of the phenomenon in each case. A kinetic analysis is also necessary in order to reveal any eve,~tual heterogeneity of the enzyme which might arise from the presence of isozymes, partially active degradation products, or even other enzymes of similar specificity. In all cases, the purity, the yield, and the chemical identity of the product of titration should be carefully determined. Finally, it should be emphasized that titration procedures, although highly accurate and theoretically straightforward, have a rather low sensitivity inherent to the method of detection. For example, most spectrophotometric titrations will require enzyme concentrations of the
6
TITRATION METHODS
[I]
order of 10-e M at least. Furthermore, even the simplest and most selective titrations will be reliable only with relatively pure protein preparations, when the concentration of interfering impurities is at a reasonably low level. Therefore it is unlikely that titration procedures will ever completely replace rate assays which can easily determine relative enzyme concentrations of the order of 10-9 M by the use of appropriate specific substrates and extremely simple experimental procedures. Titration procedures will be of practical application only with enzymes which are readily obtained in large quantities and with high purity. For other enzymes the titration of one preparation will allow one to standardize the rate assay in absolute concentrations, thereby eliminating the use of cumbersome and ill-defined enzyme units. The result of active site titrations is expressed in units of molarity of active sites. This value is independent of the purity of the protein preparation, and thus it renders possible quantitative mechanistic studies on unstable or imperfectly purified enzymes. Combination of titration data with other determinations can also yield valuable conclusions about enzyme purity and about the number of active sites and subunits per molecule. Finally, titration of pure enzymes is one of the most accurate ways of determining their molecular weights. In the examples of active site titrations which we will discuss now the emphasis will be on the use of spectrophotometric methods. Numerous other techniques have been developed for the titration of active sites, but we feel that the spectrophotometric methods are unexcelled because of their convenience, accuracy, and rapidity. In the first experimental method we will consider the use of a slowly reversible, covalently bound inhibitor. Next, we will consider the use of rapidly reversible, covalently bound substrates as titran~s. The Use of Slowly Reversible, Covalently Bound Inhibitors
Titration o] the .~ctive Site o] a-Chymotrypsin with ~-Hydroxy-5-nitro-a-toluenesul]onic Acid Sultone The titration procedure described below appears to be the most convenient method at the present time for the determination of the operational molarity of the active sites in a-chymotrypsin solutions. The reaction utilized is the extremely rapid formation of 2-hydroxy-5-nitroa-toluenesulfonyl-a-chymotrypsin from the attack of the serine hydroxyl at the active site of chymotrypsin on 2-hydroxy-5-nitro-=-toluenesulfonic acid sultone (Eq. 1).2 The sulfonyl enzyme decomposes slowly, regenerats$. H. Heidema and E. T. E:~i~r, J. Am. Chem. Boo. 90, 1860 (1968) ; ibid., ~ (1967).
460
[1]
ACTIVE SITE TITRATION OF PROTEOLYTIC ENZYMES
7
ing active enzyme, and the slowness of this desulfonylation reaction as compared to the sulfonylation process makes it possible to employ the sultone as an active site titrant.
HO--CH~ J(
)I
SO2
HO--CH2
+
%~..o / S
Ks E
~-..~o / ES
(I) HO-- CH2
O2N~"~ "oHCH~--SOsH + P
I ~ ks E
ES'
Site o/Reaction Experiments on the degradation of the sulfonyl enzyme, 2-hydroxy5-nitro-a-toluenesulfonyl-a-chymotrypsin, which were done in an attempt to demonstrate the site of attachment of the sulfonyl group to the enzyme, were unsuccessful. However, other kinds of evidence for the location of the sulfonyl group on the enzyme were obtained. The evidence that the sulfonyl group of 2-hydroxy-5-nitro-a-toluenesulfonyl-a-chymotrypsin is located on Serif5 is 3-fold. First, the sulfonyl enzyme is inactive toward both the active site titrant cinnamoyl imidazole3 and the specific ester substrate N-acetyl-L-tryptophan methyl ester. Also, the inhibition constant obtained for the inhibition by N-acetylL-tryptophan of the sulfonylation of chymotrypsin by 2-hydroxy-5nitro-a-toluenesulfonic acid sultone agrees well with inhibition constants found using other chymotrypsin substrates known to react at the active site of the enzyme? A second type of evidence implicating Ser195 rather than His57 as the active site residue which is sulfonylated by the sultone comes from spectrophotometric titration of the phenolic hydroxyl group and from the kinetics of desulfonylation of the sulfonyl enzyme (steps k-2 + k3 of Eq. 1). The titration behavior of the phenolic hydroxyl indicates that the ionization of this residue is perturbed by the ionization of another residue on the enzyme which is almost certainly the imidazole moiety of His57. Furthermore, the pH dependence of the desulfonylation reaction clearly implicates the participation of a histidine residue in this process. In addition to these observations supporting the assignment of * G. R. Schonbaum, B. Zerner, and M. L. Bender, J. Biol. Chem. 236, 2930 (1961).
8
TIZRATIOS MSTHODS
[1]
Ser195 as the site of covalent binding of the sulfonyl group, there is a third kind of evidence based on a comparison of the behavior in acidic solution of 2-hydroxy-5-nitro-a-toluenesulfonyl-a-chymotrypsin with that of atoluenesulfonyl-a-chymotrypsin. 4 In both of these cases when the sulfonyl enzymes were desulfonylated at low p H and elevated temperature (pH 1.9 at 40 °, for example) and the p H was quickly adjusted to 7 and the temperature to 25 °, the desulfonylated enzyme species obtained were nearly inactive initially against the specific substrate N-acetyl-L-tryptophan methyl ester, and they slowly regained activity. Whatever the mechanism of these reactions is, this parallel behavior of the sulfonyl enzymes offers convincing evidence that the enzyme is sulfonylated at the same site in both sulfonyl chymotrypsins. Since a variety of evidence (including X - r a y crystallographic data 5) exists demonstrating the attachment of the sulfonyl group in a-toluenesulfonyl-a-chymotrypsin to Ser195, we conclude that the sulfonyl group in 2-hydroxy-5-nitro-a-toluenesulIonyl-a-chymotrypsin is attached to the same site in the enzyme. K i n e t i c Considerations
To establish thoroughly the validity of an active site titration, it is necessary to determine the kinetics of the reaction between the titration reagent and the enzyme. This point will now be illustrated for the titration of chymotrypsin with 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone. Under conditions of So >> Eo, assuming the reaction scheme of Eq. (1), the relationship between ES' and Eo (the total concentration of active enzyme in solution) is given by Eq. (2).6 When titrations with 2-hydroxy-5-nitro-a-toluenesulfonic acid 'A. M. Gold and D. E. Fahrney, Biochemistry 3, 783 (1964); A. M. Gold, Biochemistry 4, 897 (1965); D. E. Fahrney, Ph.D. thesis, Columbia University, New York, 1963. ~B. W. Matthews, P. B. Sigler, R. Henderson, and D. M. Blow, Nature 214, 652 (1967). ' Equation (2) can be derived as follows. From Eq. (1), E = ES(K,/S). The total enzyme concentration Eo = E -}- ES -Jr ES'. Thus, ES
E0
-
ES'
1 + K./S
Making the stationary state assumption (dES'/dt) -= 0 ~-- k2ES -- ES'(/e-~- k~), the expr- ~sion ES' ffi
k2(E0 -- ES') (k_~ -{- ks)(1 ~- Ko/S) can be obtained. Rearrangement of this equation leads to Eq. (2).
[1]
ACTIVE SITE TITRATION OF PROTEOLYTIC ENZYMES ]62 k_~ + k3
(ES') (Eo)
9
1 +
+ k-2 + k3
sultone are done according to the titration procedure which will be described later, the ratio (ES')/(Eo) is ~'~ry close to 1. In other words, the titration reagent measures the active site concentrations of chymotrypsin solutions with great accuracy. Since the kinetic parameters of Eq. (2) are known, we can be confident of this point as can be shown by a sample calculation. At pH 5.9, for instance, the value of k-2 -}- k~ 2.5 X 10-2 sec-1, k2 ~ 10 sec -1 and K~ ~ 5 X 10-4 M. ~ At a typical substrate concentration of 5 X l0 -~ M used in a titration, the ratio k2/(k_~ ka) is about 4 X 103 which is enormously larger than the quantity 1 -~ (K~/S). Thus, the titration efficiency is considerably better than one part in a hundred.
Comparison of 2-Hydroxy-5-nitro-a-toluenesul]onic Acid Sultone with trans-Cinnamoyl Imidazole as an a-Chymotrypsin Titrant To justify the use of any new titrant, a comparison should be made to the use of other titrants, if they exist. Currently, trans-cinnamoyl imidazole which was introduced as a chymotrypsin active site titrant by Bender in 19612 is the most widely used reagent for this purpose. The availability of the trans-cinnamoyl imidazole titration method has contributed greatly to the rapid progress made in understanding the chemistry of chymotrypsin-catalyzed reactions. A calculation similar to that which we have performed for the sultone can be done to show that at acidic pH values (pH 5, for example) trans-cinnamoyl imidazole is a very effective titrant for chymotrypsin active sites. What then are the advantages to using the sultone? First, the sultone can be employed as a titrant over a much wider pH range than trans-cinnamoyl imidazole. Chymotrypsin can be titrated with the sultone over the pH range 5-7.5 with no difficulty since the sulfonyl enzyme is quite stable. A t pH 7.27 (0.01 M phosphate buffer), for example, the first-order rate constant 7 observed for the formation of the hydrolysis product 2-hydroxy-5-nitro-a-toluenesulfonic acid from the decomposition of 2-hydroxy-5-nitro-a-toluenesulfonyl-a-chymotrypsin is 8.1 X 10.4 sec -1. On the other hand, trans-cinnamoyl-a-chymotrypsin is considerably less stable in the neutral pH range. At pH 7.39 in phosphate buffer the first-order rate constant for the formation of trans-cinnamic 'J. H. Heidema, Ph.D. thesis, Department of Chemistry, University of Chicago, Chicago, Illinois, 1969.
I0
TITRAZION METHODS
[I]
acid from trans-cinnamoyl-a-chymotrypsin s is 7.8 X 10-8 sec-1, a value about ten times greater than that for the production of the sulfonic acid quoted above. Even above pH 7.5 it is possible to use the sultone, although the rapidity of its nonenzymatic hydrolysis begins to be a problem. A second advantage to using the sultone is that it is easier to purify and store than trans-cinnamoyl imidazole. The sultone is readily purified by crystallization from ethanol and can be stored indefinitely. However, it is hard to remove the last traces of impurity from trans-einnamoyl imidazole which can be crystallized from cyclohexane or hexane. Furthermore, the stability of this compound on storage is not good. Finally, titrations with 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone can be done at 390 nm, a wavelength where enzyme absorption is negligible. In the case of trans-cinnamoyl imidazole, if titrations are done at 310 nm, the enzyme absorption cannot be neglected (although it is not very significant at 335 nm, another recommended wavelength for titrations with trans-cinnamoyl imidazole). TABLE I CONCENTRATION OF A CHYMOTRYPSIN STOCK SOLUTION AS DETERMINED BY TITRATION WITH DIFFERENT REAGENTS AT VARIOUS p H VALUES
Reagent trans-Cinnamoyl imidazole • 2-Hydroxy-5-nitro-atoluenesulfonic acid sultone
pH
Wavelength (nm)
5.05 5.05 6.23 5.05 6.84 7.48
335 335 335 321 390 390
Buffer 0.10 0.10 0.08 0.10 0.08 0.10
M M M M M M
Acetate Acetate 2,6-Lutidine (NOB-) Acetate 2,6-Lutidine (NOa-) Tris (NO~-)
Enzyme conc." ME X 10~ (M) 9.87* 9.96 b 9.85 9.44 c 9.61 9.58
" Average of duplicate determinations. b These titrations a t pH 5.05 were performed a week a p a r t on a C T solution stored in a refrigerator a t 2 °. c Average of triplicate determinations.
Results of titrations of chymotrypsin solutions with 2-hydroxy-5nitro-a-toluenesulfonic acid sultone and trans-cinnamoyl imidazole are compared in Table I. The agreement between the active site molarities determined by the two methods is excellent. 8 M. L. Bender, G. R. Schonbaum, and B. Zerner, J. Am. Chem. Soc. 84, 2562 (1962).
[1]
ACTIVE SITE TITRATION OF PROTEOLYTIC ENZYMES
11
Titration Procedure ~ T h e c h y m o t r y p s i n ( W o r t h i n g t o n B i o c h e m i c a l Corp., F r e e h o l d , N . J . , t h r i c e c r y s t a l l i z e d ) s t o c k s o l u t i o n ~° e m p l o y e d w a s a b o u t 1.0 X 10-8 M in a c t i v e e n z y m e . T h e s t o c k s o l u t i o n of t h e s u b s t r a t e w a s 3.0 X 10 -8 M in 2 - h y d r o x y - 5 - n i t r o - a - t o l u e n e s u l f o n i c a c i d s u l t o n e a n d was p r e p a r e d in a c e t o n i t r i l e solvent. T o 3.00 m l of t h e d e s i r e d buffer in a 1-cm p a t h l e n g t h r e c t a n g u l a r s p e c t r o p h o t o m e t e r c u v e t t e w a s a d d e d 100 ~l of c h y m o t r y p s i n s t o c k solution. T h e a b s o r b a n c e A~ of t h e r e s u l t a n t s o l u t i o n a t t h e a p p r o p r i a t e w a v e l e n g t h s e r v e d as a baseline. T h e n 50 ~l of t h e r e a g e n t s t o c k s o l u t i o n w a s a d d e d w i t h stirring. T h e n e w a b s o r b a n c e of t h e s o l u t i o n , A2, w a s d e t e r m i n e d b y l i n e a r e x t r a p o l a t i o n of t h e o b s e r v e d a b s o r b a n c e b a c k to t h e t i m e of r e a g e n t a d d i t i o n . ~ W h e n t h i s p r o c e d u r e is used, E q . (3) TM gives t h e m o l a r i t y of e n z y m e ' a c t i v e sites in t h e s t o c k solution, ME. ~m. 9 This titration procedure has been described by Dr. J. H. Heidema. See footnote 7. See also J. H. Heidema and E. T. Kaiser, Chem. Commun. p. 300 (1968). Stock solutions of ehymotrypsin were usually prepared by dissolving 0,300 g of the commercially obtained, three times crystallized and lyophilized enzyme in 10.0 ml of pH 5.05 acetate buffer. Such a solution had a chymotrypsin active site molarity of about 1.0 X 10-* M. Stock solutions of chymotrypsin were always stored in a refrigerator at 2 ° . 11Absorba~ce measurements were made on a Cary 15 spectrophotometer. A chart speed of 10 seconds per division (2 inches/minute) was used. After the immediate absorbance burst there is a slow nearly linear change in absorbance with time because of the slow solvent-catalyzed desulfonylation of the sulfonyl enzyme. On the time scale employed for the titrations these slow reactions cause a virtually constant ~ t / A t and thus justify a simple linear extrapolation of the absorbanee to zero time to obtain A2 (As ~ A1 plus the absorbance burst). At pH 5 these subsequent reactions are so slow that AA/A~ is essentially negligible. At pH values above 7~5 they become sufficiently fast that lL4/At is no longer virtually constant and extrapolation to zero time must therefore be nonlinear. 1'Equation (3) results from the following considerations. Immediately after the absorbance burst the solution contains only the sulfonyl enzyme, ES' and the excess sultone S. Thus A -----~s,C~s. ~ esCs -~- ~C~ where Cx represents the concentration of X in the solution. The absorbance caused by the enzyme protein must be included since e~s, actually represents the difference in extinction coefficients between ES' and E. (At the wavelengths chosen ~C~ was very small, always less than 0.01.) Then, considering the various dilutions of the stock solutions in the procedure employed A2 = EES' X 10ME/315 + ~s(Ms/63 - 10ME/315) + 310A~/315 A2 Mp. X 10(E~s, -- es)/315 ~ 310A1/315 + ~ r s / 6 3 =
Therefore ME =
A~ - 310A1/315 -- ~Ms/63 = A2 -- 62A1/63 -- ~Ms/63 10(eEs -- Es)/315 2(eEs -- ~s)/63
12
TITRATION METHODS
[1]
and cs are extinction coefficients of the sulfonyl enzyme, 2-hydroxy-5nitro-a-toluenesulfonyl chymotrypsin, and the titration reagent, respectively, and Ms is the molarity of the reagent stock solution. Ms is known from the method of preparation of the reagent stock solution, cEs, may be found by adding a known volume of the reagent stock solution to buffer in a cuvette at the desired pH containing a molar excess of enzyme. In the presence of excess enzyme the reagent will be converted entirely to the sulfonyl enzyme. From the absorbance burst AA observed, ~.s, may be calculated simply by the use of Eq. (4). ~s may be determined likewise, but employing buffer which does not contain enzyme. The value of cs observed at a n y given wavelength does not depend on pH. However, the absorbance of 2-hydroxy-5-nitro-a-toluenesulfonyl-a-chymotrypsin depends upon the extent of ionization of a nitrophenol chromophore which changes with pH. Thus cEs, must be determined at the exact pH employed for the titration. This can be accomplished simply by repeating the titration procedure in the same buffer with the addition of only 25 ~l of the reagent stock solution. Under these conditions the enzyme remains in excess and ~.s. can be calculated using Eq. (5). ME
A2 - 62A1/63 - (~sMs/63) --2(~ES' -- ~S)/63 ~ES' = A A / ( M s X dilution factor) A s - 124A,/125 ~Es, = Ms/125
(3) (4) (5)
In practice some simplification of Eqs. (3) and (5) can be made. At all of the wavelengths used, A1, the absorbance caused by the enzyme may be so small that the small adjustments in A1, theoretically necessary because of dilution, can be neglected. Also, in titrations done at 390 nm (near neutral pH) cs can generally be neglected. Thus, under these conditions Eq. (3) can be simplified to Eq. (6) and Eq. (5) becomes Eq. (7). ME = 31.5(A~ - - A1)/eEs, ~.s' -- 125(A~ - A I ) / M s ME -- (As - A1 - 33.2 Ms)/149.7
(6)
(7) (8)
When titrations were performed at pH 5.05, the reaction was observed at 321 nm, and Eq. (8) was employed. At this pH the nitrophenol chromophore of the sulfonyl enzyme is almost entirely protonated, and the value of cEs, at 321 nm, the absorption maximum for the nitrophenol chromophore, has been found to be rather insensitive to small changes in pH. In titrations with 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone at 390 nm, an alternative method for the determination of cEs, can be
[1]
ACTIVE SITE TITRATION OF PROTEOLYTIC ENZYMES
E F F E C T OF
TABLE II pH ON THE EXTINCTION
13
COEFFICIENT OF
2-HYDROXY-5-NITRO-a-TOLUEN ESULFONYL CHYMOTRYPSIN AT
390
nm ~
pH
+ES'
pH
eES,
6.37 6.55 6.87 7.15 7.41
3,492 4,300 5,849 7,246 8,315
7.62 7.76 7.95 8.29 8.71
9,276 9,734 10,720 11,850 12,960
o Dr. John Heidema has found eEs, to be independent of the various buffer systems used in his work. used. I f the p H of the titration medium 'is accurately determined, CES' m a y be found from the data of T a b l e II. The method using Eq. (7) is expected to be more accurate since it does not require a precise p H measurement, and it is unaffected by any differences in absorbance as measured on different spectrophotometers. 13
Syntheses Preparation of ~-Hydroxy-a-toluenesul]onic Acid Sultone. 1~ Sodium 2-hydroxy-a-toluenesulfonate was prepared first. To 41.3 g (0.397 mole) of sodium bisulfite was added 49.2 g (0.397 mole) of 2-hydroxybenzyl alcohol (Eastman) and enough water to dissolve the mixture (ca. 900 ml). The solution was refluxed for 8 hours, most of the excess water was removed by distillation, and the remaining solution was evaporated to dryness on a r o t a r y evaporator, yielding a white residue which was air dried. Continuous extraction of this residue with ethanol using a Soxhlet extractor gave 78.3 g (94% yield) of sodium 2-hydroxy-a-toluenesulfonate which was isolated as a flaky, white product. The sultone was synthesized by the cyclization of sodium 2-hydroxya-toluenesulfonate using phosphorus oxychloride. To 40.0 g (0.190 mole) of sodium 2-hydroxy-a-toluenesulfonate was added 320 g (2.09 moles) of phosphorus oxychloride. No reaction occurred at room temperature, and the reaction mixture was heated slowly to 125 °. H y d r o g e n chloride fumes started to evolve at 110 °, and the mixture was refluxed at 125 °. 13We recommend avoiding the use of phosphate buffers in the neutral pH range since these buffers catalyze the hydrolysis of 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone. 1, This synthetic procedure is taken from the Ph.D. thesis of Dr. O. R. Zaborsky, Department of Chemistry, University of Chicago, Chicago, Illinois, 1968.
14
TITRATION METHODS
[1]
Heating was continued for 1 hour, the excess POC13 was removed by distillation, and the cream-colored residue was allowed to cool. The residue was ground and was transferred slowly into 600 ml of ice water. The white material was left in contact with the water for 4 hours, then suction filtered, thoroughly washed with cold water, and finally air dried. Crystallization from ethanol gave 17.6 g (54% yield) of pure, white crystals, m.p. 86.1-87.1. 2-Hydroxy-a-toluenesulfonic acid sultone is available commercially from Eastman (No. 10333).
Preparation o/ ~-Hydroxy-5-nitro-~-toluenesul]onic Acid Sultone. 15 2-Hydroxy-a-toluenesulfonic acid sultone (2.40 g, 14.1 millimoles) was dissolved in 20 ml concentrated sulfuric acid, and the solution was cooled in an ice bath. To this solution 1.07 ml (1.52 g, 16.9 millimoles) of 70.3% nitric acid was added dropwise with stirring over a period of 15 minutes. The resultant solution was pale yellow and was allowed to stand in the ice bath for 10 minutes. Small ice cubes were slowly added until no further precipitate wa~ obtained. The pale yellow precipitate was filtered on a sintered-glass funnel, washed with a small amount of ice-cold water and by suction. Recrystallization from ethanol gave very pale yellow crystals, m.p. 148.5-149.5 °. The yield was 2.85 g (94%). 2-Hydroxy-5-nitro-a-toluenesulfonic acid sultone is now available commercially from Eastman (No. 10335). We recommend that the sultone be recrystallized twice from dry ethanol before use. The Use of Rapidly Reversible Covalenfly B o u n d Substrates
Titration o/a-Chymotrypsin with p-Nitrophenyl Ester Substrates As pointed out in the introduction, optimal enzyme titrations should make use of a stoichiometric reaction between the enzyme and a specific substrate. The serine proteases offer an excellent opportunity for such a titration, since the reaction of the enzyme (E) with the substrate (S) comprises the formation of a covalent intermediate, t h e acyl enzyme (ES'), according to the reaction scheme: K,
k2
ka
E + S ~- ES--~ ES'--~ E -t- P2 + P1
(9)
where P1 and P2 are the alcohol and acid portions of the substrate respectively. It is readily apparent from this scheme that a quantitative transThis preparation has been described by Dr. K. W. Lo, Ph.D. thesis, Department of Chemistry, University of Chicago, Chicago, Illinois, 1968.
[1]
ACTIVE SITE TITRATION OF PROTEOLYTIC ENZYMES
15
formation of E into ES' is only possible when k3 ~ ks and when the substrate concentration is high enough to transform a reasonable amount of enzyme into the enzyme-substrate complex. It is also obvious that ES' will exist in the solution only as long as there is an excess of substrate present. Thus both the amount of substrate and the value of k3 will be important factors in limiting the time span during which the titration has to be performed. For these reasons it would seem that an ideal titrant would have k3 = 0; i.e., instead of being a substrate, it would be an irreversible inhibitor. Unfortunately, it would be then impossible to ascertain whether active enzyme molecules are the only ones reacting with the titrant. Therefore, titrants with a small but measurable k3 are preferred. The reaction of titration is quantitated by measuring the change in some physical property of one of the participating molecular species. The spectrophotometric determination of a change in the visible or ultraviolet absorption spectrum is ideally suited for this purpose because of its accuracy and sensitivity, and is therefore presently preferred to any other method. The absorption spectra of E, ES, and ES p are rather unsuitable for titrations with rapidly reversible covalently bound substrates because of the experimental difficulties involved in the determination of their molar absorptivity. Measurement of the disappearance of the substrafe is also subopti'~al, since the requirement of excess substrate means that one has to measure a small change of disappearing substrate against a large background of excess substrate, thereby decreasing the accuracy of the method. The measurement of the formation of P1 seems to be most satisfactory, especially when there is a spectral region where P1 alone absorbs. As early as 1954, p-nitrophenol was proposed as a P1 of choice for titrations of a-chymotrypsin.16 In its ionized form (pK~ = 7.04), p-nitrophenol has a high absorbance in the visible region of the spectrum (c4oo = 18,300)17 and, therefore, with a good spectrophotometer its concentration can be measured in a l0 -6 M solution with less than 2% error. On the other hand, p-nitrophenyl esters have a negligibly small c4oo. In solutions of pH < 6, protonated p-nitrophenol has to be measured at wavelengths where the absorbance of the ester is not negligible. In this case the choice of the wavelength will be dictated by the optimal value of (Ephenol- Eester)//~ester, which occurs at the neighborhood of X----340 nm for many p-nitrophenyl esters. Finally, p-nitrophenol is an excellent leaving group and its esters have high values of k~/k3 with chymotrypsin, while the esters are not too unstable in aqueous solutions at neutral pH values at room temperature. '~B. S. Haxtley and B. A. Kilby, Biochem. J. 56, 288 (1954). 1~F. J. K6zdy and M. L. Bender, Biochemistry 1, 1097 (1962).
16
TITRATION METHODS
[1]
When a great excess of a p-nitrophenyl ester reacts with the enzyme according to Eq. (9), the concentration of P~ as a function of time can be expressed by an equation of the form? 8-~° P1 - PXo = At + ~'(1 - e-~t)
(10)
where A = (kcatEoS0)/(S0 + K~) = Eo[k~/(k2 + k3)]2/[1 + K~/S0]" b = [(ks + k3)S0 + k ~ , ] / ( S 0 + K,) keat = k~ks/(k2 + k3) K~, = g,ka/(k2 + ks)
(11)
Equation (10) states t h a t for values of t > 5/b the production of P1 will be linear with time, i.e., P1 - PI° = At + ~
(12)
The slope of this straight line is of course equal to the steady-state reaction rate ( = V ) , from which ke,tEo (=Vmax) and Km can be calculated by routine procedures. Extrapolation of the line to t = 0 yields the value of 7r as the intercept on the P, axis. Thus, the determination of 7r is a very simple experimental procedure: After addition of the enzyme to the substrate solution one measures the steady-state production of P1 for a short period of time. When the data are plotted as P1 vs. t, a straight line should be obtained which intersects the P~ axis at v. This graphically extrapolated value of 7r is directly proportional to the active site concentration, according to Eq. (11). In the simplest possible case, when k2 >> k3 and So >> Kin, v is equal to Eo. If these simplifying conditions are not fulfilled, then determination of 7r at several initial substrate concentrations m a y be necessary. Since Eq. (11) can be transformed into the form
1 / ~ / ~ = (k~ + ks)/k3 ~¢/Eo + (K,/%/Eo)(1/S,)
(la)
a plot of 1 / ~ / ~ vs. 1/So will yield (k2 + k3)/k3~/Eoo as the intercept on the axis of the ordinates. If k2 >> k3, then Eo is determined directly from this plot. K~ is readily measured from turnover kinetics and thus a numerical ~H. Gutfreund and J. M. Sturtevant, Biochem. J. 63, 656 (1956). M. L. Bender, F. J. K~zdy, and F. C. Wedler, J. Chem. Educ. 44, 84 (1967). For the more complicated case where the formation of ES from E and S cannot be treated as a rapid equilibrium, the reader is referred to the complete steadystate derivation by L. Ouellet and J. A. Stewart, Can. J. Chem. 37, 737 (1959).
[1]
ACTIVE SITE TITRATION OF PROTEOLYTIC ENZYMES
17
correction of ~ also would lead to the correct value of active site concentrations: Eo = 9(1 + K=/S0) 2
(14)
It is much more difficult to assess the value of k3/k2. For the limited case of nonspecific substrates, when k3 is small, isolation of ES' is possible by rapid gel filtration of the reaction mixture. Then the kinetics of decomposition of ES" will yield ka, and hence from the definition of kcat we can calculate £3/k2 = (ka/kcat) - 1. But in general, detailed kinetic study of both the steady state and the presteady state will be necessary in order to determine k2 and k3 individually before ka/k2 can be calculated. Experimental Methods for Tit-rations with Nitrophenyl Esters In the following discussion a few representative titrations of a-chymotrypsin by p-nitrophenyl esters will be described. These procedures were chosen because of their simplicity and the wide range of experimental conditions and specificity of the titrants.
Materials p-Nitrophenyl acetate was synthesized by adding a stoichiometric amount of acetyl chloride in chloroform to a dioxane suspension of sodium p-nitrophenolate. The sodium chloride formed is removed by filtration and the solvent evaporated. The ester is recrystallized from chloroform-hexane, m.p. 80.0 °. p-Nitrophenyl trimethylacetate was synthesized similarly; recrystallization from ethanol yielded colorless crystals, m.p. 95 °. p-Nitrophenyl N-benzyloxycarbonyl-L-tyrosinate was a product of Mann Laboratories, Inc. p-Nitrophenyl N-acetyl-DL-tryptophanate was purchased from Cyclo Chemical Co. The commercially available esters were used without further purification. Substrate stock solutions were prepared by dissolving an appropriate amount of substrate in acetonitrile. a-Chymotryp~in (Worthington Biochemical Corp., Freehold, New Jersey) crystallized three times, was dissolved in 0.02 M acetate buffer, pH 4.0, or 0.0.5 M citrate buffer, pH 3.2, and stored on ice.
Titration Procedure Three milliliters of the appropriate buffer is pipetted into a 1-cm pathlength cuvette and placed into the thermostatted cell holder of a recording spectrophotometer. Then, 50 t~l of the substrate stock solution is added on the flattened tip of a glass rod, and the solution is thoroughly mixed. The absorbancy of the solution is recorded for a short
18
TX~R~TION METHODS
[1]
o
÷1
©
~
~
÷1
.~ ~
o0
o
g
~ O
c
o
N
~w
a6
k
c~ c~ e~
o
~
0
.~
e~
[1]
ACTXV~. SITE TITRATION OF PROTEOLYTIC ENZYM]~,S
19
period of time, in order to allow graphical extrapolation of the absorbance to the time when the enzyme will be added. This extrapolated value will be designated as A,. At time zero an aliquot (20-100 t~l) of the enzyme stock solution is added (Ve) to the reaction mixture and the time recorded. The solution is rapidly mixed, and recording of the absorbance of the solution is started at a known time t after addition of the enzyme at an appropriate wavelength. When a sufficient portion of the linear steady-state phase has been recorded, the value of rr is measured by extrapolating graphically the absorbance vs. time curve to t ~- 0. This yields Ao. The concentration of active sites in the enzyme stock solutions can then be calculated from the following equation: E~tO~k = 3.05 q- Ve X f X A0 - A,(3.05/3.05 X Ve) V. A~
(15)
The corrective factor ] is necessary because with some of the substrates the condition So >> K,~ is not fully satisfied. The value of ] is calculated from the known value of K~: f = (1 + K,~/So) ~
(16)
Experimental conditions and the accuracy of the measurements on a Cary 14 or 15 recording spectrophotometer are reported for four representative p-nitrophenyl esters in Table III. Discussion
Titration of a-chymotrypsin by the four suhstrates described yields values of concentrations of active sites which are in excellent agreement with each other and also with results obtained by other methods, such as titration by trans-cinnamoyl imidazole. For all these substrates it has been ascertained by kinetic studies that Eq. (11) is indeed the correct description of the phenomenon and that the value of k2/ka is much larger than 100. Therefore, the correction due to the factor (1 q-k3/k2) 2 is negligible. Because of the high sensitivity and specificity of the substrate, titration of ~-chymotrypsin by p-nitrophenyl N-benzyloxycarbonyl-Ltyrosinate is recommended whenever a recording speetrophotometer is available. When the absorbances are measured manually, p-nitrophenyl acetate will be a more appropriate titrant because of its much slower turnover rate.
F. J. Kfizdy, G. E. Clement, and M. L. Bender, J. Am. Chem. Boe. 86, 3690 (1964). M. L. Bender et al., Y. Am. Chem. Soc. 88, 5890 (1966).
20
TIvaAzIOS MSTHODS
[2]
"Limitations o] the Method When p-nitrophenyl CBZ-tyrosinate is used as a titrant, the pH of the enzyme stock solution should be smaller than 3.5 or greater than 6.0 in order to avoid artifacts due to the slow dedimerization of the chymotrypsin dimer which occurs in the pH range of 4 to 5. Finally, it cannot be over emphasized that titration with even the most specific substrate will not distinguish between closely related enzymes, such as ~-, •-, and $-chymotrypsin, or even chymotrypsin and trypsin. The evaluation of the concentration of individual species in such mixtures requires a lengthy kinetic analysis of the data and is far more time consuming than a routine titration. In the case of enzyme mixtures an approach based on the use of selective inhibitors can be used as a general technique to differentiating the active site concentrations of closely related enzyme species. ~3
Conclusions Historically, titration procedures for the determination of active site concentrations were first developed for chymotrypsin. Since then, in the past decade a great many papers have been devoted to finding newer and better methods and titrants not only for chymotrypsin but also for a host of other enzymes. It is the hope of the authors that the time is not far when enzyme concentrations will be routinely expressed in units of active site concentrations, and when the use of arbitrary enzyme units will be considered as an oddity of the past. ~sL. Lorand and F. V. Condit, Biochemistry 4, 265 (1965).
[ 2 ] T i t r a t i o n of T r y p s i n , P l a s m i n , a n d T h r o m b i n w i t h p - N i t r o p h e n y l p ' - G u a n i d i n o b e n z o a t e HC11
By THEODORECHASE,JR., and ELLIOTT SHAW The theory and desirability of titration methods for proteolytic enzymes has been described. 1~,~ In the case of serine proteinases, advantage is taken of the fact that in the normal catalytic process an acyl enzyme is formed as an intermediate (Fig. 1). Titrants have been de1 Research carried out at Brookhaven National Laboratory under the auspices of the U~. Atomic Energy Commission. t" F. J. K~zdy and E. T. Kaiser, see this volume [1]. z M. L. Bender, M. L. Begue-Canton, R. L. Blakeley, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. K~zdy, J. V. Kilheffer, Jr., T. H. Marshall, C. G. Miller, R. W. Roeske, and J. K. Stoops, J. Am. Chem. Soc. 88, 5890 (1966).
20
TIvaAzIOS MSTHODS
[2]
"Limitations o] the Method When p-nitrophenyl CBZ-tyrosinate is used as a titrant, the pH of the enzyme stock solution should be smaller than 3.5 or greater than 6.0 in order to avoid artifacts due to the slow dedimerization of the chymotrypsin dimer which occurs in the pH range of 4 to 5. Finally, it cannot be over emphasized that titration with even the most specific substrate will not distinguish between closely related enzymes, such as ~-, •-, and $-chymotrypsin, or even chymotrypsin and trypsin. The evaluation of the concentration of individual species in such mixtures requires a lengthy kinetic analysis of the data and is far more time consuming than a routine titration. In the case of enzyme mixtures an approach based on the use of selective inhibitors can be used as a general technique to differentiating the active site concentrations of closely related enzyme species. ~3
Conclusions Historically, titration procedures for the determination of active site concentrations were first developed for chymotrypsin. Since then, in the past decade a great many papers have been devoted to finding newer and better methods and titrants not only for chymotrypsin but also for a host of other enzymes. It is the hope of the authors that the time is not far when enzyme concentrations will be routinely expressed in units of active site concentrations, and when the use of arbitrary enzyme units will be considered as an oddity of the past. ~sL. Lorand and F. V. Condit, Biochemistry 4, 265 (1965).
[ 2 ] T i t r a t i o n of T r y p s i n , P l a s m i n , a n d T h r o m b i n w i t h p - N i t r o p h e n y l p ' - G u a n i d i n o b e n z o a t e HC11
By THEODORECHASE,JR., and ELLIOTT SHAW The theory and desirability of titration methods for proteolytic enzymes has been described. 1~,~ In the case of serine proteinases, advantage is taken of the fact that in the normal catalytic process an acyl enzyme is formed as an intermediate (Fig. 1). Titrants have been de1 Research carried out at Brookhaven National Laboratory under the auspices of the U~. Atomic Energy Commission. t" F. J. K~zdy and E. T. Kaiser, see this volume [1]. z M. L. Bender, M. L. Begue-Canton, R. L. Blakeley, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. K~zdy, J. V. Kilheffer, Jr., T. H. Marshall, C. G. Miller, R. W. Roeske, and J. K. Stoops, J. Am. Chem. Soc. 88, 5890 (1966).
[~]
TITRATION OF TRYPSIN, PLASMIN, AND THROMBIN kz k3 E + S~ E ' S ~ Acyl enzyme ~ A c i d Complex
21
+ E
Fla. 1 veloped which also utilize acyl enzyme formation. However, in these cases, deacylation is very slow and the amount of alcohol released is just equivalent to the active enzyme, providing the reagent has enough affinity to convert all active enzyme to acyl enzyme. These ideal conditions are rarely met in practice. The use of a light-absorbing or chromogenic alcohol is the analytical basis of the method. A number of titrants for trypsin have been suggested: p-nitrophenyl Na-benzyloxycarbonyl-L-lysinate HC12,3 (also used with thrombin4), Na-methyl-Na-(p-toluenesulfonyl)-L-lysine fl-naphthyl ester, 5~ p-nitrophenyl-N2-acetyl-Nl-benzylcarbazate, ~b p-nitrophenyl p'-guanidinobenzoate HC1 (p-NPGB) e,7 and p-nitrophenyl-p'-amidinobenzoate HC12 Of these, only the last two afford the high sensitivity of p-nitrophenyl esters usable above the pK~ of the product p-nitrophenol (molar extinction coefficient of p-nitrophenoxide 18,300 at 402 nm, 2 as against 6000 for the un-ionized compound2), p-NPGB is preferable for the much slower rate of hydrolysis of the resultant acyl enzyme T as well as the greater ease of synthesis of the compound. In addition, because of its high affinity, determinations are not dependent on reagent concentration when an adequate amount is used as described below. Its use has been extended to the titration of the proteolytic enzymes of the blood clotting system, thrombin ~,9 and plasmin (Fig. 2). 7
Reagents Buffer. Sodium Veronal, 0.1 M, pH 8.3; for the titration of trypsin or human thrombin the buffer should also be 0.02 M in CaC12. Sodium Veronal (sodium barbital, sodium diethylbarbiturate) is dissolved to a concentration of slightly over 0.1 M (20.6 g/liter), titrated carefully to pH 8.3 with HCI (the free acid is sparingly 3M. L. Bender, J. V. Kilheffer, Jr., and R. W. Roeske, Biochem. Biophys. Res. Commun. 19, 161 (1965) ; M. L. Bender, F. J. K6zdy, and J. Feder, J. Am. Chem. Soc. 87, 4953 (1965). 4 F. J. K6zdy, L. Lorand, and K. D. Miller, Biochemistry 4, 2302 (1965). 5D. T. Elmore and J. J. Smyth, Biochem. J. 107, (a)97, (b)103 (1968). ST. Cha~e, Jr. and E. Shaw, Bioehem. Biophys. Res. Commun. 29, 508 (1967). 7 T. Chase, Jr. and E. Shaw, Biochemistry 8, 2212 (1969). s K. Tanizawa, S. Ishii, and Y. Kanaoka, Bioehem. Biophys. Res. Commun. 32,
893 (1968). 9 j . R. Baird and D. T. Elmore, Federation European Biochem. Soc. Letters 1, 843
(1968).
22
T I T R A T I O N METHODS
[2] HOC6H4NO~
O H ~ IJ H2N,,,c/N%\//~--~ C--O --CsH4--~NO ~ +~,Hz
Cl
trypsin (plasmtn or
p -Nitrophenol
thrombin)
k2
~"
+
H /~ H2N" N --.--.(,, \C /
-
p - Nitrophenyl p ' - guanidinobenzoate NPGB
O N /)--- C --O- Enzyme
+JI-H zN /
H / ~ ,
HsN
N---~
O
/ ) - - C - - O H + Enzyme
\c / +~H 2
FIG. 2 soluble, and will precipitate if the pH is allowed to drop to 8.0) and diluted to volume. It should be stored at 4 ° . p-NPGB. The solid reagent is dissolved in dimethylformamide to a concentration of 0.05 M (16.8 mg/ml), diluted with 4 volumes of acetonitrile (final concentration of 0.01 M), and stored at 4°; it is stable for weeks in this solution (more stable than in dimethylformamide alone, as originally describede), and may be used until the solution is visibly yellow. Synthesis of p - N P G B
p-Guanidinobenzoic Acid Hydrochloride. 1° p-Aminobenzoic acid, 27.4 g (0.2 mole), and 22.8 g NH4SCN (0.3 mole) are dissolved in 185 ml H20-4-20 ml concentrated HC1. The solution is evaporated to dryness (about 4 hours) in an evaporating dish on a steam bath in the hood. The residue is dissolved in excess NaOH and the product, p-thioureidobenzoic acid, precipitated by addition of excess HC1 and chilling. The collected product is dried overnight; yield, 90%. The dried p-thioureidobenzoic acid is suspended in 200 ml absolute ethanol with 45.6 g CHsI (about 1.8 equivalents). The suspension is refluxed for 1¼ hour in the hood, chilled, and some ether added to complete precipitation. Yield of dried p-(S-methylisothioureido)benzoic acid hydroiodide is 36.9 g (61%). This material is refluxed with 75 ml concentrated NH4OH in the hood for 3 hours; some precipitation of the zwitterion of p-guanidinobenzoic acid occurs. After cooling, the pH is adjusted to about 6 to ensure full precipitation of the zwitterion. The precipitate is filtered off and dis~ H . C. B e y e r m a n a n d J. S. B o n t e k o e , Rec. Tray. Chem. Pays-Bas 72, 643 (1953).
[2]
TITRATION OF TRYPSIN~ PLASMIN, AND THROMBIN
23
solved in hot 1 N HC1 for crystallization; it should be recrystallized at least once to ensure removal of iodide. Yield of dried p-guanidinobenzoic acid hydrochloride is 13.5 g (58%; 31.5% from p-aminobenzoic acid); m.p. 2680. TM p-NPGB, p-Guanidinobenzoic acid HC1 (1.075 g, 5 millimoles) is dissolved in 7.5 ml dimethylformamide, and dicyclohexylcarbodiimide (1.083 g, 5 millimoles) is dissolved in 7.5 ml pyridine or tetrahydrofuran. The solutions are mixed and p-nitrophenol (0.725 g, 5.2 millimoles) is added. After standing overnight at room temperature the precipitated dicyclohexylurea is filtered off and the solvents removed in vacuo. The oily residue is taken up in 25 ml 0.1 N HC1 and the slurry extracted repeatedly with 25-ml aliquots of ethyl acetate, with separation of the phases by centrifugation. Extraction of the residual p-nitrophenol is monitored by shaking the ethyl acetate extract with a few milliliters of weak base ('iris buffer, pH 8); when the Tris extract is only faintly yellow the extraction is complete. The slurry of p-NPGB in HC1 is then dissolved in 300 ml water-saturated t-amyl alcohol and any residue filtered off; the t-amyl alcohol solution, after extraction with two 10-ml aliquots of 0.1 N HCI to remove p-guanidinobenzoic acid HCI, is evaporated to dryness below 40 ° . The residue is dissolved in minimum volume of boiling glacial acetic acid and crystallizes out on cooling (a little ether may be added to the cool solution to prevent freezing at 4 °) ; yield, 6872%, giving 98--99% of the expected amount of p-nitrophenoxide on hydrolysis in 0.1 N NaOH. The product shows some discoloration and decomposition at about 260 ° , but does not melt up to 300 ° except when heated rapidly. Titration
The following instructions are for use of 1-ml cells and a double-beam recording spectrophotometer, in which nonenzymatic breakdown of pNPGB can be automatically subtracted by adding an equal amount of the compound to the reference cuvette. A single-beam recording spectrophotometer can be used and the nonenzymatic hydrolysis subtracted manually; larger cells can be used, with suitable multiplication of the volumes of buffer, enzyme, and p-NPGB. Veronal buffer (1.0M) is pipetted into the reference cuvette, and enzyme sample and buffer are pipetted into the sample cuvette to a volume of 1.0 ml. (If the enzyme is at low concentration in Tris or other nucleophilic buffer, it may be desirable to include an equal amount of buffer in the reference cuvette, so that nonenzymatic hydrolysis of p-NPGB catalyzed by the buffer will be the same in both cuvettes.) The euvettes are placed in the spectrophotometer and the instrument (set at
24
TITRATION
METHODS
[2J
I.(;
0.;
;:).:
0.:
LIJ (.3 Z 0.:
0 m 3.,.;
0.7-
o.;
•.-~. I1' , N e g P
~--
D.c
°
'
TIME
(SECONDS)
Fia. 3. Titration of (left) 0.05 ml and (right) 0.08 ml of a solution of fl-trypsin. The procedure is described in the text. Extrapolation of the absorbance to time zero, which is the chart line just to the right of the initial rise in absorbance, corresponds to enzyme concentrations of 2.22 X 10-~M and 3.76 X 10-~M, respectively. The stock solution is thus 4~8 --- 0.13 X 10-4 M. 402 o r 410 n m ) zeroed, u s i n g t h e 0-1 slide wire for e x p e c t e d e n z y m e conc e n t r a t i o n (in t h e c u v e t t e ) 0.7-5 X 10 -5 M , 0-0.1 slide wire for e n z y m e c o n c e n t r a t i o n 0.1-0.7 X 10-5 M ; t h e c h a r t is run to a p o i n t 10 seconds b e y o n d a c h a r t line. T h e e u v e t t e s a r e t h e n r e m o v e d f r o m t h e i n s t r u m e n t ; 5 ~l p - N P G B s o l u t i o n is a d d e d to the reference cuvette. T h e solutions are m i x e d b y i n v e r s i o n a n d t h e c u v e t t e r e p l a c e d in the i n s t r u m e n t . As q u i c k l y
[2]
TITRATION OF TRYPSIN~ PLASMIN, AND THROMBIN
25
as possible, 5 ~l of p-NPGB solution are added to the sample cuvette, the solutions mixed and the cuvette replaced, and the instrument turned on. The optical density increased in an immediate burst, then slowly (see footnotes 2, 6, and 7 for illustrations). The response of trypsin is shown in Fig. 3. The instrument is allowed to run until a straight line of postburst nitrophenoxide production is established. This line is extrapolated back to the chart line 10 seconds before the burst (to allow for postburst nitrophenoxide production during the mixing and insertion into the instrument, which takes about 10 seconds). The concentration of active enzyme in the cuvette is then calculated from the increase in optical density thus determined and the molar extinction coefficient of p-nitrophenol at this pH and wavelength. Considerations
A pH of 8.3 was selected as the pH of titration in a compromise between two considerations: the instability of both trypsin and p-NPGB at higher pH, and the rapid change of the operational extinction coefficient at lower pH (pKa of p-nitrophenol, 7.2).11 At pH 8.3 p-nitrophenol is approximately 92% ionized; not only is the sensitivity of the titration near maximal, but only small errors are introduced by small changes of pH (___0.2 units) caused by the buffering effect of a buffered sample. At a lower pH not only is the sensitivity of the titration less because the product is present partially in the un-ionized form, but the pH must be controlled very tightly so that the correct operational extinction coefficient is used in calculation of the amount of p-nitrophenol released in the burst. 11 If the enzyme sample is buffered at a pH other than 8.3 and the volume or buffer concentration of the sample is large, it may be desirable to check the pH of the enzyme-Veronal mixture and if necessary correct the pH to 8.3 or determine the extinction coefficient of p-nitrophenol at this pH. The authors find that up to 0.5 ml trypsin in 10-3 M HC1 0.02M CaC12 can be titrated in a total volume of 1.0 ml without a significant pH drop below 8.3. Veronal was selected as the preferred buffer for the titration because the rate of nonenzymatic hydrolysis of p-NPGB is lower in it (ko in 0.1 M Na-Veronal, pH 8.3, 3.3 X l0 ~ sec-1) than in other buffers of useful capacity at this pH (/Coin Tris-C1, 0.1 M, pH 8.3, 18.3 )< l0 -5 sec -1 ; " A t pH values below 6 the now completely protonated product p-nitrophenol can be measured without severe pH effects, although the extinction coefficientis lower (e3~o~ 6000) and the acylation slower. This situation has been utilized (J. F. Hruska, J. H. Law, and F. J. K$zdy, Biochem. Biophys. Res. Commun. 36, 272 (1969)] to demonstrate the heterogeneity of trypsin preparations by observation of several different rate constants of acylation by p-NPGB.
26
TITRATION METHODS
[2]
in 0.1 M K-borate, pH 8.6, 18.0 X 10-5 sec-1) ; although the nonenzymatic hydrolysis is blanked out by adding p-NPGB to the reference cuvette as well as to the sample cuvette, any inequality of such addition will result in an erroneous rate of postburst nitrophenol production and possible error in the extrapolation to the time of addition of the reagent, and this error will be magnified if the rate of nonenzymatic hydrolysis is high; also, in the above procedure the reagent is not added to the reference and sample cuvettes at the same instant, and the amount of p-nitrophenol produced in the reference cuvette before the reagent is added to the sample cuvette will be subtracted from the true burst in the sample cuvette, so it must be kept small. After the immediate burst of p-nitrophenol produced by the stoichiometric reaction of enzyme active sites with p-NPGB, a slow further production of p-nitrophenol is observed, due either to hydrolysis of the acyl enzyme and reaction of the freed active site with a second molecule of p-NPGB ("turnover") or to nonspecifie catalysis of p-NPGB hydrolysis by other nucleophilic groups of the enzyme protein or buffer ("nonspecifie hydrolysis"). For discrimination between these, see footnote 7. The turnover rate is an inescapable property of the enzyme; but if nonspecific hydrolysis is rapid enough to make uncertain the determination of the burst by extrapolation to zero time, as is the case with many preparations of human plasmin, it may be minimized by using low concentrations of p-NPGB (10-5M) and plasmin ( ~ 1 0 - s M ) and the 0-0.1 scale. The problem is not significant with trypsin (where both types of postburst nitrophenol production are discernible but small 7) or either human or bovine thrombin (where only turnover has been detected) .T The theory of titration 1,2 shows that enzyme concentration is equal to the observed burst of p-nitrophenol v only if k2 >> k8 and the titrant concentration is much greater than the apparent Michaelis constant. If these conditions are not fulfilled, as is the case with many titrants, 2 the size of the burst will be affected by the titrant concentration, and must be determined at several concentrations and extrapolated to infinite titrant concentration (plotting 1 / ~ - ~ vs. 1/[S] 1.~) to determine the true concentration of active enzyme. Obviously, this consumes time, reagent, and enzyme; but the observation that no effect of p-NPGB concentration on burst size is observed down to [p-NPGB] = 2 X 10-e M with trypsin, while with plasmin and thrombin the effect is observed only when [p-NPGB] ~ 5 X 10- 6M, justifies titrating at only a single concentration (5 X 10-5M, or even 10-5M). For determination of the apparent k~ and relevant kinetic constants see footnote 7. A reading of 410 nm was selected and maintained as the wavelength
[2]
TITRATION OF TRYPSIN, PLASMIN~ AND THROMBIN
27
of observation of p-nitrophenol production for reasons of convenience in this laboratory; the absorbance maximum is actually 402 nm. In a n y case, it is desirable to determine the extinction coefficient with reerystallized p-nitrophenol in the instrument to be used; in our Cary 15 c41o at pH 8.3 = 16,595, so that complete hydrolysis of 5 X 10-5 M p-NPGB would lead to an optical density of 0.830.
Accuracy and Specificity The accuracy and reproducibility of the method are probably limited principally by the accuracy of pipetting the enzyme solutions; if considerable care is taken in pipetting 0.100 or 0.200 ml enzyme, with ordinary pipettes rather than micropipettes, reproducibility of ± 2 % can be obtained (using the 0-1 slide wire), although ± 5 % is more usual; with the 0-0.1 slide wire accuracy is usually limited by instrument noise. Errors due to free p-nitrophenol in the p-NPGB solution and rapid nonenzymatic hydrolysis in the cuvette are in the double-beam instrument dependent only on the variation in volume of p-NPGB solution added. p-NPGB has been used successfully for the titration of bovine trypsin, an insect trypsin, 12 bovine and human thrombin, and human plasmin; it presumably can be used for the titration of other closely related serine proteases, although not necessarily with all such, as shown by the observation that it is a normal substrate, not a titrant, for the C'la esterase of guinea pig complement (David H. Bing, personal communication). With a-chymotrypsin p-NPGB gives a burst followed by rapid turnover; it is therefore not suited for discrimination between trypsin and chymotrypsin (or between plasmin and thrombin), p-NPGB does not react with subtilisin or papain.
~J. F. Hruska, J. H. Law, and F. J. KSzdy, Biochem. Biophys. Res. Commun. 36, 272 (1969).
~]
SEmNE PRO~ASES
31
[3] Serine Proteases B y K. A. WALSH and P. E. WILCOX
Introduction Characterization of Serine Proteases
The serine proteases comprise a large group of enzymes which is distinguished by the reactivity of a serine residue in the active site. 1 A common test for these enzymes is the inhibition of their hydrolase activity by the reaction of this serine residue with diisopropylphosphorofluoridate (DFP). Although the natural substrates of the serine proteases are proteins or fragments of proteins, most studies of these enzymes have been based on their hydrolase activity toward synthetic peptides, peptide amides, and esters. All members of the group may be classified as endopeptidases since it is generally observed that the cleavage of terminal peptide bonds is inhibited by the charge on the amino or carboxyl group of a terminal residue. The serine proteases also exhibit strong esterolytic activity toward esters analogous to the specific peptide substrates. This property is useful for kinetic assay methods to be described later in this section. In addition, hydrolytic activity extends to a variety of acyl compounds in which the acyl moiety is bonded to a good leaving group, such as p-nitrophenol or imidazole. Depending upon the nature of the acyl group and the pH of the solution, rapid acylation of the enzyme may be followed by very slow hydrolytic deacylation. Reactions such as these form the basis for measurements of functional normality of the serine proteases by methods described in Section I (see this volume, F. J. K~zdy and E. T. Kaiser [1] ). Comparisons of relative rates of catalysis using a variety of synthetic substrates have led to the definition of the specificities of representative members of the class. These range from the narrow specificity of the trypsins, which is directed toward the bond on the carboxyl side of arginine and lysine, to the rather broad specificity of the subtilisins which will attack bonds between a wide variety of amino acids. It should be noted, however, that specificity toward natural protein substrates may be influenced not only by the side chain next to the peptide bond, but also by neighboring residues in the sequence and the surrounding structure of the protein. 1B. S. Hartley, Ann. Rev. Biochem. 29, 45 (1960).
32
~H~. SERINE PROTr~SES
[3]
More recently a quite different method has been utilized to further define their specificity, namely, irreversible inhibition by reaction with synthetic chloromethyl ketones which are analogs of peptide substrates. 2 The general structure may be written: X - - N H - - C H (R)--CO--CH2C1. Specificity is in large part determined by the side chain R; and X may be a blocking group, such as p-toluenesulfonyl or benzyloxycarbonyl, or may be one or more amino acid residues in a peptide chain blocked at the N-terminal. 3 The reaction of a specific chloromethyl ketone with the corresponding enzyme results in the stoichiometric alkylation of a histidine residue in the active site. These reactions provide a discriminating method for selectively blocking the action of one serine protease in the presence of another. General Distribution and Occurrence of Zymogens Serine proteases are widely distributed in nature. Historically their characteristic properties were first recognized among the digestive enzymes originating in the pancreas of mammals. More recently, examples of such digestive enzymes have been found throughout the vertebrate subphylum, in fact, in every vertebrate species investigated.4,5 Furthermore, they are not restricted to digestive functions in the intestinal tract. Thrombin and plasmin, enzymes involved in the formation and dissolution of blood clots, show these characteristic properties, and serine proteases have also been detected in mammalian mast cells2 The discovery of DFP-sensitive proteases extends to invertebrate phyla such as the Coelenterata (sea anemone, Metridium senileT), Echinodermata (starfish, Evasterias trocheliiS), and the Arthropoda (hornet, Vespa orientalisa). The isolation of proteases from bacteria, such as the subtilisins from Bacillus subtilus and related strains, is well known. These subtilisins and enzymes isolated from quite different bacteria, Streptomyces griseus 1° and Sorangium sp., 11 have also been characterized as serine proteases. 2E. Shaw, Vol. XI [80], p. 677. 3j. C. Powers and P. E. Wilcox, J. A.m. Chem. Soc. in press, 1970. * E. N. Zendzian and E. A. Barnaxd, Arch. Biochem. Biophys. 122, 690 (1967). ~H. Neurath, K. A. Walsh, and W. P. Winter, Science 158, 1638 (1967); L. B. Smillie, A. Furka, N. Nagabhushan, K. J. Stevenson, and C. O. Paxkes, Nature 218, 343 (1968). 6Z. Darzynkiewicz and E. A. Barnard, Nature 213, 1198 (1967), and references contained therein. D. Gibson and G. H. Dixon, Nature 222, 753 (1969). 8W. P. Winter and H. Neurath, Federation Proc. 27, 492 (1968). H. H. Sonneborn, G. Pfleiderer, and J. Ishay, Hoppe-Seylers Z. Physiol. Chem.
350, 389 (1969). '°S. Wahlby, Biochim. Biophys. Acta 151, 394 (1968); ibid. 185, 178 (1969). riD. R. Whitaker and C. Roy, Can. J. Biochem. 45, 911 (1967).
[3]
SERINE PROTEASES
33
In vertebrates with a distinct pancreas, the proteases are present in the gland and in the exocrine juice as inactive precursors, or zymogens, which must be activated before they can be identified by enzymatic assays. All of the known zymogens of the serine proteases are converted into active enzymes by the action of trypsin. 12 Usually the cleavage of one specific peptide bond is sufficient for this activation. Trypsin itself arises from the activation of pancreatic trypsinogen by an autolytic mechanism; in vivo this process is initiated by an intestinal enzyme, enterokinase. In teleost fishes, which do not have a distinct pancreas, serine proteases have been found in the pyloric ceca, but only in the active form. 4 It is likely that the detection and isolation of the corresponding zymogens have been prevented by technical difficulties. One elasmobranch, the spiny Pacific dogfish (Squalus acanthias) contains both a discrete pancreas and zymogens. TM The proteases involved with blood clots, thrombin and plasmin, also originate as zymogens and circulate as such in the blood. Although the conversion of these zymogens, prothrombin and plasminogen, to active enzymes may be accomplished with trypsin, activations are brought about in vivo by complex systems of blood and tissue enzymes. Procedures for the preparation of gram quantities of the various serine proteases have generally taken advantage of rich and plentiful source materials such as fresh pancreases, acetone powders prepared from them, pancreatic juices, or large-scale bacterial fermentation broths. The classical preparations of bovine trypsin, chymotrypsins A and B, and the respective zymogens from fresh pancreases have been described in Volume II, [2] and [3]. In recent times the isolation and purification of enzymes have been greatly facilitated by ion-exchange chromatography. This is notably demonstrated by the methods presented in the following sections. Not only are these chromatographic methods applicable to the large-scale preparations which are given in detail; they are also useful for the isolation and characterization of proteases in smaller amounts of source material, whether it be some animal tissue, small organism, or bacterial culture. Variants of Serine Proteases
Specific examination of the amino acid sequence of a variety of serine proteases has revealed common structural features indicating that they belong to a "family" of homologous proteins2 Each homologous structure within this group has a specific biological function, apparently ~H. Neurath, Federation Proc. 23, 1 (1964). 13j. W. Prahl and It. Neurath, Biochemistry 5, 2131 (1966).
34
THE SERINE PROTEASES
~]
acquired during a process of divergent molecular evolution from a common ancestral prototype. For example, specific serine proteases with common structural features are known which catalyze the hydrolysis of polypeptides for the varied biological purposes of digestion, blood clotting, clot lysis, sensing pain, and chemically opening insect cocoons. Some of these adaptations of an ancestral hydrolytic function have apparently occurred by selection during the evolution of species (e.g., trypsin in diverse animals). These may involve major changes in such features as the isoelectric point without alteration of the characteristic function of the protein. In contrast, in many other cases, several distinctly different but homologous enzymes occur within one species (e.g., trypsin, chymotrypsin, elastase, plasmin, and thrombin) and must have resulted from a gene duplication process preceding divergent molecular evolution. 1~ It is clear that more than one family of serine proteases exist since the subtilisins 15 bear no homologous relationship to the family of serine proteases discussed above, but are functionally serine proteases. Both the subtilisin family and the trypsin-chymotrypsin family of serine proteases operate by analogous mechanisms involving specific seryl and histidyl residues, but the amino acid sequences surrounding the serine and the histidine of one family bear no structural resemblance to those of the other family. The occurrence of several separable serine proteases in one animal species, each catalyzing the hydrolysis of the same test substrate, can lead to confusion regarding their specific identification. This multiplicity may simply reflect an overlapping of specificity of distinctly different enzymes. Alternatively, it may denote the occurrence of several allotypes within a given population (e.g., in dogfish trypsin ~e) or it may indicate that active, but partly degraded, enzyme derivatives have resulted from autolytic or proteolytic degradation. Autolyzed derivatives of enzymes have been characterized for both chymotrypsin and trypsin and almost surely exist for most serine proteases. In the case of bovine trypsin, chain cleavage without loss of function has been noted at either Arglos-Vallo8 ~7 or LySl~l-Serl~2,~8 whereas inactive products are obtained by bond cleavage at Lys49-Serso 18 I~G. H. Dixon, Essays Biochem. 2, 147 (1966). E. L. Smith, F. S. Markland, C. B. Kasper, R. J. DeLange, M. Landon, and W. H. Evans, J. Biol. Chem. 241, 5974 (1966). H. Neurath, R. A. Bradshaw, and R. Arnon, Proc. Intern. Syrup. Structure-Function Relationships o] Proteolytic Enzymes, Munksgaard, Copenhagen, in press, 1969. 1~S. Maroux and P. Desnuelle, Biochim. Biophys. Acta 181, 59 (1969). ~D. D. Schroeder and E. Shaw, J. Biol. Chem. 243, 2943 (19{}8).
[3]
SERINE PROTEASES
35
or LyslT~-Asp,7.19 The latter autolysis product, "pseudotrypsin," although devoid of activity against BAEE, still possesses the characteristics of a serine protease as indicated by stoichiometric reaction with DFP. Chain breaks have also been identified in chymotrypsinogen prior to the specific chain break inducing activity. These inactive precursors are called neochymotrypsinogens. In both the neochymotrypsinogens and the degraded enzymes, the nomenclature has been the arbitrary one of assigning Greek letters to a characterized product. It is recommended that the specific locus of chain cleavage be clearly stated in the identification of these serine protease variants, e.g., a-trypsin, which has been activated by the removal of residues 1-6 from trypsinogen, and in which LyslT6-Aspl~ has autolyzed, could be identified as a-trypsin (7-176, 177-229). G e n e r a l M e t h o d s of A s s a y
Principle
Enzymatic activity measurements generally fall into three categories, depending upon the experimental objectives: 1. Determination of the amount of a particular enzyme in an impure mixture, for example, a tissue extract. 2. Determination of the amount of activity remaining in a relatively pure enzyme sample after a given chemical or physical treatment. 3. Determination of relative activity toward various specific substrates in order to define the specificity of a given enzyme preparation. For the assay of a serine protease, any one of these objectives is usually met by using a simple synthetic substrate in which only one bond is susceptible to enzymatic hydrolysis. For such systems the kinetic parameters may be readily defined. The preferred substrates are esters of L-a-amino acids blocked at the N-terminal. Ester substrates have advantages over the corresponding amides in that Km values are lower and catalytic rate constants are higher. Furthermore, the release of at least one equivalent of protons from an ester permits the use of sensitive potentiometric methods. The general equation is: R I
R I
X--NH--CH--CO--OR' -b H~O ~ X--NH--CH--COO- q- HOR' -k H + in which X is a blocking group such as acetyl, benzoyl, tosyl, or benzylI~R. L. Smith and E. Shaw, J. Biol. Chem. 244, 4704 (1969).
36
TH~ SERINE PROTEASES
~]
oxycarbonyl; R is a side chain which meets the specificity requirements of the enzyme; and R p may be a methyl, ethyl, or p-nitrophenyl group. Potentiometric methods measure the rate of production of protons and are most conveniently accomplished in a recording pH-stat. The pH is usually set at about 8 where the serine proteases show maximal activity. Spectrophotometric methods depend upon differences in absorption between the products on the right side of the above equation and the substrate on the left. The rate of change in absorption at a selected wavelength is proportional to the rate of hydrolysis and is most conveniently determined on a recording spectrophotometer with a constant speed chart. Substrates which contain aromatic groups absorbing below 300 rim, e.g., the chymotrypsin substrate N-benzoyl-L-tyrosine ethyl ester, have difference spectra with maxima in the region of 240-260 nm. The hydrolysis of p-nitrophenyl esters results in useful difference spectra above 300 nm. Such substrates have special advantages for applications in which interference from UV-absorbing substances is to be avoided. Catalytic rate constants are of a magnitude such that p°nitrophenyl esters can be used at pH values below 6 under circumstances which make this an advantage. In recent times the general applicability of esterolytic assays has limited the use of protein substrates to special purposes. For a particular investigation the natural specificity may be of predominant interest, for example, the action of thrombin in blood clotting. In other cases, the natural, specific substrate may have some advantage in identifying a particular serine protease activity, for example, the use of elastin for the estimation of elastase in a mixture containing other proteases. A third type of application for natural substrates is represented by an investigation of tissue extracts which may contain protease activities of unknown or partially defined specificity. The rate of digestion of a denatured protein gives a rough measure of the total protease activity. That portion inhibited by D F P may then "be identified as due to serine proteases. A more complete characterization of the enzymes may be obtained by the use of specific inhibitors, such as the chloromethyl ketones. 2° Studies of the action of a protease on small natural peptides of known sequence have also been useful in obtaining a broad characterization of the enzyme specificity. After proteolytic digestion under controlled conditions, the peptides of the digest are separated and identified. In this way the bonds which are specifically cleaved become known and the relative extent of each cleavage can be estimated. For example, the specificity of bovine chymotrypsin B was characterized by investigating its action on glucagon. ~E. N. Zendzian and E. A. Barnard, Arch. Biochem. Biophys. 122, 714 (1967).
[3]
SERINE PROTEASES
37
For measurement of general proteolytic activity, the widely tested method of Kunitz is recommended. A description of this method, which uses casein as substrate and a spectrophotometric procedure, may be found in Vol. II [3]. In the sections immediately below, detailed examples are given of a potentiometric assay and a spectrophotometric assay, each using an ester substrate. Although these examples are designed for bovine trypsin and chymotrypsin A with substrates suitable for their assay, the methods are applicable in principle to the other serine proteases. Furthermore, these general methods have been adapted to a wide selection of substrates for various enzymes. Short descriptions with references to particular assay systems will be found in the sections on individual enzymes. Examples of the Potentiometric Method Assay for trypsin using a-N-benzoyl-L-arginine ethyl ester:
Reagents Buffer. 0.10 M KC1, 0.05 M CaC12, 0.01 M Tris at pH 7.75 with HCI Substrate solution. Dissolve 343 mg of a-N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) in a final volume of 100 ml of buffer. The final solution contains 0.01 M BAEE and can be used for a period of 2 days providing it is stored at 0-4 °. Enzyme. A standard solution of trypsin. It is prepared in 0.01 M HC1 at 0 ° at a concentration of approximately 10 mg per milliliter and dialyzed overnight at 4 ° against 0.001 M HCI. Any traces of insoluble material are removed by centrifugation. The precise concentration is established by measuring the absorbancy at 280 nm after 1 : 10 dilution with 0.001 M HC1 ~1~ \'~1 cm = 15.4).
Procedure Assays are performed in a pH-stat at a constant temperature (26 °) maintained in a thermostatted 4-ml vessel. The design of the vessel allows for continuous (magnetic) stirring at a rate sufficient to avoid local concentrations of titrant in the vicinity of the electrodes. With the Radiometer pH-stat, model TTTlc, a convenient electrode is the combined glass/calomel electrode, GK2302c. The syringe is filled with standardized 0.1 M N a 0 H and the titrator set to deliver with a maximum response bo small pH increments (on the Radiometer pH-stat this corresponds to a "proportional band" setting of 0.1). The chart drive is set at about 1/~ inch per minute. Place 2.0-3.0 ml of substrate solution in the pH-stat vessel (the exact volume is not critical) and allow the solution to reach thermal equilibrium. Set the titrator to raise the pH to 7.80. When this is accom-
THE SERINE PROTEASES
38
[3]
plished add an aliquot (up to 100 ~l) of enzyme solution, containing 0.001 to 0.1 mg of trypsin, directly into the substrate solution. In this particular assay the rate of acid production is constant throughout more than 90% of the hydrolysis since the K~ for BAEE is less than 10-~ M and benzoylarginine is not a strong inhibitor of the system. The Slope of the linear relationship between NaOH consumed and the time of reaction gives a direct measure of mieromoles of substrate consumed per minute.
Definition o] Unit and Specific Activity One unit of activity catalyzes the hydrolysis of 1 micromole of substrate per minute, thus the slope of the straight-line progress curve for the reaction (expressed in micromoles/minute) is equal to the number of units of the enzyme in the aliquot analyzed. T h e specific activity is expressed as units per milligram of protein. The specific activity of the best preparations of trypsin examined is 70 units per milligram. Examples of Spectrophotometric Method 1. Assay for chymotrypsin A. using N-benzoyl-L-tyrosine ethyl e s t e r . 21
Reagents Substrate solution. N-Benzoyl-L-tyrosine ethyl ester (BTEE), 0.001 M in 50% (w/w) aqueous methanol. This substrate is readily available from several commercial sources. Dissolve 15.7 mg of B T E E in 30 ml of methanol, spectral grade, and make up to 50 ml with water. Solution should be freshly prepared each day. Buffer. Tris-HC1, 0.10 M, pH 7.8, containing CaC12, 0.10M Enzyme. Prepare a standard solution of ehymotrypsin A~ in 0.001 M HC1 at a concentration of approximately 1 mg per milliliter. Determine exact concentration by absorbancy measurement at 282 nm (E 1% = 20.0). Just before use dilute this stock solution 1 :50 with 0.001 M HC1.
Procedure Assays are performed in 10-mm quartz cuvettes of 3.5 ml capacity. The euvettes are held at 30.0 ° in a thermostatted compartment. Preferably the spectrophotometer has a recording mechanism with a constant speed chart drive, for example, the Cary 15. Prepare the reference solution by mixing 1.5 ml of substrate solution and 1.5 ml of buffer in a cuvette. Place 1.5 ml of substrate solution and 1.4 ml of buffer in the assay euvette. Allow 5 minutes for the solutions to come to thermal ~B. C. W. Hummel, Can. J. Biochem. Physiol. 37, 1393 (1959).
[3]
SERINE PROTEASES
39
equilibrium in the cell compartment and zero the instrument at 256 nm, the wavelength of the maximum in the difference spectrum. At zero time add 100 ~l of the diluted enzyme solution to the assay cuvette, mix thoroughly for 5 seconds and record absorbancy difference for a period of about 5 minutes. The rate of change of absorbancy should be linear up to a reading of 0.12. For accurate measurements, a 0.0 to 0.1 range setting of the recording spectrophotometer should be used. Samples of unknown activity should be adjusted in concentration so that a 100-~1 aliquot contains an amount of enzyme corresponding to about 1 to 3 ~g of chymotrypsin A,.
Definition o/ Unit and Specific Activity Activity is calculated from the slope of the linear portion of the reaction curve. One unit is equal to the hydrolysis of 1 micromole of substrate per minute. The value of Ac (change in molar extinction coefficient) for complete hydrolysis of B T E E is 964. Specific activity is expressed as units per milligram of protein. The specific activities of various chymotrypsin A, preparations vary, largely because of the presence of 10 to 15% inert protein. Based on the determination of the functional normality of several preparations, the author has obtained an average specific activity of 48 units per milligram for fully active enzyme. 2. Assay for chymotrypsin A~ using N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester! 2
Reagents Substrate. N-Benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester (ZTNE) may be obtained from a few commercial sources. Synthesis in the laboratory may be carried out from more readily obtained N-benzyloxycarbonyl-L-tyrosine. The following general procedure may be used to prepare substrates from other blocked amino acids. Dissolve Z-L-tyrosine (1.58 g, 5 millimoles) in 15 ml of anhydrous dioxane containing 1.2 ml of tributylamine. Cool to 5 ° and maintain anhydrous conditions with stirring during the addition of a solution of ethyl chloroformate (0.65 ml in 5 ml of dioxane). After 30 minutes, add nitrophenol (0.70 g, 5 millimoles) and stir for 1 hour. Remove the solvent under vacuum in a rotary evaporator. The residual oil is diluted with chloroform and the solution is washed with cold 1 N HC1, saturated sodium bicar~' C. J. Martin, J. Golubow, and A. E. Axelrod, J. Biol. Chem. 243, 294 (1959).
40
THE SERINE PROTEASES
[3]
bonate, 1 N HC1, and water. Dry the solution over sodium sulfate and remove the solvent under vacuum as before. Add methanol to the oil and induce crystallization of the product. Recrystallize the product from hot chloroform to obtain white material melting at 158-159 ° (uncorr.). Yield, approximately 900 mg, 55%. MW----436. [~]D------16.3 ° (1.0% in acetone). Substrate solution. ZTNE, 1.2 X 10-8 M in dioxane, spectral grade. Dissolve 5.23 mg of substrate in 10 ml of solvent. Methanolic buffer. The complete buffer contains Tris-HC1, 0.03 M, pH 8.0; methanol, 12% (v/v); and CaC12, 0.1 M. Enzyme. A standard enzyme solution is prepared as described in Example 1 above. However, the dilution to be made just before calibration of the assay is 1:2500 with 0.12 M CaC12. Great care must be taken in handling very dilute solutions of the enzyme to prevent loss on the glass surfaces. Calcium ion tends to prevent losses.
Procedure The instrumentation is the same as that described in Example 1 above. Because the substrate undergoes spontaneous hydrolysis at pH 8, it is necessary to determine the rate of this background reaction and subtract it from the assay rate. Add 2.8 ml of buffer to a cuvette plus 0.10 ml of 0.12 M CaC12. Equilibrate the cuvette at 30 ° for 5 minutes in the spectrophotometer and zero the instrument at 400 m~ against water as reference. At zero time add 100 tL1 of the substrate solution, mix thoroughly for 5 seconds, and record the progress of the reaction for 3 minutes. From the initial slope obtain the rate in units of absorbancy change per minute. The assay is performed by the addition of 100 ,td of enzyme solution to 2.80 ml of buffer in a cuvette at 30 °, followed 1 minute later by the addition of 100 td of substrate solution. Since the value of K,~ for this enzymatic reaction is 3.2 X 10-5 M and the substrate concentration is 4.0 X l0 -5 M, the reaction curve is not linear. Estimate the value of the initial slope and obtain the corrected value by subtraction of the rate of spontaneous hydrolysis.
Definition oJ Specific Activity In general, investigators using this method have expressed activities in terms of specific molar rate constants, p-Nitrophenol has a molar extinction coefficient of about 18,700 under assay conditions. Using this value, the specific rate constant at maximum velocity was reported in the original work to be 545 sec -1 (recalculated for enzyme MW = 25,000).
[4]
TRYPSINOGENS AND TRYPSINS
41
The rate constant at a substrate concentration of 4 X 10-SM should therefore be close to 300 sec -1, not far from the value observed in the author's laboratory. However, the difficulties of mixed kinetics and blank corrections are such that all assays should be calibrated with known concentrations of enzyme, using a standard solution of measured functional normality when this is advisable. Comment
The method described above is the most sensitive assay for chymotrypsin that has been developed in detail. As little as 5 m~g can be measured with fair precision. Reproducibility depends greatly upon precise technique. The substrate (ZTNE) is not specific for chymotrypsin. It is known that trypsin and papain both are effective catalysts for the hydrolysis. However, the method provides a useful model for the assay of a serine protease, the identity of which is not in doubt. The method has been found to be particularly useful in studies of inhibitors under conditions which make it necessary to measure very low levels of activity. The substrate, ZTNE, may also be used for assay at pH 5 rather than at pH 8, the point of optimal enzyme activity. At the lower pH spontaneous hydrolysis is negligible relative to enzymatic hydrolysis, and no blank correction is needed. Simplification of procedure is accompanied by loss of sensitivity due to lower enzyme activity and to a lower extinction coefficient for the product, p-nitrophenol. In one investigation~s the following conditions were used: acetate buffer, 0.2M, pH 5.0; 0.65% acetonitrile (v/v); substrate, 1.5 X 10-~M; temperature, 25°; wavelength of measurement, 340 nm (maximum in difference spectrum at pH 5, ac -~ 6014). The value of kca t is given as 1.17 sec -1 and K,~ (app.) as 3 X 10-5 M. 23F. J. K6zdy, A. Thomson, and M. L. Bender, J. Am. Chem. Soc. 89, 1004 (1967).
[4] T r y p s i n o g e n s a n d T r y p s i n s of V a r i o u s Species B y K. A. W ~ s ~
The proteolytic enzyme trypsin and its inactive precursor, trypsinogen, were first obtained in crystalline form from bovine pancreatic tissue by Northrop and Kunitz. 1 Trypsinogen is transformed into trypsin as the i j. H. Northrop, M. Kunitz, and R. M. l=Ierriott, "Crystalline Enzymes," 2rid Ed. Columbia Univ. Press, New York, 1948.
[4]
TRYPSINOGENS AND TRYPSINS
41
The rate constant at a substrate concentration of 4 X 10-SM should therefore be close to 300 sec -1, not far from the value observed in the author's laboratory. However, the difficulties of mixed kinetics and blank corrections are such that all assays should be calibrated with known concentrations of enzyme, using a standard solution of measured functional normality when this is advisable. Comment
The method described above is the most sensitive assay for chymotrypsin that has been developed in detail. As little as 5 m~g can be measured with fair precision. Reproducibility depends greatly upon precise technique. The substrate (ZTNE) is not specific for chymotrypsin. It is known that trypsin and papain both are effective catalysts for the hydrolysis. However, the method provides a useful model for the assay of a serine protease, the identity of which is not in doubt. The method has been found to be particularly useful in studies of inhibitors under conditions which make it necessary to measure very low levels of activity. The substrate, ZTNE, may also be used for assay at pH 5 rather than at pH 8, the point of optimal enzyme activity. At the lower pH spontaneous hydrolysis is negligible relative to enzymatic hydrolysis, and no blank correction is needed. Simplification of procedure is accompanied by loss of sensitivity due to lower enzyme activity and to a lower extinction coefficient for the product, p-nitrophenol. In one investigation~s the following conditions were used: acetate buffer, 0.2M, pH 5.0; 0.65% acetonitrile (v/v); substrate, 1.5 X 10-~M; temperature, 25°; wavelength of measurement, 340 nm (maximum in difference spectrum at pH 5, ac -~ 6014). The value of kca t is given as 1.17 sec -1 and K,~ (app.) as 3 X 10-5 M. 23F. J. K6zdy, A. Thomson, and M. L. Bender, J. Am. Chem. Soc. 89, 1004 (1967).
[4] T r y p s i n o g e n s a n d T r y p s i n s of V a r i o u s Species B y K. A. W ~ s ~
The proteolytic enzyme trypsin and its inactive precursor, trypsinogen, were first obtained in crystalline form from bovine pancreatic tissue by Northrop and Kunitz. 1 Trypsinogen is transformed into trypsin as the i j. H. Northrop, M. Kunitz, and R. M. l=Ierriott, "Crystalline Enzymes," 2rid Ed. Columbia Univ. Press, New York, 1948.
42
THE SERINE PROTEASES
[4]
result of the cleavage of a single peptide bond (Lys6-IleT) near the N-terminus of the zymogen, 2 and the appearance of activity is accompanied by conformati0nal changes2 The activation process is catalyzed by a variety of enzymes including enterokinase, ~ mold proteases, 5 and trypsin itself. The latter autocatalytic process is accelerated by calcium ions which bind to the N-terminal region of the zymogen and promote the specific bond cleavage2 ,7 A striking feature of the enzyme is the narrow specificity of its action, which is almost exclusively directed toward L-lysyl and L-argininyl bonds of polypeptides. Biologically, trypsin serves as the activator of all the other zymogens of pancreatic tissue. Thus, the control of the activation of trypsinogen has broad consequences in terms of the formation of the endopeptidase and exopeptidase components of pancreatic juice. Methods of Assay of T r y p s i n The substrates of trypsin can be described by the general formula, R - - C O - - X , where the narrow specificity of trypsin is determined by the acyl moiety ( R - - C O - - ) and the type of bond cleaved (i.e., peptide, amide, or ester) is defined by the nature of X. M a n y assay procedures have been described using each type of catalysis. In addition, by the appropriate choice of R and X, the deacylation step of hydrolysis can be slowed so that the initial release of X is a measure of the available active sites. Such compounds serve as active site titrants 8-11 which define the true concentration of functional active sites. Esterase Activity
The most widely used assays for trypsin follow the esterase activity toward benzoyl-L-arginine ethyl ester (BAEE) 12,1~ or p-toluenesulfonylL-arginine methyl ester (TAME)1~ by potentiometric 12 or spectrophoto' E. W. Davie and H. Neurath, J. Biol. Chem. 212, 515 (1955); P. Desnuelle and C. Fabre, Biochim. Biophys. Acta 18, 49 (1955). ' H. Neurath and G. H. Dixon, Federation Proc. 16~ 791 (1957). 4M. Kunitz, J. Gen. Physiol. 22, 429 (1939). cf. T: Hofmann, this volume [25]. e T. M. Radhakrishnan, K. A. Walsh, and H. Neurath, Biochemistry 8~ 4020 (1969). 7j. p. Abita, M. Delaage, M. Lazdunski, and J. Savrda, European J. Biochem. 8, 314 (1969). 8M. L. Bender and F. J. K~zdy, J. Am. Chem. Soc. 87, 4953 (1965). 9T. Chase, Jr. and E. Shaw, Biochem. Biophys. Res. Commun. 29~ 508 (1967). ~D. T. Elmore and J. J. Smyth, Biochem. J. 107, 97, 103 (1968). I~R. J. Vaughan and F. H. Westheimer, Anal. Biochem. 29~ 305 (1969). See K. A. Walsh and P. E. Wilcox, this volume [3]. G. W. Sehwert and Y. Takenaka, Biochim. Biophys. Acta 16, 570 (1955). 14B. C. W. Hummel, Can. J. Biochem. Physiol. 37~ 1393 (1959).
[4]
TRYPSINOGENS AND TRYPSINS
43
metric procedures. Potentiometric measurement of the specific esterase activity toward BAEE under standardized conditions is described in this volume (p. 37). An alternative spectrophotometric method using TAME 14 has the dual advantage over BAEE of greater sensitivity and of greater selectivity (chymotrypsin does not hydrolyze TAME), and is described below. A buffer solution [0.04M Tris (hydroxymethyl aminomethane), 0.01 M CaC12, pH 8.1] is first prepared by dissolving 1.47 g of CaC12.2 H~O in 200 ml of 0.2M Tris, adjusting the pH to 8.1 with HC1, and diluting to 1 liter. Then 19.7 mg of TAME.HC1 is added to 50 ml of buffer to yield a substrate concentration of 1.04 X 10-~ M. The assay procedure employed is exactly analogous to that described for the assay of chymotrypsin A~ with N-benzoyl-L-tyrosine ethyl ester in this volume (p. 38). Three milliliters of substrate solution are measured into each cuvette of the spectrophotometer. Allow 3 minutes for temperature equilibration to be reached in the compartment a t 30 ° . Balance the absorbance of the two cuvettes at 247 nm. Add 0.1 ml of water to the reference cuvette and mix well. At zero time add 0.1 ml of enzyme solution (containing up to 0.45 ~g of trypsin) to the sample cuvette and mix well. The rate of increase of absorbance should be linear up to an absorbanee of 0.24 (65% hydrolysis) and this rate is directly proportional to the concentration of the enzyme. One unit of TAME activity is defined as the amount of trypsin catalyzing the hydrolysis of 1 micromole of TAME per minute. The absorbance change accompanying hydrolysis of 1 mieromole of TAME per milliliter of assay solution is 0.409 cm-1 mM -~. Trypsin, at a concentration of 0.1 ~gfml in the assay cuvette results in an absorbance change of 0.0101 absorbance units (min-~.cm-~). Thus, 1.0 mg of trypsin is equal to 247 TAME units.
Proteolytic Activity Toward Casein A method of assaying trypsin by its proteolytic activity toward casein has been described in detail by Laskowski.15
Amidase Activity Amidase activity can be determined with benzoyl-L-argininamide by the manual method of Schwert et al. TM as outlined in Vol. II [2] or by its adaptation to the automatic analytical procedure described by Lenard M. Laskowski, in Vol. II [2]. G. W. Schwert, H. Neurath, S. Kaufman, and J. E. Snoke, J. Biol. Chem. 172, 221 (1948).
44
THE SERINE PROTEASES
[4]
et al. 17 Alternatively, the colorimetric procedure of Erlanger et al. is using benzoyl-DL-arginine p-nitroanilide provides a sensitive amidase assay. A c t i v e Site Titration with p - N i t r o p h e n y l , p'-Guanidinobenzoate (NPGB) 9
This method is based on the rapid and stoichiometrie p-guanidinobenzoylation of trypsin by NPGB as measured by the extent of liberation of p-nitrophenol. An aliquot of a trypsin solution is diluted with 0.1 M Veronal buffer, pH 8.3, containing 0.02 M CaC12 to give 0.99 ml containing 0.05-2.4 mg of trypsin (2-100 X 10-6M in trypsin). This is placed in a 1-ml cuvette (1-cm light path) in a double-beam recording spectrophotometer and the instrument balanced at 410 nm against a reference cuvette containing buffer alone. Ten microliters of a 0.01 M solution of NPGB in dimethylformamide are added to the reference cuvette and the contents thoroughly mixed. This procedure is repeated with another 10-~l aliquot of the NPGB solution in the sample cuvette at zero time. The optical density is followed for 3-5 minutes and a very slow liberation of p-nitrophenol extrapolated back to zero time to give a measure of the initial burst of p-nitrophenol. The increase of absorbance above baseline during the initial burst multiplied by 6.025 X 10-5 yields the molarity of liberated p-nitrophenol (and therefore the molarity of active trypsin). Although this method is less sensitive than the rate assays described above, it has the inherent advantage of providing an absolute standardization of the concentration of active enzyme and avoiding the uncertain variables involved in rate assays. Purification of Trypsinogen The classical purification .of bovine trypsinogen by Kunitz and Northrop 19 involves acid extraction of fresh pancreas, ammonium sulfate fractionation, and removal of a-chymotrypsinogen as a crystalline byproduct. This is described in detail in Volume II [2]. Subsequent purification and crystallization of trypsinogen is described in detail in Volume II [3] and involves treatment of the mother liquor and washings from the crystallization of chymotrypsinogen with acidic 70% saturated ammonium sulfate to form a precipitate of crude trypsinogen. Two alternate methods of crystallization of trypsinogen are then possible. ~7j . Lenard, S. L. Johnson, R. W. Hyman, and G. P. Hess, Anal. Biochem. 11, 30
(1065). ~B. F. Erlanger, N. Kokowsky, and W. Cohen, Arch. Biochem. Biophys. 95, 271 (1961). ~*M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936).
[4]
TRYPSINOGENS AND TRYPSINS
45
Crystallization Procedure A
A method is outlined in detail by Laskowski in Volume II [3] involving MgS04 at pH 8.0 and yields short triangular prisms of trypsinogen containing approximately 50% MgS04 by weight. It should be noted that autocatalytic activation of trypsinogen is avoided during this procedure because pancreatic trypsin inhibitor is present during the crystallization. Attempts to recrystallize trypsinogen by this procedure result in autocatalytic activation and loss of the zymogen, although recrystallization has been accomplished by Tietze ~° using 0.01 M diisopropylphosphofluoridate (DFP) at pH 9 as described in Volume II [3]. Crystallization Procedure B
An alternate method of crystallization in ethanol-water mixtures at 4 ° is described by Balls. 21 In this procedure, 100 g of the crude trypsinogen precipitate formed in acidic 70% saturated ammonium sulfate (above) are dissolved in 300 ml of water and 105 ml of 95% ethanol. The pH is adjusted to 7.5 v¢ith 5 N NaOH. After standing for 30 minutes, the solution is filtered through Celite on a large sintered glass funnel. The volume of the filtrate is measured and to each 100 ml are added 14 ml of 95% ethanol. The filtrate is seeded (if possible) with trypsinogen crystals and stored at 4 °. After about 3 days the crystals are collected on hardened filter paper, washed with 100 ml of cold 33% ethanol, then (very rapidly) with ethanol, 1:1 ethanol-ether and finally with ether. The crystals can be dried in air and in vacuo at 55 ° over anhydrous calcium sulfate. The yield (about 11 g from 100 g of "crude trypsinogen precipitate") can be increased by 20% by leaving the alcoholic filtrate (not including the washings) at 4 ° for several more days. Further recrystallizations must be carried out at low pH since pancreatic trypsin inhibitor is no longer present. This is accomplished by suspending 1 g of dried crystals in 100 ml of 25% ethanol at 4 °. The pH is adjusted to 3.5 with 1 N HC1 and stirred occasionally for 20 minutes. Insoluble material is removed by centrifugation and discarded. Fourteen milliliters of 95% ethanol is added to raise the concentration of ethanol to 33%. This is followed by the addition of 1 ml of saturated ammonium sulfate per 100 ml of solution and the pH adjusted to 7.5 with 2 N NaOH. Traces of precipitate are rapidly removed by centrifugation, the supernatant is seeded with crystals of trypsinogen and left for 24 hours at 4 °. Crystals can be collected by filtration on hardened paper and washed with cold 33% ethanol. This recrystallization procedure can then be repeated if desired. ~F. Tietze, J. Biol. Chem. 204, 1 (1953). ~'A. K. Balls, Proc. Natl. Acad. Sci. U.S. 53, 392 (1965).
46
THE SERINE PROTEASES
[4]
Crystalline trypsinogen obtained by either procedure A or procedure B still shows minor heterogeneity during ion-exchange chromatography. A highly purified product can be obtained by chromatography on SESephadex at pH 4.2.
Chromatography o] Trypsinogen 22 SE-Sephadex is prepared by swelling 15 g of C-50 grade in three changes of 2 liters each of chromatography buffer (0.20M NaC1, 0.05 M acetic acid at pH 4.20 with NaOH at room temperature) for 1 day. Do not stir the suspension with a magnetic mixer during any of the preparatory steps as the gel beads are fragile and crush easily, resulting in poor flow rates. After swelling the SE-Sephadex, allow a slurry of the ionexchanger to settle in a graduate cylinder for 30 to 45 minutes. Remove the slower sedimenting particles with an aspirator. Repeat until all the fine particles are removed as judged by a sharply sedimenting boundary. Failure to do this results in poor flow characteristics. Check that the pH of the suspension is 4.20. Suspend the ion-exchanger in about 1 liter of buffer and pour the slurry into a 50-cm chromatography column (2.5 cm I.D.) with a nylon net support. Allow the gel slurry to settle under gravity for about 5 minutes, then, with the outlet open, allow the remainder of the slurry to settle with no more than 30 em of water pressure between the top of the buffer in the column and the end of the outlet tube. Allow the remainder of the slurry to settle. Continue to add slurry until the gel bed is 40 cm long. Place the column at 4 ° and connect to the outlet tube a peristaltic pump adjusted to deliver about 25 ml/hour. (Note: Under no circumstances should the buffer be pumped into the top of the column since it leads to packing of the gel bed and obstruction of flow.) Ideal flow behavior is obtained when the buffer reservoir and pump are at the same height. Allow the column to equilibrate with flowing buffer for 24 hours at 4 °, at which time the pH and the ionic strength of the ettiuent at room temperature should be identical with those of the chromatography buffer. Dissolve 200 mg of trypsinogen crystals obtained by procedure A or 100 mg obtained by procedure B in 7 ml of cold 10-2 M HC1 at 0% The solution should contain about 14 mg of trypsinogen per milliliter in either case. Dialyze overnight against 4 liters of 10-3 M HCI at 4 ° to remove the salts occluded in the crystals. Remove the trypsinogen solution from the dialysis sac, add dropwise with stirring 1.0 ml of 2 M NaC1 in 0.5 M acetic acid (at pH 4.20 with NaOH) and dilute to 10.0 ml with water. Remove traces of insoluble material by centrifugation and adjust the pH to exactly 4.20. m N. Robinson, M. Sanders, and K. A. Walsh, unpublished experiments.
[4]
TRYPSINOGENS AND TRYPSINS
47
This trypsinogen solution is applied to the top of the gel bed by opening the column, removing the buffer by aspiration, and adding the solution by pipette without stirring up the top of the packed gel bed. After this sample flows into the gel bed, it is washed in with several 2-ml washes of buffer and the column top is connected to the chromatography buffer which is pumped at 25 nil per hour into a fraction collector. Fivemilliliter fractions are collected and examined at 280 nm for protein. Trypsinogen begins to elute as a symmetrical peak in about 12 hours. It appears after traces of contaminants and before contaminating trypsin. The contents of tubes containing the trypsinogen are pooled, the pH adjusted to 3.0, and the solution dialyzed for 24 hours at 4 ° against two changes of 4 liters each of 10-s M HC1. The trypsinogen solution can then be lyophilized and stored at --20 °. Trypsinogen Purification ]rom Other Species
Trypsinogens have been prepared from rat, 2s goat, 24 sheep, 25 pig, ~6 horse, ~ and dogfish28 pancreas as well as from bovine pancreas. The procedures involve ion-exchange chromatography techniques and yield in all cases, material sufficiently pure for characterization of their chemical and physical properties. Several of these fractionation procedures have been summarized by Marchis-Mouren. ~Sa Properties of Trypsinogen
Bovine trypsinogen prepared as described above contains less than 0.2% contaminating trypsin. It has 229 amino acid residues and a molecular weight of 23,991. 29 It is stable at low pH and can be stored either as a lyophilized powder or as a frozen solution in 10-s M HC1. The amino acid sequence has been described and is given in Table I and Fig. 2. Activation of the zymogen to form trypsin involves the specific cleavage of the Lys6-Ile~ bond near the N-terminus. 2 The physical properties of trypsinogen are summarized together with those of trypsin in Table V. A. Vandermeers and J. Christophe, Biochim. Biophys. Acta 188, 101 (1969). ~ S. Bricteux-Gr~goire, R. Schyns, and M. Florkin, Arch. Intern. Physwl. Biochim. 76, 571 (1968). ~S. Bricteux-Gr~goire, R.. Schyns, and M. Florkin, Biochim. Biophys. Acta 127, 277 (1966). M. Charles, M. Rovery, A. Guidoni, and P. Desnuelle, Biochim. Biophys. Acta 69, 115 (1963). 2~C. I. Harris and T. Hofmann, Biochim. J. 114, 82P (1969). R. Haynes, W. P. Winter, R. Tye, and H. Neurath, personal communication, 1968. ~ G. Marchis-Mouren, Bull. Soc. Chim. Biol. 47, 2207 (1965). K. A. Walsh and H. Neurath, Proc. Natl. Acad. Sci. U.S. 52, 884 (1964).
48
THE SV.RIN~, PROT~,AS~,S
[4]
Autocatalytic Activation of Bovine Trypsinogen 3° One hundred milligrams of trypsinogen is dissolved in 4 ml of 10 -2 M HC1 and dialyzed against 10-3 M HC1 at 4 ° to remove any salts. The absorbance at 280 nm is measured (with an aliquot diluted 1:20 with 10-a M HC1) to insure that the trypsinogen concentration is in the range 10-20 mg/ml ~l~mlw1%----15.0). The volume is measured, the solution chilled to 0 ° and an equal quantity of cold 0.2M Tris buffer, pH 8.1, containing 0.1 M CaC12 is added. The addition of trypsin to a concentration of about 0.5 mg/ml (1:20 weight ratio of trypsin:trypsinogen) initiates the autocatalytic activation. The progress of trypsinogen activation can be readily followed using 5-25 /~l aliquots in the BAEE assay procedure. TM Maximum activity is seen in about 90 minutes and this is followed by a slow loss in activity, presumably the result of autolysis. To express the results of an absolute basis, the concentration of trypsin added to initiate the activation must be subtracted. The best preparations of trypsinogen give more than 95% of the stoichiometrie yield of trypsin.
Nature o/ the Activation Process During the activation of trypsinogens from all species examined, a highly charged N-terminal peptide with the sequence X-Asp4-Lys is TABLE I N-TERMINAL SEQUENCES OF VARIOUS TRYPSINOGENS
Species Cow a
Goat a Sheep c Pig d Horse" Dogfish/
N-Terminal sequence Val-Asp-Asp-Asp-Asp-LysVal-Asp-Asp-Asp-Asp-Lyst Val-Asp-Asp-Asp-Asp-LysPhe-Pro-Val-Asp-Asp-AsI>-Asp-LysPhe-Fro-Thr-Asp-Asp-Asp-Asp-LysSer-Ser-Thr-Asp-Asp-Asp-Asp-Lys~.la-Pro-Asp-Asp-Asp-Asp-Lys
a E. W. Davie and H. Neurath, J. Biol. Chem. 212, 515 (1955). b S. Bricteux-Grdgoire, R. Schyns and M. Ylorkin, Arch. Intern. Physiol. Biochim. 76, 571 (1968). " S. Bricteux-Grdgoire, R. Sehyns, and M. Florkin, Biochim. Biophys. Acta 127, 277 (1966). d M. Charles, M. Rovery, A. Guidoni, and P. Desnuelle, Biochim. Biophys. Acta 69, 115 (1963). C. I. Harris and T. Hofmann, Biochem. J. 114, 82P (1969). ] Quoted by H. Neurath, R. Arnon, and R. A. Bradshaw, Proc. Symp. StructureFunction Relationships of Proteolytic Enzymes, Munksgaard, Copenhagen, in press, 1969. ~oj. F. Pech~re and H. Neurath, J. Biol. Chem. 229, 389 (1957).
[4]
TRYPSINOGENS AND TRYPSINS
49
cleaved from the N-terminus to expose the N-terminal isoleucyl residue of trypsin. 2,24-27,21 The results of these studies are summarized in Table I. Calcium ions promote the activation reaction by binding to the N-terminal hexapeptide of bovine trypsinogen and accelerating the specific bond cleavage process. 6,7 A conformational change accompanies the appearance of activity 3 and it is not known whether this reflects an essential step in the activation or an incidental step accompanying the activation. By analogy with the activation of chymotrypsinogen, 32 it is generally anticipated that the homologous Ile-Val-Gly sequence at the N-terminus of trypsin may form a salt link with Asp18~ as an integral part of a refolding process resulting in the assembly of components of the active site. Purification of Trypsin Bovine trypsin is readily prepared from trypsinogen by autocatalytic activation and crystallization procedures. 19 The yield was improved by McDonald and Kunitz, 3~ who showed that the inclusion of calcium ions in the activation mixture minimized a side reaction leading to the formation of "inert protein." The starting material for trypsin purification is the "crude trypsinogen" obtained as a by-product of the chymotrypsinogen preparation 19 as described in detail by Laskowski in Volume II [2]. A detailed description of the procedure for crystallizing and recrystallizing trypsin is outlined by Laskowski in Volume II [3]. The crystalline product can be stored at 4 ° as a dry powder. The maximum specific activity observed, expressed in units of BAEE activity (this volume, p. 39) is 70 units per milligram. Preparations of crystalline bovine trypsin have been found to contain variable quantities of at least five molecular species, apparently resulting from partial autolysis (Table II). Functionally, these species may be completely active, partially active, or inert. Several chromatographic methods have been described to fractionate this mixture and purify the enzyme species (Table III). Of these, the method of Maroux, Rovery, and Desnuelle ~4 effectively removes chymotrypsin and inert proteins from trypsin and is described in detail in Volume XI [25]. Higher resolution of the individual components of partially autolyzed trypsin is given by the equilibrium chromatographic procedure of Schroeder and ~1Quoted in H. Neurath, R. Amon, and R. A. Bradshaw, Proc. Syrup. StructureFunction Relationships of Proleolytic Enzymes, Munksgaard, Copenhagen, in press, 1969. 32p. B. Sigler, I). M. Blow, B. W. Matthews, and R. Henderson, J. MoI. Bial. 35, 143 (1968). ~8M. R. McDonald and M. Kunitz, J. Gen. Physiol. 29, 155 (1946). 34S. Maroux, M. Rovery, and P. Desnuelle, Biochim. Biophys. Acta 56, 202 (1962).
50
~E
s ~ . m ~ PROrSAS~.S
[4]
T A B L E II NATURE OF AUTOLYSIS PRODUCTS OF TRYPSIN Internal bond cleaved
Nomenclature assigned
Active toward •
I n h i b i t e d Unreactive by ~ with~ Reference
None Lys4~-Sere Arg,06-Vall06
B-Trypsin ---
All substrates -BAEE
Lys,,1-Serl,,
a-Trypsin
BANA TAME BAEE BANA d BAEE d
Lysl~6-ASl~ and Pseudotrypsin Lysm-Sen,,
All inhibitors -TLCK DFP NPGB DFP TLCK TLCK DFP
TLCK
b c ,
--
b
TLCK
•
Abbreviations: BANA, N-a-benzoyl-DL-arginine p-nitroanilide; BAEE, N-benzoylL-arginine ethyl ester; T A M E , p-toluenesulfonyl-L-arginine methyl ester; TLCK, 1-chloro-3-tosylamido-7-amino-2 heptanone; DFP, diisopropylphosphofluoridate; NPGB, p-nitrophenyl, p'-guanidinobenzoate. D. D. Sehroeder and E. Shaw, J. Biol. Chem. 243, 2943 (1968). c S. Maroux and P. Desnuelle, Biochim. Biophys. Acla 181, 59 (1969). d The activity is greatly diminished from that of B-trypsin. , R. L. Smith and E. Shaw, J. Biol. Chem. 244, 4704 (1969).
TABLE III CHROMATOGRAPHIC SYSTEMS FOR THE PURIFICATION OF BOVINE TRYPSIN
Ion-exchanger
pH
Component preventing autolysis during chromatography
Carboxymethyl cellulose IRC-50 Carboxymethyl cellulose Carboxymethyl cellulose SE-Sephadex SE-Sephadex SE-Sephadex
3.2 6.1 6.0 5.5 2.6 7.1 3.0
Low pH 8 M urea Low pH and calcium Low pH and calcium Low pH Benzamidine Low pH
Reference o b d ° ! g
a I. E. Liener, Arch. Biochem. Biophys. 88, 216 (1960). b R. D. Cole and J. M. Kinkade, J. Biol. Chem. 236, 2443 (1961). c S. Maroux, M. Rovery, and P. Desnuelle, Biochim. Biophys. Acta 56, 202 (1962) ; Vol. X I [25]. d S. Maroux and P. Desnuelle, Biochim. Biophys. Acta 181, 59 (1969). ° S. Papiannou and I. E. Liener, J. Chromatog. 32, 746 (1968). ] D. D. Schroeder and E. Shaw, J. Biol. Chem. 243, 2943 (1968). o j. G. Beeley and H. Neurath, Biochemistry 7, 1239 (1968).
[41
TRYPSINOGENS AND TRYPSINS
51
Shaw35 which separates a-trypsin from fl-trypsin (see Table II for definition of these species).
Chromatography o] Trypsin 85 A cation-exchange column (2.2 X 85 cm) of SE-Sephadex (C-50 beaded) is prepared and equilibrated with chromatography buffer containing 0.1 M Tris chloride, 0.02 M CaC12, 10-3 M benzamidine at pH 7.1. Precautions to be observed during equilibration and packing of the column are described above in the section outlining the chromatography of trypsinogen. The flow rate is adjusted to 15-25 ml per hour at 4 ° with as little hydrostatic pressure head as possible and 9-10 ml fractions are collected. Since the chromatographic procedure is very long (5-7 days), autolysis after chromatography is excluded by acidifying the collected fractions. This can be accomplished either by providing each fraction collector tube with 0.4 ml of 1.25 M potassium formate, pH 2.9, and a mixing device or by pumping this pH 2.9 buffer into the effluent stream via a T-tube at the rate of about 1 ml per hour. The sample of trypsin is prepared by dissolving 250 mg in 25 ml of the chromatography buffer. (If the trypsin preparation contains occluded salts, they should be removed by dialysis against 4 liters of chromatography buffer at 4°). Any insoluble material should be removed by centrifugation at about 2000 g at 4 °. The clear supernatant is then applied to the column and the chromatogram developed with 3 liters of buffer. The effluent is monitored at 280 nm (measured against a blank of chromatography buffer since benzamidine absorbs light at 280 nm). A peak of inert protein appears during the first day of chromatography, a-trypsin during the fourth day, and ~-trypsin during the fifth day ~(at 20 ml per hour). Pooled fractions of a-trypsin or fl-trypsin can be concentrated to about 100 ml each by adsorption at 4° on a small SESephadex C-50 column (4.4 X 4 cm) previously equilibrated with 0.05 M Tris-formate, pH 3.0, and elution (at about 1 liter per hour) with 0.5 M Tris-chloride, pH 7.6, containing 0.02M CaCI~, 10-3M benzamidine. The concentrated trypsin fractions are then brought to pH 3 with 2 M HC1, dialyzed against 10-~ M HC1, and lyophilized.
Trypsin Purification ]rom Other Species Trypsin and trypsinlike enzymes have been prepared from 14 animal species and microbiological organisms largely by chromatographic procedures. The amino acid compositions of this related group of enzymes u D. D. Schroeder and E. Shaw, J. Biol. Chem. g43, 2943 (1968).
52
~s
C) g~ ~D
z
SER~SZ PaOTE.~SES
OQ
[4]
I4 ]
TRYPSINOGENS AND TRYPSINS
53
54
sEaI
P O ASES
[4]
are summarized in Table IV. Porcine trypsin was obtained as a crystalline product 8e,3~ and has been characterized in greatest detail. Properties of Trypsin The chemical, physical, and enzymatic properties of trypsin have been reviewed by Desnuelle 8s in 1960 and by Cunningham89 in 1965. Only certain selected features of the enzyme and its zymogen will be mentioned here as a guide to the general characteristics of trypsin. Stability O] Trypsin
Trypsin is stable at pH 3 at low temperatures where it can be stored for weeks without loss of activity. It can be reversibly heat-denatured ~° and the effect of pH on the temperature-induced reversible denaturation of both bovine trypsinogen and bovine trypsin has been studied by Lazdunski and Delaage. 41 Their state diagrams (Fig. 1) reveal that four different forms of the enzyme can be distinguished at low temperatures and reversible transformations between these forms are controlled by pH. Below pH 8 an increase in temperature results in reversible denaturation, whereas above pH 8 elevated temperatures induce irreversible denaturation. A similar state diagram is seen for bovine try psinogen. Porcine trypsin is more stable than bovine trypsin to thermal denaturation.'2 Trypsin loses enzymatic activity reversibly in high concentrations of denaturants such as 8 M urea. However, Harris has shown that the enzyme retains about 50% activity toward TAME in 4 M urea ~3 where there is apparently an equilibrium mixture of native and denatured enzymes. In 4 M urea the enzyme activity is irreversibly lost as active enzyme attacks its denatured counterpart. Delaage and Lazdunski have examined the susceptibility of the different forms of trypsin (Fig. 1) to urea denaturation and have shown that substrates or competitive inhibitors can protect trypsin from denaturation.~' The addition of calcium ions to trypsin solutions retards autolysis. Nj. Travis and I. E. Liener, J. Biol. Chem. 240, 1962 (1965). JTp. j. VanMelle, S. H. Lewis, E. G. Samsa, and R. J. WestfaU, Enzymologia 26, laa (1963). rap. Desnuelle, in "The Enzymes," (P. D. Boyer, H. Lardy, and K. Myrb~ck, eds.), Vol. 4, p. 119. Academic Press, New York, 1960. ~L. Cunniugham, in "ComprehensiveBiochemistry," (M. Florkin and E. H. Stotz, eds.) Vol. 16, p. 85. Elsevier Press, Amsterdam, 1965. M. L. Anson and A. E. Mirsky, J. Gen. Physiol. 17, 393 (1934). M. Lazdunski and M. Delaage, Biochim. Biophys. Aeta 140, 417 (1967). *~M. La~dunski and M. Delaage, Biochim. Biophys. Aeta 105, 541 (1965). a j . I. Harris, Nature 177, 471 (1956). M. Delaage and M. La~dunski, European J. Bioehem. 4, 378 (1968).
[4]
TRYPSINOGENS AND TRYPSINS
T(°C}
55
~
(a)
80 70 60 50 40 30 20 I0 0
I
2
~
4
5
6
-f
pH
8
9
I0
II
T(°C)
(b)
80
70 60 50 40
12
y
~
30 20 I0 0
2
3
4
5
6
7 pH
9
I0
II
12
Fie. 1. State diagrams of bovine trypsinogen and trypsin.~ The figures are separated into two regions by a hatched line, I', II', III', D' for trypsinogen, and I, II, III, D for trypsin represent the different molecular forms in equilibrium at acidic pH's. (Q) Spectrophotometric studies at constant pH (the inflection points of the Ae=~=m-temperature curves). ([E) Spectrophotometric studies at constant temperature (the inflection points of Ae==~, A~==, or A~nm-pH plots). ( A ) Spectropolarimetric studies. (X) Activity measurements. The curves represent, in fact, isoconcentration lines of two adjacent forms. 0nly one alkaline transition is represented in the diagrams at alkaline pH. I t is mainly due to the reversible unmasking of buried tyrosines. This unmasking is rapidly followed by an irreversible inactivation due to attack of hydroxyl ions on the disulfide bridges. Reproduced from Biochim. Biophys. Acta 140, 422 (1967).
This stabilizing effect is accompanied by a conformational change in the trypsin molecule, apparently induced by calcium and resulting in a more compact structure. 42 The protective effect of calcium ions is much more
56 Horse h S. O r i s e u s g Turkey f PorcineC, d
Dogfish e
THe. S~.aINE eROTEAS~S
[4]
ILE ILE ILE (ILE,VAL,GLY,GLY,TYR,GLX,CYS,PR0,LYS)ASX(TH~,VAL,Pfl0,TYR,HIS,GLY,ARG)
SER-MET-GLY-CYSASX-SER-GLY-TYR-HIS-PHE-CY$ASN-SER-GLY-$ER-HIS-PHE-CYSASX-VAL-GLY-TYR(HIS,PHE,CY$,
8ovinea, b
~LE-~AL~GLY-GLY-TYR-THR~CYS-GLY-ALA-A~N~THR-VAL-PR~TYR~GLN~VAL~ER-LEU-A~N-~ER~GL~.TYR~H~-PHE~CY~.
S, G r l s e u s g Dogfish e
GLY-GLY-ALA-LEU GL¥-GLX-SER-LEU GLY-GLY-SER-LEU GLY,GLY,SER)LEU,ILE-HIS-GLX-GLX
Bovlnea, ~
GLY••LY-•ER-LEU••LE•A•N•$ER••LN-TRP•VAL•VAL-•ER-ALA•ALA-H••-CYS•TYR-L••-•ER-GLY-•LE-GLN°VAL-ARG•LEU
7 Turkey f Porcine c
10
20 THR-ALA-ALA-HIS-CYS-VAL(ASN,ASN,GLY,GLY,SER,SER) ALA-ALA-HIS-CY$-TYR-LYS ALA-ALA-HIS-CYS-TYR-LYS iLE-GLN-VAL-ARG,LEU~O
Turkey f Porclne c
Dogfish e Bovine e Bovine ° Turkey F Porclne c
Dogfish e 8ovlne 4 Bovine b
Oogflsh e
o
50
ALA-LEU-THR-HIS-PRO-ASXGLY-GLX~ASX-ASX-ILE-VAL-LEU ILE-ILE-THR-HIS-PR0-ASNGLY-~t~H~-A~x-~LE-~R-ALA-A$x(GL~GL~AS~THR~GLx)TYR-~LE-A$P-$ER-SER~NET(VAL~LE)A~G'H~S-PR~A~x GLY-•LU-A$P•A$N-iLE-A•N•VAL-VAL-GLU••LY•ASP-•LU-GLN-PHE••LE-•ER•ALA-SE••LY••$ER••LE•VAL•••••PR•-•ER• GLN 60 ASN-GLN 70 80 TYR PHE ~($~R~A~X~LEU~GL~T~R`A~A~N~"~LE~T'LEU~LE)L~(LEU.L~)PR~-ALA-ALA-~R-LEU-A~X-ARG~A~P-VAL~ T•R-ASN•F•••ASN•THR•LEU)ASN-•SN-ASP-•LE-•ET-LEU-•LE-L•$-LEU-•YS-SER-ALA-ALA-SER-LEU-ASN-•ER•A•G-VAL-SER-ASN-THR-LEU" 90 I00
Bovinea, b
ASN-LEU-~LE-~ER-L~U-PR~-T~R-GL~-CY~ALA-TYR-ALA-~ET(GL~GLX)C~LEU-~LE-SER-GL~TRP-GLY(A~x.TH~A~ ALA-$~R-~LE-SER-LEU-PR~-THR-$ER-CYS-ALA-$ER-ALA-GLY-THR-GLN-¢Y$-LEU-~LE~SER-GLY-TRP~GLY-A~N-THR-L~I10 120 130
S. G r i s e u s g Do9fish e Bovlne a Bovlne b
ALA-ALA-CY$-ARG-SERGLY)HET-ALA-VAL-SEfl"GLY'ASP-GLN-LEU-GLN-CYS"LEU-ASP'ALA"PR0"VAL(ALA ,ASP-LEU,GLU. SER)CYS-LY$ ,GLY5ER-SER-GLY'THR-SER-TYR"PR0-ASP'VAL'LEU'LYS- CYS'LEU-LYS-ALA-PRO-I LE- LEU-5ER-ASP-SER-$ER-CYS-LYS- SERItIO 150 ASH
S. G r i s e u s 9
ALA'TYR-GLY-ASN'GLU
Porcine c
DogFish e 8ovine a Bovine b
$. G r i s e u s g Porcine c
DogFish e 8ovinea, b
Horse h S. G r i s e u s g Turkey f Porcine d Dogfish e Bovinea, b
GLU'ILE'CYS'ALA-GLY'TYR+ASP'THR'GLY-GLY-VAL-ASP-THR-CYS-GLN-GLY" ASN-SER-CY$-GLN-GLYALA-T~R-PR~GL~(A~N~[L~'THR~HET)A~N~h~T-HE~-~Y~-~AL~GL~-T~R~ET~GLU-GL~-GLY-L~-ASP*~(R-C~(GLN'GLY~ A~-TYR-~-GL~GLN~LE-THR-~ER~A~N~ET-P~E-~°A~A-GLy-~-LEU-G~U-G~-G~-~Y~-A~-~ER-~-GL~G~160 170 ASN 180 ASP-SER-GLY'GLY'PRO°HET-PHE TYR-GLY-CYS-ALA-ARG-PR0ASP-SER-GLY-GLY-PRO-VAL-VAL-CYS-GLY-GLN-GLN°LEU ASP.$ER,GLY,GLY,PR0,VAL)VAL'CY$ ASP••ER-GLY-GLY-PR•-VAL-•AL•CY•-SER-GLY-L••-LEU-GLN•GLY-•LE•VAL•SE•-TRP-GLY-•ER-GLY-•Y$•ALA-GLN-LYS190 200 (GLN,THR,ILE,ALA)ALA-ASN GLY-TYR-PRO-GLY-VAL SER-ASN THR(ILE,GLN)ALA'ASN THR-ILE-ALA(SER,ALAz) A$N-LYS-PR~-GLY-VAL-TY~-TH~-LY~-VAL-CYS-ASN~T~R-VAL-$ER-TRP-~LE-LY~-GLN-THR-~E-ALA-~ER-A~N 210
220
FIG. 2. Partial and complete amino acid sequences of various trypsins. The residue numbering is that for bovine trypsinogen. The structure of bovine trypsinogen was elucidated by two independent groups" b which diffeP only where indicated. Disulfides of bovine trypsin are paired I as follows: 13-143; 31-47; 115-216; 122-189; 154-168; 179-203. a. Bovine--K. A. Walsh and H. Neurath, Proc. Natl. Acad. Sci. U.S. 52, 884
(1964). b. Bovine---O. Mikes, V. Holeysovsky, V. Tomasek, and F. Sorm, B~chem. Biophys. Res. Commun. 24, 346 (1966). c. Porcine--J. Travis and I. E. Liener, J. Biol. Chem. 240, 1967 (1965); R. A. Smith and I. E. Liener, J. Biol. Chem. 242, 4033 (1967). d. Porcine--M. Charles, M. Rovery, A. Guidoni, and P. Desnuelle, Biochim. Biophys. Acta 69, 115 (1963). e. Dogfish--R. A. Bradshaw, H. Neurath, K. A. Walsh, R. W. Tye, and W. P. Winter, quoted by H. Neurath, R. A. Bradshaw, and R. Arnon in Proc. Symp. Structure-Function Relationships oJ Proteolytic Enzymes, Munksgaard, Copenhagen, in press, 1969. f. Turkey--T. Kishida and I. E. Liener, Arch. Biochem. Biophys. 126, 111 (1968).
[4]
TRYPSINOGENS AND TRYPSINS
57
pronounced with bovine and ovine trypsin than with porcine trypsin. ~s Green and Neurath have reviewed the protective effect of other cations on bovine trypsin. 4~ Self-digestion of trypsin can be minimized by chemical modification of the c-amino groups of lysyl residues. Labouesse and Gervais 46 report that c-N-acetylated trypsin does not lose activity at neutral or slightly alkaline pH during several days of storage at room temperature. Purity o] Trypsin
The variants, intrinsic in partially autolyzed preparations of trypsin, have already been discussed (see Table II). It should be emphasized that the specificity of action and catalytic parameters of these variants have not been examined in detail. An important feature of trypsin is its narrow specificity of action, which renders it suitable for specific cleavage at lysyl or arginyl residues in amino acid sequence analysis. Any contamination with chymotrypsin obviously reduces its effectiveness in this role. A method of removing traces of chymotrypsin by chromatography is described in detail by Rovery in Volume XI [25]. An alternate method of selectively inactivating the chymotryptic contaminant with tosylphenylalanine ehloromethyl ketone (TPCK) 47 is described by Carpenter in Volume XI [26]. The product of the latter treatment, "TPCK-trypsin," retains very low activity toward chymotryptic substrates, but this may be an inherent property of the trypsin molecule.4s Structure of Trypsin and Trypsinogen from Various Species
Amino acid compositions and partial amino acid sequences of various trypsins and trypsinlike enzymes are summarized in Table IV and Fig. 2. F. F. Buck, A. J. Vithayathil, M. Bier, and F. F. Nord, Arch. Biochem. Biophys. 97, 417 (1962).
aa N. M. Green and H. Neurath, in "The Enzymes," (H. Neurath and K. Bailey, eds.), Vol. II,B, p. 1057. Academic Press, New York, 1954. g. Streptomyces griseus--L. Jurasek, D. Fa~kre, and L. B. Smillie, Biochem. Biophys. Res. Co~r~mun. 37, 99 (1969). h. Horse--C. I. Harris and T. Hofmann, Biochem. J. 114, 82P (1969). i. D. Kauffman, J. Mol. Biol. 12, 929 (1965). z. May be an alternate sequence in an allotypic variant [see (e)]. * His is inserted at this point in porcine trypsin. ** Corrected sequence reported by K. A. Walsh, L. L. Houston, and R. A. Kenner, in Proc. Syrup. Structure-Function Relationships o] Proteolytic Enzymes, Munksgaard, Copenhagen, in press, 1969. ~-Pro is inserted at this point in S. griseus trypsinlike enzyme.
58
~.
s~.Ris~. PaO~TasEs
[4]
An examination of the partial sequence data reveals the high degree of homology among the structures so far examined. The similarity of amino acid compositions provides a basis for the prediction that most, if not all, of the trypsins from diverse species will possess homologous structures. Thus, it is probable that they have arisen from a common ancestor by a process of divergent molecular evolution2 ~ The recent finding of virtual identity in the three-dimensional conformation of two homologous enzymes, bovine chymotrypsin and porcine elastase, 5° provides a valid basis for attempts to predict the threedimensional structure of trypsin. Several such hypothetical structures have been described~2,51,52 and, at the least, provide a guide to the probable three-dimensional structure of trypsin in advance of its detailed determination by X-ray crystallographic techniques.
Physical Properties o] Trypsins and Trypsinogens Various physical parameters of trypsin and trypsinogen are summarized in Table V. Both the molecular weight data in this table and the amino acid composition data in Table IV indicate that the molecular weight of trypsin is approximately 24,000 regardless of the species examined. The net charge of trypsin varies widely, depending upon the species examined. Although isoionic or isoelectric points have been determined in a few cases only, it has been shown that rat, ~3 dogfish,31 and lungfish ~1 trypsins are anionic at neutral pH, whereas bovine and porcine trypsins are cationic (Table V). Thus, the net charge is not an important determinant of specific tryptic function. The physical forms of trypsin and trypsinogen at various temperatures and pH values are summarized in Fig. 1 and discussed in detail by Lazdunski and Delaage. ~x
Inhibitors oJ Trypsin Three categories of inhibitors of trypsin have been studied: (a) protein inhibitors; (b) competitive inhibitors; and (c) active site titrants. a j . Labouesse and M. Gervais, European J. Biochem. 2, 215 (1967). ~7~-(1-Tosylamido-2-phenylethyl) chloromethyl ketone as designed by G. SchoeUman and E. Shaw, Biochemistry 2, 252 (1963). T. Inagami and J. M. Sturtevant, J. Biol. Chem. 23~ 1019 (1960). H. Neurath, K. A. Walsh, and W. P. Winter, Sc/ence 158, 1638 (1967). D. M. Shotton and H. C. Watson, Proc. Roy. ~goc. (London) 8er. B, in press, 1969. ~1K. A. Walsh, L. L. Houston, and R. A. Kenner, Proc. ~ymp. ~tructure-Function Relationships o] Proteolytic Enzymes, Munksgaard, Copenhagen, in press, 1969. roB. Kefl, V. Dlouh~, V. Holey~ovsky, and F. ~orm, Collection Czech. Chem. Commun. 33, 2307 (1968).
[4]
TRYPSINOGENS
59
AND TRYPSINS
TABLE V SOME PHYSICAL PARAMETERS OF VARIOUS TRYPSINOGENS AND TRYPSINS Molecular weight Bovine trypsmogen Bovine trypsmogen Bovine trypmnogen Bovine trypsmogen Bovine trypsmogen Bovine trypsmogen Bovine trypsin Porcine trypmnogen Porcine trypsin Equine trypsmogen H u m a n trypsin Dogfish trypsmogen
s~0,w
23,700" 23,991 ~ 24,200 '~,° 24,000 ~,f 25,700.,o 22,100 d,h 24,000 ~ 25,327i 23,400 I,k 25,600 m 22,900" 24,520p
2.48"
Isoionic point
fifo
9.3 b
1.15 -
10.1 b 7.5 ~" 10.8k, z
1.2 ~
2.7 • 2.50 ~ 2.77 k 2.68 °
Optical rotatory dispersion parametersq ~(nm) ao b," Bovine Bovine Bovine Bovine
trypsin, Forml" trypsin, Form I l I trypsinogen, Form I ' trypsinogen, Form I I I '
249 237 240 236
± ± + +
3 2 3 2
--213 + 8 °
--143 + 15 °
--280 + 14 °
--143 + 18 °
" Sedimentation velocity and diffusion. F. Tietze, J. Biol. Chem. 204, 1 (1953). b N. M. Green and H. Neurath, in " T h e Froteins," (H. N e u r a t h and K. Bailey, eds.) Vol. IIB, p. 1057, Academic Press, N e w York, 1954. c Sequence analysis, K. A. Walsh and H. Neurath, Proc. Natl. Acad. Sci. U.S. 52, 884 (1964). Light scattering. * C. M. Kay, L. B. Smillie, and F. A. Hilderman, J. Biol. Chem. 236, 118 (1961). I Approach to sedimentation equilibrium. 0 Sedimentation--viscosity. h Reported b y A. K. Balls, Proc. Natl. Acad. Sci. U.S. 53, 392 (1985). Diisopropylphosphoryl trypsin. L. W. Cunningham, Jr., F. Tietze, N. M. Green, and H. Neurath, Discussions Faraday ,~oc. 13, 58 (1953). i M. Charles, M. Rovery, A. Guidoni, and P. Desnuelle, Biochim. Biophys. Acta 69, 115 (1963). * J. Travis and I. E. Liener, or. Biol. Chem. 240, 1962 (1965). l Isoelectric point. '~ C. I. Harris and T. Hofmann, Biochem. J. 114, 82P (1969). " J. Travis and R. C. Roberts, Biochemistry 8, 2884 (1969). ° Sedimentation velocity experiments a t a concentration of 2.5 mg/ml. p R. W. Tye, personal communication. M. Lazdunski and M. Delaage, Bioehim. Biophys. Aeta 140, 417 (1967). r M = 212 nm. " The various physical states of trypsin a n d trypsinogen are defined in Fig. 1.
60
szR1sz PSOZZAszs
These have provided both a variety of reagents for terminating the action of the enzyme and considerable insight into the nature of the catalytic mechanism. The protein inhibitors are discussed separately in this volume [66, 67] and provide stoichiometric inhibition by binding with characteristically large association constants. Studies with a variety of competitive inhibitors have correlated the inhibitory capacity both with the hydrophobic nature of the compound and with the specific location of a positive charge. 53-55 The characteristics of some of these inhil~itors are summarized in Table VI. Active site titrants (Table VI) provide reagents for both irreversibly inhibiting the enzyme and estimating the concentrations of active sites. In addition, active site titrants have led to direct proof of the involvement of His48 and Ser~s3 in the active center of bovine trypsin2 9,5B
Specificity o] Trypsin The specificity of trypsin is directed toward lysyl and arginyl residues 57 in natural and synthetic substrates, thus exhibiting the most restricted specificity of action of the known endopeptidases. The length of the side chain containing the positive charge is apparently an important determinant of this specificity.5s,59 Some kinetic constants for trypsins with selected substrates are summarized in Table VII. Theories of the mechanism of action of the enzyme have been reviewed by Cunningham, 39 Bender and Kaiser, e° and Bender and K~zdy.61 In general, they draw attention to the apparent analogy with the mechanism of chymotryptic action, the intermediate acylation of the enzyme and the specific involvement of Serls3 and His4e in a combination of general acid, general base, and nucleophilic catalysis. More recently it has been suggested that aspartic acidgo accentuates the nucleophilic character of Serlsa. Although the specificities of action of trypsin and chymotrypsin are quite different,57 analogy in their catalytic mechanisms is striking and is apparently dictated by homology in primary structure. It is significant in this connection that the greatest similarity in the two primary struc5sM. Mares-Guia and E. Shaw, J. Biol. Chem. 240, 1579 (1965). o4M. Maxes-Guia, Arch. Biochem. Biophys. 127, 317 (1968). MaT. Inagami and S. S. York, Biochemistry 7, 4045 (1968). ~J. D. Geratz, Arch. Biochem. Biophys. 118, 90 (1967). E. Shaw,~ Vol. XI [80]. ~' H. Neurath and G. W. Schwert, Chem. Rev. 46, 69 (1950). obj. B. Baird, E. F. Curragh, and D. T. Elmore, Biochem. J. 96, 733 (1965). ~'N. J. Baines, J . B . Baird, and D. T. Elmore, Biochem. J. 90, 470 (1964). eoM. L. Bender and E. T. Kaiser, J. Am. Chem. Soc. 84, 2556 (1962). ,1M. L. Bender and F. J. Kdzdy, Ann. Rev. Biochem. 34, 49 (1965).
TABLE VI SOME INI~IBITORS OF B O W N E TRYPSIN
KI (M)
pH
I. Competitive inhibitors a p-Aminobenzamidine 8.25 X 10-6 8.15 Benzamidine 1.66 X 10 -5 8.15 ~-Naphthamidine 1.46 X 10-5 8.15 m-Toluamidine 2.27 X 10-6 8.15 p-Toluamidine 2.65 X 10-5 8.15 Cyclohexylcarboxamidine 4.27 X 10-4 8.15 Phenylguanidine 7.25 X 10-~ 8.15 Phenylguanidine 1.4 X 10-4 7.0 a-N-Benzoyl-~-arginine 5.8 X 10-a 8.0 1-Propyl-guanidine 5.3 X 10-4 8.0 n-Butylamine 1.7 X 10-~ 6.6 Benzylamine 6.0 X 10-4 6.6 Thionine 2.5 X 10-5 7.6 Proflavin 4.0 X 10 -6 7.3 II. Active site titrants Diisopropylphosphofluoridate (DFP) 1-Chloro-3-tosylamido-7-amino heptanone (TLCK) p-Nitrophenyl-p'-guanidinobenzoate Ethyl-p-guanidinobenzoate a-N-Methyl-~-N-toluene-p-sulfonyl-L-lysine B-naphthyl ester p-Nitrophenyl N2-acetyl-Nl-benzylearbazate p-Nitrophenyl a-N-benzyloxycarbonyl-L-lysinate p-Nitrophenyl ethyl diazomalonate
Temp. (°C)
Ref.
15.0
b
15.0
c
15.0 15.0 15.0 15.0 15.0 10.0 25 25.0 25 25 24
c c c b b d e f g g h
25
i
J k l m n o
p q
a The values of Kr reported have been obtained with undefined mixtures of E-trypsin and its autolysis products. The various species of active trypsin may possess different binding affinities for the inhibitors. M . Mares-Guia and E. Shaw, J. Biol. Chem. 240, 1579 (1965). c M. Mares-Guia, Arch. Biochem. Biophys. 127, 317 (1968). d Equilibrium dialysis by B. M. Sanborn and W. P. Bryan, Biochemistry 7, 3624 (1968). • J. J. Bechet, M. C. Gardiennet, and J. Yon, Biochim. Biophys. Acta 122, 101 (1966). / T. Inagami and S. S. York, Biochemistry 7, 4045 (1968). o T. Inagami, J. Biol. Chem. 239, 787 (1964). h A. N. Glazer, J. Biol. Chem. 242, 3326 (1967). S. A. Bernhard and H. Gutfreund, Proc. Natl. Acad. Sci. U.S. 53, 1238 (1965). E. F. Jansen, M. D. F. Nutting, R. Jang, and A. K. Balls, J. Biol. Chem. 179, 189 (1949); G. H. Dixon, D. L. Kauffman, and H. Neurath, J. Biol. Chem. 233, 1373 (1958). k E. Shaw, M. Mares-Guia, and W. Cohen, Biochemistry 4, 2219 (1965); E. Shaw and S. Springhorn, Biochem. Biophys. Res. Commun. 27, 391 (1967). T. Chase, Jr., and E. Shaw, Biochem. Biophys. Res. Commun. 29, 508 (1967). M. Mares~uia and E. Shaw, J. Biol. Chem. 242, 5782 (1967). D. T. Elmore and J. J. Smyth, Biochem. J. 107, 97 (1968). ° D. T. Elmore and J. J. Smyth, Biochem. J. 107, 103 (1968). P M. L. Bender, F. J. K6zdy, and J. Feder, J. Am. Chem. Soc. 87, 4953 (1965). 1R. J. Vaughan and F. H. Westheimer, Anal. Biochem. 29, 305 (1969).
62
THe. S~.mN~ P ~ O ~ A s ~ . s
[4]
TABLE VII KINETIC CONSTANTS FOR SOME SYNTHETIC SUBSTRATES OF BOVINE TRYPSINa
Substrate Amides Benzoyl-L-argininep-nitroanilide L-Lysyl-p-nitroanilide Benzoyl-L-argininamide N-a-Benzyloxycarbonyl-L-arginTne p-toluidide Esters Benzoyl-L-arginineethyl ester Tosyl-L-argininemethyl ester a-N-Acetyl 8-(~-aminoethyl) cysteine ethyl ester
a-N-Tosyl-S-(~-aminoethyl) cysteine ethyl ester a-N-Acetyl lysine ethyl ester Ethyl p-guanidinophenylacetate Neutral substrates N-Acetyl-L-tyrosine ethyl ester N-Acetyl-glycine ethyl ester N-a-Benzoyl-L-citrullinemethyl ester N-Benzoyl-L-heptylinemethyl ester
/cm (app.) (raM)
pH
Temp. (°C)
8.15 8.65 8.35 8.0
15 15 35 25
b b c d
14.6 100.0 18.7
8.0 8.0 8.0
25 25 25
e f g
0.369
55.6
8.0
25
h
0.28 0.0115
80.0 0.047
8.0 8.0
25 25
g i
14.5 0.012 0.14
8.0 7. O 7.0
25 25 25
3" k l
7.0
25
l, m
0.939 0.364 3.1 1.4
0. 0043 0.0125 1
42.0 800.0 41.0 0.1
k~t sec-I
0.611 0.003 1.35 0.69
9.4
Ref.
° It should be noted that the kinetic constants have been obtained with undefined mixtures of ~otrypsin and its auto]ysis products. Since a-trypsin and E-trypsin have been shownu to differ in their activity toward either N-a-benzoyl-vL-arglnine p-nitroanilide or tosyl-L-arginine methyl ester, the constants should be regarded only as a guide to the probable value associated with a particular species of enzyme. b B. F. Erlanger, N. Kokowski, and W. Cohen, Arch. Biochem. Biophys. 95, 271 (1961). c j. Chevallier and J. Yon, Biochim. Biophys. Acta 112, 116 (1966). d In 5~o dimethylformamide, T. Inagami, J. Biochera. 66, 277 (1969). ° N. J. Baines, J. B. Baird, and D. T. Elmore, Biochem. J. 90, 470 (1964). I C. G. Trowbridge, A. Krehbiel, and M. Laskowski, Jr., Bioc~raistry 2, 843 (1963). Note that the kinetics are complicated by excess substrate activation in the concentration range 1-100 mM. o M. Gorecki and Y. Shalitin, Biochem. Biophys. Res. Cornrnun. 29, 189 (1967). h D. T. Elmore, D. V. Roberts, and J. J. Smyth, Biochem. J. 102, 728 (1967). M. Mares-Guia, E. Shaw, and W. Cohen, J. Biol. Chem. 242, 5777 (1967). i T. Inagami and J. M. Sturtevant, J. ~wl. Chem. 2 ~ , 1019 (1960). k T. Inagami and H. Mitsuda, J. Biol. Chem. 259, 1388 (1964). B. M. Sanborn and G. E. Hein, Biochemistry 7, 3616 (1968). mIn 18% acetonitrile.
[4]
TRYPSINOGENS AND TRYPSINS
63
tures is found in those portions of the two molecules specifically implicated in the catalytic mechanisms.29,e2 The action of trypsin toward protein substrates can be redirected by selectively blocking lysyl residues 8s or arginyl residues 6' of the substrate. Alternatively, cystinyl or cysteinyl residues can be converted to the lysyl homolog, S-(p-aminoethyl) cysteine, as detailed by Cole in Volume XI [33]. Thus, by choosing the appropriate modification procedures it is possible to use trypsin to hydrolyze selectively lysyl, cysteinyl, or arginyl residues. It has recently become clear that the accepted picture of the specificity of trypsin toward lysyl and arginyl residues is incomplete. Trowbridge et al. 65 found that high concentrations of tosyl-L-arginine methyl ester deviated from Michaelis-Menten kinetics. Their observation of activation by excess substrate was explained in terms of a ternary complex involving an auxiliary binding site. Sanborn and Hein e6 have examined the nature and significance of such a ternary complex and found that even neutral compounds can bind in an auxiliary site, resulting in cooperative interaction with the active site. They have indicated that this could be important in explaining anomalies in polypeptide hydrolyses by trypsin 87,e8 since the general environment of a given bond could satisfy secondary specificity considerations. Some synthetic neutral substrates are hydrolyzed by trypsin 66,6~ (Table VII) and apparently bind to the enzyme in a manner which overlaps the primary binding site. It is not clear what relationship exists between the auxiliary site for binding of additional cationic substrate and this neutral substrate binding site.
'~D. M. Blow, J. J. Birktoft, and B. S. Hartley, Nature 221, 337 (1969). e3R. F. Goldberger and C. B. Anfinsen,Biochemistry 1, 401 (1962). See discussion by Goldberger in Vol. XI [34]. 6' K. Toi, E. Bynum, E. Norris, and H. A. Itano, J. Biol. Chem. 240~ PC3455 (1965). a C. G. Trowbridge, A. Krehbiel, and M. Laskowski,Jr., Biochemistry 2, 843 (1963). UB. M. Sanborn and G. E. Hein, Biochemistry 7, 3616 (1968). e~H. Bachmayer, K. T. Yasunobu, J. L. Peel, and S. Mayhew, J. Biol. Chem. 243, 1022 (19{}8).
aB. V. Plapp, M. A. Raftery, and R. D. Cole, J. Biol. Chem. 242, 265 (1967). ~W. Cohen and P. P~tra, Biochemistry 6, 1047 (1967).
64
THE S E R I N E PROTEASES
[5]
[5] Chymotrypsinogens--Chymotrypsins B y P. E. WILCOX
A. Bovine Chymotrypsinogen A and Chymotrypsins A Chymotrypsinogen A ChT A, Chymotrypsinogen A
T
, ChT A,
ChT
~ ChT Aa
ChT
~ ChT A~
pH 3-4 slow ChT
' ChT A~ T
* neochymotrypsinogens --* ChT A~
ChT A~ pH ~-8 ChT Av slow
Chymotrypsinogen A is converted to active enzyme at p H 7.6 by tryptic cleavage of the bond between Arg15 and Ilels (of. sequence in Table II). A subsequent autolytic action of chymotrypsin removes the dipeptide, Ser14-Arg15, to produce chymotrypsin A~. Cleavage of two more bonds with the deletion of Thr247-Asn24s leads to chymotrypsin AT. This sequence is the normal course of events under conditions of rapid activation (trypsin-to-zymogen ratio of 1:30, 5°). The T-form of the enzyme m a y be crystallized at p H 5.6 from ammonium sulfate solution. When the protein in the activation mixture is precipitated at p H 3 with ammonium sulfate and crystallized at p H 4, the T-for m of the enzyme is transformed into monoclinic crystals of chymotrypsin A~2 The primary structures of the a- and T-forms of the enzyme do not differ, but they do differ in conformation and physical properties, though not to a large degree. 2 Under slow activation conditions (trypsin-to-zymogen ratio of l:10,000, 5 °) there is ample opportunity for chymotrypsin to attack the zymogen and produce neochymotrypsinogens. The principal N-terminal residues that appear in the neochymotrypsinogens are Thr, Ala, and Ser; therefore it is probable that these arise by cleavages of the same bonds t h a t occur in the transformation of A~ to A~ and A~ to A T. T h e neochymotrypsinogens are activated by trypsin to the various forms of the 1It has been proposed in the past that chymotrypsin A~ was derived directly from At during activation. However, no enzyme with properties distinctive for A~, in particular the ability to dimerize at pH 42i, can be found in rapid activation mixtures even after they have been allowed to stand at pH 7.6 and 5 ° for 48 hours. (T. Horbett and D. C. Teller, unpublished data.) ~H. T. Wright, J. Kraut, and P. E. Wilcox, J. Mol. Biol. 37, 363 (1968); B. W. Matthews, G. H. Cohen, E. W. Sflverton, H. Braxton, and D. R. Davies, J. Mol. Biol. 36, 179 (1968).
[~]
CHYMOTRYPSINOGENS--CHYMOTRYPSINS
65
enzyme. In a slow activation mixture at the end of 48 hours a large portion of the protein has the properties of chymotrypsin A~, although as much as 25% does not. Chymotrypsinogen A and chymotrypsin A, are available in large quantities from commercial sources. Zymogen which has been recrystallized several times is usually homogeneous. Samples of enzyme are more or less heterogeneous, depending upon the care of preparation and whether or not they have been subjected recently to purification procedures. The commercial proteins are prepared from bovine pancreas by the classical procedure of Kunitz. Briefly, the zymogen is first isolated from acid extracts of the tissue and purified by recrystallization from ammonium sulfate solution. The pure zymogen is treated with bovine trypsin under slow activation conditions and the enzyme is isolated by crystallization at about pH 4 from ammonium sulfate solution. Salt is removed by dialysis against 10`3 M HC1 and the product lyophilized. In the Kunitz procedure, chymotrypsin A~ is obtained by holding a solution of A, at pH 8 and 5 ° for 3 weeks and crystallizing the enzyme at pH 5.6 from ammonium sulfate solution. Treatment of the zymogen under conditions of rapid activation for only 60 minutes results in an activation mixture which contains chymotrypsin A~ as the principal component. This form of the enzyme cannot be isolated in a pure crystalline state. Dialyzed and lyophilized preparations contain not only A~ but also forms of the enzyme with Thr and Ala as additional N-terminal residues, and Tyr as C-terminal. Neither chymotrypsins A~ or AT, as they are available commercially, have been thoroughly characterized. Details of the procedures outlined above have been given by M. Laskowski in Vol. II [2].
Chymotrypsinogen A
Purity Zymogen which has been purified by manifold recrystallization, as many as five or seven times, appears homogeneous by physical criteria, including sedimentation analysis and free-boundary eleetrophoresis at pH 4.97. 3," Impurities unrelated to the zymogen are absent, but recrystallization is not entirely effective in removing small amounts of ehymotrypsin and neochymotrypsinogens, which are sometimes generated during early stages of the preparation. These impurities account for the faint 3p. E. Wilcox, J. Kraut, R. D. Wade, and H. Neurath, Biochim. Biophys. Acta 24, 72 (1957). W. J. Dreyer, R. D. Wade, and H. Neurath, Arch. Biochem. Biophys. 59, 145 (1955).
66
THe. S~.RIN~. PROT~.AS~.S
[5]
band that is observed in disc gel electrophoresis at pH 4.8 in addition to the sharp, heavy band of the zymogen itself. The faint band has the lower mobility, as is characteristic for the active enzymes. Test ]or Chymotrypsin. The most sensitive test is the spectrophotometric assay using N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester as substrate (see this volume [3] ). The use of 0.1 mg of zymogen in this assay permits the measurement of active enzyme in the range of 0.02% of the weight of total protein. Some commercial samples of zymogen have been found to contain more than 100 times this amount. Test ]or End Groups. Impurities may be detected by the Stark method of N-terminal analysis (Vol. XI [11]). Pure samples of zymogen give only traces of hydantoins by this method, less than 0.02 mole per mole of protein. 5 N-Terminal threonine is usually found in the highest amount. Care must be taken to obtain proper correction factors from the blank analyses. WARNING: If appreciable amounts of chymotrypsin are present in the zymogen, proteolysis may occur during the treatment with cyanate even in the presence of urea. In that event, the analysis can no longer be a true measure of the inhomogeneity of the zymogen. The difficulty can be avoided by the addition of diisopropylphosphofluoridate (DFP) or phenylmethanesulfonyl fluoride (100 moles per mole of protein) to the solution just before it is to be treated with eyanate at pH 8.
Purification by Chromatography e Principle. Chromatography on high-capacity carboxymethyl cellulose (CM-cellulose) at pH 6.2, using a linear salt gradient of low slope, yields a highly purified chymotrypsinogen. Impurities having the chromatographic properties of active enzyme and showing N-terminal threonine and alanine are reduced to less than 2%. The zymogen is treated with inhibitor before chromatography in order to prevent any enzyme action on the column or in the effluent. This procedure may also be used in the preparation of inhibited derivatives of chymotrypsin A.~ or A~ (see below) and for the purification of A~ without the addition of inhibitor. Chymotrypsin A~ does not chromatograph well in this system.
Reagents Initial eluant. Potassium phosphate buffer, 0.05M in total phosphate, pH 6.2 Final eluant. The above buffer containing KC1, 0.10 M CM-cellulose. A high capacity adsorbent is required for satisfactory G. R. Stark and G. D. Smyth, J. Biol. Chem. 238, 214 (1963). ' E. Surbeck and P. E. Wilcox, unpublished data.
[5]
CHYMOTRYPSINOGENS---CHYMOTRYPSINS
67
resolution of protein components. Whatman CM 52, microgranular, is recommended. The adsorbent is equilibrated with initial buffer before use. Inhibitor. DFP, 0.10M in isopropanol; or phenylmethanesulfonyl fluoride, 0.10 M in dioxane. Only a few tenths of a milliliter are needed for each run. Acid. Acetic acid, 1.0 M; HC1, 10-3 M
Procedure. All chromatographic operations are carried out at 5 ° . Prepare a column of CM-cellulose (2.0 X 70 cm) and wash the column with 2 liters of initial buffer. A satisfactory method for packing the column is given in Whatman Data Manual 2000. The column is fed by a two-reservoir system devised to deliver a linear gradient of buffer solution at a constant rate between 20 and 50 ml per hour. Initially each reservoir holds 3.0 liters of buffer. Dissolve 500 mg of salt-free protein in about 20 ml of initial buffer. Add 200 ~l of one of the inhibitor solutions and let the sample stand for 1 hour. Apply the sample to the column and elute the protein with a gradient running from the initial to the final concentrations of the buffers over the total volume of 6.0 liters. Fractions of about 15 ml i~re collected, and the effluent is monitored spectrophotometrically at 282 nm. Impurities emerge in the breakthrough fractions and in two or three small peaks that run before the main peak. The main peak appears at a K ÷ concentration of about 0.12 M and should be nearly Gaussian in shape. Peak fractions are pooled, the pH is reduced to 4.0 with 1.0 M acetic acid, and the solution dialyzed against 10-s M HC1. The purified protein may be recovered by lyophilization. A concentrated solution is best prepared by ultrafiltration in the Amincon Diaflo apparatus. Chemical and Physical Characteristics Amino Acid Composition. Amino acid analyses of bovine chymotrypsinogen A are compared in Table I with the composition that is indicated by the determined sequence. Early analyses gave low values for glutamic acid, probably because of some loss of this amino acid as the pyrrolidone. All analyses give somewhat low values for lysine for reasons that are not clear, although the high serine content may be implicated. Sequence. Bovine chymotrypsinogens A and B are compared in Table II. Physical Properties. Physical parameters are collected in Table III. Chymotrypsinogen A is stable in solution at room temperature between pH 3.5 and 10.5; over this range it shows no change in properties that are
68
~.
s~.~.
~,~o~As~
[~]
Z 0
z
Z
Z 0
r.
0
0
0 0
~
~
°~
[5]
CHYMOTRYPSINOGENS----CHYMOTRYPSINS •~ .
,~
,.~ " ' ~
o_= •~
~.~ 0
~
~
cot'-
o0o0
~
~ ~ ~.~ . ~
•
~
~
~.
~ = ~ =-. ~
.~
69
70
~s
C) Z
z
Z 0
r~
c~
z
SERINE P S O ~ Z S
[S]
[5]
CHYMOTRYPSINOGENS--CHYMOTRYPSINS
@
71
• "~
~ u~
~eN
~x
72
THE SERINE PROTEASES
[5]
TABLE III PHYSICAL PROPERTIES OF BOVINE CHYMOTRYPSINOGENS A AND B a
Chymotrypsinogen Parameter
A
B
(ml/g) [7] (ml/g) 8°.w pH 3.0 pH 5.0 pH 7.5 D~0,w × 107 (cm~ sec-1) c --~ 0 pH 3.0 pH 7.5 Molecular weight (daltons) Light scattering Sedimentation rate--diffusion Approach to sedimentation equil. Osmotic pressure Amino acid analysis X-ray diffraction Sequence Optical rotatory dispersion
0.733 2.18
0. 721 2.26
2.49 S
2.58 S 2.60
)~c (nm)
ao bo [m']231.b Absorbancy coefficients E X 10-4 (282 rim) E~m (282 nm)
2.58 9. 009 9.48O 26,100 24,200
25,081 25,000 25,635
25,000 24,700 24,000 24,940 25,717
231.8
231.1
--436 ° --86 ° -- 3300 °
--465 ° --97 ° -- 3640 °
5.13 20.0
4.81 18.7
Values for the parameters in this table have been taken from L. B. Smillie, A. G. Enenkel, and C. M. Kay, J. Biol. Chem. 241, 2097 (1966). Additional references to the source of some of the parameters will be found in that paper. s e n s i t i v e to c o n f o r m a t i o n . T h e s e d i m e n t a t i o n c o n s t a n t is i n d e p e n d e n t of p H in t h i s r a n g e a t ionic s t r e n g t h s a b o v e 0.1; t h e p r o t e i n does n o t t e n d to d i m e r i z e in t h e a c i d r a n g e as is t h e case for c h y m o t r y p s i n A~. A t p H 2 t h e p r o t e i n a s s u m e s a different c o n f o r m a t i o n a l s t a t e . T h i s p r o c e s s is r e v e r s i b l e in d i l u t e solutions, b u t a t c o n c e n t r a t i o n s a b o v e 2 m K / m l a n d in t h e p r e s e n c e o f salt's such as s o d i u m s u l f a t e , a g g r e g a t i o n of t h e p r o t e i n p r e v e n t s r e t u r n to t h e n a t i v e c o n f o r m a t i o n . T h e r e v e r s i b l e t r a n s i t i o n b e t w e e n t h e two c o n f o r m a t i o n a l s t a t e s is l i n k e d to t h e i o n i z a t i o n of a single g r o u p w i t h p K = 3.0. 7 Binding of Ca2+.7 C h y m o t r y p s i n o g e n b i n d s one C a 2÷ s t r o n g l y a n d M. Delaage, J. P. Abita, and M. Lazdunski, European J. Bioche,m. 5, 285 (1968).
[5]
CHYMOTRYPSINOGENS--CHYMOTRYPSINS
73
thereby becomes stabilized against denaturation. The association constant for the ion at pH 8.4, ionic strength 0.4, and 20 ° is 1.0 X 108. This constant was derived from the effect of Ca 2÷ concentration on the first-order rate constant for denaturation in urea solution. Denaturation was followed by measuring the change of absorbance at 298 nm, the wavelength at which a maximum occurs in the difference spectrum for the reaction. The presence of Ca 2÷ has no effect on the rate of activation of the zymogen by trypsin. Chymotrypsins A
Assay Methods Kinetic methods are generally suitable for a variety of experimental purposes in studies of the chymotrypsins. However, the specific activity of chymotrypsin changes during activation and further autolytic modification. Some of this change is due to inherent differences among the different species of chymotrypsin, some is due to inhibition by products of autolysis, and some to the formation of inert protein fragments. Therefore, it is advisable for some purposes to determine the functional normality of the sample of enzyme and to calibrate the kinetic assay on the basis of the concentration of active molecules. (See this volume [1].)
Potentiometric Method Procedure. The principle and procedure generally applicable to the serine proteases may be found in an earlier chapter (K. A. Walsh and P. E. Wilcox [3] ). For chymotrypsin the substrate of choice is N-acetylL-tyrosine ethyl ester (ATEE) at an initial concentration of 0.01 M. The assay solution should always contain 0.10M CaC12. Methanol, ethanol, or acetonitrile at a concentration of 5% may be used to increase the solubility of the substrate without affecting the assay significantly. The optimal enzyme concentration in the assay solution is 1.0 ~ m l when 0.5 N base is used as titrant in the pH-stat. The optimal concentration can be reduced to 0.2 tLg/ml by using 0.1 N base, but with some loss of precision. Total protein concentration in stock solutions of chymotrypsin is determined spectrophotometrically at 282 nm. The assumption is made that the molecular extinction coefficient for all species is the same as that for zymogen (c = 5.00 X 104, 1~.1~ = 20.5 for A~) The value of K,~ for the enzymatic reaction is 0.0007 M; therefore the observed velocity is about 93% of V.... For practical purposes the rate of hydrolysis is constant until 25% of the ATEE is hydrolyzed. Specific Activity. The specific rate constant (kcat) for the hydrolysis of ATEE by chymotrypsin A, has been reported to be 193 sec-1 at 25 ° ~ l ~ m
•
74
THe. S~.RINE PRO~aSES
[5]
and 295 sec-1 at 31 ° (pH 8.6, 1.8% acetonitrile)2 These constants were calculated on the basis of the normality of active enzyme. This determination is not far from an earlier value reported by Cunningham and Brown/ namely 170 see-1 (pH 8.0, 25 °, 0.1 M Ca2+; recalculated for E ~ ----20.5), and the agreement is exact if the enzyme used in the earlier work contained 10% inert protein. The amount of inert protein in commercial samples of A~ is generally found to run between 10 and 15~. Expressed in standard units of enzyme activity, a rate constant of 193 see-~ corresponds to 464 micromoles per minute per milligram of enzyme. When samples of enzyme are taken from a rapid activation mixture, the specific activity measured by the ATEE assay is considerably higher than the value for chymotrypsin A~. Chymotrypsin A~ preparations show an activity of about 530 units per milligram (kcat-~-220 see-~) in assays at 25 ° . In the early phase of rapid activation, the peak activity may reach over 600 units/rag, reflecting the long-known fact that chymotrypsin A~ is the most active of all forms of the enzyme. Spectrophotometric Method
The application of the spectrophotometrie method to the assay of chymotrypsin has been presented as two examples in a preceding chapter (this volume [3]). The definitions of unit and specific activity for assays using N-benzoyl-L-tyrosine ethyl ester as substrate are also given. Differences in the specific activity between samples of chymotrypsin A~ and At have been found in spectrophotometric assays, as well as in the potentiometric measurements noted above. Using the substrate BTEE, the activity of a freshly prepared solution of Aa was 130% of the activity of a commercial sample of A~, 59 units per milligram compared to 45 units. TM I t has also been reported that the amide substrate, N-acetyl-L-tryptophan amide, is more effectively hydrolyzed by A~ than by A~. The respective values of K~ (app.) are 0.002 M and 0.004 M, and the values for kcat a r e 0.07 sec-1 and 0.05 sec-1.11 Comments
The various forms of chymotrypsin A are basic proteins and therefore tend to be adsorbed on glass surfaces as monolayers. Contact of dilute solutions with glass surfaces should be kept to a minimum, otherwise large errors are introduced into the measurements and precision is poor. 8M. L. Bender, F. J. K~zdy, and C. R. Gunter, Y. Am. Chem. Soc. 86, 3714 (1964). °L. W. Cunningham and C. S. Brown, J. Biol. Chem. 221, 287 (1956). D. D. Miller and D. C. Teller, unpublished data, "A. Hilmoe, B. C. Park, and G. P. Hess, J. Biol. Chem. 242, 919 (1967).
[5]
CHYMOTRYPSINOGENS----CHYMOTRYPSINS
75
A high concentration of Ca 2÷, at least 0.1 M, appears to help overcome this problem. Some investigators have suggested the use of poor substrates for assay purposes, for example, acyl-L-valine esters 12 or fatty acid esters of o-hydroxybenzoic acid, Is thereby permitting the use of higher concentrations of enzyme. Advantages of such methods are lessened by the reduced sensitivity of the assay and by self-aggregation of the enzyme which complicates the kinetic behavior of the system. Another approach has been to add serum albumin as a protective agent. ~4 A procedure for measuring the amidase activity of chymotrypsin, using the substrate N-acetyl-L-tryptophan amide has been recently reported. ~1 The rate of the reaction is obtained from the increase in free ammonia, which is determined by means of a Technicon Autoanalyzer. PURIFICATION OF CHYMOTRYPSIN A,
Preparations of active chymotrypsin have never been found to be homogeneous, as demonstrated by free-boundary and disc gel electrophoresis and by end-group analysis (see Table IV). Crystalline chymotrypsin A~ contains variable amounts of inert protein (sometimes as much as 15%) and low molecular weight material. Reerystallization is not effective in lowering the level of impurities. In fact, heterogeneity may be increased because autolysis proceeds even at low pH, although slowly, and some impurities appear to be concentrated by cocrystallization. It has been reported that ninhydrin-positive material, probably autolysis products, are bound to the enzyme and interfere with some of its kinetic properties. 15 These low molecular weight impurities can be removed by gel filtration through Sephadex G-25. Gel Filtration Through Sephadex G-~5.15 All operations are carried out at 4 °. A column of Sephadex G-25 (fine), 4.0 X 45 cm, is equilibrated with 10-3 M HC1. About 300 mg of chymotrypsin A~ are dissolved in 15 ml of 10-3 M HCI and the pH is adjusted to 3.0. The sample is applied to the column and eluted with 10-~ M HC1 at a flow rate of 20 ml per hour. Fractions of 5 ml each are collected and monitored by spectrophotometry at 282 nm. The enzyme appears in about five tubes as a sharp peak. Tests of these fractions show a low ninhydrin color reaction. Emerging after the main peak is a peak of small-sized material giving a strong ninhydrin reaction. The tubes containing the high enzyme concentrations are pooled, but the last one or two tubes on the trailing edge =T. H. Applewhite, H. Waite, and C. Niemann, J. Am. Chem. 8oc. 80, 1465 (1958). UB. H. J. Hofstee, Biochim. Biophys. Acta 32, 182 (1959). 14B. H. J. Hofstee, J. Am. Chem. Soc. 82, 5166 (1960). A. Yapel, M. Han, R. Lumry, A. Rosenberg, and D. F. Shiao, J. Am. Chem. Soc. 88, 2573 (1966).
76
T H ~ SERINE P R O T E A S E S
O
[5]
O e~
o
O < •
.
.
.
.
D as would be expected from their amino acid compositions and chromatographic properties on D E A E . The pepsins which are generated from the purified precursors have molecular weights of 34,000 to 36,000 by ultracentrifugal analysis, indicating a loss of fragments with molecular weights of approximately 7000 during the conversion process. The molecular weight of pepsinogen B was 96,000 from sedimentation-equilibrium runs and 45,000 from molecular sieve chromatography on a calibrated Sephadex G-100 column. It is more basic than pepsinogens D, A, and C. Stabilities. The dogfish enzymes are more stable than swine pepsin at neutral pH. Lyophilization of both precursors and enzymes should be avoided since it leads to aggregation with loss in potential enzymatic or enzymatic activities. Specificities. Dogfish pepsins A, D, and C are active on protein substrates at acid p H only and show no activity toward the synthetic substrate, N-Cbz-L-Glu-L-Tyr. Pepsin A has a broader pH optimum
372
THE
ACIDIC P R O T E A S E S
[25]
than swine pepsin using hemoglobin as a substrate. Differences in specificities exist since dogfish pepsins A, D, and C digest various protein substrates differently from each other and from swine pepsin. Their specificities on protein substrates of known sequence are unknown. Pepsin B has no activity toward hemoglobin. There is, however, a second pepsinogen B fraction in which the activities toward N-CbzL-Glu-L-Tyr and hemoglobin have proven inseparable by the chromatographic methods used thus far. Pepsin B is active only at acid pH and can hydrolyze the N-Cbz derivatives of L-Tyr-L-Tyr, Gly-L-Tyr, L-Glu-L,Phe, Gly-L-Phe, L-Phe-L-Tyr, L-Tyr-L-Phe, and L-Phe-L-Phe in addition to the test substrate N-Cbz-L-Glu-L-Tyr.
[25] Microbial Acid Proteinases By J. ~ODEK and T. HOFMANS
Introduction The presence of proteolytic enzymes with a p H optimum in the acid p H range (pH I-5) has been reported in a variety of microorganisms where they occur both intracellularly and extracellularly.The enzymes from Eumycota (true fungi) have been studied most extensively and many have been isolated and purified. Acid proteinases are also found in many protozoa, both as intracellularand extracellular enzymes? The intracellular enzyme from Tetrahymena pyri]ormis has been partially purified? On the other hand, there are only few reports of the occurrence of acid proteinases among the bacteria: Several strains of Clostridia (acetobutylicum and butyricum) 3 and lactobacilli ~ have been shown to produce weak proteinase activity with an acid pH optimum. No information appears to be available on acid proteinases in algae. The only enzymes which have been fully purified are of fungal origin and are extracellular. The intracellular acid enzyme (proteinase A) from bakers' yeast was first noted by Dernby 5 and described by Lenney6 and was partially purified by Hata et al/ 1M. Muller, in "Chemical Zoology" (G. W. Kidder, ed.), Vol. I, p. 374. Academic Press, New York, 1967. N. Dickie and I. E. Liener, Biochim. Biophys. Acta 64, 41 (1962). s F. Uchino, K. Miura, and S. Doi, J. Ferment. Technol. 46, 188 (1968). ' V . Bottaszi, Intern. Dairy Congr. Proc. 16th Copenhagen 2, 522 (1962). s K. G. Dernby, Biochem. Z. 81, 107 (1917). 6j. F. Lenney, J. Biol. Chem. 221, 919 (1956).
' T. Hata, R. Hayashi, and E. Doi, Agr. Biol. Chem. 31, 357 (1967).
372
THE
ACIDIC P R O T E A S E S
[25]
than swine pepsin using hemoglobin as a substrate. Differences in specificities exist since dogfish pepsins A, D, and C digest various protein substrates differently from each other and from swine pepsin. Their specificities on protein substrates of known sequence are unknown. Pepsin B has no activity toward hemoglobin. There is, however, a second pepsinogen B fraction in which the activities toward N-CbzL-Glu-L-Tyr and hemoglobin have proven inseparable by the chromatographic methods used thus far. Pepsin B is active only at acid pH and can hydrolyze the N-Cbz derivatives of L-Tyr-L-Tyr, Gly-L-Tyr, L-Glu-L,Phe, Gly-L-Phe, L-Phe-L-Tyr, L-Tyr-L-Phe, and L-Phe-L-Phe in addition to the test substrate N-Cbz-L-Glu-L-Tyr.
[25] Microbial Acid Proteinases By J. ~ODEK and T. HOFMANS
Introduction The presence of proteolytic enzymes with a p H optimum in the acid p H range (pH I-5) has been reported in a variety of microorganisms where they occur both intracellularly and extracellularly.The enzymes from Eumycota (true fungi) have been studied most extensively and many have been isolated and purified. Acid proteinases are also found in many protozoa, both as intracellularand extracellular enzymes? The intracellular enzyme from Tetrahymena pyri]ormis has been partially purified? On the other hand, there are only few reports of the occurrence of acid proteinases among the bacteria: Several strains of Clostridia (acetobutylicum and butyricum) 3 and lactobacilli ~ have been shown to produce weak proteinase activity with an acid pH optimum. No information appears to be available on acid proteinases in algae. The only enzymes which have been fully purified are of fungal origin and are extracellular. The intracellular acid enzyme (proteinase A) from bakers' yeast was first noted by Dernby 5 and described by Lenney6 and was partially purified by Hata et al/ 1M. Muller, in "Chemical Zoology" (G. W. Kidder, ed.), Vol. I, p. 374. Academic Press, New York, 1967. N. Dickie and I. E. Liener, Biochim. Biophys. Acta 64, 41 (1962). s F. Uchino, K. Miura, and S. Doi, J. Ferment. Technol. 46, 188 (1968). ' V . Bottaszi, Intern. Dairy Congr. Proc. 16th Copenhagen 2, 522 (1962). s K. G. Dernby, Biochem. Z. 81, 107 (1917). 6j. F. Lenney, J. Biol. Chem. 221, 919 (1956).
' T. Hata, R. Hayashi, and E. Doi, Agr. Biol. Chem. 31, 357 (1967).
[25]
MICROBIAL ACID PROTEINASES
373
The following enzymes will be described: (a) the trypsinogen-activating enzyme from Pencillium janthinellum s (penicillopepsin, previously called peptidase A) ; (b) penicillocarboxypeptidase from the same organismg; (c) "trypsinogen kinase" from AspergiUus oryzae (Takadiastase) lo; (d) Candida albicans acid proteinase11; (e) Paecilomyces varioti acid proteinasel~; (f) Rhizopus chinensis acid proteinase. 13 The trypsinogen-activating acid proteinase from Aspergillus saitoi ~ (aspergillopeptidase A) and the milk-clotting enzymes from Bndothia parasitica ~5,16 and from Mucor pusillus (mucor rennin) 1T,ls are described elsewhere in this volume (Articles [26], [29], and [30], respectively). The acid proteinase from Penicillium notatum has been partially purified. It rapidly and extensively digests a variety of different proteins at pH 3.8-4.2.19 The acid proteinase from Trametes sanguinea has been purified and crystallized ~° but no information is available on its properties. Two comments on the action of fungal acid proteinases may be made at this point. First, the enzymes described are all endopeptidases with the exception of penicillocarboxypeptidase (p. 385). In spite of an extensive search, no small synthetic substrates have been found for penicillopepsin 2°~ (p. 383) and aspergillopeptidase A (see this volume [26]). Although it has been reported that the "trypsinogen-kinase" from Aspergillus oryzae (p. 386), the Paecilomyces variot~~1 acid proteinase (p. 391), and the Rhizopus chinensis, acid proteinase (p. 391) hydrolyze di- and tripeptides, the rates of hydrolysis are very low and require incubation periods up to 24 hours. It is probable that these activities are due to contaminations with penicillocarboxypeptidaselike enzymes and ST. Hofmann and R. Shaw, Biochim. Biophys. Aeta 92, 543 (1964). ' R. Shaw, Biochim. Biophys. Acta 92, 558 (1964). " K . Nakanishi, J. Biochem. (Tokyo) 46, 1263 (1959). "H. Remold, H. Fasold, and F. Staib, Biochim. Biophys. Acta 167, 399 (1968). ~J. Sawada, Agr. Biol. Chem. 27, 677 (1964). laj. Fukumoto, D. T's~ru, and T. Yamamoto, Agr. Biol. Chem. 31, 710 (1967). 1,F. Yoshida and M. Nagasawa, Bull. Agr. Chem. Soc. Japan 20, 257 (1956). l~J. L. Sardinas, Appl. Microbiol. 16, 248 (1968). K. Hagemeyer, I. Fawwal, and J. R. Whitaker, J. Dairy Sci. 51, 1916 (1968). 17j. Yu, S. Iwasaki, G. Tamura, and K. Arima, Agr. Biol. Chem. 32, 1051 (1968). G. A. Somkuti and F. J. Babel, J. Baeteriol. 95, 1407 (1968). ~W. E. Marshall, R. Manion, and J. Porath, Biochim. Biophys. Acta 151, 414 (1968). "~K. Tomoda and H. Shimazono, Agr. Biol. Chem. 28, 770 (1964). ~" A current study of the action of penicillopepsin on synthetic peptides shows that it will hydrolyze the Phe.Phe bond in CbzGly-Gly.Phe.Phe.4-(propyloxy) pyridine, a recently described pepsin substrate. [G. P. Sachdev and J. S. Fruton, Biochemistry 8, 4231 (1969).] 21j. Sawada, Agr. Biol. Chem. 28, 869 (1964)..
374
THE ACIDIC PROTEASES
[2S]
other enzymes. On the other hand, the hydrolysis of m a n y proteins by the fungal acid endopeptidases is extensive s and indicative of a low degree of specificity. The failure to find small synthetic substrates indicates that these enzymes require a yet-unknown minimal length of peptide chain for activity, and unlike pepsin and such alkaline endopeptidases as chymotrypsin, subtilisin, and many others, are unable to act on di- and tripeptides. Second, special mention should be made of the unique ability of several fungal acid proteinases to activate pancreatic trypsinogen from several species 22 at pH 3-4, an activity first discovered by Kunitz 28 for an unidentified strain of Penicillium. Since then this activity has been demonstrated in Aspergillus oryzae, 1° in Aspergillus carbonarius, 24 and in the following penicillin: two strains of P. janthinellum, and in brevi-compactum, expansum, italic~m, notatum, spinulosum, stipitatum, vermiculatum, and wortmanii (all reported by Hofmann2~). I t is probable that all strains of aspergilli and penicillin and other ascomycetes produce an acid proteinase with this activity. Recently, this activity has also been detected ~ in the commercially available acid proteinase from Rhizopus chinensis, a fungus from the class Phycomycetes. On the other hand, another species of Aseomycetes, Saccharomyces cerevisiae,25 does not produce a trypsinogen-activating enzyme. The acid proteinase from Mucor pusill~, a fungus closely related to Rhizopus, also lacks this ability. ~6 The mechanism of activation of bovine trypsinogen by the mold enzymes is identical with t h a t of autocatalytic activation and involves the cleavage of the lysine-6 isoleucine-7 bond with the liberation of the hexapeptide valyl(aspartyl)41ysine. 2~-a° In addition, however, breakdown products of the hexapeptide are formed. These differ for the different Although most of the studies have been carried out with the bovine zymogen it has been demonstrated that equine (Harris and Hofmann, to be published) and dog-fish trypsinogen (Prahl and Hofmann, unpublished observation) are also activated by penicillopepsin. n M. Kunitz, J. Gen. Physiol. 21, 601 (1938). 2'T. Hofmann, Pharm. Acta Helv. 38, 634 (1963). s j. ~odek and T. Hofmann, to be published. *IS. Iwasaki, T. Yasui, G. Tamura, and K. Arima, Agr. Biol. Chem. 31, 1421 (1967). 2~E. W. Davie and H. Neurath, J. Biol. Chem. 212, 515 (1955). 2ST. Hofmann, Bull. Soe. Chim. Biol. 42, 1279 (1960). Recent experiments (G. Mains and T. Hofmann, unpublished) show that the highly purified enzyme liberates only the hexapeptide, Val.Asp.Lys~, and does not produce smaller peptides. It is possible that the AspergiUus trypsinogen activating enzymes also specifically liberate the hexapeptide. In each case the production of the smaller peptides could then be attributed to the action of contaminant peptidases. inK. Nakanishi, J. Bioehem (Tokyo) 46, 1553 (1959). inC. Gabeloteau and P. Desnuelle, Bioehim. Biophys. Acta 42, 230 (1960).
[25]
MICROBIAL ACID PROTEINASES
375
enzymes (Penicillium janthinellum:valyl-aspartyl-aspartic acid and (aspartyl) Jysine, 28,~8a Aspergillus oryzae: valine and (aspartyl) ~aspartic acid and lysine, 29 and Aspergillus saitoi: valyl-(aspartyl)2-aspartic acid and aspartyl-lysine3°). The activation of trypsinogen by these enzymes is very specific; bovine chymotrypsinogen is activated by aspergillopeptidase A at a rate which is only about 2% of that of the trypsinogen activation 8° while porcine proelastase is not activated by penicillopepsin. 31 The trypsinogen-activating property is specific for only one of the several peptidases which many fungi secrete 3~ and thus provides a convenient assay for this proteinase which can be used even when other acid and alkaline peptidases are present. This assay is also very sensitive and allows the detection of a few nanograms of the enzyme. The acid proteinases from Mucor species a3,s* and Endothia parasitica ~5,1~ are being studied for their ability to clot milk; they are potentially useful for replacing calf rennin in the cheese-making process. Nomenclature
A few remarks about the nomenclature of the microbial acid proteinases may be appropriate. I t is very likely that at least some of the acid proteinases will turn out to be homologous. The enzyme from PenicilIium janthinellum has m a n y properties in common with porcine pepsin 35 and, like pepsin, has an aspartic acid residue at the active site2 ~a The sum of these properties suggests that this enzyme is homologous to pepsin. As mentioned above, it is one of the trypsinogen-activating enzymes and it is likely that all these enzymes are related to each other. I t is therefore imperative for the sake of clarity t h a t there should be uniformity of nomenclature among these enzymes. The enzyme from Aspergillus saitoi has been named aspergillopeptidase A (EC 3.4.4.17) by the International Commission on Enzymes26 Similarly, the Penicillium janthinellum enzyme has been called Penicillium janthinellum $1y. Birk and A. Gertler, Abstr. Fed. European Biochem. Soc. 6th Meeting, Madrid, No. 469 (1969). = The neutral proteinase from Aspergillus oryzae has a weak trypsinogen-activation activity at pH 5~5, but not at 3A. S. Iwasaki, G. Tamura, and K. Arima, Agr. Biol. Chem. 31, 546 (1967). 14G. H. Richardson, J. H. Nelson, R. E. Lubnow, and R. L. Schwarberg, J. Dairy Sci. 50, 1066 (1967). =J. ~odek and T. Hofmann, J. Biol. Chem. 243, 450 (1968). The sequence of the active site peptide which contains this aspartic acid has recently been determined. ~ It is Ile-Ala.Asp.Thr.Gly.Thr.Thr.Leu. and shows extensive homology to the active site peptide of porcine pepsin which has the sequence Ile. Val. Asp"Thr. Gly- Thr. Ser.=~ J. ~odek and T. Hofmann, Can. J. Biochem. 48, 425 (1970). R. S. Bayliss, J. R. Knowles, and G. B. Wybrandt, Biochem. J. 113, 377 (1969).
376
THE ACIDIC PROTEASES
[25]
peptidase A. s This nomenclature is likely to lead to confusion. Thus, the name aspergillopeptidase B has been given to an alkaline serine proteinase from Aspergillus oryzae 87 and the name penicillium peptidase B to an acid carboxypeptidase from Penicillium janthinellum 9 (see also p. 384). We should like to suggest here that the giving of letters and trivial names to acid proteinases should be avoided and that for those enzymes where there is sufficient evidence of homology with a well-known enzyme, such as pepsin, the trivial name of the enzyme should be added after the generic name or after the name of the organisms, e.g., penicillopepsin or aspergillopepsin, or if the species is to be identified, Penicillium janthinellure pepsin or Aspergillus oryzae pepsin. The acid proteinase from Mucor pusillus has been named in a similar manner mucor rennin, '~ but this was done solely on the basis of its ability to clot milk. No other evidence for a relationship to rennin is a~ present available. I t is interesting to note that this principle for nomenclature was used by D e r n b y 5 who called the acid proteinase in a yeast autolyzate "Here pepsin." The nomenclature suggested would be in accordance with the practice in mammalian enzymes and would be based on firm molecular criteria. Penicillopepsin (formerly Peptidase A) from Penicillium janthinellum (C. M. I. 75589 -----N R R L 905) Assay M e t h o d s
Principle. The assay for penicillopepsin is based on its ability to activate trypsinogen at p H 3.4. The trypsin formed is determined by one of the standard methods at p H 8.0; for routine work, the most convenient one is that described by Kunitz 89 with casein as substrate. The complete assay given below is based on that of Kunitz 23 with the modifications given by Hofmann and Shaw? The enzyme can also be assayed with bovine serum albumin at p H 2.6. TRYPSINOGEN ACTIVATION ASSAY
Reagents Activation step Sodium citrate buffer at p H 3.4, 0.1 M KH2P04, 0.1 M ~Report Commission on Enzymes, Intern. Union Biochem., p. 114. Macmillan (Pergamon), New York, 1961. ~TA. R. Subramanian and G. Kalnitsky, Biochemistry 3, 1861 (1964). ~8K. Arima, J. Yu, S. Iwasaki, and G. Tamura, Appl. Microbiol. 16, 1727 (1968). M. Kunitz, J. Gen. Physiol. 30, 291 (1947).
[25]
MICROBIAL ACID PROTEINASES
377
HC1, 0.0025 M Trypsinogen, 10-s M. Commercial trypsinogen, 5.0 mg (containing 50% MgSO4), are dissolved in 10 ml of 0.0025 M HC1. This is stable under refrigeration for 1 day. Enzyme Dilutions. These are made in 0.1 M KH2P04. Trypsin assay Sodium phosphate buffer at pH 8.0, 0.1 M Tris [tris- (hydroxymethyl) amino-methane], 0.37 M 8.5% w/v Trichloroacetic acid (TCA) 2% Casein in phosphate buffer at pH 8.0. Suspend 2 g casein (Hammarsten) in 100 ml of the phosphate buffer (pH 8.0). Heat for 15 minutes in a boiling water bath with occasional stirring, cool, and adjust the pH to 8.0. This solution can be kept for many months at --20 °. 0.05% Trypsin in 0.0025 M HC1 for preparing a standard curve. The concentration of active enzyme is determined by active site titration with carbobenzoxy-L-lysine p-nitrophenyl ester at pH 3.0. 40 The following solutions are prepared daily: Solution A--1 volume trypsinogen plus 2 volumes citrate buffer, pH 3.4. Solution B--1 volume casein plus 1 volume Tris buffer. A mixture of 3 volumes A, two volumes B, and 1 volume KtI2PO~ should give pH 7.9-8.1. If this pH is not obtained, the concentration of the Tris can be adjusted so that the mixture will give this pH, which is essential for the trypsin assay. Procedure
Preparation of Trypsin Standard Curve. Tris buffer (0.37 M, 20 ml) is mixed with 2% casein (20 ml), citrate buffer (0.1 M, pH 3.4, 20 ml), and 0.1M KH2P04 (10 ml). Aliquots of 3.5 ml of this solution are pipetted into ten test tubes and warmed to 36 °. At 1-minute intervals, 0.5 ml trypsin standard solutions (containing 6-140 fig trypsin per milliliter) are added. After exactly 10 minutes, undigested casein is precipitated with TCA (2 ml). The tubes are left at 36 ° for 10 minutes and filtered through Whatman No. 42 (7 cm) filter paper. The extinction values at 280 nm are read against a blank prepared without trypsin. They are plotted as a function of the total amount of trypsin (in nanomoles) in each tube. ,o M. L. Bender, M. L. Begue°Canton, R. L. Blakeley, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. Kezdy, J. V. Killheffer,T. H. Marshall, C. G. Miller, R. W. Roeske, and J. K. Stoops, J. Am. Chem. ~oc. 88, 5890 (1966).
378
THe. ACIDIC PROTEASES
[2S]
Assay Activation Step. Solution A (1.5 ml) is warmed to 36 °. Five-tenths milliliter of enzyme solution in 0.1 M KH2P04 or the growth medium containing the equivalent of 5-20 ng of the pure enzyme is added to start the reaction. Trypsin Assay. After exactly 10 minutes, solution B (2 ml) is added and after a further 10 minutes the undigested casein is precipitated by 8.5% TCA (2 ml). After 10 minutes of standing at 36 °, the precipitate is filtered through Whatman No. 42 (7 cm) filter paper. The extinction values at 280 nm are read against a blank containing the reagents. The number of nanomoles of trypsinogen activated is obtained from the standard curve. Definition o] Unit and Specific Activity. One unit of penicillopepsin activity is defined as the amount of enzyme required to activate 1 micromole trypsinogen per minute at 25 °, pH 3.4, and saturating substrate concentration. Routine assays are carried out, however, at 36 ° with suboptimal substrate concentrations so that a conversion is required to obtain the defined units. The conversion factor is calculated from the known maximum velocities at 25 ° and 36 ° (420 and 685 micromoles trypsinogen activated per micromole penicillopepsin per minute) to obtain the temperature correction. The correction for nonsaturating conditions is calculated from the Michaelis-Menten equation using a value for K~ ---- 7.6 X 10-8 M. The combined conversion factor = 0.405. Thus the defined units---- (micromoles trypsinogen activated at 36 °) X 0.405. The specific activity is taken as the number of units of activity per milligram of enzyme. ASSAY WITH BOVINE SERUM ALBUMIN
Reagents 1% Bovine serum albumin (fraction IV, B-grade, Calbiochem) in McIlvaine's citric acid-phosphate buffer~1 at pH 2.6 8.5% Trichlor0acetic acid (TCA)
Procedure The solution of bovine serum albumin (1 ml) is warmed to 36 °. The enzyme solution (50 #l) containing the equivalent of 1 to 25 ~g of the pure enzyme is added to start the reaction. After exactly 10 minutes, undigested albumin is precipitated with TCA (2 ml). Ten minutes later 'IR. G. Bates, in "Handbook of Biochemistry" (H. Sober, ed.), p. J-197. The Chemical Rubber Co., Cleveland, Ohio, 1968.
[25]
MICROBIAL ACID PROTEINASES
379
the precipitate is filtered (Whatman No. 42, 7 cm). The extinction values at 280 nm are read against a blank prepared without enzyme. The enzyme concentration is read off a standard curve. It may be mentioned that the reading for 5 ~g of the purest enzyme preparation is 0.200 OD units. Purification P r o c e d u r e
Step 1. Cultivation o] Penicillium janthinellum. The medium used for growing the organism is that described previously s where stationary cultures were used. However, higher enzyme levels are obtained in a shorter time with submerged cultures. The medium is made up as follows: 7.2 g sucrose, 3.6 g glucose, 1.23 g MgS04.7 H20, 13.62 g KH2P04, 2 g KN03, 1.1 g CaC12.6 H20, and 1 liter of distilled water. The following trace elements are added to 1 liter of medium to provide for better growthS: 1.5 mg ZnS04"6 H20, 2.8 mg Fe(NH4)2(SO,)2"6 H20, 0.32 mg CUS04"5 H20, 0.14 mg MnC12'4 H20, and 0.08 mg (NH4)2Mo04. Usually four l-liter culture flasks, each containing 400 ml of the complete medium, are used to grow an inoculum for a 10-liter cultivation in a Microferm apparatus (New Brunswick Scientific Co., New Brunswick, N.J.). The medium and apparatus are sterilized by autoclaving at 115 psi for 30 minutes. Separately sterilized lactic acid (10% v/v) is then added to the medium (100 ml per liter) to adjust the pH to approximately 3.0. The culture flasks are seeded with spores from an agar slant. The original cultures are obtainable from the Commonwealth Mycological Institute, Kew, Surrey, U.K. (strain 75589) or from the Northern Utilization Research Branch, U.S.D.A., Peoria, Ill. (strain 905). These two strains are identical. Another strain (CMI 75588 = N R R L 904) produces lower enzyme levels in stationary culture. 24 Incubation is carried out on a mechanical shaker operating at 180 cycles/minute and at 25 °. When a good growth of viable mycelium has been produced (2-3 days), the contents of the flasks are used to inoculate the medium contained in the Microferm. Rapid growth is attained at 28 ° with continuous stirring at 200-300 rpm. Vigorous and uninterrupted aeration (4-6 liters/minute) is essential for the production of penicillopepsin. Maximum enzyme production is usually reached after 4 days. The initial addition of lactic acid is usually sufficient to give a pH between 5.0 and 5.6 when maximum enzyme levels are reached. This pH range provides optimal conditions for adsorbing the enzyme on DEAE-cellulose. If the pH rises too quickly, however, further additions of lactic acid may be required as the enzyme is unstable above pH 6.0. At harvest the
380
THE ACmIC rROTSASES
[25]
culture medium is filtered twice through Whatman No. 1 filter paper in preparation for adsorption on DEAE-cellulose. Step 2. Adsorption on DEAE-Cellulose. The penicillopepsin contained in the culture medium (about 10 liters) is concentrated by adsorption on DEAE-cellulose (Whatman DE-11). Approximately 45 g of resin, sufficient to pack a column (4 X 28 cm) with a bed volume of about 400 ml is washed successively with 0.5 M N a 0 H , water, 0.5 M HC1, and then equilibrated to pH 5.2 with 0.005 M acetate buffer. After the column is packed at 4 °, the whole medium is passed through the bed. The enzyme is almost quantitatively adsorbed. It is eluted with a mixture of equal volumes of 0.2 M citrate buffer and 0.2 M ammonium sulfate to give a final pH of 3.2. Most of the penicillopepsin activity is eluted in 300-400 ml and is thus concentrated approximately 30 times. Further concentration is achieved with Diaflo apparatus using a UM-1 membrane (Amicon Corp., Cambridge, Mass.) until the volume is reduced to approximately 40 ml. Step 3. Chromatography on Aminoethyl Cellulose (AE-Cellulose). Before adsorption on AE-cellulose, the concentrated enzyme is dialyzed free of ammonium sulfate. At this stage the preparation still contains high cellulase activity and it is advisable to use double dialysis tubing. When a test of the dialysis water with acidified BaC12 indicates that it is free of S04, the material is equilibrated to pH 3.8 with 0.002 M citrate buffer. The AE-cellulose (Whatman AE-11, 25 g), after being washed as described for the DEAE-cellulose, is equilibrated with the same citrate buffer at pH 3.8, and packed into a column at 4 ° (2 X 40 cm). The enzyme is applied and is adsorbed almost completely. Two elution buffers are then used consecutively. The first, 0.1 M citrate, pH 5.0, elutes a protein band essentially free of penicillopepsin. When all this has been removed, the second buffer, 0.25 M ammonium sulfate, adjusted to pH 3.6 with H2S04 is passed through the column. This brings out a single protein peak containing the total penicillopepsin activity. The enzyme is concentrated again by Diaflo filtration to a volume of approximately 5-10 ml. Step 4. Chromatography on Phosphocellulose. The enzyme is again dialyzed free of ammonium sulfate and then equilibrated with 0.005 M acetate buffer, pH 3.8. Meanwhile, unused phosphocellulose (Whatman P-11, 2.5 g) is directly equilibrated with the same buffer and packed into a column (0.5 X 20 em) in the cold. The penicillopepsin passes through the column while the remaining impurities are retarded. Penieillocarboxypeptidase is the most likely contaminant since it chromatographs closely behind penicillopepsin. Its presence can be detected by assaying with carbobenzoxyglutamyl tyrosine. The purification procedure is summarized in Table I.
[25]
MICROBIAL ACID PROTEINASES
381
TABLE I SUMMARY OF PURIFICATION PROCEDURE OF PENICILLOPEPSIN
Fraction Medium DEAE-cellulose eluate Diafio I AE-ceUulose eluate Diaflo II Phosphocellulose eluate
Volume (ml) 10,000 153 41 45 7 11.0
Protein cone. (mg/ml)
Specific activity A c t i v i t y (units/rag Yield (units/ml) protein) (%)
0.416 0.801
0.1 5.0
2.90 1.72 8.88 2.48
17.9 15.4 79.2 43.7
0. 244 6.25 6.2 8.99 8.9 18.0
100 76 72 69.4 57 48
The pure enzyme can be crystallized directly or precipitated with 60% ammonium sulfate and stored under refrigeration. It can also be freeze dried with little loss of activity. A successful preparation has also been made in cooperation with the Connaught Medical Research Laboratories in a 1500-liter fermentor using essentially the conditions and methods described 35b above. The yield was 2.6 g. Properties
Stability: The stability of purified penicillopepsin has been determined under various conditions of pH and temperature? ~b It is completely stable at pH 4.9 for 70 hours. The enzyme is most stable toward higher temperatures at pH 4.9, retaining full activity after 1 hour at 55 °. In the crystalline state at 20 °, it retains full activity for several weeks. Purity. Penicillopepsin from the phosphocellulose column is homogeneous as shown by moving-boundary electrophoresis at pH 4.25, by electrophoresis in acrylamide at pH 8.6, and in starch gel at pH 3.0, 4.5, 6.0, and 8.6.8 Boundary analysis according to the Fu)ita equation of a sedimenting peak in the Model E Ultracentrifuge also indicates homogeneity? Molecular Properties The enzyme has the following physical constantsS: molecular weight ---- 32,000; sedimentation constant S2o,~= 3.1 X 10-1~ sec; diffusion constant D2o,~ -----8.4 (___0.23)X 10-7 cm2 see-l; partial specific volume ~? ---- 0.718 (calculated from the amino acid composition) ; electrophoretic mobility (in 0.22 M acetate, pH 4.25) is/~ -------4.47 -+- 0.02 cm2V-1 see-~ ;
382
THE ACIDIC PROTEASES
[25]
FiG. 1. Crystals of penicillopepsin from (NH,)~SO~ (0.3 saturated) pH 4.4. Reproduced by permission of the National Research Council of Canada from the Canadian Journal o] Biochemistry 48, 430 (1970). isoelectric point below pH 3.8; molecular extinction at 280 nm is ~, -43,200; specific extinction ~1~lr~/m]cm= 1.35. Amino Acid Composition. g Lys~, His3, Asps6, Thr2g, Ser~2, Glu~8, Pro12, Gly89, Ala2s, Va122, Ile12-18, Leu2o, Tyro4, Phel~, Trp,_~, (NH~)48. There is no arginine, half-cystine, or methionine. Alanine is both iN-terminal and C-terminal25b An essential aspartie acid residue is at the active site.~, 35a Crystals. ~2 The enzyme crystallizes from ammonium sulfate (0.3 saturated) at pH values between 3.5 and 6. Crystals for X-ray are obtained at pH 4.4 and are shown in Fig. 1. The crystals are monoclinic, space group C2. The unit cell dimensions are a ~ 97.81 ti, b = 46.73 .~, c ---- 65.69 A, and fl ---- 115.55 °. The unit cell contains 4 molecules. Enzymatic Properties Inhibitors. Penicillium janthinellum pepsin is inhibited competitively by the pepsin and carboxypeptidase A substrates carbobenzoxyglutamyl "N. Camerman, T. Hofmann, S. Jones, and S. C. Nyburg, J. Mol. Biol. 44, 569 (1969).
[25]
MICROBIAL ACID PROTEINASES
383
tyrosine (K~ app. 5 X 10.5 M), carbobenzoxyglutaminyl tyrosine, carbobenzoxyvalyl tyrosine, and also by carbobenzoxyglutamic acid, but not by carbobenzoxytyrosine and carbobenzoxyphenylalanine2 The pepsin inhibitors diazoacetyl norleucine methyl ester and diazoacetyl phenylalanine methyl ester in the presence of Cu 2÷ inhibit the enzyme by reacting covalently with an aspartic acid residue. ~,42a Another pepsin inhibitor, p-bromophenacylbromide does not inhibit. Millimolar concentrations of diisopropylphosphofluoridate, p-hydroxymereuribenzenesulfonic acid, iodoacetic acid, ethylenediaminetetraacetic acid, F 1-, Fe ~÷, Cu 2÷, and Mn 2÷ have no effect on the enzyme. Specificity. Penicillopepsin shows a specificity similar to pepsin toward the B-chain of S-sulfo insulin. It cleaves about 15% of the bonds in native bovine serum albumin. 8 It does not act significantly on Congo red elastin, polyglutamic acid, polyalanine, and polyglycine; polylysine is slowly attacked, and the copolymer of glutamic acid, tyrosine, and lysine is readily hydrolyzed23 It. does not hydrolyze any of some fifty N-substituted and nonsubstituted di- and tripeptides, amino acid esters, and amino acid amidesS; among them are the commonly used, commercially available proteinase and peptidase substrates. It is especially notable that it has no effect on carbobenzoxyhistidylphenylalanyl tryptophan ethyl ester, 43 a recently described excellent pepsin substrate. 2°a,44 The enzyme appears to have a high specificity with regard to size of the peptide, but relatively low specificity for the bonds involved. The most specific substrates are trypsinogens from various species. Bovine trypsinogen is activated rapidly with the specific cleavage of the bond lysine-6-isoleucine-7. Chymotrypsinogen A and proelastase are not activated. pH Optimum. Optimal trypsinogen activation is obtained at pH 3.48; the pH optimum of hydrolysis of bovine serum albumin is 2.6. 25 Kinetic Constants. K,~ for trypsinogen activation is ( 7 . 6 _ 2 ) X 10.6 M at 0 °, pH 3.4? The molecular activities (turnover numbers) for trypsinogen activation are listed in Table II. It should be noted that these measure specifically the cleavage of the activating peptide bond. 42aOther enzymes inhibited by diazoketones are pepsin, cathepsin D, an enzyme from Aspergillus awamori and thyroid acid proteinase. [T. G. Rajagopalan, W. H. Stein, and S. Moore, J. Biol. Chem. 241, 4925 (1966) ; V. M. Stepanov, V. N. Orekhovich, L. S. Lobareva, and T. I. Mzhel'skaya, Biokhimiya 34, 209 (1969) ; V. M. Stepanov, L. S. Lobareva, N. F. Frolova, and L. I. Oreshenko, Izv. Akad. Nauk SSSR, Set. Khim. 12, 2840 (1968); G. D. Smith, M. A. Murray, L. W. Nichol, and V. M. Trikojus, Biochim. Biophys. Acta 171, 288 (1969); respectively.] ~3G. Mains and T. Hofmann, unpublished observations. K. Inouye, I. M. Voynick, G. R. I)elpierre, and J. S. Fruton, Biochemistry 5, 2473 (1966).
384
THE ACIDIC PROTEASES
[25]
TABLE II MOLECULAR ACTIVITIES OF PENICILLOPEPSINa Temperature (°C)
Trypsinogen activated (micromoles/minute/micromole enzyme)
0 10 15 20 25 30 35
64 105 180 260 420 640 660
Conditions: 0.01 M sodium citrate, pH 3.4; trypsinogen = 10-4 M.
PeniciUocarboxypeptidase, (formerly Peptidase B) ~ from Penicillium janthinellurn This enzyme has been described by Shaw? It is obtained in low yield as a byproduct at the last stage of the purification of penicillopepsin described in the preceding section. Assay Method
Principle. The enzyme hydrolyzes carbobenzoxyglutamyl tyrosine and similar compounds. The hydrolysis can be followed by a ninhydrin procedure? a Reagents Substrate. Carbobenzoxy-L-glutamyl-v~-tyrosine (2 X 10-~ M) in 0.02 M sodium acetate, pH 4.7. Sodium acetate buffer, 4 M, pH 5.3, containing 2 X 10-~ M NaCN. (Prepared fresh daily from 4.08M Na-acetate and-10-2M NaCN.) 3 ~ Ninhydrin in Methyl Cellosolve Isopropanol-water, 1 : 1
Procedure. Equal volumes (0.5 ml) of the enzyme in distilled water or very dilute buffers and the substrate are incubated at 35 ° for 10 minutes. Ninhydrin (0.25 ml) and cyanide-acetate buffer (0.25 ml) are added. The mixture is heated to 100 ° for 20 minutes. After adding 2.5 ml isopropanol-water the extinction is read at 570 nm. In order to avoid confusion with the enzyme aspergillopeptidase B which is a DFP-inhibited alkaline endopeptidase and because of its enzymatic properties, which are those of a carboxypeptidase, the term peptidase B will be dropped. '~H. Rosen, Arch. Biochem. Biophys. 60, 10 (1957).
[25]
MICROBIAL ACID PROTEINASES
385
Definition o] Units. One unit is defined as the amount of enzyme which liberates 1 micromole tyrosine per minute under the standard assay conditions. Purification Procedure Penicillocarboxypeptidase is eluted from the phosphocellulose column (last stage of penicillopepsin) by sodium acetate (0.01 M, pH 4.0), as a single peak in about 6% yield as measured from the second purification stage (chromatography on DEAE-cellulose, see Table I). Properties Penicillocarboxypeptidase is homogeneous as judged by movingboundary and paper electrophoresis? The optimum for hydrolysis of earbobenzoxyglutamyl tyrosine is pH 4.7. K,, is 5 X 10-~ M.
Inhibitors. The enzyme is inhibited by Fe s*, to a small extent by Ba ~÷ and Cd 2÷, but not by Ca 2÷, Zn2+, and Mg 2÷ (all 5 X I0 -a M). F a t t y acids, most di- and tricarboxylic acids and divalent inorganic anions show inhibition at a concentration of 5 X 10-2 M. The inhibition by aliphatic monocarboxylic acids increases with increasing chain lengths. It is about 40% with butyric acid (10 -2 M) and approaches 100% with heptanoic and octanoic acid (10-2 M). It is not affected by thiol reagents (10-8 M). Specificit9. The enzyme is a carboxypeptidase with limited specificity as shown in Table III. Most notable is the absence of activity on a substrate with C-terminal arginine and on substrates with glycine as the residue next to the C-terminal. It does not act on bovine serum albumin.8 TABLE III ACTION OF PENICILLOCARBOXYPEPTIDASE ON PEPTIDES a
Cbz-Glu-Tyr Cbz-Glu-Phe Cbz-Tyr-Glu Cbz-Glu(NH2)-Tyr Cbz-Val-Tyr Cbz-Tyr-Glu(OMe)~ Tosyl-Lys-Gly Cbz-Phe-Ser Tosyl-Arg-Gly Cbz-Gly-Phe Cbz-Gly-Glu C1Ae-Gly-Leu Cbz-Leu-Arg
100 105 72 55 48 33 17 12 8 0 0 0 0
Leu-Tyr GlyoPhe Gly-Leu Ala-Gly Leu-Gly-Gly Gly-Tyr Glutathione Glu-Asp Gly-His Gly-Asp(NH~)
"Values are relative to that for Cbz.Glu.Tyr(= 100). b-y-Methylester.
17 0 0 0 0 0 0 0 0 0
386
THE ACIDIC PROTEASES
[25]
Trypsinogen-Kinase from Aspergillus oryzae ( T a k a d i a s t a s e ) l° A s s a y Method
The assay of this enzyme using trypsinogen activation is carried out as described for penicillopepsin (p. 376). As an alternative, casein digestion at pH 3.0 can be used. Purification Procedure The enzyme is prepared from commercially available Takadiastase by extraction with water (1500 ml per 30 g powder). After adjustment of the extract to pH 3.5, the enzyme is adsorbed on a Duolite CS 101 column (column volume 300 ml) at pH 3.5. The enzyme is eluted with 0.5M sodium acetate (355 ml) and precipitated with (NH4)~S04. The fraction between 0.6 and 0.8 saturation contains the enzyme. After dialysis, the enzyme is precipitated with ethanol between 50 and 60~. Properties The enzyme gives essentially one peak on paper electrophoresis at pH 5.5 and 7.4. It hydrolyzes casein and, in contrast to penicillopepsin, a number of small peptides with a low degree of specificity~7: carbobenzoxyglutamyl tyrosine, carbobenzoxyleucyl arginine, glutathione, L-leucyl-t,-arginine, benzoyl-L-arginyl-L-leucine, and L-phenylalanyl-Larginine. However, the turnover numbers for these substrates are very low and incubation times of 24 hours are used to measure their hydrolysis. As mentioned in the introduction, this activity could be due to contamination by an enzyme like penicillocarboxypeptidase (p. 385). Similar Enzymes Bergkvist ~8 has partially purified an acid proteinase ("proteinase III") from a strain of Aspergillus oryzae. Two types of acid proteinases were isolated from Aspergillus niger var. macrosporus. ~9 It is not known whether these enzymes activate trypsinogen. Aspergillopeptidase A from Aspergillus saitoi is described elsewhere in this volume (article [26] ). Candide albicans Acid Proteinase
The enzyme is purified from the culture medium of Candida albicans strain C.B.S. 2730.11 ~TK. Nakanishi, J. Bioehem. (Tokyo) 47, 16 (1960). R. Bergkvist, Acta Chem. Scand. 17, 1541 (1963). ~Y. Koaze, H. Goi, K. Ezawa, Y. Yamada, and T. Hara, Agr. Biol. Chem. 28, 216 (1964).
[25]
MICROBIAL ACID PROTEINASES
387
Assay Method Principle. The acid proteinase activity is determined by digestion of bovine serum albumin at pH 3.2. The extent of hydrolysis is derived from the amount of TCA-soluble material remaining after digestion. Reagents 1% w/v Bovine serum albumin in 0.05M sodium citrate buffer, pH 3.2 5% w/v Trichloroaeetic acid Procedure. Enzyme samples (0.5 ml) are incubated at 37 ° with 2.0 ml of the 1% bovine serum albumin substrate (pH 3.2) for 60 minutes. The reaction is terminated by the addition of 5% TCA (5 ml) and the precipitated albumin removed by centrifugation. Two milliliters of the supernatant is taken to determine the amount of TCA-soluble products by the method of Lowry et al? ° Specific Activity. The unit of proteinase activity has not been defined and thus specific activity is reported in 'arbitrary units. Purification Procedure Step 1. Cultivation. The culture medium used to provide good proteinase production consists of the following; 0.5 g MgS04, 1 g KH~P04, 20 g glucose, 2 g human .or bovine serum albumin, and 1.25 ml Protovita (polyvitamin preparation, Hoffmann-La Roche Co.) in 1 liter of water. The pH is adjusted to 4.0 and the total medium sterilized by filtration. The medium is inoculated to contain 500 viable cells per milliliter and incubated with shaking at 26 ° for 3-5 days. During this time the pH usually decreases to 3.0-3.2. In order to harvest, the medium containing the enzyme is filtered to remove cellular material. Step 2. Concentration. The medium containing 600 mg protein per liter is concentrated to a workable volume by pervaporation21 The volume is reduced from 1 liter to .approximately 40 ml. Step 3. Gel Filtration. The concentrated protein is applied to a column of Sephadex G-75 (3 X 100 cm) equilibrated to pH 4.0 with 0.05M sodium citrate buffer. Three major protein peaks are eluted after 380 ml, 450 ml, and 560 ml. The second of these contains the acid proteinase activity. It is pooled (approx. 70 ml) and dialyzed against 0.01 M sodium citrate buffer (pH 6.5). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). '~This step is probably replaceable by a step using a Diaflo (Amicon Corp.) apparatus.
388
THe. ACIDIC PROTEASES
[25]
Step 4. Chromatography on DEAE-Sephadex A-25. The dialyzate is applied to a DEAE-Sephadex A-25 column (0.8 X 10 cm). Elution with the equilibration buffer removes a peak with no activity. After approximately 15 ml, a linear gradient from 0.01 M sodium citrate to 0.2 M sodium citrate using a gradient volume of 100 ml is applied. This elutes the enzyme as two partially separated peaks. Rechromatography of each of the peaks under the same conditions produces in each case a major protein peak and a minor peak with identical specific activities and pH optima. They appear to be two very similar forms of the enzyme and can be separated by acrylamide gel electrophoresis. The purification is summarized in Table IV. TABLE IV PURIFICATIONOF Candida albicans ACID PROTEINASE
Fraction
Protein concentration (mg/ml)
Volume (ml)
Specific activity (arbitrary units)
Culture medium Conc. medium Sephadex G-75 eluate DEAE-Sephadex eluate
0.6 8.8 0.93 0.27
1000 39 102 7.9
1 1.4 4.1 140
Properties
Stability. Gradual loss of activity occurs on standing in solution in the cold or when frozen. Activity is rapidly lost on repeated freezing and thawing and lyophilization. Purity. The two acid proteinases appear homogeneous on acrylamide disc electrophoresis at pH 4.5 and pH 8.0 and give a single peak in the ultracentrifuge. Molecular Properties. Molecular weight determined by gel filtration (Sephadex G-75 and 100): 42,000; sedimentation constant: S~o,~= 3.5 X 10-13 seconds (density gradient) or 3.2 X 10-13 seconds (sedimentation velocity). Enzymatic Properties. pH optimum: for bovine serum albumin: pH 3.2. Inhibition. Millimolar concentrations of EDTA, mercaptoethanol, CaC12, MgCl~, MnC12, (NH4)~SO4, p-chloromercuribenzoate, kallikrein (equal weight to enzyme) do not affect the activity. The two pepsin inhibitors p-bromophenacylbromide and diazoacetylnorleucine methyl ester were without effect.
[25]
MICROBIAL ACID PROTEINASES
389
Specificity. The Candida albicans acid proteinase cleaves 9% of the peptide bonds in bovine serum albumin. Digestion of the B-chains of insulin indicates a preference for peptide bonds involving the carboxyl group of amino acids with hydrophobie side chains. However, there are no indications of any strict specificity. Paecilamyces varioti Acid Proteinase 12 Assay Procedure The enzyme is assayed with a 0.6% casein solution in Sorensen buffer41 at pH 3.0, 40 °. Undigested casein is precipitated with trichloroacetic acid (0.2 M final concentration) and centrifuged. Digested casein in the supernatant is determined with the Folin-Ciocalteau reagent? 2 Definition o] Units. One unit is equal to the amount of enzyme which liberates the equivalent of 1 microgram of tyrosine per minute. Purification Procedure TM The organism, Paecilomyces varioti Bainier TRR-220, is grown on wheat bran (10 kg) and rice husks (3 kg) mixed with 5 liters water at 30 ° . After 60 hours the culture is extracted with 50 liters water. After centrifugation the enzyme is precipitated from the supernatant with 2 volumes ethanol precooled to --25 °. The precipitate is dried in vacuo. It is extracted with water (1 liter for 200 g) and centrifuged. The residue is washed with 200 ml water. Both supernatants (950 ml) are adjusted to pH 4.0 with cone. HC1; cold ethanol (475 ml) is added slowly so that the temperature is maintained below 0 °. Cornstarch (300 g) is stirred in at --5 ° and removed after 1 hour by centrifugation. The ethanol concentration of the supernatant is increased to 40% and the cornstarch treatment repeated. (This treatment serves to remove amylase.) After removal of the starch, the ethanol concentration is raised to 65%. The precipitate is centrifuged off and dissolved in 0.1 M sodium acetate buffer, pH 3.0 (250 ml). Insoluble material is centrifuged off and discarded. The superntant (240 ml) is adjusted to pH 4.0; calcium acetate (1 M, 80 ml) is added and a precipitate removed. The supernatant is dialyzed against running water, centrifuged, and brought to 40% saturation with ammonium sulfate at pH 5.3. After removing a precipitate, the ammonium sulfate concentration is increased to 85% saturation and the pH adjusted to 3.5. The precipitate is centrifuged off and dissolved in 0.005 M KH2PO~ (pH 5.0, 50 ml). Ammonium sulfate is removed on a Sephadex G-25 column (3 X 70 cm) equilibrated with 0.005 M KH2PO,. 5~M. L. Anson, J. Gen. Physiol. 20, 561 (1937) ; ibid., 22, 79 (1938).
390
THE ACIDIC PROTEASES
[2S]
The fraction containing the enzyme is adjusted to pH 3.0, a small amount of precipitate is removed, and after adjusting to pH 4.0 cold acetone is added to 60%. The precipitate is removed, dissolved in 0.005 M KH2PO, at pH 3.5 to give a 2% protein concentration and centrifuged to clarify. After adjusting to pH 4.0 the enzyme is crystallized by adding acetone to 50~. On recrystallization 960 mg of the proteinase crystals are obtained. The purification is summarized in Table V. TABLE V Paecilomyces PROTEINASE
SUMMARY OF PURIFICATION OF
Fraction
Specific activity (units/rag protein)
Yield (%)
Crude extract First starch treatment Second starch treatment Ethanol precipitate 85 % Saturated (NH4)~S04precipitate 60% Acetone precipitate First crystals Second crystals
378 670 810 1100 1460 1600 1650 1940
100 88.5 84.0 82.4 51.9 46.2 19.2 14.3
Properties
Stability. The enzyme is stable between pH 3 and 6.5. It is most stable to elevated temperatures at p H 5.0. Purity. The crystalline enzyme gives a single sedimenting boundary in the ultracentrifuge and single bands on continuous paper electrophoresis at pH 7.35 and 8.25. Molecular Properties28 Molecular weight 37,300; sedimentation constant at 15 °, $i~ : 2.75 X 10-is seconds; isoelectric point 3.8; amino acid composition: Lys28, HisT, Arg3, Asp4~, Thr2~, Ser89, Glu23, Pro2o, Gly33, Ala2e, CySH1, Valls, Meh, Ilel~, Leu21, Tyr16, Phe14, Trp3. There is evidence for the presence of one SH group. Leucine or isoleucine is N-terminal, alanine C-terminah Enzymatic Properties. pH optimum: This is 3.0 for the digestion of casein , ovalbumin, and fibrin; between 3.0 and 4.5 for hemoglobinS~; 3.5 for carbobenzoxy-L-glutamyl-L-tyrosine and 5.5 for benzoyl-L-arginine amide. Inhibitors. The enzyme is inhibited by 5 X 10-3 M Hg 2÷ but not by other metal ions. It is partially inhibited by p-hydroxymercury benzoic acid and iodoacetic acid, and by bacitracin, tetracycline, and nitrofurylacrylamide. The effect of many other compounds on the activity is also u j. Sawada, Agr. Biol. Chem. 30, 393 (1966). ~J. Sawada, Agr. Biol. Chem. 28, 348 (1964).
[25]
MICROBIAL
ACID PROTEINASES
391
given by Sawada24 Many divalent metal ions increase the heat stability of the enzyme. Specificity. The enzyme readily digests casein and hydrolyzes some peptides, notably, carbobenzoxy-L-glutamyl-L-tyrosine (I) and benzoylL-arginine amide (II). Approximate turnover numbers calculated from Sawada's paper 2~ are 0.05 to 0.1 micromoles substrate per minute per micromole enzyme. As with the Aspergillus oryzae enzyme (this volume [26] ) it is possible that the hydrolysis of these small substrates is due to contaminating enzymes. Kinetic Constants. K,~ for (I) = 4.1 X 10-3M; for (II) = 6.6 X 10.8 M. Acid Proteinase from Rhizopus chinensis The acid proteinase has been purified by Fukumoto et al. 13 from the culture medium of Rhizopus chinensis Saito. It is also available commercially as a 3 X crystallized preparation from Miles Laboratories, Elkhart, Ind. and from Seikagaku Kogyo Ltd., Tokyo, Japan.
Assay Method Principle. The activity of the enzyme is determined using casein at pH 3.1 as a substrateJ 3,55 Alternatively, the enzyme may be assayed by the trypsinogen-kinase assay described on page 376, by digestion of bovine serum albumin (p. 378) or by a milk-clotting assay. Reagents 0.6% Casein in 5 X 10-2 M lactic acid, pH 3.1 Trichloroacetic acid (TCA) precipitant: 0.22 M TCA-0.33 M acetic acid-0.11 M sodium acetate
Procedure. The enzyme solution (1 ml) is added to the casein (5 ml) at 30 °. After 10 minutes, 1 ml TCA precipitant is added. The precipitate is filtered off after 30 minutes at 30 ° and the extinction at 275 nm of the filtrate measured. Definition o] Units. One unit is defined as the amount of enzyme which liberates extinction units equivalent to 1 ~g tyrosine per milliliter per minute. Specific activity is defined as the enzyme units per milligram protein. Purification Procedure
Method o] Fukumoto et al. ~3 Rhizopus chinensis Saito is cultured by the Koji method. The culture medium, which consists of 1 kg of wheat bran (Senkan) and 100 g of cornstarch in 1 liter of tap water, is sterilized D. Tsuru, T. Yamamoto, and J. Fukumoto, Agr. Biol. Chem. 30, 651 (1966).
392
T~. AcmIc Pao1~,s~.s
[25]
at 120 ° for 30 minutes. The mixture is spread on a pan, inoculated with spores, and left under a cover at 25 ° for 70 hours. The medium containing the enzyme is extracted with 8 liters of deionized water, pH 5.8, with occasional stirring for 1.5 hours at 30 °. Insoluble material is removed with a filter press and benT.alkonium chloride added to give a concentration of 0.2~ at pH 5.2. After standing overnight, a precipitate forms which is removed by filtration through a layer of Celite. The filtrate is passed through a column of Asmit 173 N (4 X 70 cm) equilibrated to pH 5.2 with 10-2 M acetate buffer to remove colored impurities. The total effluent, including 500 ml equilibration buffer, is collected. Ammonium sulfate is added to the combined effluent to 70% saturation. After standing for 5 hours, the precipitate is collected by centrifugation and dissolved in 10-2 M acetate buffer (500 ml), pH 5.4. The presence of cellulase in this solution prevents dialysis. Sulfate ions are therefore precipitated with barium acetate and after adjusting the pH to 5.4, the barium sulfate precipitate is centrifuged off. The supernatant is diluted with deionized water until the concentration of ammonium acetate is 5 X 10 -~ M.
The proteinase is next adsorbed on a Duolite A-2 column (4 X 70 cm), equilibrated with ammonium acetate at pH 5.4. The cellulase, glucoamylase, and some hemicellulases pass through. One liter of equilibration buffer is used to wash the column before the proteinase is eluted with 0.3N acetic acid (1.5 liters). The proteinase emerges when the pH drops below 4.2. Ammonium sulfate is added to 70% saturation. The precipitate formed overnight is centrifuged off and dissolved in 10-~ M acetate buffer (30 ml), pH 4.5 and applied to a Sephadex G-100 column (2.8 X 80 cm) equilibrated with the same buffer. The column is operated at a flow rate of 30 ml/hour and the en~.ymatic fraction (150 ml) collected. The desalted material is adsorbed on a CM-cellulose column (2.3 X 70 cm) equilibrated with 10-~ M acetate buffer, pH 4.5. After washing the column with 200 ml of the equilibration buffer, the acid proteinase is eluted with a linear sodium chloride gradient (0 to 0.4 M, prepared from 1 liter buffer and 1 liter buffer-0.4M sodium chloride) at a flow rate of 30 ml/hr. The proteinase activity emerges as a single peak at 0.2 M sodium chloride. The combined fractions are brought to 75% saturation with ammonium sulfate; the precipitate is collected by centrifugation and dissolved in 10-2 M acetate buffer, pH 4.2 (170 ml). The acid proteinase can be crystallized at this stage by dialyzing against the acetate buffer containing 2 X 10 -~ M calcium acetate and stirring for 24 hours. Acetone is added until the material becomes slightly cloudy. After leaving at 0 °,
[25]
MICROBIAL ACID PROTEINASES
393
needle crystals appear in 10 minutes and additional drops of acetone will crystallize out most of the remaining enzyme within 24 hours. The crystals are collected by centrifugation, washed twice with small quantities of 2 X 10-8 M calcium chloride solution, pH 5.0, and then dissolved by the dropwise addition of 0.2 N ammonium hydroxide, bringing the pH to 7.0. Any insoluble material is removed by centrifugation and the supernatant adjusted to pH 4.8--5.4 with 0.2 N acetic acid before dialyzing against 10-8 M calcium chloride solution, pH 5.2. Pillar-shaped crystals form 1 or 2 days after dialysis. Dropwise addition of acetone to a final concentration of 20% completes the crystallization. The purification is summarized in Table VI. T A B L E VI PURIFICATION OF Rhizopus chinensis ACID PROTEINASE
Fraction Crude extract Benzalkonium chloride treatment Asmit 173-N eluate a Barium acetate t r e a t m e n t Duolite A-2 eluatea Sephadex G-100 eluate CM-cellulose eluate o Acetate buffer dialyzate Acetone crystals Recrystallization Lyophilized product
Volume (ml)
Total activity × 10-~ (units)
Specific activity (units/mg)
8000 8000
344 320
-50
8500 6000 900 150 170 35 ----
290 270 130 115 • 89 70 65 62 60
90 230 615 1140 1840 2140 2200 2200 2200
Yield (%) 100 93 84.4 78.5 37.8 33.4 25.9 20.4 18.9 18.0 17.4
a After concentration by ammonium sulfate precipitation.
Purification o] Commercial Enzyme? s The acid proteinase is available as a 3 X crystallized product from Miles Laboratories. It is prepared in a manner similar to that described in the preceding paragraphs27 The preparation shows three major components on both acrylamide disc electrophoresis at pH 9.0 (Fig. 2; A,0) and analytical isoelectric focusing5s,s9 (Fig. 2; B,0). It can be purified by isoelectric focusing on a preparative scale by the method of Svensson2 ° J. ~odek and T. H o f m a n n , to be published. sTD. Tsuru, personal communication. ~ H. Svensson, Acta Chem. Scand. 15, 325 (1961). mA. Dale, and A. L. Latner, Lancet, p. 847 (1968). e°H. Svensson, Arch. Biochem. Biophys. Suppl. I, 132 (1962).
394
THE ACIDIC PROTEASES
[2S]
8
~H
+ 0
t
2
0
~
2
A
3
B
FIG. 2. Electrophoresis of commercial Rhizopus chinensis acid proteinase on acrylamide gel, pH 9.0 (A) and by analytical electrofocusing (B). (0) Starting material (3 X crystallized preparation) (1-3) Peaks I, II, and III, respectively, after purification by preparative electrofocusing.
I
t2 1.0 =
[!, / LO
~6 0.6 Q
o
/
Ii', r'l
0.8
8 (kl ! X
0.4
8 pH
o.6} ~. o...~t...~r ' ' ~ ' ~
iv,, I--,J ....
:~
•~il--f
102
,.._,_,.._,_,~,,~,
• ~'~,A J
Io
-
I I I
''
~2
•
i
•
n
|
Froctionnumber
FIG. 3. Electrofocusing of commercial Rhizopus chinensis acid proteinase on a preparative LKB electrofocusing column 8101 (110 ml). Fractions 14 ml. (O O) pH, (X X) OD at 280 nm, (A A) trypsinogen-activating activity.
[25]
MICROBIAL ACID PROTEINASES
395
The preparative method is carried out on an LKB electrofocusing column (No. 8101) using 2% carrier ampholyte (pH range 5-8) in a sucrose gradient. The freeze-dried Miles preparation is dissolved in distilled water (approximately 20 mg in 2 ml of water) and dialyzed against distilled water overnight. It is added to those sucrose solutions that will form the midregion of the gradient to prevent the protein from being exposed to either anode or cathode buffer solution. A constant voltage (500 V) is applied over a period of 24 hours. During this time the current passing through the column drops to zero as the pit gradient is formed and the proteins focus at their respective isoelectric points. The sucrose gradient is eluted in 1.5 ml fractions; the pH, optical density at 280 nm, and the activity are determined (for a typical run see Fig. 3). One peak is eluted from the acidic region of the gradient; two major peaks with isoelectric point 5.2 (peak I) and isoelectric point 6.0 (peak II) and a minor peak (III, isoelectric point = 5.45) are eluted subsequently. Peak I and peak II have comparable specific activities using both bovine serum albumin and trypsinogen as substrates. Peak III and the other, unlabeled peaks appear to be inactive in both assays. Stability. 13 Rhizopus chinensis acid proteinase is stable for 24 hours between pH 2.8 and 6.5 at 30 °. At pH 3.1, the enzyme retains full activity after 15 minutes of exposure to temperatures up to 60 ° . Purity. Fukumoto Preparation. Ultracentrifugation and behavior on elution from sulfoethyl Sephadex suggest homogeneity of the enzyme. TM On disc electrophoresis, isoelectric focusing, and moving-boundary electrophoresis, a minor component accounting for less than 5% of the total protein is occasionally observed. 57 Purified Commercial Preparation. Peak I and peak II appear as a single band on acrylamide disc eleetrophoresis (Fig. 2; A, 1 and 2) and on analytical isoelectric focusing (Fig. 2; B, 1 and 2).
Molecular Properties The enzyme has the following physical constants61: molecular weight = 35,000; sedimentation constant, S2o,w = 2.83 X 10-13 seconds; partial specific volume, V = 0.73 (calculated from the amino acid composition) ; isoelectric point, 5.2; specific extinction, ~l~g/r~m = 1.29. The optical rotary dispersion parameters are [ a ] , = - - 3 3 , ao = --200, bo = 0, )~c = 208 nm and [m'] 227 n m = --2.100, suggesting the absence of a-helix structure in the molecule. Peak I from the commercial preparation is identical in molecular weight, partial specific volume, isoelectric point, and specific extinction. Amino Acid Composition. The amino acid composition of the Rhizopus
396
TH~. ACIDIC PROTEASES
[2S]
TABLE VII AMINO ACID COMPOSITIONSOF Rhizopus chinensis ACID PROTEINASE FUKUMOTO AND PURIFIED "MILES" PREPARATIONS
Rhizopus chinensis" residues"
Miles P I b residues
P IIb residues
12 0 9 43 32 26 21 14 39 23 19 3 21 23 14 16 5 4 32
13 0 9 43 30 27 21 13 46 22 20 3 21 21 15 17 d 4 d
14 0 8 40 28 30 21.5 14 46 22 21 1.4 20 21 12 16 d d d
Lysine I=Iistidine Arginine Aspartie acid Threonine Serine Glutamic acid Proline Glyeine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan tIalf-cystine Amide NH~
D. Tsuru, A. Hattori, H. Tsuji, and J. Fukumoto, J. Biochem. (Tokyo), 67, 415 (1970). J. ~odek and T. Hofmann, to be published. "Number of residues per molecule (MW = 35,000). Not determined.
"
acid proteinase 61 is listed in Table VII. Also listed for comparison are the composition for Miles peak I and peak II. 56 A noticeable feature in all three is the absence of histidine. In spite of the clear electrophoretic difference between peaks I and IT, there are only minor differences in composition between them. Peak I is probably identical to the Fukumoto enzyme. Alanine is the N-terminal amino acid.
Enzymatic Properties Inhibition. Millimolar concentrations of E D T A , o-phenanthroline, CuS04, HgCl~, FeSO4, Pb-acetate, monoiodoacetic acid, p-chloromercuribenzoate, and ascorbic acid have no appreciable affect on the activity? s Ferric sulfate, 2.0 mM, inhibits approximately 75%. is The pepsin inhibitor, diazoacetyl norleucine methyl ester in the e'D. Tsuru, A. ttattori, H. Tsuji, and J. Fukumoto, J. Biochem. (Tokyo), 67, 415 (1970).
ACID PROTEINASE OF Aspergillus saitoi
[26]
397
presence of Cu 2÷, completely inhibits activity of the Miles peak I and peak H when assayed with serum albumin or with trypsinogen as substrate2 ~ In each case, 1.3 and 2.0 residues, respectively, of norleucine per molecule are incorporated. Specificity. The digestion of the B-chain of oxidized beef insulin by Rhizopus chinensis acid proteinase is similar to that obtained by pepsin22 During the digestion of casein about 15% of the peptide bonds are broken. The enzyme acts on polyglutamic acid and polylysine.6z The following di- and tripeptides are hydrolyzed6~ (after incubation for 18 hours at 37 °) : lysyl-tyrosyl-glutamic acid, carbobenzoxyglutamyl-tyrosine, carbobenzoxyglutamyl-phenylalanine, carbobenzoxyphenylalanyl-tyrosine, carbobenzoxy-glycyl-phenylalanine, glycyl-leucyl-tyrosine, glycyl-glutamyttyrosine, leucyl-glycyl-phenylalanine, glutamyl-glycyl-phenylalanine, leucyl-tyrosine, glutamyl-tyrosine, and leucyl-phenylalanine. Except for the first substrate, where both peptide bonds are cleaved and transpeptidation occurs, a C-terminal aromatic amino acid is liberated in all cases. Other di- and tripeptides lacking the C-terminal aromatic amino acid are not hydrolyzed2~ The enzyme also has milk-clotting activity. pH Optimum. The pH optimum of casein digestion is between 2.9 and 3.3.18 Similar Enzymes. A milk-clotting acid proteinase from Rhizopus oligosporus (NRRL-3271) has been partially purified23 Acknowledgments The authors are most grateful to Dr. Tsuru for making available unpublished observations. Experimental work by the authors reported here was supported by the Medical Research Council (Canada) Grants MT-1982 and MA-2438. J. S. is the holder of an M.R.C. Studentship. D. Tsuru, A. Hattori, H. Tsuji, T. Yamamoto, and J. Fukumoto, Agr. Biol. Chem. 33, 1419 (1969). ~3H. L. Wang, D. I. Ruttle, and C. W. Hesseltine, Can. J. Microbiol. 15, 99 (1969).
[26] P u r i f i c a t i o n a n d M o d e of A s s a y f o r A c i d P r o t e i n a s e of Aspergillus saitoi
By EIJI ICHISHIMA Assay Method
Principle. Methods developed by Anson, Kunitz, and Hagihara I are modified for estimating the extracellular acid proteinase of Aspergillus
ACID PROTEINASE OF Aspergillus saitoi
[26]
397
presence of Cu 2÷, completely inhibits activity of the Miles peak I and peak H when assayed with serum albumin or with trypsinogen as substrate2 ~ In each case, 1.3 and 2.0 residues, respectively, of norleucine per molecule are incorporated. Specificity. The digestion of the B-chain of oxidized beef insulin by Rhizopus chinensis acid proteinase is similar to that obtained by pepsin22 During the digestion of casein about 15% of the peptide bonds are broken. The enzyme acts on polyglutamic acid and polylysine.6z The following di- and tripeptides are hydrolyzed6~ (after incubation for 18 hours at 37 °) : lysyl-tyrosyl-glutamic acid, carbobenzoxyglutamyl-tyrosine, carbobenzoxyglutamyl-phenylalanine, carbobenzoxyphenylalanyl-tyrosine, carbobenzoxy-glycyl-phenylalanine, glycyl-leucyl-tyrosine, glycyl-glutamyttyrosine, leucyl-glycyl-phenylalanine, glutamyl-glycyl-phenylalanine, leucyl-tyrosine, glutamyl-tyrosine, and leucyl-phenylalanine. Except for the first substrate, where both peptide bonds are cleaved and transpeptidation occurs, a C-terminal aromatic amino acid is liberated in all cases. Other di- and tripeptides lacking the C-terminal aromatic amino acid are not hydrolyzed2~ The enzyme also has milk-clotting activity. pH Optimum. The pH optimum of casein digestion is between 2.9 and 3.3.18 Similar Enzymes. A milk-clotting acid proteinase from Rhizopus oligosporus (NRRL-3271) has been partially purified23 Acknowledgments The authors are most grateful to Dr. Tsuru for making available unpublished observations. Experimental work by the authors reported here was supported by the Medical Research Council (Canada) Grants MT-1982 and MA-2438. J. S. is the holder of an M.R.C. Studentship. D. Tsuru, A. Hattori, H. Tsuji, T. Yamamoto, and J. Fukumoto, Agr. Biol. Chem. 33, 1419 (1969). ~3H. L. Wang, D. I. Ruttle, and C. W. Hesseltine, Can. J. Microbiol. 15, 99 (1969).
[26] P u r i f i c a t i o n a n d M o d e of A s s a y f o r A c i d P r o t e i n a s e of Aspergillus saitoi
By EIJI ICHISHIMA Assay Method
Principle. Methods developed by Anson, Kunitz, and Hagihara I are modified for estimating the extracellular acid proteinase of Aspergillus
398
THE ACIDIC PROTWSES
[25]
saitoi (EC 3.4.4.17, aspergillopeptidase A). A convenient procedure is based on the determination of split products, soluble in 0.4 M trichlor(~acetic acid, of a standard protein. The optimal pH for milk casein digestion is in the range of 2.5 to 3.0.
Reagents HC1-CHsCOONa buffer, 0.1 M, pH 2.7 Two percent milk casein solution at pH 2.7 is prepared by suspending 2.0 g milk casein (Hammarsten, E. Merck Co.) in about 10 ml of water. This suspension is allowed to stand for about 10 to 15 minutes at 30 °. The mixture is prepared by kneading in a volume of about 60 ml of water. To the milk casein suspension is added 1.5 ml 1.0 N HC1 and the mixture is dissolved by stirring on a boiling water bath. The solution is then adjusted to pH 2.7, made up to 100 ml, and stored in the refrigerator. Triehloroacetic acid, 0.4M Na~C08, 0.4 M Folin-Ciocalteau reagent. 2 The commercially available Folin-Ciocalteau phenol reagent is diluted 1:5 with distilled H20 before use.
Standard Tyrosine Solution. The tyrosine standard is prepared by dissolving 18.10 mg of pure, dry tyrosine in I liter of 0.2 M HCI. I
Procedure. One milliliter of a 2% milk casein solution of pH 2.7 is digested for I0 minutes at 30 ° by 1 ml of a suitably diluted acid proteinase solution. The enzyme solution is diluted with 0.1 M sodium acetate buffer at pH 2.7. The reaction is terminated by the addition of 2 ml of 0.4M trichloroacetic acid and the mixture is filtered through Toyo filter paper No. 2 or Whatman paper No. 2. The tyrosine and tryptophan in an aliquot of the filtrate are determined by the blue color given with the Folin-Ciocalteau phenol reagent in alkaline solution. The color is compared against a standard tyrosine solution. One milliliter of the tricholoroacetic acid filtrate is placed in a 25-mi test tube and 5 ml of 0.4M Na~C03 and l ml of the diluted FolinCiocalteau phenol reagent are added while the solution is kept at 30 ° during addition of the phenol reagent. After standing for 20 to 30 minutes, the color is read at 660 nm against a standard prepared in the same manner. A blank is run with each series of determinations. Determination o] Units and Specific Activity. One unit of the acid ZB. Hagihaxa, Ann. Rept. Sei. Works, Fac. Sci. Osaka Univ. 2, 35 (1954). • ' O. Folin and V. Ciocalteau, 3. Biol. Chem. 73, 627 (1927).
[25]
ACID PROTEINASE OF Aspergillus saitoi
399
proteinase is defined as the amount of enzyme which yields the color equivalent to 1 micromole of tyrosine per minute in 2 ml of digestion mixture at pH 2.7 and 30 °. The protein nitrogen of the trichloroacetic acid-precipitated fraction of the enzyme solution is determined by the micro-Kjeldahl method. Using a 5-ml beaker, 0.5 ml of 21% trichloroacetic acid is added to 1.0 ml of the enzyme solution. The mixture is allowed to stand for about 30 minutes to obtain complete precipitation of the protein. 1 The specific activity of acid proteinase per milligram enzyme is expressed in terms of mieromoles of tyrosine equivalents produced per minute during casein hydrolysis at pH 2.7 and 30 °, as determined by the increase of the absorbancy at 660 nm through application of the Folin-Ciocalteau reagent. Purification Procedure
Cultivation o/Aspergillus saitoi Cultivation of molds of the genus Aspergillus is carried out both in solid culture (Koji-culture) and liquid culture (surface or submerged culture) .3 The first extracellular acid proteinase of Aspergillus saitoi was found by Yoshida. 4 The production of acid proteinase either on solid or in liquid media involves many variable factors: medium components, sterilization of media, size of inoculum, type of antifoam reagents, temperature control, aeration and agitation, and sterility control during the operation. Organism. The organism used in the preparation of this enzyme is Aspergillus saitoi R-3813 (ATCC-14332) of black Aspergillus. 5'6 In all the strains of black AspergilIus isolated by Sakaguchi, Iizuka, and Yamazaki ~,6 (A. saitoi, A. usamii, A. inuii, A. aureus, A. awamori, and A. nakazawai), the characteristics of the conidial wall surface are quite different from those of ordinary A. niger. In black AspergiUus the wall could be either rough in appearance or perfectly smooth, while in A. niger it is distinctly echinulated or with colored bars, as mentioned by Thorn and Raper. 7 The nature of the eonidial wall as well as the size, shape, and color of conidia of Aspergillus are the essential features used in the 3K. Arima, in "Global Impacts of Applied Microbiology" (M. P. Starr, ed.), p. 277. Almgvist and Wikselle Boktryckeri Aktiebolag, Uppsala, Sweden, 1964. 4F. Yoshida, J. Agr. Chem. Soc. Japan 28, 66 (1954). K. Sakaguchi, H. Iizuka, and S. Yamazaki, J. Agr. Chem. Soc. Japan 24, 138 (1951). *H. Iizuka, J. Gen. Appl. Microbiol. (Tokyo) 1, 10 (1955). C. Thom and K. B. Raper, in "A Manual of the Aspergilli," p. 214. Williams & Wilkins, Baltimore, Maryland, 1945.
400
THE ACIDIC PROTEASES
[26]
taxonomy of the genus Aspergillus. ~,8 The electron microscope is an indispensable tool for studying the conidial wall of Aspergillus? The mold A. saitoi is maintained on Koji agar slant at 10° and transferred twice per year. Czapeck's medium and the modified Czapeck medium, containing glucose and peptone, can be substituted for the Koji agar slant. A. saitoi grows well in a solid or liquid medium, with added wheatbran or/and defatted soybean. K o j i Culture. 4,9 A wheat bran medium is prepared by kneading 5 g of wheat bran and 3.5 ml of tap water in a 150-ml Erlenmeyer flask and autoclaving at 15 lb pressure for 30 minutes. The production of acid proteinase is increased by about 60% by adding 50 mg NH4C1.9 The sterilized bran is inoculated with spores of A. saitoi and is then incubated at 30 ° for about 60 hours or more. A large number of molds grow satisfactorily on this medium. For the sake of temperature control it is a good procedure to shake and ,crush germinating or growing myeelium twice a day. Contamination should be avoided. Submerged Culture. ~°, 1~ A liquid medium prepared from wheat bran, defatted soybean, and NH4C1 is used. Wheat bran, 1.8 g, and crushed defatted soybean, 1.2 g, are mixed with 3 ml of tap water containing 0.03 ml conc. HC1, and are then sterilized at 15 lb for 2 hours. The sterilized mixture with or without 1 g of NH~C1 is prepared in 100 ml tap water in a 500-ml flask and is autoclaved at 15 lb pressure for 2 hours. For the germination of black Aspergillus ~°,~ the pH is adjusted to about 5.5. After inoculation with spores, a culture is incubated at 30 to 35 ° and is shaken continuously at 140 7-cm strokes per minute for 60 hours or more in a reciprocating-type shaker. The production of acid proteinase falls by 30 to 50% if the 1% NH~C1 is omitted from the medium.~°,1~ The described fermentation unit accommodates flasks from 500-ml shake types to the 20-liter Waldhof's type ~2,~3 jar fermentor. ~1 A suspension of rapidly growing cells is used for inoculation at a level of 5%. This suspension is obtained by inoculating 100 ml of inoculum medium into a 500-ml shake flask and incubating for 24 hours on the shaker at 35 °. Fermentation is carried out at 35 ° in a base medium consisting of 0.66% wheat bran, 1.33% defatted soybean, and 1% NH4C1 at pH 5.5. 8K. Sakaguchi and K. Yamada, J. Agr. Chem. Soc. Japan 20, 65, 141 (1944). *E. Ichishima and F. Yoshida, Agr. Biol. Chem. (Tokyo) 26, 547 (1962). I°E. Ichishima and F. Yoshida, Agr. Biol. Chem. (Tokyo) 26, 554 (1962). 1~E. Ichishima, Y. Gomi, T. Watarai, and F. Yoshida, Agr. Biol. Chem. (Tokyo) 0.7, 302 (1963). K. Yamada, T. Ito, A. Koyama, and Y. Handa, J. Agr. Chem. 8oc. Japan 25, 173 (1951). K. Yamada, T. Ito, Y. Handa, and A. Koyama, J. Agr. Chem. 8oc. Japan 26, 335 (1952).
[25]
ACID PROTEINASE OF Aspergillus saitoi
401
In all cases, 10 liters of inoculated medium are stirred and aerated in a 20-liter fermentor. The rate of agitation and aeration are 300 to 400 rpm and 2.5 to 5 liters/minute. Sterilized soy sauce oil,1~,1~ rapeseed oil, or soybean oil at a concentration of 0.5% is used as a defoaming agent. 1~ Maximal production of acid proteinase is resched in 60 to 90 hours.
Isolation and Purification o] Acid Proteinase of Aspergillus saitoi Preparation o] Crude Enzyme ]rom Culture Filtrate o] Koji or Submerged Cultures. The major part of acid proteinase produced in the Koji culture is extracted with 10 volumes of tap water. The mixture is adjusted to pH 4.0 with 1 N HC1 and is allowed to stand in the cold room for 30 to 60 minutes. TM After adjusting the culture filtrates to pH 4.0, the acid proteinase is precipitated with cold (5 °) ethyl alcohol to obtain a final ethanol concentration of 70%. The precipitate is separated by centrifugation and dried in vacuo or lyophilized (fraction M1). TM The acid proteinase can be separated from the liquor by methyl alcohol, isopropyl alcohol, or acetone, and can be salted out by addition of 53 g solid (NH4)2SO4 per each 100 ml of solution. 16,~7 The dried material is ground up in a mortar and stored in the cold. Chromatographic Purification o] Acid Proteinase. TM Chromatographic elution is accomplished at low temperature (e.g., 5°). Six grams of the crude enzyme preparation are added to l0 ml of water, and the mixture is prepared by kneading and is added to about 30 ml of water. The enzyme solution is adjusted to pH 3.5 with 1 N HC1 in 60 ml; it is allowed to stand for 1 hour in the refrigerator and is subsequently filtered through Celite. The filtrate containing the acid proteinase is dialyzed against 0.02 M sodium acetate buffer at pH 3.5. Commercial cation-exchange resin, Duolite CS-101 or Amberlite CG-50, 100 mesh, has been found suitable for chromatographic separation of the acid proteinase from culture filtrates. TM Commercial Duolite CS-101 or Amberlite CG-50 should be thoroughly washed before use. The dry material is suspended in 1.0 N NaOH. The suspension is filtered and washed with additional 1 N NaOH. The Na form of the resin is filtered and washed with water until no more alkali is removed. This is followed by the addition of sufficient 1 N HC1 to make a strongly acid suspension, which is filtered and washed free of acid with water. The filter cake is adjusted to the pH of the starting buffer. The column (3 X 60 cm) is 1~S. Uchida, J. Chem. Soc. Japan 37, 442 (1934). T. Kubo, J. Agr. Chem. Soc. Japan 23, 335 (1947). 16E. Ichishima, M. Funabashi, and F. Yoshida, Agr. Biol. Chem. (Tokyo) 27, 310 (1963). 1TF. Yoshida and M. Nagasawa, Bull. Agr. Chem. Soc. Japan 20, 252 (1956). E. Ichishima and F. Yoshida, Biochim. Biophys. Acta 99, 360 (1965).
402
THE ACIDIC PROTEASES
[26]
first equilibrated with 0.02 M sodium acetate buffer at pH 3.5. Fifty milliliters of the dialyzed enzyme filtrate is applied to the column. Elution is performed with 0.15 M sodium acetate buffer at pH 5.2 with increasing concentration of salt and a pH gradient, using a mixing chamber of 1000 ml. The effluent is collected in 10-ml fractions at a flow rate of 30 ml per hour. The absorbance at 280 nm is used to ~'ca:~ the eluate and to locate the protein-containing fractions. The acid proteinase activity is determined according to the method described. It has been found that the stepwise effluent fraction 19 of the acid proteinase on Duolite CS-101 is contaminated with a cellulase and on testing with free boundary electrophoresis reveals several components. 18 The modified procedure using gradient elution chromatography is more successful. In this chromatography the mobility of the acid proteinase is greater than that of cellulase. The yield of the acid proteinase in this step is more than 70% and the specific activity increased about 4 times with respect to the fraction M~. The active fraction is dialyzed against 0.005 M sodium acetate buffer of pH 4.1. The further purification on a DEAE-cellulose column (3 X 70 em) yields a further enriched active fraction. Commercial cellulose absorbent, DEAE-cellulose, is prepared and packed into a column according to Peterson and Sober. 2° Elution is performed with 0.01 M sodium acetate buffer, pH 4.1, with increasing concentrations of NaC1, using two 500-ml mixing chambers. The sodium acetate buffer of pH 4.1 in the reservoir, containing 0.1 M NaC1, is tightly connected to the mixing chambers. Ten-milliliter portions of the effluent are collected and the flow rate is about 30 ml/hour. Proteolytic activity is not found in any other fraction except in the last emerging peak. This procedure results in a 2- to 3-fold purification and a 98% yield of the acid proteinase. The active fraction is dialyzed against (}.01 M acetic acid or 0.02 M ammonium formate buffer2~ and is lyophilized. In electrophoresis, the enzyme preparation obtained with DEAE-cellulose at pH 5.0 to 7.0 showed two peaks, the major one containing about 90% of the protein. In contrast, the preparation appeared to be homogeneous on ultracentrifugation. Repeated purification of 50 mg of the partially purified lyophilized preparation is attempted using a column (2 X 50 cm) of SE-Sephadex at pH 4.1. The SE-Sephadex is prepared by the method of Peterson and Sober. 2° The salt gradient procedure is the same as that used for DEAEcellulose chromatography. Ten-milliliter portions of the effluent are ~*F. Yoshida and M. Nagasawa, Bull. Agr. Chem. Soc. Japan 0.2, 32 (1958). ~oE. A. Peterson and H. A. Sober, Vol. V, p. 3. ~1C. H. W. Him, S. Moore, and W. H. Stein, J. Biol. Chem. 195, 669 (1952).
[26]
ACID PROTEINASE OF AspergiUus saitoi
403
collected at a flow rate of 30 ml/hour. A major enzymatically active component and a minor inactive component are separated by this procedure. This ionic strength gradient at pH 4.1 is more successful for the separation of the contaminated compound than the pH gradient procedure. A symmetrical peak is obtained by chromatography on SESephadex. The active fraction is dialyzed against 0.01 M acetic acid or 0.02 M ammonium formate buffer 21 and is lyophilized. The lyophilized preparation is further purified on commercial Sephadex G-100 with 0.01 M acetic acid or 0.02 M ammonium formate buffer to separate the minor autodigestive products formed. The specific activity is about 150 times that of the initial culture filtrate. The preparation is stored in a desiccator in the refrigerator. Properties Stability. There is no appreciable difference in the residual activities of the acid proteinase of A. saitoi on Koji or submerged culture filtrates kept for 24 hours at pH 3.0 to 5.0 and at.temperatures of 5 ° to 30 °.le The acid proteinase of A. saitoi in the culture filtrate appears to be most stable at pH 4.0, TM which strongly indicates the possibility of concentrating the filtrate at pH 4.0. TM At pH 5.5 and at 22 ° to 25 °, no autodigestion of aspergillopeptidase A occurs. After 16 hours, the loss of the enzymatic activity is about 5%? 2 At pH 2.7 and 30 °, there is a drastic decrease in enzymatic activity of aspergillopeptidase A. It is likely that this decrease in enzymatic activity may be due to autolysis of the proteinase during incubation. The decrease in enzymatic activity of aspergillopeptidase A as a function of time has been observed to parallel the increase of amino nitrogen liberated. These results indicate that presence of the substrate prevents autodigestion to a considerable extent. The optical rotatory dispersion properties of aspergillopeptidase A in the far-ultraviolet region do not change in the pH range of 2.7 to 5.5. 23 At pH 2.4, the optical rotatory dispersion of aspergillopeptidase A shows a small pH dependence. 23 The conformational change of aspergillopeptidase A starts at pH 2.0. 23 Denaturation and inactivation of aspergillopeptidase A begins above pH 6.0 to 7.0.1~,22,23 At pH 7.0 and 22 ° the Cotton effect above the 210 nm zone is no longer detected in the optical rotatory dispersion spectrum; it is replaced by a smooth (featureless) dispersion curve. 23 When examined at pH 7.0 and 0 °, a strong effect of 2 M NaC1 has been observed
= E. Ichishima and F. Yoshida, Biochim. Biophys. Acta 19.8, 130 (1966). 2aE. Ichishima and F. Yoshida, Biochim. Biophys. Acta 147, 341 (1967).
404
THE ACIDIC PROTEASES
[25]
in the stabilization of the enzymatic activity of aspergillopeptidase A.~4,2~ The enzymatic activity completely disappears when an enzyme solution of pH 5.5 is maintained at 55 ° for 20 minutes. ~3 The ultraviolet optical rotatory dispersion curves obtained on heat denaturation show a plain dispersion curve similar to that obtained in the presence of weakly alkaline solutions of pH 8.0. 53 Ca 2÷ does not prevent heat inactivation of the enzyme.26 The degree of inactivation in the presence of 5 M urea of an apparent pH of 5.5 has been 46% after 30 minutes and 59% after 1 hour's incubation. 22 At low concentration of urea, neither a conformational change nor inactivation of the enzyme occurs. The decrease in the enzymatic activity of aspergillopeptidase A as a function of the urea concentration parallels the behavior of the --ao value and the behavior of the difference spectra at 294 nm, in the region of tryptophan absorption. 22 Purity and Physical Properties. Purified aspergillopeptidase A appears to be homogeneous on free boundary electrophoresis over a pH range of 2.0 to 10.0.lg The sedimentation patterns show aspergillopeptidase A to be monodisperse; the sedimentation constant s°0.wis 3.33 S? 8 The isoelectric point is at pH 3.65. Is The molecular weight of aspergillopeptidase A is in the range of 34,900 to 34,200, calculated with the aid of the Scheraga-Mandelkern formula and the Yphantis procedure, respectively.27,28 Using the method of Andrews a molecular weight of 34,500 has been estimated. ~9 It is apparent that these three values of the molecular weight are in good agreement. The conformation of aspergillopeptidase A has been investigated in aqueous solution. 22,23,3° The optical rotation, [a]o, is --35 °. The optical rotatory dispersion constant, X¢, is 207 nm, and the Moffitt-Yang parameter, bo, is zero. The --ao value in the Moffitt-Yang parameter or levorotation of the aspergillopeptidase A molecule increases markedly in the presence of urea, while the value of bo remains unchanged. In the native state in the ultraviolet optical rotatory dispersion, aspergillopeptidase A has a minimum at 220 nm with [m'] = --2400 and a maximum 203 nm with [m'] ---- 900, respectively,s3,8° The conformation of aspergillopeptidase A is apparently converted 2, E. Ichishima and F. Yoshida, Nature 215, 412 (1967). E. Ichishima and F. Yoshida, in "Microbial Enzymes," (K. Tsuda, ed.), p. 186. Tokyo Univ. Press, Tokyo, 1967. 2, F. Yoshida and M. Nagasawa, Bull. Agr. Chem. Soc. Japan 20, 257 (1956). ~TE. Ichishima and F. Yoshida, Nature 207, 525 (1965). 28E. Ichishima and F. Yoshida, Biochim. Biophys. Acta 110, 155 (1965). ~K. Hayashi, D. Fukushima, and K. Mogi, Agr. Biol. Chem. (Tokyo) 31, 1171 (1967). ~oE. Ichishima and F. Yoshida, Agr. Biol. Chem. (Tokyo) 31, 507 (1967).
[26]
ACID PROTEINASE OF Aspergillu8 saitoi
405
by the anionic detergent, sodium lauryl sulfate, into an a-helical conformation, as judged from the optical rotatory dispersion curve in the ultraviolet region. ~3,~° The [m']233 and [m']lg8 values have been found to be --3400 and 11600, respectively. ~3,3° It is concluded that aspergillopeptidase A is devoid of a complete a-helical strand as judged from the visible and ultraviolet optical rotatory dispersion. 22,23,3° The infrared result indicates that the deuterium-exchanged aspergillopeptidase A exists in the antiparallel fl structure. 2~ The location of an amide I band at 1632 cm -1 has been observed. The spectrum has shown the presence of a weak band around 1685 cm-1.~2 Aspergillopeptidase A has the following amino acid composition and a total of 283-289 residues: Lys11, His3, Argl, Asp34-35, Thr2~, Ser42-4~, i
I
Glu~2, Prolo, Glyal-32, Ala2o, Cys CySl, Va122, Meto, I1e11-12, Leuig-2o, Tyr~7_~8, Phe~3, Trpl, amide-ammonia31_32. 28 The molecule of aspergillopeptidase A consists of a single polypeptide chain with serine as the NH2-terminus and with alanine as the COOH-terminal residue? ~ Activators and Inactivators. No activators are required for this acid proteinase. Aspergillopeptidase A is inhibited 50% by a 100 M excess of N-acetylimidazole in 0.033M phosphate buffer containing 2.0M NaC1, pH 7.0. 25,27,32 The degree of inactivation is 30% after 1 hour and 60% after 5 hours inactivation with a 1000 molar excess of iodine at pH 7.0, 0°. 25,s~ The enzymatic activity of aspergillopeptidase A is completely destroyed in 20 minutes if 40 moles of N-bromosuccinimide are present for each molecule of enzyme at 0 ° and pH 4.1. 25,27,a2 Aspergillopeptidase A is inactivated by photooxidation at pH 5.5. 20,2~,~2 Aspergillopeptidase A is rapidly inactivated by methyl alcohol-HC1, 33 which suggests that the carboxyl group of the enzyme may be esterified. 2~,~2 The residual activity of aspergillopeptidase A falls to zero at sodium lauryl sulfate concentrations between 5 and 10-3M at pH 5.5. 22 The conformational transition of aspergillopeptidase A by the detergent has been observed. 23 Specificity. Aspergillopeptidase A is capable of activating trypsinogen and chymotrypsinogen A at acidic pH range 3~ (optimum pH = 3.5). This activation of trypsinogen is associated with the usual cleavage of the Lys-Ile bond and liberation of the hexapeptide. Aspergillopeptidase A does not hydrolyze any of the small synthetic ~IE. Ichishima and F. Yoshida, J..Biochern. (Tokyo) 59, 183 (1966). E. Ichishima and F. Yoshida, ~nd Intern. Symp. Gtirungsind. Tech. Mikrobiol. Enzymol. Leipzig May, 1968, pp. 166, 572. H. Fraenkel-Conrat, Vol. IV, p. 247. '* C. Gabeloteau and P. Desnuelle, Biochim. Biophys. Acta 42, 230 (1960).
406
THE ACIDIC PROTEASES
[27]
peptides, amides, and esters. ~2 The acid proteinase is inactive in the milk clotting test25 Kinetic Properties. The activation system of trypsinogen follows Michaelis-Menten kinetics, giving K,, app ----1.25 X 10-5 M (at 30 °, pH 3.5) and a Vmax =- 1.5 X 10-2 M/minute/rag aspergillopeptidase A. Initial velocity, v, is measured as ~moles trypsin/minute/mg aspergillopeptidase A. Kinetic properties have also been obtained by Abita et al. ~6 These two values of the kinetic data are in good agreement. Distribution. The culture products of Aspergillus usamii, A. inuii, A. aureus, A. awamori, A. nakazawai, A. niger, A. oryzae, A. sojae, and other strains of genus Aspergillus also catalyze the hydrolysis of proteins at low pH range. 9-1a,1~, 37 In enzymatic properties, Penicillium janthinellum peptidase A, 38-4° PaeciUomyces varioti acid proteinase, 41 Rhizopus chinensis proteinase, 35 and Trametes sanguinea acid proteinase 42 resemble aspergillopeptidase A. u j . Fukumoto, D. Tsuru, and T. Yamamoto, Agr. Biol. Chem. (Tokyo) 31, 710
(1967). J. P. Abita, M. Delaage, M. Lazdunski, and J. Savrda, European J. Biochem. 8, 314 (1969). aTK. Nakanishi, J. Biochem. (Tokyo) 46, 1263 (1959). 38T. Hofmann and R. Shaw, Biochim. Biophys. Acta 92, 543 (1964). s~A. Thangamani and T. Hofmann, Can. J. Biochem. 44, 579 (1966). J. SSdek and T. Hofmann, J. Biol. Che.m. 243, 450 (1968). 41j . Sawada, Agr. Biol. Chem. (Tokyo) 27, 677 (1963). K. Tomoda and H. Shimazono, Agr. Biol. Chem. (Tokyo) 28, 770 (1964).
[27] Gastricsin and Pepsin B y JORDAN TANG
Assay Methods Hemoglobin Assay This assay can be used for both pepsin and gastricsin. The method is essentially that of Anson and Mirsky 1 but modified slightly to give more sensitivity and reproducibility. Principle. Acid-denatured bovine hemoglobin is used as substrate in this assay. The small peptides, which are produced in the proteolytic digestion, are measured in the filtrate of trichloroacetic acid by means of their absorption at 280 nm. 1M. L. Anson and A. E. Mirsky, J. Gen. Physiol. 16, 59 (1932).
406
THE ACIDIC PROTEASES
[27]
peptides, amides, and esters. ~2 The acid proteinase is inactive in the milk clotting test25 Kinetic Properties. The activation system of trypsinogen follows Michaelis-Menten kinetics, giving K,, app ----1.25 X 10-5 M (at 30 °, pH 3.5) and a Vmax =- 1.5 X 10-2 M/minute/rag aspergillopeptidase A. Initial velocity, v, is measured as ~moles trypsin/minute/mg aspergillopeptidase A. Kinetic properties have also been obtained by Abita et al. ~6 These two values of the kinetic data are in good agreement. Distribution. The culture products of Aspergillus usamii, A. inuii, A. aureus, A. awamori, A. nakazawai, A. niger, A. oryzae, A. sojae, and other strains of genus Aspergillus also catalyze the hydrolysis of proteins at low pH range. 9-1a,1~, 37 In enzymatic properties, Penicillium janthinellum peptidase A, 38-4° PaeciUomyces varioti acid proteinase, 41 Rhizopus chinensis proteinase, 35 and Trametes sanguinea acid proteinase 42 resemble aspergillopeptidase A. u j . Fukumoto, D. Tsuru, and T. Yamamoto, Agr. Biol. Chem. (Tokyo) 31, 710
(1967). J. P. Abita, M. Delaage, M. Lazdunski, and J. Savrda, European J. Biochem. 8, 314 (1969). aTK. Nakanishi, J. Biochem. (Tokyo) 46, 1263 (1959). 38T. Hofmann and R. Shaw, Biochim. Biophys. Acta 92, 543 (1964). s~A. Thangamani and T. Hofmann, Can. J. Biochem. 44, 579 (1966). J. SSdek and T. Hofmann, J. Biol. Che.m. 243, 450 (1968). 41j . Sawada, Agr. Biol. Chem. (Tokyo) 27, 677 (1963). K. Tomoda and H. Shimazono, Agr. Biol. Chem. (Tokyo) 28, 770 (1964).
[27] Gastricsin and Pepsin B y JORDAN TANG
Assay Methods Hemoglobin Assay This assay can be used for both pepsin and gastricsin. The method is essentially that of Anson and Mirsky 1 but modified slightly to give more sensitivity and reproducibility. Principle. Acid-denatured bovine hemoglobin is used as substrate in this assay. The small peptides, which are produced in the proteolytic digestion, are measured in the filtrate of trichloroacetic acid by means of their absorption at 280 nm. 1M. L. Anson and A. E. Mirsky, J. Gen. Physiol. 16, 59 (1932).
[27]
GASTRICSIN AND PEPSIN
407
Reagents 10% Hemoglobin Solution. This solution is stable for several months when stored at 4 ° . Therefore, if the assay is carried out routinely, it should be prepared in a larger quantity. Bovine hemoglobin easily forms lumps and is difficult to dissolve. A 50-ml glass tissue grinder (Kontes K-88545) is a useful tool in speeding the solution of the hemoglobin. HC1 solution, 0.3 N Sodium citrate buffer, 0.1 M, pH 3.1 5% Trichloroacetic acid (TCA)
Procedure. The acidified hemoglobin substrate was freshly prepared by addition of 7 ml of 0.3 N HC1 to 20 ml of 10% hemoglobin solution. This solution is diluted with distilled water to 100 ml. The final pH of the solution should be pH 3.1 (adjust to that pH value using small aliquots of NaOH or HC1 if the final pH is off by more than 0.1 unit). The sample to be assayed is diluted with water so that each 0.5 ml contains approximately 2 to 15 #g of gastricsin (or 5 to 30/Lg of pepsin). All the solutions should be warmed to 37 ° in the water bath prior to the mixing. To 0.5 ml of enzyme solution, 0.5 ml of 0.1 M sodium citrate buffer, pH 3.1, and 5 ml of acidified hemoglobin solution are added. The solutions are rapidly swirled by a Vortex mixer and placed in a water bath at 37 °. After 10 minutes of incubation, the reaction is stopped by the addition of 10 ml of 5% TCA. After filtering through Whatman No. 50 filter paper (d = 7 cm), the optical density of the filtrate at 280 nm is determined with a speetrophotometer. A blank should be run in which the TCA is added before the enzyme solution. The blank usually has an optical density at 280 nm of about 0.2 to 0.3. All determinations and blanks are run in duplicates and averages are used. Calculation and Cautions. The specific activity which is calculated as AOD 280 nm/10 minutes incubation//~g enzyme, is 0.015 for pepsin from human and hog and 0.050 for gastricsin from human. It is important to adjust the enzyme quantities in the assay so that ~OD 280 nm values are below 1.0. Above that value, the linear relationship between absorbancy and enzyme amount is no longer held. Determination of Pepsin in a Mixture Containing Gastricsin Using Synthetic Substrate Principle. The quantity of pepsin in a mixture can be determined by using N-acetyl-L-diiodotyrosine (APDT) as substrate, which is not hydrolyzed by gastricsin. ~ The amount of pepsin is determined by the 2L. Chiang, L. Sanchez-Chiang, S. Wolf, and J. Tang, Proc. Soc. Exptl. Biol. Med. l~,, 700 (1966).
408
THE ACIDIC PROTEAS~.S
[27]
amount of free diiodotyrosine produced during the incubation, which is analyzed by using the ninhydrin reaction of Rosen2
Reagents HC1, 0.01 N N-acetyl-L-phenylalanyl-L-diiodotyrosine (Cyclo Chemical Corp.) 0.002 M, in 0.005 M NaOH (freshly prepared before assay) Sodium acetate buffer, 4 M, pH 5.3 Sodium cyanide solution, 0.01 M 3% Ninhydrin in ethyleneglycol monomethyl ether 50% Isopropanol in water
Procedure. To 50 ~l of enzyme solution, 0.7 ml of 0.01 N HC1 and 0.25 ml of APDT solution are added. (For the analysis of pepsin in human gastric juice, 1 ml of sample is mixed with 6.5 ml of 0.01 N HC1. An aliquot of 0.75 ml is taken and mixed with 0.25 ml of APDT.) The mixture is then incubated in a water bath at 37 ° for 1 hour. The incubation mixture is then analyzed for ninhydrin color using the procedure of Rosen 8 as in the following. The acetate-cyanide buffer is made by the addition of 0.2 ml of sodium cyanide to 10 ml of sodium acetate buffer. To each tube, containing 1 ml incubation mixture, 0.5 ml of acetate-cyanide buffer and 0.5 ml of ninhydrin solution are added. The tubes are heated in a boiling water bath for 15 minutes. At the end of incubation, 5 ml of 50% isopropanol is added, which is followed by vigorous shaking for about 30 seconds. After the tubes are allowed to cool to about room temperature, the optical density at 570 nm is determined in a spectrophotometer. Calculation and Cautions. The optical density at 570 nm produced from the substrate by 1 ~g of human pepsin was found to be 0.0111. However, porcine pepsin hydrolyzes APDT at a somewhat faster rate and gives a higher value. ~ Therefore, when pepsin from a source other than human is used the new specific activity should be determined using pure enzyme. Samples of human gastric juice contain small amounts of ninhydrin-positive substances. ~ A blank run for zero time incubation usually corrects this problem. Determination of Gastricsin in a Mixture Containing Pepsin Using Hemoglobin and APDT Assays The hemoglobin assay described above determines the sum of activities of pepsin and gastricsin in a mixture. On the other hand, APDT ~H. Rosen, Arch. Biochem. Biophys. 67, 10 (1957). 4L. Chiang, L. Sanchez-Chiang, J. Mills, and J. Tang, J. Biol. Chem. 242, 3098 (1967).
[27]
GASTRICSIN AND PEPSIN
409
assay determines the amount of pepsin in the mixture. The amount of gastricsin in the sample, therefore, can be calculated by using the results of both assays. 2 /'specific activity \ [~g of pepsin \ ~g of Total AOD ~80~m -- [of pepsin in | X ~found in APDT] gastricsin in _ in hemoglobinassay \hemoglobin assay/ \assay / the incubation specific activity of gastricsin in hemoglobinassay mixture 50D 280 nm in hemog, assay - (0.015 × j~gpepsin APDT assay) 0.050
Resolution and Determination o] Human Pepsin and Gastricsin ]rom a Mixture Using a Small Column o/Amberlite IRC-60 (CG-50) Principle. Since human pepsin and gastricsin can be easily separated on a column of Amberlite IRC-50 (CG-50), quantitative measurement of the enzymes can be made using this process2 The elution of ~ e enzymes from the column is pH dependent. Therefore, the stepwise elutions are used in the recovery of the enzymes and the activity of each is determined using the hemoglobin assay. Reagents Amberlite IRC-50 (CG-50) resin (Mallinckrodt ~3341, 200-400 mesh) Sodium citrate buffer, 0.2 M, pH 3.0 Sodium citrate buffer, 0.2M, pH 4.16 Sodium citrate buffer, 0.2 M, pH 4.60 The reagents used in the hemoglobin assay
Procedures. A column of 0.4 X 5.5 cm is prepared and washed with 20 ml of 0.2 M sodium citrate buffer, pH 3.0. A convenient way of preparing the column, especially when a large number is needed, is to use commercially available disposable capillary Pasteur pipettes (such as Fisher No. 13-678-5, 53~ inches long). The tips of the capillary is cut to about 1 cm in length and a small plug of fine glass wool is inserted into the bottom of the column before the resin is poured. A sample containing 0.1 to 0.3 mg of a mixture of the two enzymes is dissolved in 2 to 5 ml of the same buffer. If the sample has been previously dissolved in another solution, it is necessary to dialyze it in the cold against several changes of citrate buffer, pH 3, before subjecting it to chromatography. The solution is then allowed to pass into the column at an eluate flow rate of approximately 1 ml per 10 minutes. After the enzymes have been absorbed on the resin, an additional 5 ml portion of 0.2 M sodium citrate buffer, pH 3, is passed through the column. Human pepsin is then eluted ~J. Tang and K. Tang, J. Biol. Chem. 238, 606 (1963).
410
ZHE ACIDIC PROTEASZS
[27]
with 20 ml of citrate buffer, pH 4.16, and gastricsin is eluted with 20 ml of citrate buffer, pH 4.6. The amount of enzyme in the eluants is then analyzed using the hemoglobin assay as described above. Cautions. Since the eluants contain considerable buffer capacity, the aliquots taken for analysis in the hemoglobin assay should be as small as possible. The error of this method should be about 5 ~ . Purification Procedure PURIFICATION OF H U M A N
GASTRICSIN AND PEPSIN
H u m a n gastricsin and pepsin have been purified from gastric juicee,~ and human gastric mucosa extract2 Only human gastricsin has been obtained in crystalline form. 7 Starting Material. The best source of human gastricsin and pepsin is gastric juice. For a large-scale preparation, convenient collections are made overnight in the surgical ward of the hospital where gastric juices are removed from patients scheduled for stomach operations on the following day. This "overnight juice" usually has a green color, due to the contamination of bile pigments during the collection. In a large hospital, it is not difficultto collect several liters of gastric juice in a week. Each liter of such iuice usually contains about 300-500 m g of pepsin and 100-200 mg of gastricsin. There are two common methods of storing gastric juice. If the volume involved is quite small, it can be filtered through fine glass wood, dialyzed against several changes of distilled water, and lyophilized. Gastricsin and pepsin are stable in lyophilized gastric juice powder at 4 ° for months. However, the recovery of these enzymes after a storage of a few years can be considerably less. The second method of storage is to pool and filter the gastric juice through glass wool, then store in 2-liter polyethylene bottles at --30 ° . This method is suitable for handling large quantities of juice. Fractionation o] Human Gastricsin and Pepsin on a Column o] Amberlite IRC-50. T w o sizes of columns are most commonly used. A column of 4.5 X 30 cm can fractionate up to 3 g of lyophilized gastric juice powder. A large column 8 X 25 cm, could be used to fractionate up to 10 g of lyophilized gastric juice powder, or 3 liters of gastric juice if that is the starting material (see below). A typical fractionation procedure is described in the following: Three grams of lyophilized gastric juice powder is dissolved in 30 ml 6V. Richmond, J. Tang, S. Wolf, R. E. Trucco, and R. Caputto, Bioch~rn. Biophys. Acta 29, 453 (1958). J. Tang, S. Wolf, It. Caputto, and R. E. Trucco, J. Biol. Chem. 234, 1174 (1959).
[2 7]
GASTRICSIN AND PEPSIN
411
of 0 . 2 M sodium citrate buffer, pH 3.0. The solution flows freely and should not have a jellied appearance. If the latter is the case, more of the pH 3.0 buffer can be used to dilute the starting material. The solution is then centrifuged in a Spinco model L centrifuge at 30,000 rpm for 30 minutes. The supernatant is retained while the mucouslike sedimenting material is discarded. The resin Amberlite IRC-50 (CG-50) is treated according to the procedure outlined by Hirs et al. 8 and equilibrated with the starting buffer, 0.2M sodium citrate, pH 3.0. The column chromatography is operated at room temperature. After the buffer level in the column is brought to the same height as that of the resin, the supernatant of the gastric juice is carefully layered on the top of the resin bed and allowed to flow slowly into the resin. The column is then washed with 300 ml of the same pH 3.0 citrate buffer. The flow rate of the column is approximately 1 ml per minute and fractions of 10 ml per tube are collected. The influent buffer is then changed to 0.2 M sodium citrate, pH 3.80. The elution with this buffer is continued until the effluent pH has reached 3.80 and its optical density at 280 nm is less than 0.1. The elution buffer is then changed to 0.2M sodium citrate buffer, pH 4.2, with which human pepsin is eluted in a sharp peak. The effluent at the top of the human pepsin peak should be pH 4.0. After the effluent pH has reached 4.2, the elution buffer is changed to 0 . 2 M sodium citrate, pH 4.6. Human gastricsin is eluted from the column by this buffer at an effluent pH of 4.4. A typical chromatographic pattern is shown in Fig. 1. The material under the enzyme peaks is pooled, dialyzed in cold against at least three changes of distilled water, and lyophilized. The enzymes prepared in the fractionation should be free from cross contamination and have purities better than 80%. If the large-sized column is used, the procedures are the same. However, gastric juice samples can be directly absorbed onto the column. Three liters of glass wool filtered gastric juice are measured into a flask containing 80.6 g of citric acid and 51.9 g of sodium citrate. After complete solution, the gastric juice should have a pH of about 3.0. Solutions of 0.2M citric acid or 0.2M sodium citrate can be used to adjust the gastric juice to the starting pH. The starting sample is then allowed to flow slowly through the column. Nearly all the proteolytic activity is retained by the column. The elution and resolution of pepsin and gastricsin are usually not satisfactory after this absorption procedure. Instead, the two enzymes are eluted together with pH 4.60 buffer into a flask. After dialysis and lyophilization, the enzymes are separated ' C. H. W. Hirs, S. Moore, and W. H. Stein, d. Biol. Chem. 200, 493 (1953).
412
[27]
THE ACIDIC PROTEASES
Influent pH --~--3.0 Effluent pH -"~-5.0
4.18 5.8
4.6 410
4.18
4.4 2.0
2.0
~ 1.5
.,5 u
o
c~ 1.0
a o 0.5
0.5
0 ILl
0
I
1000
2000
3000
|1|
a.
0
4000
Effluent, ml
Fro. 1. Chromatography of dialyzed human gastric juice on a column of Amberlite IltC-50 (CG-50). Solid line: protein concentration. Broken line: proteolytic activity. by repeating the column chromatography using the small size column (4.5 X 30 cm) as described above. Crystallization of. Human Gastricsin. The gastricsin solution obtained from the Amberlite column is dialyzed against distilled water and lyophilized. About 20-30 mg of the powder obtained is dissolved with 2.0 ml of distilled water at 4 ° and centrifuged at 2000 rpm for 5 minutes. The supernatant solution is poured into a centrifuge tube, put into an ice bath, and 0.26 g of crystalline (NH4)2S0, is added. The protein precipitate formed is centrifuged at 2000 rpm for 10 minutes, and the supernatant solution discarded. The precipitate is dissolved with 2.0 ml of water and the previous precipitation repeated. The precipitate is dissolved with 2 ml of cold 0.2 M sodium acetate buffer (pH 5.0). A clear solution is obtained to which crystalline (NH4)~SO4 is added in small portions until the first indication of protein precipitation is observed [approximately 0.2 g of (NH4)2S04 is required]. The tube is then placed in a water bath at 20 ° and left for 5 minutes. If the turbidity disappears, drops of saturated (NH4)2S0~ solution are added until it reappears. The tube is then moved to a 40 ° water bath, in which, after 5 minutes, the turbidity usually disappears. When it does not, however, the insoluble material is removed by centrifugation. The tube is placed in a beaker containing 2 liters of water at 30 ° and moved into the 4 ° cold room where, after 6-8 hours, a white precipitate is formed. The first precipitate, however, is mostly noncrystalline and is removed by centrifugation in the cold room and at a low speed. The supernatant solution is left stand-
[27]
GASTRICSIN AND P E P S I N
413
FIG. 2. Crystalline human gastricsiu at 480 X magnification. ing at 4 ° for a period of 20-30 hours during which crystals are formed. T h e crystals of gastricsin, shown in Fig. 2, are transparent, and the microscopic observations are best made in dim light. Seeding of crystalline gastricsin reduces the time required for the crystals to appear. After 2-3 days, the first collection of crystals is made by centrifugation at low speed. The supernatant solution still contains enzyme activity, and the formation of more crystals can be obtained by further addition of drops of saturated (NH4)2S04 solution. T a b l e I shows a protocol of purification procedure. The specific TABLE I PURIFICATION AND CRYSTALLIZATION OF GASTRICSIN a
Freeze-dried gastric juice Chromatographic fraction First crystals Second crystals
Dry weight (rng)
Specific activity (AOD~ nm/mg protein)
Total activity (%)
2000 36 7.2 6.7
4.6 b 153.6 227.5 242.5
100 60 17.8 17.6
The activities were measured according to the method of Anson and Mirsky, J. Gen. Physiol. 16, 59 (1932). b The value for gastricsin given in this table was calculated from the total proteolytic activity recovered from the column after adding the values of both peaks and then assuming that the percent recoveries for pepsin and gastricsin were the same.
414
THE ACIDIC PROTF~SES
[27]
activities of the crystalline gastricsin at 25 to 3 0 ~ higher than that of the lyophilized materials obtained by chromatography, and the yields, related to the same starting material, vary from 20 to 30~. The shape and the activity of the crystals remain unaltered after storage for a long time (up to 2 years), and this is the best way to preserve the enzyme. Further Purification o] Human Pepsin. Human pepsin obtained from the Amberlite IRC-50 (CG-50) column is usually not free of contamination (see below), especially the material obtained from the fractionation on the large column. Repeating the fractionation on a column of Amberlite IRC-50 is necessary to obtain a pure preparation.
Purification o] Human Gastricsin and Pepsin ]rom Gastric Mucosa. Human gastricsin and pepsin can be obtained from the acidified gastric mucosa2 About 100 g of human gastric mucosa are minced and homogenized in a Waring blendor with 0.1 M NaHCOs solution. The operation should be carried out in the cold. The homogenate is centrifuged at 30,000 g for 60 minutes. The supernatant is dialyzed against distilled water which has been adjusted to pH 8 with ammonium hydroxide and lyophilized. The powder of mucosa extract is then dissolved in 0.2 M sodium citrate buffer, pH 3.0, in a concentration of about 100 mg/ml. The solution is allowed to stand in room temperature for 30 minutes and is then transferred to a column of Amberlite IRC-50. The fractionation procedure for gastricsin and pepsin is the same as described above. However, if the starting material is small in quantity, the size of the column should also be reduced. PURIFICATION OF PORCINE GASTRICSIN Gastricsin can be isolated from porcine gastric mucosa or commercial crude pepsin preparations. 4 Starting Material. Whole porcine stomachs can be obtained from slaughterhouses. They are placed in an ice chest immediately after removal from the hogs. The fundus mucosae is separated from each stomach, and the samples which are not processed the same day are frozen and stored. Crude porcine pepsin (1:10,000) can be purchased from commercial companies. Extraction. The fundus mucosae from the hog stomachs are ground in a chilled meat grinder. The ground tissue (1 to 2 kg, wet weight) is extracted with four times its weight of 0.45 saturated ammonium sulfate (at 25 °) in 0.1 M sodium bicarbonate. The suspension is filtered. Ammonium Sul]ate Fractionation. The filtrate is brought to 68% saturation of ammonium sulfate by the addition of 458 g of the salt to each liter of filtrate. The resulting precipitate is recovered by filtra-
[27]
GASTRICSIN AND PEPSIN
415
tion, then dissolved in 0.02 M phosphate buffer, pH 7.0, and dialyzed at 4 ° against distilled water adjusted to pH 8.0 with KOH. The solution is dialyzed at 4 ° against several changes of 0.2 M sodium citrate buffer, pH 4.2, for 24 hours in order to activate the zymogens. Fractionation on Amberlite IRC-50 (CG-50) Column. The enzyme solution obtained by the above procedure is equilibrated with 0.2M sodium citrate buffer, pH 2.1, by dialysis overnight and then passed into a column (2.5 X 20 cm) of Amberlite IRC-50 resin which has been equilibrated with the same buffer. Porcine pepsin, which accounted for about 95% of the proteolytic activity of the starting material, passes through the column unabsorbed. The column is then washed with 0.2 M sodium citrate buffers of pH 3.8 and 4.2. Proteolytic activity should not appear in the effluent of either of these buffers. After the effluent pH has reached 4.2, porcine gastricsin is eluted from the column by 0.2 M sodium citrate buffer, pH 4.6, as shown in Fig. 3. The fractions containing porcine gastricsin are pooled, dialyzed against distilled water, and lyophilized. Fractionation of Sephadex G-75 Column. The lyophilized enzyme
~.1
3.8
4.2
4.6
~
Effluent
pH
1.4i J~ 1
1.2
I I I I
1.0 E 0 GO
i. I
0.8
0.6 0
(14 Q2 ",,,, 01.1
0
1000
2000
3000
Effluent, ml
Fro. 3. C h r o m a t o g r a p h y of porcine gastricsin on a column of Amberlite I R C 50 (CG-50). Porcine gastricsin is eluted in a sharp peak at 2700 ml of the effluent.
416
ThE ACIDIC PROTF_,ASBS
[27]
powder is dissolved in 3 ml of 0.05 M citrate buffer, pH 5.0, and fractionated on a column (2 X 150 cm) of Sephadex G-75. The enzyme is eluted at about 220 ml of efltuent with the same buffer. Usually only minor contaminating peaks can be seen in the chromatogram.
Purification o] Porcine Gastricsin ]rom Commercial Crude Pepsin Preparations. One hundred grams of commercial crude pepsin (1:10,000) are dissolved in 100 ml of distilled water. The solution is adjusted to pH 2.1 with 3 N HC1. The solution is then dialyzed against 0.2M sodium citrate buffer, pH 2.1. The solution is then transferred onto a column of Amberlite IRC-50 and fractionated as described in the preceding paragraphs. Properties There are considerable similarities in the enzymatic properties of gastricsin and pepsin from human and porcine sources. The properties reviewed in the following paragraphs, therefore, will be made in a comparative manner. General Enzymatic Properties. Gastriesin as well as pepsin exhibit milk-clotting activity in addition to proteolytic activity. The former can be tested by using the method of Berridge2 The specific activity in milk-clotting for gastricsin and pepsin is about the same, however, either one is only about 45% of the specific activity of rennin/ The enzymes have somewhat different optimal pH in proteolysis when bovine hemoglobin is the substrate2 The optimal pH for gastricsin is 3.0, while for pepsin it is 1.9. The specific proteolytic activity for the enzymes is dependent on the nature of the protein substrate. Using hemoglobin as substrate, the specific proteolytic activity of pepsin is only about 74% of that of gastricsin. However, in the case of egg albumin substrate, pepsin hydrolyzes the substrate more than twice as fast as gastricsin. 1° Their comparative enzymatic properties are shown in Table lI. Stability. Gastricsin as well as pepsin is stable in acidic solutions. However, both lose activity rapidly in neutral or alkaline solutions. The enzymes are rather stable at room temperature. Column chromatography for the fractiontiaon of the enzymes can be carried out at room temperature without significant loss of activity. The lyophilized powder of the enzymes can be stored at 4 ° indefinitely. However, prolonged storage (over a year) often results in decreasing amounts of soluble enzyme when one attempts to redissolve the powder. The best storage form of gastricsin is its crystal. The suspension of enzyme crystals in saturated ' N. J. Berridge, Vol. II, p. 69. ~*J. Tang, J. Mills, L. Chiang, and L. deChaing, Ann. N.Y. Acad. Sci. 140, 688 (1987).
[27]
GASTRICSIN
~o
~
o
0
~w ~Q
0
0
0 r~ o Z
o r,.)
~
o
©
P.
AND
PEPSIN
417
418
THE ACIDIC PROTEASES
[27]
ammonium sulfate can be stored at 4 ° for several years without significant loss of activity. The heat stability of gastricsin and pepsin is somewhat dependent on the pH of the solution. ~ Both enzymes are more heat stable at pH 2 than at pH 3.2. Purity and Physical Properties. The 2>( crystalline human gastricsin shows a single band in starch gel electrophoresis and a single sedimenting boundary in the schlieren pattern of an ultracentrifuge. 7 It shows a single amino terminal residue, serine. 10 The 2>( chromatographed human pepsin (see purification above) satisfies the same homogeneity criteria. The human enzymes obtained from a single column chromatography on Amberlite IRC-50, however, are not free of impurities. This is evident since gastricsin increases its specific activity in the first crystallization. Also, human pepsin from the first column chromatography shows additional bands in electrophoresis. 7 Porcine gastricsin, after chromatographic separation on columns of Amberlite IRC-50 and Sephadex G-75, shows one boundary in ultracentrifugation and a single band in electrophoresis. 4 The molecular weights for human pepsin and gastricsin are about 34,000 and 31,400, respectively, as determined by the sedimentationdiffusion method, sedimentation equilibrium studies, and amino acid analysis data. 1~ The sedimentation coefficient, s2o,w, are 3.14 S for human pepsin and 3.32 S for gastricsin. The sedimentation coefficient for gastricsin is concentration dependent, which is consistent with the high intrinsic viscosity observed (Table II). The diffusion coefficients D2o,w were found to be 8.7 X 10-7 cm 2 sec-~ and 9.6 X 10 -7 cm 2 sec-1 for human pepsin and gastricsin, respectively. The partial specific volume calculated from the amino acid composition is 0.724 ml/g for both enzymes. The intrinsic viscosity as well as the axial ratio for gastricsin are considerably higher than those of pepsin (Table II). Porcine gastricsin appears to have a somewhat larger molecular weight, 32,500, than human gastriesin, 31,400. ~ Activators and Inactivators. There is no activator known fo," human gastricsin and pepsin. A number of pepsin inhibitors, diazoacetylglycine ethyl ester and phenylbenzoyldiazomethane 12 and diphenyldiazomethane, 13 also inhibit human gastricsin to various degrees. ~4 Specificity. Both human gastricsin and pepsin show broad specificity in the hydrolysis of peptide bonds in protein and peptide substrates. Additionally, these specificities in two enzymes are qualitatively quite 11j. N. Mills and J. Tang, J. Biol. Chem. 242, 3093 (1967). L. V. Kozlov, L. M. Ginodman, and V. N. Orekhovich, Biokhimiya 32, 1011 (1967). " G . R. Delpierre and J. S. Fruton, Federation Proc. 54, 1161 (1965). 1, j. Mills, personal communication, 1969.
[27]
419
GASTRICSIN AND PEPSIN
TABLE III THE HYDROLYSIS OF SYNTHETIC DIPEPTIDES BY HUMAN GASTRICSIN AND PEPSIN
Specific aetivity a Substrate
Gastricsin
Pepsin
Zb-L-Tyrosyl-L-alanine Z-L-Tyrosyl-L-threonine Z-L-Tyrosyl-L-leucine Z-L-Tyrosyl-L-serine Z-L-Tyrosyl-L-valine Z-L-Tyrosyl-L-tyrosine Z-L-Tyrosyl-L-phenylalanine Z-L-Tyrosyl-L-glycine Z-L-Seryl-L-tyrosine Z-L-Alanyl-L-tyrosine Z-L-Phenylalanyl-L-serine Z-L-Tryptophanyl-L-alanine Z-L-Tryptophanyl-L-serine Z-L-Tryptophanyl-L-leucine Z-L-Glutamyl-L-tyrosin e Z-Glycyl-L-phenylalanine Z-L-Glutamyl-L-phenylalanine N-Acetyl-L-phenylalanyl-L-tyrosine N-Acetyl-L-phenylalanyl-b-diiodotyrosine
91.0 52.1 27.2 19.3 8.4 7.6 2.1 0 0 0 0 40.0 < 2.0 0 17.0 2.0 1.0 230.0 0
000 ml
FIG. 1. Chromatographic fractionation of chymosin. Sample: 750 mg of chymosin. Column: DEAE-cellulose 2 cm in diameter 20 cm high. Elution: linear gradient of 1105 ml 0.2 M sodium phosphate buffer, pH 5~, and 1105 ml 0.3 M phosphate buffer, pH 5.6, flow rate 30 ml per hour. Solid line shows absorbancy at 278 nm. Circles indicate the ratio between milk-clotting activity and Am. ~'B. Foltmann, Acta Chem. Scand. 14, 2059 (1960).
430
THE ACIDIC PROTEASES
[28]
and the absorbancy at 278 nm. The fractions containing the central part of each peak are pooled as indicated by the double-headed arrows. Each of the pooled solutions of chromatographically purified chymosin is diluted with a 3-fold volume of distilled water. From these solutions chymosin is absorbed on short columns of DEAE-cellulose equilibrated with 0.1 M phosphate buffer, pH 5.8, and chymosin is again eluted with 1 M NaC1 in 0.01 M phosphate buffer, pH 6.0. Most of the chymosin will be eluted with a concentration higher than 2 mg/ml. From such solutions the chromatographically purified components may crystallize again. Preliminary experiments have shown that it is possible to proceed directly from step 4 of the purification of chymosin to the chromatographic fractionation. In this case salt is removed by dialysis against 0.05 M phosphate buffer, pH 5.9. The dialyzed solution of partly purified ehymosin is applied to a column of DEAE-cellulose equilibrated with 0.1M phosphate buffer, pH 5.8. Nonenzymatic material is washed through the column with 0.1 M phosphate buffer, and subsequently the chymosins are eluted with a linear gradient of sodium phosphate buffer from 0.1 to 0.3 M. Preparations with yields of more than 50 mg of the individual components have not yet been performed. Properties
Stability and Activation o] Prochymosin. Solutions of prochymosin are reasonably stable in the pH range of 5.5 to 9. 4 At pH below 5 prochymosin is converted into chymosin by a limited proteolysis during which a peptide segment is cleaved from the N-terminus. The N-terminal residue of prochymosin is alanine 12,15 while that of chymosin is glycine. Table II118 shows a balance sheet of the amino acid compositions of chromatographically purified preparations t)f prochymosin and chymosin and of peptides released during the activation of prochymosin. The reaction is very dependent on pH, salt concentration, and temperature. At pH 5 and room temperature the activation is completed in 2 or 3 days, 14 while at pH 2, room temperature, and ionic strength of 0.1, the activation is completed in 5-10 minutes. 13 If the increase of milk-clotting activity is plotted against time, the course of the activation at pH 4.5 to 5 appears as more or less S-shaped curves, indicating that the reaction is at least partly autocatalytic. Experiments carried out at lower pH show a different course of reaction. The lower the pH, the higher is the initial rate of the reaction and the Sshape of the curve disappears. Some results are illustrated in Fig. 2. ~ ~B. Foltmann, Compt. Rend. Tray. Lab. Carlsberg 34, 275 (1964).
[28]
PROCHYMOSIN AND CHYMOSIN
431
TABLE I I I AMINO ACID COMPOSITION OF PROC~YMOS~N-B, CHYMOSIN-B, AND OF PEPTIDES SPLIT OFF DURING THE ACTIVATION OF PROCHYMOSIN
Residues per molecule Prochymosin-B Residues per Nearest 36200 g integer Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Amide N
12.97 5.01 7.01 33.37 20.42 30.56 36.31 13.98 28.49 15.01 5.08 22.80 6.76 19.04 25.56 18.34 16.45 4.05 34.03
13 5 7 33 20 31 36 14 28 15 6a 23 7 19 26 18 16 4 34
Molar ratio of Difference amino acid in between Chymosin-B activation proposed peptides cal- formulas of Residues culated prochymosin per Nearest relative to and 30700 g integer arginine chymosin 8.14 4.10 4.85 29.99 18.45 26.68 28.93 12.02 24.50 12.82 4.88 20.90 6.66 15.16 18.54 15.38 13.79 4.19 30.70
8 4 5 30 18 27 29 12 25 13 6a 21 7 15 19 15 14 4 31
4.91 1.11 2.00 3.40 2.48 4.44 7.20 1.96 4.89 2.87 b 1.99 0.14 3.42 6.50 2.79 2.43 b
5 1 2 3 2 4 7 2 3 2 0 2 0 4 7 3 2 0
From sequence studies. b Not determined. I h a v e o b t a i n e d t h e h i g h e s t y i e l d of c h y m o s i n b y a r a p i d a c t i v a t i o n a t p H 2, while R a n d a n d E r n s t r o m p r e f e r a slow a c t i v a t i o n a t p H 5.14 F o r a discussion of t h e a c t i v a t i o n k i n e t i c s see f o o t n o t e s 4 a n d 5. Stability o] Chymosin. Cheese r e n n e t p o w d e r a n d s u s p e n s i o n s of c h y m o s i n c r y s t a l s a p p e a r to be v e r y s t a b l e w h e n s t o r e d a t t e m p e r a t u r e s b e l o w 5 °. S o l u t i o n s of c h y m o s i n h a v e o p t i m u m s t a b i l i t y a t p H b e t w e e n 5.3 a n d 6.3. A t p H a r o u n d 3.5 a n d a b o v e 7 t h e e n z y m e r a p i d l y loses its a c t i v i t y . A t p H 2, h o w e v e r , c h y m o s i n s o l u t i o n s a r e r e l a t i v e l y stableS; if t h e s o l u t i o n s a r e s a l t f r e e , t h e s t a b i l i t y is r e d u c e d b y i n c r e a s i n g s a l t c o n c e n t r a t i o n . 19 Solubility o] Chymosin. M o s t d e t e r m i n a t i o n s of t h e s o l u b i l i t y of c h y m o s i n h a v e been c a r r i e d o u t in s o l u t i o n s of NaC1. A t a b o u t p H 5.5 'gR. Mickelsen and C. A. Ernstrom,
J. Dairy Sci. 50, 645 (1967).
432
~Z~E ACZDICPROT~.ASES
[28]
CU/rnl
A27s I00 80
60 40 20 ,
I
I
I
I
I
I
5
I
I
I
I
I
Io
24
hours
FiG. 2. Activation of prochymosin. Prochymosin concentration ca. 1 mg per ml reaction mixture. Temperature 25°. Experiment I (A): 1 ml prochymosin solution-~-1 ml 02M acetate pH 4.7. Experiment II (O): 1 ml prochymosin solution-~-1 ml 0.025N HC1, pH 2.0. Experiment III (O): 1 ml prochymosin solution ~ 1 ml 0.025N HCI/02 M NaCI, pH 2.0. crystalline chymosin has a solubility less than 1 mg/mi in solutions containing more than 0.5 M NaC1. Crystalline chymosin prepared in the author's laboratory has been soluble by dialysis against distilled water, while Berridge 8 has reported that his crystals were insoluble in distilled water. In equilibrium with amorphous precipitates chymosin is very soluble in solutions of 1 M NaC1 and pH about 5.5. In 2 M solutions of NaC1 the solubility is of the order 20 mg/ml and it is less than 1 mg/ml in saturated solutions of NaC1. In salting out experiments, amorphous precipitates of chymosin are more soluble at 2 ° than at 25 °. At pH between 5 and 6 chymosin has a strange solubility minimum at ionic strength of 0.1. Thus a transient amorphous precipitate is observed when NaC1 is added during the recrystallization.2° Chymosin has an isoelectric point of about pH 4.6. At ionic strength of 0.005, chymosin is very insoluble from pH 4.4 to 4.8, the solubility being increased by increasing ionic strength. Molecular Weights, Amino Acid Compositions, and Primary Structures. The data from ultracentrifugation, diffusion, and gel filtration have been compared with the minimum molecular weights obtained from the amino acid composition,ls,21 Rounded off to the nearest thousand, ~B. Foltmann, Acta Chem. Scand. 13, 1936 (1959). ~1R. Djurtoft, B. l%ltmann, and A. Johansen, Compt. Rend. Tray. Lab. Carlsberg 34, 287 (1964).
[28]
PROCHYMOSIN AND CHYMOSIN
433
molecular weights of 36,000 and 31,000 are found for prochymosin and chymosin, respectively. The individual fractions of chymosin cannot be distinguished by their molecular weights. (See Table IV.) On the basis of sedimentation equilibrium studies Cheeseman 22 has recently reported t h a t at p H 3.1 solutions of chymosin contain a monomer of molecular weight about 30,000 while at p H 5.8 to 6.5 a dimer is mainly present (apparent molecular weight about 64,000). TABLE IV MOLECULAR WEIGHT OF PROCHYMOSIN AND CHYMOSIN
Mol. wt. (Dalt ons)
S~.,,, (S)
Method
D.IO-7 (cms sec-1)
(ml.g-I)
Ref.
ca. 8.5
0.73
a a b
9.5 6.8 8.5
0.75
c d a a e f b f
Prochymosin
36,500 36,000 36,200
Sedimentation/diffusion Gel filtration Amino acid composition
40,000 34,400 34,000 33,000 31 ,O00 34,000 30,700 30,400
Sedimentation/diffusion Sedimentation/diffusion Sedimentation/diffusion Gel filtration Gel filtration Gel filtration Amino acid composition Amino acid composition
3.5
Chymosin
4 2.6 3.2
0.73
a R. Djurtoft, B. Foltmann, and A. Johansen, Compt. Rend. Tray. Lab. Carlsberg $4, 287 (1964). bB. Foltmann, Compt. Rend. Tray. Lab. Carlsberg 34, 275 (1964). c H. Schwander, P. Zahler, and Hs. Nitschmann, Helv. Chim. Acta 35, 553 (1952). a L. Friedman, Dissertation Abstr. 20, 4510 (1960). • P. Andrews, Biochem. J. 91, 222 (1964). / p. J. de Koning, Thesis, Univ. of Amsterdam, The Netherlands, 1967. Within the experimental error of the amino acid analyses published so far, it has not been possible to show any significant differences between amino acid compositions of total crystalline chymosin and chromatographically purified chymosin-B. The results of analyses of chromatographically purified preparations are illustrated in Table III, which also shows the amino acid composition of the peptides liberated during the limited proteolysis of the a c t i v a t i o n process. Most of the sequence studies carried out until now have been performed with total crystalline chymosin. The results from some major G. C. Cheeseraan, J. Dairy Res. 36, 299 (1969).
434
~ H E ACIDIC PROTEASES
[~8]
I 0 01 I®
I,~ ol I.~ ~ I L~-- _ ~
rE-~-]
o
Z
F~T-m]
0
r..?
I~, ~1 L~__~J
t~
iT M
~I
z L "~ - __~J
.~
~
~
Z
N
r~ -~
L~__~j
_~j
I F~--~ 0
F~--~ :~
~. L~,
I
I
r m - -~n
r~
-~. (J
0 ~e
!i °~
d~
E~
.~ ~
•
~
.
[28]
PROCHYMOSIN AND CHYMOSIN
435
fragments are illustrated in Table V. It should be emphasized that peptide patterns obtained from chromatographically purified chymosin-B are consistent with the results shown in Table V. To what extent the different chymosins are identical remains to be investigated. General Proteolytic Activity and Specificity. All the chromatographic fractions of chymosin show general proteolytic activity around pH 3.5. It should be noted, however, that the ratio between the milk-clotting and the proteolytic activity is lower for chymosin-C than it is for chymosin-A, and -B. 11 The proteolytic specificity has been tested with the B-chain of oxidized insulin as substrate. It has not been possible to demonstrate any differences between the proteolytic specificities of the individual fractions by this method. The specificity has some resemblance to that of pepsin. 23'24 Homology with Pepsin. Distribution. In many respects the properties of chymosin are similar to those of pepsin. Both are acidic gastric proteases of similar molecular weights, and they are secreted as precursors which are converted into active enzymes by a limited proteolysis from the N-terminus. The similarities between chymosin and pepsin probably arise from similarities in primary structures. We are able to compare only relatively short sections of the amino acid sequences of pepsin and chymosin, but three of the peptide fragments illustrated in Table V show a pronounced homology. In the C-terminal sequence, and the sequences around the S-S bridges marked B and C the amino acid residues outside the frames are identical in the two enzymes, the differences are marked in the frames. The amino acid sequences around the third S-S bridge (A) show a faint degree of homology, this is not marked in the table. However, the Nterminal amino acid sequence of chymosin shows no obvious resemblance to any known sequence from pepsin. The amino acid sequence around the active center of chymosin is not yet known, but after chymotryptic digestion a peptide has been found which is very similar to the active center peptide from pepsin found recently. 25 The homology suggests that the chymosin peptide in fact also represents the active center. The results are shown in Table VI. The similarities between chymosin and pepsin are pertinent for an evaluation of the distribution of the enzyme. Chymosin has only been isolated from calf, but the scattered information suggests that a proteoly'~J. C. Fish, Nature 180, 345 (1957). :*V. Bang-Jensen, B. Foltmann, and W. Rombauts, Compt. Rend. Trav. Lab. Carlsberg 34, 326 (1964). R. S. Bayliss, J. R. Knowies, and G. Wybrandt, Biochem. J. 113, 377 (1969).
436
[29]
T H E ACIDIC PROTEASES
TABLE VI HOMOLOGY BETWEEN AN ACTIVE CENTER PEPTIDE FROM HoG PEPSIN a AND A CHYMOTRYPTIC FRAGMENT FROM CHYMOSINb
Pepsin Chymosin
Ile
Val
Asp Asp
Thr Thr
Gly Gly
Thr Ser
Ser Ser
Asp
Phe
a R. S. Bayliss, J. R. Knowles, and G. Wybrandt, Biochem.J. 113, 377 (1969). bB. Foltmann, unpublished results, 1969. * Indicates the active aspartic acid residue in pepsin. tic enzyme with a high milk-clotting activity is characteristic for the gastric juice of the young ruminants. As the animal grows up, a change takes place from' predominant chymosin to predominant pepsin secretion. The possibility exists that a similar change in the composition of the gastric juice may occur during the ontogenesis of other animals, but it has not been noticed since it is not accompanied with an easily observable change in milk-clotting activity. M y opinion is, that had it not been because of ~heese-makingl chymosin would have been known as calf pepsin with addition of some odd letter.
[ 2 9 ] P r o t e a s e of Endothia parasitica
By JOHN R. WHITAKER Assay Method
Principle. The renninlike protease produced by the fungus Endothia parasitica catalyzes a specific proteolysis leading to clotting of milk as well as a general proteolysis of proteins. Under carefully standardized conditions, the primary phase (proteolysis) is the rate-determining step in milk-clotting and measurement of the clotting time can be used as a very sensitive, quick, and reliable assay for monitoring the purification procedure. A criterion that the enzymatic phase, and not the coagulation phase, is rate-determining is that a plot of clotting time versus reciprocal of enzyme concentration is a straight line with an intercept of zero. When greater precision is required, the assay method based on the initial rate of hydrolysis of casein or hemoglobin is recommended. MILK-CLOTTING ASSAY
Reagents Nonfat dry milk powder. Carnation Instant Nonfat Dry Milk powder is excellent. The product should be as fresh as possible
436
[29]
T H E ACIDIC PROTEASES
TABLE VI HOMOLOGY BETWEEN AN ACTIVE CENTER PEPTIDE FROM HoG PEPSIN a AND A CHYMOTRYPTIC FRAGMENT FROM CHYMOSINb
Pepsin Chymosin
Ile
Val
Asp Asp
Thr Thr
Gly Gly
Thr Ser
Ser Ser
Asp
Phe
a R. S. Bayliss, J. R. Knowles, and G. Wybrandt, Biochem.J. 113, 377 (1969). bB. Foltmann, unpublished results, 1969. * Indicates the active aspartic acid residue in pepsin. tic enzyme with a high milk-clotting activity is characteristic for the gastric juice of the young ruminants. As the animal grows up, a change takes place from' predominant chymosin to predominant pepsin secretion. The possibility exists that a similar change in the composition of the gastric juice may occur during the ontogenesis of other animals, but it has not been noticed since it is not accompanied with an easily observable change in milk-clotting activity. M y opinion is, that had it not been because of ~heese-makingl chymosin would have been known as calf pepsin with addition of some odd letter.
[ 2 9 ] P r o t e a s e of Endothia parasitica
By JOHN R. WHITAKER Assay Method
Principle. The renninlike protease produced by the fungus Endothia parasitica catalyzes a specific proteolysis leading to clotting of milk as well as a general proteolysis of proteins. Under carefully standardized conditions, the primary phase (proteolysis) is the rate-determining step in milk-clotting and measurement of the clotting time can be used as a very sensitive, quick, and reliable assay for monitoring the purification procedure. A criterion that the enzymatic phase, and not the coagulation phase, is rate-determining is that a plot of clotting time versus reciprocal of enzyme concentration is a straight line with an intercept of zero. When greater precision is required, the assay method based on the initial rate of hydrolysis of casein or hemoglobin is recommended. MILK-CLOTTING ASSAY
Reagents Nonfat dry milk powder. Carnation Instant Nonfat Dry Milk powder is excellent. The product should be as fresh as possible
[29]
PROTEASE
OF Endothia parasitica
437
and it should be stored at 2 ° , protected from moisture after opening. Sodium acetate buffer, 0.67 M, pH 4.60 Procedure. 1,~ Twenty grams of milk powder are dissolved in 50 ml distilled water at room temperature. Care should be taken that all the milk powder is dissolved at this point. Then 30 ml of sodium acetate buffer, pH 4.60, are added slowly from a pipette with continuous stirring and with care to prevent excess foaming. The solution is made to 100 ml in a volumetric flask with water. The pH should be 5.10 ___0.05. The substrate solution is permitted to stand at room temperature for not less than 45 minutes before use. It should not be stored in a refrigerator or used for more than 1 day. One-milliliter aliquots of substrate are pipetted into the bottom of 13 X 125-mm Pyrex text tubes which are then incubated in a water bath at 35.0 ° for not less than 5 minutes or more than 15 minutes. An aliquot (preferably 0.05 ml) of the enzyme solution is added rapidly from a 0.10 ml pipette (mark to mark) by placing the tip of the pipette just below the milk surface. A timer, calibrated to 0.1 second, is started immediately, the contents of the tube are mixed thoroughly but gently so as not to cause a bubble, and the tube is rotated slowly around the short axis while held at a 30o-40 ° angle. The film of milk around the wall of the tube is observed and the end point is the first appearance of a grainy texture. For greater precision, an enzyme concentration should be chosen which gives a clotting time of not less than 50 seconds and not more than 100 seconds. The determination is done in triplicate and the clotting times should agree within 2%. For the greatest precision, a high-intensity light mounted immediately above the water bath and a test tube rack (wire) so positioned in the bath that when the tube rests against one of the openings in the rack and the side of the bath it forms a 30 ° angle are helpful. Unit and Specific Activity. One unit is defined as that amount of enzyme which Will clot 1 ml of milk in 60 seconds. Specific activity is expressed as units per milligram protein. Remarks. Lower concentrations of substrate can be used with a proportional decrease in clotting time at a fixed enzyme concentration2 Clot formation can be detected even at 2% substrate concentration. However, at lower substrate concentrations the coagulation phase (nonenzymatic) becomes more important and, because of the large number
~J. 1~. Whitaker, Food Technol. 13, 86 (1959). 2A. K. Balls and S. R. Hoover, J. Biol. Chem. 121, 737 (1937). s M. K. Larson, "Some Chemical and Enzymatic Properties of Endothia parasitica Protease." M.S. thesis, Univ. of California, Davis, 1969.
438
THE ACIDIC
PROTEASES
[29]
of factors which affect the coagulation phase, the precision of the method is decreased. PROTEIN HYDROLYSIS METHOD
Reagents 1% Casein or hemoglobin. One gram of casein (Hammarsten quality) is dissolved in 25 ml 0.2 M phosphoric acid, 55 ml of distilled water are added, and the pH is adjusted with 1 N NaOH to 2.50 using a pH meter. The solution is diluted to 100 ml with distilled water. For activity determination at pH 6.0, 1 gram casein is dissolved in 25 ml 0.2 M sodium acetate by brief warming in a boiling water bath, the solution is cooled, 55 ml distilled water are added, and the pH is adjusted with 1 N HC1 to 6.0. The solution is diluted to 100 ml with water. The hemoglobin must be denatured before use." Two grams of Hemoglobin Substrate Powder (Mann Research Lab.) are dissolved in 75 ml of 0.06 N HC1, the pH is adjusted to 1.8 with 0.5 N HC1, and the solution is held at room temperature for 20-24 hours. After exhaustive dialysis of the solution against distilled water at 2 °, it is diluted to 100 ml with water. The stock solution is stable for several weeks in the refrigerator. For hydrolysis at pH 2.5, 25 ml of 0.2 M phosphoric acid are added to 50 ml of stock hemoglobin solution, the pH is adjusted to 2.5 with 1 N NaOH, and the solution is diluted to 100 ml. For hydrolysis at pH 6.0, 25 ml of 0.2 M acetic acid are used in place of phosphoric acid and the pH is adjusted to 6.0. 5% Trichloroacetic acid (TCA)
Procedure2 Fifteen milliliters of substrate are placed in a 20 X 175mm test tube and equilibrated for 10 minutes at 35.0 ° in a water bath. An aliquot of enzyme (0.05 ml, equivalent to 0.03 mg purified enzyme) is added, mixed, and 2-ml aliquots are removed at zero time and subsequently at 5-minute intervals into 3 ml of 5% TCA. The contents are mixed thoroughly, allowed to stand at room temperature for 1 hour, centrifuged at 2000 rpm (International Clinical centrifuge) for 20 minutes, and the absorbance of the supernatant liquid read at 280 nm. Alternatively, 0.10-ml aliquots may be removed at intervals into 0.90 ml 0.1 N HC1 and the number of peptide bonds split determined ' M. L. Anson, J. Gen. Physiol. 22, 79 (1938). s M. Kunitz, J. Gen. Physiol. 30, 291 (1947).
[29]
PROTEASE OF Endothia parasitica
439
with the quantitative ninhydrin reagent 6 using 0.1-0.2-ml aliquots of the HCl-diluted sample. Unit and Specific Activity. One unit is defined as that amount of enzyme which will produce a change of 0.001 in absorbance per minute measured as an initial rate. Specific activity is expressed as units per milligram protein. Remarks. The progress curve is not linear at either pH 2.5 or 6.0. The nonlinearity is much greater at pH 6.0. Only the initial rates of hydrolysis are linearly related to enzyme concentration. Purification Procedure
Production o] Enzyme. 7 Cultures s of the organism are maintained on potato dextrose agar; the organism will grow in a medium containing a source of carbohydrate, nitrogen, and inorganic salts. For the production of protease the following procedure has been successful. The growth medium contains (per liter): 5.0 g N-Z amine B (hydrolyzed casein preparation, Sheffield Chemical Co., Inc.), 2.0 g soya protein digest (Soytone, Difco Lab.), 2.0 g yeast extract, 10.0 g soluble starch, 5.0 g D-mannitol, 15.0 mg FeS04.7 H20, and 1.0 ml of trace elements--Fe [as Fe(NH4)2(S04)2], 1.0 mg/ml; Zn (as ZnSO~), 1.0 mg/ml; Mn (as MnSO4), 0.50 mg/ml; Cu (as CuS04), 0.08 mg/ml; Co (as COS04), 0.10 mg/ml; B (as H3BO~), 0.10 mg/ml. One liter of the medium is placed in a 2.8-liter Fernbach flask and sterilized in an autoclave for 45 minutes at 121% The organism is washed from the potato dextrose agar slant into the flask under sterile conditions. The flask is then incubated in a rotating shaker at 28 ° for 96 hours. The enzyme production medium contains (per liter): 30.0 g soybean meal, 10.0 g Cerelose (glucose), 10.0 skim milk, 3.0 g NAN03, 0.50 g KH2P04, and 0.25 g MgSO~.7 H20. The pH of the medium is about 6.8. The medium is sterilized in 2-liter portions in 4-liter fermentation vessels in an autoclave for 1 hour at 121 °. After cooling the enzyme production medium, 100 ml of the growth medium (inoculum) are added to each 2 liters of sterile growth medium. The cultures are agitated at 1700 rpm at 28 ° while air is introduced at the rate of 0.5 volumes air per volume medium per minute. After 48 hours of incubation, 0.5 ml of the medium will clot 5 ml sweet milk within 3 minutes. Preparation o] Crude Enzyme. 7 The culture medium (2 liters) is 6S. Moore and W. H. Stein, J. Biol. Chem. 211, 907 (1954). 7j. L. Sardinas, U~S. Pat. 3,275,453, Sept. 27, 1966. (Assigned to Charles Pfizer and Co., New York.) 8The organism is available from American Type Culture Collection, Washington, D.C. where it is designated as ATCC 14729.
440
THE ACIDIC PROTEASES
[29]
filtered on a Biichner funnel containing a 0.5-cm layer of Hyflo Super-Cel and the filter cake is washed with 100 ml of deionized water. The filtrate is freeze dried and the freeze-dried solids (clotting specific activity ~2.6 units/mg) are reconstituted in 400 ml deionized water. In the subsequent steps nitrogen is flushed over the container. After cooling the solution to 1°, 2 volumes of acetone, at --25 °, are added slowly with constant stirring. The acetone-water layer is decanted from the congealed solid, the excess acetone--water is removed from the solid by pressing with a spatula, and most of the acetone is removed in a rapid stream of nitrogen. Immediately after, the solid is suspended in an equal volume of deionized water and freeze dried. The freeze-dried solids, containing 80-85% of the original activity, are dissolved in water to give a 15% (w/v) solution. The solution is cooled to 1° and acetone (approx. 1.5 vol), at --25 °, is added slowly with continual stirring until the precipitate just begins to congeal. The precipitate is allowed to settle and the liquid is removed by decantation. An additional 0.75 volumes of acetone are added slowly to the supernatant liquid. The liquid is removed and the solids are freeze dried as described for the first acetone precipitation. The yield is about 40% of the original activity with a clotting specific activity of about 15 units/mg. Exhaustive dialysis of the solids (dissolved in an equal volume of water) against deionized water increases the specific activity to about 30-32 units/rag. Enzyme of this purity is available from Charles Pfizer and Co., Milwaukee, Wis.
Purification o] Enzyme ~ Further purification of the enzyme can be obtained by (NH4)~S04 precipitation, Sephadex G-100 filtration, and chromatogralbhy on DEAEcellulose. All subsequent steps are carried out at 0 ° to 2 °. Centrifugation is performed at 10,400 g in a Sevall refrigerated centrifuge for 20 minutes. Dialysis is carried out against 0.05 M acetate buffer, pH 4.60. The dialyzed solution of the crude enzyme (above) is diluted to give a 2.8% solution (protein concentration). Solid (NH4)~SO~ i s added slowly to the solution (fraction 1, Table I) to 60~ saturation (0°). After 1 hour the suspension is centrifuged and the supernatant liquid (fraction 3) is discarded. The precipitate is dissolved in water to give a 3.5% solution and dialyzed for 48 hours (fraction 2). Solid (NH4)2SO, is added to fraction 2 to 40% saturation and the suspension is centrifuged after 1 hour. The precipitate (fraction 4) is discarded. Additional (NH4)2S04 is added to the supernatant liquid to 55% saturation and the suspension centrifuged after 1 hour. The supernatant liquid (fraction 6) is dis9 K. Hagemeyer, I. Fawwal, and J. R. Whitaker, J. Dairy 8c/. 51, 1916 (1968).
[29]
PROTEASE OF Endothia parasitica
441
carded. The precipitate is dissolved in 0.05 M acetate buffer, pH 4.6, to give a 3.5% solution and is dialyzed overnight (fraction 5). At this stage the preparation still contains a brown pigment which is not removed by Polyclar AT or by charcoal but is readily removed by filtration on Sephadex G-100. Fifty-milliliter aliquots of fraction 5 may be added at one time to a 4.1 X 45-cm column equilibrated with 0.05 M acetate buffer --0.4 M NaC1, pH 4.60. The column is irrigated with the same buffer. All the milk-clotting activity is obtained in the major peak (280 nm absorbance) eluted at 2.5 times the void volume of the column while the pigment is eluted as a separate peak at 4.5 times the void volume. The tubes containing the majority of the enzyme are combined (fraction 7) and precipitated with (NH4)~S04 to 90% saturation. The supernatant liquid (fraction 9) is discarded and the precipitate (very white) is dissolved in 0.05M acetate buffer, pH 4.6, to give a 3.5% solution. After dialysis against 0.2 M acetate buffer, pH 4.6, up to 0.25 g of fraction 8 are added to the top of a 2.1 X 45-cm column of DEAEcellulose equilibrated against the same buffer. DEAE-cellulose is prepared for chromatography by grinding through an 80-mesh sieve of a Wiley Mill, removing of fines, washing with 0.25 M NaC1-0.25 M NaOH, with water until free of Cl-, with 0.25 M HC1, with water until free of C1- and then five times with the starting buffer. The column is poured in one segment by means of an extension tube and, after placing in a cold room, the column is washed with a minimum of 3 void volumes of starting buffer before use. The protein is eluted from the column with a linear gradient. The mixing vessel contains 500 ml of 0.2 M acetate buffer, pH 4.6, and the second vessel contains 500 ml of 0.2 M acetate buffer0.6M NaC1, pH 4.6 (0.6M acetate buffer, pH 4.6, may also be used). Ten-milliliter fractions are collected at a rate of 3 fractions per hour. Several minor protein components (up to 4) are eluted in rapid succession from the column. The major protein peak, and the only one with milk-clotting activity, begins to come off at fraction 55 and reaches a maximum concentration in fraction 60. The fractions containing the majority of the enzyme are combined, and the enzyme precipitated by addition of (NH4)2SO4 to 90% saturation. The precipitate, after recovery by centrifugation, is dissolved in a minimum volume of water and the solution is dialyzed against 0.33M acetate buffer, pH 4.6 (fraction 10). Fraction 10 is rechromatographed on a DEAE-cellulose column (2.1 X 45 cm) equilibrated with 0.33M acetate buffer, pH 4.6. The protein is eluted with the starting buffer. A minor inert protein peak is eluted first, followed by the enzyme which is eluted as a single peak (center of peak is at 2.5 times the void volume of the column). The
442
THE ACIDIC PROTEASF,S
[29]
fractions containing the majority of the activity are combined, and the enzyme is precipitated with (NH4)2SO~ to 90% saturation. After 1 hour the precipitate is removed by centrifugation and is dissolved in a minimum volume of water and dialyzed against 0.05 M acetate buffer, pH 4.60 (fraction 11). Our best preparations of enzyme have 12-17~ the specific activity in clotting milk of our crystalline rennin preparations. A typical purification is presented in Table I. Crystallization. A typical example is given. Cold (0 °) isopropanol is added dropwise to 10 ml of a preparation equivalent to fraction 11 (Table I) which contains 33.5 mg/ml protein until the first trace of turbidity is seen. This requires an isopropanol concentration of 28.6%. The solution is allowed to stand at 2% After 24 hours there is some amorphous precipitate but no crystals. After 48 hours the tube is filled with crystals. After 96 hours, the needle-shaped crystals are collected by eentrifugation, washed with a little cold 28.6% isopropanol (isopropanol diluted with 0.05M acetate buffer, pH 4.60) and dried in a desiccator over P~O~ at 2% The yield is 72% with a specific activity of 125 units/mg. Crystallization of the enzyme is extremely easy. We have never failed to obtain crystals and even less pure preparations may be crystallized. However, the specific activity is always decreased about 1012% by the crystallization procedure. For this reason, storage of the enzyme solution at --15 ° or lyophilization of the enzyme is preferred. When the enzyme solution is equilibrated against 0.01 M acetate buffer, pH 4.6, and frozen at --25 ° to --30 ° it may be lyophilized with no loss in activity. Properties
Stability2 The enzyme is maximally stable at pH 3.8 to 4.5. At 50 °, only 30% activity is lost in 30 minutes in this pH range. Below pH 2.5 loss in activity is associated with an increase in ninhydrin-reactive groups, fragmentation, and an activation energy of inactivation, Ea, of 14,100 eal/mole. Above pH 6.5 activity is lost rapidly (almost instantaneously above pH 8) and is associated with no increase in ninhydrin-reactive groups, a decrease in solubility, and an Ea of 51,500 cal/mole. Purity2 Physical and Chemical Properties. Fraction 11 (Table I) is at least 95% chromatographically pure by starting buffer elution on an analytical DEAE-cellulose column. It migrates as a single band on cellulose acetate electrophoresis at pH 5. The molecular weight has been reported to be 37,500 and 34,000 as determined by Sephadex G-1009 and Bio-Gel P-1001° filtration, respectively. The isoelectric point appears •J. L. Sardinas, Appl. Microbiol. 16, 248 (1968).
laROT:EASE OF Endothia parasitica
[29]
443
o
~0~,0
C~O0
eo
0"301
0
~=~
oo== 0
o~
Z 0 0
~k~k~ ~ k~
h~
0
T H E ACIDIC PROTEASES
~
T A B L E II AMINO ACID CO~POSITm~ OF E. parasitica P R O T E A S E
[29]
a
Residues per 37,500
Amino acid
Determinations ~
Aspartic acid Threonine c Serine* Glutamic acid Proline Glycine Alanine Valine Half-cystine~ Methionine Isoleueine Leucine Phenylalanine Tyrosine Ammoniac Lysine Histidine Arginine Tryptophan *
11 12 12 13 9 12 12 12 11 11 13 12 13 13 12 12 12 10 2
25.7 50.0 43.9 14.6 13.2 37.6 28.6 22.2 1.62 0.435 17.4 18.7 13.1 19.4 31.1 11.6 3.05 1.70 3.18
Residues
Integer
± 1.2
26 50 44 15 13 38 29 22 2 1 17 19 13 19 31 12 3 2 3
± 0.8 ± 1.4 ± 2.7 :[: 1.7 _ 2.4 + 0.71 ± 0.180 ± 1.5 ± 0.9 5= 1.4 ± 1.4 (18.5)" ± 1.2 ± 1.02 _ 0.35
a Hydrolysis performed by method of Moore and Stein (Vol. VI, p. 819) and analysis performed on Technicon Auto Analyzer. Hydrolyses performed for 20, 40, and 70 hours. Corrected for destruction by extrapolation to zero time. Determined both as eystine and as cysteic acid. • Determined spectrophotometrically hy method of Fraenkel-Conrat (Vol. IV, p. 252). t o be b e l o w p H 4.6 since t h e e n z y m e is a d s o r b e d s t r o n g l y to D E A E cellulose in 0.2 M a c e t a t e buffer, p H 4.6. 9 H o w e v e r , S a r d i n a s 1° h a s rep o r t e d t h a t his p r e p a r a t i o n of e n z y m e b e h a v e d as a c a t i o n a t p H 4.5 on cellulose p a p e r . T h e a m i n o a c i d c o m p o s i t i o n of t h e e n z y m e is shown in T a b l e I I . 11 Activators and Inhibitors2 T h e e n z y m e is n o t i n h i b i t e d b y s o d i u m t e t r a t h i o n a t e (1 X 10 - 3 M ) , HgC12 (1 X 10 - 3 M ) , N - e t h y l m a l e i m i d e (2 X 10 -3 M ) , i o d o a c e t a m i d e (2 X 10 -3 M ) , p - c h l o r o m e r c u r i b e n z o a t e (1 X 1 0 - 3 M ) , c y s t e i n e (1 X 1 0 - 2 M ) , T P C K 12 (1 X 1 0 - 3 M ) , a n d T L C K TM (1" X 10 -3 M ) . Specificity. T h e e n z y m e does n o t h y d r o l y z e b e n z y l o x y c a r b o n y l - L J. R. Whitaker, unpublished data. n The chloromethyl ketones derived from N-tosyl-L-phenylalanine and N-tosylL-lysine, respectively. E. Shaw, Vol. XI, p. 677.
[29]
PROTEASE OF Endothia parasitica
445
RENNIN
E. PARASITICA PROTEASE
@
0
Q
0
©
T
>-r O. "G 20
io. 4 io.
2
m0 c,) I
I00 200 Frocfion number
FIG. 2. Chromatographic pattern of mucor rennin on DEAE-Sephadex A-50 (6 X 24 cm). ( 0 ) Milk-clotting activity, (O) OD 280 nm; concentration of KC1. solid line. Elution buffer, 0.05 M sodium acetate buffer (pH 5.0); eluted volume, 20 ml.
452
[30]
~H~. ACIDIC PROTEASE8 o
% -
O
3O 4
-G
10
2
20
40
\x,.;3 80 I00
60
Fractionnumber
120
Fia. 3. Chromatographic pattern of mucor rennin on DEAE-Sephadex A-50 (4 X 29 cm). ( 0 ) Milk-clotting activity, (O) OD 280 nm; concentration of KC1, solid line. Elution buffer, 0.05 M sodium acetate buffer (pH 5.0); elution volume, 20 ml.
of 70~. After standing at 5 ° for 10 hours, the precipitate is collected by centrifugation at 10,000 g for 20 minutes at 0 °. The sedimented material is dissolved in a minimal amount of 0.05 M sodium acetate (pH 5.0). The solution is then passed through a Sephadex G-100 column, using 0.05 M sodium acetate buffer of pH 5.0 for elution. As seen in Fig. 4, a portion of the pigment fraction is separated by this procedure. Repeated gel filtration on Sephadex G-100 is used to remove remaining pigment residues. The active fractions (20.31 X 106 units) are pooled and ammonium sulfate is admixed slowly to 70% saturation. After 10 hours at 0 °, the precipitate is collected by centrifugation and is dissolved in 0.1 M sodium acetate buffer of pH 5.0 to a 2-3% protein concentration.
/°\
~- ~ 40
°
~
.~t~!!.~ Pigm__nt .
.
t~,,,~
o
40 80 Fraction number
120
Fro. 4. Column, ehromatogram of mueor rennin on Sephadex G-100 (3 X 8 era). ( 0 ) Milk-clotting activity, (O) OD 280 nm. Elution buffer, 0.05 M sodium acetate buffer (pH 5.0).
[30]
MILK-CLOTTING
ENZYME FROM M. pusillus
453
Saturated ammonium sulfate solution is then added with gentle stirring until a slight turbidity is formed. This solution is clarified by centrifugation. The ammonium sulfate concentration of the supernatant is slowly increased to 50% saturation by adding further increments of a saturated ammonium sulfate solution. Crystallization is allowed to take place during storage at 3 0 4 ° and occurs as the concentration of both ammonium sulfate and enzyme increase by evaporation (ca. 2 months). It can be greatly hastened, however, by seeding the solution with enzyme crystals obtained earlier. With such seeding crystallization starts in about 10-15 hours. Various crystal forms were observed (Fig. 5). Nevertheless, all crystalline forms have the same specific activity. It is conceivable that the actual shape of the crystal depends on the method employed for crystallization. The mentioned procedure of evaporation over a period of 2 months in the refrigerator produced elliptical crystals. A crystallization method based on dialysis induces the formation of different crystals. The enzyme solution (2-3% protein) is put into a cellophane bag for dialysis against 0.1M sodium acetate buffer which is 50% saturated with ammonium sulfate. Saturated solution of ammonium sulfate is then added dropwise to the outside dialyzing
Fro. 5. Different forms of crystalline mucor rennin.
454
Tn~, ACIDIC PROZ~,ASrS
[30]
TABLE III PURIFICATION FROCESS OF MUCOR BENNIN CRYSTAL
Specific activity OD 280 nm Determination Crude enzyme Amberlite CG-50 Amberlite CG-50 DEAE-Sephadex .%-50 DEAE-Sephadex A-50 (NH4)~SO4, saturated 0.7 Sephadex G-100 Sephadex G-100 (l~I-I4)~SO4
Mucor rennin crystal, ~ 2.35 g
OD 280 nm
Clotting activity Ratio of OD purifi280 nm cation
Yield (%)
X 10a Yield Units (%)
113,400 100 24,000 21.5 14,000 12.4 6,660 5.9 4,220 3.7
52,000 100 51,000 99.3 40,240 77.3 29,667 57.2 25,480 49.0
460 2,140 2,870 4,450 6,040
1.0 4.7 6.2 9.1 13.2
23,320 20,310 19,300 17,200
6,050 6,650 6,670 6,670
13.3 14.4 14.4 14.5
3,850 3,050 2,910 2,580
3.4 3.0 2.6 2.6
44.8 38.7 37.1 33.1
a The crystals were prepared by the dialysis crystallization method described in the text. fluid to give a final saturation of 70%. As the concentration of ammonium sulfate inside the cellophane bag slowly rises, crystals of the enzyme form. The crystals m a y be collected in about a week. The enzyme crystals are separated by centrifugation at 10,000 g for 10 minutes and are dissolved in distilled water. Following dialysis against water for 24 hours, the solution is freeze dried and yields 2.35 g of enzyme p o w d e r (corresponding to 2580 OD2s0 units). The recovered total milkclotting activity is 172 X 10' units with a specific activity of 6670 units per OD~go. The purification procedure is summarized in Table III. Properties
Stability25,s6 As judged by retention of activity at 60 ° over a 10minute period, mueor rennin appears to be most stable at p H 5.0. I t is recalled t h a t the calf enzyme is stable in a similar p H range. Purity and Physical Properties28 Homogeneity of the crystalline enzyme (in 0.1 M sodium acetate buffer, p H 5.0) was attested both by Tiselius electrophoresis and by ultracentrifugal analysis. Electrophoresis was performed at 90 V and 5 mA. The enzyme migrated as a single symmetrical peak. The ultracentrifugal analysis was carried out at 55,400 rpm at 11.3 ° for 3 hours and 17 minutes. The sedimentation pattern showed only a single symmetrical peak. Relation o] Milk-Clotting to Proteolytic Activity26 Ratio of the milk-clotting activity of mucor-rennin crystals to their proteolytic activ-
MILK-CLOTTING ENZYME FROM M .
[30]
pusiUus
455
TABLE IV RATIO OF CLOTTING ACTIVITY TO PROTEOLYTIC ACTIVITY OF VARIOUS PROTEASES• Protease
Clotting activity (units/ml)
Rennin Mucor rennin crystal Pfizer microbial rennin Papain Trypsin Molsin Ficin Biodiastase
293 511 750 216 1.6 1.3 267 115
Proteolytic activity Ratio (OD 660 nm) (units/OD 660 nm) 0.04 0.11 0.29 0.59 0.44 0.18 0.68 0.83
7350 4650 2590 367 4 7 393 138
ity was compared with those of other proteases. Values for this ratio, expressed as milk-clotting units per the OD66o's obtained in the proteolyric measurements, were as follows. Calf rennin, 7350; mucor rennin, 4650; Pfizer rennin from Endothia parasitica, 2590. Molsin (Kikkoman Co. Ltd.), ficin, trypsin, papain, and Biodiastase (Amano Co. Ltd.) gave a ratio of less than 393 (Table IV). Optimum pH ]or Proteolytic Activity. Optimum pH is 4.5 for r-casein and 4.0 for hemoglobin. For Hammarsten casein it is around pH 3.5. Thus the optimum pH of activity of the crystalline mucor rennin enzyme is similar to that of calf rennin. ~a-~5 Ef]ect o/ pH on Milk Coagulation Activity. The crystalline mucor enzyme clots milk most rapidly at pH 5.5. Clot formation at pH 7.0 is greatly delayed. Similar results are also obtained with the crude enzyme.~°,46 Calf rennet activity 46 is likewise pH dependent and is reported to be weaker in alkaline conditions than in acidic conditions.
Physical Properties and Amino Acid Composition o/Crystalline Mucor Rennin. ~9 SEDIMENTATION, DIFFUSION, AND FRICTIONAL COEFFICIENTS. Though a sedimentation value for the purified enzyme21 was reported earlier, no correction was made to standard conditions. We now measured an s°.w of 2.39S for the crystalline protein in 0.1M sodium acetate buffer, pH 5.0. Density measurements indicate a partial specific volume of 0.74. Using the maximum ordinate method and the maximum ordinate area method,47 a diffusion D2°o.,, of 7.9 ~ 0.3 X 10-7 cm 2 sec-1 was obtained. ~ M. L. Anson, J. Gen. Physiol. 22, 79 (1938). " H . Schwander, P. Zahler, and H. Nitschman, Helv. Chem. Acta 25, 553 (1952). '~B. Foltmann, Acta Chem. Scan& 13, 1927 (1959). "*G. H. Richardson and J. H. Nelson, J. Dairy Sei. 50, 1066 (1967). ,7 S. Mizushima and S. Akabori, Chem. Protein 2, 383, 385 (1954).
456
ThE ACIDIC PROTF.ASES
[30]
Frictional ratio is 1.33. Calculation of molecular weight according to the Svedberg equation~8 gives a value of about 29,000_ 1400. Using the Yphantis method,49 the molecular weight of crystalline mucor rennin is 30,600. Amino acid analysis for crystalline mucor enzyme is given in Table V and is compared with those of rennin and prorennin2° The 277-281 TABLE V AMINO ACID COMPOSITION OF MICROBIAL RENNIN~ PRORENNIN~ AND RENNIN Microbial rennin
Rennin. MW 31,100
Prorennin ° MW 36,200
Amino acid
MW 30,600
Residues
Half-cys Met Asp Thr Set Glu Pro Gly Ala Val Ile Leu Tyr Phe His Lys Arg Trp
2.31 3.17 43.6 21.2 22.1 19.7 14.1 33.5 16.4 23.8 11.8 14.8 12.8 18.9 1.42 11.4 4.1 2.45
2 3 44 21 22 20 14 34 16-17 24 12 15 13 19 1-2 11-12 4 2-3
7 31 28 27 29 12 25 13 21 15 19 15 14 4 8 7
7 33 21 31 36 14 29 15 23 19 26 18 14 5 13 5
277.52 1.4.3
277-281
263
313
NHz Ref. 11.
residues in mucor rennin are distributed as follows: 1/~Cys2, Mets, Asp44, Thr21, Ser22, Glu~o, Pro14, Glys4, Ala16_~, Va124, Ilel2, Leu15, Tyr18, Phe~9, Hiss_2, LyS~l_12, Arg4, and Trp2-3. Elementary analysis gives C, 49.2449.59%; H, 6.99 and 7.44~; N, 13.86-14.19~; S, 0.57-0.65~. These values agree with the theoretical values obtained from amino acid composition and NH~. For purposes of comparison, some data regarding the physical prop~8T. Svedberg and K. O. Pedersen, "The Ultracentrifuge," Clarendon, Oxford, 1940. ~°D. A. Yphantis, Ann. N.Y. Acad. Scl. 88, 586 (1960). ~oH. Schwander, P. Zahler, and H. Nitschman, Helv. Chem. Acta 35, 553 (1952).
MILK-CLOTTING ENZYME FROM M .
[30]
pusillus
457
TABLE VI SUMMARIZED PHYSICAL PROPERTIES OF RENNIN AND MUCOR RENNIN
Value Property
Mucor rennin
Frictional coefficient Sedimentation coefficient
fifo so,,
1.33 2.39 X 10-1~
Diffusion coefficient
D°,,
7.9 X 10-'
Partial specific volume Molecular weight Svedberg Yphantis Andrews Amino acid analysis
O. 742 29,000
Rennin ~ 0.98 b 3.2 X 10-is 4 X 1 0 -13~ 9 X lO-V 9.5 X 10-Tb O. 749 ~ 31,100, 40,000 ~
Prorennin a
3.5 X 10-1'
36,200
30,600 32,500 29,690 30,213 (approx.)
~Ref. 11. Ref. 21.
erties of rennin are given in Table VI. Foltmann and Hartley ~5,51 reported on the amino acid composition of this enzyme. It could also be mentioned that the optimum pH for hydrolysis of both casein52 and hemoglobin*s by rennin is pH 3.7. Altogether, the general properties of mucor rennet ~°,46 and the mucor crystalline rennin a4-4~ differ only slightly from those of animal rennet and rennin. Active Site oj the Enzyme and the Role o] Ca-Ions in Milk Coagulation) 8 Inhibition of the enzyme activity by iodine is pH dependent. Almost complete inhibition (>90 °) occurs at pH 6.7, but there is little loss of activity ( ~ 1 0 ~ ) if the treatment is carried out in the 3.0-5.0 pH range. Figure 6 shows the loss of activity of the enzyme caused by photooxidation and also indicates the concomitant changes in the tyrosine, tryptophan, and histidine content. The loss of histidine parallels the inactivation of the enzyme. Treatment of mucor rennin by p-chloromercuribenzoic acid, iodoacetamide, or N-ethylmaleimide does not affect the milk-clotting activity. B. Foltmann and S. Hartley, Biochem. J. 104, 1064 (1967). mE. Schram, S. Moore, and E. J. Bavel, J. Dairy Sci. 49, 700 (1966). N. J. Berridge, Biochem. J. 39, 179 (1945).
458
T~.
ACIDIC PROTEASES
[30]
I00, o~
75
0
=
50
a:
25 00
I
I
l
I
Ill
400 200 Time (rain)
360
Fie. 6. Relation between activity of mucor rennin and residual amino acid by photooxidation. ( 0 ) Histidine, (~7) tyrosine, (X) tryptophan, (O) residual milkclotting activity.
The use of diazonium-lH-tetrazole (DHT) for the spectrophotometric determination of histidine residues in protein has been applied to several proteins. 5. When D H T is added to a solution of mucor rennin, optical, density at 480 nm increases 55 and the milk-clotting activity is completely lost. One of the two histidine residues of the protein is suggested to be critical for activity. While Ca ions do not affect the proteolytic activity of mucor rennin, they enhance the apparent activity in the milk-clotting assay (Fig. 7). Calcium ions seem to influence the clotting of casein p e r se rather than affecting the enzyme reaction itself. S p e c i f i c i t y . ~2 Among 58 synthetic peptides tested, the following were
$~D
800
C3
0.6
"; 600 .1.¢...1 0
o
o.4
~. 4 0 0 .~_
L)
0
0.2
7 200
g 0
2
4
6 8 x t0-3M
t0
o (3.
FIe. 7. Effect of CaCh on clotting and proteolytic activity. ( 0 ) Final concentration of CaClffi milk-clotting activity, (O) proteolytic activity,u H. Horinishi, Y. Hachimori, K. Kurihara, and K. Shibata, Biochim. Biophys. Acta 86, 447 (1964). ~J. Yu, G. Tamura, and K. Arima, will be published in Agr. Biol. Chem. (1970).
[30a]
THE ACID PROTEASE OF M. miehei
459
hydrolyzed by mucor rennin: Z-L-Glu-L-Tyr-0H, A-L-Glu-L-Phe-0H, Z-L-Phe-L-Tyr-OH, Z-L-Phe-L-Leu-OH, Z-L-Tyr-L-Leu-OH, L-Leu-GlyOH, L-Leu-Gly-Gly-0H, and Z-Gly-Pro-L-Leu-Gly-0H. Altogether, the specificity of mucor rennin appears to be similar to that of pepsin and calf rennin but somewhat narrower than that of Aspergillus saitoi and Streptomyces griseus proteases.
[ 3 0 a ] T h e A c i d P r o t e a s e of M u c o r rniehei By MARTIN OTTESEN and W. RICKERT A milk-clotting acid protease which resembles pepsin, rennin, and the protease from Mucor pusiUus, 1 has recently been isolated from a strain of Mucor miehei, CBS number 370.65. 2 The enzyme was obtained from commercially available culture concentrate TM by ammonium sulfate fractionation, batchwise adsorption on DEAE-Sephadex, SE-Sephadex, and DEAE-Sephadex chromatography and gel filtration. Although the enzyme has not as yet been crystallized, it appeared homogeneous by paper electrophoresis within the pH range 4.0-8.0, by free moving boundary electrophoresis in the pH range 5.2-3.2, and during ultracentrifugation within the pH range 5.0-8.0. The isoelectric point was approximately 4.2 in acetare buffer, and the sedimentation coefficient, s°0.w, 3.35 Svedberg units. Boundary spreading in the ultracentrifuge indicated a value for the diffusion coefficient, D O 20,W, of approximately 7.6 X 10-7 cm 2 see-~. From this value, and an estimate of 0.72 for the partial specific volume, a molecular weight of around 38,000 was indicated. Optical rotatory dispersion measurements indicated the Moffitt parameter bo to be close to zero in agreement with the find!ngs of other acid proteases. The amino acid composition expressed as moles per 100,000 grams of protein was as follows: Asp~lo-m Thr72_,3 Ser88-91 Glu81-62 Pro4~-~ Glys~-84 Ala6~-66 1/2Cys9-1o Val~3-~4 Mete5_16 Ileu~6~, Leu4~-5o Tyr48-49 Phe~2-54 TrypT-8 Lys~2-23 His4_5 Arg15-~6. Although no covalently bound phosphorus was detected in the enzyme preparation, the Mucor miehei protease did have a 6% carbohydrate content made up of hexosamine and neutral hexoses. Since neither heat denaturation nor exposure of the enzyme to 8 M urea diminished the carbohydrate content, it appeared to be covalently bound to the enzyme. The carbohydrate content, the sedimentation coefficient, and the amino i j. Yu, G. Tamura, and K. Ariraa, Biochim. Biophys. Acla 171, 138 (1969). 1~"Rennilase," Novo Industry A/S, Copenhagen, Denmark. 2M. Ottesen and W. Rickert, Compt. Rend. Tray. Lab. Carlsberg 37, 301 (1970).
[30a]
THE ACID PROTEASE OF M. miehei
459
hydrolyzed by mucor rennin: Z-L-Glu-L-Tyr-0H, A-L-Glu-L-Phe-0H, Z-L-Phe-L-Tyr-OH, Z-L-Phe-L-Leu-OH, Z-L-Tyr-L-Leu-OH, L-Leu-GlyOH, L-Leu-Gly-Gly-0H, and Z-Gly-Pro-L-Leu-Gly-0H. Altogether, the specificity of mucor rennin appears to be similar to that of pepsin and calf rennin but somewhat narrower than that of Aspergillus saitoi and Streptomyces griseus proteases.
[ 3 0 a ] T h e A c i d P r o t e a s e of M u c o r rniehei By MARTIN OTTESEN and W. RICKERT A milk-clotting acid protease which resembles pepsin, rennin, and the protease from Mucor pusiUus, 1 has recently been isolated from a strain of Mucor miehei, CBS number 370.65. 2 The enzyme was obtained from commercially available culture concentrate TM by ammonium sulfate fractionation, batchwise adsorption on DEAE-Sephadex, SE-Sephadex, and DEAE-Sephadex chromatography and gel filtration. Although the enzyme has not as yet been crystallized, it appeared homogeneous by paper electrophoresis within the pH range 4.0-8.0, by free moving boundary electrophoresis in the pH range 5.2-3.2, and during ultracentrifugation within the pH range 5.0-8.0. The isoelectric point was approximately 4.2 in acetare buffer, and the sedimentation coefficient, s°0.w, 3.35 Svedberg units. Boundary spreading in the ultracentrifuge indicated a value for the diffusion coefficient, D O 20,W, of approximately 7.6 X 10-7 cm 2 see-~. From this value, and an estimate of 0.72 for the partial specific volume, a molecular weight of around 38,000 was indicated. Optical rotatory dispersion measurements indicated the Moffitt parameter bo to be close to zero in agreement with the find!ngs of other acid proteases. The amino acid composition expressed as moles per 100,000 grams of protein was as follows: Asp~lo-m Thr72_,3 Ser88-91 Glu81-62 Pro4~-~ Glys~-84 Ala6~-66 1/2Cys9-1o Val~3-~4 Mete5_16 Ileu~6~, Leu4~-5o Tyr48-49 Phe~2-54 TrypT-8 Lys~2-23 His4_5 Arg15-~6. Although no covalently bound phosphorus was detected in the enzyme preparation, the Mucor miehei protease did have a 6% carbohydrate content made up of hexosamine and neutral hexoses. Since neither heat denaturation nor exposure of the enzyme to 8 M urea diminished the carbohydrate content, it appeared to be covalently bound to the enzyme. The carbohydrate content, the sedimentation coefficient, and the amino i j. Yu, G. Tamura, and K. Ariraa, Biochim. Biophys. Acla 171, 138 (1969). 1~"Rennilase," Novo Industry A/S, Copenhagen, Denmark. 2M. Ottesen and W. Rickert, Compt. Rend. Tray. Lab. Carlsberg 37, 301 (1970).
460
PROTEASES RELEASING COOH-TERMINAL AMINO ACIDS
[31]
acid composition distinguished the M u c o r miehei protease from the M u c o r pusillus protease described by Arima et al. 1 M u c o r miehei protease was remarkably stable. After 8 days of incubation at 38 ° between pH 3.0 and 6.0 more than 90% of the activity was retained, and in 8 M urea at pH 6 no detectable loss in activity occurred after 11 hours incubation. The pH optimum for the enzyme was close to 4, when the B-chain of oxidized insulin was used as substrafe. Splittings of the substrate were observed at the bonds Phe(1)Val (2), Leu (15)-Tyr (16), Tyr (16)-Leu (17), Phe (24)-Phe (25), and Phe (25)-Tyr(26). This indicated that among the bonds cleaved by both pepsin and rennin, only those bonds involving aromatic residues were hydrolyzed by the M u c o r miehei protease. 3 J W. Rickert, Campt. Rend. Tray. Lab. Carlsberg 38, 1 (1970).
[31] Bovine Procarboxypeptidase and Carboxypeptidase A
B y PmLIP H. P~TRA
Bovine Procarboxypeptidase A Bovine procarboxypeptidase A (PCP-A) occurs in the pancreas as a zymogen from which the active form can be generated by limited proteolysis.The existence of the zymogen in vivo was firstinferred by Anson I in 1935 and was, established 20 years later by Keller, Cohen, and Neurath. s,3 Some of the properties of this early preparation of the zymogen have already been reviewed.* In recent years, much work has been directed toward the elucidation of the activation processS-9;however, up to the present, the definition of the molecular events describing the generation of carboxypeptidase A activity is incomplete. The reasons for this difficultycan be stated as follows. First, bovine procarboxypeptidase A, unlike trypsinogen and chymotrypsinogen, exists in aggregate forms. 1M. L. Anson, Sc/ence 81, 467 (1935). 2p. j. Keller, E. Cohen, and H. Neurath, J. Biol. Chem. 223, 457 (1956). IP. J. Keller, E. Cohen, and It. Neurath, J. Biol. Chem. 230, 905 (1958).
4H. Neurath, Vol. II, p. 77. 5H. Neurath, Proc. Intern. Corr. Biochem. 7th, Tokyo, 1967. ' H. Neurath, R. A. Bradshaw, P. H. P~tra, and K. A. Walsh, Phil. Trans. Proc. Roy. Soe. (London) B 257, 159-176 (1970). ~J. H. Freisheim, K. A. Walsh, and H. Neurath, Biochemistry 6, 3020 (1967). 8j. H. Freisheim, K. A. Walsh, and H. Neurath, Biochemistry 6, 3010 (1967). 'P. Piras and B. L. VaJlee, Biochemistry 6, 348 (1967).
460
PROTEASES RELEASING COOH-TERMINAL AMINO ACIDS
[31]
acid composition distinguished the M u c o r miehei protease from the M u c o r pusillus protease described by Arima et al. 1 M u c o r miehei protease was remarkably stable. After 8 days of incubation at 38 ° between pH 3.0 and 6.0 more than 90% of the activity was retained, and in 8 M urea at pH 6 no detectable loss in activity occurred after 11 hours incubation. The pH optimum for the enzyme was close to 4, when the B-chain of oxidized insulin was used as substrafe. Splittings of the substrate were observed at the bonds Phe(1)Val (2), Leu (15)-Tyr (16), Tyr (16)-Leu (17), Phe (24)-Phe (25), and Phe (25)-Tyr(26). This indicated that among the bonds cleaved by both pepsin and rennin, only those bonds involving aromatic residues were hydrolyzed by the M u c o r miehei protease. 3 J W. Rickert, Campt. Rend. Tray. Lab. Carlsberg 38, 1 (1970).
[31] Bovine Procarboxypeptidase and Carboxypeptidase A
B y PmLIP H. P~TRA
Bovine Procarboxypeptidase A Bovine procarboxypeptidase A (PCP-A) occurs in the pancreas as a zymogen from which the active form can be generated by limited proteolysis.The existence of the zymogen in vivo was firstinferred by Anson I in 1935 and was, established 20 years later by Keller, Cohen, and Neurath. s,3 Some of the properties of this early preparation of the zymogen have already been reviewed.* In recent years, much work has been directed toward the elucidation of the activation processS-9;however, up to the present, the definition of the molecular events describing the generation of carboxypeptidase A activity is incomplete. The reasons for this difficultycan be stated as follows. First, bovine procarboxypeptidase A, unlike trypsinogen and chymotrypsinogen, exists in aggregate forms. 1M. L. Anson, Sc/ence 81, 467 (1935). 2p. j. Keller, E. Cohen, and H. Neurath, J. Biol. Chem. 223, 457 (1956). IP. J. Keller, E. Cohen, and It. Neurath, J. Biol. Chem. 230, 905 (1958).
4H. Neurath, Vol. II, p. 77. 5H. Neurath, Proc. Intern. Corr. Biochem. 7th, Tokyo, 1967. ' H. Neurath, R. A. Bradshaw, P. H. P~tra, and K. A. Walsh, Phil. Trans. Proc. Roy. Soe. (London) B 257, 159-176 (1970). ~J. H. Freisheim, K. A. Walsh, and H. Neurath, Biochemistry 6, 3020 (1967). 8j. H. Freisheim, K. A. Walsh, and H. Neurath, Biochemistry 6, 3010 (1967). 'P. Piras and B. L. VaJlee, Biochemistry 6, 348 (1967).
[31]
PROCARBOXYPEPTIDASE AND CARBOXYPEPTIDASE
461
One of these is an aggregate of two subunits (PCPA-S5) 1° and the other of three subunits (PCPA-S6).11,~2 Subunit I is common to both forms of the zymogen and is the immediate precursor of the enzyme. So far it has not been possible to isolate subunit I in its native state. Second, the peptide fragment released upon activation contains approximately 60 amino acids as compared to the dipeptide or hexapeptide released during the activation of chymotrypsinogen or trypsinogen. And third, at least three forms of the active enzyme, differing in the structure of their amino terminal region, can be generated from the zymogen. The relative proportion of these three forms depends upon the method of activation. Brown et al. 12 have proposed a mechanism of activation shown in Fig. 1. The process can be divided into two steps involving the tryptic activation of the endopeptidase, subunit II, which then acts in concert with trypsin to convert subunit I into carboxypeptidase. Subunit III of PCPA-S6 has not yet been fully characterized, but preliminary chemical evidence suggests that it may be an inactive form of subunit II. Purification of Procarboxypeptidasc S t e p 1. Extraction. ~2,~3 One hundred grams of pancreatic acetone
(
~
Trypsin 1:100 0 =, ! hr
pH6.5 ~Tr ypsin1:5 37° 4 hrs "~kpH 6.5
PCPA-S6
~
+ Activotion
products
~37Torypsin CP-A1:5
Trypsin 1:100 0 °, I hr
~
pH
4 hrs 6.5
pH 65
PCPA-S5 FIG. 1. Schematic diagram describing the mechanism of activation of procarboxypeptidase A-S5 and A-S6, adapted from J. It. Brown et al? 2 ~oj. R. Brown, M. Yamasaki, and H. Neurath, Biochemistry 2, 877 (1963). ~J. R. Brown, D. J. Cox, R. N. Greenshields, K. A. Walsh, M. Yamasaki, and H. Neurath, Proc. Natl. Acad. Sci. U.S. 47, 1554 (1961).
462
PRO~AS~S
[31]
RELEASING COOH-TERMINAL AMINO ACIDS
powder1~ (which can be prepared according to published methods2,15) is suspended in 1 liter of cold distilled water containing 10-3 M diisopropylphosphofluoridate (DFP). (One gram of powder is equivalent to approximately 5 g of ground pancreas.) After extraction for 4--12 hours in the cold room using gentle stirring, the suspension is centrifuged for 20 minutes in the Spinco Model L centrifuge (No. 21 rotor) at 20,000 rpm. The residue is discarded and the supernatant directly chromatographed on DEAE-eellulose. Step ~. Chromatography of Crude Extract. ~3,~5 DEAE-cellulose (Selectacel-DEAE standard type no. 70 purchased from Schleicher and Schuell Co.) is washed with 0.25M NaC1, 0.5M NaOH, followed by 40-
a.
0. a_
:300 GO o4
~
20-
-0.2
I0-
-0.1
o
( 10-1~ seconds, ST. Jovin, A. Chrambach, and M. A. Naughton, Anal. Biochem. 9, 351 (1964); B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964).
550
P R O T E A S E SRELEASING NH2--TERMINAL AMINO ACIDS
[37a]
D 20,w O = 5.0 ± 0.25 X 10-~ em ~ see-t, Ms, n = 36,500 + 4000). On the basis of the amino acid analysis (protein concentration) and the molecular weight, the extinction coefficient was found to be 10.2 and the molar extinction coefficient E28o = 4.1 X 1@ (.05 M Tris-HC1 buffer, pH 7.2, 0.001 M CoC12). Stability. The purified, native AP I is stable in solution (0.05 M TrisHC1 buffer, pH 7.2, 0.001 M CoC12) for several months if kept in a refrigerator. The enzyme is stable for 15 hours at 80 °. The activity increases by 20 to 30% within 30 minutes (increase in Vm~x, Km is constant). In cobalt-free buffer the thermostability of AP I is markedly reduced (1 hour, 80°: 35% residual activity). The apoenzyme is also very thermolabile. In 8 M urea (at pH 7.2) AP I is not denatured (in gel eleetrophoresis the activity is only slightly, if at all, reduced in the hydrolysis of peptides). After treatment with 0.3% sodium dodecylsulfate, the activity is reduced by 8%. Enzyme Structure. See Table II for the amino acid composition of B. stearothermophilus. AP I is a metal enzyme (complex), Co s÷ ions conferring the highest activity upon activation ~see Stability) and upon recombination from apoenzyme with Co s÷ ions. The Co s÷ ions are strongly bound at pH 8.1; the metal enzyme complex is fairly stable up to an EDTA concentration of 0.01 M (only a slight decrease in activity oc2 curs). At a pH of less than 6.0 the metal enzyme is converted into a stable, inactive apoenzyme following dialysis against 0.01 M EDTA. Specificity. The specificity of AP I is similar to that of other known aminopepfidases (broad substrate specificity). It releases not only neutral but also acid and basic amino acids, including proline, from the amino end. In contrast to leucine aminopeptidase 2 from pig kidneys or eye lenses, it splits leucine-p-nitroanilide relatively more slowly than the peptides [specific activity: (a) leucine-p-nitroanilide hydrolysis, 6.6 units rag-l; (b) hydrolysis of Leu-Gly, 400 units mg-1; hydrolysis of GlyLeu-Tyr (splitting of Gly), 900 units rag-l]. Besides rapidly hydrolyzing aliphatic amino acid residues from the amino end, it also quickly splits off aromatic amino acid residues. Kinetic Properties. The pH range of maximum activity for the substrate Gly-Leu-Tyr is between 9.2 and 9.4 and for the substrate leucinep-nitroanilide between 7.5 and 8.0. The optimum temperature for the hydrolysis of Leu-Gly (pH 7.2) is 90 ° (10 minutes) (activation energy 16,300 cal mole-l). At pH 7.2 the Km was found to be 95 mM for Leu-Gly and 8 mM for leucine-p-nitroanilide (65°). Assuming that AP I has a Dp. Moser, G. Roncari, and H. Zuber, Intern. J. Protein Res., in press (lgT0). E. J. Cohn and J. T. Edsall, "Proteins, Amino Acids and Peptides," p. 375. Reinhold, New York, 1943.
[37a]
THERMOPHILIC
AMINOPEPTIDASES
4~
~
O h~ O
O Z
( 95 cm column of the Recychrome type? For large-scale operations, a large column (7.5 X 115 cm) is used which gives an identical separation pattern as the smaller one. The proteinase extract (10 ml) is applied to the column by means of a peristaltic pump. Elution is carried out by pumping the buffer upward through the column at a flow rate of 20 ml/hour. Fractions of 6 ml are collected and the distribution of material analyzed spectrophotometrically at 280 nm. Activity for each fraction is also determined. Four distinct peaks appear and activity is localized solely to peak III which is eluted after about 450 ml of buffer has passed through the s The tannin precipitated culture fluid from P. notatum is provided as a dry powder by Astra AB, SSdertAlje, Sweden. 4j. Porath, Biochim. Biophys. Acta 39, 193 (1960). sj. Porath and H. Bennich, Arch. Biochem. Biophys. Suppl. 1, 152 (1962).
578
SOME BACTERIAL AND MOLD PROTEASES
[40]
column. The activity curve does not coincide exactly with the protein peak, indicating heterogeneity. The active fractions are pooled (about 130 ml) and precipitated with (NH~)2S04 (80% saturation). After 4 hours or overnight, the precipitate is centrifuged (as in step 1). The sediment is dissolved in a small amount of buffer (or distilled water) and kept at ~ 4 ° or deep-frozen at --15 ° until used. Step 3. Ion-Exchange Chromatography. The ion exchanger is prepared as follows: About 30 g of SE-Sephadex C-50 is allowed to swell in a large excess of distilled water overnight and fine particles removed by decantation. The swollen gel is transferred to a Biichner funnel and 2 liters of 0.5 M HC1 passed quickly through the gel, followed by washing with about 2 liters of distilled water. It was then washed with 2 liters of 0.5 M NaOH, and again washed with about 2 liters of distilled water. Finally, the gel is continuously washed with about 10 to 15 liters of 0.1 M sodium acetate buffer, pH 4.5, with continuous stirring until the pH and conductivity of the washings and equilibrating buffer match exactly. The equilibrated ion exchanger is then packed into a column (3.5 X 30 cm) of the type described above. A larger column (7 X 30 cm) can be used for large-scale preparations since the separation pattern obtained is comparable to that obtained using the smaller column. I f necessary, additional buffer is pumped through the column until the pH and conductivity of the emuent buffer matches exactly with that used for elution. The active material obtained from step 2 is desalted on a column of Sephadex G-25 equilibrated with 0.1 M sodium acetate buffer, pH 4.5. Of the desalted sample, 20 ml is applied to the ion-exchange column. Since the gel shrinks with increase in the ionic strength of the eluant buffer, elution is done by pumping the buffer downward at a flow rate of 18 ml/hour. Fractions of about 6 ml are collected and after passage of 400 ml of buffer, the adsorbed material is displaced by using 500 ml of 0.3 M sodium acetate buffer, pH 4.5. The distribution of material and activity in the fractions is determined. The active peak appears after passage of a total of about 500 ml of buffer. At this stage, the protein and activity curves correspond well. The fractions containing the proteinase are pooled (about 30 ml) and precipitated with (NH4)~S04 up to 80% saturation. The sediment obtained after centrifugation, as in step 1, is dissolved in a small amount of distilled water. The solution is desalted on a 1 X 50 cm column of Sephadex G-25 equilibrated with 0.025 M Tris-acetate buffer, pH 8.0. The fractions containing the active material are pooled and concentrated to a volume of about 3 ml in a collodium bag subjected to mild pressure on the outside. Care must be taken that the pressure is not very low since the activity will diminish considerably.
[40]
EXTRACELLULARPROTEINASE FROM P. notatum
579
Step 4. Column Zone Electrophoresis. Cellulose powder* was used as a support and anticonvection media. It was packed in a glass column (2 X 50 cm) and in a manner similar to that described elsewhereY The buffer used is 0.05 M Tris-acetate, pH 8.0. The homogeneity of the packed bed of cellulose is tested by passing a solution of a marker dye, dinitrophenyl-ethanolamine, through the column. The buffer above the bed is removed. The sample (3 ml), which is equilibrated with 0.025 M Tris-acetate buffer, pH 8.0, so as to get a narrow starting zone,8 is carefully layered on top of the bed. Electrophoresis is carried out for about 60 hours (anode direction downward) with an applied voltage of 800 V and with a current of 20 to 22 mA. The temperature of the cooling water circulating through the external jacket is ~-8 °. After eleetrophoresis, the fraetionated material is eluted with fresh buffer at a flow rate of 10 ml/hour and 2.5 ml fractions are collected. For each fraction the distribution of material and enzyme activity are determined. The enzyme activity is located in the major peak eluted between fractions 37-48. At this stage, the protein and activity peaks coincide exactly. The other peaks eluted before the proteinase peak are inactive and are comparatively low in concentration. A summary of the purification procedure is outlined in the table. Properties
Stability. The enzyme is stable in the pH range of 4 to 10, but is inactivated below pH 3. It is not markedly affected by solutions of 7 M urea or 5 M guanidium chloride in the pH range of 5 to 8. The enzyme solution slowly loses its activity on storage at ~ 4 °, but at pH values around 5 it is stable for at least 2 weeks in the presence of (NH4)2S04. Lyophilization causes a marked decrease, or complete loss in its activity. It can be kept at room temperature for 48 hours without any detectable loss in activity. Purity and Physical Properties. The purified enzyme did not contain protein impurities as tested by the following criteria of purity: 1. It showed only one band after electrophoresis in analytical polyacrylamide gels 9 and on agarose slides 1° at pH 4.5 and 8.0. The same result is obtained after electrophoresis in starch gels using 7 M urea 1~ Obtained from Grycksbo Pappersbruk AB, Grycksbo, Sweden. J. Porath and S. Hjert4n, in "Methods of Biochemical Analysis" (D. Glick, ed.), Vol. IX, p. 194. Wiley (Interscience), New York, 1962. 8H. Haglund and A. Tiselius, Acta Chem. Scand. 4, 957 (1950). 98. tIjert~n, S. Jerstedt, and A. Tiselius, Anal. Biochem. 11, 219 (1965). 1,S. Hjert~n, Biochim. Biophys. Acta 53, 514 (1961). 11G. M. Edelman and M. D. Poulik, J. Exptl. Med. 113, 861 (1961).
580
SOME BACTERIAL
AND MOLD PROTEASES
aO
o0
°~ v
C~ v
~
[40]
[41]
ALKALINE PROTEINASES FROM Aspergillus
581
and at pH 8.0, but at pH 3.5 and under the same conditions, four bands are obtained. 2. It was monodisperse in the ultracentrifuge. Crystals are obtained when the enzyme is concentrated at pH 4.5 and at -}-4°. 3. It showed only one peak when run in an isoelectric focusing apparatus. TM The enzyme has a sedimentation constant of 1.6 and a molecular weight close to 20,000 which is in approximate agreement with that calculated from its amino acid composition. It is acidic with an isoelectric point of 4.92 as determined by the isoelectric focusing apparatus. The enzyme contains no carbohydrate. pH Optimum. Using denatured hemoglobin as substrate, the pH optimum lies in the range of pH 7.5 to 9.5. Activators and Inhibitors. Trypsin inhibitors from egg white and soy bean had no inhibitory effect. Other chemicals such as EDTA, Ca ~+, glutathione, or hydroxylamine did not activate or inhibit it. However, a 1 0 M excess of diisoprophylphosphofluoridate (DFP) irreversibly inhibits the enzyme. Specificity. The exact bond specificity of the enzyme is not known. However, it attacks different kinds of protein substrates such as denatured hemoglobin, albumin, and conjugated proteins such as ceruloplasmin and glycoproteins. It also attacks synthetic substrates, e.g., benzoylarginine ethyl ester. It hydrolyzes the oxidized B-chain of insulin to about 7 components not yet identified. Such results indicate that the enzyme is probably not specific in its action. A similar conclusion has been reached by Marshall et al. 18 when investigating the properties of a proteinase from a different strain of the same organism. O. Vosterberg and It. Svensson, Acta Chem. Scand. 4, 957 (1960). W. E. Marshall, R. Manion, and J. Porath, Biochim. Biophys. Acta 151, 414 (1968).
[41]
Alkaline Proteinases from Aspergillus B y YASUSHI NAKA~AWA
It has been reported that Aspergillus oryzae produces three types of proteolytic enzymes with acid, neutral, and alkaline pH-activity optima, respectively) Ichishima describes a procedure for the isolation of the acid proteinase from A. saitoi.~ The following section deals with the isolation and purification of alkaline proteinase. Recently, reports have been published on the isolation, purifica-
[41]
ALKALINE PROTEINASES FROM Aspergillus
581
and at pH 8.0, but at pH 3.5 and under the same conditions, four bands are obtained. 2. It was monodisperse in the ultracentrifuge. Crystals are obtained when the enzyme is concentrated at pH 4.5 and at -}-4°. 3. It showed only one peak when run in an isoelectric focusing apparatus. TM The enzyme has a sedimentation constant of 1.6 and a molecular weight close to 20,000 which is in approximate agreement with that calculated from its amino acid composition. It is acidic with an isoelectric point of 4.92 as determined by the isoelectric focusing apparatus. The enzyme contains no carbohydrate. pH Optimum. Using denatured hemoglobin as substrate, the pH optimum lies in the range of pH 7.5 to 9.5. Activators and Inhibitors. Trypsin inhibitors from egg white and soy bean had no inhibitory effect. Other chemicals such as EDTA, Ca ~+, glutathione, or hydroxylamine did not activate or inhibit it. However, a 1 0 M excess of diisoprophylphosphofluoridate (DFP) irreversibly inhibits the enzyme. Specificity. The exact bond specificity of the enzyme is not known. However, it attacks different kinds of protein substrates such as denatured hemoglobin, albumin, and conjugated proteins such as ceruloplasmin and glycoproteins. It also attacks synthetic substrates, e.g., benzoylarginine ethyl ester. It hydrolyzes the oxidized B-chain of insulin to about 7 components not yet identified. Such results indicate that the enzyme is probably not specific in its action. A similar conclusion has been reached by Marshall et al. 18 when investigating the properties of a proteinase from a different strain of the same organism. O. Vosterberg and It. Svensson, Acta Chem. Scand. 4, 957 (1960). W. E. Marshall, R. Manion, and J. Porath, Biochim. Biophys. Acta 151, 414 (1968).
[41]
Alkaline Proteinases from Aspergillus B y YASUSHI NAKA~AWA
It has been reported that Aspergillus oryzae produces three types of proteolytic enzymes with acid, neutral, and alkaline pH-activity optima, respectively) Ichishima describes a procedure for the isolation of the acid proteinase from A. saitoi.~ The following section deals with the isolation and purification of alkaline proteinase. Recently, reports have been published on the isolation, purifica-
582
SOME BACTERIAL AND MOLD PROTEASES
[41]
tion, and characterization of alkaline proteinases from A. flavus/ A. oryzae/-e A. sojae/ and A. sydowi. S These isolation and purification methods are similar to each other and two are shown here as examples. These enzymes have p H optima in the alkaline r a n g e / b u t there are some differences in the inhibitory effects of metal ions, substrates specificity, and amino acid compositions. Further studies will be required to identify these alkaline proteinases.
Assay Method Principle. The proteolytic activity is determined by measuring the increase in optical density at 280 nm of a trichloroacetic acid filtrate based on liberation of tyrosine and t r y p t o p h a n by the enzymatic hydrolysis of hemoglobin or casein. 1° In an alternate procedure, the trichloroacetic acid filtrate is allowed to react with the phenol reagent (Folin-Ciocalteau reagent) and the color developed is measured at 660 or 750 nm. Reagents Hemoglobin solution: Prepared by a modification of the method by Anson. 11 "Hemoglobin substrate powder" (Worthington Biochemical Corp., Freehold, N . J . ) ; 2 g is dissolved in 100 ml of 0.1 M sodium phosphate buffer, p H 7.5, and filtered through glass wool. Casein solution: 0.5% solution of casein (Hammarsten) 12 1B. Hagihara, in "The Enzymes" (Boyer, Lardy, and Myrb~ck, eds.) Vol. 4, p. 193. Academic Press, New York, 1960. s E. Ichishima, this volume, [26]. aj. TurkovA, O. Mike~, K. Gan~ev, and B. Boublik, Biochim. Biophys. Aeta 178, 100 (1969). "R. Bergkvist, Acta Chem. 8cand. 17, 1521 (196~). ~A. R. Subrama~ian and G. Kalnitaky, Biochemistry 3, 1861 (1964). *A. Nordwig and W. F. Jahn, European J. Biochem. 3, 519 (1968). 'K. Haya~hi, D. Fukushima, and K. Mogi, Agr. Biol. Chem. (Tokyo) 31, 1237 (1967). a G. Danno and S. Yoshimura, Agr. Biol. Chem. (Tokyo) 31, 1151 (1967). °A pH-activity optimum occasionally changes depending on a substrate and a medium used (i.e., hemoglobin, synthetic peptides, or a type of buffer solution) or during the process of purification,e 1' The use of casein as substrate is limited since the isoelectric point of casein is pH 4.6. u M. L. Anson, J. Gen. Physiol. 22, 79 (1938) ; J. H. Northrop, M. Kunitz, and R. M. Herriott, "Crystalline Enzymes," p. 303. Columbia University Press, New York, 1946. ~M. Laskowsky, Vol. II [2], [3].
[41]
ALKALINE PROTEINASES FROM
Aspergillus
583
Trichloroacetic acid solution: 5% (w/v) aqueous solution Buffer solution: 0.1 M sodium phosphate solution, p H 7.518 Procedure. A 2-ml aliquot of hemoglobin solution is added to a test tube (14 X 2 cm) and equilibrated in a 37 ° bath for 5 minutes. To the hemoglobin solution is added 0.5 ml of a properly diluted enzyme solution and the mixture is incubated at 37 ° for 10 minutes. At the end of the incubation period the reaction is terminated by addition of 4 ml of 5% trichloroacetic acid. After standing for 5 minutes the suspension is filtered through thick filter paper [e.g., W h a t m a n No. 3 or S & S (Schleicher & Schuell) No. 576] or is centrifuged. The optical density at 280 nm of the clear solution is measured in a silica cuvette with 1-cm pathlength. A zero time incubation b l a n k is prepared by reversing the sequence of the addition of the enzyme solution and the 5% trichloroacetic acid. F o r the blank solution 0.5 ml of water is substituted for the enzyme solution. In the second procedure an aliquot of trichloroacetic acid filtrate is allowed to react with the phenol reagent 14 and the color developed is measured at 660 or 750 nm. 15 In this case the absorption must not exceed 0.8-0.92 An 0.1 unit absorption is-equivalent to 0.035 ~eq of tyrosine/minute2 The protein concentration is determined by the micro-Kjeldahl method or by absorption at 280 nm. Definition o] a Unit. The hemoglobin activity is conventionally expressed as a one-unit increase in the optical density at 280 n m / m g of enzyme per 10 minutes of incubation. This value can be converted to express the results in micromoles of tyrosine e, 1~ or leucine. TM
Purification Procedure P r o d u c t i o n of E n z y m e s 17
An aqueous solution containing 1-1.5% soybean meal or peanut meal, 2-4% carbohydrate, and magnesium and phosphorus salts is adjusted 13G. Gomori, Vol. I, [16]. 14The phenol reagent is commercially available from Fisher Scientific Co. Several modified procedures of this method were reported. 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); W. Rick, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), p. 808. Academic Press, New York, 1963; C. E. McDonald and L. L. Chen, Anal. Biochem. 10, 175 (1965). leT. G. Rajagopalan, S. Moore, and W. H. Stein, J. Biol. Chem. 241, 4940 (1966). ~ "Fungal protease" prepared from A. oryzae is commercially available from Miles Chemical Co., Inc., Clifton, N.J.~
584
[41]
SOME BACTERIAL AND MOLD PROTEASES
to pH 6.5 to 7.0,4 or the following synthetic medium is also usedS,18: glucose, 3%; milk casein, 0.5%; yeast extract, 0.1%; NH4N08, 0.2~; K2HP04, 0.1%; KH2P04, 0.05~; MgS04"7 H20, 0.05%; FeS04"7 H20, 1 rag/100 ml; and ZnS04.7 H20, 1 rag/100 ml. One hundred milliliters of this solution is placed in a 500-ml flask and 1 g of sterilized CaC08 is added before inoculation. After sterilization and cooling, the medium is inoculated with spores of A. oryzae (or other species) and incubated by shaking at 35 ° for 4 days. At the end of the incubation, the broth is filtered through four layers of gauze, followed by filtration through a 1-inch layer of Celite. The filtrate is used for the isolation of alkaline proteinase. Isolation o/ the Enzyme M E T H O D OF BERGKVIST 4
The flow sheet shows the isolation steps. A Separation Method by Bergkvtet 4 Step 1
Dissolve 500 g of tannin-precipitated protease mixture ~g in 250 liters of water and adjust the pH to 5.5 (protein concentration 2 g/liter)
Step 2
Add 500 g of CM-cellulose previously equilibrated to pH 5.5 with
t
0.01 M sodium phosphate buffer Stir 60 minutes at 20 ° Filter
t
I contains p r o t e a ~
Step 3
CM-Cellulo~:
Step 4
Wash with 20 liters of 0.01 M sodium phosphate buffer, pH 5.5 Filter [
Step 5
CM-Cellulose: contains protease I
Step 6
Elute with 3 liters of 0.05 M sodium phosphate buffer, pH 7.0 Filter [
f
I Step 7
I
CM-CeUulose: (to be regenerated)
t Elute 1: contains protease I
I
Filtrate 1: Contains protease II and m together with impurities
I
Filtrate 2: Contains impurities r e tained on CM-cellulose
I
I
The combined filtrates are adjusted to pH 4.5 i I
The procedure is repeated from Step 2 at pH 4.5 for the isolation of p r o t e g e H I I
The procedure is also repeated from Step 2 at pH 3.0 for the isolation of protease HI ~ A s l i g h t l y different s y n t h e t i c m e d i u m is u s e d b y W . G. C r e w t h e r a n d F . G, L e n n o x , Australian J. Biol. 8ci, 6, 410 (1953).
[41]
ALKALZN~.PROTEINASES FROM AspergiUus
585
Finally each enzyme fraction is purified on a DEAE-cellulose column (3 X 30 cm). Protease I is eluted from the column with an 0.01 M sodium phosphate buffer, pH 6.0. Protease II is isolated from the impure proteins by washing the column with 0.01 M sodium phosphate, pH 6.0, and then 0.10M sodium phosphate buffer, pH 6.0. Protease III is purified by washing the column with 0.01 M and 0.1 M sodium phosphate buffers, pH 6.0. It is eluted with 0.50 M phosphate buffers, pH 6.0. METHODS OF SUBRAMANIAN AND KALNITSKY5
Step 1. Five hundred grams of "fungal protease ''17 is dissolved in 2 liters of ice-cold water with gentle stirring. Centrifuge the solution and wash the precipitate twice with 200 mt of cold water. The combined supernatant and washings are dialyzed against cold water for 24 hours. Step ~. Three hundred grams of anion-exchange cellulose (ECTEOLA, 0.48 meq/g, Bio-Rad Laboratories, Richmond, Calif.) is added and stirred for 20-30 minutes, and is subsequently filtered through a Biichner funnel with slow suction to prevent foaming. The cellulose is washed twice with 200 ml of water. To remove most of the pigments this step is repeated using 200 g of ECTEOLA-cellulose (ECTEOLA-cellulose:protein -~ 3:1 was found to be the best). Step 3. The volume of the filtrate is measured and ammonium sulfate is added to reach 75% saturation. 2° (It is not necessary to adjust the pH of the solution.) After addition of ammonium sulfate, the solution is kept at 0 ° for 6-10 hours, and is subsequently centrifuged. Ammonium sulfate is added to the supernatant to bring the ammonium sulfate concentration to 85% saturation. The suspension is centrifuged and the precipitated protein is dissolved in distilled water. The enzyme solution is then dialyzed against water until no sulfate can be detected and is lyophilized. Step ~. The enzyme is dissolved in 0.01 M sodium phosphate, pH 6.5 (protein concentration is adjusted to 1.5 g in 25 ml). The solution is chromatographed on an Amberlite CG-50 column (40 X 6.5 cm). The enzyme is eluted in a cold room with the same buffer at a flow rate of 180 ml/hour. The elution pattern is shown in Fig. 1. 1°The enzymes are precipitated from the broth by the slow addition of 10% tannin solution to make the final concentration of 3.0 g of tannin per 100 ml of the filtrate at pH 5~5. After 2 hours the precipitated protein is recovered by centrifugation. The t-q.nnluis removed by washing several times with acetone, and repeating the centrifugation. The precipitate is dried in vacuum. This step retains 80-100% of the original proteinase in the culture filtrate.4 A. A. Green and W. L. Hughes, ¥ol. I [10]. The required amount of ammonium sulfate is also estimated by the formula given by E. A. Noltmann, C. G. Gubler, and S. A. Kuby, J. Biol. Chem. 236, 1225 (1961).
586
[41]
SOME BACTERIAL AND MOLD PROTEASES
4.0 30 2.0 ---
Prolein Activity
7140
1.8
I
I I
A
1.6
",20 I
0
I I
0O
1 ,,
14
-~ I00
o
1.2
D
I I
\
1.0
!
c
'6
- ~ 6 0 ~,
0.8 06
--440 I I I
04
1
n 20 0.2
i t
1 I
I
400
800
1200
1600
2000
Effluent volume, ml
Fro. 1. Chromatography of aspergillopeptidase B on Amberlite CG-50, pH 6~5. A 40 X 6.5 cm preparative column was prepared in 0.01 M sodium phosphate buffer, pH 6~5. The enzyme was eluted with the same buffer at the rate of 180 ml/hour in the cold room. Reprinted from Biochemistry 3, 1861 (1964). Copyright (1961) by the American Chemical Society. Reprinted by permission of the copyright owner.
Step 5. T h e Amberlite C G - 5 0 column c h r o m a t o g r a p h y (30 X 0.8 cm) is repeated with 0.01 M sodium phosphate, p H 7.5 at a flow rate of 6.6 ml/hour. The c h r o m a t o g r a m is shown in Fig. 2. T h e enzyme eluant is dialyzed against cold water for 24-36 hours and is lyophilized. See T a b l e I for a s u m m a r y of this purification. Properties
Stability. T h e alkaline proteinase (protein concentration of 0.002~) of A. sydowi is completely inactivated after incubation in 0.01 M sodium phosphate, p H 7.5, at 55 ° for 10 m i n u t e s : The enzyme is most stable at p H 7.0, 35 °. Alkaline proteinase from A. sojae has a p H activity o p t i m u m at 9-10. 21 I1K. Hayashi, D. Fukushima, and K. Mogl, Agr. Biol. Chem. (Tokyo) 31, 642 (1967).
[41]
ALKALINE PROTEINASES FROM
Aspergillus
587 F
44
150
"lO0
I I I
I I I I I I
1.0
80
Protein
------ Activity
I
0.8
60
E
in
E
O CO Cxl
5
>:
06
~ 0.4
ill V
~
0.2
20
0
-~'=;'5
I0
15
20
"-
~'0
25
Effluent volume, ml
FIG. 2. Chromatography of aspergillopeptidase B on Amberlite CG-50, pH 7~5. A 30 X 0~ em column was prepared in 0.01 M sodium phosphate buffer, pH 7,5. Proteinase was eluted with the same buffer at the rate of 6.6 ml/hour. Activity recovered, 96%; protein recovered, 96%. Reprinted from Biochemistry 3, 1861 (1964). Copyright (1961) by the American Chemical Society. Reprinted by permission of the copyright owner. TABLE I PURIFICATION OF ASPERGILLOPEPTTDASE 8 6
None Dialysis Anion celhflose AmmSO4, 75-85% Amberlite 0.01 M phosphate, pH 6.5 Step 5. Amberlite 0.01 M phosphate, pH 7.5
Step Step Step Step
1. 2. 3. 4.
Weight (g)
Total activity (units X 10~)
Purity (units/ OD~ nm)
Yield (%)
Purification
500 150 55 6.5 1.1
203 199 171 75 30
5.6 8.0 39 106 295
100 98 84 37 15
1 1.5 7.0 19 53
0.9
28
310
14
55
588
SOME BACTERIAL
A N D MOLD P R O T E A S E S
[41]
Bergkvist reported that Protease I is stable between pH 5 and pH 8.5, Protease II has a broader stability range from pH 4.5 to pH 10.5, and Protease I I I is stable between pH 3 and pH 6.22 Three enzymes are completely inactivated in less than 2 minutes at 60 °, but lose their activities slowly at 40 ° . Purity and Physical Properties. The purity of alkaline proteinase (aspergillopeptidase B) prepared by the method of Subramanian and Kalnitsky was examined by moving-boundary and paper electrophoresis, sedimentation velocity ultracentrifugation, and chromatography. These methods showed the preparation was homogeneous2 These techniques were also used to demonstrate the purity of alkaline proteinase from A. sojaeY Polyaerylamide gel electrophoresis of the enzyme from A. sydowi showed a single band at pH 6.0, 7.6, and 8.6, respectively,s The homogeneity of the alkaline proteinase from A. flavus was confirmed by disc electrophoresis in Tris-glycine buffer and by immunoelectrophoresis.3 The molecular weights reported for alkaline proteinases are as follows: Alkaline proteinase from A. flavus 8 Aspergillopeptidase B (A. oryzae) 28 Aspergillopeptidase C (A. oryzae) 5 Alkaline proteinase from A. sojae ~
18,000 18,000 19,650 25,500
The optical rotatory dispersion of alkaline proteinase from A. sojae was measured at pH 7.0~4: [a]~ )~.
= -- 16.5° = 256 nm ao = - - 78 bo -- -- 74 [M']~oo-- 11,700 [M']~8 = --2,400 From the bo value the a-helix content was estimated as 12%. The apparent isoelectric pH of this enzyme is 5.1.24 Both alkaline proteinases from A. flavus 8 and A. oryzae (aspergillopeptidase B) 2s had an ~1% .a~:lem 9.04 and 9.0 at 280 nm and pH 5.0, respectively. R. Bergkvist, Acta Chem. ~eand. 17, 1541 (1963). ~SA. R. Subramanian and G. Kalnitsky, Biochemistry 3, 1868 (1964). K. Hayashi, D. Fukushima, and K. Mogi, Agr. Biol. Chem. (Tokyo) 31, 1171 (1967).
[41]
ALKALINE PROTEINASES FROM Aspergillus
589
TABLE II AMINO ACID COMPOSITIONS OF ALKALINE PROTEINASES
Amino acid
Aspergillopeptidase B
Aspergillopeptidase C
Alkaline proteinase
Alkaline proteinase
(A. oryzae)2*
(A. oryzae)6
(A. sojae)~
(A. flavus) s
12 4 3 21-22 13 23 13 5-6 21 23 0 16 1 10-11 10 4-5 6 2 -187-191 ----
14 5 3 31 18 28 19 6 27 32 2 18 2 14 14 8 7 2 20 250 --No sugar
11 3-4 2 21 11 20 12-13 4-5 20 23 0 15 1 9-10 9 5 5 2 17 173-177 Glycine Alanine 1 mole as ribose per mole of protein
Lysine 11-12 Histidine 4 Arginine 2 Aspartic acid 21 Threonine 11 Serine 19 Glutamic acid 12 Proline 4 Glycine 19 Alanine 23 Half-cystine 0 Valine 15 Methionine 0 Isoleucine 9-10 Leucine 9 Tyrosine 5 Phenylalanine 5 Tryptophan 2 Amide-NH, 15 Total 171-173 N-terminal Glycine C-terminal Alanine Carbohydrate 1-2% as mannose
Chemical Properties. T h e a m i n o acid compositions, N a n d C t e r m i n i , a n d c a r b o h y d r a t e c o m p o n e n t s are s u m m a r i z e d i n T a b l e I I . T h e active site sequence of a l k a l i n e p r o t e i n a s e has been s t u d i e d u s i n g s 2 P - d i i s o p r o p y l p h o s p h o r y l d e r i v a t i v e s . T h u s the sequence for the a l k a l i n e p r o t e i n a s e from A. flavus is - G l y - T h r - S e r ~ - M e t - A l a - ,2~ a n d for t h a t of A. oryzae it is - T h r - S e r ~ - M e t - A l a - . 26 Activator and Inhibitor. A n a c t i v a t o r for a l k a l i n e p r o t e i n a s e from Aspergilli has n o t been reported. H o w e v e r , the collagenase a c t i v i t y of a s p e r g i l l o p e p t i d a s e C increases with Ca2+. 6 Also Ca 2+ a t c o n c e n t r a t i o n of 10 .4 to 10 - ~ M p r o t e c t s a p r o t e o l y t i c a c t i v i t y of the e n z y m e from A. sydowi a t p H 7.1 t o 7.8W ~ O. Mike§, J. Turkov~, N. Bao Toan, and F. ~orm, Biochim. Biophys. Acta 178, 112 (1969). ~'F. Sanger, Proc. Chem. Soc. p. 76 (1963). 27G. Danno and S. Yoshimura, Agr. Biol. Chem. (Tokyo) 31, 1159 (1967).
590
[41]
SOME BACTERIAL AND MOLD PROTEASES
Alkaline proteinases from A. oryzae, A. sydowi, and A. flavus have no free sulfhydryl groups and, therefore, are "serine enzymes. ''28 T h e y are not inhibited by sulfhydryl reagents, p-chloromercuribenzoate, cysteine, or KCN. However, they are inhibited by diisopropylphosphofluoridate, and N-bromosuccinimide. Nordwig and Jahn 6 showed the inhibition of aspergillopeptidase C by phenylmethanesulfonyl fluoride s~ and diphenylcarbamyl chloride 3° (inhibitors for serine-195 of a-chymotrypsin). The potato inhibitor 31 is reported to be an inhibitor of alkaline proteinase from A. s y d o w i 2 E D T A does not inhibit the enzymes which have been examined. (The collagenase activity of aspergillopeptidase C is decreased 40% by the addition of 1 m M of E D T A 2 ) The differences between these alkaline proteinases are observed in the inhibition of their activity by divalent metal ions. The results are summarized in Table III. TABLE III ~,FFECTS OF METAL IONS ON ALKALINE PROTEINASE ACTIVITYa
Protease Alkaline proteinase
Metal ion Ca 2+ Ni2+ Cu'+ Zn2+ Hgz+
AspergilloAspergillo- peptidsse C (A. sydowi) 8 peptidase B (A. oryzae)e (10-* M) (A. oryzae) b (10-2 M) 95% 84 96 83 60
99% +
48 22 65
I
II
III
(.4. oryzae) ~
(10-z M) 95% 54 3 10
41% 17 8 7
80% 66 12 3
6 The activity is expressed in relative activity as percent of control.
b From A. 1R. SubranmRlan, S. Spadari, and G. Kalnitsky, Federation Proc. 24, 593 (1965). The proteinases prepared by Bergkvist are somewhat different from other alkaline proteinases. The easeinolytic activity of Aspergillus Proteases I and I I I were only slightly inhibited by ascorbic acid; however, Protease I I was inhibited. E D T A and L-cysteine were inhibitors for Protease II, but these had no effect in Proteases I and III. Although mM. L. Bender and F. J. K6zdy, Ann. Rev. Bioehem. 34, 49 (1965). hA. M. Gold and D. Fahrney, Biochemistry 3, 783 (1964). nB. F. Erlanger and W. Cohen, J. Am. Chem. Soc. 85, 348 (1963). n K. Matushima, J. Agr. Chem. Soc. Japan 29, 883 (1955); Chem. Abstr. 51, ~a (1957).
[42]
MYXOBACTER AL-1 PROTEASE
591
Proteases I and I I I were inhibited by sodium laurylsulfonate, Protease I I was not. However, laurylamine inhibited all these proteinases. 22
Source As already mentioned, A. oryzae, A. sojae, A. flavus, and A. sydowi have been used as sources of mold alkaline proteinases. In addition to these Aspergillus species, A. ]umigatus/2 Penicilium cyaneo-]ulvum/~ Alternaria tenuissima/" and Gliocladium rose~m 8s produce alkaline proteinases. Acknowledgments The author wishes to acknowledge the kindness of Dr. G. Kalnitsky who made available the use of his figures, lie also wishes to thank Dr. R. Bergkvist for giving permission to use his data. :~A. G. JSnsson and S. M. Martin, Apr. Biol. Chem. (Tokyo) 28, 734 (1964). 3~K. Singh and S. M. Martin, Can. J. Biochem. Physiol. 38, 969 (1960). HA. G. JSnsson and S. M. Martin, Agr. Biol. Chem. (Tokyo) 29, 787 (1965). ~T. Kishida and S. Yoshimura, Agr. Biol. Chem. (Tokyo) 30, 1188 (1966).
[42 ] Myxobacter
AL- I Protease
By RICHARD L. JACKSON and GARY R. MATSUEDA I n 1965 Ensign and Wolfe 1 reported the isolation of an organism which was capable of lysing cell walls of Arthrobacter crystallopoietes. F r o m the culture media of the organism, called M y x o b a c t e r strain AL-1, a protease was purified which was able to hydrolyze the interglyean peptides in the cell walls of A. crystallopoietes 2 and Staphylococcus aureus2 For convenience the enzyme is called the AL-1 protease.
Assay M e t h o d D u e to an apparent substrate size requirement 4 (cf. Substrate Specificity, p. 598), a small soluble substrate has not yet been found for the AL-1 protease. Therefore, one must rely upon general methods of proteolytic assays. 1j. C. Ensign and R. S. Wolfe, J. Bacteriol. 90, 395 (1965). 2T. A. Krulwich, J. C. Ensign, D. J. Tipper, and J. L. Strominger, J. Bacteriol. 92, 734 (1967). 3j. M. Ghuysen and J. L. Strominger, Biochemistry 2, 1110 (1963). ' R. L. Jackson and R. S. Wolfe, J. Biol. Chem. 243, 879 (1968).
[42]
MYXOBACTER AL-1 PROTEASE
591
Proteases I and I I I were inhibited by sodium laurylsulfonate, Protease I I was not. However, laurylamine inhibited all these proteinases. 22
Source As already mentioned, A. oryzae, A. sojae, A. flavus, and A. sydowi have been used as sources of mold alkaline proteinases. In addition to these Aspergillus species, A. ]umigatus/2 Penicilium cyaneo-]ulvum/~ Alternaria tenuissima/" and Gliocladium rose~m 8s produce alkaline proteinases. Acknowledgments The author wishes to acknowledge the kindness of Dr. G. Kalnitsky who made available the use of his figures, lie also wishes to thank Dr. R. Bergkvist for giving permission to use his data. :~A. G. JSnsson and S. M. Martin, Apr. Biol. Chem. (Tokyo) 28, 734 (1964). 3~K. Singh and S. M. Martin, Can. J. Biochem. Physiol. 38, 969 (1960). HA. G. JSnsson and S. M. Martin, Agr. Biol. Chem. (Tokyo) 29, 787 (1965). ~T. Kishida and S. Yoshimura, Agr. Biol. Chem. (Tokyo) 30, 1188 (1966).
[42 ] Myxobacter
AL- I Protease
By RICHARD L. JACKSON and GARY R. MATSUEDA I n 1965 Ensign and Wolfe 1 reported the isolation of an organism which was capable of lysing cell walls of Arthrobacter crystallopoietes. F r o m the culture media of the organism, called M y x o b a c t e r strain AL-1, a protease was purified which was able to hydrolyze the interglyean peptides in the cell walls of A. crystallopoietes 2 and Staphylococcus aureus2 For convenience the enzyme is called the AL-1 protease.
Assay M e t h o d D u e to an apparent substrate size requirement 4 (cf. Substrate Specificity, p. 598), a small soluble substrate has not yet been found for the AL-1 protease. Therefore, one must rely upon general methods of proteolytic assays. 1j. C. Ensign and R. S. Wolfe, J. Bacteriol. 90, 395 (1965). 2T. A. Krulwich, J. C. Ensign, D. J. Tipper, and J. L. Strominger, J. Bacteriol. 92, 734 (1967). 3j. M. Ghuysen and J. L. Strominger, Biochemistry 2, 1110 (1963). ' R. L. Jackson and R. S. Wolfe, J. Biol. Chem. 243, 879 (1968).
592
SOME BACTERIAL AND MOLD PROTEASES
[42]
Cell Wall Lyric Assay: Turbidimetrie 5 At time zero an aliquot of suspended cell walls of A. crystallopoietes is added to a buffered enzyme solution. As the peptides linking the carbohydrate chains together are hydrolyzed, the reaction mixture clears.
Reagents Tris-HC1, 0.05 M, pH 9.0, containing 10-s M EDTA
A. crystallopoietes cell wall suspension with a turbidity of 2.0 at 660 nm. The cell walls are prepared by sonication of a wet cell mass. After centrifugation at 13,000 g, collection is accomplished by gently washing the white flocculent upper layer of the pellet into another centrifuge tube. The enriched cell wall fraction is resuspended in distilled water and recentrifuged. The last traces of intact cells disappear after collection and centrifugation have been repeated about ten times.
Procedure. To a culture test tube (13 X 100 ram), 0.2 ml of buffer solution and an aliquot of enzyme solution are added. The volume is then adjusted to 1.20 ml with distilled water. After 0.3 ml of the cell wall suspension is added, the timer is started as the turbidity at 660 nm is read. The blank, prepared with 0.2 ml buffer and 1.0 ml of water, is similarly treated. After a 15-minute incubation at 38 ° the turbidity of the reaction mixture is measured at 660 nm relative to the blank. Specific Activity. A unit of enzyme activity is arbitrarily defined as that amount of enzyme required to decrease the ODeeo by 0.001 absorbance units. One milligram of pure AL-1 pro~ease will give approximately 175,000 units under the described conditions. Azocoll Assay: Colorimetric ~ Azocoll is collagen complexed with an azo dye to provide an insoluble substrate. Proteases digesting this insoluble substrate release color to the solution. The reaction is terminated by simply filtering off the substrate. The absorbancy of the filtrate measured at 580 nm is an index of proteolytie activity.
Reagents Azocoll (Calbiochem) used without purification Tris-HC1, 0.05 M, pH 9.0, containing 10-~ M EDTA
Procedure. The reaction is conveniently carried out in a 25-ml Erlenmeyer flask containing 1.0 ml buffer solution and 1.5 ml of enzyme s j . C. Ensign and R. S. Wolfe, J. Bacteriol. 90, 395 (1965).
[42]
MYXOBACTER A~-I PROTF~SE
593
solution. The reaction is started by adding l0 mg of Azocoll. After shaking for exactly 10 minutes at 38 °, the reaction mixture is filtered through a sintered glass filter. The absorbancy of the filtrate is then measured at 580 nm relative to a blank to which no ehzyme had been added. Specific Activity. A unit of activity is arbitrarily defined as that amount of enzyme required to increase the OD,so by 0.001 absorbance units. One milligram of AL-1 protease will give approximately 130,000 units under the described conditions. Purification
The Myxobacter strain AL-1 is mass cultured using standard aseptic techniques. 4 The organism is transferred from a solid stock culture to 300 ml of sterile 1% yeast extract (Difco) in a 500-ml Erlenmeyer flask to produce an actively growing culture after shaking for 24 hours at 30 °. The culture is then transferred to a 3-liter Erlenmeyer flask containing 1 liter of sterile 1% yeast extract, and shaking at 30 ° is continued for 12 hours. Finally 15 liters of sterile 1% yeast extract in a 20liter carboy is inoculated with the l-liter culture while vigorously aerating the medium through a sterile glass-wool plug to two sterile Nalgene gas dispersion tubes at a rate of approximately 12 liters/minute. After 1 ml of sterile polypropylene glycol has been added as antifoam agent, sterile aeration is continued for 30-36 hours at 30 ° . The cells are subsequently harvested with a 10-liter Sharpies centrifuge, and the effluent liquor containing the AL-1 protease is collected in the rinsed culture carboys and stored at 4 °. To the cooled growth liquor, about 60 ml of 3 M HC1 is added dropwise until pH 6.7 is attained on a pH meter. Meanwhile, 350 ml of 10% (w/v) ZnC12 is heated on a steam bath to facilitate solubilization and is then filtered while hot. As this filtrate is added with stirring to the acidified growth liquor, certain proteins including the AL-1 protease begin to precipitate. Precipitation is completed with a 30-minute cooling period at 4 °. The zinc precipitate is collected using the 500-ml Sharples centrifuge. A typical purification yields 27 g of zinc precipitate per 14 liters of growth liquor. At this point the zinc precipitate may be conveniently frozen at --20 ° until a sufficient supply has accumulated. In the next step, 30 g of thawed zinc precipitate is first homogenized with 900 ml of 0.05M Tris-HCl-10-3M EDTA, pH 9.0, with short bursts in a Waring Blendor. During a 30-minute slow stirring period, more of the contaminating proteins are solubilized. The remaining zinc precipitate should be then easily collected by centrifugation at 10,000 g. In the following crucial and sometimes temperamental citrate extraction step, the AL-1 protease is solubilized. First the collected zinc precipitate
594
SOME BACTERIAL AND MOLD PROTEASES
[42]
is roughly divided into thirds. Approximately one-third of the Tris-extracted zinc precipitate is returned to the blender for another quick homogenization with 900 ml of 0.15 M Na citrate, pH 5.0. The homogehate is transferred to a beaker for 5 minutes of slow stirring at 0 °, after which the remaining chunks of zinc precipitate are allowed to settle for 5 minutes. To the decanted amber solution, solid (NH4)~S04 is added with gentle stirring at 0 ° to 70~ saturation. After 5 minutes, the (NH4)2S0~ precipitate which contains the AL-1 protease is collected at 9000 g. The other two portions of Tris-extracted zinc precipitate are similarly treated, and the ammonium sulfate precipitates from each of the three portions combined. The AL-1 protease is resolubilized by treating the combined ammonium sulfate precipitates with 250 ml of 0.025 M Tris-HC1, pH 7.5, and stirring for 1 hour at 0 °. After centrifugation, the amber supernatant is dialyzed for 2 hours against 14 liters of 0.025 Tris-HC1, pH 7.5, in preparation for carboxymethyl cellulose batch chromatography. The dialyzed solution is divided among three CM-cellulose columns (3.5 X 1.5 cm) each equilibrated with 0.025 M Tris-HC1, pH 7.5. After sample application, the columns are washed with the same buffer until the efftuent OD2so is less than 0.1. The AL-1 protease-containing fraction is then eluted with 0.05 M glycine-NaOH-0.3M NaC1, pH 10.0. The eluants from the three columns are combined and the protein precipitated with solid (NH~)2S04 at 90% saturation. After centrifugation the ammonium sulfate precipitate is resuspended in 25 ml of 0.025M Tris-HC1, pH 7.5, and stirred until dissolved at 0 °. The solution is quantitatively transferred to a dialysis tubing and dialyzed for 2 hours against 10 liters of 0.025 M Tris-HC1, pH 7.0, in preparation for the final column chromatography. Alternatively, the solution may be exhaustively dialyzed against distilled water and lyophilized for convenient storage. At this point, the slightly brown preparation has been purified 65 times over the growth liquor, and has a specific activity approaching 160,000. To obtain a homogeneous enzyme preparation, gradient column chromatography is necessary. Lyophilized enzyme obtained from 15 liters of growth liquor is dissolved in 10 ml of 0.025 M Tris-HC1, pH 7.0, and dialyzed against the same buffer for 4 hours. The dialyzed sample is quantitatively applied on an equilibrated low-capacity carboxymethyl Biogel-30 (Bio-Rad) column (2.0 X 20 cm) and washed into the resin with the starting buffer, 0.025 M Tris-HC1, pH 7.0. Immediately a pH gradient is started from 0.025 M Tris-HC1, pH 7.0, to 0.025 M glycine-NaOH, pH 10.0 using 400 ml of each buffer. When the pH gradient is complete, a linear salt gradient is immediately started from 0.025 M glycine-NaOH,
[42]
MYXOBACTER AL-1 PROTEASE
595
• 2.0
1.0 0.8
1.Z 8
J,o
0.4
/4
x ~ x ~ x
0.2 MNaCl
0.4
0.2
0
0.8 .~
Iv I
Y
10
~
~
I
20
~
3O
I
I
4O
~
I
50
I0
6O
Fraction no. (7.5ml per tube)
FIG. 1. CM-Biogel column chromatography of the CM-ceUulose batch eluted
AL-1 protease. For the experiment, 1.87 X 10~ units of enzyme in 10 ml was applied to the column ( L S X l l cm) equilibrated with 0,025M Tris-HC1, pH 7.0. A pH gradient (X) from 0.025M Tris-HC1, pH 7.0 to 0.025M glycine-NaOH, pH 10.0 was immediately started. Upon completion of the pH gradient, a salt gradient ( ) was quickly started from 0.025M glycine-NaOH, pH 10.0 to 0.025M glycine-NaOH-02 M NaCI, pH 10.0. In this step, 100 ml of each buffer was used. Cell wall lytic activity (vertical solid bars) was determined with aliquots from each tube under the peaks of the protein profile (O). p H 10.0, to 0.025 M glycine-NaOH-0.2 M NaCl, p H 10.0 using 400 ml of each buffer. For a typical run, 5 hours is required to complete each gradient. The fraction corresponding to the last peak in Fig. 1 is now free from amber proteins and exhibits proteolytic as well as cell wall lytic activity. The pooled enzyme is dialyzed against distilled water until the dialyzate gives a negative chloride test with 0.03 M AgNOa. The dialyzed enzyme is then lyophilized and stored at --20 ° over a desiccant. Table I summarizes yield data for a typical purification which takes 3 working days. Four days are required for the mass culture and harvest to produce the growth liquor. P u r i t y and Physical Properties Three criteria have been used to judge the homogeneity of enzyme preparations4: (1) electrophoresis on cellulose acetate strips at p H 7; (2) Ouchterlony double diffusion against rabbit antibody; and (3) poly-
596
S O M E B A C T E R I A L A N D MOLD P R O T E A S E S
v M
×~
~
o0
J
-ij O
o
•6
U
w
[42]
[42]
MYXOBACTER AL-1 PROTEASE
597
TABLE II PHYSICAL CHEMICAL PROPERTIES OF THE
AL-1
Property 280 nm E1 omtat % 8~0,w Do20,w
[~], intrinsic viscosity (f/fo), frictional coefficient MW, amino acid composition MW, sedimentation diffusion Hexose Metal content N-terminal amino acid of carboxymethylated protein C-terminal amino acid
PROTEASE 4,7
Value 15.8 2.40 S 14 X 10-z cms sec-1 1.66
_ 0.1mlg-1
1.0 13,407 14,340 0.88 mole/mole protein Insignificant; Cu and Zn 0.27 moles/mole protein Undetected by DNFB, DNS-C1 Valine by hydrazinolysis
acrylamide disc electrophoresis at p H 6.6. Disc tinely used to check the purity of p e a k tubes chromatography. T a b l e I I lists the physieochemical properties T a b l e I I I contains amino acid composition data
electrophoresis is roufrom gradient column of the AL-1 protease. for the AL-1 protease.
Enzymatic Properties p H Optimum. A sharp p H optimum at 9.0 is observed with both the cell wall lytic and Azocoll assays.5 Stability.5 At 4 ° in 0.02 M Tris-HCl, p H 9.0, the AL-1 protease is stable for at least 8 hours and retains 9 3 % of its cell wall lytic activity after 48 hours at 4 ° . At 35 ° under identical conditions, 8 7 % of the original activity remains after 8 hours. From 40 ° to 50 ° enzymatic activity is slowly lost, such that 3 0 % of the original activity remains after 8 hours at 50 °. "From p H 6.5 to p H 9.5, the enzyme exhibits m a x i m u m stabilitywhen incubated for 1 hour at 45 ° in 0.02 M buffers ranging from p H 3.0 to p H 11.0. At p H 3, less than 5 % of the initial cell wall lytie activity remains. Inactivators.5 Studies with inhibitors have not revealed any outstandingly potent enzyme inactivators. Heavy metal ions at approximately 10-8 M concentrations quench activity, but this is not surprising as zinc chloride at 10-3M efficientlyprecipitates the enzyme in the purification scheme. What is perhaps most revealing is the ineffectiveness of such compounds as p-chloromercuribenzoate, iodoacetic acid, diisopropylphospho-
598
SOME BACTERIAL AND MOLD PROTEASES
[42]
TABLE I I I AMINO ACID COMPOSITION OF THE
AL-1
Amino acid
Assumed no. of residues
Lysine Histidine Arginine Cys-SOa a CM-Cys b Aspartic acid Threonine Serine Glutamic acid Proline Glycine" Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan ~ Total Amides" Cysteine as CM-Cysf
PROTEASE 4
2 6 3 2 2 16 11 17 7 5 17 10 3 2 3 7 8 4 3 128
MW 13,500
15 0
Determined with performic oxidized protein. b Determined with carboxymethylated protein. ' Determined with trichloroacetic-acid-precipitated protein.~ Method of Spies and Chambers. s ' Method of Johnson 9 using amino acid analyzer. I Determined by alkylation of unreduced protein with iodoacetic acid in 8 M urea and by method of Boyer. 1° fluoridate ( D F P ) a n d dinitrofluorobenzene in the 10 -4 M range a n d metal chelators like citrate a n d E D T A in the 10 -2 M range. S p e c i f i c i t y . E x p e r i m e n t s to date h a v e not been able to establish a n y well-defined specificity. H o w e v e r , the d a t a indicate an affinity t o w a r d peptide bonds formed b y at least one h y d r o p h o b i c amino acid in a struct u r e at least as large as a tetrapeptide. A case in p o i n t : T h e A l a - L e u bond in the B - c h a i n of insulin is h y d r o l y z e d ; but C B Z - A I a - L e u - N H 2 ' G. R. Matsueda, unpublished data. 8j. R. Spies and D. C. Chambers, Anal. Chem. 21, 1249 (1949). o M. J. Johnson, J. BioG. Chem. 137, 575 (1941). P. D. Boyer, J. Am. Chem. Soc. 76, 4331 (1954).
[43]
a-LYTIC PROTEASE
599
is not. 4 Furthermore, pentaglycine is readily hydrolyzed; tetraglycine is hydrolyzed with difficulty, and tri- and diglycine are not attacked at all. 4 Such a size requirement would be useful in amino acid sequence work where large peptides must be degraded, preferably to tetra- and pentapeptides. Distribution Inhibition studies 5 suggest that the AL-1 protease mechanism of action is unlike any proposed by Hartley. 11 Present data eliminate mechanism of action proposed for the sulfhydryl proteinases, the acid proteinases, the metal proteinases, and the serine proteinases if sensitivity to DFP is a criterion. In this respect, the AL-1 protease is similar to the zinc containing p-lyric protease from Sorangium. ~2 ~B. S. Hartley, Ann. Rev. Biochem. 29, 45 (1960). D. R. Whitaker, L. Jurasek, and C. Ray, Biochem. Biophys. Res. Commun. 24,
173 (1966).
[ 4 3 ] T h e a - L y t i c P r o t e a s e of a M y x o b a c t e r i u m
By D. R. WHITAKER Introduction The bacterium, "Myxobacter 495," which produces this enzyme was isolated in Ottawa during a study of Myxobacterales from local soil. 1,2 Its taxonomy is still under investigation, 3 but it has been reported to be a species of Sorangium. 2 Culture filtrates of the organism have strong bacteriolytic activity toward a number of other soil bacteria 2 and lyse soil nemat:odes.1 Two extracellular, basic proteases, which have been designated the "a-lytic protease" and the "fi-lytic protease" are responsible for most of the bacteriolytic activity. 4 The two enzymes can be readily distinguished: the fi-enzyme is not a serine protease and, for example, on electrophoresis at pH 8 in cellulose acetate it migrates just behind egg-white lysozyme whereas the a-enzyme migrates slightly ahead of egg-white lysozyme. The organism produces several other proteases as well and some, like the a-enzyme, are serine proteases but they are 1H. Katznelson, D. C. GiUespie, and F. D. Cook, Can. J. Microbiol. 10, 699 (1964). D. C. Gfllespie and F. D. Cook, Can. J. Microbiol. 11, 109 (1965). a F. D. Cook, personal communication. ~D. R. Whitaker, Can. J. Biochem. 43, 1935 (1965).
[43]
a-LYTIC PROTEASE
599
is not. 4 Furthermore, pentaglycine is readily hydrolyzed; tetraglycine is hydrolyzed with difficulty, and tri- and diglycine are not attacked at all. 4 Such a size requirement would be useful in amino acid sequence work where large peptides must be degraded, preferably to tetra- and pentapeptides. Distribution Inhibition studies 5 suggest that the AL-1 protease mechanism of action is unlike any proposed by Hartley. 11 Present data eliminate mechanism of action proposed for the sulfhydryl proteinases, the acid proteinases, the metal proteinases, and the serine proteinases if sensitivity to DFP is a criterion. In this respect, the AL-1 protease is similar to the zinc containing p-lyric protease from Sorangium. ~2 ~B. S. Hartley, Ann. Rev. Biochem. 29, 45 (1960). D. R. Whitaker, L. Jurasek, and C. Ray, Biochem. Biophys. Res. Commun. 24,
173 (1966).
[ 4 3 ] T h e a - L y t i c P r o t e a s e of a M y x o b a c t e r i u m
By D. R. WHITAKER Introduction The bacterium, "Myxobacter 495," which produces this enzyme was isolated in Ottawa during a study of Myxobacterales from local soil. 1,2 Its taxonomy is still under investigation, 3 but it has been reported to be a species of Sorangium. 2 Culture filtrates of the organism have strong bacteriolytic activity toward a number of other soil bacteria 2 and lyse soil nemat:odes.1 Two extracellular, basic proteases, which have been designated the "a-lytic protease" and the "fi-lytic protease" are responsible for most of the bacteriolytic activity. 4 The two enzymes can be readily distinguished: the fi-enzyme is not a serine protease and, for example, on electrophoresis at pH 8 in cellulose acetate it migrates just behind egg-white lysozyme whereas the a-enzyme migrates slightly ahead of egg-white lysozyme. The organism produces several other proteases as well and some, like the a-enzyme, are serine proteases but they are 1H. Katznelson, D. C. GiUespie, and F. D. Cook, Can. J. Microbiol. 10, 699 (1964). D. C. Gfllespie and F. D. Cook, Can. J. Microbiol. 11, 109 (1965). a F. D. Cook, personal communication. ~D. R. Whitaker, Can. J. Biochem. 43, 1935 (1965).
600
SOME BACTERIAL AND MOLD PROTEASES
[43]
much less basic and have different sequences around their reactive serine residue. The unique feature of the a-enzyme is that it is a bacterial enzyme with amino acid sequences around its reactive serine residue5 and around its single histidine residue 6 which classes it as a homolog of the pancreatic serine proteases. Its enzymatic properties closely match those of porcine elastase. A s s a y Methods
Three assay methods are listed below. The first two are not specific for the a-enzyme but are useful routine assay methods for production and purification work. The third method gives a precise measure of esterase activity. Proteolytic Activity Toward Casein Five milliters of a 0.2% solution of casein (Hammarsten quality) in 0.025 M Tris buffer of pH 8.5 is mixed at 25 ° with 20-150 ~g of enzyme in 1 ml of the same buffer. The reaction is stopped after 10 minutes by the addition of 10 ml of 5% trichloroacetic acid. The subsequent analysis with Folin-Ciocalteau reagent is exactly as described by Herriott. ~ The color yield per 100 ~g of a-enzyme is equivalent to a production of 3.4 micromoles of tyrosine per 20 mg of casein. At least five other proteases in the culture filtrate have caseinase activity. Bacteriolytic Activity Toward Arthrobacter globiformis Cells 8 Cells for this assay are obtained from 24-hour culture of Arthrobacter globi]ormis (Canada Department of Agriculture, Microbiology Research Institute, Strain No. 616) in a basal salts medium9 supplemented by 1% sucrose and 0.2% casamino acids. The culture solution is centrifuged and the sediment of packed cells is thoroughly mixed with 11 volumes of acetone at --15 ° . The cells are left to settle at room temperature, washed with ether, and freed of solvent in vacuo. The dried cells are mixed with distilled water to give a thick suspension, and the treatment with acetone and ether is repeated. For assays of bacteriolytic activity, 5.0 ml of a 0.03% suspension of A. globi]ormis cells in 0.025 M Tris-0.004 M KC1-HC1 buffer of pH 9.0 5D. R. Whitaker and C. Roy, Can. I. Biochem. 45, 911 (1967). e L. B. SmiLlieand D. R. Whitaker, Y. Am. Chem. Soe. 89, 3350 (1967). ' R. Herriott, Vol. II, p. 3. sD. R. Whitaker, F. D. Cook, and D. C. Gillespie, Can. J. Biochem. 43, 1927 (1965). °The salt composition is the same as that of the medium for Myxobacter 495 with the addition of CaCh (02 g/liter) and NaC1 (0.1 g/liter). [A. G. Lockhead and M. O. Burton, Can. J. M/crob/ol. 1, 319 (1955).]
[43]
a-LYTIC PROTEASE
601
is mixed at 25 ° with 0.5-6 9g of a-enzyme in 200 ~1 of the same buffer. The absorbance per centimeter at 660 nm is measured at 5-minute intervals. The rate of change of the reciprocal of the absorbance, i.e., dA-l/dt, is a linear function of the enzyme concentration. The value of dA-I/dt per microgram of enzyme varies with the cell preparation; a representative value is 0.01 reciprocal absorbance units per minute per microgram. The fl-enzyme is the only other protease of Myxobacter 495 which seriously interferes with the assay.'
Esterase Activity Toward N-Benzoyl-L-alanine Methyl Ester ~° The substrate can be prepared by the following modification of the method of Hein and Niemann. ~ Seventeen milliliters of methanol is cooled to --10 ° in an ice-salt bath and stirred continuously while 4.4 ml of thionyl chloride is added dropwise. Five grams of L-alanine is added in small portions. The temperature is slowly raised to 40 ° and maintained at 40 ° for 2 hours. During this period, the alanine dissolves completely. The methanol is then removed in vacuo. The residue of crude ester hydrochloride is dissolved in 20 ml of water. The solution is brought to pH 8.0 in a pH-stat by titration with 1 N NaOH and maintained at pH 8.0 while 6.6 ml of benzoyl chloride is slowly added. The N-benzoyl ester separates from the solution as a milky white liquid and is crystallized from high-boiling petroleum ether. Yield: 5.4 g (47%). Melting point: 580_590; [~]2~- = +30.8 o. Esterase activity is measured in a pH-stat maintained at pH 8.0 with 0.02N N a 0 H . Suitable reaction conditions are: Solvent, 0.10M KC1; initial substrate concentration ([S] o), 5 X 10-3 M; enzyme concentration ([E]), from 10-s to 10-~ M, i.e., 0.02-2 ~g/ml. The rate of hydrolysis is directly proportional to [S]o and [E]. The second-order velocity constant, TM i.e., moles of alkali consumed per liter per second/ [E]o" [5]0, is equal to 720M -1 second -1. Production and Purification
Production The production of enzyme in a 130-liter fermentor has been described 9 but substantial amounts of enzyme can be obtained with much simpler ~oH. Kaplan and D. R. Whitaker, J. Am. Chem. Soc. 89, 3352 (1967) ; ibid., Can. J. Biochem. 47, 305 (1969). u G . E. Hein and C. Niemann, J. Am. Chem. Soc. 84, 4487 (1962). As the overall kinetics are Michaelis-Menten kinetics, the second-order velocity constant is equal to k~ot/Km where kcat is the catalytic rate coefficient, i.e., Vmax/[E], and Km is the Michaelis-Menten constant.
602
SOME BACTERIAL AND MOLD PROTEASES
[43]
equipment. 's The final stage of the procedure described below requires a rotary shaker capable of holding four 40-liter carboys, but it can be scaled down to the capacity of any rotary shaker. Culture on Agar Slopes. Slopes of 0.2% Difco tryptone-l% agar are inoculated and incubated at 25 ° for 48 hours. The slopes should be used immediately if they are to provide the inocula for the first stage of shake cultures, but can be stored for several dr,ys at 4 ° if they are merely to provide the inocula for other slopes. If prolonged storage is required, cells from the slopes can be dispersed in steriJ~ 15% glycerol and stored at --40 °.1~ Shake Cultures. The composition of the culture medium is as follows: Casamino acids 15 Glucose K~HPO4 KNO3 MgSO4.7 H20 FeClr6 H~O ZnS04
10 1 1 0.5 0.2 0.01 0.001
g/liter g/liter g/liter g/liter g/liter g/liter g/liter
The glucose is autoclaved separately; the solution containing the other components is adjusted to pH 7.0-7.1 with N a 0 H before it is autoclaved. It is immaterial whether technical grade or "vitamin-free" casamino acids are used, but if a "salt-free" grade is used, sodium chloride (3.5 g/liter) should be added to the medium. The organism is grown at 25 ° in three stages of shake culture. The first two stages are in flasks with cotton-wool plugs. The third stage is in 40-liter carboys plugged by rubber stoppers pierced by air-inlet and air-outlet tubes, the former extending through the stopper to within a few inches of the surface of the medium. The air-inlet and outlet tubes are connected to air filters (glass bulbs packed with glass wool) which are autoclaved in situ; before entering the inlet filter, the air is scrubbed in a carboy containing 0.2~ sulfuric acid and then freed of entrained acid in a spray trap. The rotary shaker for the third stage is set to describe a 1-inch circle at about 150 rpm. The first stage of shake culturc 24-hour growth in 500-ml flasks containing 75 ml of medium--provides the inoculum for the second stage--24-hour growth in 10-liter flasks containing 2 liters of medium. ~I). R. Whitaker, Can. J. Biovhem. 45, 991 (1967). M. T. Clement, Can. J. Microbiol. 10, 613 (1964). Casamino acid is a neutralized, acid-hydrolyzate of casein.
[43]
a-LYTIC PROTEASE
603
The second stage provides the inoculum for two 40-liter carboys, each containing 11 liters of medium. Four such carboys are shaken for 48 hours with continuous aeration at approximately 1 liter of air per minute per carboy. The medium is virtually exhausted of nutrients by the end of the third stage and growth is in stationary phase. The absorbance at 660 nm per centimeter of culture solution is 4-5. Cell Removal. The solution is freed of cells by passage through a Sharples supercentrifuge. This operation should not be delayed unduly or the cells may autolyze. The absorbance of the cell-free solution depends to some extent on the casamino acid preparation used in the medium; typical values for solutions from a "salt-free" easamino acid medium are: A2so~m= 3.8-4.1 per centimeter, A~o~m ---- 3.6-3.7 per centimeter. The pH is 8.1-8.5.
Purification The procedures of this laboratory were designed to isolate the flenzyme as well as the a-enzyme and might be simplified if only the latter were required. The two enzymes are displaced selectively from the cation-exchange resin, Amberlite CG-50. An earlier procedure 4 used a concentration gradient of buffer to effect this separation. It is a more informative procedure than the stepwise displacement described below but the latter is simpler and requires less attention, la The Amberlite IR-45 and IR-120 resins used in the initial treatment of the filtrate are analytical grade, 16-50 mesh resins and require no special treatment, but the Amberlite CG-50 resin is 400-600 mesh and must be completely cleared of fines before it can be used in columns. Fines are removed in this laboratory by stirring the resin in a large volume of solution (40 liter per pound of resin) and leaving it to settle for various periods before the supernatant solution is discarded. A typical sequence of settling times is: 5 hours, 2½ hours, overnight, 2 hours, and 11/~ hours in 0.5% NaOH-0.25% sodium citrate in tapwater; overnight in 2.5% acetic acid (in distilled water); 2 hours in 0.5% N a 0 H , overnight in water; 11/~ hours in 1% HCI; 8 hours in 0.5% sodium citrate-0.05% Versene and overnight in 0.5% NaOH. The resin particles from this operation had a diameter of 29 ___2 ~. The resin is equilibrated with a given buffer, e.g., acetic acid-0.10 N NaOH of pH 4.95 by dispersing the resin in a large volume of 0.10 N NaOH, adiusting the pH of the dispersion to 4.95 with acetic acid, redispersing the resin in a large volume of buffer, and readjusting the pH, if necessary, with acetic acid. A suitable support for the resin can be made from layers of acid-washed beads of graded diameter (e.g., 40 ~, 100 ~, and 500 ~ Bal-
604
SOME BACTERIAL AND MOLD PROTEASES
[43]
lotini) above a small disk of porous polyethylene in the outlet tube of the column. Step 1. Mixed-Bed Ion Exchange. Five hundred milliliters (settled volume) of Amberlite IR-45 (acetate) and 400 ml of Amberlite IR-120 (NH +) are stirred in the cell-free culture solution and the dispersion is left to cool in a cold room (2°-5°). All subsequent steps are in the cold room. The dispersion is filtered through a Biichner funnel containing 100 ml of each resin on a pad of nylon cloth. The resin is drained and washed with a liter of water. Step 2. Adsorption on Amberlite CG-50 at pH 5.0. The filtrate from step 1 is brought to pH 4.95 with 20% acetic acid (roughly 1 liter). Eight hundred milliliters (settled volume) of Amberlite CG-50, equilibrated with sodium hydroxide-acetic acid buffer which is 0.10M with respect to sodium and of pH 5.00, is added in small portions with vigorous stirring and then left to settle overnight. The supernatant solution is removed and the resin is dispersed in 7 liters of the above-mentioned acetate buffer and again left to settle. Finally it is washed once more with 4 liters of acetate buffer. Step 3. Titration and Washing o] the Resin at pH 6.25. If several batches of shake culture are being prepared, it is convenient to combine two sets of resin from step 2 at this point. The ]oIlowing procedure assumes that this has been done. The resins from step 2 are dispersed i n 3 liters of cold sodium hydroxide-citric acid buffer which is 0.033M with respect to citrate and of pH 6.25. The dispersion is titrated to pH 6.4 by the addition, with stirring, of 0.5 M sodium hydroxide (approximately 1800 ml) through a capillary tube. The dispersion is added to a large column (12 cm diameter) which contains 1 liter, of the abovementioned citrate buffer above a 2-cm bed of resin which has been equilibrated with similar buffer. The column is left to drain until there is no supernatant solution above the settled resin. Step $. Stepwise Displacement o] the a- and fl-enzyme. The subsequent inputs to the column are: (a) 4.2 liters of sodium hydroxide-citric acid buffer which is 0.160 M with respect to sodium and of pH 5.88 -(b) 1 liter of sodium hydroxide-citric acid buffer which is 0.210 M with respect to sodium and of pH 6.00 (c) 4.5 liters of sodium hydroxide-citric acid buffer which is 0.270 M with respect to sodium and of pH 6.20 The last 3 liters of effluent from (a) plus the first 500 ml from (b) contain most of the fl-enzyme with minor contamination by less basic proteases. The last 500 ml of effluent from (b) contain small and roughly
[43]
c~-LVTIC PROTEASE
605
equal amounts of both the a- and B-enzyme. The effluent from (c) contains practically all the a-enzyme with slight contamination by Benzyme. Step 5. Re]ractionation. The enzyme in the 4.6-liter eluate from buffer (c) is refractionated by the same procedure as was used for the initial fractionation. The solution is brought to pH 5.0 with acetic acid and mixed with 500 ml (settled volume) of Amberlite CG-50 in acetate buffer. The resin is washed with acetate buffer, dispersed in 0.33 M citrate buffer, and titrated to pH 6.4 with 0.5 M NaOH. The dispersion is added to a column containing two layers of resin: a lower layer (2 cm) of resin which had been equilibrated with 0.033 M citrate buffer of pH 6.25 and an upper layer (7 em) of resin which had been equilibrated similarly but, before addition to the column, had been titrated to pH 6.4 with 0.5 M sodium hydroxide. The column is left to drain as before and the contaminating B-enzyme is displaced with 1 liter of the 0.160 M citrate buffer. The a-enzyme is then displaced with 6 liters of 0.270 M citrate. The effluent from this buffer is collected in 50-100 ml portions at the start and in 100-200-ml portions toward the end; effluent with an absorbance less than one-third the maximum absorbance at 280 nm is discarded; the remainder for the next step. Step 6. Precipitation and Dialysis. The solution from step 5 is adjusted to pH 8 with dilute ammonia, brought to roughly 60% saturation with "enzyme grade" ammonium sulfate (455 g/liter of solution), and left in the cold room for 48 hours. The precipitated enzyme settles cleanly and most of the supernatant solution can be siphoned off; the remainder is removed by high-speed centrifugation. The sediment is dissolved in 0.1 M KC1 and recentrifuged (30 minutes at about 20,000 g) to remove trace amounts of insoluble material. The solution is dialyzed in 3- to 4-foot lengths of 1/~-inch dialysis tubing which are suspended in glass columns in which a slow downward flow of 0.1 M KC1 is maintained for 24 hours, and then followed by a flow of glass-distilled water for 48 hours. The resulting salt-free solution is freeze-dried. A typical yield is about 2 g. Crystallization. '6 Crystals suitable for X-ray diffraction studies can be obtained by dialysis in capillary tubes such as those described by Zeppezauer et al. 17 The capillary, loaded with a 1% solution of enzyme in glass-distilled water, is immersed in 1.7 M ammonium sulfate which = L. B. Smillie, personal communication. "M. Zeppezauer, H. Eklund, and E. S. Zeppezauer, Arch. Biochem. Biophys. 126, 561 (1968).
606
SOME BAC~EmAL
A N D MOLD P R O T E A S E S
o
'2 O Z
cq
w
[43]
[43]
a-LYTIC PROTEASE
607
has been adjusted to p H 7.35 with ammonia. The deposition of crystals commences in about 10 days. A m i n o A c i d S e q u e n c e a n d R e l a t e d Properties
The amino acid sequence of the enzyme is in Table I, the amino acid composition in Table II. The following points are noteworthy: The sequence around the active serine residue, S e r l , , i.e., Gly-Asp. S e r , G l y - G l y , is identical with the active serine sequences of the panereato-peptidases. The sequence around the single histidine residue, His3e, i.e., V a l . T h r A l a . G l y . H i s . C y s . G l y is almost identical with the sequence around the catalytically functional histidine residue of chymotrypsin, i.e., V a l . T h r . Ala. Ala- His- Cys. Gly. Only one aspartic acid residue, Asp63, is available to function in a charge-relay system such as that which Blow et al. have postulated for chymotrypsin.18 The molecular weight of the enzyme was estimated to be 19,10019,300 from the rate of approach to sedimentation equilibrium and 19,100 from the elution volume for Sephadex G-75.19 The molecular weight calculated from the sequence in Table I is 19,778. I t is apparent from the amino acid composition that the isoelectric point of the enzyme will tend to be very high. In Tris buffer of p H 9, the mobility is still strongly positive. TABLE II AMINO ACID COMPOSITION O1~ a-LYTIC PRO~ASE
Amino acid Histidine Lysine Arginine Aspartic acid Asparagine Glutamic acid
Glutamine Serine Threonine Proline
No. of residuesa 1 (1.06 2 (2.03 12 (12.08 25 13 ~ (15.75 4{ 95 (13.07
± ± ± ±
0.09) 0.03) 0.12) 0.41)
± 0.13)
20 (20.85 ± 1.22) 18 (16.47 ± 0.91) 4 (4.94 + 0.31)
Amino acid
No. of residues~
Glycine Alanine Valine Isoleucine Leucine Phenylalanine Tyrosine Tryptophan Methionine Half-cystine Amide groups
32 (32.2 ± 0.82) 24 (23.89 ± 0.31) 19 (18.63 4- 0.22) 8 (8.00 + 0.09) 10 (9.95 ± 0.11) 6 (5.96 + 0.24) 4 (3.95 ± 0.06) 2 2-4 2 1.84 + 0.04 6 5.95 22 19.5
The values in parenthesis are the estimates from the original amino acid analyses. [L. Jur~ek and D. R. Whitaker, Can. J. Biochem. 45, 917 (1967).] The estimate for proline and the higher value for tryptophan are known to be overestimates. ~D. M. Blow, J. J. Birktuft, and B. S. Hartley, Nature 221, 337 (1969). L. Jura~ek and D. R. Whitaker, Can. J. Biochem. 43, 1955 (1965).
608
SOME BACTERIAL AND MOLD PROTEASES
[43]
Properties
Stability. Freeze-dried preparations can be stored at --10 ° for at least 4 years without a detectable loss in activity. Frozen solutions of the enzyme are also stable although repeated freezing and thawing leads to losses in activity. The enzyme is subject to autodigestion if it is dissolved in concentrated urea at a pH above pH 5 and, if a chemical modification entails treatment with concentrated urea, the enzyme's initial exposure should be at a pH below 5. Purity. Enzyme prepared by the foregoing procedures satisfies the following criteria. It is homogeneous (a) on eleetrophoresis in cellulose acetate in a variety of buffers ranging in pH from 3.0 to 9.3, including buffers containing 7 M urea, 19 (b) on electrophoresis in starch gel at pH 8.0,19 (c) on electrophoresis at pH 4.3 in polyacrylamide gels prepared from 7.5% and from 15% solutions of polyacrylamide, with and without 8 M urea, ~° (d) on sedimentation in the ultracentrifuge in buffers of pH 5.0 and 8.0,19 and (e) on chromatography in columns of hydroxylapatite and of Sephadex G-75.19 A single N-terminal residue is detectable with 1-fiuoro-2,4-dinitrobenzene,1° no anomalous peptides were detected in the course of the determination of the sequence and the amino acid composition assigned by the sequence is in good agreement with the composition deduced from the original amino acid analyses of the enzyme (Table II). Physical Properties. The enzyme is very soluble in distilled water. The sedimentation coefficient, S2o,w, is approximately 2.2 Svedberg units. 1~ The ultraviolet absorption spectrum has no unusual features; the absorbanee at 280 nm/cm of solution can be related to enzyme concentration by the equation, [E] = 5.16 X
lem 10-5 X A -~280
where [E] is the enzyme concentration in moles/liter. I~ The optical rotatory dispersion spectrum at wavelengths above 220 nm has one major Cotton effect with its trough at 230-232 nm and small Cotton effects with troughs at 290-291 nm and at 283-285 nm. 21 Its most notable feature is the low magnitude of the rotations, for example, [M']~ao, the reduced mean residue rotation at the trough of the main Cotton effect, is approximately--1650 ° cm2/decimole (cf. poly-L-lysine, where [MP]233 = - - 1 8 0 0 ° when the content of a-helix is 0% and [M']238 =--16,000 ° when it is 100%.) The ORD spectrum is not pH-dependent from pH 5 to 10. ~G. M. Paterson, personal communication. The buffer system used was that described by R. A. Reisfeld, O. J. Lewis, and D. E. WiUiams, Nature 195, 281 (1962). ~1G. M. Paterson and D. R. Whitaker, Can. J. Biochem. 47, 317 (1969).
a-L~rIc PROT~SE
[43]
609
Crystallographic Properties. 2~ The following data refer to crystals obtained by the procedure of L. B. Smillie.
Crystal system Crystal habit Cell dimensions Space group
trigonal hexagonal prism and dipyramid elongated along the C axis a = b - 66.5~k,C = 80.2/~ P3121 or P3~12
A c t i v a t i o n and Inactivation. The enzyme does not require any specific activator. It is inactivated rapidly by diisopropylphosphorofluoridate (DFP) and isopropyl methylphosphonofluoridate (sarin), e.g., treatment of 2.4 X 10-* M enzyme at pH 7.7 with 6 X 10-4 M D F P at 25 ° gives a 95% inhibition in 10 minutes2 Inhibition is accompanied by irreversible esterification of Ser14~. By analogy with the inhibition of chymotrypsin by chloromethyl ketones derived from phenylalanine and the inhibition of trypsin and subtilisin by chloro- or bromomethyl ketones derived from lysine, the a-enzyme's specificity would suggest that chloro- or bromomethyl ketones derived from neutral, aliphatic amino acids might be effective inhibitors. However, tests with chloro- and bromomethyl ketones derived from N-tosyl glycine and N-tosyl valine have been uniformly negativc even after prolonged incubation (18 hours) with up to a 100-fold excess of reagent. 1°,23 Tests on porcine elastase 24 were also negative. Specificity. The bacteriolytic activity of the a-enzyme is derived from its ability to cleave the peptides which cross link chains of amino sugars in the cell walls of susceptible bacteria. 25 It is not a specific property of the a-enzyme; for example, porcine elastase can lyse A r t h r o b a c t e r globi]ormis cells, its specific activity being about half that of the a-enzyme.23 Conversely, the a-enzyme has appreciable activity toward elastin, its specific activity in assays with orcein-impregnated elastin 26 being from 30-50% of that of porcine elastase. 23 Table I I I lists the amino acids on either side of linkages which are
M. N. G. James and L. B. Smillie, Nature 224, 694 (1970). 23It. Kaplan, V. B. Symonds, H. Dugas, and D. R. Whitaker, Can. J. Biochem. 48, in press. 2,References to porcine elastase refer to crystalline elastase prepared from "Trypsin 1-300" by Smillie and Hartley's modificationof the method of Lewis et al. [L.B. Smfltie and B. S. Hartley, Biochem. J. 101, 232 (1966).] C. S. Tsai, D. R. Whitaker, L. JurA~ek, and D. C. Gillespie, Can. J. Biochem. 43, 1971 (1965). L. A. Sachar, K. K. Winter, N. Sichler, and S. Frankel, Proc. Soc. Exptl. Biol. Med. 90, 323 (1955).
SOM~. BACTERIAL AND MOLD PROTEASES
610
[43]
TABLE I I I AMINO ACID RESIDUES ( ~ R C O ' - - AND - N H R ) ~T PEPTIDE LINKAGES CLEAVED BY ot-LYTIC PRO~ASE ~
--.NH. R - ~R.CO
Ala
Ser Val
Group 1
Group 2
In A-Chain of Performate-Oxidized Bovine Insulin~: Ser~ Val~0, Leu~, Cys~ (SO, H) Glu~ In B-Chain of Pefformate-Oxidized Bovine Insulinb: Leu~
Ser Val Gly Leu Ala Gly Val Ser Thr Ile Leu Met
Hiss0 Glu~, Cys~,(SO,H) Ser~ Tyr~ In ~-Aminoethyl a-Lytic Protease ~ Vails, ArgsT, GlnT,, Glnl~, Glu~6, Ala05, Ash2, ASh81, Asntm Ser~75, Glyss, Lysm, TrpN, Phe~, Val~, Arg,~ Phe~l Asn62, Ala~, Alal~, Glye, Gly4s, Thrm, Leum Ala~, Cysm(--(CH=)d~lH2), Gly~0, Asn4~, Args0, Va161, Thr~ Gly6=, Glyz Leu,~, AlaT~, Alatsl, Gly, o4, Gly,~ Asnl~,-Glnlg0,Leu~, Leulg~ Ala,, Alal~, Argo, Lys~, Phe~, Ala~, Alam, Glnm, Val~ Ser~l, Ser~, Leu~6 Leul~ Ph~ Serm
• The experimental conditions for these digestions were as follows. (1) For digestion of the A- and B-chains of insulinb: [•]0 ----0.8 raM, [E] ffi 0.02 raM, solvent: 0.025 M ammonium carbonate at 25 °. Peptides were isolated by high-voltage electrophoresis. Cleavages are listed in Group 1 if the relevant peptides were major components of 10-minute digests and in Group 2 if the peptides were major components of 60 minutes but not of 10-minute digests. (2) For digestion of S-aminoethyl a-lyric protease~: [8]0 --- 0.5 raM, [E] -0.02 raM, solvent: 0.001 M ammonium chloride maintained at pH 8.0 and 25 °, reaction period: 60 minutes. The peptides were isolated by extensive column chromatography and high-voltage electrophoresis. Cleavages are listed in Group 1 if the relevant peptides were isolated in yields exceeding 6% and in Group 2 if the yields were less than 6% but greater than 0.6%. b D. R. Whitaker, C. Roy, C. S. Tasi, and L. Jur~ek, Can. J. Biochem. 43, 1961 (1965). c M. O. J. Olson and L. B. Smillie, personal communication.
[43]
a-LYTIC PmyrF~S~.
611
known to be cleaved during digestions of three polypeptides by the a-enzyme. A comparison of the data for the insulin chains with that of Naughton and Sanger 2~ for digestions by porcine elastase indicates that the a-enzyme is essentially elastaselike in specificity but it cleaves the chains more selectively than elastase. For example, the fast cleavages at Vallo and at Cysl~ of the A-chain a n d at Glu~ and Leu~5 of the B-chain are alternative, not consecutive, fast cleavages---if one linkage is cleaved, the other is cleaved much more slowly. The third substrate provides a much more thorough test of specificity. Clearly, linkages cleaved by the a-enzyme are linkages which involve the carbonyl group of neutral, aliphatic amino acids. TABLE IV ESTERASE ACTIVITYOF a-LYTIC PROTEASE AND PORCINE ELASTASE TOWARD METHYL ESTERS OF 2V-SuBsTITUTEDAMINOACIDSAT pH 8.0 AND 25.0 ° ~ a-Lytic Pr(~tease
Porcine elastase
k~.J K ,.
k.t/ K.
Acyl group of
K=
k~t
(M-1
K.
k~t
(M -1
methyl ester
(raM)
(sec -1)
sec -1)
(raM)
(sec -1)
sec -1)
17
21 19 640
Benzoyl glycine
8.1
Benzoyl L-leucine Benzoyl L-valine Benzoyl L-alanine
35
R-oxycarbonyl-t~danine R~-----phenyl R-~methyl R==ethyl
R~-----n-propyl R~---n-butyl R-~-isobutyl R-~4-butyl
2.9
23
28 230 720
20
38 32 26
6.4 1.4 10
170 43 390
13 13 22
3.9 0.44 0.99
300 33 46
20 23 26 8.8
13 9.4 11 11
650 410 430 1200
21 21 36 41
3.5 5.5 7.8 4.7
160 260 220 120
The same specificity is shown in hydrolyses of amino acid esters. Substrates for trypsin such as N-benzoyl arginine methyl ester and substrates for chymotrypsin such as N-acetyl tyrosine methyl ester and N-acetyl-tryptophan methyl ester are not hydrolyzed by the a-enzyme; neither is N-benzoyl-n-alanine methyl ester, the D-isomer of one of the best substrates of the enzyme.1° As shown in Table IV, the enzyme is sensitive to the nature of the N-substituent; hydrolyses by the a-enzyme and porcine elastase show quite similar trends in the values for koat but = M. A. Naughton and F. Sanger, Biochem. J. 78, 156 (1961).
612
SOME BACTERIXL XND MOLD PROTF~SES
[43]
opposing trends are evident in the values for K~. (See Note Added in P~oof, p. 613.) Kinetic Properties. 1° The more general kinetic properties of the enzyme between pH 4 and 10 can be summarized as follows: (1) the Michaelis-Menten constant, K,~, is independent of pH (2) the catalytic rate coefficient, k~t, is dependent on an ionization with a pK of 6.7 in H~0 and of 7.3 in H~0 (3) A(log kcat/Km)/ApH for this ionization is equal to 1.0 (4) kcat/Km is reduced by 50% when H20 is replaced by H20 (5) when p-nitrophenyl trimethyl acetate is hydrolyzed by the enzyme, the initial burst of p-nitrophenol is proportional to the enzyme concentration; the magnitude of the proportionality factor and the rate of attainment of a steady state are consistent with the condition, k2 ~ ks where k2 is the rate of acylation of the enzyme and k3 is the rate of deacylation. Property (1) is held in common with elastase ~s but not with chymotrypsin and trypsin. In agreement with the indication that substrate binding is uninfluenced by ionizations of a-amino or ~-amino groups, the a-enzyme and elastase retain their esterase activity when these groups are completely acetylated by acetic anhydride. 29 As previously mentioned, the optical rotatory dispersion spectra of the enzyme also shows no evidence of pH-dependent changes in the range of pH under consideration. Properties (2)-(5) are held in common with all the panereatopeptidases; the first three are consistent with general basic catalysis by a single, unprotonated histidine residue; the last property may merely indicate that esters of p-nitrophenol and its derivatives are powerful acylating agents for all these enzymes. The catalytic processes of the a-enzyme are quite efficient as evident from the values of kcat in Table IV, and from the rate at which a steady state is attained when p-nitrophenyl trimethyl acetate is the substrate. For example, under reaction conditions which give a transient phase of roughly 400 seconds for chymotrypsin and 150 seconds for elastase, the transient phase for the ~-enzyme is no more than about 40 seconds. 1° Distribution. At present, Myxobacter 495 is the only bacterium known to produce an enzyme with such a combined functional and structural relationship to the pancreatopeptides. However the trypsinlike component of "Pronase," a protease preparation from Streptomyces griseus, H. Kaplan and H. Dugas, Biochem. Biophys. Res. Commun. 34, 681 (1969). n The enzymes were acetylated at 0 ° in a pH-stat, maintained at p H 6~ while the acetic anhydride was slowly added, and then dialyzed. The acetylated enzymes must n o t be freeze dried.
[44]
COLLAGENASES
613
has been shown recently to be a homolog of trypsin2 ° Like the a-enzyme, it has only one histidine residue. Note Added in Proof
Narayanan and Anwar have recently demonstrated that porcine elastase is more selective than was previously thought. When thoroughly purified, it cleaves the B chain of insulin rapidly at two linkages--between the Ala14 Leu15linkage and Vah8 and cysteic acidlo. [A. S. Narayanan and R. A. Anwar, Biochem. J. 114, 11 (1969).] The evidence in Table IV of a very high specificity for alanine supports their findings. L. Jur~ek, D. Fackre, and L. B. Smillie, Biochem. Biophys. Res, Commun. 37, 99 (1969).
[44] Collagenases 1
By SAM SEIFTER and ELVIN HARPF_~ Native collagen -4- H~O --* peptides and/or larger fragments Introduction B y definition, collagenases are enzymes that catalyze the hydrolytic cleavage of undenatured collagen. This is a broad definition permitting inclusion of enzymes that make either few or multiple scissions. The newer knowledge of the collagen molecule TM introduces novel considerations into a discussion of collagenases. The basic collagen unit, the tropocollagen molecule, consists of three chains (al, a2, and a second a l or an a3), each of approximately 95,000 molecular weight. These are intertwined into the characteristic triplestranded collagen helix. The structure is stabilized by hydrogen bonding, pyrrolidine residue stabilization, and occurrence of interchain covalent cross links. Among the latter, in certain vertebrate collagens, is an aldol condensation product of two residues of a-aminoadipie acid 8-semialdehyde. In certain invertebrate collagens, such as the collagen of the cuticle of Ascaris, disulfide bonds occur linking smaller peptide lengths into a larger structure of approximately 900,000 molecular weight. Accordingly, depending on their mode of attack, collagenases con1This chapter was written while the authors were under the auspices of grants from the United States Public Health Service, AM 3172 and AM 3564. One of us (EH) is a Research Fellow of The Arthritis Foundation. 1"S. Seifter and P. M. Gallop, in "The Proteins," 2nd Ed. (H. Neurath, ed.), Vol. IV, p. 293. Academic Press, New York, 1966.
[44]
COLLAGENASES
613
has been shown recently to be a homolog of trypsin2 ° Like the a-enzyme, it has only one histidine residue. Note Added in Proof
Narayanan and Anwar have recently demonstrated that porcine elastase is more selective than was previously thought. When thoroughly purified, it cleaves the B chain of insulin rapidly at two linkages--between the Ala14 Leu15linkage and Vah8 and cysteic acidlo. [A. S. Narayanan and R. A. Anwar, Biochem. J. 114, 11 (1969).] The evidence in Table IV of a very high specificity for alanine supports their findings. L. Jur~ek, D. Fackre, and L. B. Smillie, Biochem. Biophys. Res, Commun. 37, 99 (1969).
[44] Collagenases 1
By SAM SEIFTER and ELVIN HARPF_~ Native collagen -4- H~O --* peptides and/or larger fragments Introduction B y definition, collagenases are enzymes that catalyze the hydrolytic cleavage of undenatured collagen. This is a broad definition permitting inclusion of enzymes that make either few or multiple scissions. The newer knowledge of the collagen molecule TM introduces novel considerations into a discussion of collagenases. The basic collagen unit, the tropocollagen molecule, consists of three chains (al, a2, and a second a l or an a3), each of approximately 95,000 molecular weight. These are intertwined into the characteristic triplestranded collagen helix. The structure is stabilized by hydrogen bonding, pyrrolidine residue stabilization, and occurrence of interchain covalent cross links. Among the latter, in certain vertebrate collagens, is an aldol condensation product of two residues of a-aminoadipie acid 8-semialdehyde. In certain invertebrate collagens, such as the collagen of the cuticle of Ascaris, disulfide bonds occur linking smaller peptide lengths into a larger structure of approximately 900,000 molecular weight. Accordingly, depending on their mode of attack, collagenases con1This chapter was written while the authors were under the auspices of grants from the United States Public Health Service, AM 3172 and AM 3564. One of us (EH) is a Research Fellow of The Arthritis Foundation. 1"S. Seifter and P. M. Gallop, in "The Proteins," 2nd Ed. (H. Neurath, ed.), Vol. IV, p. 293. Academic Press, New York, 1966.
614
COLnAGENASES
[44]
ceivably may cause proteolytic scissions in a single a-chain without necessarily causing collapse of the whole collagen structure. Alternatively, a collagenase could cleave simultaneously across three chains in a lateral fashion to yield segments of the triple-stranded structure retaining features of the collagen fiber observable in electron photomicrographs. At the other end of the range of action_ of collagenases is a group of enzymes, originally described for microorganisms but now also being detected in animal tissues, that cleave undenatured collagen more extensively, yielding small molecular weight peptides as well as certain larger ones. The newer knowledge of the structure of collagen also explains the limited action of certain common proteases on this protein. The terminal portions of a-chains have characteristics differing from those of the main chain portions, i.e., they are almost globular and contain little if any of the specific collagen helix. Trypsin, chymotrypsin, pepsin, and pronase, among other common proteases, can promote a limited digestion of these ends without disrupting the main collagen structure. In this sense they could be classified as collagenases, but one prefers to state that they are proteases with a limited action on undenatured collagen. The true collagenases would then be required, by definition, to attack the characteristic helical regions of the collagen molecule. Thus several enzymes are known that can attack undenatured collagens, and a range of specificities is encountered. On the other hand, almost all proteases can attack denatured collagens (gelatins), i.e., collagens in which the native triple-stranded structure has been melted out. The occurrence of proline and hydroxyproline regularly throughout the amino acid sequence of a-chains in gelatin may impose some restriction on the action of certain proteases, but these do not seem to be too severe. The actions of several exopeptidases, such as earboxypeptidase and amino acid peptidases, would be expected to be limited by the large content of imino acids in the substrate. The speeifieities of some of the collagenases have been defined by the nature of terminal and penultimate amino acids appearing in peptides after cleavage of the collagen molecule. In the case of clostridial collagenase, and perhaps in the case of certain recently described animal collagenases, specificity has also been determined by action on synthetic peptides of known amino acid sequence. The synthetic peptides often resemble sequences occurring in a-chains of collagen, but the action of an enzyme on such peptides should not be used as a sole criterion for collagenases. The sine qua n o n for a collagenase must remain its capacity for attack on undenatured collagen, including attack on the portion of the collagen structure characterized by the polyproline-type of helix.
[44]
COLLAGENASES
615
Another problem in definition of a collagenase arises from the nature of the collagen used for study. Although collagens throughout the animal kingdom have certain features in common, there are widely disparate aspects of their composition. Thus a single collagenase could act with different rates on different species of collagen, and in certain cases with a modified specificity. A few words should be said about the inferred functions of collagenases. Most of the described collagenases are found as secreted extracellular enzymes. In higher organisms, some collagenases purportedly are lysosomal in origin, although again their action on tissue collagen may be extracellular. Generally speaking, the microorganisms producing collagenases are host-invasive, and presumably the enzymes contribute to pathogenecity by allowing the organisms to penetrate a connective tissue barrier. In this group of organisms are the Clostridia, M. tuberculosis, and certain fungi. In higher organisms, collagenases appear to be elaborated by specific cells involved in repair and remodeling processes; thus we have the classic example of a collagenase involved in resorption and remodeling of the back skin and tail of the tadpole. The elaboration of collagenases, whether by microbes as a mechanism of invasion or by higher organisms undergoing remodeling, generally is accompanied by elaboration of other enzymes capable of acting on different connective tissue components or membrane structures. Thus collagenases may occur together with elastases, specific peptidases, hyaluronidases, and phospholipases. The collagenases have proved to be of considerable use to biochemists and biologists in general as investigative tools. In the field of collagen research, the highly specific clostridial collagenase has proved of inestimable value in mapping out the repeating nature of amino acid sequences in a-chains. It has been used to localize the aldehyde and carbohydrate functional groups of the tropocollagen molecule. The tissue collagenases have provided limited fragments of collagen amenable to sequence studies in connection with the cyanogen bromide cleavage technique. These fragments have helped to define the nature of collagen dissolution in repair and remodeling processes. Another important use of collagenases has been for the preparation of single cell types; for example, the employment of bacterial collagenase to liberate ~-cells of the pancreatic islet tissue has aided in proving the elaboration of a proinsulin. In accord with the definition of collagenases used here, we have suggested that definitive assays ultimately must be based on the use of undenatured collagen as a substrate. Generally, physical criteria for collagenolytic activity must be used; these represent either dissolution
616
COLL~ENAS~S
~.~ ~
[44]
~~.~
.~
r~
H
¢1
~t
~" .~, ~ . ~
~ ~.~'~.~ 0
0
o
i
o
"B o
[44]
COLLAGENASES
617
,.-, v
v •
~'~
~
o
!
r,.)
.-
p.,
o ~,..~
=..~
c~'~
~o'~,
~
,.., ®
~ .-a "a
2 ~'~
~.~
0~
~'..
.'7 ;a 23 © o~
•
•~ ~ k o ~
~
~
~, ~.~
"~.
8- ' " o~ ,-¢s
.~
~'
g.
:~
o~
o'= I ~
t
•
t
r,~
1.4
(..)
g,
g r,.)
r,,.)
I::1 Q:,
I ~ ..c:I
618
[44]
COLLAG~.N.~SF~
~
Cq
co o¢ v
.~-
~
~.~
~ ~ °.~
~
@
,~
0
r..}
ce
f
~
N g
0.~
I~
[44]
CO~,h~GE~ASES
~
.~.~ .. ~ .~
~ ..~
'~' ~ -~
O~
o ~~ ~
~
. ~ -~ ~
.
-.= 0
-~
~~ ~
•
,,:
,.
~= ~ . = ~ ~ ~
.~
~
~
~0
~'~ ~'~
~
O~
~ ~ 0 . ~ - o
~ ~
~_~r~ .
.
~.
619
~
~ '~
~
~
,.~ , ~
~
~'~ ~ q
"
~
-o ® o ~~ _ ~~ - ~ ~ ~ " • "
~
"
~
~
~o
"
~
o
• - ,~~
~ •
~
" ~-~ ~
~
.
.
~:
-~
" "~
'
~.~
•
620
COLLAGENASES
~4]
of insoluble collagen or a change in one of the physical properties of collagen in solution. Dissolution can be quantified by examining the supernatant fluid either with a biuret-phenol reagent or by ninhydrin reactivity. Action on ~eollagen in solution can be measured most conveniently by a decrease in viscosity as the enzyme digests the substrate, although change in optical rotatory activity could also be used. A word of caution is i;equired regarding assay of collagenases in the impure form. 2,3 Tissues elaborating a collagenase generally also contain peptidases that carry out the further degradation of peptides produced by the primary collagenolytic action. These peptidases ultimately should prove to have specificities for amino acid sequences found in a-chains, and their presence may be detected by action on synthetic peptides with sequences of this nature. Thus, in course of the preparation of the tadpole collagenase, one can separate a peptidase that cannot act on collagen but can cleave the synthetic peptides, Z-Gly-Pro-Gly-Gly-Pro-Ala-OH and PZ-Pro-Leu-Gly-Pro-D-Arg-OH, both characterized by sequences similar to those found in collagen. 2 Table I lists a group of enzymes that may qualify as collagenases according to the criteria discussed above. Only a few of these have been isolated in fairly purified form, and the others have been detected by one or another of the assay procedures used for collagenases. The only collagenase available commercially, and indeed the only one prepared thus far in quantity, is the collagenase of Clostridium histolyticum. In the following sections we describe: (1) a general method employed for screening and preparation of eollagenases in a variety of animal tissues, namely a surviving cell tissue culture method; (2) the preparation and properties of purified tadpole collagenase obtained by use of the tissue culture method; and (3) the preparation and properties of the collagenases of Clostridium histolyticum. Surviving Cell Tissue Culture M e t h o d
This method was introduced by Gross and Lapiere ~8 in 1962. Direct extraction procedures for collagenases, although applicable in a few cases involving animal tissues, 26 frequently fail because the enzyme is either absent or present in minute quantities unless its synthesis is induced or stimulated. Extraction also may be difficult because of the tight binding of collagenases to insoluble matrices of collagenous material. In the tissue culture method, the medium employed contains balanced salts, glucose, and antibiotics to limit bacterial contamination. When a s E. Harper and J. Gross, Biochim. Biophys. Acta 198, 286 (1970). 3 H.-G. Heidrich, D. Prokopov£, and K. Hannig, Z. Physiol. Chem. 350, 1430 (1969).
•-~TSee Table I, p. 619. ~J. Gross and C. M. I~piere, Proc. Natl. Acad. Sci. U,S. 48, 1014 (1962).
[44]
COLLAGENASES
621
tissue capable of producing a collagenase is maintained in this medium, the enzyme is detectable on the third day of incubation. The enzyme probably digests endogenous collagen in the tissue and is then leached into the medium. The method in this or somewhat modified form has been used to demonstrate the production or preparation of specific collagenases in a number of tissues, including bullfrog tadpole tail fin, back skin and gills, 2° newt regenerating limbs, 37 goat bone, 2s rabbit and guinea pig wound tissues, 33 human bone, 2s human synovium, 29,39 cholesteotoma, 4° postpartum uterus, ~1 and skin. ~7 The general method is described below in its specific application for the preparation of bullfrog tadpole collagenase. 2°
Preparation of Collagenase from Bullfrog Tadpoles Bullfrog tadpoles (Rana catesbiana), ranging in length from 3 to 5 inches, are exposed to a mixture of penicillin (300,000 units), streptomycin (0.25 mg), and chloramphenicol (100 mg) per liter of aquarium water for 24 hours prior to removal of back skin and tail fins under sterile conditions. These tissues are then cut into thin strips about 1 mm wide, spread upon filter paper disks floating in 10 ml amphibian Tyrode's solution in 105 mm petri dishes, and cultivated at 37 ° in a imoist atmosphere containing 5% C02-95% 05. The tissues from four animals fill one dish; the tail fins and back skins are kept separate. The culture medium has the following composition: NaC1, 8 g; KC1, 200 mg; CaCl~, 200 rag; NaH2P04, 50 mg; NaHC03, 1 g; glucose, 2 g; penicillin, 230,000 units; streptomycin, 200 mg; and water, 1500 ml. At intervals of 24 hours the fluid in each dish is assayed for collagenolytic activity and examined for bacterial contamination by use of a phase microscope and by bacterial culture. Contaminated cultures are discarded. Enzyme activity usually reaches a peak after 3 days of incubation, and the medium at this time is processed for preparation of the enzyme. The medium is centrifuged at 25,000 g for 15 minutes at 2 °. The medium is collected and replenished for each 24 hours subsequent to the first collection. The centrifuged medium is dialyzed against 0.01 M Tris buffer containing 0.001 M CaCl2, pH 7.4. The material is then lyophilized. Crude lyophilized powder (1 g) is dissolved in 50 ml of Tris buffer, 0.05 M, pH 7.4, containing 0.005 M CaC12 (temperature about 2°). The solution is centrifuged in the cold at 10,000 g for 10 minutes and the residue discarded. To the clear amber supernatant solution in an ice ~J. M. Evanson, J. J. Jeffrey, and S. M. Krane, J. Clin. Invest. 47, 2639 (1968). 4oM. Abramson, Ann. Olol. Rhinol. Laryngol. 78, 112 (1969).
622
COLhAG~.NASF_,S
[44]
bath, solid ammonium sulfate is added to 20~ saturation, and the preparation is allowed to stand for 30 minutes. Any visible precipitate is removed by centrifugation. More ammonium sulfate is added to 50~ saturation and the suspension allowed to stand for 1 hour before removing the precipitate in the centrifuge at 10,000 g for 10 minutes. The supernatant solution is discarded. The precipitate is dissolved in 5--10 ml of Tris-CaCl2 buffer, dialyzed against another Tris buffer, 0.01 M, pH 7.4, containing 0.2M NaCl and 0.005 M CaCl2. The material is then centrifuged to remove any undissolved residue. Gel filtration is then carried out on this solution. This is accomplished in a water-jacketed column (2 °) of Sephadex G-200 or 8% Agarose. The column dimensions are 1.5 X 65 cm. Tris buffer, 0.01 M, pH 7.4, containing 0.2M NaC1 and 0.005 M CaCl~, is used for elution. The eftiuent is monitored at 230 and 280 nm, respectively. Collagenolytic activity is determined for each tube, and the active contents are pooled. The final purification steps require the use of the gel slab electrophoresis method of Raymond 41 using the buffer and gel systems of Davis '2 and Ornstein, ~s but substituting riboflavin (0.0005%) for am-. monium persulfate as described by Brewer '~ and by Fantes and Furminger. ~5 The enzyme solution obtained after the gel filtration step is layered on the gel slab and run at 4 ° and 10 mA. The slab is then cut into slices at the left and right ends and in the center to provide guide strips for staining. The removed strips are stained with amido black, washed and then returned to their positions in the gel slab to permit marking out of the protein bands. The protein bands from the unstained portions are removed and eluted with Tris-CaCl~ buffer of pH 7.4. The materials are then assayed for collagenolytic activity. The enzyme has also been purified using DEAE-cellulose columns,s° but appears to be less stable under these conditions. The enzyme appears unstable to manipulations such as lyophilization and, in solution, to repeated freezing and thawing.
Assay Systems for Collagenases Prepared in Surviving Cell Cultures At least three types of assay systems have been described. These include one type, not elaborated in this discussion, called the "change of opacity assay". 2° In this assay, collagen is acted on by the collagenase and extent of activity determined by the subsequent failure of the collagen, under defined conditions, to form fibrils. ~18. Raymond, Clin. Chem. 8, 455 (1962). B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964). 4SL. Omstein, Ann. N.Y. Avad. Sci. 121, 321 (1964). ~J. M. Brewer, Science 156, 256 (1967). K. H. Fantes and I. G. S. Furminger, Nature 215, 750 (1967).
[44]
COLLAGENASES
623
Viscosity Assay. This is a variation of the viscosity method introduced by Gallop et al. 4e and described in detail by Seifter and Gallop in another volume of this series. 4 The modification employed usually utilizes guinea pig skin collagen (acid-extracted) dissolved to a concentration of 0.15% in buffer, pH 7.4, containing 0.05 M Tris and 0.4 M NaC1. The reaction is conducted at 27 °, and the drop in specific viscosity with time is recorded. The assay does not provide a measure of units of enzyme activity. If a measure of specific units is required, considerations described by Seifter and Gallop 4 must be employed. Assay Based on Release o] 14C-Glycine-Containing Peptides. This assay, designed by Gross and his colleagues, 18,~° is rapid and extremely useful, but requires use of 14C-labeled collagen and counting equipment. The substrate is prepared from the skins of guinea pigs previously injected with 14C-glycine. The collagen is isolated from the skins by standard procedures of extraction with acetic acid2 s Gels prepared from 200 ~l of collagen solution containing about 400 ~g of collagen (8001000 cpm) in 1 ml plastic centrifuge tubes are incubated for 12 hours at 37 ° in a water bath. The tubes must be scrupulously clean so that the resultant gels will be equally opaque. The gels are disrupted with a steel needle, diluted with 200 ~l of 0.1 M Tris buffer, pH 7.4, containing 0.001 M CaC12, and then reincubated for 1 hour at 37 °. The enzyme solutions to be tested are then added, and the tubes gently agitated and reincubated for various times. The reaction is stopped by addition to each tube of 100 ~l of 0.6M EDTA; incubation is then continued for 1 hour. The tubes are centrifuged at room temperature for 15 minutes at 20,000 g to separate the undissolved collagen. An aliquot of the solution is assayed for radioactivity in a scintillation or gas flow counter. The activity is expressed as counts per minute released in the reaction mixture minus the counts per minute in the supernatant fluid of control gels. A solution assay with ~4C-collagen can also be employed. ~° The preparation of the assay mixture is identical with the fibril method, except that preincubation at 37 ° to form the gel is omitted. The enzyme is added to the collagen solution and incubation is carried out at 33 ° in solution phase rather than in gel phase. At the end of the prescribed periods of time, phosphotungstic acid is added to a final concentration of 0.01 N and HC1 to a final concentration of 0.5 N. The unreacted collagen is precipitated and products of enzymatic digestion remain in solution. The material is centrifuged at 15,000 g for 5 minutes, and the supernatant fluid is counted for radioactivity. Again, enzymatic activity is expressed in terms of counts per minute released in the reaction mixture minus the counts per minute in the supernatant fluids of control gels. P. M. Gallop, S. Seifter, and E. Meilman, J. Biol. Chem. 227, 891 (1957).
624
COLLAGENASES
[44]
Properties of Tadpole Collagenase The purified enzyme has an optimum activity between pH 8 and 9.20 As prepared, 2o the enzyme contains a small amount of caseinolytie activity, presumably an impurity, but curiously inhibited in a parallel manner by conditions causing inhibition of collagenolytic activity. Both, for instance, are inactivated by heating for 10 minutes at 50 ° and 60 °, and both are irreversibly destroyed at pH 3.5. Both activities are inhibited by 0.002 M EDTA, the inhibition being reversible by dialysis against solutions of CaC12. Cysteine, 0.005 M, reversibly inhibits both activities. Diisopropylphosphorofluoridate in concentrations as high as 10-2 M has no effect on either collagenolytie or caseinolytie activity. 2° In all of these properties the tadpole enzyme fully resembles the collagenase of Clostridium histolyticum, discussed below. Specificity o] Tadpole Collagenase. Unlike the eollagenase of Clostridium histolyticum which degrades collagen or gelatin to a great number of small peptides, the tadpole enzyme cleaves the collagen molecule across the three strands at one point in each strand. 21 Thus, native rat skin collagen is cleaved into two. unequal pieces, both retaining the triple helical collagen structure. 21 The larger piece, called TC A (denoting a portion of the tropocollagen molecule containing the end designated as A by electron microscopists), represents 75% of the molecule, while the smaller piece, TC B (arising from the B end of the tropocollagen molecule as designated by electron microscopists), represents 25% of the original molecule. The two pieces of the cleaved collagen molecule can be separated or, alternatively, the mixture can be denatured by mild heat and fractionated to yield corresponding pieces of cleaved a-chains, named according to the chain from which they derive and the end of the molecule (A or B). Thus, the smaller pieces, designated al B and a2 B, are each 24,000 molecular weight, and the 'larger pieces, al A and a2 A, are each 71,000 molecular weight. The A ends contain the original N-terminals of the a-chains. Figure 1 demonstrates exquisitely the action of tadpole collagenase on collagen as viewed by electron microscopy. The technique of using enzymes to cleave collagen, then reconstituting the pieces as in this instance, and finally aligning electron photomicrographs as shown here, is proving of inestimable aid in locating the site of enzymatic action. Nagai et al29 reported that the action of the tadpole collagenase on native collagen liberated only C-terminal glycine and N-terminal leucine and isoleucine. On denatured collagen (gelatin), they found more extensive cleavage and liberation of N-terminal leucine, isoleueine, valine, alanine, and phenylalanine; glycine was the only C-terminal amino acid
[44]
COLhAOENASES
625
A
b 22 - -
B FIo. 1. Electron microgaph of reconstituted SLS (segmented long spacing) fibers of collagen before and after the action of tadpole collagenase. The photograph to the left shows the alignment of complete tropocollagen molecules with A and B ends labeled. The photograph to the right shows the TC A fragment obtained after action of the enzyme, and demonstrates that the tropocollagen molecule is cleaved to yield a segment from the A end comprising about 75% of the molecule. Another fragment, not shown here, is obtained and comprises the remaining 25% from the B end (TC~). (Courtesy of Jerome Gross.) found in this instance also. Certain tripeptides with a G l y - L e u - X sequence were found to be weakly cleaved at the G l y - L e u linkage. Synthetic peptides known to be cleaved b y clostridial collagenase were not cleaved b y the tadpole enzyme. 2'19 However, the exact specificity of the tadpole collagenase is y e t to be determined since it is not definite t h a t all peptidases are excluded from the preparation. Collagenases of Clostridiurn histolyticum This enzyme has been described in an earlier volume of this series. 4 I n this article we shall present newer methods for the purification of collagenases A and B, and newer information concerning their properties and mode of action. We shall not include a description of the viscosity
626
COL~ENAS~.S
[44]
assay fundamental for the study of collagenases here since the method as presented in the earlier volume 4 can be used without modification. Nor shall we report the method of preparation of the collagen substrate since that also is given in another volume of this series27 Culture of Clostridium ldstoiyticum In our laboratory we have maintained and used a stock culture of Clostridium h i s t o l y t ~ m 47Q5 obtained from Dr. George Warren of Wyeth Laboratories. The bacteria were grown in the medium of Warren and Gray 48 with modifications introduced by Takahashi and Seifter 49 as described below. Other strains of the organism have been used for commercial preparations of collagenase. Takahashi and Seifter 4~ have found that the sodium thioglycolate used~ as a reducing agent in the culture medium can be replaced altogether or in part by other reducing agents or conditions. Thus, an atmosphere of nitrogen, an atmosphere of hydrogen generated with sodium borohydride, or inclusion of sodium bisulfite can promote growth of the organisms and production of collagenase. Since the organism generates its own reducing medium after a lag period, one may even omit reducing substances other than the glucose of the medium. For best results, however, the medium of Warren and Gray ~8 modified to include 0.5% sodium hisulfite and 0.5% oxidized sodium thioglycolate, should be used. The use of this medium yields collagenases of uniformly high specific activity and considerably diminished amounts of general, so-called "nonspecific," proteases. This latter point is of great importance, since the most difficult problem in purification of bacterial collagenase is separation from other proteases in the culture medium. An inoculum is prepared in 500 ml of medium, and after 24 hours it is added to 3 liters of fresh medium. After incubation for 48 hours at 24°-25% maximum yield of enzyme is obtained. Preparation and Purification of Collagenases A and B
The following procedure, taken from the doctoral dissertation of Harper, 11 can be used for 8--10 liters of culture medium after growth of the organisms. The bacterial filtrate or centrifugal supernatant fluid after 48 hours growth is treated with solid ammonium sulfate to 90% saturation. The mixture is allowed to stand overnight at 4 ° and then either filtered through Celite 545 filter aid at room temperature or centrifuged at 13,200 g for 20 minutes at 10 °. The resulting precipitate is dialyzed against successive changes of water at 4 ° until free of am4' P. M. Gallop and S. Seifter, Vol. VI [196]. eG. H. Warren and J. Gray, Nature 192, 755 (1961). 4oS. Takahashi and S. Seifter, Bioehim. Biophys. Acta, in press.
[44]
COLLAGENASES
627
monium sulfate, lyophilized, and then dissolved in distilled water. Alternatively, the precipitate is not dialyzed but dissolved in water and clarified by centrifugation. In either case the resulting aqueous solution is then placed on a column (2.5 X 85 cm) of Sephadex G-50 (fine). The column is eluted with water at room temperature and 5-ml fractions are collected at the rate of 60 ml/hour. Elution is monitored by measurement for protein at 280 nm and for enzymatic activity. The screening assay consists of incubation with 2 ml of 2% gelatin in water in 13 X 100 mm test tubes for 15 minutes at 37 °, and observing the degree of gelation subsequent to immersion in an ice bath for 5 minutes. If enzyme is present, proteolysis occurs and gelation does not take place. Fractions containing enzymatic activity by this assay are pooled, lyophilized, and the pool assayed viscometrically. The protein is dissolved in 0.06M sodium-phosphate buffer, pH 7.4, and the solution placed on a DEAE-cellulose column of 1.5 X 17 cm. Elution is then conducted using the same phosphate buffer followed by 0.6 M acetate buffer of pH 5.6. Fractions (2 ml) are collected at the rate of 60 ml/hour. Two enzymatically active protein fractions, labeled respectively collagenase A and collagenase B, are obtained. These fractions are dialyzed against distilled water at 4 °, lyophilized, and again assayed for enzymatic activity. Table II summarizes a typical purification by this method. The preparation is free of contaminating activities against casein, denatured hemoglobin, benzoyl arginine amide, and several glycosides. ~ Collagenase A, prepared by this method from a culture medium containing sodium thioglycolate but no sodium bisulfite, often is obtained in specific activity of 600 viscometric units ~ per milligram of protein, and collagenase B with specific activity of 300 to 400 units per milligram protein. Takahashi and Seifter, '9 starting with a culture filtrate from a medium containing both sodium bisulfite and oxidized sodium thioglycolate, were consistently able to obtain collagenase A with a specific activity of 1100 viscometric units per milligram of protein. The modified procedure used by them, starting with 10 liters of growth medium, is as follows. The bacterial filtrate or centrifugate obtained after 48 hours' growth is treated with solid ammonium sulfate to 90% saturation. The mixture is allowed to stand overnight at 4 ° and then filtered through Celite 545 filter aid. The filtrate is discarded, and the cake is extracted 3 times with water to give a total extract of about 50 ml. The extract is then subjected to pressure dialysis at 4 ° using an Amicon membrane to exclude material of 50,000 molecular weight. The concentrate (about 30 ml) is placed on * Note added in proof. A personal communication from Dr. M. Bernfield indicates that the preparation is active against chondroitin sulfate.
628
COLLAOENASES
[44]
TABLE II SUMMARY OF PURIFICATION PROCEDURE
Total protein (nag) ~
Crude (supernatant fluid) 90% saturation with (NI-h)~S04 (precipitate) Sephadex G-50 (eluate) D E ~ l l u l o s e (eluate) Fraction A (elution with phosphate) Fraction B (elution with acetate)
Units per mg
protein
Initial yield Total units (% of units)
834 650
28 30
23,400 19,500
100 83
125
98
12,250
52
12
525
6,300
27
10
286
2,860
12
Protein determined using method of Lowry et al. (See text footnote 61.) a column of Sephadex G-100 (3 X 100 era), and eluted with 0.005 M Tris buffer, pH 7.5, containing 0.005 M CaC12. Elutjon is at room temperature, and 5-ml fractions are collected. Fractions are monitored for protein content and screened for enzymatic activity as described above in the first method of preparation. The pattern of elution from the Sephadex column is characterized by four major protein peaks, the second of which contains most of the collagenolytic activity. The contents of the tubes comprising this peak are pooled and concentrated by pressure dialysis using an Amicon 50,000 membrane. The concentrate (approximately 30 ml) is placed on a DEAE-cellulose column (1.5 X 17 cm) and eluted with a gradient of Tris buffer, pH 7.5, constantly 0.005 M with respect to CaCI2; the Tris concentration varies from 0.01 to 0.2 M. Collagenase k elutes in the region of 0.1 M 'iris, and the contents of the peak in this region are combined. The pooled material is then concentrated by pressure dialysis, again using the 50,000 Amicon membrane. The concentrate is then lyophilized, although it has been kept active in solution at 2 ° for 1 month. Collagenase A, prepared in this manner, is free of contaminating "nonspecifie" proteases, of amidase, and of certain glycosidases. It resembles fully the collagenase A prepared by the first method, except that it consistently has the higher specific activity of 1100 viscometric units per milligram of protein. Collagenase B can also be obtained from the DEAE-cellulose column, but its characteristics after this kind of separation have not yet been studied. Physical Properties of Collagenases A and 13
Prepared by the method described by Harper et al., collagenase A was shown by sedimentation equilibrium analysis to have a molecular
[44]
COLLAGENASES
629
weight of 105,000 and collagenase B to have a molecular weight of 57,400. 50 Thus, collagenase A is similar in molecular weight to preparations described by Seifter et al., 6 Mandl et al., s Yoshida and Noda, 1° Strauch and Grassmann, 51 and Levidkova et al22 Yoshida and Noda 1° described a second clostridial collagenase, perhaps identical with collagenase B, to which they assigned a molecular weight of 79,000. Seifter et al., using another method of preparation, had obtained a collagenase, most probably collagenase A, with a sedimentation constant of s°20.w -= 5.4S and a diffusion constant of D°0.~ ----4.3 X 10-7 cm2/ second. An isoelectric point of 8.6 has been reported by Nordwig. 5 Using another preparation, Levdikova et al. ~2 reported a diffusion constant of 5 )< 10-~ cm2/second. Possibility of Subunits of Collagenase A
Levdikova et al2 ~ suggested the possibility that clostridial collagenase is composed of subunits. They presented evidence to show that a basic structural unit of the enzyme is an inactive "monomer" of molecular weight of 25,000. Thus, in their hands, a collagenase preparation of 100,000 molecular weight could be dissociated into 4 molecules of 25,000 molecular weight. This dissociation was effected by exposure of the enzyme ~o either pH 3 or 12, and perhaps by the use of chelating agents. Harper et al2 pursued the idea of subunits of collagenases A, suggesting that indeed collagenase B could be an active dimer (MW 57,400) of the 25,000 molecular weight "monomer" of Levdikova et al. Evidence for the relationship between collagenases A and B is similarity of amino acid composition (see below), immunological cross reactivities of the purified enzymes with antisera prepared to either enzyme, molecular weight relationship between A and B, and, under certain conditions, the binding of a specific inhibitor (cysteine) in the molecular ratios of 2:1 with collagenase A and 1:1 with collagenase B. 11,53 Arguments against a subunit relationship of B to A are primarily against the notion of identical subunits. These are that the amino acid compositions are similar but not identical, and that significant differences exist between the number of charged amino acid residues in collagenases B and A. However, it must be noted that neither enzyme has yet been prepared in crystalline form, and reproducibility of preparations used E. Harper and S. Seifter, J. Biol. Chem., in press. ,1L. Strauch and W. Grassmann, Z. Physiol. Chem. 344, 140 (1966). G. A. Levdikova, N. Orekhovich, N. I. Solov'eva, and V. O. Shpikiter, Dokl. Akad. Nauk S.S.R. 153, 725 (1963); English Transl. 153, 1429 (1964). E. Harper and S. Seifter, Federation Proc. 24, 359 (1965).
630
[44]
COLLAGENASES
for amino acid analysis has not always been of high order. The likelihood is great that the collagenases have undergone some degree of degradation or deamidation by the potent "nonspecific" proteases and amidases in the growth medium, leading to difficulty both in obtaining crystals and obtaining preparations without some degree of difference in composition. Thus the problem of subunit relationships awaits further standardization of the B as well as the A enzyme preparation. Amino Acid Compositions of C o l l a g e n a s e s Table III shows the composition of collagenases A and B prepared by the first method presented here. Similar results were reported by Harper et al2 Generally the two enzymes show few differences in composition, although the differences may prove to be significant. Their contents of glycine, tyrosine, phenylalanine, lysine, and histidine are almost identical. Among the preparations analyzed, differences in serine contents were notable but cannot be evaluated at present since hydrolysis was done only at one time. A large difference in proline contents between collagenases A and B was noted, and as yet has not been rationalized on the parent-subunit hypothesis. Of special significance for many studies with the collagenases is the absence, first noted by Grant and A l b u r n / o f sulfur-containing amino TABLE III AMINO ACID COMPOSITIONS OF COLLAGENASES A AND B a
Aspartic acid Threonine Serine Glutamic acid Proline
Glycine Alanine Valine Isoleucine
Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan Amide
Collagenase A
Collagenase B
159 54 27 85 56 96 73 66 56 73 50 50 103 16 37 7 67
170 60 32 93 40 96 68 60 61 74 48 49 103 18 29 6 not done
a Residues per 1,000 total residues; uncorrected for losses during hydrolysis.
[44]
COLLAGENASES
631
acids. Thus inactivation of the enzymes by reagents containing - - S H groups cannot be attributed to disulfide exchange or reduction. Functions of C a 2+ Ions in Collagenolytic Action
Ca 2+ ions are required for both the binding of the enzyme to the collagen substrate and for full catalytic activity. Magnesium ions cannot substitute for calcium ions.4~ Thus agents such as E D T A inhibit collagenolytic activity in the first instance by binding calcium46; this is true for both eollagenase A and B? M a n y studies indicate that calcium ions are required to keep the enzyme in a conformation necessary for its catalytic function. Thus Takahashi and Seifter54 showed that collagenase A can be photoinactivated in the presence of methylene blue, and that enzymatic activity is fully lost after destruction of 4 of the total of 16 residues of histidine; calcium ions, but not magnesium ions, were shown to offer strong protection against this inactivation. Furthermore, as will be discussed under inhibitors, the inactivation of coUagenase A by cysteine proceeds differently in the presence or absence of calcium ions, being in the one case reversible by dialysis and in the other case seemingly irreversible. Also, as will be discussed, there is much evidence that an intrinsic metal component, most likely a zinc atom, is present at the active site of collagenase. Taken with the facts already presented, it would seem that calcium ions make the enzyme protein conform around the putative zinc atom so that the latter is chelated with two or three amino acid residues in a manner resembling that of zinc in carboxypeptidase A. If calcium ions are not present in sufficient amount, the protein conformation will be different, and perhaps only one or two of the chelating positions of the zinc atom are occupied by amino acid residues of the enzyme; this would leave two or three positions around the zinc atom able to bind other substances. Presumably one or two of these positions ordinarily binds to the substrate. One m a y understand, then, that if a multifunctional inhibitor such as cysteine is brought into the environment in absence of calcium ions, its binding would be more firm, because a different species of enzyme inhibitor should have been formed. Thus, inhibition would be less effective in the presence of calcium ions, a matter that appears to be experimental fact. Inhibition by Cysteine Maschmann 5s originally described inhibition of collagenase by cysteine, and Seifter et al2 and Harper and Seifter53 studied details of the inhibition. In addition to its inhibition by cysteine, clostridial collagenase aS. Takahashi and S. Seifter,Federation Proc. 28, 407 (1969); Biochim. Biophys. Acta 198 (1970) in press. mE. Maschmann, Biochem. Z. 295, 391 (1938); ibid. 300, 89 (1938).
632
COLLAGE~ASES
~4]
is inhibited by reduced glutathione, 56 by 2,3-dimercaptopropanol, and by dithiothreitol. It is poorly inhibited by ---SH containing substances that do not contain a second functional group capable of chelating strongly2 Seifter et al2 suggested that cysteine and similar reagents inhibit by their properties of chelation, and that they were acting on a probable zinc component in the enzyme. At pH 7.4, 0.01 M cysteine is required for inhibition. At pH 9.0, only 0.001 M cysteine is required, showing that more of the chelating species is present at the higher pH. One can infer, from the pH data, that - - S H and --NH2 groups of cysteine are most important for chelation to the proposed metal component of the enzyme. It has also been found5s that under optimum conditions approximately two molecules of cysteine chelate with one of collagenase A, and that approximately one molecule of cysteine chelates with one molecule of collagenase B. In either case the inactivated enzyme can be recycled through a column of DEAE-cellulose, and the modified enzyme, with bound cysteine, isolated. The enzyme in this condition can no longer bind to the substrate at 0 °, as can active collagenase, showing that the cysteine covers the binding site (probably the zinc atom). On the other hand, titration of the cysteine-enzyme with Elman's reagent or with p-hydroxymercuribenzoate does not show an available ---SH group, demonstrating that the cysteine is bound through that group to the enzyme. Recently, Seifter et al26a have performed the following experiment offering the strongest evidence yet that cysteine binds to zinc in collagenase. Collagenase A was prepared from a clostridial culture medium in which 85Zn was incorporated. The purified enzyme contained radioactivity and could be followed in the course of treatment with chelating agents. The esZn-collagenase was first treated with 2,3dimercaptopropanol and the solution was then passed through a Sephadex column that resulted in a eSZn-free protein, enzymatically inactive, and 65Zn-thiol complex. The zinc-free, inactive collagenase was then treated with 35S-cysteine and this solution then passed through a column of Sephadex. The elution pattern showed recovery of nonradioactive, enzymatically inactive collagenase and of all the cysteine added. Thus, prior removal of zinc from collagenase rendered it incapable, subsequently, to bind with cysteine. Inhibition by Histidine, Imidazole, and Related Compounds
Harper and Seifteff8 have demonstrated that 0.1 M to 0.05 M histidine at pH 7.4 can inhibit either collagenase A or B. Imidazole also inhibits, but less effectively. This is also true of imidazole derivatives NA. A. Tytell and K. Hewson, Proc. Soc. Exptl. Biol. Med. 74, 555 (1950). ~'S. Seifter, S. Takahashi, and E. Harper, Biochim. Biophys. Acta 198 (1970), in press.
[44]
COLLAGENASES
633
containing fewer possible chelating groups than does histidine. Inhibition by histidine is reversible by dialysis against water or by passage through suitable columns. Most importantly, inhibition by histidine appears to be competitive with a collagen substrate, so that in a viscometric assay in which histidine concentration is kept constant but collagen concentration varied, one gets typical competitive inhibition kinetics. It would seem that histidine and the substrate compete for the intrinsic metal atom of the enzyme. The degree of inhibition of the collagenases and the ease of reversibility of inhibition follow closely the stability constants known for the various inhibitors in combination with zinc as chelates. Other Inhibitors of CoUagenase Both collagenases A 6 and B are inhibited by a number of metalchelating agents, including o-phenanthroline, a~-bipyridyl, and 8-hydroxyquinoline. These are generally effective in concentrations ranging from 0.01 to 0.001 M. Their action would seem to be on the proposed metal component other than calcium. Diisopropylphosphorofluoridate does not inhibit collagenase, e eliminating this enzyme from the class of "serine proteases." Effects of Cobalt and Manganese
Yagisawa et al2 ~ had suggested some degree of activation of collagenase by cobalt. Takahashi and Seifter .9,54 have now studied the effects of metals in detail with the following conclusions. First, if cobalt ions are incorporated into the culture medium, enzyme with specific activity 1.8 times higher than that obtained in the usual culture medium can be prepared. Second, if collagenase A, prepared from the usual culture medium, is subjected to equilibrium dialysis in the presence of both calcium ions and cobalt ions, the activity of the enzyme is increased 1.8 times. Significant, but less striking results are obtained with manganese and iron ions in place of cobalt ions (Co 2÷, Mn 2÷, Fe2÷). Thus, as in the case of other zinc-containing enzymes, collagenase activity is improved by substitution of cobalt or manganese; in this case, however, calcium ions exert a strong directing effect on the replacement of the metal atom. Isolation of Peptides from the Active Center
Harper and Seifter ~ and Takahashi and Seifter ~9 have been using a double-label technique to isolate portions of the collagenase molecule, 57S. Yagisawa, F, Morita, Y. Nagai, H. Noda, and Y. Ogura, J. Biochem. (Japan) 58, 407 (1965). t~E. Harper and S. Seifter, Abstr. Am. Chem. Soc. 15~nd Meeting ~ept. 1966 p. C-39.
634
COLLAGENASES
[44]
presumably at its active center. 85Zn-collagenase A is obtained by purification of growth medium in which this isotope has been present. The purified enzyme is then reacted with 35S-cysteine, and from the mixture an inactive enzyme isolated that contains both esZn and 35S-cysteine. This is digested with trypsin and chymotrypsin, and labeled peptide fragments are isolated by column and paper electrophoretic techniques. Several relatively small peptides have been i.~olated which contain both radioactive labels; they are characterized by inclusion of residues of lysine and glutamic acid and, in one case, lso of histidine. Thus it would seem that the zinc atom is chelated to one or more basic amino acid residues and perhaps a glutamic acid residue. More work has yet to be performed to show the direct linkage of the metal atom to one or more of these residues. Binding of Collagenases to Collagen at Low Temperature Collagenase has been purified partially by taking advantage of its capacity to bind firmly at 0 ° to a suspended collagen substrate. "8 The binding is dependent on presence of calcium ions and is inhibited by EDTA. In the presence of calcium and suspended or insoluble collagen, the enzyme is adsorbed, and can be isolated as a complex which, on raising of the temperature, becomes the kinetic complex and exhibits dissolution of the collagen. Using 14C-labeled collagenase A, Takahashi and Seifter 49 have been able to isolate enzyme-substrate complexes at 0 ° which appear to be saturated when the ratio is 17 collagen molecules to 1 enzyme molecule. This is the lowest figure obtained in many experiments and is achieved by reacting enzyme and substrate in presence of the reversible inhibitor, histidine, and then dialyzing against 0.002M phosphate buffer of pH 8. The dialysis allows collagen fibrils to form in the presence of enzyme. Specificity of Collagenases A and B
Both enzymes act against native and denatured collagens to yield seemingly identical peptide mixtures. Both act against synthetic peptide substrates designed for screening collagenolytic activities, Z-Gly-ProGly-Gly-Pro-Ala-OH 59 and PZ-Pro-Leu-GIy-Pro-D-Arg-0HY ~ In general, but perhaps in not all cases, the specificity resides in a sequence, common in the collagen molecule, of -P-X-Gly-P-Y-, enzyme action occurring between -X-Gly- residues to yield a peptide with C-terminal X and always peptides with N-terminal glycin e as reviewed elsewhere. 4,5 "E. Wiinsch, Z. Phys/o/. Chem. 332, 295 (1963).
[45]
CLOSTRIPAIN
635
P (either pro or hypro) seems to be required in the 1 and 4 positions of the defined sequence. It is of interest that the collagen of the earthworm cuticle, which contrary to most collagens, contains more hypro (17%) than pro (about 1%), is also digested by clostridial collagenase, albeit to a different end point2 ° One might conjecture that the requirement for pyrrolidine residues in positions 1 and 4 of a susceptible sequence is related to a positioning of the metal atom of the enzyme between them. To sum up the status of specificity, one could say that the general sequence required has been defined but experiments with different types of collagens and with synthetic peptides demonstrate that this specificity is not so absolute that minor escapes are not encountered. A full discussion of specificity is given by Seifter and Harper? 1 Kinetics of Collagenase Action This topic was briefly considered in the chapter on collagenases in the previous volume of this series.' Yagisawa et al2 ~ have considered the kinetics of action of collagenase on synthetic substrates. Seifter and Harper 61 have reviewed in detail the kinetics of action on collagen and gelatin substrates. '*D. Fujimoto, Biochim. Biophys. Acta 154, 183 (1968). elS. Seifter and E. Harper, in "The Enzymes" (P. Boyer, H. Lardy, and K. Myrbiick, eds.), Vol. IV. Academic Press, New York, 1971.
[45] Clostripain B y WILLIAM M. MITCHELL and WILLIAM F. HARRINGTON
Introduction Clostripain (clostridiopeptidase B, EC 3.4.4.20) is a sulfhydryl proteolytic enzyme from the culture filtrate of Clostridium histolyticum. The enzyme possesses esterase, amidase, and protease activity with a highly limited specificity directed primarily at the carboxyl linkage of arginine. Although the remarkable specificity of clostripain was not reported until 1953 by Ogle and Tytell, 1 the existence of this protease 1j. D. Ogle and A. A. Tytell, Arch. Biochem. Biophys. 42, 327 (1953).
[45]
CLOSTRIPAIN
635
P (either pro or hypro) seems to be required in the 1 and 4 positions of the defined sequence. It is of interest that the collagen of the earthworm cuticle, which contrary to most collagens, contains more hypro (17%) than pro (about 1%), is also digested by clostridial collagenase, albeit to a different end point2 ° One might conjecture that the requirement for pyrrolidine residues in positions 1 and 4 of a susceptible sequence is related to a positioning of the metal atom of the enzyme between them. To sum up the status of specificity, one could say that the general sequence required has been defined but experiments with different types of collagens and with synthetic peptides demonstrate that this specificity is not so absolute that minor escapes are not encountered. A full discussion of specificity is given by Seifter and Harper? 1 Kinetics of Collagenase Action This topic was briefly considered in the chapter on collagenases in the previous volume of this series.' Yagisawa et al2 ~ have considered the kinetics of action of collagenase on synthetic substrates. Seifter and Harper 61 have reviewed in detail the kinetics of action on collagen and gelatin substrates. '*D. Fujimoto, Biochim. Biophys. Acta 154, 183 (1968). elS. Seifter and E. Harper, in "The Enzymes" (P. Boyer, H. Lardy, and K. Myrbiick, eds.), Vol. IV. Academic Press, New York, 1971.
[45] Clostripain B y WILLIAM M. MITCHELL and WILLIAM F. HARRINGTON
Introduction Clostripain (clostridiopeptidase B, EC 3.4.4.20) is a sulfhydryl proteolytic enzyme from the culture filtrate of Clostridium histolyticum. The enzyme possesses esterase, amidase, and protease activity with a highly limited specificity directed primarily at the carboxyl linkage of arginine. Although the remarkable specificity of clostripain was not reported until 1953 by Ogle and Tytell, 1 the existence of this protease 1j. D. Ogle and A. A. Tytell, Arch. Biochem. Biophys. 42, 327 (1953).
636
OTHER PROTEASES
~S]
has been known for many years. 2-11 Subsequent studies on the purification and properties of the enzyme from bacterial culture have been reported by Labouesse et al. ~2-~ Despite the unusual behavior and possible significance of such a highly specific enzyme in peptide sequence work, the difficulties inherent in handling potentially pathogenic bacterial cultures has inhibited more intensive investigation of clostripain. Recently, clostripain has been purified to apparent homogeneity from a crude commercial collagenase preparation, allowing a more readily available source of the enzyme. ~8 The following discussion outlines in detail the methods for purification from the commercial source, a simple and accurate assay procedure, and a summary of the known properties of elostripain. Standard Assay A sensitive and accurate assay of clostripain activity is obtained by measurement of the initial hydrolytic rate against benzoylarginine ethyl ester (BAE). Since the esterolysis of BAE results in an increased ultraviolet absorption by the product, benzoylarginine, a convenient means of following the initial rate is by monitoring the change in optical density at 253 nm with time. ~7 Although manual spectrophotometric determinations m a y be utilized, much greater accuracy and speed is realized by the utilization of an automatic recording spectrophotometer. Standard reaction conditions consist of sodium phosphate buffer, pH 7.8, 1~[2---0.05, BAE, 2.5 X I0 -~ M; and dithiothreitol, 2.5 X I0-3M at 25 °. If an automatic recording spectrophotometer is used, a recorder full range of 0.2 optical density units is most appropriate. A straight edge is used to isolate the initial zero-order kinetic element of the reaction~8; a molar W. Kochalaty, L. Weil, and L. Smith, Biochem. J. 32, 168,5 (1938). s E. Maschmann, Bioehem. Z. 295, 391 (1938). ~W. E. Van Heyningen, Biochem. J. 34, 1540 (1949). R. C. Bard and L. S. McClung, J. Bacteriol. 56, 665 (1948). e W. Kochalaty and L. E. Krejci, Arch. Bioehem. 18, 1 (1948). ~C. L. Oakley and G. H. Warrack, J. Gen. Microbiol. 4, 365 (1950). 81. H. Lepow, S. Katz, J. Pansky, and L. Pillemer, J. Immunol. 60, 435 (1953). oR. DeBellis, I. Mandl, J. D. M~cLennan, and E. L. Howes, Nature 174, 1191 (1954). ioj. D. MacLennan, I. Mandl, and E. L. IIowes, J. Gen. Microbiol. 18, I (1958). 11A. Nordwig and L. Strauch, Hoppe-Seylers Z. Physiol. Chem. 330, 145 (1963). ~R. Monier, G. Litwack, M. Somlo, and B. Labouesse, Biochim. Biophys. Acta 18, 71 (1955). ~B. Labouesse and P. Gros, Bull. Soc. Chim. Biol. 42, 543 (1960). 14p. Gros and B. Labouesse, Bull. Soc. Chim. Biol. 42, 559 (1960). ~B. Labouesse, Bull. 8oc. Chim. Biol. 42, 1293 (1960). W. M. Mitchell and W. F. ttarrington, J. Biol. Chem. 243, 4683 (1968). I~G. W. Schwert and Y. Takenaka, Biochim. Biophys. Acta 18, 5?0 (1955).
[4S]
CLOSTRIPAIN
637
a b s o r p t i v i t y difference of 1 1 5 0 M -1 c m -1 is u s e d t o express t h e r e s u l t s as m i c r o m o l e s of B A E h y d r o l y z e d p e r m i n u t e a t 25°. TM W i t h i n t h e e n z y m e c o n c e n t r a t i o n s a p p l i c a b l e for t h e s p e c t r o p h o t o m e t r i c a s s a y , t h e i n i t i a l r a t e s of h y d r o l y s i s a r e p r o p o r t i o n a l to c o n c e n t r a t i o n . l~urification P r o c e d u r e A l t h o u g h c l o s t r i p a i n m a y be i s o l a t e d d i r e c t l y f r o m t h e c u l t u r e illt r a t e ~° of C. histolyticum, a m o r e c o n v e n i e n t m e t h o d TM is afforded t h r o u g h h y d r o x y l a p a t i t e a n d S e p h a d e x c o l u m n c h r o m a t o g r a p h y b y u t i l i z a t i o n of t h e c o m m e r c i a l l y a v a i l a b l e W o r t h i n g t o n collagenase. Y i e l d s v a r y g r e a t l y , d e p e n d i n g on t h e lot. H o w e v e r , u n u s u a l l y high y i e l d s h a v e been o b t a i n e d recently from a "high protease" eollagenase preparation (Worthington L o t No. 95). A l l p r o c e d u r e s a r e c a r r i e d o u t a t r o o m t e m p e r a t u r e e x c e p t for t h e p r e s s u r e c o n c e n t r a t i o n a n d c o l u m n effluents w h i c h a r e collected ~ I f the enzyme is in the oxidized state prior to assay, a 30-60 second lag will generally be observed. l~j. R. Whitaker and M. L. Bender, J. Am. Chem. Soc. 87, 2728 (1965). ~0Method of Labouesse and Gros (footnote 13): Bacteria are grown anaerobically at27 ° for 25-28 hours at pH 7.4 (initial) in 10 liters of medium consisting per liter of: peptone, 60 g; KH2P04, 1.9 g; Na~HPO~.12 H20, 22 g; FeSO4"7 H~O, 0.6 g; MgS04, 12 mg; MnCl~, 6 rag. The cultures are chilled and bacteria removed by centrifugation for 15 minutes at 3500 g. Clostripain is precipitated from solution by the addition of solid ammonium sulfate to 70% saturation at 0--5° . The precipitate is suspended in 500 ml of 40% ammonium sulfate, pH 7.2, plus 20 g Hyflosupercel (Johns-Manville, New York). The suspension is filtered through a Biichner funnel and extracted with 40% ammonium sulfate until no clostripain activity is obtained. The pooled active solution (approximately 2 liters) is brought to 70% saturation and clostripain again precipitated. This precipitate is dissolved by dialysis against water and subsequently incubated in 50 mM cysteine and I0 mM CaCh at 35 ° for 4-6 hours. This incubation is terminated by increasing the temperature to 60 ° for 5 minutes. The precipitate is removed by centrifugation, with the major portion of clostripain activity remaining in the supernatant. The latter is dialyzed against pH 7.2, 25 mM Veronal buffer containing 10 mM CaCh and passed through a DEAE-cellulose column (15 X 2.5 cm) equilibrated with the same buffer. This step retains the culture pigment on the column while allowing the enzyme to pass through the column. The column wash is passed over a second identical DEAE column, dialyzed against water, and concentrated approximately 10-fold in a rotary evaporator. The concentrated solution is brought to a 1 mM final concentration of phosphate buffer, pH 7.0, and passed through a hydroxylapatite column (10 X 2 cm) which is equilibrated with the same buffer. Stepwise buffer molarity increments in 1 mM, 10 mM, and 100 mM are made every 150 ml with clostripain emerging on the final 100 mM step increase. This procedure realizes an approximately 50% yield with a 500-fold purification. The reported purification profile may be artificially high since clostripain is easily oxidized and the large purification realized by the cysteine incubation step may be secondary to clostripain reduction and concomitant activation.
638
OTHER PROTEASES
[4S]
in a refrigerated fraction collector and monitored at OD28o. A 1- or 2-g sample of the crude collagenase is solubilized, giving a 2% solution with 0.10M sodium phosphate buffer, pH 6.7. The resulting solution is clarified by low-speed centrifugation in a clinical centrifuge and applied to a hydroxylapatite column, 2.5 X 12 cm. After several column volumes of buffer are passed through the hydroxylapatite gel, the crude enzyme preparation is pumped onto the surface of the column at 60 ml per hour. This method of application prevents cracking of the gel, provided that the protein concentration is not greater than 2%. Following emergence of the initial peak, and after the optical density (~----280 nm) has decreased to about 0.1 absorbance unit, the concentration of the eluting buffer is increased to 0.20M sodium phosphate (pH 6.7) in order to elute the desired column-bound components. The pooled fractions are concentrated under N2 in an Amicon pressure cell (Amicon Corp., Lexington, Mass.) using an XM-50 filter at 5 ° and chromatographed on a column, 3.5 X 125 cm, of Sephadex G-75 in 0.05 M sodium phosphate, pH 6.7, at a flow rate of 50 ml per hour. Two distinct, well-separated peaks emerge. The first peak is eluted in the column void volume (i.e., at the position of the blue dextran marker) and contains various collagenolytic enzymes. The second peak consists of clostripain. Although the clostripain fraction at this point will generally be of high purity, some lots of Worthington collagenase contain a pigment which is not completely resolved by the above procedure and which yields enzyme preparations of low specific activity (i.e., 60 micromoles BAE hydrolyzed per minute per milligram enzyme). This situation may be circumvented by the following procedure. The pooled clostripain fraction is dialyzed for approximately 12 hours at 5 ° against 0.1 M, pH 7.8, sodium phosphate buffer containing 10-3 M dithiothreitol and applied to a second hydroxylapatite column as previously described. After initial loading, a linear gradient (0.10 to 0.30 M sodium phosphate, pH 6.7) in a total volume of 800 ml is developed. A single peak is resolved which is pigment free and of high specific activity. 21 Clostripain prepared in the above manner exhibits a high degree of purity 22 as judged by sedimentation velocity and sedimentation equilibrium ultracentrifugation, immunodiffusion, immunoelectrophoresis, and polyacrylamide gel electrophoresis. Erroneous conclusions of heterogenity may arise in polyacrylamide gel electrophoresis and gradientdeveloped hydroxylapatite columns if precautions are not taken to insure that the enzyme is fully reduced.21,22a 21Failure to fully reduce clostripain results in double peaks of identical although diminished specific activity. Although purified clostripain appears highly homogenous with respect to physical
[4S]
CLOSTRIPAIN
639
Properties of the Enzyme
pH Optimum and Ion Ef]ects The pH optimum for hydrolysis of L-arginine methyl ester in phosphate buffer is about 7.2 whereas maximum activity against a-benzoylarginine ethyl ester occurs in the range pH 7.4--7.8 in Tris-HC1 buffer. Calcium is unique among metal ions in enhancing the esterase activity (50% increase in initial rate of cleavage at 10-2M) and also acts to depress thermal inactivation. The work of Labouesse and Gros indicates that binding of calcium ion is an essential prerequisite of activity1~ whereas significant inhibition of esterase activity is observed in the presence of other cations such as Co 2÷, Cu 2÷, Cd 2÷, Na +, and K÷.TM Conversely, EDTA completely inhibits enzymatic activity at concentrations as low as 10 pJl/. Citrate, borate, Veronal, and Tris anions partially inhibit esterase activity. Sul]hydryl Requirement. The enzymatic activity of clostripain is rapidly lost on incubation in the presence of oxidizing agents such as hydrogen peroxide, but activity may be regenerated completely by reduction with dithiothreitol.TM Labouesse and Gros 1~ have investigated the effect of cysteine concentration on the esterase activity of the enzyme and report that one molecule of cysteine is required per mole of enzyme for activation; their studies also demonstrate inactivation of the enzyme by p-chloromercuribenzoate (PCMB) with complete loss of activity occurring at a 1:1 molar ratio of enzyme to inhibitor. Physical Properties. The physical properties of clostripain are listed in Table I. The molecular weight of 50,000 g/mole is large for a protease, TM but is about half the value reported by Labouesse and Gros ~3 from sedimentation-diffusion data. Since the higher value was obtained in a low salt environment, this discrepancy may be the result of dimerization in the low-salt medium. This interpretation is consistent with the measured sedimentation coefficients: s°0.w =4.43 S (high salt) TM and s 2o0 , W --6.70 S (low salt). ~3 Hydrodynamic evidence and electron -microscopy reveal the enzyme to be a globular protein of approximately 33-35.~ radiusY 3 Clostripain possesses an acid isoelectric point 24 and a low a-helix content. TM The amino acid content TM per thousand residues is structure by all analytical procedures employedto date, recent studies (W. H. Porter and W. M. Mitchell) with affinity labeling (tosyllysine chloromethyl ketone) of the active site of clostripain suggest that only a fraction of the molecules are fully active. ~a W. M. Mitchell, Biochem. Biophys. Acta 147, 171 (1967). ~W. M. Mitchell and J. J. Schmidt, Biochim. Biophys. Acta 175, 207 (1969). 5, W. M. Mitchell, Biochim. Biophys. Acta 178, 194 (1969).
640
OTHER eROTEASES
[4S]
TABLE I PHYSICAL PROPERTIES OF CLOSTRIPAIN
Molecular weight~ Sedimentation coefficient(S~o,w)• Partial specificvolume, V, calculated Intrinsic viscosity, [,1] Particle shape Particle radius (from hydrodynamicmeasurements and electron microscopy) Isoelectric point (8°) Optical rotatory dispersion (4.5°) Cotton effects minimum b0 (Moflitt) (one term Drude) % a-Helicity, estimated Helix-coil transition t e m p e r a t u r e
50, O00 4.43 S O.72 ml/g o. o53 dl/g ~spherical ~-~33-35/~ 4.8--4.9 233 in~ --19.7 ° 229 ° 30% ~50 °
a See text.
as follows: Asp14s, Thr~9, SerT,, Glu99, Pro~o, Glys2, Ala~l, Val~, Isl,~, LeuT,, Tyr35, Phe39, Lysg~, His24, Arg24, Trp~9, ½-Cyst2, Met27.
Substrate Specificity Synthetic Esters and Amides. The relative rate of hydrolysis of a number of simple substrates is presented in Table II. Among the synthetic esters and amides which have been examined, arginine-containing substrates are much more rapidly hydrolyzed than identically substituted lysine compounds. The cleavage rate of L-lysine methyl ester is anomalously low and the amides of L-phenylalanine, L-leucine, glycine, and L-proline as well as the peptides L-leueylglycine, glycylglyeine, and L-leucylglycine are not attacked. No demonstrable activity can be detected against the standard chymotryptie substrates N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester. Thus the enzyme appears to have a specificity similar to that of trypsin in that cleavage of the simple substrates occurs at the carboxyl terminal bonds of arginine and lysine but a striking preference is shown for cleavage at arginine residues. Synthetic Homopolymers. Polyarginine is completely resistant to hydrolysis by clostripain although the homopolymer is digested to diand tripeptides by trypsin. ~ This phenomenon may be explained by the observed isoelectric points of elostripain and trypsin (4.9 and 11, respectively). ~ At the pH of digestion (pH 8), polyarginine is positively charged while trypsin carries a formal positive charge and clostripain a negative charge. Thus at the pH optimum of hydrolysis, a strong electrostatic interaction of t~e charged homopolymer with clostripain may
[45]
CLOSTRIPAIN
641
TABLE II COMPARISON OF ESTERASE AND AMIDASE ACTIVITIES
FOUND IS
C. histolyticum CULTURE FXLTRATES Esters or amides
I a,~
Arginine N-Benzoyl-L-arginine ethyl ester N-Benzoyl DT.arginine naphthyl amide N-Benzoyl-L-arginine amide N-Benzoyl-L-arginine methyl ester p-Toluene sulfonyl-L-arginine methyl ester L-Arginine methyl ester D-Arginine methyl ester N-Benzoyl-L-arginine benzyl ester N-Benzoyl-L-arginine isopropyl ester
IIb
IIIb
IV
V +
+
+ + + +
+ +
+ -}-t+ -t-
+
+
+
+
Lysine p-Toluene-sulfonyl-L-lysine methyl ester L-Lysine ethyl ester L-Lysine methyl ester
_
(+)c
(+) a Others
N-Acetyl-L-tyrosine ethyl ester N-Benzoyl-L-tyrosine ethyl ester D,L-Phenylalanine methyl ester N-Benzoyl-D,L-phenylalanine ethyl ester N-Benzoyl-D,L-phenylalaninamide L-Leucine methyl ester L-Leucinamide Benzoylglycinamide Glycylglycine ethyl ester L-Proline benzyl ester L-Prolinamide Benzoylglycine phenylalaninamide Benzoylglycine leucinamide I = Nordwig and Strauchn; II = Ogle and Tytelll; I I I = DeBellis et al); IV = Mitchell and Harrington, TMV -- Gros and Labouesse?4 b Assays on relatively crude preparations. • Conversion a t 8 % the rate of the arginine homolog. d Conversion was found with this substrate. However all other hydrolyses were reported virtually complete so that initial rates and relative magnitudes of activity cannot be compared. p r e v e n t hydrolysis. T h e like charge of p o l y a r g i n i n e and t r y p s i n u n d e r n o r m a l h y d r o l y t i c conditions prevents association and thus allows cleavage to occur. T h e specificity regarding arginine versus lysine R groups is also found in the synthetic h o m o p o l y m e r s . A l t h o u g h neither polyarginine nor polylysine are h y d r o l y z e d by clostripain, both are effective inhibitors
642
OTHER PROTEASES
[45]
of the esterase activity against BAE. However, polyarginine functions as an inhibitor some two orders of magnitude more efficiently than polylysine, a phenomenon consistent with a preferential binding of the arginine moiety to the active site of the enzyme. ~6 Heteropolymers. The preference of clostripain for an arginine moiety in synthetic esters, amides, and homopolymers is also present in naturally occurring polypeptides and proteins where hydrolysis occurs most rapidly at the carboxyl terminal peptide bond of arginine. Although cleavage also occurs at lysine residues, hydrolytic rates are generally one to two orders of magnitude less. Moreover, this unusually specific protease is distinguished by its ability to hydrolyze the arginylproline peptide bond/~ a bond usually resistant to proteolysis. Conclusion Clostripain is a unique enzyme with regard to its specificity. This property may be of advantage in protein sequence work where large initial peptide fragments are desired as well as in active site studies regarding the origin of its arginine specificity. The stringent sulfhydryl conditions may prove of great interest with regard to the active site chemistry of the enzyme as well as in evolutionary comparisons with the plant sulfhydryl proteases. u W. M. Mitchell, Science 162, 374 (1968).
[46] Purification and
Assay of Thermolysin
B y HIROSHI MATSUBARA
Assay Method Principle. Two procedures will be described--casein digestion and hydrolysis of synthetic substrates. The first method is based on that of Kunitz 1 which involves digestion of casein by thermolysin under defined conditions. The digestion mixture is treated with trichloroacetic acid (TCA) to remove undigested casein. The extent of digestion is measured by the amount of unprecipitated products which contain tyrosine and tryptophan, and absorb at about 280 nm. 2 The second method uses synthetic peptides, such as carbobenzoxy(Cbz)-glycyl-L-phenylalaninamide and Cbz-L-threonyl-T,-leucinamide, as the substrates. The extent
1M. Kunitz, J. Gen. Physiol. 30, 291 (1947). The reader may refer to the methods described previously: M. Laskowski, Vol. II, p. 8; B. Hagihara, H. Matsubara, M. Nakai, and K. Okunuki, J. Biochem. 45, 185 (1958); N. C. Davis and E. L. Smith, Methods Biochem. Anal. 2, 215 (1955).
642
OTHER PROTEASES
[45]
of the esterase activity against BAE. However, polyarginine functions as an inhibitor some two orders of magnitude more efficiently than polylysine, a phenomenon consistent with a preferential binding of the arginine moiety to the active site of the enzyme. ~6 Heteropolymers. The preference of clostripain for an arginine moiety in synthetic esters, amides, and homopolymers is also present in naturally occurring polypeptides and proteins where hydrolysis occurs most rapidly at the carboxyl terminal peptide bond of arginine. Although cleavage also occurs at lysine residues, hydrolytic rates are generally one to two orders of magnitude less. Moreover, this unusually specific protease is distinguished by its ability to hydrolyze the arginylproline peptide bond/~ a bond usually resistant to proteolysis. Conclusion Clostripain is a unique enzyme with regard to its specificity. This property may be of advantage in protein sequence work where large initial peptide fragments are desired as well as in active site studies regarding the origin of its arginine specificity. The stringent sulfhydryl conditions may prove of great interest with regard to the active site chemistry of the enzyme as well as in evolutionary comparisons with the plant sulfhydryl proteases. u W. M. Mitchell, Science 162, 374 (1968).
[46] Purification and
Assay of Thermolysin
B y HIROSHI MATSUBARA
Assay Method Principle. Two procedures will be described--casein digestion and hydrolysis of synthetic substrates. The first method is based on that of Kunitz 1 which involves digestion of casein by thermolysin under defined conditions. The digestion mixture is treated with trichloroacetic acid (TCA) to remove undigested casein. The extent of digestion is measured by the amount of unprecipitated products which contain tyrosine and tryptophan, and absorb at about 280 nm. 2 The second method uses synthetic peptides, such as carbobenzoxy(Cbz)-glycyl-L-phenylalaninamide and Cbz-L-threonyl-T,-leucinamide, as the substrates. The extent
1M. Kunitz, J. Gen. Physiol. 30, 291 (1947). The reader may refer to the methods described previously: M. Laskowski, Vol. II, p. 8; B. Hagihara, H. Matsubara, M. Nakai, and K. Okunuki, J. Biochem. 45, 185 (1958); N. C. Davis and E. L. Smith, Methods Biochem. Anal. 2, 215 (1955).
[45]
PURIFICATION AND ASSAY OF THERMOLYSIN
643
of hydrolysis is measured by a colorimetrie ninhydrin method based on that of Moore and Stein 3 which estimates the number of a-amino groups liberated after hydrolysis of the glycylphenylalanine or threonylleucine bond. ~ The enzyme units are calculated for each case from a standard curve.
Reagents Casein Digestion--Method A Tris-HC1 buffer, 0.01 M, pH 8.0, with 0.01 M CaCl~ NaOH, 0.2 N CH3COOH, 0.2 N 2% Casein Solution. Two grams of casein (Hammarsten) are dissolved in 90 ml of 0.1 M Tris with the aid of 0.2 N NaOH; the pH of the solution is then adjusted to 8.0 by addition of 0.2 N CH3COOH with stirring. The total volume of the solution is adjusted to 100 ml with water. The solution may be stored at --10 ° for at least several weeks. Trichloracetic acid (TCA), 1.2 M Hydrolysis of Synthetic Substrates--Method B Tris-HC1 buffer as described above. CH3COOH, 0.1 N Substrates (5 X 10-3M). Cbz-Gly-L-Phe-amide (Mann Research Lab.) (7.1 mg) or Cbz-L-Thr-L-Leu-amide (Cyclo Chemical) (7.3 mg) are dissolved in 0.2 ml of ethanol by warming in a bath at 60 ° for 1-2 minutes. To the solution is added 3.8 ml of 0.01 M Tris-HC1 buffer equilibrated to 40 °. The final solution is kept in a bath at 40 °. Ninhydrin solution. This solution is made according to Moore2 Other preparations 6,7 give similar results. 50% Ethanol solution
Procedure Casein Digestion. The casein solution is incubated in a water bath at 35 ° for at least 5 minutes. Thermolysin solutions,8 appropriately diluted with 0.01 M Tris-HC1 buffer, pH 8.0, are pipetted into test tubes 3S. Moore and W. H. Stein, J. Biol. Chem. 176, 367 (1948). 4Refer also to the methods described previously: H. Neurath, Vol. II, p. 77; N. C. Davis and E. L. Smith, Methods Biochem. Anal. 2, 215 (1955). S. Moore, J. Biol. Chem. 243, 6281 (1968). 6 S. Moore and W. H. Stein, J. Biol. Chem. 211, 907 (1954). ~D. H. Spackman, W. H. Stein, and S. Moore, Anal. Chem. 30, 1190 (1958), To make a stock solution of thermolysin, the following procedure is convenient. About 10 mg of crystals are suspended in 4 ml or 0.01 M Tris-HC1 buffer containing 0.01 M CaCh, pH 8, in an ice bath and dissolved with 0.2 N NaOH (0.5-1.0 ml). The
644
OTHER PROTEASES
[45]
and incubated at 35 °. One milliliter of casein solution is pipetted into tubes with enzyme at proper intervals, e.g., at every 30 seconds. The solutions are mixed well and the reaction is carried out for 10 minutes, at the end of which 2 ml of 1.2 M TCA is added and, after shaking, the tubes are kept in the bath for about 30 minutes. The precipitate is filtered off through Whatman No. 1 filter paper, 7 cm, and the absorbance of the filtrate is measured at 280 nm against the blank. The blank is prepared by first mixing the casein solution with TCA and then adding the enzyme solution to the casein-TCA mixture. The standard curve for thermolysin is illustrated in Fig. 1. Two curves are shown for different temperatures. Hydrolysis o/ Synthetic Substrates. The synthetic peptide solution (0.5 ml) is incubated with the enzyme solution 8 (0.5 ml) in a test tube at 40 °, for 10 minutes. At the end of the reaction, 0.5 ml of 0.1N C H a C 0 0 H is added. Next 1 ml of ninhydrin solution is added and the mixture is placed in a boiling water bath for 15 minutes. It may be p i l l cas..35_~280 ~J pmole T~r 0.I 0.2 0.3 |
I
I
i
I
0.4 I
I
1.0 o
~o.8 .~0.6
.- 0.4
~ 0.2
SY I
/
y, I
I
1.0 I
05
I
I
3.0 I
I
I
5.0 I
B
I
1.5 ?-5 Therm01ysin,mg x I03
A
ii
FIo. 1. Standard curve for thermolysin activity using casein substrate. (A) 60 °, (B) 35 o.
p H of the solution is immediately adjusted to 8.0 with 0 2 N CH~COOH (1.0-L5 ml) and the solution is kept at --10% The activity of thermolysin remains nearly cons t a n t for over 1 month. The solution with an absorbance of 5 X 10-~ at 277 nm is suitable for the assay by m e t h o d (A), and 1 X 10-= for method (B).
[46]
PURIFICATION AND ASSAY OF THERMOLYSIN
645
D ill CGPA,40",Nin -Vipmole Leu x I02 0.5
E
1.0
1.5
1.0
•
O
i
~o.8 "o
. /i,
~o.6
/
L/J
i
Bf
i _/ i
o
= 0.4
I
. . . . . . . .
Q
I
0
I
~ ~0.2
2.0
,
2.0 4.0 6.0 Thermolysin,mg x 103
Fro. 2. Standard curve for thermolysin activity using synthetic ~ubstrates. (A) Cbz-L-Thr-L-Leu-amide, (B) Cbz-Gly-L-Phe-amide. convenient to put a small glass funnel or a marble on the top of the tube to lessen evaporation of water from the reaction mixture. The tube is cooled in an ice bath and the solution is diluted with 2 ml of 50% ethanol solution. The absorbance of ninhydrin color at 570 nm is measured against the blank. The blank is made by first mixing substrate with acetic acid and then adding the enzyme solution to the mixture. The standard curves are shown in Fig. 2.
Definition oJ Unit and Specific Activity Casein Digestion. One unit of thermolysin activity is defined as that liberating material which has absorbance at 280 nm in the casein digest equivalent to 1 micromole of tyrosine in 1 minute at 35 °. The unit is corrected by a standard curve and represented by rDTT1 t ~ ] / a m o .... l e35°,280 Tyr • Specific activity is expressed as the unit activity per milligram of protein. 9 It is calculated to be 2060 for pure thermolysin. Hydrolysis o] Synthetic Substrates. One unit of thermolysin activity is defined as liberating the a-amino groups from Cbz-Gly-L-Phe-amide equivalent to 1 micromole of leucine in 1 minute at 40 ° assayed by the ninhydrin method and represented by rP L TTlcGPA'4°°nin ~Jpmole, Leu • Specific activity is expressed as the unit activity per milligram of protein 9 and calculated to be 3.0 for pure thermolysin. 'Y. Ohta, Y. Ogura, and A. Wada, 3. Biol. Chem. 241, 5919 (1966). The present author recalculated F_~= to be 18,3 from the data reported.
646
OTHER PROTEASES
[45]
Purification Procedure
Production. The following brief procedure for the enzyme production is that described by Endo I°,11 for a semimanufacturing scale. About 40 liters of culture medium containing 3% wheat flour, I% lactose, 1.5% soy bean cake, and 0.3% casein (pH 6--7) are sterilized at 120 ° for 30 minutes in a 70-1iter tank. After cooling to 55 °, about 100 ml of a culture of Bacillus thermoproteolytics Rokko TM are added and incubated at 55 ° for 13 hours with stirring (400 rpm) and aeration (30 liters per minute). Thermolysin is excreted from the cells. The presence of zinc ion in the medium stimulates enzyme production. 13 Purification o] Thermolysin. The purification procedure is essentially that described by Endo. ~° As crystalline material is now commercially available, I' only recrystallization is required for further purification. AMMONIUM SULFATEFRACTIONATION.The culture solution (10 liters) is filtered through a filter press with the aid of radiolite (or Celite) and the enzyme in the filtrate is precipitated with solid ammonium sulfate (45% saturation at pH 7.0). The p.recipitate is dissolved in 0.02M Ca-acetate (1.5 liters) and the solution is brought to 30% saturation with solid ammonium sulfate. After centrifugation, the supernatant solution is brought to 45% saturation with solid ammonium sulfate (pH 7.0). The precipitate is dissolved in 0.02M Ca-acetate (950 ml) at pH 7.0 and the solution is dialyzed overnight against 0.01 M Ca-acetate, pH 7.0 at 5 ° . ACETONE FP,ACTIONATION AND CRYSTALLIZATION.T o the dialyzed solution (1030 ml) chilled acetone (824 ml) is gradually added and the viscous precipitate formed is removed by centrifugation. The enzyme in the supernatant solution is precipitated by the addition of acetone (750 ml). The precipitate is collected by centrifugation and dissolved in 0.02 M Ca-acetate (200 ml). Crystals appear within 30 minutes and crystallization is completed by standing at 5 ° overnight. Total recovery of enzyme activity is about 25% (approximately 1.5 g). RECRYSTALLIZATION.The crystals (or commercial preparation) (about 1 g) are suspended in 40 ml of cold 0.02 M Ca-acetate, pH 7.0, in an ice bath, and the pH of the suspension is adjusted to 11.515 by dropwise addition of cold 0.2 N NaOH (4-5 ml). The crystals dissolve completely and if any precipitate appears, the solution is quickly centrifuged (3000 g, loS. Endo, J. Ferment. Technol. 40, 346 (1962) (in Japanese). 1, S. Endo and Y. Noguchi, Japanese Patent, 5447, 1964. " A new species isolated from soil. Not registered. ,s S. Endo, personal communication. I, Calbiochem., Los Angeles; grade B (1968). pH test papers (e~., Hydrlon pH papers) are convenient.
[46]
PURIFICATION AND ASSAY OF THERMOLYSIN
647
2 minutes). The pH of the clear solution is then adjusted to 8.5-9.0 with cold 0.2 N CH3C00H added dropwise. Any precipitate formed is centrifuged off16 and the clear solution is adjusted to pH 7.0-7.2 with 0.2 N CHsCOOH. The crystals appear and grow on standing in a refrigerator (5 ° ) overnight. They grow very rapidly when the solution is kept at room temperature (25°). The crystals are collected by eentrifugation and suspended in 0.02M Ca-acetate (about 30 ml). Recrystallization is repeated as described above three or four times. The recovery of thermolysin after four recrystallizations is about 200 mg. The suspension may be lyophilized. Properties Stability. The lyophilized enzyme is very stable for months if stored in a refrigerator. The enzyme solution can be kept for weeks in a frozen state without marked loss of activity. At pH 6.5 to 8.5, full activity is observed in the presence of 0.002 M Ca-acetate even after treatment at 60 ° for 1 hour?,1°,17 The half-life of thermolysin activity at 80 ° is about 1 hour. 1°'" Without calcium ion, thermolysin is very unstable. 1° The enzyme is stable in 8 M urea, 20% ethanol or methanol, and 0.12% eetyl trimethylammonium bromide with 100% activity at room temperature, but these reagents promote thermal denaturation of the enzyme,is Slight inactivation, about 5-10%, occurs in n-butanol or n-propanol, is Physical and Chemical Properties. Free boundary electrophoresis/, lo sedimentation/ Sephadex gel filtration/ polyacrylamide gel electrophoresis, 19 and amino terminal analysis19 showed that the crystalline preparation of thermolysin was homogeneous. Ohta et al2 determined the molecular weight of the enzyme to be 37,500 by a sedimentation equilibrium method using an assumed partial specific volume of 0.73 cmS/g; the sedimentation constant, s°w----3.54-3.63 X 10-2 seconds; and the molar extinction coefficient at 280 nm is 66,300. The amino acid composition9 was reported to be Asp4s, Thr2s, Ser2s, Glu2o, Pros, Gly38, Ala2s, 1/2-Cyso, Vale, Met2, Ilels, Leu17, Tyr29, Phe~o, Lys~2, Hisg, Arglo, Trps, (NH3)ss. Another composition was also reported on the basis of a higher molecular weight. 17 The amino terminal sequence is Ile-Thr-Gly-Thr-? 9 The precipitate as well as mother liquor at every crystallization step may contain thermolysin and be saved for further purification,e.g., acetone or ammonium sulfate fractionation. I'H. Matsubara, in "Molecular Mechanism of Temperature Adaptation" (C. L. Prosser, ed.), p. 283. American Association for the Advancement of Science, Washington, D.C., 1967. 1,y. Ohta, J. Biol. Chem. 241, 509 (1966). It. Matsnbara, unpublished results.
648
OTHEa PaOT~ASES
[45]
I n h i b i t o r s . E D T A and 1,10-phenanthroline were found to be strong inhibitors. 1°,2° The enzyme activity is lost completely with 0.005M E D T A at 40 ° in 3 minutes. Oxalate, citrate, and phosphate also have inhibitory effects. Mercuric chloride and silver nitrate in concentrations of 0.005 M cause complete inactivation. 1° No inhibition was observed with diisopropylphosphofluoridate ( D F P ) ,17,20 T P C K (L-tosylamido-2phenyl) ethyl chloromethyl ketone, TM dibromoacetophenone, 19 diphenylcarbamyl chloride, ~9 potato inhibitor, 1°,2° soybean trypsin inhibitor, 2° eysteine, 2° or sodium cyanide. 2° S p e c i f i c i t y . A broad survey of the substrate specificity of thermolysin was carried out by hydrolyzing proteins, ~7,2°-24 and it was concluded that in general thermolysin hydrolyzes peptide bonds involving the amino groups of hydrophobic amino acid residues with bulky side chains, 2~ e.g., isoleucine, leucine, valine, phenylalanine, methionine, and alanine. However, it is not strictly limited to these amino acid residues. The amino sites of tyrosine, glycine, threonine, and serine residues were also found to be susceptible in some cases, although to a minor extent. These latter cases were especially observed when a longer reaction time or a higher enzyme-substrate ratio was employed to hydrolyze proteins or peptides. There is so far no clear example of the hydrolysis of t r y p t o p h a n site. The table summarizes the peptide bonds hydrolyzed by thermolysin in various proteins and peptides. Besides these common cases, several unusual hydrolyses were also observed at the bonds, Tyr-Glu, TM ThrHis, 23 Thr-Asn, 24 Ser-CySO3H, 2~ Glu-CySO3H, 24 Gly-His, 2' Arg-His, 2~ His-Asn, 26 Tyr-Asn, ~ and Glu-Thr. 28 However, it is not clear at the present time whether these hydrolyses were caused by thermolysin or b y contaminating enzymes. Several studies were conducted with synthetic substrates ~9,29-s5 and
K. Morihara and T. Tsuzuki, Biochim. Biophys. Acta 118, 215 (1966). 21H. Matsubara, A. Singer, R. M. Sasaki, and T. It. Jukes, Biochem. Biophys. Res. C6mmun. 21, 242 (1965). 22H. Matsubara, R. M. Sasaki, A. Singer, and T. H. Jukes, Arch. Biochem. Biophys. 115, 324 (1966). ~ It. Matsubara and R. M. Sasaki, J. Biol. Chem. 243, 1732 (1968). 2~R. P. Ambler and R. J. Meadway, Biochem. J. 108, 893 (1968). 2sR. J. Delange, R. M. Fambrough, E. L. Smith, and J. Bonner, J. Biol. Chem. 244, 319 (1969). '*A. Tsugita, M. Kobayashi, T. Kajihava, and B. Hagihara, J. Biochem. 64, 727 (1968). ~'D. M. Blow, J. J. Birktoft, and B. S. Hartley, Nature 221, 337 (1969). K. Dus, K. Sletten, and M. D. K.amen, J. Biol. Chem. 243, 5507 (1968). Y. Ohta and Y. Ogura, J. Biochem. 58, 607 (1965). ~H. Matsubaxa, Biochem. Biophys. Res. Commun. 24, 427 (1966).
PURIFICATION AND ASSAY OF THERMOLYSIN
[46]
649
AMINO ACID RESIDUES INVOLVED IN THERMOLYSIN HYDROLYSISa-p R~q:
Ile Leu Val Phe Ala Met Tyr Thr Set Gly
R1 Lys,Arg.Cys(Cm),'Asp,Glu,Asn,Gln,Thr,Ser, Pro,Gly,Ala,Val,Met. Ile,Leu,Tyr, Phe Lys, H is,Asp, Glu,Asn, Gin,Thr, Ser, Gly, Ala,Val, Ile, Leu, Tyr, Phe, Trp Lys,Arg,Cys(Cm),Asp,Glu,Asn,Gln,Thr,Ser, Pro,Gly,Leu,Tyr,Phe, Trp,Lys(Me) Lys,Arg, Asp,Glu,Gln,Thr, Ser, Gly, Ala,Met,Leu,Phe Lys,Arg, CySOsH,Asp,Asn, Thr, Ser, Ala, Tyr,P he" Lys,Asp, Gly, Ala, Leu,Tyr, Gln Arg, Gln,Tyr, Phe, Lys,Ala Lys, His,Arg, Ser, Tyr Arg.Ser, Ala, Tyr, Phe Thr
a H. Matsubara, in "Molecular Mechanism of Temperature Adaptation," (C. L. Prosser, ed.), p. 283. American Association for the Advancement of Science, Washington, D.C., 1967. b K. Morihara and T. Tsuzuki, Biochim. Biophys. Acta 118, 215 (1966). c H. Matsubara, A. Singer, R. M. Sasaki, and T. H. Jukes, Biochem. Biophys. Res. Commun. 21, 242 (1965). d H. Matsubara, R. M. Sasaki, A. Singer, and T. H. Jukes, Arch. Biochem. Biophys. 115, 324 (1966). H. Matsubara and R. M. Sasaki, J. Biol. Chem. 243, 1732 (1968). I R. P. Ambler and R. J. Meadway, Biochem. J. 108, 893 (1968). g K. Sugeno and H. Matsubara, Biochem. Biophys. Res. Commun. 32, 951 (1968). h A. Benson and K. T. Yasunobu, J. Biol. Chem. 244, 955 (1969). ~J. N. Tsunoda, K. T. Yasunobu, and H. R. Whiteley, J. Biol. Chem. 243, 6262 (1968). i R. J. Delange, D. M. Fambrough, E. L. Smith, and J. Bonnet, J. Biol. Chem. 244, 319 (1969). k T. C. Vanaman, S. J. Wakil, and R. L. Hill, J. Biol. Chem. 245, 6409, 6420 (1968). l T. Ando and K. Suzuki, Biochim. Biophys. Acta. 140, 375 (1967). ~' A. Tsugita, M. Kobayashi, T. Kajihara, and B. Hagihara, J. Biochem. 64, 727 (1968). n D. M. Blow, J. J. Birktoft, and B. S. Hartley, Nature 221, 337 (1969). ° W. Rombauts and H. Fraenkel-Conrat, Biochemistry 7, 3334 (1968). p K. Dus, K. Sletten, and M. D. Kamen, J. Biol. Chem. 243, 5507 (1968). q R~ and R2 represent the amino acid residues involved in thermolysin hydrolysis of a peptide bond, NH2 . . . R1 -- R~ . . . COOH. " Cys (Cm), S-carboxymethylcysteine; Lys(Me), ~-N-methyllysin. "Italics represents minor hydrolysis. 31H. 34, K. 3~K. ~K. K.
Matsubara, A. Singer, and R. M. Sasaki, Biochem. Biophys. Res. Commun. 719 (1969). Morihara and M. Ebata, J. Biochem. 61, 149 (1967). Morihara, Biochem. Biophys. Res. Commun. 26, 656 (1967). Morihara, H. Tsuzuki, and T. Oka, Arch. Biochem. Biophys. 123, 572 (1968). Morihara and T. Oka, Biochem. Biophys. Res. Commun. 36, 625 (1968).
650
OTHER PaOTEASES
[4~
supported the general conclusion obtained with natural substrates. Thermolysin requires L-configuration at the sensitive residue22 Complete absence of a free amino group in this area is essential. Slightly less essential is the absence of free carboxyl groups from the immediate vicinity of the sensitive peptide bond. 3°,32 Thus thermolysin is an endopeptidase. However, ~-amino or carboxyl groups alter the activity only slightly. ~4 The appearance of specificity is affected not only by the sensitive residue but also by the nature of at least five residues in its neighborhood. 3s This might lead to unexpected cleavages in proteins. Thermolysin does not have amidase and esterase activity, 17,2° but it has a strong elastase activity. 2° Thermolysin does not cleave the peptide bond at the amino site of a hydrophobic amino acid residue which has a proline residue at the carboxyl site, 17,~4 regardless of the presence or absence of the second residue attached to the carboxyl group of proline. 31 The specificity of thermolysin was shown to be the same at extremely different temperatures.~7, ~ The unique specificity of thermolysin led to its broad and effective use in studies of protein sequences. 23 Kinetic Properties. The Michaelis-Menten constant (K,~) and maximum velocity (Vmax) determined at 0.1 X 10-6 M enzyme, pH 8.0, and 40 °, are 19.2 X 10-3M and 161.3 X 10-6M per minute per milligram protein N, respectively, for Cbz-Gly-L-Pro-L-Leu-Gly-L-Pro; 26 X 10-3 M and 178.7 X 10-6 M per minute per milligram protein N for Cbz-GlyL-Leu-amide22 The maximum velocity of native and heat-treated (80 °, 1 hour) enzyme was the same, but the Michaelis constant is different: heat-treated enzyme showed a larger value than that of native enzyme, suggesting that no large alteration appeared to occur in its conformation. 9 Distribution. The enzymes having a similar specificity are found in various organisms: B. subtilis neutral proteinase, 36'37 Pseudomona aeruginosa elastase, 38 Staphylococcus griseus neutral proteinase, 34 Aspergillus oryzae neutral proteinases, 34 snake venom proteinase, 39 hog thyroid proteinase, 49 and Bacillus megaterium proteinase. .1
s j . D. McConn, D. Tsuru, and K. T. Yasunobu, J. Biol. Chem. 239, 3706 (1964). "J. Feder and C. Lewis, Jr., Bioehem. Biophys. Res. Commun. 28, 318 (1967). ,5K. Morihara and It. Tsuzuki, Arch. Biochem. Biophys. 114, 158 (1966). G. Pfliederer and A. Krauss, Biochem. Z. 342, 85 (1965). ~°L. F. Kress, R. J. Peanasky, and H. M. Klitgaard, Biochim. Biophys. Acta 113, 375 (1966). "J. Millet and R. Acher, Biochim. Biophys. Acta 151, 302 (1968).
~7]
PRONASE
651
N o t e A d d e d in P r o o f Readers should also refer to a spectrophotometric assay procedure which was originally applied to the Baceillus subtilis neutral protease [J. Feder, Biochem. Biophys. Res. Commun. 32, 326 (1968)]. An example of the hydrolysis at a tryptophan residue by thermolysin was observed during the sequence study of carboxypeptidase A JR. A. Bradshaw, Biochemistry 8, 3871 (1969)].
[47] Pronase
By YOSHIKO NARAHASHI Pronase is a mixture of several proteolytic enzymes, including endopeptidases and exopeptidases which are produced by a strain of Streptomyces griseus K-1.1-8 Those that have been ascertained are neutral and alkaline proteinases, aminopeptidases, and carboxypeptidase. 7 Methods for the determination of activity of these enzymes will be described below. Assay Method DETERMINATION OF PROTEINASE ACTIVITY BY THE CASEIN DIGESTION METHOD
Pr~ncip~. The casein-275 nm method, based on the method of Kunitz s and modified by Hagihara et al2 is used with further modification for the determination of both neutral and alkaline proteinases. This method determines the activity producing trichloroacetic acid-soluble product from casein using extinction at 275 nm. For the activity of both neutral and alkaline proteinases, pronase solution is directly used without pretreatment which involves inactivation of only neutral proteinases by EDTA. The distribution ratio of neutral and alkaline proteinases entails 1y. Narahashi and M. Yanagita, Sc/. Papers Inst. Phys. Chem. Res. (Tokyo) 59, 44 (1965). 2y . Narahashi and M. Yanagita, J. Biochem. (Tokyo) 62, 633 (1967). ~A. Hiramatsu and T. Ouchi, J. Biochem. (Tokyo) 54, 462 (1963). ' M . Nomoto, Y. Narahashi, T. Ouchi, and A. Hiramatsu, Abstr. Intern. Congr Biochem. 6th New York 4, 123 (1964). S. Wiihlby, Biochim. Biophys. Acta 151, 394 (1968). 6 M. Trop and Y. Birk, Biochem. J. 109, 475 (1968). Y. Narahashi, K. Shibuya, and M. Yanagita, J. Biochem. (Tokyo) 64, 427 (1968). 8 M. Kunitz, J. Gen. Physiol. 30, 291 (1947). 9B. Hagihara, Ann. Rept. Sci. Works Osaka Univ. 2, 35 (1954).
~7]
PRONASE
651
N o t e A d d e d in P r o o f Readers should also refer to a spectrophotometric assay procedure which was originally applied to the Baceillus subtilis neutral protease [J. Feder, Biochem. Biophys. Res. Commun. 32, 326 (1968)]. An example of the hydrolysis at a tryptophan residue by thermolysin was observed during the sequence study of carboxypeptidase A JR. A. Bradshaw, Biochemistry 8, 3871 (1969)].
[47] Pronase
By YOSHIKO NARAHASHI Pronase is a mixture of several proteolytic enzymes, including endopeptidases and exopeptidases which are produced by a strain of Streptomyces griseus K-1.1-8 Those that have been ascertained are neutral and alkaline proteinases, aminopeptidases, and carboxypeptidase. 7 Methods for the determination of activity of these enzymes will be described below. Assay Method DETERMINATION OF PROTEINASE ACTIVITY BY THE CASEIN DIGESTION METHOD
Pr~ncip~. The casein-275 nm method, based on the method of Kunitz s and modified by Hagihara et al2 is used with further modification for the determination of both neutral and alkaline proteinases. This method determines the activity producing trichloroacetic acid-soluble product from casein using extinction at 275 nm. For the activity of both neutral and alkaline proteinases, pronase solution is directly used without pretreatment which involves inactivation of only neutral proteinases by EDTA. The distribution ratio of neutral and alkaline proteinases entails 1y. Narahashi and M. Yanagita, Sc/. Papers Inst. Phys. Chem. Res. (Tokyo) 59, 44 (1965). 2y . Narahashi and M. Yanagita, J. Biochem. (Tokyo) 62, 633 (1967). ~A. Hiramatsu and T. Ouchi, J. Biochem. (Tokyo) 54, 462 (1963). ' M . Nomoto, Y. Narahashi, T. Ouchi, and A. Hiramatsu, Abstr. Intern. Congr Biochem. 6th New York 4, 123 (1964). S. Wiihlby, Biochim. Biophys. Acta 151, 394 (1968). 6 M. Trop and Y. Birk, Biochem. J. 109, 475 (1968). Y. Narahashi, K. Shibuya, and M. Yanagita, J. Biochem. (Tokyo) 64, 427 (1968). 8 M. Kunitz, J. Gen. Physiol. 30, 291 (1947). 9B. Hagihara, Ann. Rept. Sci. Works Osaka Univ. 2, 35 (1954).
652
OTHER
PROTEASES
~7]
two determinations, one with pronase dissolved in a buffer, which gives the sum of neutral and alkaline proteinases, the other with the pronase solution dialyzed against 0.05 M ethylenediaminetetraacetic acid disodium salt (EDTA), which measures the alkaline proteinases. The difference in the two values is the neutral proteinases.
Reagents Substrate, 2% casein. The substrate solution is made by suspending 2 g of casein (Hammarsten, "Merck" Ltd.) in 80 ml of 0.1 M sodium borate. The solution is heated for 10 minutes in boiling water and its pH is adjusted to 7.5 with 0.5 N hydrochloric acid. The volume of the solution is adjusted to 100 ml with distilled water. This casein solution should be stored in a freezer. Prior to the assay, the frozen casein is dissolved in a water bath at 40 °. Trichloroacetic acid (TCA), 0.11 M, containing 0.22M sodium acetate and 0.33 M acetic acid. Enzyme solution containing 4 to 40 ~g of pronase per milliliter in 0.1 M sodium borate~0.05 M hydrochloric acid buffer containing 5 mM calcium chloride, pH 7.5. (For the determination of neutral and alkaline proteinases.) Pronase solution dialyzed against 0.05 M EDTA. The concentration of pronase before dialysis is about 0.001-0.010%. (For the determination for alkaline proteinases.)
Procedure. To 2 ml of 2% casein in a test tube, equilibrated at 40 °, is added 2 ml of a suitably diluted enzyme solution, also equilibrated at 40 °. Four milliliters of 0.11 M TCA solution is added to the reaction mixture exactly 10 minutes after addition of the enzyme. The resulting suspension is kept at 40 ° for 20 minutes and is then filtered through Toyo No.-5B filter paper. The filter paper used should not contain materials giving absorption at 275 nm. The optical density of the filtrate is read at 275 nm. The reading is corrected for the value of blank which is mixed with enzyme solution and TCA solution before the addition of substrate. Definition o] Activity Unit. One unit of caseinolytic activity is defined as the quantity of an epzyme giving an absorbancy equivalent to 1 ~g tyrosine per minute under the conditions described. The unit is determined by the standard curve shown in Fig. 1, and represented by 1Cas.275 J ' ~ J J~g Tyr " PTT
According to this method, pronase-P, also known as pronase-research grade, shows 1,250,000 rpTTlc~.276 per gram. Several types of product L ~ " ~ J/~g Tyr
~]
PRONASE
653
0.8
~ ~ f .
eJ
0.1849 =
"~
/
.~- 0.4
=, ul
0.2
0
"
ml,-ry
•
.......
~ rlDil'lCos,275 ~-" ~ J ~g pTry
rt
8
16
24
32
40
Pronose-P ( # g / m l ) °
Fio. 1. Standard activity curve of pronase-P by casein-275 nm method. Indicated concentrations of pronase represent the concentrations of enzyme solution used for the assay, not those of enzyme in the reaction mixture.
such as pronase-E, -research grade, -AS, -AF, and -D are commercially available and graded by the Kaken Chemical Co., Ltd., Tokyo, Japan. For instance, pronase-research grade has been shown to have 45,000 PUK/g. PUK is an abbreviation for proteinase activity unit of Kaken with casein as substrate at pH 7.4. One unit of PUK has been defined as the activity of enzyme which liberates, per minute at 40 °, a digestion product equivalent to 25 ~g of tyrosine. DETERMINATION OF THE ESTERASE ACTIVITY OF PROTEINASES BY A T I TRIMETRIC METHOD
The esterase activity of pronase results from three alkaline proteinases; one of them hydrolyzes benzoyl-L-arginine ethyl ester (BAEE) and the other two enzymes hydrolyze aetyl-L-tyrosine ethyl ester (ATEE), and, therefore, this method is the most useful when alkaline proteinases are isolated from pronase. W~hlby" measured the activity of alkaline proteinases which hydrolyze acetyl-L-tyrosine ethyl ester, using p-nitrophenyl acetate as a substrate, by means of the spectrophotometric method of Huggins and Lapides. 1° Principle. The potentiometric determination of esterase activity of 1oC. Huggins and J. Lapides, J. Biol. Chem. 170, 467 (1947).
654
OTHER PROTEASES
[47]
trypsin, which was first described by Schwert et al., 11 is applied with small modification for pronase. The principle of the method is a continuous titratiton of liberated carboxyl groups from an appropriate ester, using a potentiometer. In our laboratory, Radiometer Model TTTlc in conjunction with a TTA31 titration assembly and a type SBR2 recorder (Radiometer, Copenhagen) and 0.1 N NaOH as titrating agent have been used. Reagents
Substrates. 0.1 M BAEE in distilled water or 0.1 M ATEE in ethanol. Buffer. 0.005 M Tris (hydroxymethyl) aminomethane-hydrochlorie acid buffer containing 0.04 M sodium chloride and 0.02 M calcium chloride, pH 8.0. Enzyme solution. 1% (for hydrolysis of ATEE) or 0.2% (for hydrolysis of BAEE) pronase dissolved in 1 mM hydrochloric acid. Titrating agent. 0.1 M sodium hydroxide. Procedure. All the measurements are made at pH 8.0 and at 37 °. Three milliliters of the buffer (pH 8.0) and 0.3 ml of 0.1 M BAEE or 0.3 ml of 0.1 M ATEE are pipetted into a 5-ml reaction vessel with a jacket for circulating thermostatted water and the solution is stirred with a small mechanical stirrer. A slow stream of nitrogen that had been washed in a medium similar to that in the reaction vessel is used to expel carbon dioxide. After standing for 5 minutes, the solution is adjusted to pH 8.0 by the addition of 0.1 M sodium hydroxide, and 0.1 ml of a suitably diluted enzyme solution is added to the reaction vessel. Esterase activity by this method is expressed as the amount of reacted BAEE or ATEE in micromoles per minute per milliliter of enzyme solution. Other substrates used for BAEE are tosyl-L-arginine methyl ester, benzoyl-L-arginine methyl ester, and tosyl-L-lysine methyl ester. Since the hydrolysis of ATEE is apparently of first order, the results are calculated on the basis of the initial slope. Esterase activity with BAEE may be determined by both potentiometric 12 and the spectrophotometric methods. 1~ However, the other esterase activity toward ATEE should not be determined by the spectro"G. W. Schwert, H. Neurath, S. Kaufman, and J. E. Snoke, J. Biol. Chem. 172, 221 (1948). " M . Laskowski, Vol. II, p. 36. "B. C. W. Hummel, Can. J. Biochem. Physiol. 37~ 1393 (1959).
[47]
PRONASE
655
photometric method of Schwert and Takenaka 14 because of the high blank value resulting from low affinity of the enzyme toward ATEE and of inactivation of enzyme by ethanol or methanol used for dissolving the substrate. When the concentration of ethanol or methanol in the reaction mixture is above 10%, the enzyme activity is inhibited. Therefore, it is impossible to use benzoyl-L-tyrosine ethyl ester for ATEE as a substrate since 50% methanol has to be used for dissolving the substrate. 13 DETERMINATION OF AMINOPEPTIDASE ACTIVITY OF PRONASE
Principle. It appears that aminopeptidases present in pronase have a substrate specificity similar to that of the leucine aminopeptidase from swine kidney, ~,15 and hydrolyze amino acid amides and peptide bonds of N-terminal amino acids of di-, tri-, and polypeptides whose amino group must be free. The sensitive and convenient substrate for this enzyme is L-leucylglycine. Hydrolysis of the substrate is measured by the ninhydrin method of Yemm and Cocking is for the determination of amino groups liberated. Reagents Substrate. 4.8 mM L-leucylglycine in distilled water, adjusted to pH 8.0 with sodium hydroxide. Enzyme contained 2 to 10 ~g pronase per milliliter in 0.05M Veronal sodium-0.05 N HC1 buffer containing 0.01 M calcium chloride and 0.001 M cobaltous chloride, pH 8.0. Hydrochloric acid, 0.05 N Citrate buffer, 0.2 M, pH 5.0 Citric acid (C6HsOT-H~O, 21.008 g) is dissolved in 200 ml of distilled water, mixed with 200 ml of 1 N sodium hydroxide, and diluted to 500 ml. Store in the cold with a little thymol. Potassium cyanide, 0.01 M Ethanol in water, 60% by volume Potassium cyanide-methyl Cellosolve solution. 5 ml of 0.01 M potassium cyanide are diluted to 250 ml with methyl Cellosolve. Methyl Cellosolve-ninhydrin solution. 5% (w/v) solution of ninhydrin in methyl Cellosolve is prepared. Potassium cyanide-methyl Cellosolve-ninhydrin solution. 50 ml of the methyl Cellosolve-ninhydrin solution are mixed with 250 14G. W. Schwert and Y. Takenaka, Biochim. Biophys. Acta 26, 570 (1955). 1~E. L. Smith and R. L. Hill, in "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrb~ick, eds.), Vol. 4, p. 37. Academic Press, New York, 1960. HE. W. Yemm and E. C. Cocking, Analyst 80, 209 (1955).
656
OTHER PROTEASES
~?]
ml of the potassium cyanidc methyl Cellosolve solution. The resulting solution is at first red, but soon becomes yellow. It should be stored overnight before use. Procedure. One milliliter of suitably diluted enzyme solution is incubated at 40 ° with 1 ml of L-leucylglycine soultion and the enzyme reaction is terminated exactly 10 minutes later by the addition of 1 ml of 0.05 N hydrochloric acid. After 8-fold dilution of the reaction mixture with distilled water, 1 ml of it is mixed with 0.5 ml of citrate buffer and 1.2 ml of the potassium cyanide--methyl Cellosolve-ninhydrin solution is added. The mixed solution is heated at 100 ° for 15 minutes, cooled for 5 minutes in running tap water, 3 ml of ethanol are added, and the color intensity is read at 570 nm. The reading is corrected for the. value of blank which is mixed with enzyme solution and 0.05 N hydrochloric acid before the addition of substrate. One aminopeptidase unit is defined as the amount of enzyme which catalyzes the hydrolysis of 1 micromole of L-leucylglycine (LG) per minute under the conditions described. As shown in Fig. 2, 1 g of pronase-P, research grade, has 25,000 aminopeptidase units. Aminopeptidase activity of pronase can also be determined by a method of Goldbarg and Rutenburg, using L-leucyl-•-naphthylamide as a substrateY
0.4 E 0 f,..
~58=~0m"Iciromoel
0.3
~>.
/ , ^. /m,, ,ly)~
.//
,/.
./
0.2 0.1 , ~ °
.
I unit
I
8
I
I0
Pronase- P ( ~g/rnl) FIG. 2. Standard curve of aminopeptidase activity of pronase-P.
1,j. A. Goldbarg and A. M. Rutenburg, Cancer I1, 283 (1958).
[47]
PRONASE
657
DETERMINATION OF CARBOXYPEPTIDASEACTIVITY OF PROI~ASE
Principle. This enzyme hydrolyzes carbobenzoxy-glycyl-L-leucine at the glycylleucyl linkage, z,7 Assay is conducted by the ninhydrin method of Yemm and Cocking 16 for liberated amino groups. Reagents Substrate. 0.04M carbobenzoxy-glycyl-L-leucine (CGL). In 10 ml of 0.1 M sodium borate containing 0.01 M calcium chloride, pH 7.6, is dissolved CGL (322.3 mg), adjusted to pH 7.6 with 1 N sodium hydroxide, and diluted to 25 ml with distilled water. Enzyme sample contained 10 to 100 #g of pronase per milliliter of 0.1 M sodium borate-0.05 M hydrochloric acid buffer containing 0.01 M calcium chloride, pH 7.6.
Procedure. Five-tenths milliliter of 0.2 M citrate buffer, pH 5.0, and 1.2 ml of potassium cyanide-methyl Cellosolve-ninhydrin solution are pipetted into the test tube provided with an aluminum cap. To 1 ml of 0.04 M CGL preineubated at 40 ° 1 ml of enzyme solution is added at zero time. At zero time and at 5-minute intervals, a 0.5 ml aliquot of the reaction mixture is removed and pipetted into the test tube containing ninhydrin solution (prepared previously). The procedure of color development by the ninhydrin reagent is the same as that for aminopeptidase assay. A blank value is estimated by the reaction mixture pipetted at zero time. One carboxypeptidase unit is defined as the amount of enzyme which hydrolyzed 1 micromole o.f CGL per minute under the conditions described. Figure 3 shows the standard curve of carboxypeptidase activ-
E 0.6
_/0.01 miCromole / 0.1038-t--~/ml,
\ Leu)
. /
°
c:
/./
0 I'-,i..0
0.4
./°
g .c 0.2 "/ c -
0
20
0.01 unil corboxypepfidase ,
,
,
,
40
60
80
Ioo
Pronase- P (/.~g/ml)
FIG. 3. Standard curve of carboxypeptidase activity of pronase-P.
658
OTHER PROTEASES
[47]
ity of pronase, and usually pronase-P has 610 units carboxypeptidase activity per gram. Purification Procedure Pronase is a partially purified preparation obtained from the culture broth of Streptomyces griseus K-l, purified on an industrial scale at Kaken Chemical Co., Ltd. The isolation and purification of each enzyme, neutral and alkaline proteinase, aminopeptidase, and carboxypeptidase, from pronase will be described below. Step I. Initial Group SeparationJ The best initial purification procedure developed for group separation of these enzymes is chromatography of pronase on CM-cellulose column. A column (4.1 X 40 ,cm) of CM-cellulose (100 g by dry weight, Serva, 0.57 meq/g) is equilibrated with 0.01 M sodium acetate buffer containing 5 mM calcium chloride, pH 5.2. In the pH range of 5.0 to 5.5, chromatographic behavior of pronase is independent of pH. Unless the equilibrium of CM-cellulose with the buffer is sufficient with respect to both pH and calcium ion, neutral proteinases and aminopeptidases become inactive during the chromatography. After application of 200 ml of 10% pronase solution dialyzed against the same buffer, the column is washed with about 500 ml of the starting buffer until the absorption of the effluent at 280 nm will return nearly to a base line value. After appearance of the breakthrough peak, the remaining enzymes are eluted from the column by linear gradient developed by use of 0.25 M sodium chloride in the starting buffer, as shown in Fig. 4. All procedures should be performed at 4o-5 °, and a flow rate is 12 ml/cm2/hour. Highly reproducible results are obtained under the conditions described and four protein peaks having enzyme activity are resolved. The first peak which is not adsorbed on CM-cellulose consists of neutral proteinase and an aminopeptidase. The aminopeptidase overlaps the protein peak of neutral proteinase but is chromatographically distinct from it. The second peak appears immediately after the beginning of sodium chloride gradient and contains neutral proteinase together with a small amount of aminopeptidase and carboxypeptidase. The third peak includes carboxypeptidase and two different kinds of proteinase, one of which hydrolyzes acetyl-L-tyrosine ethyl ester and the other hydroly~.es benzoylL-arginine ethyl ester. The fourth peak contains mainly a proteinase which hydrolyzes acetyl-L-tyrosine ethyl ester. Proteinases present in peaks I and II are neutral proteinase in contrast to peaks III and IV in which alkaline proteinases are eluted. Three alkaline proteinases with esterolytic activity are designated in the order of their elution from the column of CM-cellulose as alkaline pro-
[47]
6~
PRONASE
(I)
16
12
ic
/"X
:o
(m}
I"
O (xl
J
~6
.
-
3OO 200
.IQ
I00 i
4oc A ~,.30C
A~ - - - . - - ~ ' f z ~ ,
///4
,~'-'~, -~ ,-"-" A !d 40- -40
I/ 'A
-
'
," ~ ~ - ~ .
\
1,' t-.2,,~,"~_. ", ,~-,o Froction number
FIG. 4. Chromatography on CM-cellulose of pronase-P. Two hundred milliliters of 10% pronase dialyzed against 0.01 M sodium acetate buffer containing 5 mM calcium chloride, pit 52, were applied on a column (4.1 X 40 cm) of CM-cellulose equilibrated previously with the same buffer. After washing the column with 500 ml of the same buffer, a linear gradient was developed using 025 M sodium chloride in the starting buffer, pH 52. Effluent of 20 ml was collected in one tube. The top graph gives the locations of the protein, CGL and LG hydrolyzing activities. The bottom chart gives the caseinolytic activity and the esterase activities toward BAEE and ATEE. The symbols are as follows: ( 0 ) absorbance at 280 nm. ( - O - ) activity toward LG, (11) activity toward CGL, (A) caseinolytic activity, ( - O - ) activity toward BAEE, (X) activity toward ATEE.
OTItER PROTEASES
660
[47]
teinase a, b, and c; proteinase a is an enzyme which hydrolyzes A T E E and appears in peak III, proteinase b hydroly~es BAEE, and c is present in peak IV and hydrolyzes A T E E . Not only the separation of neutral and alkaline proteinases but also that of aminopeptidase and earboxypeptidase is achieved by this first group separation. The recoveries of protein, caseinolytic, BAEE-hydrolyzing, ATEE-hydroly~ing, aminopeptidase, and carboxypeptidase activities are about 90, 80, 75, 75, 90, and 75%, respectively. Step ~. Isolation o] Carboxypeptidase ]rom Alkaline Proteinases. ~ The fractions of peak I I I in the previous step are combined and dialyzed against 0.05 M sodium acetate buffer containing 0.01 M calcium chloride, p H 5.2. A column of Amberlite CG-50 resin (Rohm & Haas) is equilibrated with 0 . 0 5 M sodium acetate buffer containing 0.01 M calcium chloride, p H 5.2, and washed with distilled water. The dialyzed enzyme
I
It
/TJ
II
I
M
II
8 o
~'. . . . . . . . . .
-f
i.500
'0 JO
16o ,~o Fraction number
FIG. 5. Chromatographic separation of carboxypeptidase from alkaline proteinases on Amberlite CG-50 resin. One-sixth of the combined solution obtained from peak III in the previous step was applied on the column (13 X 25 cm) of Amberlite CG-50 resin. After washing the column with 1 liter of the starting buffer, the elution was developed by a stepwise increase of sodium acetate (0.4 M, 1 M) at flow rate of 10 ml/hour. Effluent of 5 ml was collected in one tube. Only the section of the elution pattern where the enzyme activities appeared is shown in the figure. ( 0 ) Absorbance at 280 nm, ( I ) caxboxypeptidase activity, (A) caseinolytic activity.
[47]
PRONASE
661
solution is applied to the column, the column is first washed with the same buffer, and the enzymes adsorbed are eluted by a stepwise increase in sodium acetate concentration. Proteinases are first eluted at the concentration of 0.4 M sodium acetate, carboxypeptidase is eluted at the concentration of 1 M sodium acetate. Step 3. Separation and Purification o] Alkaline Proteinase. 7 B y the methods of the first and second steps, alkaline proteinases are freed from aminopeptidase, neutral proteinases (step 1), and carboxypeptidase (step 2). However, two (a, b) of the three alkaline proteinases still remain inseparable from each other in step 2. Complete separation of these alkaline proteinases, a and b, is achieved by subsequent CM-Sephadex chromatography of the proteinase sample obtained from the previous step, and proteinase c is further purified by rechromatography on CMcellulose under the same conditions as step 1. The fractions with proteinases obtained by step 2 are combined and
E
5¸ T E
oE
Z !'~ 13D "
4!
80C ~
c 0
~3
0.3 i60C 60
~6
~2 JQ
x
I I0
20
~Z)
40
Frocf~on
,~)
60
x -x
"tO
0.2 4 0 C
40
Q I - 20C
20
0-0
0
~b 9b
number
Fro. 6. Chromatographic separation of alkaline proteinases a and b on CMSephadex. The combined enzyme solution obtained from peak III on CM-cellulose (610 ml) was dialyzed against 0.01 M sodium acetate buffer containing 0.07 M sodium chloride, pH 42. Dialyzed enzyme solution was applied on the column (2.8 X 40.5 cm). After washing the column with 250 ml of the same buffer, a linear gradient was developed with sodium chloride. Effluent of 10 ml was collected in one tube at flow rate of 50 ml/hour. ( 0 ) Absorbancy at 280 nm, (O) activity toward BAEE, (X) activity toward ATEE.
662
OTHER PROTEASES
[47]
dialyzed against 0.01 M sodium acetate buffer containing 0.07 M NaC1, pH 4.2. The fractions of peak III obtained from step 1 may also be used, and carboxypeptidase which coexists with alkaline proteinases in peak H I is denatured and removed by extensive dialysis of the combined solution against 0.01 M sodium acetate buffer, pH 4.2. The dialyzed enzyme solution is applied to a column of CM-Sephadex (C-50, capacity; 4.5 ± 0.5 meq/g, Pharmacia) previously equilibrated with the same buffer. After being washed with the same buffer, the column is eluted with a linear gradient of 0.07 M to 0.35 M sodium chloride in 0.01 M sodium acetate buffer, pH 4.2. By using this chromatographic step, alkaline proteinase a which hydrolyzes acetyl-L-tyrosine ethyl ester is first eluted and is completely separated from the other alkaline proteinase b which hydrolyzes benzoyl-L-arginine ethyl ester and is eluted at a higher concentration of sodium chloride. Recoveries of alkaline proteinase a and b in this step are about 70 and 80%, respectively, and by the use of the above steps, purified alkaline proteinase b is obtained in approximate yield of 50-60% of pronase and is purified 12-fold over pronase-P. W~ihlby ~ reported purification of three alkaline proteinases of pronase by consecutive chromatography on CM-cellulose and phosphorylated cellulose. Our effort for separation of these alkaline proteinases by using phosphorylated cellulose, sulfoethytcellulose, or rechromatography on CM-cellulose did not effect complete separation but chromatography on CM-Sephadex did. For further purification, molecular sieving with Sephadex G-100 or G-75 is recommended for each of the separated " alkaline proteinases. Step ~. Separation o] Neutral Proteinases ]rom Aminopeptidases. 7 In the CM-cellulose chromatography of pronase (step 1), a greater part of neutral proteinase was found together with aminopeptidase in the nonadsorbed fractions. For separation of aminopeptidases from neutral proteinases, the fractions with neutral proteinase in peak I are combined and the pH of the enzyme solution is adjusted to 7.0 with sodium hydroxide. The enzymes are salted out with ammonium sulfate (0.5 saturation). The precipitate is collected, dissolved in 0.005M Veronal sodium-hydrochloric acid buffer containing 1 mM calcium chloride, pH 7.6, and dialyzed against the same buffer. The dialyzed enzyme solution is then concentrated by lyophilization. The lyophilized enzyme is dissolved in the same buffer (about 5%) and is applied to a column of DEAE-Sephadex A-50 (capacity; 3.5 ± 0.5 meq/g, size; 40-120#, Pharmacia) which was equilibrated with the same buffer. The equilibrium should be checked by determining both the pH and the counter-ion concentration of the washings. Otherwise, reproducible results may not
[47]
PRONASE
663 o
q
~q 120-
~
~oo-
.~ =
l
(J
=~5
E
5
~0
~0 ~0
ee
.__
!
c
E c o
8~
:~ 0
Q~
o-
c
d °
)c
~8
c
6~
0
-5
~ 40-
~, 2~-
i
Oc
4
I
oe
/
]
0-
b
20
4'0
do
~o
~bo ,~o
Fraction
~ho t~o
~ho 2bo 2~o
number
Fro. 7. ChromatograpbJc separation of neutral proteinases from aminopeptidases. The lyophilized enzyme obtained from peak I (200 rag) was dissolved in 10 ml of 0.005M Veronal sodium-hydrochloric acid buffer containing 1 m M calcium
chloride, pH 7.6, and the enzyme solution was applied on the column (2.0 X 55 cm) of DEAE-Sephadex equilibrated with the with 1 liter of the same buffer, enzymes effluent of 10 ml was collected in one tube ancy at 280 nm, ( A ) caseinolytic activity,
same buffer. After washing the column were eluted by a stepwise method. An at flow rate of 25 ml/hour. (Q) Absorb( O ) LG-hydrolyzing activity.
be expected. After washing the column with the same buffer, the elution is carried out by a stepwise increase of concentration of the buffer and change of pH as shown in Fig. 7. The neutral proteinase found in the nonadsorbed fractions (peak I) on CM-cellulose chromatography is separated from amino peptidase and is shown to be further fractionable into three proteinase components. The amino peptidase is also separated into two fractions. The recoveries of protein, caseinolytic, and amino peptidase activities are about 90, 70, and 100%, respectively. The use of DEAE-cellulose instead of DEAE-Sephadex to separate amino peptidase and neutral proteinase was not successful. Properties 2, 7 Purity of each enzyme has not yet been established. Recently, in our laboratory, alkaline proteinase a which hydrolyzes acteyl-L-tyrosine ethyl ester was crystallized in columns, 's and its homogeneity was confirmed in free-boundary electrophoresis and column chromatography. Y. Narahashi, M. Yanagita, and K. Sibuya, Sym. Enzyme Chemistry Japan, p. 168 (196g).
664
OTHER PROTEASES
[47]
The neutral proteinases are stable in a narrow pH range of 7.0-7.5 in the absence of calcium ion, but highly stable at pH of 5.0-9.0 in the presence of calcium ion. Addition of calcium ion to the solution in which neutral proteinase is dissolved protects the enzyme from denaturation during purification procedures such as dialysis, dhromatography, and salting out, etc. The alkaline proteinase b which hydrolyzes benzoyl-Larginine ethyl ester is the most stable in 1 mM hydrochloric acid and is also stable in a buffer of pH 4.0-5.0, and becomes unstable above pH 5.5. Alkaline proteinases a and c are stable at a pH range of 4.0 to 6.5 and become unstable at pH below 4.0 and above 7.0. Aminopeptidase and carboxypeptidase are stable at pH 5.0-8.0 in the presence of calcium ion. Aminopeptidase and carboxypeptidases are both metalloenzymes, and the binding of activating metal with EDTA renders the enzyme inactive. Upon reactivation by calcium and cobalt ions, both activities are restored. Aminopeptidase is heat-stable below 80 ° but is very labile on dialysis against distilled water. Carboxypeptidase is less heat-stable but is stable on dialysis against distilled water. Neutral proteinases are completely inhibited by EDTA but not by diisopropylphosphorofluoridate (DFP), while three alkaline proteinases are inhibited by D F P but are not affected by EDTA. The alkaline proteinase which hydrolyzes benzoyl arginine ethyl ester is inhibited by 1-chloro-3-tosylamido-7-amino-2heptanone (TLCK) but other two alkaline proteinases which hydrolyze acetyl-L-tyrosine ethyl ester are not inhibited by either tosylyhenylalanine chloromethyl ketone (TPCK) or TLCK, which are specific inhibitors for chymotrypsin and trypsin, respectively.1~,2° Reporting the substrate specificity of one of the neutral proteinases by using synhhetic substrates, Morihara 21 indicated that the enzyme hydrolyzes the peptide bond containing the amino group of leucine, phenylalanine, or tyrosine. Independently, Narahashi found that the enzyme is.capable of hydrolyzing the peptide bond containing the leucine~ amino group. Some kinetic paramters of alkaline proteinase b which hydrolyze benzoyl-L-arginine ethyl ester have been studied. The enzyme hydrolyzes, in general, substrates similar to trypsin in pancreas. The K~ values are 9.0 X 10-6M for BAEE, 7.7 X 10-sM for TAME, and 1.4 X 10-SM for BAPNA.
G. Schoellmann and E. Shaw, Biochemistry 2, 252 (1963). ~E. Shaw, M. Marse-Guia, and W. Cohen, Biochemistry 4, 2219 (1965). 21K. Morihara, H. Tsuzuki, and T. Oka, Arch. Biochem. Biophys. 123, 572 (1968).
[48]
URINARY PLASMINOG~,NACTIVATOR
665
[48] Urinary Plasminogen Activator (Urokinase)1 By WILFRID F. WHITE and GRANT H. BARLOW Assay Methods Principle. Two general types of assay are available, based on (1) the specific activator of plasminogen and (2) the esterolytic activity of urokinase toward synthetic substrates. We have used an adaptation of the former method to monitor the purification process and have used the latter method for a precise evaluation ~f the finished products.
First Method (Fibrin Tube) Reagents Sodium barbital buffer, 0.05 M, pH 7.6 Bovine thrombin (17 NIH units/ml) Fibrinogen (bovine fraction I), 16 mg/ml in barbital buffer
Procedure. Transfer 0.2 ml of ten different dilutions (made with 0.1% human serum albumin, Fr. V, Pentex, Inc., Kankakee, Ill.) of the urokinase to be assayed to separate tubes. Add to each, in order, 0.3 ml bovine thrombin and 1.0 ml of fibrinogen solution. Incubate Me tubes at 37 ° for 16 hours. If the proper dilutions have been used, a graded series of clots will remain, ranging from unchanged to complete lysis. The level of urokinase resulting in 50% clot lysis is estimated and the activity of the original sample is calculated against a standard of assigned potency, which is run simultaneously. Second Method (Esterolytic) Reagents Phosphate (0.06 M) NaC1 (0.09 M) buffer, pH 7.5 Perchloric acid (0.75 M) Methanol-spectroanalyzed 0.1 M Potassium permanganate 2% Sodium sulfite 10% Sulfuric acid 67% (v/v) N-a-Acetyl-L-lysine methyl ester hydrochloride (ALME) (Cyclo Chem. Corp., Los Angeles, Calif.) 0.24% I W. F. White, G. H. Barlow, and M. M. Mozen, Biochemistry 5, 2160 (1966).
666
OTHER PROTEASES
[48]
Chromotropic acid reagents: 200 mg of 4,5-dihydroxy-2,7-naphthalene-disulfonic acid disodium (Eastman Kodak Co., Rochester, N.Y.) dissolved in 10 ml of distilled water and then mixed with 90 ml of sulfuric acid reagent.
Procedure. A standard methanol curve is made by diluting the methanol stock with phosphate buffer to give samples in the range 0.25 to 1.5 micromoles/ml. A 1-ml aliquot of each dilution is transferred to a tube containing 0.5 ml of 0.75 M perchloric ,cid; a blank is prepared in the same manner using phosphate buffer: The contents of each tube are mixed and 1 ml from each is transferred to a test tube suitable for boiling. To each tube is added 0.1 ml KMnO4 close to the fluid surface. The contents are mixed well, and exactly 1 minute is allowed for the oxidation of methanol to HCHO; this is followed by the addition of 0.1 ml sodium sulfite to each tube which is immediately shaken vigorously to insure thorough mixing and decolorizing of the solution. Four milliliters of chromotropic acid reagent is added to each tube, and the contents mixed well, preferably on a mechanical mixer because of the corrosive and viscous nature of the acid. The tubes, covered with marbles or small beakers, are placed in a boiling water bath for 15 minutes, then cooled to room temperature and the absorbancy read in a spectrophotometer at 580 nm. With the standard urokinase a dilution series is made containing from 200 to 1000 CTA units per milliliter. An 0.2 ml aliquot of each dilution is mixed with 2 ml ALME, and the mixtures are incubated at 37 °. After 2 and 32 minutes incubation, respectively, a 1-ml aliquot is removed and added to 0.5 ml of 0.75 M perchloric acid and kept cold until all tubes are ready for centrifugation. From each supernatant, 1-ml aliquots are transferred to boiling tubes and treated in the same manner as described above for methanol. The absorbancy of the 32-minute sample is read using the corresponding 2-minute sample as a blank. An unknown urokinase preparation is assayed in the manner described above and the activity is found by direct extrapolation to the control curve and multiplication of the dilution factor. Definition o] Unit. The unit of urokinase activity, referred to as the CTA unit, was adopted in 1964 by the Committee on Trombolytic Agents, National Heart Institute. This unit is based on the activity of a working standard urokinase preparation 2 which was independently assayed in several laboratories. The standard urokinase preparation was also as2 The working standard urokinase is available from W H O International Laboratory for Biological Standards, National Institute for Medical Research, London, England.
[48]
URINARY PLASMINOGEN ACTIVATOR
667
sayed by its ability to split the synthetic substrate N-acetyl-L-lysine methyl ester (ALME). One unit of urokinase activity releases 5 X 10-~ micromoles/CTA unit/minute at 370.8 Purification Procedure Freshly collected urine from healthy young males is transported rapidly to the proeessing area and cooled quickly to less than I0 ° by pumping through a heat exchanger into a tank 4 where the temperature is maintained at 10° until enough urine is collected for foaming. This is conveniently done in batches of 900-1400 liters by the use of a Model N33 G-300, 3 hp, Lightning Fixed Mounted Agitator. With the agitator blades 15-30 cm below the urine surfaee a vortex will be created when the agitator is turned on. After 20 minutes agitation, the foam layer is allowed to collect on the liquid surface for 10 minutes. The urine is rapidly drained from the bottom of the tank through a 2-inch pipe fitted with a Teflon diaphragm valve and a sight glass. When the foam level reaches the sight glass, the valve is quickly closed. The foam is then allowed to break down further until an additional 25 minutes has elapsed, when the urine layer is again drawn off. The foam is then liquefied by the addition of a minimal amount of octyl alcohol. Foam concentrates are frozen and stored at --20 ° unless further processing is carried out immediately. Step I. Isolation o] Crude Urokinase. 5 The activity is precipitated from fresh or newly thawed foam concentrate by adding (NH4)2S04 to 65% saturation (2°). The precipitate is recovered by centrifugation or filtration, dissolved in 3% NaCl in a volume 1/200 of the original urine volume, and dialyzed overnight against 12 volumes (two changes) of 0.1 M phosphate buffer, pH 6.5, containing 0.1% EDTA. The bag contents are clarified by eentrifugation (30,000 g for 30 minutes). This solution, designated crude urokinase, has a specific activity of about 250 units/A2so. Step 2. Purification on IRC-50 Resin. Amberlite IRC-50 resin (previously equilibrated agains t 0.1 M phosphate buffer, pH 6.5, containing 0.1 M NaC1 and 0.1% disodium versenate) is poured as a slurry into a column tall enough to give a bed height of 100 cm and having a cross' This value appears in article by A. J. Johnson, D. L. Kline, and N. Alkjaersig, Thromb. Diath. Haemorrhag. 21, 259 (1969). However, other values ranging from 6.9 to 8~ X 10.4 have been reported. It is essential that a CTA unit be defined in each laboratory in terms of the standard preparation. • Equipment used during the processing of urokinase is either of stainless steel, polyethylene, or ceramic. ' Unless otherwise stated, all purification procedures are carried out at 4°.
668
OTHERPROTEASES
[48]
sectional area of about 1 cm -2 per 600,000 CTA units of crude urokinase. The crude urokinase is introduced into the column, which is then washed with distilled water until the optical absorbancy of the effluent 280 nm is about 0.06. The adsorbed urokinase is eluted with 3% NaC1 solution containing 0 . 1 ~ E D T A . Suitable fractions are collected and a plot is made of the optical absorbancies at 280 nm. The major portion of the peak usually contains 50 to 60% of the starting crude urokinase activity at a specific activity of about 10,000 CTA units/A~8o. I t is usually considered unrewarding to attempt to work up the tail of the peak, which contains only about 10% of the starting activity. A pool is made of the fractions from the main part of the elution peak and it is either lyophilized directly or first concentrated by ultrafiltration and then lyophilized. Step $. Gel Filtration on Sephadex G-IO0 Columns. Step 2 material is further purified by gel filtration in a column of Sephadex G-100. The column is poured to a height of 100-110 cm in a column with a crosssectional area of about 20 cm -2 per gram of step 2 material. Figure 1 shows a typical large-scale run made in a standard 4-foot length of
E0.7t
~
I
I
A
0.6
0.5
N 0.4|
0'21 0.1 I
6
8 10 Effluent volume (liters)
12
Fro. 1. Gel filtration of step 2 urokinase. Column size: 152 X 110 cm. Equilibrated to pH buffer (0.1M NaC1; 0.1M phosphate). Sample (5 g step 2 fraction) applied in volume of 73 ml. Flow rate: 200 ml/hour. Fractions cut at low points between peaks except on left side of C1, where 280 ml was rejecfed to reduce contamination by peak B. An additional peak, designated D, emerges beyond the righthand limit of this figure. Reprinted from Biochemistry 5,~2160 (1966). Copyright (1966) by the American Chemical Society. Reprinted by permission of the copyright owner.
[481
URINARY PLASMINOGEN ACTIVATOR
669
6-inch Pyrex pipe, using a 325 mesh stainless steel screen supported by a perforated Teflon plate at the bottom. The clarified sample of step 2 material (having been equilibrated by dialysis against the pH 6.5 buffer, is layered carefully on top of the column, the surface of which is stabilized by means of a Whatman No. 1 paper disk held in place by a lightweight perforated Lucite plate. Filtration is done by gravity alone with a buffer head of about 40 cm above the top of the Sephadex bed. In addition to the peaks shown in Fig. 1, continued elution results in the emergence of a fifth peak, the beginning of which is apparent in the righthand edge of the figure. Bioactivity is found only in the peaks labeled C-1 and C-2. The total yield of activity is in the range of 80 to 85%. The cutting of fractions from the column depends on the use to which the product will be put. It is likely that the smaller molecular weight material (C-2) is produced by enzymatic degradation 1 of the larger material either during the storage of the urine in the bladder or during processing, perhaps both. Therefore, the C-2 material is usually disregarded in the further workup of the product even though it is active in lysing clots as well as offering an interesting subject for study by protein chemists desiring to work with the smallest molecule possessing activator activity. In working up to the main peak (C-l), a few tubes on either edge of the peak are rejected in order to restrict contamination by adjacent peaks. After pooling the tubes from the central region, the C-1 product is concentrated by ultrafiltration. Ordinarily, C-1 type material constitutes at least 70% of the total biological activity contained in the parent step 2A fraction and exhibits a specific activity in the range of 30,000 to 35,000 units/A28o. Step ~. Re/ractionation o] Sephadex G-IO0. For final removal of contaminants (especially the inert proteins of peak B), fraction C-1 is usually subjected to refiltration on Sephadex G-100. This is done either by combining four or five batches of step 3 material and using the same column as employed in step 3 or by running a single batch of step 3 material in a column having a cross-sectional area one-fifth that of the step 3 column. In either case a large, symmetrical peak of 280 nm absorbancy emerges with the only evidence of impurities being a small peak or shoulder at the leading edge of the main peak. Ninety percent or more of the optical density applied to the column is contained in the main peak, which emerges at an effluent volume equal to 50% +__1% of the total volume of the column. Expressed in another way, the ratio of the emergence volume for urokinase to that of egg albumin is 0.92. The material from the main peak is concentrated by ultrafiltration, dialyzed against 0.05 M sodium chloride containing 0.1% EDTA, and lyophilized. This step 4 product has a potency in the range of 50,000 to 75,000 CTA units/A28o. It has been found to be suitable for clinical use as well as for laboratory experiments in the activation of plasminogen.
670
OTHER PROTEASES
[48]
Physical Properties Purity. Despite the symmetrical appearance of C-1 peak after rechromatography on Sephadex G-100, examination of the step 4 product by polyacrylamide disc electrophoresis and by immunochemical methods suggested the presence of two subtypes of urokinase. A separation into two active types, designated S1 and $2, has been achieved by chromatography on hydroxylapatite. 1 A list of physical constants of these two types is given in the table, e Despite the difference in the molecular weights PHYSICAI~ C O N S T A N T S ON U R O K I N A S E
S1 Sedimentation coefficient (s°,a), S pH 2.6 pH 4.5 pH 6.5 pH 8.5 Heterogeneity constant (p) pH 4.5 pH 6.5 pH 8.5 Diffusion coefficient (D°0.w), cm2/sec pH 6.5 Partial specific volume, a IT, ral/g Frictional r a t i o Molecular weight S and D Equilibrium Electrophoretie mobility X 10~ cm2/V sec (pH 4.8, acetate buffer, U 0.1) E1% l¢~a 280 nm, pH 6.5
--
2.75 2.66 2.71 0.28
10-18 0.35 0.32
$2
3.18
3.33 3.27
X
7.41 X 10-7 0. 724 1.35
m
0.728
31,700 31,300 43.5
54,700 -t-2.2
13.2
13.6
"Calculated from amino acid composition data. Reprinted from Biochemistry 5, 2160 (1966). Copyright (1966) by the American Chemical Society. Reprinted by permission of the copyright owner. * An alternative method for the purification of urokinase has been given by A. Lesuk (U~. Patent 3,355,361), whose crystalline product has been described [Sc/ence 147, 880 (1965)]. Ultracentrifugal methods have given a molecular weight of approximately 54,000 and the product shows an activity of about 104,000 CTA units per milligram protein. These values agree closely with our values for type $2 material, leaving unexplained the origin of our S1 species. Lesuk et al. Thromb. Diath. Haemorrhag. I8, 293 (1967) have shown that enzymes such as trypsin can effectively reduce the molecular size of urokinase. However, Burges et al., Nature 208, 894 (1965) have shown by chromatography in calibrated Sephadex columns that the activator activity of urine and of crude concentrates from urine shows a molecular weight of 34,500--+ 2000, which agrees with our value for type S1.
[48]
URINARY PLASMINOGEN ACTIVATOR
671
obtained by ultracentrifugation as shown in the table, the two forms do not show a separation on Sephadex G-100 on which separately their effluent volumes differ by only 6%. The fibrinolytie activity for S1 is 120,000 CTA units per A28o or about 200,000 CTA units per milligram protein, 7 while for $2 the values are 60,000 CTA units per unit A2so and 103,000 units per milligram protein. S t a b i l i t y . Urokinase is a moderately stable enzyme, showing no appreciable loss in activity over years in lyophilized form or over months in sterile solutions at 1 mg/ml or more at refrigerator temperatures. However, in dilute solution it has been found advisable to maintain a concentration of 0.1% EDTA when dialyzing or holding for more than a few hours. Stability is decreased at salt concentration below 0.03 M sodium chloride, and precipitation with loss in activity occurs at very low salt concentrations. In diluting to the levels of activity measured in the fibrinolytic assay, it is advisable to add a protein such as human serum albumin fraction V or gelatin to prevent surface denaturation. Activators and Inhibitors Unlike numerous analogous systems in the field of enzymes, no proenzyme has been found for urokinase. Apparently urokinase is excreted into the urine in fully activated form. Human plasma (and serum) contains an inhibitor to urokinase which, in certain pathological conditions, reaches levels ten to twenty times normal2 The inhibitor is destroyed by heating at 56 ° for 30 minutes and is contained in Cohn's fraction IV-I. Synthetic ~-aminocaproic acid inhibits the action of urokinase on plasminogen9 as well as on synthetic ester substrates, l° Specificity Urokinase resembles trypsin and plasmin in its activity against simple synthetic substrates, although there are differences in the relative rates for the three enzymes, depending on the substrate. However, urokinase is much more fastidious in its action on naturally occurring proteins, plasminogen being the only protein substrate against which it has been shown to have any activity. By contrast, both trypsin and plasmin attack a variety of proteins including casein, the plasma proteins, ACTH and 7Protein determination made by the Folin-Ciocalteau method [O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951)]. 8I. M. Nilsson, H. Krook, N. H. Sternby, E. Soederberg, and N. Soderstrom. Acta Med. Scand. 1{}9,323 (1961). 9N. Alkjaersig, A. P. Fletcher, and S. Sherry, J. Biol. Chem. 234, 832 (1959). loL. Lorand and E. V. Condit, Biochemistry 4, 265 (1965).
672
OTHER PROTEAS~S
[49]
fl-MSH, all of which resist the action of urokinase. Robbins et al. u have shown that the activation of plasminogen to plasmin by urokinase is due to the cleavage of a single arginyl-valine bond. Distribution Activator activity is found in most tissues and in plasma. However, despite circumstantial evidence that the kidney is primarily involved in regulating the level of circulating activator, Kucinski et al. ~ were unable to detect the presence of urokinase in renal venous blood by immunochemical means and also established lack of identity between urokinase and tissue activator. Since the activator produced in culture of human kidney cells is immunochemically identical to urokinase TM and shows molecular size contrasts indistinguishable from urokinase,1" it appears reasonable to postulate at this time that urokinase is synthesized in the kidney and is secreted only in the urine. K. C. Robbins, L. Summaria, B. Hsieh, and R. J. Shah, J. Biol. Chem. 242, 2333 (1967). C. S. Kucinski, A. P. Fletcher, and S. Sherry, J. Clin. Invest. 47, 1238 (1968). M. Bernik and H. C. Kwaan, J. Lab. Clin. Med. 70, 650 (1967). 14G. H. Barlow and W. F. White, unpublished observations.
[49] Mouse Submaxillary Gland Proteases 1 B y MILTOn LEVY, L. FISHMAN, and I. SCH~.NXEIN
Mouse submaxillary glands contain at least four enzymes capable of hydrolyzing tosylarginine methyl ester (TAM), a substrate hydrolyzed by several proteolytic enzymes, including trypsin and thrombin. We will describe a method for the isolation of two of the mouse enzymes, " A " and " D , " in pure form from the glands of mature male mice. TM The size of the glands and their enzyme contents are controlled by sex hormones. Our preparations were primarily directed toward the isolation of "nerve growth factor ''2 and as a consequence there may be some steps not needed when only the enzymes are wanted. The enzymes A and D are related but not identical by immunological tests. Using TAM hydrolysis as the criterion, A has much the higher specific activity. On the same basis both are more active than trypsin. 1EC 3.4.4--. 1"I. Schenkein, M. Boesman, E. Tokarsky, L. Fishman, and M. Levy, Biochem. Biophys. Res. Commun. 36, 156 (1969). I. Schenkein, M. Levy, E. D. Bueker, and E. Tokarsky, Science 159, 640 (1968).
672
OTHER PROTEAS~S
[49]
fl-MSH, all of which resist the action of urokinase. Robbins et al. u have shown that the activation of plasminogen to plasmin by urokinase is due to the cleavage of a single arginyl-valine bond. Distribution Activator activity is found in most tissues and in plasma. However, despite circumstantial evidence that the kidney is primarily involved in regulating the level of circulating activator, Kucinski et al. ~ were unable to detect the presence of urokinase in renal venous blood by immunochemical means and also established lack of identity between urokinase and tissue activator. Since the activator produced in culture of human kidney cells is immunochemically identical to urokinase TM and shows molecular size contrasts indistinguishable from urokinase,1" it appears reasonable to postulate at this time that urokinase is synthesized in the kidney and is secreted only in the urine. K. C. Robbins, L. Summaria, B. Hsieh, and R. J. Shah, J. Biol. Chem. 242, 2333 (1967). C. S. Kucinski, A. P. Fletcher, and S. Sherry, J. Clin. Invest. 47, 1238 (1968). M. Bernik and H. C. Kwaan, J. Lab. Clin. Med. 70, 650 (1967). 14G. H. Barlow and W. F. White, unpublished observations.
[49] Mouse Submaxillary Gland Proteases 1 B y MILTOn LEVY, L. FISHMAN, and I. SCH~.NXEIN
Mouse submaxillary glands contain at least four enzymes capable of hydrolyzing tosylarginine methyl ester (TAM), a substrate hydrolyzed by several proteolytic enzymes, including trypsin and thrombin. We will describe a method for the isolation of two of the mouse enzymes, " A " and " D , " in pure form from the glands of mature male mice. TM The size of the glands and their enzyme contents are controlled by sex hormones. Our preparations were primarily directed toward the isolation of "nerve growth factor ''2 and as a consequence there may be some steps not needed when only the enzymes are wanted. The enzymes A and D are related but not identical by immunological tests. Using TAM hydrolysis as the criterion, A has much the higher specific activity. On the same basis both are more active than trypsin. 1EC 3.4.4--. 1"I. Schenkein, M. Boesman, E. Tokarsky, L. Fishman, and M. Levy, Biochem. Biophys. Res. Commun. 36, 156 (1969). I. Schenkein, M. Levy, E. D. Bueker, and E. Tokarsky, Science 159, 640 (1968).
[49]
MOUSE SUBMAXILLARY GLAND PROTEASES
673
D is strongly activated by all amino acids (both d and l) as well as by tosylarginine. 1 The enzymes act on protein substrates such as protamines, histones, and lysozyme but not in proportion to their "tamase" activity as compared with trypsin. They seem to have a strong preference for arginyl bonds. A s s a y of E n z y m e s
The assay reaction is C~HTSO~NH--CH[--(CH2)r-NH--C(=NH)--NHa+]--COOCHs + OHC~HTSO~NH--CH[--(CH~)a--NH--C(~NH)--NH~+]--COO - + CHaOH
It is followed by measurement of the alkali uptake in the pH-stat technique2 A manual method is described in Vol. II and spectrophotometric * and fluorometric 5 methods have been described for the same reaction with trypsin. With the addition of glycine to the substrate solution (insuring the activation of D) these can be used for the assay of submaxillary enzymes. A unit of enzyme catalyses the hydrolysis of 1 micromole of TAM per minute at 37 ° and pH 8.0. The apparatus used consists of the Radiometer (Copenhagen) Titrator TTTlc, which controls the Titragraph (SBR 2c) and associated syringe buret (SBUla). The syringe delivers alkali into the reaction mixture to maintain the pH at 8.0 on signal from the Titrator. The Titragraph chart moves at uniform speed and makes a record of the alkali delivered as a function of time. We have found Hamilton syringes 8 less subject to leakage than glass plunger syringes. They have the nominal disadvantage of not delivering volumes with whole number ratios to the chart paper divisions. We standardize the solutions on the Titragraph scale and label them in terms of micromoles delivered per chart division. This is called the D value. Thus a 0.2 D alkali delivers 0.2 micromoles of reagent per chart division. The actual volume delivery is about 0.44 ml/100 chart divisions with the syringe in use. A thermostat operated at 37 ° is used for temperature control. As suggested by Jacobsen et al2 the calomel electrode is also thermostated by immersion in this bath. A cross section of this part of the apparatus is shown in Fig. 1. The reaction vessel is a 12-ml weighing bottle with 3C. F. Jacobsen, J. Leonis, K. LinderstrCm-Lang, and M. Ottesen in "Methods of Biochemical Analysis" (D. Glick, ed.), Vol. 4, p. 171. Wiley (Interscience), New York, 1957; M. Laskowski, Vol. II [3]. 4 M. A. Seligman, A. S. Carlson, and T. Robertson, Arch. Biochem. Biophys. 97, 159 (1962). 6V. M. Saresai and H. S. Provido, J. Lab. Clin. Med. 64, 1023 (1965). ' Hamilton Company, Whittier, Calif.
674
OTHER PROTEASES
To motor
[40]
J
.,~ To meter
KOH. II IIII
Sotd.
KCl
Colomel, Hg,KCI FIG. I. Cross section of titration vessel and calomel electrode. Not to scale. PE, Polyethylene tubing; PVC, polyvinylchloride tubing. The rubber stopper has a hole (not shown) about 5 m m in diameter for the introduction of sample. N2 is passed through a saturator immersed in the bath before entering the vessel. The calomel and glass electrode parts are supported independently. The wavy line indicates the bath water level.
a 24/12 internal grinding ~ fitted onto a rubber stopper carrying the items shown and having an addition hole through which the enzyme solution may be added. It is mounted on a clamp and rod by which it may be lifted and rotated out of the bath for the manipulation of renewing the liquid junction and the substrate solution. The flexible polyvinylchloride KCI bridge allows the calomel electrode to remain in the thermostat. The liquid junction is made at the small U-shaped glass tubing as shown. Only the end of this tube is glass. The main part passing through the stopper is polyethylene tubing. It has sufficient ~Kimble Products Cat. #15145 25 X 40 ram.
[49]
MOUSE SUBMAXILLARY GLAND PROTEASES
675
flexibility to avoid breakage. The openings in the liquid junction and diameter oi tubing are about 1 m m ( ~ in. in the P V C section). Openings that are too small m a y result in too great resistance in the circuit. For the same reason even partial bubble blockage is not tolerable in the tubings. The calomel electrode is constructed from a three=way T-bore Teflon plug stopcock, a screw cap test tube, a rimmed test tube, and a tube carrying a sealed wire to the mercury, calomel, KCI section. The latter is held by a tightly fitting rubber stopper which is waxed in place with the calomel electrode partly filled.The fillingis completed with saturated KCI. Air is removed through the stopcock by appropriate manipulation. The substrate solution contains 2 m g of T A M HCI and 0.2 m g of glycine per milliliterin 0.1 M KCI. Three millilitersof this solution is measured into one of the weighing bottles. A few drops of KCI are permitted to flow through the system from the reservoir and the stopcock turned to the position shown. External KCI solution is wiped from the U-tube with a bit of absorbent paper and the weighing bottle put on the stopper. The rod carrrying the glass electrode, motor, etc., is raised, rotated, and the reaction vessel lowered into the thermostat. The stirrer is started, the tip of the buret placed in the solution, the equipment set for pH-stat operation at a p H of 8.0 and after a few minutes for temperature equilibration, the addition of alkali begun by the use of the appropriate switch. The p H will come to 8.0 and the titragraph trace then becomes practically vertical. After 1 or 2 minutes to demonstrate this stability,the enzyme solution is added, usually in a volume of 1 to 200 ~l, using a constriction pipette,s In the presence of enzyme, alkali Uptake will begin and continue at a linear rate for 30-70% of complete hydrolysis. If the alkali is 0.2 D and the chart speed 0.5 cm per minute, then the units of enzyme added are equal to 0.1 the number of vertical lines crossed on the chart between two of the horizontal lines. Runs of 4-16 minutes are usual and the rates can be measured to about 1 % accuracy. The zero enzyme rate should be determined and subtracted. It is about 0.015 micromoles per minute. At the end of the run the vessel is lifted and rotated out of the bath, the electrode and fittings rinsed with distilled water, blotted with a cellulose wipe, the liquid junction renewed, and a new weighing bottle with substrate solution put in place. Rates are proportional to enzyme concentration from 0.2 to 1.5 micromoles per minute. Qualitative Test. A useful qualitative test for activity consists of a solution of TAM, 1 mg/ml, and glycine, 0.1 mg/ml, containing phenol 9 M. Levy, Compt. Rend. Tray. Lab. Carlsberg, Set. Chim. pp. 21-101 (1936).
676
OTHER PROTEASES
[49]
red and adjusted to a clear red by addition of alkali. A drop of enzyme solution added to 0.5 ml of this solution soon changes the color from red to yellow. The time required is roughly a measure of enzyme concentration. The test is used to test the effluents from chromatographs for activity.
Preparation of Enzymes Raw Material. The submaxillary glands of sexually mature male mice (6 weeks and older) are dissected and placed in a minimal volume of cold 0.1 M phosphate buffer, pH 7.4. The mixture is then frozen with dry ice and may be held for some months in this condition. Several commercial sources (Pel-Freez Inc., St. Louis Serum Co.) can supply this material. Extraction. Our standard preparation uses 2000 submaxillary glands (1000 mice). The frozen glands are partly thawed and then homogenized for 3 minutes at 30-5 ° in a Waring blendor run at high speed with enough water to make a liter. The resultant mixture is centrifuged at 10,000 g for 10 minutes. The supernatant fluid is collected. Purification. A 0.2 M solution of streptomycin sulfate is adjusted to pH 7.4 with sodium hydroxide. To each volume of the supernatant liquid one-ninth volume of the streptomycin solution is added with stirring. After storage for 3 hours at 30-5 ° the cold mixture is centrifuged at 10,000 g for -10 minutes. The supernatant fluid is collected. Seven milliliters of absolute alcohol at --15 ° is added slowly with stirring to each 100 ml of the fluid. After storage at --15 ° for 15 minutes, it is centrifuged at 10,000 g for 10 minutes. The supernatant solution is collected. Fortyeight milliliters of ethanol at --15 ° is added, with stirring, per 100 ml of the supernatant solution obtained from the streptomycin precipitate. The mixture is stored for 2 hours at --15 ° and the fluid decanted from the gummy red precipitate. The supernatant contains considerable enzymatic activity and appears to be richer in A than is the precipitate. The precipitate is dissolved in 500 ml of water, and 24.5 g of ammonium sulfate is added per I00 ml (35% saturation). The pH is kept at 7.4 by addition of Tris buffer. After 30 minutes at 3o-5 ° the mixture is centrifuged for 10 minutes at 10,000 g. The supernatant solution is brought to 80% saturation with ammonium sulfate by addition of 31.5 g per 100 ml of the original solution. After 2-4 hours the mixture is centrifuged for 15 minutes at 10,000 g. The precipitate is dissolved in 200 ml of water and the solution dialyzed in regenerated cellulose casing 9 with changes of external water every 2 hours until no sulfate is detectable in the external solution by addition of 1% BaC12 solution. oA. H. Thomas Cat. #4465-A ~ inch.
[49]
MOUSE SUBMAXILLARYGLkND PROTEASES
677
Chromatography on CM-Cellulose. Twenty grams of Whatman CM23 carboxymethyl cellulose is stirred with 0.5 M NaOH containing 0.5 M NaC1 and filtered on a flitted glass Biichner funnel. This is washed with water until the washings are free of alkali and finally with 0.005 M NaC1. A jacketed column kept at 3o-4 ° and 2 cm in diameter is provided. The cellulose derivative is poured to form a column 60 cm long and the 0.005 M NaC1 pumped through the column at about 4 ml per minute until in and out flow have the same pH and conductivity. The entire dialyzed solution is applied to the column and eluted with distilled water at 3-4 ml per minute. The first three column volumes contain nerve growth factor and enzymes. The collected fluid is lyophilized. The white material, designated CMI, is stable for some months at --50 °. Chromatography on DEAE-Sephadex. A jacketed column 5 cm in diameter and capable of holding a column 89 cm long is loaded with DEAE-Sephadex A-25 (Pharmacia Inc.). The material is prepared by washing 500 g in succession with 0.5 M NaOH, water, 2 hours with 0.5 M HC1 and finally with water until free of acid. To the suspension in water solid Tris is added until the pH is stable at 7.45 for an hour while stirring. The material is then suspended in buffer (0.05 M Tris-HC1, pH 7.45) and decantation used to remove fines. The suspension is cooled to 3o-4 ° and poured to form the column. Buffer is pumped overnight at 4 ml per minute with the column kept at 3o-4 °. The column should now be at equilibrium with the buffer in that the in and out flow are at the same pH. Two grams of CMI is dissolved in 20 ml of the buffer and dialyzed against it overnight. The resultant volume of solution (about 21 ml) is layered on the column and the elution begun with the buffer at 780 inl per hour. Twenty-six milliliter fractions are collected. The elution diagram is given in Fig. 2. The location of enzyme is highly dependent on elution rate but the qualitative test enables the enzyme to be detected quickly. Disc electrophoresis on this material shows 4 or 5 bands. The appropriate fractions are combined, dialyzed against water, and lyophilized. Electrophoretic Separation. The final step of purification involves the use of a preparative acrylamide gel apparatus 1° (Fractophorator, Joseph Buchler, Inc.). The gel tube of the apparatus is extended by a piece of polyvinylchloride (Tygon) tubing about 6 cm long which in turn is closed by a rubber stopper (Fig. 3). The tube is clamped in a vertical position. The gelation mixture is made in a buffer containing 0.0125 M Tris and 0.096M glycine which is also used as collecting fluid and wI. Schenkein, M. Levy, and P. Weis, Anal. Biochem. 25, 387 (1968).
678
OTHER P~OT~AS~S
[49]
1.0 /
i 0(~t~ I
I
I
1 I I00
Froction Number
FIo. 2. Elution from DEA~,-SephadexA-25. See text for details. Fractions are 26 ml each. electrode wash. To 100 ml of buffer 7.5 g of acrylamide, 0.2 g of bisacrylamide, and 0.06 ml of T E M E D (NNN'N'-tetramethylethylene diamine) are added. This solution is deaerated. Ammonium persulfate (0.7 mg/ml) is added to a sut~cient volume of the gel mixture and poured into the closed gel tube to form a meniscus 3.5 cm above the end of the glass. A layer of buffer with a tracer dye is carefully placed above the heavy solution to flatten the meniscus. After the gel has set, the extender tube and gel are cut through at the end of the glass with a razor to produce a cut surface for the exit of constituents. The apparatus is kept at about tube
tt line ~tender topper
FIG.3. Preparationof gel tube for Fractophorator operation. The extender is polyvinylchloridetubing.
[49]
MOUSE SUBMAXILLARY GLAND PROTEASES
679
10°. The gel tube is placed in the apparatus and prerun for an hour at 15 mA (about 400 V). The sample is prepared for electrophoresis by dissolving about 100 mg of lyophilized DS II in 1.75 ml of 0.00125 M Tris and 0.0096 M glyeine buffer (1-10 dilution of standard buffer). About 200 mg of sucrose is dissolved in the solution. The protein solution is layered above the gel through the supernatant buffer. For the initial 15-20 minutes of run the current is held to 5 mA and the electrode wash buffer is not circulated. A zone of protein forms and enters the gel. When this has happened, the electrode buffer circulation is begun and the current raised to 15 mA. The volume of each collection is kept at about 3 ml and the time per fraction is 3 minutes. A typical result is shown in Fig. 4. In this run some dye and other impurities emerged before the enzymes. Appropriate combining of fractions as indicated in Fig. 4 gives pure A and D as shown by analytical disc electrophoresis on acrylamide gels. The combined fractions are freed of buffer using Sephadex G-25 and lyophilized. For testing, the solids are dissolved in 0.001 M HC1 at about 0.2 mg/ml. The preparations are pure white powders which on disc electrophoresis show only one band each and which when tested by immunoelectrophoresis and by double diffusion against an antiserum to DS
1.0
0 QO O4
c o ,0
0
,
I
0
I0O Froction Number
Fro. 4. Record of electrophoretic separation on "Fractophorator." Fractions at 3-minute intervals. Total load 100 mg fraction DS II. The vertical bars indicate enzyme tests on a 0~4 scale by the qualitative test described in the text.
680
OTHER PROTEASES
[49]
TABLE I PREPARATION OF MOUSZ SUBMAXILLARYPROTEASE Step
Protein (mg)
Homogenate 85% Ammonium sulfate ppt CM I DS II "D" Fractophorator "A"
24,800 3,680 2,362 400 174 86
Enzymes Specific activity (units X 10-~) (~/mg) 3.65 2.66 2.32 0.72 0.10-0.07 0.26
150 725 1,000 1,800 400-600 l 3,000 )
Yield (%). 100 73 64 20 10
Several enzymes with the same qualitative activity are involved. The yields are therefore recoveries of activity, not of substances. II show only a single precipitation line for each preparation. When tested against DS II itself five precipitation lines appear with the antiserum. Table I is a record of a preparation made with 2000 glands. Enzymatic Properties The optimum pH is 8.0-8.5 for either enzyme. The acid branches of the activity-pH curve show a midpoint at 6.5 for A and 7.1 for D. All measurements are made in the presence of glycine. In the absence of activator (glycine in the assay system) the hydrolysis curve is sigmoid with D. This results from the activating effect on D of the tosylarginine liberated. The initial rate is increased 2- to 4-fold by the activators. The K,~ for TAM is 6.5 X 10~4M for A and 2 X 10-4 M for D. K,~ for the hydrolysis of protamine sulfate is 0.63 g/liter. Chemical Properties The ultraviolet absorbance curves for the two enzymes show rather wide flat peaks with maxima at 276 and 275 nm. At 280 nm a solution containing 0.16 mg of nitrogen (presumed 1 mg of protein) has an optical density of 2.49 for A and 2.59 for D. The enzymes are inhibited by diisopropylphosphofluoridate and by 1-chloro-3-tolsylamido-7-amino-2-heptanone hydrochloride (TLCK) at second-order rates considerably less than those for trypsin. D is denatured by urea, reversible at low (4M) and high (10M) concentrations but irreversibly at 6 M urea. The irreversible loss at 6 M is second order and thus appears to be self-hydrolysis of a denatured form by a still-active form. The sedimentation patterns are symmetrical and lead to $ = 3.1 for A and S = 2.8 for D. These are consistent with the summation of residues from the amino acid analyses given in Table II. Analytically significant differences in numbers of residues are present only for Pro, Val, Met, Ile,
[SO]
GLANDULAR KALLIKREINS
681
TABLE I I COMPOSITIONS OF MOUSE SUBMAXILLARY GLANDPROTEXSES
Residues~/molecule Amino acid
A
D
Asp Thr Ser Glu Pro Gly Ala Cys
31 16 18 22 21 30 15 9
Val
13 6
Met Ile Leu Tyr Phe Lys His Arg Trp b
30 16 17 21 16 29 16 9 9
(4) 6 26 9
11 30 8 9 20 6 (4) 12
~ residue wt.
29, 700
7
22 7 5 7 27 )900
a Rounded off to nearest whole numbers, averages of seven analyses. The calculation base is in parenthesis in each ease. b Calculated from tyrosine contents and ratio of tryptophan to tyrosine from ultraviolet absorption.
Leu, and Trp. These are all less in D than in A. A few amino acids are present in (doubtfully) greater amounts in D than in A (Ala, Tyr, Lys, His, Arg).
[50] Glandular Kallikreins from Horse and H u m a n Urine and from H o g Pancreas 1
By MARION E. WEBSTF~ and ELINE S. PaADO kallikrein W kininogen --* kinin T h e k a l l i k r e i n s ( E C 3.4.4.21) h a v e been defined as e n d o g e n o u s enzymes which rapidly and specifically liberate the polypeptides called k i n i n s f r o m t h e s u b s t r a t e s in p l a s m a called k i n i n o g e n s , la P e r h a p s m o r e
[SO]
GLANDULAR KALLIKREINS
681
TABLE I I COMPOSITIONS OF MOUSE SUBMAXILLARY GLANDPROTEXSES
Residues~/molecule Amino acid
A
D
Asp Thr Ser Glu Pro Gly Ala Cys
31 16 18 22 21 30 15 9
Val
13 6
Met Ile Leu Tyr Phe Lys His Arg Trp b
30 16 17 21 16 29 16 9 9
(4) 6 26 9
11 30 8 9 20 6 (4) 12
~ residue wt.
29, 700
7
22 7 5 7 27 )900
a Rounded off to nearest whole numbers, averages of seven analyses. The calculation base is in parenthesis in each ease. b Calculated from tyrosine contents and ratio of tryptophan to tyrosine from ultraviolet absorption.
Leu, and Trp. These are all less in D than in A. A few amino acids are present in (doubtfully) greater amounts in D than in A (Ala, Tyr, Lys, His, Arg).
[50] Glandular Kallikreins from Horse and H u m a n Urine and from H o g Pancreas 1
By MARION E. WEBSTF~ and ELINE S. PaADO kallikrein W kininogen --* kinin T h e k a l l i k r e i n s ( E C 3.4.4.21) h a v e been defined as e n d o g e n o u s enzymes which rapidly and specifically liberate the polypeptides called k i n i n s f r o m t h e s u b s t r a t e s in p l a s m a called k i n i n o g e n s , la P e r h a p s m o r e
682
OTaER PROTEASES
[50]
comprehensively they might be defined as those proteolytic enzymes of animal origin which release a kinin from kininogen but do not readily cleave proteolytic bonds in other proteins. The kallikreins, due to their different properties, have been separated into two classes' those derived from glandular sources and those derived from plasma. Each kallikrein derived from different sources or different species varies not only in structure 2 but in the proteins from which it must be separated and, therefore, purification techniques for individual kallikreins are somewhat different. As methods for purification of plasma kallikreins are only now being developed, ~ this chapter will be restricted to the preparation and properties of three of the glandular kallikreins--that is, those derived from horse and human urine, and from hog pancreas.
Assay Methods The enzyme activity of the kallikreins can be estimated either by measuring the quantity of kinin formed from kininogen or by measuring their ability to cleave synthetic N-substituted arginine esters. The former method, once the technique has been mastered, provides a rapid method for the identification of the active fractions. The latter method, although more precise, is not as specific since other proteolytic enzymes cleave these ester bonds. Either method can be used for following the purification of horse or human urinary kallikreins, but the method based on formation of kinins should be employed when purifying hog pancreatic kallikrein, particularly during the early steps of the purification. F o r m a t i o n of Kini~s Principle. Cleavage of the kininogens in plasma by the kallikreins releases the biologically active polypeptides called kinins. The amount of kinin generated from excess kininogen is a reflection of the quantity of enzyme and may be quantitated by direct bioassay on a number of biological preparations or by radioimmunoassay.4 As the latter technique has not as yet been developed to the point at which it can be applied to biological material, direct bioassay of the kinins utilizing one or more of the available pharmacological methods is currently the method of
1Supported in part by Funda~o de Amparo h Pesquisa do Estado de S~o Paulo, Grant No. 68/638; National Institutes of Health, Special Fellowship, Grant No. 1 F03 AM 42717-01 and BSlsa Chefe de Pesquisas, Conselho Nacional de Pesquisas. 1, M. E. Webster, in "Hypotensive Peptides" (E. G. ErdSs, N. Back, F. Sicuteri, and A. F. Wilde, eds.), p. 648. Springer Verlag, New York, 1966. 2H. Moriya, J. V. Pierce, and M. E. Webster, Ann. N.Y. Acad. Sci. 104, 172 (1963). E. Habermann and W. Klett, Biochem. Z. 346, 133 (1966). R. C. Talamo, E. Haber, and K. F. Austen, J. Immunol. 101, 332 (1968).
[50]
GLANDULAR KALLIKREINS
683
choice. Direct measurement of the decrease in the carotid blood pressure of the anesthetized dog following injections of varying quantities of kallikrein as initially proposed 5 is a useful technique but has the disadvantage that some heterologous kallikreins, such as horse urinary kallikrein,6 do not readily liberate kinins from dog kininogens. Recording the height of contraction in vitro of various isolated smooth muscles such as the rat uterus or guinea pig ileum provides an alternate method for measuring the kinin which is formed. Although the rat uterus will detect smaller amounts of kinin, the guinea pig ileum has a more quantitative response and this method has been chosen to be described in detail. In order to minimize the destructive effects of the kininases, enzymes which cleave the kinins at various peptide bonds thus destroying their biological activity, varying amounts of kallikrein are added to a constant amount of plasma directly in the tissue bath. Whenever possible, it is recommended that the plasma from the same species as the kallikrein be used as substrate. However, dog plasma is a much better substrate for hog pancreatic kallikrein than is hog plasma. Reagents
Tyrode's Fluid Solution I
Solution II
NaC1, 200 g/liter KC1, 5 g/liter CaC12, 5 g/liter MgC12-6 H20, 2.5 g/liter NaH2PO4"H20, 2.5 g/liter NaHC03, 50 g/liter
All solutions are made with recently glass-distilled water. The stock solutions may be stored at 4 ° for at least 2 months. On the day of assay, 40 ml solution I, 20 ml solution II, 1 g glucose, and 500 ~g atropine sulfate are added to 400-500 ml water and diluted to a final volume of 1 liter. Plasma. Citrated blood (0.2 ml 20% sodium citrate/10 ml blood) is taken in silicone-coated (Siliclad, Clay-Adams, Inc., New York) containers, the plasma separated and either used the same day or frozen in silicone-coated tubes. The plasma should be frozen and thawed only once as repeated freezing and thawing will cause loss of substrate. The use of silicone-coated glassware, E. K. Frey, H. Kraut, and E. Werle, "Das Kallikrein-Kinin-Systemund seine Inhibitoren." Ferdinand Enke Verlag, Stuttgart, 1968. ~E. S. Prado and J. L. Prado, Experientia 17, 31 (1961); E. S. Prado, J. L. Prado, and C. M. W. Brandi, Arch. Intern. Pharmacodyn. 137, 358 (1962).
684
OTHER PROTEASES
[SO]
although preferable, is not essential if sufficient kininogen remains in the plasma following its contact with glass. Also, the plasma may be heated to 60 ° for 1/~ hour. Following this treatment, it can usually be dialyzed against running tap water at 4 ° without loss of substrate. Heating plasma to this temperature is thought to destroy the plasma prekallikrein and, while eliminating the possibility that activators of prekallikrein will be identified as kallikreins, also prevents their discovery. Procedure. Guinea pigs (150-250 g) are stunned by a blow on the head and exsanguinated by cutting the blood vessels on the neck. The guinea pig is washed thoroughly with water, the abdomen opened and a 10-15 cm portion of the terminal ileum is removed, care being taken to prevent contact of the intestine with the outer surface of the guinea pig. The lumen is washed several times with approximately 20 ml Tyrode's solution warmed to 37 °. The washed ileum i s allowed to equilibrate for 15-30 minutes in warm Tyrode's solution and a portion (2-3 cm) is tied at both ends with surgical needle and thread as shown in Fig. la and cut as shown in Fig. lb so that the bath fluid will circulate within the lumen freely. Forceps should be used in handling the tissue and stretching should be avoided. One end of the tissue is tied to the hook at the bottom of the muscle chamber and the other is suspended from the muscle lever as shown in Fig. 2. Tension is applied and the tissue allowed to equilibrate for approximately 1~ hour before starting the assay. During this period both the sensitivity of the tissue and the proportionality of its response to varying amounts of a standard kallikrein preparation may be determined. For these determinations, a known quantity of kallikrein (e.g., 0.2 KL units of horse urinary kallikrein) is added to the bath for 1 minute, followed by 0.4 ml plasma and the contraction is recorded for 2 minutes. The Tyrode's solution in the muscle chamber is changed twice at 1-minute intervals and 1 minute allowed for the tissue to relax. Each new addition to the bath occurs in exactly
I \
(o)
(b).
Fro. 1. Method for tying and cutting guinea pig ileum.
[50]
GLANDULAR KALLIKREINS
685
Tyrode's Recorder f . As
Fro. 2. The tissue bath (7-8 ml capacity) can be purchased from various suppliers of pharmacological equipment such as Phipps and Bird, Inc., Richmond, Va., or C. F. Palmer, Ltd., London, England, or may be made by simply inserting a rubber stopper in a glass cylinder (12 X 70 mm). Air is bubbled slowly through the chamber and can be supplied by an aquarium air pump (vibrator-type). It can be admitted to the muscle chamber by inserting a hypodermic needle through the rubber stopper. Removing the head from a hypodermic needle and bending the shaft to insert it into the rubber stopper provides a method for supplying the hook to which the tissue is tied at the bottom of the muscle chamber. For direct recording, a magnification of 6-7 times is usually employed and can be achieved by tying the muscle 3-4 cm from the axle and having the recording tip, preferably equipped with an all-metal frontal writing point, at 21-24 cm. Tension is applied by adding weight (e.g., a piece of modeling clay) to the shorter arm to exactly balance the longer arm of the rod and placing the required additional weight on the longer arm at a point equidistant from the place the muscle is tied. Usually 1 g of tension is employed and this weight is increased somewhat (0.2-0.3 g) in those tissues which show normal contraction. Recording speed, 6 mm/minute. Bath temperature, 37°. 5 minutes and this r h y t h m is maintained throughout the day. As individual tissues can v a r y in sensitivity to the kinin over a 4-fold range, increasing or decreasing concentrations of kallikrein (e.g., 0.4 or 0.1 unit) are added to the bath with the same amount of plasma until the height of contraction of the larger dose corresponds to less than 80% of the maximum muscle contraction. After several such contractions, when it has been shown that the height of contraction of at least two concentrations of kallikrein is reproducible and related to the quantity of kallikrein added to the bath, unknown solutions m a y be bioassayed. A typical dose-response curve is shown in Fig. 3 as given by 0.1 and 0.2 units of horse urinary kallikrein when added to 0.4 ml horse plasma. The plasma by itself should cause no contraction of the ileum and this should
686
OTHER PROTEASES
[50]
2 3 4 5 Fio. 3. Record showing the contractions of the isolated guinea pig ileum induced by the addition of 0A ml horse plasma to horse urinary kallikrein. 1 and 3, 02 units of enzyme. 2 and 4, 0.1 units of enzyme. 5, 0.4 ml horse plasma. Height of contraction given by 02 units----56, 52 mm, respectively. I
be demonstrated with each tissue both initially and throughout the day. If contraction should occur, the volume of plasma can sometimes be reduced, the plasma may be heated to 60 ° and dialyzed, or another plasma or tissue substituted. The height of contraction given by the various concentrations of unknown and standard are usually measured in millimeters and have been found to be linearly related to the logarithm of the dose. A four-point assay is to be preferred for those samples where precise quantitation is desired and details of the statistical treatment of the data for bradykinin have previously been published. 7 However, for routine assay of a large number of samples, the addition of two concentrations of unknown solutions (e.g., 0.1 and 0.2 ml) in a volume up to 0.5 ml, one of which has a height of contraction which falls between that of the standards, usually furnishes a sufficiently accurate estimate to permit location of the active fractions during purification. Definition of Unit. During the purification of hog pancreatic and human urinary kallikreins as reported in this paper, the original kallikrein unit (KU) was employed. This unit was defined as the decrease in the blood pressure of the dog following intravenous injection of 5.0 ml of dialyzed normal urine which had been taken from a pool of 50 liters of human urine. Varying pools of urine of this magnitude, however, have not always contained one kallikrein unit per 5.0 ml and the standard unit has been maintained by exchange of biologically active material. This unit of activity can only be measured using the dog's blood pressure as the method of bioassay and reflects the ability of the various kallikreins to release kinin only from dog kininogen. As this substrate was unsuitable for horse urinary kallikrein, one KL unit of horse urinary kallikrein was arbitrarily defined as that amount of activity per milligram of a crude ' M. Rocha e Silva, Acta Physiol. Latinoam. 2, 238 (1952).
[50]
GLANDULAR KALLIKREINS
687
preparation (Table I) and gives a calculated value of 0.25-0.75 K L units/ml horse urine. Recently, a new unit (KaU) has been proposed 8 wherein one milliunit of kallikrein is arbitrarily defined as that amount of enzyme which will release in 1.0 minute at 30 ° 1 nanomole of bradykinin equivalent from an amount of citrated plasma which has been heated for 1/2 hour at 60 ° and which contains an amount of substrate to the enzyme being assayed which is equivalent to 5 nanomoles of bradykinin. This provisional unit, however, has only recently been proposed and cannot be determined by direct bioassay as detailed above. However, it is recommended that in future publications, wherever possible, the units used for purification be standardized in terms of KaU.
Cleavage of Synthetic Esters Principle. Many methods are available for following the enzymatic cleavage of ester bonds. The method which has been chosen to be presented in detail is a modification of the Hestrin method9 and is based on the determination of residual ester by formation of a ferric complex with the hydroxamic ester resulting from the reaction of an ester with alkaline hydroxylamine.
Reagents Buffer. 0.1 M Tris, pH 8.5 Substrate. 0.12 M p-toluenesulfonyl-L-arginine ethyl ester (TAME). Dissolve 45.4 mg/ml distilled water and store at 4 ° when not in use. Alkaline hydroxylamine solution. 2 M (13.9%) aqueous hydroxylamine hydrochloride is mixed with an equal volume of 3.5 M NaOH just before use. Store 2 M hydroxylamine at 4 °. Hydrochloric acid, 3M. Dilute 1 volume of concentrated hydrochloric acid with 3 volumes distilled water. Ferric chloride, 10%. Dissolve 100 g FeCl.~.6 H~O in 1 liter 0.1 M HC1. Trichloroacetic acid (TCA), 7.5%
Procedure. Tubes containing 0.1 ml substrate and 1.5-1.8 ml buffer are placed in the water bath at 30 ° for exactly 5 minutes followed by the addition of 0.4-0.1 ml enzyme solution. The enzyme-substrate mixture M. E. Webster, in "Bradykinin, Kallidin und Kallikrein," Handbook of Experimental Pharmacology (E. G. ErdSs, ed.), Vol. XXV, p. 659. Springer Verlag, Heidelberg, 1970. ~P. S. Roberts, J. Biol. Chem. 232, 285 (1958); M. E. Brown, J. Lab. Clin. Med. 55, 616 (1960).
688
OTHER PROTEASES
[50]
is incubated at 30 ° for varying periods of time at one concentration of enzyme or for 1 hour with at least three dilutions of enzyme. The reaction is stopped by the addition of 2.0 ml 7.5% TCA, allowed to stand at room temperature for at least 30 minutes, and the precipitate removed by filtration or centrifugation. To 2.0 ml of the supernatant is added 2.0 ml of the alkaline hydroxylamine solution. After standing at room temperature for at least 25 minutes, 1.0 ml 3 M HCI and 2.0 ml 10% ferric chloride are added to the mixture and the color which develops is measured in exactly 15'minutes at 540 nm. Formation of gas bubbles in the colorimeter cell is largely prevented if the solution is thoroughly mixed and transferred to the cuvette just prior to reading. The samples are read against a reagent blank prepared by substituting buffer for enzyme and TAME. A blank of TAME should also be included which is prepared b y substituting buffer for enzyme and appropriate blanks of the enzyme solution can be prepared either by adding the enzyme to one series of tubes after the addition of the TCA or by substituting buffer for TAME. As the kallikreins are purified, little or no precipitate is obtained on the addition of the TCA. The procedure, therefore, can be simplified and the sensitivity increased by reducing the concentration of substrate to 0.06 M and stopping the enzyme reaction by the addition of the alkaline hydroxylamine. In fact, even with crude solutions, provided no protein precipitates when the acid and iron additions are made, the step involving precipitation with TCA can be omitted with consequent economy in substrate and effort. Definition o] Unit. Various authors have employed a number of different units for measurement of this enzyme activity. In future studies it is suggested that a unit based on the recommendations of the International Union of BiochemistryTM be employed. One unit would therefore be defined as that amount which will catalyze the transformation of 1 micromole of substrate per minute at 30 ° under the above standard conditions. Purification Procedures Horse Urinary Kallikrein 11 Step 1. A m m o n i u m Sul]ate Fractionation. Freshly collected horse urine (20-30 liters) can be stored at 4 ° and is fractionated with ammonium sulfate at this same temperature. The pH is adjusted to 7-7.5 with 10 M
~"Enzyme Nomenclature," p. 6. Elsevier, New York, 1965. 11E. S. Prado, J. L. Prado, and C. M. W. Brandi, Arch. Intern. Pharmacodyn. 137, 358 (1962); J. L. Prado, E. S. Prudo, C. M. W. Brandi, and A V. Katchburian, Ann. N.Y. Acad. Sci. 104, 186 (1963).
[50]
GLANDULAR KALLIKREINS
689
TABLE I PVmF~CXTmN OF HORSE URINARY KALUXREIN Specific activity Purification steps Horse urine, dialyzed 1. Ammonium sulfate fractionation, 0.4-0.6 saturation 2. Acetone fractionation, pH 6.5,
45-60% 3. DEAE-ceUulose chromatography, peak of activity 4. Acetone fractionation, pH 5.9, 60-75% 5. Sephadex G-75 gel filtration, peak of activity
KLU°/liter urine 520 315
KLU/mg protein b 0.37 1.3
EU'/mg protein 0.12
220
25
2.3
114
260
24
80
330
30
24
800
65
KLU (kallikrein liberating units) are defined as that amount of activity per milligram found in a crude 0.4-0.7 saturated ammonium sulfate fraction. The kinin liberated was measured by direct bioassay on the guinea pig ileum. A recent determination would suggest that one of these units is equivalent to approximately 1.0 mKaU as determined on the ~uinea pig ileum. b Protein measured by method of Lowry et al. (see text footnote 13). c EU (esterase units) are defined as that amount of kaUikrein which will hydrolyze 1 micromole of TAME per minute at 30° and pH 8.5. N a O H , solid a m m o n i u m sulfate (223 g/liter) is added with stirring and the solution stored overnight. Most of the clear supernatant is removed by siphoning and the remaining solution clarified by centrifugation. The precipitate (0.4 saturation), which contains less than 3% of the kallikrein activity of the urine, is discarded and additional a m m o n i u m sulfate (132 g/liter of supernatant) is added and the mixture again allowed to stand overnight. The precipitate is collected, suspended in the smallest possible volume of water, and dialyzed overnight against running tap water. The dialyzed solution is centrifuged to remove insoluble material and the s u p e r n a t a n t frozen until several batches have been accumulated. The total mixture (I50 liters of urine) contains about 60% of the kallikrein activity of the starting urine with a specific activity of 1.3 (Table I ) . Step 3. Acetone Fractionation, pH 6.5. The combined solution from step 1 is diluted to approximately 30 m g / m l protein, solid sodium chloride is added to m a k e the solution 0.1 M, and the p H is adjusted to 6.5. Acetone is added with stirring at room t e m p e r a t u r e to a final concentration of around 45% ( v / v ) . After standing for 30 minutes at room tern-
690
OTHEa r~OTF~SES
[50]
perature, the precipitate, which should contain about 15~ of the enzyme activity and 75~ of the protein, is removed by centrifugation and discarded. The concentration of acetone in the supernatant is raised to around 60% (v/v) and after 1 hour the precipitate, which should contain 70~ of the enzyme and 3 ~ of the protein, is separated by centrifugation, allowed to drain briefly at room temperature to remove traces of acetone, and dissolved in the smallest possible volume of 0.075 M sodium phosphate buffer, pH 5.9. The concentration of acetone necessary to achieve this purification may vary somewhat from preparation to preparation and preliminary small-scale trials (in increments of 5% acetone) should be conducted. Step 3. DEAE-Cellulose Chromatography. The enzyme solution is dialyzed for 16 hours at 4 ° against 20 times its volume of 0.075 M sodium phosphate buffer, pH 5.9, and added at room temperature to a column (2.7 X 20 cm) of DEAE-cellulose which had been previously equilibrated with the same buffer. TM The column is washed with the same buffer (about 600 ml) until the E2so (extinction coefficient in a 1-cm cell at 280 nm) becomes relatively constant (0.2 E2so) and 46% of the protein and 0.8% of the enzyme activity has been removed. The column is eluted stepwise with about 250 ml 0.1 M sodium phosphate buffer, pH 5.9, only until the E2so again comes to a value of 0.2. This eluate should contain about 7.8~ of the protein and 15% of the enzyme activity. The major portion of the enzyme activity is eluted from the column with 300 ml 0.125 M sodium phosphate buffer, pH 5.9, collected in 50-ml fractions and the enzyme activity and specific activity of the various fractions determined. Pooling the fractions with the highest specific activity should result in the recovery of about 50~ of the starting activity with a specific activity of 260. Step 4. Acetone Fractionation, pH 5.9, The enzyme activity in the pooled fractions from step 3 is rapidly concentrated by fractionation with acetone. In this procedure solid sodium chloride is added to the solution to make a final concentration of 0.03M and 11/~ volumes of acetone added at room temperature. After 60 minutes, the mixture is centrifuged and the precipitate discarded. The concentration of acetone in the supernatant is increased to 75% (v/v), and the mixture allowed to stand 1 hour. The precipitate is removed by eentrifugation and dissolved in the smallest possible volume of water. The solution is dialyzed overnight against several changes of distilled water at 4 ° and freeze dried to yield 75 mg of dry powder which can be stored in a dessicator at 4 °. Horse urinary kallikrein prepared in this manner is quite stable. However, further purification or, for example, preparation of horse urinary kalliE. A. Peterson and H. A. Sober, Vol. V, p. 3.
[50]
GLANDULAR KALLIKREINS
691
1.5
1500 i I I
I000 C
o
"~ 0.5
500
U
t
I0
15
20 Effluent
25
50
55
volume , mt
Fro. 4. Sephadex G-75 chromatogram of partially purified horse urinary kallikrein. Protein measured by method of Lowry et al? 3
krein from a DEAE-cellulose column employing a gradient rather than the stepwise procedure recommended above, yields a product which rapidly loses activity. Therefore, until methods are devised to stabilize this kallikrein, it is recommended that when more highly purified kallikrein is required for a particular experiment, it should be prepared as detailed in step 5 only in the amounts necessary and on the same day as the experiment. Step 5. ~ephadex G-75 Gel Filtration. The freeze-dried material from step 4 (17 mg) is dissolved in 1.0 ml distilled water and added at 4 ° to a 1.2 X 29 cm column of Sephadex G-75. It is washed through the column with distilled water (30 ml) at a flow rate of 4 ml/hour, collecting fractions of 0.5 ml. The enzyme activity is associated with the initial protein peak as shown in Fig. 4 and 55% of the activity can be recovered by pooling the main fractions. Those fractions with the highest specific activity (800) can be usually recovered in a 30% yield of the starting activity.
Human Urinary Kallikrein 14 Step 1. Amberlite IRC-50 Adsorption. Normal male human urine is collected daily and stored at 4 ° until about 180 liters is accumulated. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. B i o l . Chem. 193, 265 (1951). 14H. Moriya, J. V. Pierce, and M. E. Webster, Ann. N . Y . Acad. ~ / . 1{)4, 172 (1963).
692
OTR~n
[50]
PROZEASZS
To 174 liters of urine in a 200-liter tank is added 1.8 kg Amberlite IRC-50 (150-325 mesh) in the acid form 15 and the pH adjusted to 4.0 with approximately 2 liters of concentrated HC1. The mixture is stirred vigorously for 2 hours at 16 °, the resin recovered by filtration, washed on the funnel with 5 liters 0.1 M sodium acetate buffer, pH 4.0, suspended in 10 liters with distilled water and made 0.1 M phosphate and pH 6.0 with tribasic potassium phosphate and 10M N a 0 H . The suspension is stored overnight at 4 °, stirred for 1 hour at 15 °, and filtered. The resin is washed on the funnel with several liters of buffer and the combined eluates contain 80--100% of the enzyme activity of the urine (Table II). TABLE I I PURIFICATION OF HUMAN URINARY KALLIKREIN
Purification steps Human urine, dialyzed 1. IRC-50 adsorption 2. DEAE-cellulose batchwise adsorption 3. Acetone precipitation, pH 5.4, 50-60% 4. Ammonium sulfate fractionation, 69-83% 5. 8ephadex G-75 gel filtration 6. DEAE-Sephadex A-50 chromatography: peak of activity
EU"/liter urine
Specific activity EU/E~
120b 100 b 75 56 21 16 7.2
0.04 b 0.1 b 2.2 15 29 6O 23O
o EU (esterase units) were determined by measuring the hydrolysis of TAME at, pH 8.5, 37 ° and defined as that amount of activity given by one KU. A recent determination on a crude human kallikrein preparation would indicate that 1 EU will hydrolyze 0.02 micromoles of TAME per minute at 30 °, pH 8.5. The ratio of activity of EU/KU, where KU was determined by measuring the increase in arterial blood flow of the dog, was approximately 1.0 at all levels of purification. b These results were averages obtained on earlier pools of urine which had been dialyzed, freeze dried, and concentrated prior to assay. The bioassay employing the guinea pig ileum more readily determines the activity of these dialyzed fractions.
Step 2. DEAE-Cellulose Batchwise Adsorption. The eluates from s t e p 1 a r e d i l u t e d to 40 liters w i t h t a p w a t e r , 70 g D E A E - c e l l u l o s e {0.45 m e q / g ) a d d e d , t h e p H a d j u s t e d to 6.7, a n d t h e m i x t u r e s t i r r e d for 1 h o u r a t r o o m t e m p e r a t u r e . T h e a d s o r b e n t is r e c o v e r e d b y f i l t r a t i o n , w a s h e d on t h e funnel w i t h 600 ml 0.1 M s o d i u m p h o s p h a t e buffer, p H 5.8, c o n t a i n i n g 0.1 M NaC1 to r e m o v e i n a c t i v e m a t e r i a l s , a n d e l u t e d on t h e funnel w i t h 700 m l 0.5 M s o d i u m p h o s p h a t e buffer, p H 6.3. T h e l a t t e r e l u a t e c o n t a i n e d 5 5 - 8 5 % of t h e e n z y m e a c t i v i t y in t h e u r i n e a n d 15C~ H. W. Hirs, Vol. I, p. 113.
[50]
GLANDULAR KALLIKREINS
693
is dialyzed overnight against running tap water and freeze dried to afford 3.84 g of material which had been purified 55 times. Step 3. Acetone Precipitation. The dried material from step 2 is dissolved at 5 mg/ml in 0.065 M sodium acetate containing 0.035 M NaC1 and the pH adjusted to 5.4 with 5 M acetic acid. The solution is cooled in an ice bath and an equal volume of acetone added dropwise while stirring. After standing overnight at 0o-4 ° , the precipitate which contained 12% of the activity and 50% E~o is removed by centrifugation at 4 °, and the concentration of acetone in the supernatant increased to 60% (v/v). The following day the precipitate is removed by centrifugation, allowed to drain briefly at room temperature to remove traces of acetone, dissolved in 200 ml distilled water, and the pH adjusted to 7.0. Step 4. Ammonium Sulfate Fractio~ation. The above solution (E2so/ ml ----3) is cooled in an ice bath and a saturated solution of ammonium sulfate at 25 °, previously adjusted to pH 7.0 with ammonium hydroxide, is added dropwise with stirring to make 55% saturation. The mixture is allowed to stand overnight at 4 ° and the precipitate is removed by centrifugation at 4 °. In this manner, precipitates are removed at 60%, 65%, 69%, and 83% saturation. The fraction which precipitated between 69% and 83% contained 37% of the activity with a 2-fold increase in purity. The earlier fractions contain 53% of the starting activity at concentrations from 1.3--12 EU/liter and a specific activity of 2.7-19, respectively. The volume of the 69-83% fraction (10 ml) is reduced to 2.5 ml by placing the fraction in a dialysis bag and surrounding the bag with Sephadex G-75 in a suitable container. The level upper surface is covered with a flat plate, and weights are applied to increase the rate of water removal. TM After 4 hours at 4 ° the volume is usually reduced and, if not, the damp Sephadex can be peeled from the dialysis bag and replaced with fresh. The Sephadex is recovered by suspending in water, filtering, washing slowly on the funnel with distilled water and absolute ethanol (carefully layered on top of the gel), removing excess ethanol by suction, and air drying. Step 5. Sephadex G-75 Gel Filtration. The concentrated solution from step 4 (E28o/ml = 40) is added to a 1 X 46 cm column of Sephadex G-75 and washed through the column with approximately 100 ml of distilled water at 4 ° (flow rate, 4 ml/hour). Two E2so peaks are obtained, the second of which contained the enzyme activity. The three 4-ml fractions (48-60 ml effluent) comprised 75% of the starting activity with a 2-fold purification and are eluted from the column just ahead of the ammonium sulfate. H. Palmstierna, Biochem. Biophys. Res. Commun. 2, 53 (1960).
694
OTHER PROTEASES
[50]
Step 6. DEAE-Sephadex A-50 Chromatography. The pooled fractions from step 5 are concentrated to 2.5 ml (E28o/ml = 17) by dialysis against dry Sephadex G-75 as described in step 4 and ammonium formate, pH 6.7, is added to make a final concentration of 0.2 M. The solution is added to a 1 X 25 cm column of DEAE-Sephadex A-50 which has been previously equilibrated with the same buffer. The column is washed with 40 ml of the same buffer and a gradient of 0.2 to 0.SM ammonium formate and of pH 6.7 to 6.85 (125 ml constant-volume mixing chamber) is applied (flow rate, 5 ml/hour). The enzyme activity is eluted just slightly after the largest protein peak and pooling of the four best 2.5-ml fractions (70-80 ml of gradient effluent) gives a 45% recovery of enzyme activity with a 4-fold increase in purity. Based on the nondialyzable solids found in human urine, a 650-fold purification is realized. Hog Pancreatic Kallikrein 17 Step 1. Extraction. Hog pancreas, which had been obtained as fresh as possible from the slaughterhouse, is stored at --20 °. The glands (500 g) are thawed, cleaned of extraneous fat and minced in a meat grinder. The suspension is allowed to autolyze at 15°-25 ° for 18-24 hours, distilled water (2500 ml) is added, and the pH of the mixture adjusted to 6.0 with 6 M HC1. The mixture is stirred at room temperature for 5 hours, heated at 55 ° for 5 minutes, allowed to cool to room temperature, and the pH adjusted to 4.5 with 1 0 M HCI. The suspension is cooled in an ice bath and one-third the volume of acetone (30% v/v) added dropwise while stirring. Addition of the acetone directly to the suspension greatly improved the rate of filtration when compared to the aqueous extract and the precipitate is rapidly removed by filtration at 4 ° through filter paper placed on a Biichner funnel. The clear supernatant contains 50-75,000 KU at a purity of 0.4-1 KU/mg (Table III). Step 2. DEAE-Cellulose Batchwise Adsorption. A portion of the clear supernatant from step 1, corresponding to the extract from 100 g, is diluted 20-fold with tap water and 3.0 g DEAE-cellulose added. The pH of the suspension is adjusted to 7.0 with 1 M NaOH and the mixture stirred vigorously at 10°-20 ° for 2 hours, the pH being readjusted if necessary. The adsorbent is recovered by.filtration on a large glass funnel, transferred to a small glass funnel, and eluted on this funnel with about 60 ml 0.6 M ammonium formate, pH 6.0. The filtrate from the DEAEcellulose adsorption contains no detectable enzyme activity and 80% or 1, I'I. Moriya, K. Yamazaki, H. Fukushima, and C. Moriwaki, J. Biochem. (Tokyo) 58, 206 (1965); H. Moriya, A. Kayto, and It. Fukushima, Biochem. Pharmacol. 18, 549 (1969).
[50]
GLANDULAR KALLIKREINS
695
TABLE III PURIFICATION OF HOG PANCREATIC KALLIKREIN
Specific activity Purification steps 1. 2. 3. 4. 5. 6.
Extraction DEAE-cellulose batchwise adsorption Acrinol precipitation Precipitation at pH 4.2 Acetone precipitation, 0-67% Hydroxyapatite chromatography, peak of activity 7, Sephadex G-100 gel filtration, peak of activity
KUa/g tissue
KU/mg b
EUc/mg
125 110 70 30 30 15
0.7 10 45 75 140d 900d
-3 15 54 58d 220~
10
1200d
260d
a KU (kallikrein units) were determined by measuring the decrease in the blood pressure or increase in arterial blood flow of the dog. b Dry weight. c EU (esterase units) were determined by measuring the hydrolysis of TAME at pH 8.5, 37° and defined as that amount of activity given by one KU of human urinary kallikrein. These units will hydrolyze approximately 0.02 mieromoles of TAME per minute at 30° and pH 8.5. d Proteifi estimated by measuring E~, purest material 1 E~0 = 0.5 mg protein as estimated by Lowry et al., TM bovine serum albumin used as the standard. more of the enzyme is recovered in the eluate together with 4% of the nondialyzable solids. This step is repeated until all of the supernatant from step 1 has been adsorbed and eluted from DEAE-cellulose. Step 3. Acrinol Precipitation. The eluates from the DEAE-cellulose adsorption are pooled and the p H adjusted to 5.0 with 1 M formic acid. T h e solution is cooled in an ice b a t h and 30 ml 3% (w/v) aqueous acrinol (2-ethoxy-6,9-diaminoacridinium lactate) (Pfaltz & Bauer, Inc., Flushing, N.Y.) is added while stirring vigorously. The suspension is allowed to equilibr~ite for 4 hours at 4 ° and the supernatant clarified by centrifugation (10,000 rpm, 15 minutes, 4°). The p H of the supernatant is adjusted to p H 7.8 with 1 M a m m o n i u m hydroxide and additional acrinol (45 ml) added and the mixture allowed to stand overnight at 4 °. The precipitate, which contains the activity, is washed with 0.01 M a m m o nium acetate, p H 8.0, and extracted twice by stirring for 30 minutes with 125 and 50-ml portions of 0 . 6 M a m m o n i u m acetate, p H 4.5, in an ice bath. After centrifugation, the yellow supernatants are pooled, cooled in an ice bath, and precipitated by the addition of 3 volumes of acetone added dropwise with stirring. The suspension is stored overnight at 4 ° and the precipitate is collected by centrifugation, washed with acetone, allowed to drain briefly at room t e m p e r a t u r e to remove traces of acetone,
696
OTHER PROTEASES
[50]
and dissolved in distilled water. The slightly yellow solution contains 50-80~ of the starting activity and only 10-15% of the nondialyzable solids. Step ~. Precipitation at pH ~2. The pH of the aqueous solution from step 3 is adjusted to pH 4.2 with 1 M acetic acid and the suspension allowed to stand for 2 hours at 4 °. The voluminous inactive precipitate which forms is removed by centrifugation for 15 minutes at 10,000 rpm, 4 °. The clear supernatant is immediately adjusted to pH 6.5 with 1 M N a 0 H and contains 50-70% of the enzyme activity and 15-40% of the nondialyzable solids to give an average increase in purity of 2-fold or more. Step 5. Acetone Precipitation. Sodium acetate and sodium chloride are added to the supernatant from step 4 to give a final concentration of 0.065 M and 0.035 M, respectively. The pH of the solution is adjusted to 7.5 with 5 M NH4OH and precipitated by the addition of 2 volumes of acetone under the conditions described in step 3. The precipitate is suspended in the smallest possible volume of distilled water and kept frozen or freeze dried, if necessary, to give an average yield of around 60'mg (E28o/mg = 0.6). In subsequent purification steps the material can no longer be dried as it is unstable under these conditions. Step 6. Hydroxyapatite Chromatography. The concentrated solution from step 5, obtained from three 500-g batches of hog pancreas, is added to a 1.6 X 25 cm column of hydroxyapatite which has been previously equilibrated with 0.01 M sodium phosphate buffer, pH 6.8. The column is washed with 60 ml of the same buffer and a gradient of 0.01 to 0.15 M phosphate buffer (150 ml constant-volume mixing chamber) is applied. A flow rate of 6.0 ml/hour is used and fractions of 5.0 ml volume are collected. The enzyme activity is eluted in one peak (5 fractions) between 0.08 and 0.1 M buffer with 60--80% of activity recovered at a purity of 700-1000 KU/E28o. Step 7. Sephadex G-IO0 Gel Filtration. The active fractions from step 6 are pooled and concentrated to a volume of 1-3 ml by dialysis against dry Sephadex G-100 as described in step 4 of the purification of human urinary kallikrein or by pressure dialysis at 4 °. The solution is added at 4 ° to a 2.5 X 40 cm column of Sephadex G-100, washed through the column with distilled water (180 ml) at a flow rate of 12 ml/hour, collecting fractions of 4 ml. The enzyme activity is eluted in one main peak (50-70 ml effluent) and can be concentrated by the methods described above. The final product, which represents 80% of the starting activity, had a purity of 1,200 KU/E~8o and is obtained in a 5-8% yield having been purified approximately 2000-fold as calculated from the initial extract.
[50]
GLANDULAR KALLIKREINS
697
Properties Stability. Highly purified horse urinary kallikrein is unstable in solution, an instability which can be detected only if the kinin-forming or esterase activity is determined immediately. Storage of the enzyme in water or phosphate buffer, pH 8.0, even though the solution is mainrained most of the time in the frozen state, does not prevent the gradual loss of activity to a value around 500 KL units/rag protein. Human urinary kallikrein, at the level of purity so far attained, appears to be quite stable if maintained in solution at neutral pH (6-8). The hog pancreatic kallikrein prepared by this procedure is unstable in the dry form and is maintained in Tris buffer in the frozen state. Other highly purified preparations of this same enzyme have resulted in material which can be maintained in the dry form at --20 ° for several months. TM These latter preparations are stable for at least 5 hours at 20 °, pH 7-9.5, and even at pH 4.4 the enzyme shows little loss of activity in 1 hour, Purity and Physical Properties. Human urinary kallikrein migrated as a single band in electrophoresis on polyacrylamide gel at pH 8.7 and a molecular weight of 40,500 was found by short column sedimentation equilibrium. The hog pancreatic kallikrein prepared by this procedure migrated as a single sharp band in disc electrophoresis and immunoelectrophoresis. A molecular weight for this enzyme of 25,200 was estimated by sedimentation equilibrium. Other preparations, ls,~ have had molecular weights of 25,000 or 33,000 and at least two different kallikreins (A and B) have been separated by eleetrophoresis near pH 8.0 or chromatography on DEAE-cellulose. Even these highly purified kallikreins could again be separated into several active fractions which differed in their content of sialie acid. Whether the formation of some of these multiple forms of kallikrein depends on the degree of autolysis or on contamination of the autolyzates with a neuraminidase-containing bacteria from the intestine has not been clearly established. However, preliminary evidence suggests that two different prekallikreins, which may correspond to forms A and B, can be detected in pancreatic ~H. Fritz, J. Eckert, and E. Werle, Hoppe-Seylers Z. Physiol. Chem. 348, 1120 (1967) ; F. Fiedler and E. Werle, Hoppe-Seylers Z. Physiol. Chem. 348, 1087 (1967) ; F. Fiedler, Hoppe-Seylers Z. Physiol. Chem. 349, 926 (1968); F. Fiedler and E. Werle, European J. Biochem. 7, 27 (1968). lg H. Moriya, J. Pharm. Soc. Japan 79, 1451 (1959); H. Moriya, J. V. Pierce, and M. E. Webster, Ann. N.Y. Acad. Sci. 1{}4, 172 (1~3). E. Habermann, HoppeSeyler8 Z. Physiol. Chem. 328, 15 (1962); I. Trautschold, H. Fritz, and E. Werle, in "Hypotensive Peptides" (E. G. ErdSs, N. Back, F. Sicuteri, and A. F. Wilde, eds.), p. 221. Springer ¥erlag, New York, 1966.
698
OTHER PROTEASES
[50]
homogenates is and Moriya, in recent unpublished studies, has found that the preparation described in this paper can also be separated by isoelectric focusing into two or more active components. Activators and Inactivators. Following exhaustive dialysis, horse urinary kallikrein can no longer form kinins from dialyzed horse plasma and it has been shown that Na ÷, K ÷, Li ÷, Rb ÷, or Ca ÷ are required for its activation. 2° The velocity of the reactior of a preparation of hog pancreatic kallikrein with BAEE has been shown to increase up to a sodium chloride concentration of 0.05 M. 18 T.~ activation reported for this latter enzyme by sulfydryl compounds and chelating agents has been interpreted as due to a reversal of heavy metal inhibition. The esterolytic activity of both of the above kallikreins, as well as the formation of kinins by horse urinary kallikrein, is inhibited by heavy metal ions such as Co 2÷, Hg 2÷, Cu 2÷, Ni 2÷, and Mn ~÷ while Ca 2÷ and Mg ~÷ are without effect. All of the kallikreins are inhibited by certain trypsin inhibitors [diisopropylphosphofluoridate, benzamidine, pancreatic inhibifor (Kunitz), and Trasylol] but not by others [soybean, ovomucoid, and TLCK (1-chloro-3-tosylamide-7-amino-2-heptanone)]. Egg white inhibits human urinary kallikrein but not hog pancreatic kallikrein. Antibody to human urinary kallikrein prevents the formation of kinins by human urinary and pancreatic kallikreins but not hog pancreatic kallikrein. Other inhibitors have been found in plasma, various animal tissues, and plants and recently a number of synthetic trypsin inhibitors have been tested for their ability to inhibit some of the glandular kallikreins. Specificity. All three glandular kallikreins release kallidin from the kininogens in plasma. All are capable of cleaving various N-substituted arginine and lysine esters, the velocity of the lysine esters lower than the corresponding arginine ester. Although these enzymes do not usually cleave protein substrates such as casein and hemoglobin, horse urinary kallikrein at high concentrations has been shown to cleave the arginine bonds in salmine and in poly-L-arginine but it does not hydrolyze polyL-lysine. Kinetics. The rate at which the kallikreins cleave the various synthetic esters varies. For example, horse urinary kallikrein is only slightly more active on BAEE than on TAME, human urinary kallikrein is less active while a hog pancreatic kallikrein preparation was 30 times more active. The purest preparations of horse urinary kallikrein, human urinary kallikrein, and hog pancreatic kallikrein will hydrolyze, respec~oj. L. Prado, A. V. Katchburian, J. Mendes, and E. S. Prado, Acta Physiol. Latlnoam. 15, 386 (1965); C. Kramer, E. S. Prado, and J. L. Prado, Med. Pharmacol. Exp. 15, 389 (1966).
[51]
RENIN
699
tively, 65, 9, and 5 micromoles of TAME per milligram per minute at 30 °, pH 8.5. A K~ of 1.3 X 10-3 and 6 X 10`5 on this same substrate has been reported for horse urinary kallikrein and hog pancreatic kallikrein. Other kinetic values on synthetic substrates for a hog pancreatic kallikrein preparation have been given by Fiedler. TM Calculated values for the initial rate of kinin formation by horse urinary kallikrein under conditions of zero-order kinetics are 800 nanomoles equivalent of bradykinin per minute per milligram at 37 ° from heated and dialyzed horse plasma 2° and 140 nanomoles from a partially purified bovine kininogen.~1 Chemical Composition and Active Centers. Hog pancreatic kallikrein is a glycoprotein .containing glucosamine, galactose, fucose, mannose, glucose, and sialic acid? s This same preparation (a mixture of A and B forms) contained 283 amino acid residues and 19-21 sugar residues to give a calculated molecular weight of 33,400-33,800. Both a seryl and a histidyl residue are thought to be present in the active center of the kallikreins. Distribution. All exocrine glands, with the notable exception of the liver and gastric glands, contain a kallikrein ~ i c h is excreted by all of the glands except the pancreas in a free active form. A more comprehensive discussion and bibliography on the kallikreins can be found in following reviews. 22,23 21C. R. Diniz, A. A. Pereira, J. Barroso, and M. Mares-Guia, Biochem. Biophys. Res. Commun. 5, 448 (1965). ..,2E. K. Frey, H. Kraut, and E. Werle, "Das Kallikrein-Kinin-System und seine
Inhibitoren," Ferdinand Enke Verlag, Stuttgard, 1968. 23"Bradykinin, Kallidin and Kallikrein," Handbook of Experimental Pharmacology (E. G. ErdSs. ed.), Vol. 25. Springer Verlag, Heidelberg, 1970.
[51]
Renin
By R. R. SMEBY and F. M. BvMeus Renin substrate (angiotensinogen)
[Renin] * angiotensin I
Assay Principle. Renin has been assayed directly by its pressor effect on test animals or indirectly by measuring the amount of angiotensin generated by incubation in vitro. The direct assay has previously been described in this series? Recently, major interest has been shown in meas1E. Haas, Vol. II, p. 124.
[51]
RENIN
699
tively, 65, 9, and 5 micromoles of TAME per milligram per minute at 30 °, pH 8.5. A K~ of 1.3 X 10-3 and 6 X 10`5 on this same substrate has been reported for horse urinary kallikrein and hog pancreatic kallikrein. Other kinetic values on synthetic substrates for a hog pancreatic kallikrein preparation have been given by Fiedler. TM Calculated values for the initial rate of kinin formation by horse urinary kallikrein under conditions of zero-order kinetics are 800 nanomoles equivalent of bradykinin per minute per milligram at 37 ° from heated and dialyzed horse plasma 2° and 140 nanomoles from a partially purified bovine kininogen.~1 Chemical Composition and Active Centers. Hog pancreatic kallikrein is a glycoprotein .containing glucosamine, galactose, fucose, mannose, glucose, and sialic acid? s This same preparation (a mixture of A and B forms) contained 283 amino acid residues and 19-21 sugar residues to give a calculated molecular weight of 33,400-33,800. Both a seryl and a histidyl residue are thought to be present in the active center of the kallikreins. Distribution. All exocrine glands, with the notable exception of the liver and gastric glands, contain a kallikrein ~ i c h is excreted by all of the glands except the pancreas in a free active form. A more comprehensive discussion and bibliography on the kallikreins can be found in following reviews. 22,23 21C. R. Diniz, A. A. Pereira, J. Barroso, and M. Mares-Guia, Biochem. Biophys. Res. Commun. 5, 448 (1965). ..,2E. K. Frey, H. Kraut, and E. Werle, "Das Kallikrein-Kinin-System und seine
Inhibitoren," Ferdinand Enke Verlag, Stuttgard, 1968. 23"Bradykinin, Kallidin and Kallikrein," Handbook of Experimental Pharmacology (E. G. ErdSs. ed.), Vol. 25. Springer Verlag, Heidelberg, 1970.
[51]
Renin
By R. R. SMEBY and F. M. BvMeus Renin substrate (angiotensinogen)
[Renin] * angiotensin I
Assay Principle. Renin has been assayed directly by its pressor effect on test animals or indirectly by measuring the amount of angiotensin generated by incubation in vitro. The direct assay has previously been described in this series? Recently, major interest has been shown in meas1E. Haas, Vol. II, p. 124.
700
OTHER PROTEAS~S
[51]
uring renin activity in plasma of patients with hypertensive disease and most work has centered on indirect measurement of the plasma enzyme, since the amount of enzyme in plasma is too small to measure by direct assay. Many indirect assay procedures have been used, but most work has been done with the method reported by Helmer and Judson 2 or modifications of their procedure. Therefore, thiq assay and the modified methods of Pickens et al2 and Boucher eta[. ~ will be presented. Although these methods were developed for measurement of the enzyme in plasma, they can be used for the measurement of r,. in in kidney extracts if suitable substrate is supplied. A s s a y of H e l m e r and Judson 2 Reagents
NaC1, saturated Crude human renin prepared as described earlier in this series 1 by the first procedure Procedure. Venous or arterial blood is collected using heparin as an anticoagulant and chilled immediately with ice. The sample is centrifuged, the plasma separated and adjusted to pH 5.5. The plasma is transferred to dialysis bags and dialyzed against cold running tap water (13 ° ) for 18-20 hours. After dialysis, the pH is again adjusted to 5.5 and the precipitated proteins removed by centrifugation. The supernatant is made isotonic by the addition of 0.26 ml of saturated NaCl per 10 ml plasma. Renin substrate is determined by adding 3 ml saline containing 0.2 Goldblatt units 1 of human renin per milliliter of dialyzed plasma. This ~mount of renin provides an excess so that all substrate is converted to angiotensin. The samples are incubated for 1 or 2 hours at pH 5.5 and 37 ° and the reaction stopped by heating at 100 °. The amount of angiotensin produced can be measured by assay with a spirally cut strip of rabbit aorta, ~,e or a 48-hour bilaterally nephrectomized, pithed cat, * or t h e ganglion-blocked, vagotomized rat? 50. M. Helmer and W. E. Judson, Circulation 27, 1050 (1963). s p. T. Pickens, F. M. Bumpus, A. M. Lloyd, R. R. Smeby, and I. H. Page, Circulation Res. 17, 438 (1965). R. Boucher, R. Veyrat, J. deChamplain, and J. Genest, Can. Med. Assoc. J. 90, 194
(1964). 50. ~¢I.Helmer, Am. J. Physiol. 188, 571 (1957). e R. F. Furchgott and S. Bhadrakom, J. Pharmacol. Exptl. Therap. 108, 129 (1953). R. E. Shipley, O. M. Helmer, and K. G. Kohlstaedt, Am. J. Physiol. 149, 708
(1947).
[51]
RENIN
701
Calculation o] Activity. Activity is expressed in terms of the firstorder reaction constant calculated as: [2 - log 1oo (M - x / M ) ] 2.303 [ t J
(1)
Where M is the amount of angiotensin produced by incubation with excess renin, X is the amount of angiotensin produced by renin present in the plasma, and t is the time of incubation in minutes.
Assay of Pickens et al. s Reagents Disodium ethylenediaminetetraacetate (EDTA), 2.2 g/liter Diisopropylphosphofluoridate (DFP), 1 g dissolved in 19 ml isopropanol NaC1, 0.9% NaOH, 0.1 N 0.1% acetic acid HC1, 0.1 N Heparinized plasma (10 ml) is adjusted to pH 6.5, dialyzed against EDTA solution for 24 hours, and then against distilled water for 24 hours at 4 °. The precipitate is removed by centrifugation, 1 drop of DFP solution is added to the supernatant, and the pH adjusted to 5.5 with 0.1 N HC1. It is incubated for 4 hours at 37 °, then 1 ml 0.9% saline added, the volume adjusted to 20 ml with distilled water, the pH adjusted to 5.0, and the sample heated in a boiling water bath for 10 minutes. The protein is removed by centrifugation, the pH adjusted to 7.2 with 0.1 N NaOH, and back to 6.8 with 0.1% acetic acid and the solution taken to dryness in vacuo. The residue is redissolved in 1 ml of distilled water and the amount of angiotensin produced assayed with a ganglion-blocked, vagotomized rat. Expression o] Renin Activity. The amount of renin in the plasma is expressed as nanograms angiotensin generated per ml plasma per 4-hour incubation period.
Assay of Boucher et al. ~ Reagents Ammonium EDTA prepared by stirring 3.8 g of EDTA in 100 ml water and adjusting the pH to 6.5 with ammonium hydroxide
702
OTHER PROTEASES
[Sl]
Dowex resin 50W-X2, 100-200 mesh. Five hundred grams of resin are washed with 2 liters 4 N NaOH, then 1 liter water, 1 liter 2 N HC1, 2 liters water, and then with 0.2 N ammonium acetate, pH 6, until the eluate reaches pH 6. Ammonium acetate, 0.2 N, pH 6 Diethylamine, 0.1 N Ammonium hydroxide, 0.2 N Procedure. Blood is collected with ammonium EDTA as anticoagulant, chilled immediately to 0.5 ° and centrifuged. The pH of plasma is adjusted to 5.5 with 1 N HC1 and the plasma filtered through glass wool. To 10 ml plasma in a 50-ml Erlenmeyer flask is added 4 ml of moist, suspended Dowex 50W-X2 (NH4÷) resin. The mixture is incubated for 3 hours at 37 ° with vigorous shaking. Following incubation, the mixture is transferred to a glass column (1 X 10 cm) containing 1 ml resin. The column is washed with 15 ml ammonium acetate (pH 6) and 15 ml water. These washes are discarded. Angiotensin is eluted with 15 ml of 0.1 N diethylamine followed by 15 ml of 0.2N ammonium hydroxide into a flask containing a small amount of concentrated acetic acid. The solution is taken to dryness below 50 ° in vacuo. The residue is dissolved in 2 ml of 2 0 ~ aqueous ethanol and evaporated to dryness in vacuo. This is repeated 5 times to remove ammonium acetate. The final residue is dissolved in 1 ml of 20% aqueous ethanol for assay of angiotensin. Renin activity is expressed as nanograms angiotensin generated per 100 ml plasma per 3 hours of incubation. C o m m e n t s on Renin Assay. In these procedures, renin activity found may be modified by the presence of renin inhibitors s or activators present in plasma. The result has 'been termed "effective renin activity." To avoid this, Lever et al? isolated the renin from plasma before assay and Skinner 1° attempted to destroy these factors. The results of these assays have been mistakenly referred to as "renin concentration." Both types of assay have led to the same clinical conclusions and it is not known which may prove to be the most useful type of assay for the clinician.
Preparation of Hog Renin The procedure which yielded renin of 780 U/mg protein has previously been given in this series. 1 A simpler, chromatographic procedure s S. Sen, R. R. Smeby, and F. M. Bumpus, Biochemistry 6, 1572 (1967). 9A. F. Lever, J. I. S. Robertson, and M. Tree, Biochem. J. 91, 346 (1964). S. L. Skinner, Circulation Res. 20, 391 (1967).
[51]
RENIN
703
has recently been described by Skeggs et al. 11 which gave renin of 454 U/mg protein. During this purification four forms of the enzyme were observed and termed renins I, II, III, and IV from the order of elution from a DEAE-cellulose column. Renin I seems to be the major renin present in kidney with the other forms being formed during dialysis at pH 5. The procedure of Haas yields a mixture of renins II, III, and IV while the procedure of Skeggs et al. ~ given below yields renin II. P r o c e d u r e . The entire procedure is conducted at temperatures of 5 ° or less. Fresh, frozen hog kidneys (4.55 kg) are partially thawed and passed through an electric meat grinder into 5 liters of cold distilled water. The mixture is stirred for 1 hour and then filtered through several layers of cheesecloth. The kidney pulp is stirred an additional hour with a second 5-liter portion of cold water. The mixture is strained as before and the pulp discarded. The two filtrates are combined and shaken thoroughly with 2 liters of toluene. After standing for 1 hour the major portion of the upper layer is discarded and the lower layer centrifuged at 2500 rpm in a large centrifuge for 2 hours. The upper jellylike toluene layer and the gray bottom sediment are also discarded. The red, turbid, middle layer is diluted with 6 volumes of cold distilled water and the protein content determined. For each gram of protein in the solution, 15 g of a well-squeezed but moist filter cake of the free base form of DEAE-cellulose is added. The mixture is stirred for 3 minutes, then filtered on a large vacuum funnel and the DEAE pads squeezed by means of a rubber dam. The inactive filtrate is discarded. The DEAE pad is stirred with an original volume of cold water and pH adjusted to 4.6 with 1 N acetic acid. The pH is measured on a smalI portion of filtrate to which one-tenth volume of 1 M sodium chloride has been added. After adjusting the pH, the batch is stirred an additional 10 minutes and then filtered without further delay. The inactive filtrate is discarded. The DEAE pad is immediately suspended in a volume of cold 0.1 M sodium chloride which is one-third of the volume originally treated with DEAE cellulose. The pH is adjusted to 4.6 with 1 N acetic acid and the mixture filtered after stirring for 10 minutes. The DEAE pad is extracted once more in an identical fashion with 0.1 M sodium chloride. The two eluates are combined and solid ammonium sulfate added to obtain 2.5 M concentration. After stirring, the batch is allowed to stand in the refrigerator for 48 hours and the precipitate recovered by centrifugation and dissolved in 400 ml of cold water. At this point a typical preparation contains 14,000 Goldblatt units with a specific activity of about 1 U/mg protein. 'IL. T. Skeggs, K. E. Lentz, J. R. Kahn, and H. Hochstrasser, Circulation Res. Suppl. II 21, 91 (1967).
704
OTH~.S PROTSASZS
[51]
The enzyme is reprecipitated with ammonium sulfate by adjusting this solution to pH 7.5. Ammonium sulfate is then added to 1 M concentration, the mixture stirred thoroughly, and the precipitate removed by centrifugation and discarded. Sufficient ammonium sulfate is added to the supernatant to make the concentration 2.0 M. The mixture is stirred for a few minutes, centrifuged, and the supernatant discarded. The precipitate is dissolved in cold distilled water, dialyzed against cold distilled TABLE I SUMMARY OF REPORTED MICHAELIS CONSTANTS FOR VARIOUS RENINS Source of renin Hog kidney Hog kidney Human kidney Human kidney Human kidney Hog (I) kidney Hog (I) kidney Hog (I) kidney Hog (I) kidney Hog (I) kidney Hog (I) kidney Hog (II) kidney Hog (III) kidney Hog (IV) kidney Rabbit kidney Rabbit plasma Dog kidney Dog plasma Human kidney Human plasma Human urine Human amniotic fluid
Source of substrate Hog Rat Human Human Hog Hog (A) Hog (BI)
Hog (B2) Hog (C1) Hog (C~) Synthetic tetradeca Hog Hog Hog Ox Ox Ox Ox Ox Ox Ox Ox
pH
Ionic environment
Michaelis constant (ng/ml) Ref.
7.0 7.0 5.5 6.9 7.4 7.5 7.5 7.5 7.5 7.5 7.5
Tris, 0. 066 M Tris, 0. 066 M Salt free NaCl, 0 . 9 ~ Phosphate, 0.1 M Phosphate, 0.05 M NaC1 0.1 M NaC1 0.1 M NaCI 0.1 M NaC1 0.1 M NaC1 0.1 M
0.71 2.5 142 185 413 1724 742 742 1380 1254 1724
a a b c d c c e e e e
7.5 7.5 7.5 5.7 5.7 5.7 5.7 5.7 5.7 5.7 5.7
NaCI 0.1 M NaCI 0.1 M NaCI 0.1 M 0.15 M phosphate/saline 0.15 M phosphate/saline 0.15 M phosphate/saline 0.15 M phosphate/saline 0.15 M phosphate/saline 0.15 M phosphate/saline 0.15 M phosphate/saline 0.15 M phosphate/saline
1066 1066 1200 620 550 550 530 590 530 510 530
e e e f f f f f f f f
P. Blaquier, Am. J. Physiol. 208, 1083 (1965). b p. T. Pickens, F. M. Bumpus, A. M. Lloyd, R.R. Smeby, and I. H. Page, Uircula,. t/on Res. 17, 438 (1965). ' E. Haas and H. Goldblatt, Circulation Res. 20, 45 (1967). d A. B. Gould, L. T. Skeggs, and J. R. Kahn, Lab. Irwest. 15, 1802 (1966). ° L. T. Skeggs, K. E. Lentz, J. R. Kahn, and H. Hochstraaser, Circulation Res. Suppl. II 21, 91 (1967). ] R. R. Smeby and F. M. Bumpus, in "Renal Hypertension" (I. H. Page and J. W. McCubbin, eds.), p. 26. Year Book Medical Publishers, Chicago, IllinoiS, 1968.
[51]
RENIN
705
water containing 0.5 ml of 1 M sodium bicarbonate and 0.5 ml of 1 M sodium acetate per liter and having a pH of 7. Excess toluene is added to the buffer to act as a preservative. In this way the pH of the dialyzate was maintained between pH 7.0 and 7.5. Following dialysis, the solution is adjusted to pH 5 and the precipitate which formed is quickly removed by centrifugation. The pH of the supernatant is immediately adjusted to 7 and stored in the frozen state until used. A typical preparation contains about 3800 U of activity with a specific activity of 2.33 U/rag protein. This preparation is purified further by chromatography on a DEAEcellulose column. The column is 1.15 em in diameter by 55 cm high and equilibrated with 0.025 M sodium acetate buffer, pH 5.35. The pH of the enzyme sample is adjusted to 5.35 and diluted if necessary to achieve an ionic strength no greater than that of the 0.025M sodium acetate buffer. After application of the sample, the 0.025 M buffer, pH 5.35, is pumped through the column at a rate of 0.48 ml/minute until ten 5-ml fractions are collected. Buffer was then pumped into the column from a cylindrical gradient mixing chamber which initially contained 500 ml. At the same time, 0.025 M acetic acid was added to the chamber with mixing at 0.24 ml/minute. Thus, a descending pH gradient was formed which usually reads 3.8 after about 200 5-ml fractions are collected. Renin I is eluted from such a column at pH 5.16, renin II at pH 4.72, renin III at pH 4.53, and renin IV at pH 4.25. Renin I represents the largest renin activity peak. Renin I peak is dialyzed at pH 5.0 for 45 hours at 5 ° during which renin I is converted to renin II. This is rechromatographed on a column as described above and renin II represents the main renin TABLE II pH OF'rlmA FOR
RENIN FROM DIFFERENT SOURCES
Source of renin
Substrate
pH Optimum
Ref.
Rabbit Human Human Human Hog
Ox Human Ox Hog Synthetic
5.4--6.2 5.5 6. O 7-8 6.5-7
a b c d e
a A. F. Lever, J. I. S. Robertson, and M. Tree, Biochem. J. 91, 346 (1964). P. T. Pickens, F. M. Bumpus, A. M. Lloyd, 1~. R. Smeby, and I. H. Page, Circulation Res. 17, 438 (1965). c j. j. Brown, D. L. Davies, A. F. Lever, J. I. S. Robertson, Can. Med. Assoc. 90, 201 (1964). A. B. Gould, L. T. Skeggs, and J. R. Kahn, Lab. Invest. 15, 1802 (1966). • D. Montague, B. Riniker, and F. Gross, Am. J. Physiol. 210, 595 (1966).
706
OTHER PROTEASES
[52]
activity. The specific activity of this fraction is usually about 450 U/rag protein. Properties. The Michaelis constants for renins from a number of sources and under a variety of conditions have been reported. These are summarized in Table I. The molecular weight of hog renin has been determined by Sephadex chromatography to be between 40,000 and 50,000.12,13 Highly purified hog renin is highly unstable in the cold. 12 The pH optima for renin from several species are given in Table II. W. S. Peart, A. M. Lloyd, G. N. Thatcher, A. F. Lever, N. Payne, and N. Stone,
Biochem. J. 99, 708 (1966).
1, E. Kemp and I. Rubin, Acta Chem. Scand. 18, 2403 (1964).
[52]
Staphylokinase
By CHARLES H. LAC~ and KXTTY L. A. GLANVILLE Staphylokinase is an enzyme, produced by many strains of M. Staph-
ylococcus aureus, which converts plasminogen to plasmin. A s s a y Method •
Principle. All methods available for staphylokinase assay depend on the initial activation of plasminogen to plasmin by staphylokinase under standard conditions followed by measurement of the fibrinolytic or proteolytic activity of the plasmin formed. Most fibrinolytic methods are based on visual observation of clot lysis time and suffer from poor reproducibility. The fibrin plate method 1 is a convenient, simple procedure for screening activator activity. For a fuller discussion of assay methods, see Chapter by F. B. Taylor and R. H. Tomar on streptokinase [63] in this volume. Casein is a less sensitive substrate for plasmin than fibrin, but the results obtained with casein are more reproducible and lend themselves to rate determination. The degree of casein digestion can be easily determined from the extinction at 280 nm of the perehlorie acid-soluble fraction of the digest. The method described was developed by Davidson 2 and is a modification of a technique reported by Norman 3 for the assay of plasminogen. 1T. Astrup and S. Mullertz, Arch. Biochem. Biophys. 40, 346 (1952). 2F. M. I)avidson, Biochem. J. 76, 56 (1960). ' P. S. Norman, J. Ezptl. Med. 106, 423 (1957).
706
OTHER PROTEASES
[52]
activity. The specific activity of this fraction is usually about 450 U/rag protein. Properties. The Michaelis constants for renins from a number of sources and under a variety of conditions have been reported. These are summarized in Table I. The molecular weight of hog renin has been determined by Sephadex chromatography to be between 40,000 and 50,000.12,13 Highly purified hog renin is highly unstable in the cold. 12 The pH optima for renin from several species are given in Table II. W. S. Peart, A. M. Lloyd, G. N. Thatcher, A. F. Lever, N. Payne, and N. Stone,
Biochem. J. 99, 708 (1966).
1, E. Kemp and I. Rubin, Acta Chem. Scand. 18, 2403 (1964).
[52]
Staphylokinase
By CHARLES H. LAC~ and KXTTY L. A. GLANVILLE Staphylokinase is an enzyme, produced by many strains of M. Staph-
ylococcus aureus, which converts plasminogen to plasmin. A s s a y Method •
Principle. All methods available for staphylokinase assay depend on the initial activation of plasminogen to plasmin by staphylokinase under standard conditions followed by measurement of the fibrinolytic or proteolytic activity of the plasmin formed. Most fibrinolytic methods are based on visual observation of clot lysis time and suffer from poor reproducibility. The fibrin plate method 1 is a convenient, simple procedure for screening activator activity. For a fuller discussion of assay methods, see Chapter by F. B. Taylor and R. H. Tomar on streptokinase [63] in this volume. Casein is a less sensitive substrate for plasmin than fibrin, but the results obtained with casein are more reproducible and lend themselves to rate determination. The degree of casein digestion can be easily determined from the extinction at 280 nm of the perehlorie acid-soluble fraction of the digest. The method described was developed by Davidson 2 and is a modification of a technique reported by Norman 3 for the assay of plasminogen. 1T. Astrup and S. Mullertz, Arch. Biochem. Biophys. 40, 346 (1952). 2F. M. I)avidson, Biochem. J. 76, 56 (1960). ' P. S. Norman, J. Ezptl. Med. 106, 423 (1957).
[52]
STArI~VLOrJNASE
707
Reagents
(1) Human plasminogen, prepared by the method of Kline. ~ This is assayed by Davidson's 2 casein method which is carried out under identical conditions to the activator assay described below except that various dilutions of plasminogen are activated by 60 Christensen units of streptokinase for 5 minutes at 37 ° before casein solution is added. To determine plasmin contamination in the preparation of plasminogen, the assay is carried out using buffer in place of activator. Activity is expressed in terms of an "extinction" unit which gives, under the conditions of the test, an increase in E at 280 nm of 1.00/ minute of digestion. Therefore the activity of a solution of plasminogen assayed will be R / ( V )~ 30) units, where R is the extinction reading, after subtraction of the appropriate blank value, for the particular volume V of plasminogen assayed. (2) Palitzsch's borate saline buffer, pH 7.42 Boric acid 0.2 M containing 50 mM NaC1 is mixed with sufficient 50 mM sodium borate to give the required pH. (3) Casein solution. 4% Light white soluble casein (B.D.H. Poole, Dorset) is used without further purification. The casein is dissolved in Palitzsch's buffer, pH 7.4, by stirring at 37 ° or 56 °. The solution is stored at 4 ° and is made up fresh about every 2 weeks. (4) Perchloric Acid. A one in ten aqueous dilution of the 6 0 ~ solution obtained commercially is used. Procedure. METHOD. Several volumes of the staphylokinase solution to be assayed are incubated in duplicate in a 37 ° water bath in the presence of 50 milliunits of human plasminogen (about 0.1 ml) in a total volume of 1.1 ml, the volume being made up with Palitzsch's buffer, pH 7.4. After exactly 30 minutes, 1 ml of casein solution is added to each tube as substrate for the plasmin formed, and incubation at 37 ° is continued for exactly 30 minutes. Undigested casein is then precipitated by the addition of 3 ml of perchloric acid. The precipitate is allowed to form for 1 hour or more before it is removed by filtration on Whatman No. 1 filter paper (5.5 cm). The extinction coefficient (E) at 280 nm of the PCA-soluble digestion products contained in the filtrates is measured in a spectrophotometer. Readings are made against a "blank" filtrate prepared by adding 1 ml of casein and 3 ml of perchloric acid to 1.1 ml of buffer. The assay includes a plasminogen blank and staphylokinase blanks in which the activator or plasminogen respectively is replaced by Palitzsch's buffer. Both blank values are subtracted from the corresponding assay reading. Davidson determined the activity by interpolation. It
D. L. Kline, J. Biol. Chem. 204, 949 (1953).
708
OTaER rR~F~SES
[52]
was preferred, however, to read the activity off a standard graph prepared previously, since the curve is sigmoid in shape and it is thus easier to assess within which range of staphylokinase concentration used, the readings are reliable. Definition o] Unit and Specific Activity. A unit of staphylokinase activity is defined as that activity which gives rise, under the conditions of the assay described, to an increase in E. of 0.300. This unit is found to be approximately equal to one third of a Christensen unit? The specific activity of a preparation is expressed as units per milligram protein2 Production and Purification Procedure A strain of Staphylococcus aureus, which has been selected for its high yield of staphylokinase production,e is stored in the freeze-dried state and fresh cultures are prepared as required. These are used to inoculate a casein hydrolyzate medium~ which is shaken for 7 hours at 37 ° .2 Two-liter batches of broth culture containing staphylokinase were obtained by this process. Step 1. Preparation o] Culture Supernatant Fluid. Cells are removed from the culture by eentrifugation and the supernatant fluid is heated at 75 ° in a water bath for 30 minutes to kill any that remain. This treatment has little effect on staphylokinase activity2 Step 2. Ammonium Sul]ate Precipitation o] Active Material? The culture supernatant fluid is brought to 75% saturation with solid (NH,)~SO, (i.e., 3.07M) and allowed to stand at 4 ° overnight. The precipitate is separated by centrifuging, and washed with a saturated (NH4)~SO4 solution. It is then suspended in a suitable volume of water and dialyzed at 4 ° against running tapwater for 3 days, followed by distilled water, until free from sulfate ions. This step serves mainly to concentrate staphylokinase from a large volume without considerably increasing the specific activity. Loss of activity at this stage is variable (26--80%). Step 3. Chromatography on a CM-Cellulose Column? The staphylokinase concentrate is purified by chromatography on a CM-cellulose column1° at room temperature in one of two ways: 50. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). e C. H. Lack, J. Clin. Pathol. 10, 208 (1957). ' E. S. Duthie and G. Haughton, Biochem. J. 70, 125 (1958). 8E. B. Gerheim, J. H. Ferguson, B. L. Travis, C. L. Johnston, and P. W. Boyles, Proc. Soc. Exptl. Biol. N.Y. 68, 246 (1948). DK. L. A. Glanville,Biochem. J. 88, 11 (1963). E. A. Peterson and H. A. Sober, Vol. V, p. 3.
[5 2 ]
STAPHYLOKINASE
709
(1) When a concentrated staphylokinase solution of only moderate purity is required, the material is adsorbed on a column at pH 4 and eluted at pH 7 as follows: The dialyzed staphylokinase suspension is dissolved at pH 4 by the addition of an equal volume of 0.2 M citric acid-0.4 M phosphate buffer at pH 4, the alkaline component of the buffer system being added first to aid solution at the higher pH value. After addition of the citric acid component, the pH is checked and adjusted to 4 if necessary. Any undissolved material is removed by eentrifugation. The volume of the supernatant fluid is usually about one-tenth that of the broth culture from which it was derived. The supernatant fluid at pH 4 is applied to a CM-eellulose column (e.g., 1.7 X 11 cm) equilibrated with 0.1M citric acid-0.2 M phosphate buffer at pH 4 at the maximum flow rate obtained by gravity. Under these conditions all staphylokinase activity is adsorbed on the column; the highly pigmented effluent contains 50% or more of the protein applied. The column is washed with pH 4 buffer until the effluent is free of protein, as indicated by measurement of E2~on~,. Elution is carried out at pH 7 using the same buffer system. Most of the pigment has been removed and recovery of activity from the column is high, purification with respect to protein being 5- to 9-fold. Staphylokinase cannot be separated satisfactorily from hemolysin by this method. As the kinase moves with the pH 7 solvent front together with a faint pigment, the active fraction can be easily collected and only a minimum of control assays have to be carried out. (2) To obtain a more highly purified staphylokinase preparation free from hemolysin, a technique is used similar to that above, but employing stepwise elution with pH 6 sodium phosphate buffers of increasing molarity as follows: The staphylokinase concentrate is adsorbed on the column, equilibrated with the starting buffer either at pH 4 as described for (1) or at pH 6 from 15 mM sodium phosphate buffer. The column is developed by stepwise. increase of molarity of the pH 6 phosphate buffer solution, i.e., 15 mM, 45 mM, and 0.25 M. Hemolysin, being more strongly adsorbed than staphylokinase, can thus be completely removed from the preparation. Staphylokinase is eluted by 45 mM buffer solution whereas hemolysin is collected either by prolonged elution with the same buffer strength, or by increasing the molarity of the buffer to 0.25 M. Forty-four-fold purification of staphylokinase with respect to protein could be achieved by this method. Step 4. Precipitation by Ethanol. 11 Staphylokinase is purified further n K. L. A. GlanviUe,Ph.D. thesis, London University, England, 1966
710
OTHER
[52]
PROTEASES
O
v
I
°°
o
O O
O
8 o
[S 2 ]
STAPHYLOKINASE
711
by precipitation with an equal volume of absolute ethanol. The precipitate that is allowed to develop for about 20 hours at 4 ° is separated by centrifugation, washed once with 50% ethanol and dissolved in a suitable volume of distilled water. Recovery ranged from 50 to 100% and purification of staphylokinase, with respect to protein, varied from 3-to 7-fold. Freeze Drying. The purified staphylokinase solution is distributed in ampoules in 1 or 2 ml-aliquots and freeze dried. Loss on freeze-drying was negligible under these conditions and no activity was lost on storage of the freeze dried material for 2 years at 4 °. Degree o/Purification. The highest degree of purification achieved by this method, using elution method (2) in step 3, was 100-fold-purification of the (NH,)2S0~ precipitated concentrate (Table I). Modification. This method has been modified by Dr. K. Sargeant of the Microbiological Research Establishment, Porton, England, who carried out purification of 100-liter batches of culture supernatant fluid. The dialysis step to remove (NH4)2S0~ was replaced by deionization on Sephadex G-50 in 25 mM phosphate buffer at pH 6. For the chromatographic separation, CM-cellulose was replaced by CM-Sephadex G-50 equilibrated with 50 mM citric acid-phosphate buffer at pH 5. The active fraction, adjusted to pH 5 with McIlwaine's buffer at pH 4.4, was filtered and the filtrate applied to the column which was washed with 50 mM citric acid-phosphate buffer at pH 5. Elution was carried out using a gradient obtained by means of a two-chamber system containing 50 mM citric acid-phosphate buffer at pH 5 and 0.2 M citric acid-phosphate buffer at pH 6. The bulk of staphylokinase activity was eluted after the main protein peak and had a specific activity of 1000--3000 U/mg. A different method for the purification of staphylokinase involves fractional precipitation with (NH4)~S04, dialysis, precipitation at pH 5.5, and chromatography on Amberlite XE-64. TM Properties
Stability. Staphylokinase is relatively heat stable. 8 Maximum stability of the purified solution is at pH 6 to 8. At this pH no activity of the semisterile solution was lost in 24 hours at 37°; when heated to 100 ° for 45 minutes the solution lost about 40% of its activity. Stability is greater in concentrated than in diluted solutions, particularly in the presence of neutral buffer. Solutions in Palitzsch's buffer at pH 7.4 were stored for over 2 weeks at 4 ° without loss of activity. At --20 ° solutions in buffer containing above 40 U/ml were stable for more than 4 months. u E. Soru and M. Sternberg, Enzymolo~ia 25, 231 (1963).
712
OTHER PROTEASES
[52]
Dilute solutions of purified staphylokinase usually lost some activity when being pipetted. This could not be remedied by silicone treatment of the glassware but was prevented by addition of 0.2% (v/v) of Triton X-100, a nonionic detergent, to the staphylokinase solution. As this treatment reduced the stability of purified staphylokinase on storage, it seemed advisable to add Triton X-100 to the solution only shortly before it was pipetted out for an experiment. When assaying staphylokinase containing 0.2% (v/v) Triton X-100, the activator activity appeared to be enhanced.11 Purity and Physical Properties.~ A solution of staphylokinase (specific activity 900 U/rag), purified as described in the text using method (2) in step 3 still contains a protein impurity as shown by: (1) Starch gel electrophoresis. Two protein bands could be observed, the stronger band 'being associated only with activator activity. (2) Gel filtration on Sephadex G-100. The main protein peak which contained the activator activity was closely followed by an inactive smaller protein peak. This method could be used for further purification of staphylokinase. (3) Immunodiffusion on agar gel. Using rabbit antiserum prepared against an impure staphylokinase preparation, one main line of precipitation was obtained together with an extremely faint line. No coagulase, leukocidin, a-, fl-, or &hemolysin were detected in the preparation. The freeze-dried material is a white powder soluble in water near or above neutral pH. Buffer solution enhances the solubility. The ultraviolet absorption spectrum of the solution is that of a protein. The approximate molecular weight of staphylokinase as determined by gel filtration on a calibrated column of Sephadex G-100 is 22,500, while that of streptokinase determined on the same column is 53,000. The isoelectric point was found by paper electrophoresis to be near pH 6.7. This value is only approximate because of the limitations imposed by electrophoresis on a supporting medium. Inactivators. STAPHYLOKINASEANTIBODY.Staphylokinase is antigenic. ~8 A wide range of inhibitor level in the population has been demonstrated ~ and a rise in antistaphylokinase titer in nine out of thirty-five cases of staphylococcal osteomyelitis was observed. 15 Antibody can be obtained in serum of rabbits sensitized experimentally to staphylokinase over a period of 35 days. ¢-A~INOCAPROIC ACID (¢-ACA). ¢-ACA is an inhibitor of plasmin F. Aoi, Kitasoto, Arch. Exptl. Med. 9, 171 (1932). 14B. Sweet, G. P. McNicol, and A. S. Douglas, Clin. Sci. 29, 375 (1965). C. H. Lack and A. G. Towers, Brit. Med. J. 2, 1227 (1962).
[52]
STAPHYLOKINASE
713
activity TM but its effect on plasminogen activation b y streptokinase as a competitive inhibitor is much greater. 17 Activation of human plasminogen by staphylokinase is also inhibited by ~-ACA, but to obtain comparable inhibition of plasminogen activation about ten times higher c-ACA concentrations are required for staphylokinase than for streptokinase activation. At very low c-ACA concentration (e.g., 20 mM) its stabilizing effect on plasmin m a y be more pronounced than its inhibitory action. 11 CASEIN. Activation b y staphylokinase is inhibited in the presence of 2% casein, or an impurity in the casein used. 2 S p e c i f i c i t y . The only known action of staphylokinase is that of plasminogen activation. It does not itself hydrolyze casein and has no esterolytic effect using T A M E as substrate. 14 Unlike streptokinase, staphylokinase can activate the plasminogen of a number of animal species without the addition of human globulin as source of proactivator, but in contrast to urokinase, it does not activate ox plasminogen. Table II shoWs the results obtained by a number of workers when activating plasminogen prepared from various animal species. The resistance of bovine plasminogen to activation by staphylokinase is not due to the presence of an inhibitor of staphylokinase since the activation of human plasminogen by staphylokinase is not affected by the addition of ox euglo'bulin. 2 Cliffton and Cannamela TM noted a difference in the fibrinoly.tic and proteolytic activities of the plasmin derived from staphylokinase activation of certain animal serum plasminogens. TABLE II STAFHYLOKINASE ACTIVATION OF DIFFERENT PLASMINOGENS
Activated
References
Not activated
References
Human Monkey Dog Cat Rabbit Guinea pig
19,20,21 18 18,20,21 20 18,19,20,21 18,19,20,21
Rat Ox Chicken Horse Sheep Pig
18,20,21 18,19,20,21 18,21 18,21 21 18
1, S. 0kamoto and F. Nagasawa, Patent Specification 770, 693, London, England. The Patent Office, Oct. 21st (1954). p. 9 Granted, 1957. 17N. Alkjaersig, A. P. Fletcher, and S. Sherry, J. Biol. Chem. 234, 832 (1959). 1~E. E. Cliffton and D. A. Cannamela, Blood 8, 554 (1953). F. M. Davidson, Nature 185, 626 (1960). ~oj. H. Lewis and J. H. Ferguson, Am. J. Physiol. 166, 594 (1951). ~1E. B. Gerheim and J. H. Ferguson, Proo. Soc. Exptl. Biol. Med. 71, 261 (1949).
714
OTHER PROTEASES
IS2]
K i n e t i c Properties. The maximum rate of activation of human plasminogen by staphylokinase is between 37°-45 °1~ and at p i t 8.6.11 The resulting plasmin, however, is not s t a b l e under these conditions unless a stabilizing agent, e.g., glycerol,22 is added. The enzymatic nature of staphylokinase has been demonstrated for the activation of dog 2° and human ~ plasminogens. A free a-amino group in the staphylokinase molecule is essential for its activator activity. ~2 H u m a n plasminogen can be activated as completely by staphylokinase as by streptokinase, 2 yielding a plasmin whose molecular weight and amino acid composition are of the same order as those of streptokinase or urokmase-actlvated plasmin. ~s
Distribution Staphylokinase is usually produced by eoagul.ase-positive strains of staphylococcus of human origin• Christie and Wilson s' found that 92 out of 99 coagulase positive but none of 12 eoagulase-negative strains produced fibrinolysis. The coagulase-positive strains which failed to produce lysis were strong producers of fl-hemolysin. The production of fl-hemolysin is stated to exclude the production of staphylokinase. 25 Strains have been isolated which produce staphylokinase but no coagulase or hemolysins. 26 Of the strains isolated from human infections 96.4%, 2~ 70%, 2s 60%, 29 and 16~'o~e were reported to be ~fibrinolytic, whereas 100% s° and 50% 26 isolated from the dog and only 5% s8,s~ isolated from the ox were found to be fibrinolytie.
'~ S. Shulman, N. Alkjaersig, and S. Sherry, J. Biol. Chem. 233, 91 (1958). E. Soru, Rev. Roum. Biochim. 5, 17 (1968). 2~R. Christie and H. Wilson, Australian J. Ezptl. Biol. Med. 8ci. 19, 329 (1941). P. M. Rountree, Australian J. Exptl. Biol. Med. ~qc/.25, 359 (1947). x G. lq'entschel and H. Blobel, Zentr. Bakteriol. Parasitenk, Abt. L 0r/9. 206, 193 (1968). A. Kaffka, Zentr. Bakteriol. Parasitenk. Abt. I, Orig. 108, 381 (1957). ~8C. H. Lack and D. G. Wailling, J. Pathol. Bacteriol. 68, 431 (1954). G. H. Chapman, J. Bacteriol. 43, 313 (1941). H. W. Smith, J. Comp. Pathol. 57, 98 (1947). ~ St. H. George, K. E. Russell, and J. B. Wilson, J. In]ect. Diseases 110, 75 (1962).
[53]
SNAKE VENOM PROTF-JkSES
715
[53] Snake Venom Proteases Which Coagulate Blood
By M. P. ESNOUF Malayan Pit Viper (Ancistrodon rhodastoma) Venom Assay Method ~ The coagulant fraction of this venom, while acting almost exclusively on fibrinogen, has in addition a slight factor X converting activity. 2 The thrombinlike activity of the fraction is conveniently assayed by adding different dilutions of the fraction to a standard solution of fibrinogen and recording the clotting times. The clotting times are then compared with those obtained with dilutions of the crude venom.
Reagents Fibrinogen: A bottle of human fibrinogen (grade L from A.B. Kabi, Stockholm, Sweden) was dissolved in 100 ml distilled water and then diluted with an equal volume of 0.15 M Tris and the pH adjusted to 7.5 with hydrochloric acid.
Method. The fibrinogen solution (0.3 ml) is incubated at 37 ° with 0.1 ml of the buffer in 2½ inch X 3/8 inch glass tubes. The venom solution (0.! ml) is added, and the time from the addition of the venom to formation of the clot is recorded. Under these conditions a venom solution (30 ~gfml) gives a clot in about 20 seconds. The factor X converting activity of the venom fraction is readily demonstrated by first incubating the venom fraction with A. rhodastoma antivenom until the thrombinlike activity has been lost and then adding the venom to factor X-deficient plasma which will give a clotting time of at least 3 minutes compared with a clotting time of about 20 seconds when factor X and calcium ions are added to the factor X-deficient plasma. Assay Method 2
In addition to the coagulation assay, the purified fraction can be assayed by its esterase activity on various synthetic substrates, such as the esters of arginine or the p-nitrophenyl esters of carbobenzoxy derivatives z M. P. Esnouf and G. W. Tunnah, Brit. J. Haematol. 13, 581 (1967). L. Nahas, If. W. E. Denson, and R. G. Macfarlane, Thromb. Diath. Haemorrhag. 12, 355 (1964).
716
OTHER PROTEASES
[S3]
of the aromatic amino acids. The method using benzoyl arginine ethyl ester~ (BAEE) will be described. Reagents Benzoyl arginine ethyl ester is dissolved in 0.05 M Tris adjusted to pH 8.0 with hydrochloric acid, to give a final concentration of ester of 1.5 mM. Venom solution. Only the purified coagulant fraction can be assayed by this procedure, because there are other components in the venom which will hydrolyze these synthetic substrates but which have no activity on fibrinogen. Method. Two milliliters of the 1.5 mM benzoyl arginine ethyl ester solution was incubated at 37 ° in a 1-cm spectrophotometer silica cuvette; after a few minutes a sample (1 ml) of the venom fraction in 0.05M Tris-hydrochloric acid buffer, pH 8.0, was added. The change in the optical density of the mixture was measured at 253 nm. If p-toluenesulfonyl arginine ester is to 'be used as the substrate instead of BAEE, the reaction is followed at 247 nm. The rate of hydrolysis obtained in this way is corrected for the rate of spontaneous hydrolysis of these esters at pH 8.0. Under these conditions the rates of hydrolysis of benzoyl arginine ethyl ester and p-toluenesulfonyl arginine are about 100 millimicromoles/ minute/~g and 40 millimicromoles/minute/~g, respectively. Units. Since the venom contains other esterases, this latter type of assay can only be used to relate the different activities of purified preparations. Because of this, the clotting assay, though less precise, is more specific, and can be used in computation of specific clotting activity relative to the crude venom. However, since different batches of venom vary in their content of thrombinlike activity, the most convenient unit to use is based on equivalent thrombin units. Purification The purification consists of a two-step chromatography process. In the first stage the crude venom is chromatographed on triethylaminoethyl (TEAE) cellulose, and the coagulant fraction rechromatographed on Sephadex G-100. Fractionation o] the Crude Venom on TEAE-Cellulose. The TEAEcellulose (Serva Entwicklungs Labor., Heidelberg, Germany) is suspended in 2 M sodium chloride buffered with 0.1 M Tris adjusted to pH 6.0 with phosphoric acid. The slurry is poured into a glass column 3.6 cm in diameter, until the packed height of the column is 20 cm. The column * G. W. Schwert and Y. Takenaka, Biochlm. Biophys. Acta 16, 570 (1955).
[53]
SNAKE VENOM PROTEASES
717
is then washed with a further 2 liters of the same solvent and then equilibrated with 0.01 M Tris adjusted to pH 8.5 with phosphoric acid. The crude venom (350 mg) is dissolved in the 20 ml of 0.01 M Trisphosphate, pH 8.5, and centrifuged to remove the insoluble material. The clear supernatant is applied to the column. The venom is washed onto the column with a further 50 ml of the same buffer. After the venom has been washed onto the column, the buffer is changed to 0.01 M Tris-phosphate, pH 7.0, and the fractionation is carried out at room temperature at a flow rate of 90-100 ml/hour. After 1200 ml of this solvent, the eluting buffer is changed to 0.02 M Tris-phosphate, pH 6.0, and the fractionation continued until the pH of the effluent has reached pH 6.0, which occurs after about 600 ml of this solvent has been passed through the column. The solvent is now changed to 0.04 M Tris-phosphoric acid buffer at the same pH and the column eluted with about 600 ml of this solvent, when no further protein will be eluted with this solvent. The buffer is then changed to 0.1 M Tris-phosphoric acid buffer, pH 6.0, and it is with this solvent that the coagulant fraction is eluted. The recovery of the thrombinlike activity from the column is about 60% of the applied activity. Fractionation o] the Thrombinlike Activity on Sephadex G-IO0. The Sephadex G-100 after standing in 0.1 M sodium chloride buffer with 0.1 M sodium phosphate, pH 7.0, for 48 hours is packed into a column 5.8 cm in diameter until the Sephadex reaches a packed height of 97 cm. The void volume of the column is about 700 ml. The fraction from the TEAE-cellulose is concentrated about 20-fold and dialyzed against 0.1 M sodium chloride buffered with 0.1 M sodium phosphate and applied to the column, which is eluted with the same buffer at a flow rate of 80 ml/hour. The coagulant activity is eluted after 940 ml of this solvent. The venom fraction is sufficiently stable for it to be concentrated by rotary evaporation. Some slight losses in activity are observed when the fraction is concentrated by ultrafiltration, although this may depend on the nature of the membrane used. Properties
Stability. Dilute solutions (100 ~g/ml) of the venom dissolved in 0.1 M Tris-phosphate, pH 7.0, have remained stable for many months at --25 ° or at -~5 °. Purity and Physical Properties. The coagulant fraction obtained as described above gives only a single precipitin arc on immunoelectrophoresis 4 against the specific horse. A. rhodastoma antivenin; it also gives 4j..j. Scheideg~er, Intern. Arch. Allergy Appl. Immunol. 7, 103 (1955).
718
OTHER PROTEASES
IS3]
a single band after eleetrophoresis in 7% polyacrylamide gel2 The electrophoretic mobility of this protein at pH 7.0 is 3.9 X 10~ V/cm~/second in a moving-boundary electrophoresis apparatus. The partial specific volume V of the protein is 0.69, which suggests that the protein contains carbohydrate; this has been confirmed by chemical analysis. 1 The sedimentation coefficient so20, w of 3.35 S was found for the protein at a concentration of 4.86 mg/ml, and the diffusion coefficient D°0 is 4.81 )< 10-7 cm2/seeond at the same concentration. The approach to equilibrium method for the determination of the molecular weight gives a range of values from 37,000 to 44,000; the curvature of the plots obtained indicates that the material is polydisperse. The same conclusion is reached from low speed equilibrium experiments, in which plots of log c/r ~ (see footnote 6) have a distinct upward curvature. The weight average molecular weight for the total material present in the centrifuge cell is 45,000. The value of the Z average molecular weight Mz calculated from schlieren photographs taken during the same experiment7 is 51,000. High-speed equilibrium experiments for the calculation of the number average molecular weight Mn 8 give a value of 41,500. Assuming that the protein is biologically homogeneous, these results suggest that under the conditions of these experiments in which the protein was dissolved in 0.1 M sodium chloride buffered with 0.04 M sodium phosphate, the protein is a mixture of monomers and dimers, the monomer having a molecular weight of 30,000. Activators and Inactivators Biological. Unlike thrombin, this venom protein is unaffected by inhibitors such as heparin, hirudin, soya bean trypsin inhibitor, or by the various plasma antithrombins. The enzymatic activity is inhibited by the specific antivenin. Chemical. The esterase and coagulant activity of this protein are inhibited by 5 X 10-8 M diisopropylphosphofluoridate and by 5 >( 10-8 M toluenesulfonyl fluoride, the latter causing complete inhibition after 10 minutes of incubation at room temperature, whereas diisopropylphosphofluoridate gave only 68% inhibition after 40 minutes. Specificity. The specificity of this venom protein for macromolecular substrates seems to be confined to fibrinogen, although it does have slight 5B. J. Davis, Ann. N.Y. Acad. $ci. 121, 404 (1964). eW. D. Lansing and E. O. Kraemer, d. Am. Chem. foe. 57, 1369 (1935). O. Lamm, Z. Physik. Chem. 143, 177 (1929). s D. A. Yphantis, Biochemistry 3, 297 (1964).
[53]
SNAKE VENOM PROTEASES
719
factor X converting activity. It appears to be without activity on the other clotting factors, and does not activate plasminogen directly. The enzyme, like trypsin, hydrolyzes the two arginine esters, benzoyl arginine ethyl ester, and p-toluene arginine methyl ester. It will not hydrolyze acetyl tyrosine ethyl ester, the synthetic substrate for chymotrypsin. The venom protein will hydrolyze p-nitrophenyl esters of the carbobenzoxy derivatives of both aromatic and aliphatic amino acids. Russell's Viper ( V i p e r a russellii) V e n o m 1 Assay Method
Principle. The coagulant protein of this snake venom is specific for one coagulation factor, namely factor X, and can be readily assayed using normal citrated plasma as the indicator. The clotting time of the plasma after the addition of the venom is proportional to the rate of factor X activation and hence the amount of venom added to the plasma. Phospholipid is also added to the plasma at the same time as the venom, to ensure that the rate of thrombin formation is not a rate-determining reaction. Reagents Normal citrated plasma. This is obtained by collecting either human or bovine blood into a one-tenth volume of 0.1 M sodium citrate. The blood is then centrifuged to remove the formed elements and the supernatant plasma is stored in plastic tubes in a deep freeze. Phospholipid emulsions. A dry powder of the acetone-insoluble fraction of human brain is extracted with 15 volumes of chloroformmethanol (1:1 v/v). The extract is washed twice with water and the solvent evaporated in a rotary evaporator at 30 °. The residue is redissolved in benzene and stored at --20 °. The emulsion is prepared from the phospholipid solution by first evaporating the benzene, by blowing a stream of nitrogen over the solution, and redissolving the lipid residue in peroxide-free ether. 0.02 M Trishydrochloride acid buffer pH 7.5 is then added and the ether is removed by bubbling nitrogen through the mixture for 30 minutes at room temperature. Calcium chloride solution. This is a 0.05 M solution of calcium chloride buffered with 0.02 M Tris-hydrochloric acid, pH 7.5. Venom. Crude or purified coagulant protein is dissolved in 0.02 M Tris-hydrochloric acid, pH 7.5.
720
OTHER PROTEASES
[S3]
Procedure. It should be stressed that none of the solutions used in this assay should contain phosphate, because factor X or activated factor X is readily adsorbed by very small amounts of precipitated calcium phosphate. The citrated plasma (0.1 ml) is incubated at 37 °, with 0.1 ml of the venom solution and 0.1 ml of the phospholipid emulsion (50 mg phospholipid/ml). To this mixture is added 0.1 ml 0.05 M calcium chloride and the clotting time of the plasma is recorded from the addition of the calcium chloride. In this assay crude venom solutions of 10 ~g/ml give a clotting time of about 11 seconds. The same clotting time is given the purified fraction at a concentration of 0.7 ~g/ml. A plot of the clotting time against decreasing concentrations of the venom solution gives a straight line on double logarithmic paper. Williams and Esnouf 9 found that the coagulant protein of this venom possessed esterase activity, and that synthetic substrates such as ptoluenesulfonyl arginine methyl ester would competitively inhibit the coagulant activity of this. However, recently D. J. Hanahan (private communication) has been able to separate the esterase activity from the coagulant activity of the venom. At present the only explanation for these apparently conflicting results is the possibility that the venom of the same species of snake, living in different regions, may be different. In view of this uncertainty of the esterolytic activity of this, an assay based on the esterase activity of the protein will be omitted.
Purification Procedure In the original method 9 the venom was fractionated on Whatman DEAE-cellulose (DE 50). Similar results have been obtained on the more recent version of this cellulose (DE 52). The description of the method is based on Whatman DE 50. Twenty-five grams of the dry cellulose powder is suspended in 2 M sodium chloride buffered with 0.01 M Tris and the pH adjusted to 6.0 with phosphoric acid. The slurry is poured into a glass column 3.4 cm in diameter and the column is packed under pressure until the height of the packed column reaches about 30 cm. The column is then washed with a further 2 liters of the same solution, and equilibrated with 0.01 M Tris-phosphoric acid buffer, pH 8.5. The crude venom is dissolved in this buffer (10 mg/ml). The insoluble material is removed by centrifugation, and 30 ml of the supernatant is applied to the column. The column is then eluted with 500 ml of the pH 8.5 buffer. The buffer is then changed to 0.01 M Tris-phosphoric acid pH, 7.0, and the column eluted with this buffer until the pH of the effluent has reached oW. J. Williams and M. P. Esnouf, Biochem. J. 84, 52 (1962).
[53]
SNAKE VENOM PROTEASES
721
7.0. The column is then eluted with 0.01 M Tris-phosphoric acid buffer, pH 6.0, until the column is equilibrated at pH 6.0. The column is now eluted with 0.15 M sodium chloride dissolved in the same buffer, and about 90~o of the applied coagulant activity is reoovered from the column with this solvent. The chromatography is carried out at room temperature at a flow rate of 75 ml/hour. The fractions with coagulant activity can be concentrated by ultrafiltration. Examination of the concentrated protein in the ultracentrifuge shows that two components are present. These are readily separated by centrifugation into a linear density gradient of sucrose (5-20% w/v) dissolved in 0.1 M sodium phosphate buffer, pH 7.0. This gradient (4.2 ml) is formed in each of three 2 inch by ½ inch cellulose nitrate centrifuge tubes, and 0.6 ml of a solution containing not more than 30 mg/ml protein in 0.1 M phosphate buffer, pH 7.0, is carefully layered onto the gradient. The centrifuge tubes are then placed into a previously cooled swing-out rotor (Beckman SW 39) and centrifuged for 40 hours at 36,000 rpm at about 3 °. At the end of this period the rotor is allowed to decelerate without the brake, so as not to disturb the gradient. The contents of the tubes are then fractionated by piercing the bottom of the centrifuge tube with a pin and collecting 2 drops of solution into a series of test tubes. The optical density of the samples are measured at 280 nm after adding 2.5 ml of 0.15 M sodium chloride to each tube. The fractions containing the faster component (coagulant protein) are pooled and dialyzed against distilled water and freeze dried. Properties
Stability. The freeze-dried protein kept in the deep freeze remains fully active for many years. Solutions of the protein in buffered saline at concentrations of 100 g/ml, or greater, are stable for many weeks when deep frozen or kept at 1% Dilute solutions of the protein 10 g/ml, or less, remain fully active for only a few hours. Purity and Physical Properties. The coagulant protein obtained by the method described above does not possess any of the several enzymatic activities known to be present in the crude venom. Electrophoresis into 7% acrylamide gel TM gives only one component and similar observations have been made in free boundary electrophoresis between pH 5.5 and pH 7.0. The coagulant protein has an s°2o,wof 5.05 S and a D o20, w of 4.20 X 10.7 cm2/second - The molecular weight, as calculated by the procedure of Archibald, 11 is 105,000. 1°B. d. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964). W. T. Archibald, J. Phys. Chem. 51, 204 (1947).
722
PEPTIDASF_~
[S4]
Activators and Inhibitors
Activators. The rate at which factor X is converted to the activated form by the venom protein is influenced by the concentration of calcium ions. The optimum rate of activation is obtained in the presence of 7 mM calcium chloride, while in the absence of calcium ions there is no reaction. Calcium can 'be replaced by other divalent metal ions, although they do not activate to the same extent as calcium. They can be written in a descending order of activity as Ca > Mn ~ Sr ~ Ba ~ Mg. Inldbitors. Apart from the specific antiserum, and reagents which bind calcium ions, the venom is uninfluenced by inhibitors such as the various trypsin inhibi.tors, although competitive inhibition of the coagulant activity 9 has been obtained in the presence of p-toluenesulfonyl arginine. Specificity. The purified coagulant protein is specific for factor X present in human, bovine, and rabbit plasma, and it appears to be without any activi.ty on the other coagulation factors. The venom fraction has a slight hydrolytic activity on gelatin in the presence of calcium ions. In addition to the esterolytic activity on TAME, for which it has a K , of 5.9 X 10-4 moles, it also slowly hydrolyzes lysine ethyl ester, although, as mentioned earlier, it has to be resolved whether the coagulant protein is always associated with esterolytic activity.
[54] Renal Dipeptidase
By BENEDICT J. CAMPBELL Glycylphenylalanine -b H20 ---*glycine -}-phenylalanine Glycyldehydrophenylalanine -{-H20 ---*glycine + phenylpyruvic acid -{-NI-h
The above equations demonstrate the hydrolysis of a saturated dipeptide and an unsaturated dipeptide, of which glycylphenylalanine and glycyldehydrophenylalanine are representative. Assay Method
Principle. Renal dipeptidase isolated from fresh hog kidney exhibits activity against a variety of dipeptides.I B y means of using the unsaturated dipeptide, glycyldehydrophenylalanine, enzymatic hydrolysis may be conveniently followed by observing the fall in optical density at 275 nm. This assay can be modified for application to particulate enzyme IB. J. Campbell, Y. C. Lin, R. V. Davis, and E. Ballew, Biochim. Biophys. Acta 118, 371 (1966).
722
PEPTIDASF_~
[S4]
Activators and Inhibitors
Activators. The rate at which factor X is converted to the activated form by the venom protein is influenced by the concentration of calcium ions. The optimum rate of activation is obtained in the presence of 7 mM calcium chloride, while in the absence of calcium ions there is no reaction. Calcium can 'be replaced by other divalent metal ions, although they do not activate to the same extent as calcium. They can be written in a descending order of activity as Ca > Mn ~ Sr ~ Ba ~ Mg. Inldbitors. Apart from the specific antiserum, and reagents which bind calcium ions, the venom is uninfluenced by inhibitors such as the various trypsin inhibi.tors, although competitive inhibition of the coagulant activity 9 has been obtained in the presence of p-toluenesulfonyl arginine. Specificity. The purified coagulant protein is specific for factor X present in human, bovine, and rabbit plasma, and it appears to be without any activi.ty on the other coagulation factors. The venom fraction has a slight hydrolytic activity on gelatin in the presence of calcium ions. In addition to the esterolytic activity on TAME, for which it has a K , of 5.9 X 10-4 moles, it also slowly hydrolyzes lysine ethyl ester, although, as mentioned earlier, it has to be resolved whether the coagulant protein is always associated with esterolytic activity.
[54] Renal Dipeptidase
By BENEDICT J. CAMPBELL Glycylphenylalanine -b H20 ---*glycine -}-phenylalanine Glycyldehydrophenylalanine -{-H20 ---*glycine + phenylpyruvic acid -{-NI-h
The above equations demonstrate the hydrolysis of a saturated dipeptide and an unsaturated dipeptide, of which glycylphenylalanine and glycyldehydrophenylalanine are representative. Assay Method
Principle. Renal dipeptidase isolated from fresh hog kidney exhibits activity against a variety of dipeptides.I B y means of using the unsaturated dipeptide, glycyldehydrophenylalanine, enzymatic hydrolysis may be conveniently followed by observing the fall in optical density at 275 nm. This assay can be modified for application to particulate enzyme IB. J. Campbell, Y. C. Lin, R. V. Davis, and E. Ballew, Biochim. Biophys. Acta 118, 371 (1966).
[54]
RENAL DIPEPTIDASE
723
fractions. 2 For saturated dipeptides the colorimetric ninhydrin assay 1,3 and a titrimetric assay 4 have been employed. In the isolation procedure to be described below, the speetrophotometric assay using glycyldehydrophenylalanine as substrate was followed.
Reagents 0.025 M Tris-HC1 buffer, pH 8.0 Glycyldehydrophenylalanine (0.052 raM)
Synthesis o] Substrate. A solution of 20 g of phenylserine is prepared in 80 ml of 1 M NaOH. This solution is treated alternatively with 8 g of chloroacetylchloride and 180 ml of 1 M NaOH. Each is added in 20 equal portions with shaking after each addition. The temperature is maintained at 0 ° in an ice bath during reaction. After I hour the reaction mixture is acidified with 5 N HCI. Crystallization of chloroacetylphenylserine occurs overnight. The yield is 19.8 g (70%), and the melting point of the crystals is 152°-155 °. A reaction mixture of 5 g of ehloroacetylphenylserine in 50 ml of acetic anhydride is heated on the steam bath for1 hour. The solution is then filtered and vacuum distilled to concentrate the reaction product. Crystallization occurs after most of the liquid has been removed. Evaporation is again carried out after the addition of 13 ml of CCI~. The solution is then allowed to stand overnight at --20 °. The azlactone is filtered off the next morning as yellow crystals. The yield is 1.2 g (24~o), and the product melts at 102°-107 °. A mixture of 1.8 g of the azlaetone of chloroacetylphenylserine, 25 ml of acetone, and 13 ml of water is refluxed for 2 hours. After cooling, the mixture is shaken twice with petroleum ether, and crystals of chloroacetyldehydrophenylalanine form in the aqueous portion. The crude product is recrystallized from distilled water. The weight of the purified crystals is 1.1 g (60%), and the crystals melt at 185°-188 °. A saturated aqueous solution of NH3 is prepared by bubbling NH3 through 4 ml of water maintained at 0 ° in an ice and salt bath. To-the NH3 solution is added 80 mg of chloroacetyldehydrophenylalanine, and NH~ is bubbled through the solution at 0 ° for an additional 30 minutes. The mixture is allowed to come to room temperature and shaken at 5minute intervals until all of the chloroacetyldehydrophenylalanine dissolves. The solution is kept at 4 ° for 4 days and then concentrated by directing an air stream over its surface. A crude precipitate is collected 2A. M. Ren~ and B. J. Campbell, J. Biol. Chem. 244, 1445 (1969). s A. T. Matheson and B. L. Tattrie, Can. J. Biochem. Physiol. 42, 95 (1964). G. F. Bryce and R. B. Rabin, Biochem. J. 90, 509 (1964).
724
P~,P'rIDASSS
IS4]
and reerystallized from water. A yield of 48 mg is obtained (52~) and the final product, glycyldehydrophenylalanine, melts at 2500-257 °. The unsaturated peptide exhibits a molecular extinction coefficient of 1.53 X 10~ at 275 nm. Procedure. To a euvette with a 1-cm light path is added a solution of 2.40 ml of 0.052 mM glycyldehydrophenylalanine in 0.025 M Tris-HC1 buffer at pH 8.0. Then 0.10 ml of enzyme solution is added by means of an adder-mixer2 A cuvette employed as a speetrophotometric blank contains 2.40 ml of 0.025 M Tris-HC1 buffer at pH 8.0 and 0.10 ml of enzyme solution. The initial rate is measured as the fall in optical density at 275 nm as the reaction proceeds. The temperature of reaction is controlled, usually at 35 °, by means of a temperature-controlled jacket through which water is circulated from a Brinkmann-Haake ultra thermostat type F. Definition o] Unit and Specific Activity. One unit of renal dipeptidase activity is defined as the amount that catalyzes the hydrolysis of 1 micromole of glycyldehydrophenylalanine per minute when the substrate is at a concentration of 0.050 mM. Specific activity is expressed as units per milligram of protein. The protein concentration is determined by the Folin-Lowry method, e and the protein standard employed is 3 X crystallized bovine serum albumin. Purification Procedure Renal dipeptidase was first partially purified from swine kidney by Robinson, Birnbaum, and GreensteinJ The method described below includes additional steps and modification of earlier techniques. 2 Step 1. Homogenization. Fresh kidney cortex (1.5 kg) are cut into pieces and homogenized in a Waring blendor with 2 volumes of ice water. The homogenate is centrifuged at 1200 g for 20 minutes to remove cellular debris. Step 2. Precipitation at pH 5. The supernatant is chilled to 0 ° in a cold bath and then adjusted to pH 5 by the careful addition of 1 N HC1. The resulting thick suspension is centrifuged immediately at 0 ° and 3000 g for 30 minutes. The supernatant fluid is discarded. Step 3. Washing. The pH 5 precipitate is washed from the tubes with an equal volume of 0.066 M phosphate buffer at pH 7. The last traces of 5p. D. Boyer and H. L. Segal, in "The Mechanism of Enzyme Action" (W. D. MeElroy and B. Glass, eds.), p. 520. Johns Hopkins Press, Baltimore, Maryland, 1954. ' O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). D. S. Robinson, S. M. Birnbaum, and J. P. Greenstein, J. Biol. Chem. 202, 1 (1953).
[54]
RENAL DIPEPTIDASE
725
protein in the centrifuge tubes are removed with the same volume of 0.1 M KC1 solution and added to the first washings. The lumpy suspension is homogenized briefly in the blender and frozen at --10% After thawing, the suspension is again adjusted to pH 5 with 1 M HC1 and centrifuged for 30 minutes at 0 ° and 3000 g. The sediment is taken up with 0.066 M phosphate buffer at pH 7 and briefly homogenized. Cold acetone (0 °) is added to the suspension and stirred to a concentration of 70% (v/v). The reaction mixture is centrifuged at 0 ° and 8000 g for 40 minutes. The supernatant is carefully decanted and discarded. The sediment is washed from the tubes with an equal volume of 0.066 M phosphate buffer a t pH 7. Precipitation at pH 5 and washing with phosphate buffer is repeated until the supernatant fluid is proved to be protein-free by testing it with 10~ trichloroacetic acid solution. Step ~,. Solubilization with n-Butanol. The washed pH 5 precipitate is suspended in 1 liter of distilled, demineralized water, n-Butanol, chilled to 0 °, is added to a concentration of 20% by volume with rapid stirring. The suspension is stirred with a magnetic stirrer for 1 hour at 2 °. The resulting emulsion is then dialyzed for 18 hours against several changes of distilled water, until all butanol is removed. The dialyzate is adjusted to pH 5 by dropwise addition of 1 M HC1 at 0 °, and the resulting mixture centrifuged at 3000 g for 30 minutes at 0 °. The sediment is discarded. A solution of 0.1 M Tris-HC1 buffer at pH 7 is added to the supernatant in the amount of 1% by volume, and the mixture is adjusted to pH 7.- The volume of the preparation at this stage is approximately 2 liters. Lyophilization is employed to reduce this volume to about 400 ml. The insoluble material which appears following lyophilization is centrifuged and discarded. The solution is then dialyzed against 0.(}02 M Tris-HC1 buffer at pH 7. Step 5. Fractionation with Ammonium Sul]ate. To the aqueous solution containing solubilized protein is added solid ammonium sulfate to 50% saturation at 0 °. The precipitate which is formed after standing overnight at 0 ° is collected by centrifugation at 4500 g for 1 hour and disoarded. Solid ammonium sulfate is then added to the supernatant to 75% saturation, and after standing for 48 hours in the cold, the suspension is centrifuged at 10,000 g for 1 hour. The sediment is dissolved in 8 ml of 0.002 M Tris-HC1 buffer at pH 8.0 and dialyzed against the same buffer for 18 hours with four changes of dialyzing buffer. Step 6. Gel-Filtration with Sephadex G-150. Pharmacia Sephadex G-150 (Lot No. 8511, water regain, 15 ± 1.5 g/g, particle size 40-120 ~) is equilibrated in 0.002 M Tris-HC1 containing 0.01 mM ZfiC12 at pH 8.0. The equilibrated gel is poured to form a column of dimensions 2.5 cm X 90 cm. In a typical preparation 10 mg of the dialyzed ammonium sulfate
726
PEPTIDASES
[54]
fraction in 2 ml of 2 mM Tris-HC1 buffer at pH 8.0 are applied to the column. Elution is carried out with the initial buffer at a flow rate of 25 ml/hour, and fractions are collected as 5 ml of effluent per tube. The absorbance of each tube at 280 nm and 260 nm is measured. The enzyme activity is measured by the spectrophotometric assay. The elution pattern from the column exhibits four absorption peaks. The second peak which is eluted before the first peak reaches base line and which also forms a shoulder for the third peak contains the enzyme activity. It is necessary to assay all tubes in this region to obtain a complete enzyme yield from the column. The tubes containing enzyme activity are pooled, and the resulting solution lyophilized to a concentration appropriate for step 7. Step 7. Carboxymethylcellulose Chromatography. CM-Cellulose (Schleieher and Schuell Co., Lot No. 1310, Capacity 0.8 meq/g) is treated as outlined by Peterson and Sober2 The water-washed CM-eellulose is suspended in 5 mM ammonium acetate buffer containing 0.25 mM ZnCl~ at pH 4.6 and poured to form a column of dimensions 3.9 X 9 cm. A sample containing 64 mg of protein in 21.7 ml of Tris-HCl buffer from the Sephadex G-150 column is dialyzed .against distilled, demineralized water for 3 hours with three changes of water. One volume of 0.01 M ammonium acetate at pH 4.6, containing 0.5 mM ZnC12 is added and mixed with the sample immediately before application to the column. Star~ing buffer, 5 mM ammonium acetate at pH 4.6, containing 0.25 mM ZnC12 is allowed to pass through the column at a rate of 4 ml per minute. As soon as the first protein peak is eluted, the second buffer, 0.05 M ammonium acetate at pH 6.0, containing 0.25 nM ZnC12 is applied. As the tubes containing enzyme activity are collected, 0.1 M Tris is added dropwise immediately to bring the pH of the eluted material to 8. The tubes in the active peak are pooled and neutralized to pH 8. All of the peptidase activity is eluted in the second peak. The resulting solution is concentrated by lyophilization prior to the final purification step. Step 8. Gel Filtration with Sephadex G-~A~O. Pharmacia Sephadex G-200 (Lot No. 3177, water regain, 20 ± 2 g/g, particle size 40-20 ~) is equilibrated in 2 mM Tris-HC1 buffer at pH 8.0 containing 0.01 mM ZnC12. The equilibrated gel is poured to form a column of dimensions 2.5 X 92 cm. In a typical experiment 6 mg of CM-cellulose purified enzyme in 4 ml of 2 mM Tris-HC1 buffer at pH 8.0 containing 0.01 mM ZnC12 are applied to the column. Elution is carried out with the same buffer at a flow rate of 8 ml per hour, and 3 ml of effluent are collected per tube. In the first gel filtration purification three absorbance peaks are eluted from the column. The second peak which contains the enzyme BE. A. Peterson and H. A. Sober, see Vol. V [1], p. 3.
[54]
RENAL DIPEPTIDASE
727
activity is collected and concentrated by lyophilization. Refiltration of this material on the same column is carried out using the same conditions after prewashing the column overnight with the same buffer. The absorbance and activity peaks coincide in this final purification step. The purified renal dipeptidase is concentrated to approximately 1 mg/ml and stored frozen in a Tris-HCl buffer at pH 8.0. The overall purification produced an enzyme approximately 500 times more active than source material. From 1.5 kg of kidney cortex, 132 g of protein homogenate were obtained, and the complete purification from this homogenate yielded 5.8 mg of peptidase. A summary of the solubilization and purification of renal dipeptidase is shown in Table I. TABLE I FURIFICATION OF RENAL DIPEPTIDASE
Fraction Kidhey homogenate First pH 5 precipitate Washed precipitate Solubilized enzyme Ammoniumsulfate fraction Sephadex G-150 OarboxymethylceUulose chromatography Sephadex G-200 Sephadex G-200 refiltration
Total Volume activity (ml) (units) 3000 2000 1000 1400 40 40 30 40 40
Total protein (mg)
6 2 , 0 0 0 132,000 100,000 100,000 6 6 , 0 0 0 55,000 12,600 2,100 5,800 200 1,600 38.4 1,360 24.0 1,440 1,410
6.0 5.8
Specific activity X 100
Yield (%)
0.47 1.0 1.2 6.0 29.0 41.7 56.6
100 161 106 20 9 2.6 2.2
240 243
2.3 2.3
Properties
Stability. Renal dipeptidase may be concentrated by lyophilization without loss in activity if the lyophilizing flasks are kept cold in an ice bath and if the material is shell frozen in Tris-HC1 buffer at pH 8. Extended dialysis against nonchelating buffers does not result in loss in activity. The enzyme is rapidly inactivated below pH 5 in ammonium acetate buffer but is stable at pH 8 in Tris or phosphate buffers. If the enzyme is stored frozen in dilute solution (0.13 mg/ml Tris-HC1 buffer, pH 8), it loses approximately two-thirds of its activity over a period of 6 months. Purity and Physical Properties. The purifed enzyme exhibits only one symmetrical peak after ultracentrifugation at 59,780 rpm for 1 hour, and migration of the enzyme by acrylamide-gel electrophoresis at pH 8.0 indicates that the purified peptidase is monophoretic. It has been estimated
728
PEPTIDASES
[54]
from approach to equilibrium measurements that the molecular weight is approximately 47.200. But more recent results obtained by sedimentation equilibrium and by gel filtration indicate that the enzyme exists in a form with molecular weight 87,000-93,000. Activators. An ~ncrease in zinc content is observed with increasing purification of the peptidase until a 1:1 mole ratio of zinc to enzyme is reached if the molecular weight is assumed to be 47,200. Peptidase activity can be reduced by removal of zinc using o-phenanthroline dialysis and can 'be restored by dialysis against zinc-containing buffers. It is suggested that renal dipeptidase is a metalloenzyme with the zinc atom strongly bound to the protein portion of the enzyme. The peptidase does not exhibit absolute specificity for zinc ion activation. Preincubation of the purified native enzyme with Mg 2÷ or Co 2÷produced a 24% activation, whereas the same treatment with Ni ~÷ or Cd 2÷ leads to a 2 9 ~ inhibition in each oase. Inhibitors. A time-dependent inhibition of the enzyme is produced upon incubation with o-phenanthroline. This inhibition can only be reversed by dialysis to remove o-phenanthroline followed by dialysis against zinc-containing buffers. The peptidase is inhibited by inorganic phosphate as well as nucleotides. This inhibition is not time dependent and is completely reversible upon dilution or dialysis. The degree of inhibition for the adenine series is in the order ATP > ADP > AMP > inorganic phosphate. Inhibition is also produced by treatment with monovalent anions. This inhibition is competitive and is in the order GN- > SCN- > N.- > I- > NO.- > H C 0 . - > Br- > 0Ac- > C1- > F-. Specificity. Renal dipep~idase acts upon a variety of dip@tides to produce the constituent amino acid products. Early work using the colorimetric ninhydrin assay suggested that glycylglycine was the most readily hydrolyzed substrate, 1 but recent measurements using the titrimetric assay 4 indicate that L-alanylglycine breaks down most rapidly at a rate of 229 _ 19 micromoles/minute/mg enzyme under conditions of substrate-satura:ted enzyme. The Cerminal amino group of the dipeptide substrate cannot be 'blocked by groups such as the earbobenzoxy group, but methylation of the amino group yields derivatives that are hydrolyzed at reduced rates. The enzyme requires that the N-terminal amino acid of the dipeptide be in the L-configuration. However, activity is exhibited against dipeptides in which the C-terminal amino acid is in the D- as well as the L-configuration. The enzyme has no esterase activity, nor does it act against leucinamide, tripeptides, or proteins {casein, hemoglobin). Kinetic Properties and pH Dependence. The kinetic properties and the effect of pH on enzyme activity were determined by measurements of
[54]
RENAL DIPEPTIDASE
729
peptidase-catalyzed hydrolysis of glycyldehydrophenyalanine. The K,~ for this substrate was 1 m M at the optimum p H of 7.60 and 35 ° Proton dissociations from the enzyme-substrate complex occur during peptidase catalysis at pK's of 6.9 and 8.5. Amino Acid Composition. The amino acid composition of renal dipeptidase is given in Table II. TABLE II AMINO ACID COMPOSITION OF RENAL DIPEPTIDASE
Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ammonia Zinc Total Recovery %
Amino acid residues 7.88 2.91 5.63 9.51 4.72 5.17 11.77 4.32 3.03 4.82 3.72 5.67 1.95 3.60 9.83 4.15 5.62 4.34 0.40 0.14 99.18
Nitrogen (g/100 g protein) 1.70 0.88 2.03 1.15 0.65 0.85 1.27 0.62 0.74 0.95 0.51 0.79 0.22 0.45 1.22 0.35 0.54 0.68 0.33
Assumed R e s i d u e s " residuesper per molecule molecule 28 ..65 9.93 17.08 38.83 21.92 28.41 42.60 20.85 25.04 31.93 17.24 26.52 7.31 15.10 40.96 11.90 17.98 11.22 10.99 1.01
15.93 100.82
29 10 17 39 22 28 43 21 25 32 17 27 7 15 41 12 18 11 11 1 414
Calculated on the basis of a molecular weight of 47,200.
Distribution. The complete purification of renal dipeptidase as reported above has only been applied to hog kidney tissue; however, enzymatic activity against glycyldehydrophenylalanine by crude extracts of other mammalian tissues has been reported2
9j. p. Greenstein, Adv. Enzymol. 8, 117 (1948).
730
PE~rIDASES
[55]
[55] fl-Aspartyl Peptidase 1
By EDWARD E. HALEY Assay Method A Principle. The amino acids produced in tile reaction were estimated by a photometric ninhydrin method. A ninhyJrin-cyanide reagent was used which gives quantitative color yields ~l~h free amino acids, but much lower yields with most di- and tripeptides. ~ This reagent was used in a more sensitive assay for peptidase activity. The assay method was used for all experiments, except those to determine the effect of substrate concentration on hydrolysis. Reagents
Tris-HC1 buffer, 0.05 M, pH 8.1 fl-Aspartylleucine, 0.01 M, in 0.05 M Tris-HC1 buffer, pH 8.1. The peptide was dissolved in water and the solution adjusted to p H 8.1 with NaOH, then to 0.02 M with water. An equal volume of 0.1 M buffer was added to the peptide solution. This solution was used as the substrate. fl-Aspartylleucine, 1.25 mM Aspartic acid, 2.5 mM Leucine, 2.5 mM Trichloroacetic acid, 1.2M Sodium citrate buffer, 0.067 M, pH 5.0 Ninhydrin-potassium cyanide-methyl Cellosolve reagent. The reagent was prepared by mixing 1 volume of ninhydrin dissolved in methyl Cellosolve, 5 g per 100 ml, with 5 volumes of potassium cyanide in methyl Cellosolve. The latter solution was made by dilu:ting 2 ml of 0.01 M aqueous potassium cyanide to 100 ml with methyl Cellosolve. The final reagent should be stored overnight before use to ensure low blank readings2 Ethyl alcohol, 6 0 ~ Procedure. The reaction mixtures were made in 12-ml conical centrifuge tubes and had a total volume of 0.20 ml. The standard substrate,
1E. E. Haley, J. Biol. Chem. 243, 5748 (1968). 2A. T. Matheson and B. L. Tattrie, Can. J. Biochem. 42, 95 (1964); E. W. Yemm and E. C. Cocking, Analyst 80, 209 (1955). s Room temperature stabilities are: ninhydrin-potassium cyanide-methyl Cellosolve, 1 week; ninhydrin-methyl CeUosolve, 1 month; potassium cyanide-methyl Cellosolve, 1 week; and aqueous potassium cyanide, 1 week.
[55]
~-ASPARTYL PEFTIDASE
731
0.05 ml of fl-aspartylleucine solution, was evaporated to dryness under vacuum in the tube to enable the entire 0.20-ml reaction volume to be made up of enzyme and other reagent solutions. The total buffer content was 6.5 micromoles of Tris-HC1, pH 8.1. The reaction was initiated by the addition of the enzyme solution, and incubation was at 37 ° for 1 hour. Controls without substrate and without enzyme were included. The reaction was stopped by the addition of 10 ~l of trichloroacetic acid solution (TCA) and the tubes were centrifuged when required. The extent of hydrolysis was estimated by mixing 0.05-ml aliquots of the reaction mixture with 1.5 ml of citrate buffer and 1.2 ml of ninhydrin-potassium cyanide-methyl Cellosolve reagent, and heating in a boiling water bath for 71/2 minutes. The tubes were immediately chilled in an ice bath for 5 minutes and the contents diluted by the addition of 7.3 ml of 60% ethyl alcohol. Absorbance at 570 nm was measured in the Coleman Junior Spectrophotometer. A standard curve was obtained by preparing mixtures of fl-aspartylleucine, aspartic acid, and leucine solutions to simulate 0, 10, 20, 40, and 80% hydrolysis. Controls containing samples of Tris buffer were included. The response was linear with enzyme concentration and with time for the first 60 minutes, when the extent of hydrolysis did not exceed 50~. Assay Method B
Princ@le. The leucine formed in the enzymatic reaction was estimated by the ninhydrin method after separation of the leucine from the peptide and aspartic acid by ion exchange. This method was used in the tests to determine the effect of substrate concentration on hydrolysis. At high substrate concentrations the micromoles of peptide hydrolyzed could be determined more accurately by this method than by Method A, which determines percent hydrolysis of the peptide. Method B could be used generally, but is more lengthy. Reagents Tris-HC1 buffer, 0.05 M, pH 8.1 fl-Aspartylleucine, 0.01 M, in 0.05 M Tris-HC1 buffer, pH 8.1 Trichloroacetic acid, 1.2 M Dowex 50W-X4, 100-200 mesh, hydrogen form Dowex 2-X8, 100-200 mesh, acetate form Pyridine, 2 M Citrate buffer, 0.067 M, pH 5.0 Ninhydrin-potassium cyanide-methyl Cellosolve reagent Ethyl alcohol, 60%
Procedure. The reaction mixtures were made in conical centrifuge tubes and had a total volume of 1.0 ml. The mixture consisted of the
732
PEPTII)ASES
[5S]
enzyme sample, 20 micromoles of Tris-HC1 buffer and 0.2 to 7.5 micromoles of pep~ide in the tests to determine the effect of substrate concentration, or would be 2.5 micromoles of peptide in other tests. Incubation was at 37 ° for 30 minutes, and the reaction was stopped by the addition of 0.05 ml of TCA. All of the reaction mixture, or an aliquot of the supernatant if centrifugation was required, was added to a 1 X 2.5 era column of Dowex 50W-X4. The column was washed with 8.5 ml of water and eluted with 10 ml of pyridine solution. The pyridine eluate was evaporated to dryness under vacuum and the residue dissolved in 5 ml of water. This solution was added to a 1 X 2.5 cm column of Dowex 2-X8, and the column was washed with 15 ml of water. The effluent was evaporated to dryness, and the residue dissolved in 0.2 ml of water, An aliquot, 0.1 ml, was analyzed by the ninhydrin method described in Method A, except that the experimental samples and samples of leucine for a standard curve were heated at 100 ° for 5 minutes rather than 7½ minutes. Definition o] Unit o] Enzyme Activity. One unit of enzyme activity is defined as that amount required to hydrolyze 1 micromole of fl-aspartylleucine per minute under the conditions of the assay. Purification Procedure
Step 1. Extraction and Ammonium Sul]ate Fractionation. All the operations in this step were carried out in the cold. Escherichia coli strain B, midlogarithmic harvest, was obtained commercially.~ The cells, 67 g of frozen paste, were thawed and homogenized in a Waring blendor with 900 ml of 0.05 M Tris-HC1 buffer, pH 8.1, for 30 seconds. The suspended cells were disrupted in a French pressure cell at 15,000 to 20,000 psi, and the suspension separated in a Spinco model L ultracentrifuge at 23,000 X g for 30 minutes. Manganese chloride, 0.05 volume of 1 M concent)ration, was added to the supernatant with stirring in an ice bath2 The mixture was stirred for 40 minutes, then centrifuged as described above. Solid (NH,)2S0~ was added to the supernatant to 90% saturation at 0 °, and 10 ml of 7.4 M NH40H was added to readjust the pH to 8.1. The mixture was stirred in an ice bath for 3 hours and centrifuged as described above. The precipitate was dissolved in 0.05 M Tris-HCl buffer, pH 8.1, to a volume of 90 ml and dialyzed against the same buffer until free of (NH4)~SO4. The enzyme solution was concentrated to about two-thirds of its volume by dialysis against 500 ml of 15% polyvinylpyrrolidone (PVP) in 0.05 M Tris-HC1 buffer, pH 8.1, with no loss of activity. ~The enzymatic activity in the supematant of disrupted cells harvested in late logarithmic phase was almost identical to that for the midlogaxithmicphase. 5L. T. Mashburn and J. C. Wriston, Jr., Arch. Biochem. Biophys. 105, 450 (1964).
[SS]
~-ASPARTYL PEPTIDASE
733
Step ~. Sephadex G-~O0 Chromatography. Due to the stability of the enzyme and the relatively short time the enzyme was on the column, this step could be carried out at room temperature. The (NH4)2SO, precipitate concentrate above, 59 ml, was passed through a 4.7 X 29 am column of Sephadex G-200, 40 to 120 ~ particle size, prepared from gel swelled in 0.05M Tris buffer, pH 8.1, at 100 ° for 5 hours. The column was equilibrated and eluted with the same buffer at a flow rate of 30 ml per hour. The effluent passed through the ultraviolet absorption meter, and 3-ml fractions were collected and refrigerated. Two peaks of enzymatic activity were detected. Fractions representing the center of the principal, earlier p e a k were combined and dialyzed against 15% PVP in 0.02M Tris buffer, pH 8.1. The final volume was 20 ml. Step 3. DEAE-Cellulose Chromatography. The most effective purification step was chromatography on DEAE-cellulose, which served also to separate asparaginase and other peptidases. The Sephadex enzyme described above, 19 ml, was applied to a 0.6 X 137 cm column of Whatman DE-32 equilibrated with 0.02M Tris-HC1 buffer, pH 8.1, at 4 °. The column was eluted at approximately 13 ml per hour, first with 40 ml of ,the above buffer, then with a 300-ml linear gradient of NaC1 in the buffer to 0.4 M. Four milliliter fractions were collected. Elution was continued with 190 ml of 0.02 M buffer and 0.4 M NaC1, then with 80 ml of 0.02 M buffer and 0.8 M NaC1. Protein concentration was measured by absorbance at 280 nm. Two peaks of enzymatic activity were detected in this fractionation step also. The principal one began from the point at which the eluting buffer reached 0.3 M in NaC1. The activity reached its peak just as the gradient was completed. The active fractions were combined and concentrated to 10 ml with 15% PVP in 0.02 M buffer. The other enzyme activity was eluted with 0.02 M buffer and 0.8 M NaC1. Further purification and testing was with the principal enzyme, fractions 80 through 96. Step 4. Polyacrylamide Gel Electrophoresis. Enzymatic activity for a-aspartyl dipeptides was removed from the fl-aspartyl peptidase by electrophoresis in polyacrylamide gels. Additional purific'ation by this method was only about 2-fold. Separation of the proteins in the DEAEcellulose enzyme in limited quantity was accomplished in 5% polyacrylamide gel in a pH 8.9 to 9.1 buffer composed of 10.3 g of Tris, 1.30 g of disodium EDTA, and 0.76 g of boric acid per liter in a vertical gel apparatus2 Solid sucrose was added to the 0.3-ml sample of DEAE enzyme to give a 10% solution, and bromophenol blue was added as a marker. The sample was introduced beneath the buffer into a 3 X 78 mm gel slot. a Apparatus for vertical gel and horizontal electrophoresis was obtained from E-C Apparatus Corp., Philadelphia, Pa.
734
PErrmASES
[SS]
TABLE I PURIFICATION OF PEPTIDASE
Fraction Disruption supernatant (NH~)~SO~ ppt. soln. Sephade~ G-200 DEAE-cellulose
Volume (ml)
Protein (mg)
Total enzyme (units)
Specific activity (units/rag)
Yield~ (%)
Purification (-fold)
910
6000
42.0
0.007
100
1.0
121
1920
44.2
0.023
105
3.1
129 63
1070 28.9
38.5 21.7
0.036 0.75
95 57
5.1 107
Corrected for sampling.
Electrophoresis was carried out at 300 V (20 V/cm) and 140 to 160 mA for 2-3 hours. A sample of the eleetrophoretogram was stained with amido black, and the protein pattern was used as a guide for the extraction of sections of the untreated portion of the gel. This portion had been kept frozen during the staining and destaining procedure. The enzyme was removed from the gel by one of two methods: (1) homogenization of sections in 0.05M Tris buffer, pH 8.1, followed by dialysis of the homogenate at room temperature for 2 hours and then overnight in the cold against the same buffer, then separation by centrifugation; or (2) electrophoretic migration out of the gel seotions, which had been put into dialysis sacs with the Tris-EDTA-borate buffer. The sacs were placed side by side in a trough in the horizontal apparatus, and each sac was separated by sections of polyurethane foam saturated with the buffer. Migration into the buffer required 3 hours at 400 V (13 V/cm) TABLE II ELECTROPHORETIC PURIFICATION OF DE/~E-CELLULOSE ENZYME
Fraction DEAE-cellulose PVP conc. DEAE-cellulose PVP conc. ~ Polyacrylamide gel conc. ~
Volume (ml) 10
Protein (mg)
Total enzyme (units)
28.4
21.3
Specific activity Yield~ (units/rag) (%)
Purification (-fold)
0.45
34
64
0.6
1.70
0.78
0.45
34
64
4.5
1.03
0.79
O. 77
34
110
Corrected for sampling. Results from purification of two 0.3-ml portions of DEAE-cellulose enzyme whose active gel fractions were combined.
[55]
~-ASPARTYL P]~PTIDASE
73~
and 8 mA. The sacs were dialyzed as above, and active fractions were concentrated by dialysis against PVP solution. The quantities of protein were so low in the gel fractions that protein estimation was made by ~mino acid analysis of an acid hydrolyzate of a sample. The total amino acid residue value was related to that of the DEAE-cellulose enzyme hydrolyzate, which had been determined also by the method of Lowry et al. 7 A summary of the purification data is given in Tables I and II. Properties
Stability. The enzyme lost no detectable activity in Tris-HC1 buffer, pH 8.1, stored in the freezer for several months, nor at pH 8.1 for 3 days at room temperature. Some instability was observed closer to the pH optimum, at pH 7.5, where 17% of the activity was lost during 24 hours at room temperature. Recovery of activity was good throughout the purification procedure. Purity and Physical Properties. The preparation represented a ll0fold purification over the disrupted cell supernatant, and was, of course, not pure. The enzyme was extracted with several protein bands from the polyacrylamide gel electrophoretogram. Insufficient amounts of this material were available for analytical studies, but the material was free of asparaginase activity and peptidase activity for substrates other than fl-aspartyl dipeptides. Molecular weight estimation on the cruder DEAEcellulose enzyme on a calibrated Sephadex G-200 column was 120,000. Specificity. The results of tests on the limited types of compounds tested as substrates for the enzyme showed that the activity was restricted to fl-aspartyl dipeptides. The action of the enzyme on various types of substrates is shown in Table III. Not all fl-aspartyl dipeptides are substrates, and the tripeptides fl-aspartylleucylglycine, fl-aspartylglycylalanine, and fl-aspartylglycylglycine were not hydrolyzed, v-Glutamylleucine, a homolog of fl-aspartylleucine, was unaffected as were other leucine dipeptides, a-aspartylleucine, asparaginylleucine, glycylleucine, and leucylglycine. Activators and Inhibitors. The peptidase does not require metal ions for activation. Of the metals tested Mn 2÷, Co 2÷, and Zn 2÷ were inhibitory, and Ca 2+ and M g 2+ seemed slightly stimulatory. Sulfhydryl reagents p-hydroxymercuribenzoate, iodoacetamide, and o-iodosobenzoate were not inhibitory. pH Optimum and Bu]]er Ef]ects. The pH optimum is 7.8 to 8.0 in ' O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
736
PEPTIDASES TABLE
[55]
III
ACTION OF PEPTIDASE ON VARIOUS TYPES OF SUESTRATESa
Substrate
Relative hydrolysis
B-Asp-Leu B-Asp-Ser B-Asp-Met B-Asp-Val B-Asp-GIn B-Asp-Phe B-Asp-Ala B-AsP-IIe
100 82 68 56 48 38 33 19
B-Asp-Thr B-Asp-Asn
18 10
B-Asp-Gly B-Asp-His B-Asp-Gly-Ala B-Asp-Gly-Gly B-ASp-Leu-Gly
0 0 0b 0 0
Substrate
Relative hydrolysis
a-Asp-Leu a-Asp-Ser a-Asp-Phe a-Asp-Ala a~Asp-Ile w-AsP-Asn a-Asp-Gly-Ala a-Asp-Leu-Gly
0 0 0 0 0~ 0 0~ 0
N-Acetyl Met Asn
0" 0d
B-Asp-glucosylamine ~-Glu-Leu Gly-Leu Leu-Gly Carnosine Asn-Leu
0, 0 0 0/ 0 0
a Extent of hydrolysis of the substrate with reference to B-aspartylleucine as 100. Assay Method A unless otherwise indicated. The enzymes were the PVP concentrates of the gel electrophoresis fractions unless otherwise indicated. b With DEAE-celltdose enzyme, PVP concentrate. c With DEAE and gel enzymes at pH 8.1 and pH 7.2. d Asparaginase assay method of L. T. Mashburn and J. O. Wriston, Jr., Biochvm. Biophys. Res. Commun. 12, 50 (1963). ° Two enzyme levels tested, one which hYdrolyzed 0.11 micromole of B-Asp-Leu and 5 times that level which hydrolyzed 0.40 micromole. Assay Method A. / Tested in buffer with and without magnesium acetate at 0.01 M. See S. Simmonds, J. Biol. Chem. 241, 2502 (1966). three 0 . 0 5 M buffers: Tris-HC1, Tris-maleate, and sodium phosphate. The enzyme activity at the p H optimum was greatest in T r i s - m a l e a t e and least in phosphate, the ratio between t h e m being about 2 to 1. Substrate A]finity. T h e Michaelis constant for fl-aspartylleucine is 8.1 X 1 0 -4 M . Distribution. Other microorganisms have not been tested for fl-aspartyl peptidase activity. This activity in low level has been reported also in r a t tissues, s but its specificity is m a r k e d l y different t h a n t h a t of Escherichia coli B. For example, fl-aspartylglycine is the substrate most readily hydrolyzed by the rat enzyme, and the tripeptides fl-aspartylglycylglycine and fl-aspartylglyeylalanine are hydrolyzed at the aspartyl-glycyl bond. Both enzymes lack metal ion requirements and have the same p H optis F. E. Dorer, E. E. Haley, and D. L. Buchanan, Arch. Biochem. Biophys. 127, 490 (1968).
[56]
~-ASPARTYL PEPTIDASE FROM RAT LIVER
737
mum, but the rat enzyme is more active in phosphate than in Tris buffers. Enzyme activity was not detected in human serum.
[56] ]~-Aspartyl Peptidase from Rat L i v e r 1 B y EDWARD E. HJa~EY
Assay Method Principle. The glyeine formed in the enzymatic reaction was estimated by a photometric ninhydrin methodY Assay Method B for fl-aspartyl peptidase from Escherichia coli was adapted from this assay method with slight modifications. Reagents
Sodium phosphate buffer, a molar mixture of 3 parts Na2HP04 and 1 part NaH2P0~. The pH depends on the dilution. fl-Aspartylglycine, 0.02 M Dowex 50W-X4, 100-200 mesh, hydrogen form Dowex 2-X8, 100-200 mesh, acetate form Pyridine, 2 M Citrate buffer, 0.067 M, pH 5.0 Ninhydrin-potassium cyanide--methyl Cellosolve reagent 3 Ethyl alcohol, 60% Procedure. The reaction mixtures had a total volume of 2.0 ml and consisted of the enzyme sample, 100 micromoles of sodium phosphate buffer, and 5 micromoles of fl-aspartylglycine. The peptide was omitted from the blank. The final pH was 7.2. Incubation was at 37 ° for 2 hours and the reaction was stopped by heating in a boiling water bath for 5 minutes. After centrifugation 1.5 ml of the supernatant was added to a 1 X 2.5 cm column of Dowex 50W-X4. The column was washed with 8.5 ml of water and eluted with 10 ml of pyridine solution. The pyridine eluate was evaporated to dryness under vacuum and the residue dissolved in 5 ml of water. This solution was added to a 1 X 2.5 cm column of Dowex 2-X8, and the column was washed with 15 ml of water. The effluent was evaporated to dryness, and the residue analyzed for glycine by the ninhydrin method described in Method A for the E. coli enzyme.
F. E. Dorer, E. E. Haley, and D. L. Buchanan, Arch. Biochem. Biophys. 127, 490 (1968). *A. T. Matheson and B. L. Tattrie, Can. J. Biochem. 42, 95 (1964); E. W. Yemm and E. C. Cocking,Analyst 80, 209 (1955). s Prepared as described in the chapter on fl-aspartyl peptidase from E. coli.
[56]
~-ASPARTYL PEPTIDASE FROM RAT LIVER
737
mum, but the rat enzyme is more active in phosphate than in Tris buffers. Enzyme activity was not detected in human serum.
[56] ]~-Aspartyl Peptidase from Rat L i v e r 1 B y EDWARD E. HJa~EY
Assay Method Principle. The glyeine formed in the enzymatic reaction was estimated by a photometric ninhydrin methodY Assay Method B for fl-aspartyl peptidase from Escherichia coli was adapted from this assay method with slight modifications. Reagents
Sodium phosphate buffer, a molar mixture of 3 parts Na2HP04 and 1 part NaH2P0~. The pH depends on the dilution. fl-Aspartylglycine, 0.02 M Dowex 50W-X4, 100-200 mesh, hydrogen form Dowex 2-X8, 100-200 mesh, acetate form Pyridine, 2 M Citrate buffer, 0.067 M, pH 5.0 Ninhydrin-potassium cyanide--methyl Cellosolve reagent 3 Ethyl alcohol, 60% Procedure. The reaction mixtures had a total volume of 2.0 ml and consisted of the enzyme sample, 100 micromoles of sodium phosphate buffer, and 5 micromoles of fl-aspartylglycine. The peptide was omitted from the blank. The final pH was 7.2. Incubation was at 37 ° for 2 hours and the reaction was stopped by heating in a boiling water bath for 5 minutes. After centrifugation 1.5 ml of the supernatant was added to a 1 X 2.5 cm column of Dowex 50W-X4. The column was washed with 8.5 ml of water and eluted with 10 ml of pyridine solution. The pyridine eluate was evaporated to dryness under vacuum and the residue dissolved in 5 ml of water. This solution was added to a 1 X 2.5 cm column of Dowex 2-X8, and the column was washed with 15 ml of water. The effluent was evaporated to dryness, and the residue analyzed for glycine by the ninhydrin method described in Method A for the E. coli enzyme.
F. E. Dorer, E. E. Haley, and D. L. Buchanan, Arch. Biochem. Biophys. 127, 490 (1968). *A. T. Matheson and B. L. Tattrie, Can. J. Biochem. 42, 95 (1964); E. W. Yemm and E. C. Cocking,Analyst 80, 209 (1955). s Prepared as described in the chapter on fl-aspartyl peptidase from E. coli.
738
PEFrIDASES
[56]
Definition o] Unit of Enzyme Activity. One unit of enzyme activity is defined as that amount required to hydrolyze 1 micromole of fl-aspartylglycine in 1 minute at 37 ° under the conditions of the assay. Purification Procedure All operations except CM-cellulose and hydroxylapatite chromatography and the heat treatment were carried out in the cold. Step 1. Extraction. Albino male rats, 300-400 g, which h'ad been maintained on Purina chow pellets, were killed by decapitation, and the livers quickly removed. About 50 g of liver was blended with 3 volumes of cold 0.1 M sodium phosphate buffer (a molar mixture of 3 parts Na2HP04 and 1 part NaH2P0~) for 30 seconds in a prechilled stainless steel Waring blendor. The homogenate was centrifuged to remove cell debris? The supernatant (fraction A, Table I) was centrifuged for 1 hour at 105,000 g in a Spinco Model L ultracentrifuge. Step 2. Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to the supernatant (fraction B, 18 mg of protein/ml) to 40% saturation (0.243 g/ml). The mixture was stirred for another 30 minutes and the suspension was centrifuged. More ammonium sulfate was added to the supernatant to 55% saturation (0.097 K/ml). The mixture was stirred an additional 30 minutes and the suspension was centrifuged. The precipitate was dissolved in 20 ml of 0.1 M sodium phosphate buffer and dialyzed against 5 liters of this buffer for 16 hours. Step 3. Heat Treatment. The dialyzed solution (fraction C) was immersed in a water bath at 60 ° for 5 minutes, cooled, centrifuged, and the supernatant dialyzed against 5 liters of 0.005 M sodium phosphate buffer for 16 hours. The turbid solution was centrifuged. Step 4. CM-Cellulose Chromatography. The supernatant from above (fraction D) was applied to a 2.3 X 23 cm column of Whatman CM 32 equilibrated with 0.005 M sodium phosphate buffer. The column was eluted with the same buffer. The fractions from a wide yellow band were combined and adjusted to 0.01 M sodium phosphate buffer concentration. Step 5. Hydroxylapatite Chromatography. The above eluate solution (fraction E) was applied to a 2.3 X 11 cm column of Bio-Gel HTP 5 equilibrated with 0.01 M sodium phosphate buffer. The column was eluted with 0.05 M sodium phosphate buffer and the first 120 ml of efltuent discarded. The fractions from a narrow yellow band, which was eluted with 1 M potassium phosphate buffer, pH 7.5, were combined and dialyzed against 5 li.ters of 0.01 M sodium phosphate buffer for 16 hours (fraction F). ' Centrifugation was at 600 g for 1 hour in the cold unless otherwise stated. Purchased from Bio-Rad Laboratories, New York, New York.
[$5]
~-ASPARTYL PEPTIDAS~. FROM RAT LIVER
789
TABLE I PARTIAL PURII~ICATION OF ~-AsP.CRTYL PEFrIDASE FROM RAT LIVER"
Specific Fraction A. "Low-speed" supernatant B. 105,000 g Supernatant C. Ammonium sulfate (40-55% satn) D. Heat at 60° E. CM-cellulose F. Hydroxylapatite
Protein ~ (rag)
Activity (units)
activity (units/mg)
Yield (%)
Purification (-fold)
3300
1.55
0.00047
(100)
1.0
2370 570
0.55 0.70
0.00065 0. 0012
100 45
1.4 2.6
216 147 72
0.60 0.55 0.50
0.0028 0. 0037 0. 0070
39 35 32
6.0 8.0 14.9
, Starting with 50 g (wet weight) of liver. Protein was determined by the method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). A summary of the purification data is given in Table I. Properties
Stability. The enzyme activity is stable for as long as 5 days at room temperature, and is stable to freezing and thawing in 0.01 M sodium phosphate buffer. Stability is dependent upon the presence of sodium ions. The activity is unstable to dialysis against water or 0.01 M Tris-HC1 buffer, pH 8.0. Activity cannot be restored by the addition of NaC1, but if NaC1, 0.01 M, is included in the dialysis solution, 807~ of the activity is recovered. Purity. The enzyme preparation, fraction F, represents a 15-fold purification over the liver homogenate. The extent of purification with regard to separation from a-aspartylglyeine and leucylglycine hydrolyzing activities, however, was 500- and 40-fold, respectively. Attempts to purify the fl-aspartyl peptidase activity further by DEAE-cellulose chromatography were unsuccessful due to loss of activity on the column. Specificity. The hydrolysis of peptides other than fl-aspartyl peptides appears to be due to contaminating enzymes present in the preparation, because of the relative enrichment toward fl-aspartylglycine activity in the course of the purification. In Table II is shown the action of the enzyme on various types of substrates, and the relative difference in activities between fractions B and F toward fl-aspartyl peptides and other peptides. The fl-aspartyl tripeptides, fl-aspartylglycylglycine, -glycylalanine, and -glycylvaline, were cleaved at the aspartyl bond. Activators and Inhibitors. The peptidase appears to require sodium or potassium for maximal activity, but not alkaline earth or transition
740
PEPTIDASES
[55]
TABLE II HYDROLYSIS OF PEPTIDES BY RAT LIVER PREPARATIONS Relative hydrolysis~ (% of ~-aspartylglycine)
Peptide ~-Aspartylglycine ~-Aspartyla]anine ~-Aspartylvaline ~-Aspartylleucine ~-Aspartylisoleucine ~-Aspar tylserine ~-Aspartylthreonine ~-Aspartylmethionine 8-Aspartylglycylglycine~ fl-Aspartylglyeylalanine ~ fl-Aspar tylglycylvaline a-Aspartylglycine a-Aspar tylalanine a-Aspar tylleucine a-Aspartylserine a-Aspartylglycylglycine a-Aspar tylglycylalanine v-Glutamylleucine a-Glutamylleuc~ne Asparagine Glycylglycine Glycylalanine Glycylvaline Glycylleucine Leucylglycine Glycylglycylglycine ~-Alanylglycine N-Acetylglycine Carnosine
Fraction Bb
Fraction F~
Method of analysis
(100)
(100) 51 2865 37 56 29 82 95 55 13 3 10 9 10 3 3 0 5
• e e e e e e e e e e e e e e • e e e
0
f
59 47
1500
2000 1000
40 4O Trace Trace 50 30 0 0 0
g g g g g g g g g
a All incubations were carried out as described for ~-aspartylglycine under assay method and included a substrate blank for each peptide to correct for any free amino acid present in or formed from substrate in the absence of enzyme. b Enzyme fractions are described in Table I. c At the end of the incubation of ~-aspartylglycylglycine, glycylglycine was identified by paper chromatography IF. E. Doter, E. E. Haley, and D. L. Buchanan, A n a l Biochem. 19, 366 (1967)] in a desalted aliquot of the incubation mixture. Glycylalanine from ~oaspartylglycylalanine was identified as above. • The extent of hydrolysis was determined by ninhydrin reaction of the Dowex 2 effluent as described for ~-aspartylglycine under assay method. The results were corrected for differences in the ninhydrin color values of the various substrate C-terminal amino acids or dipeptides.
[57]
PEPTIDE HYDROLASES
741
metal ions. There was a slight activation by mercaptoethanol, while inhibition was caused by p-hydroxymercuribenzoate but not by iodoacetamide. pH Optimum and Buf]er Ef]ects. The pH optimum is 7.5-8.0 in sodium phosphate buffer. There is a broad pH optimum between 7 and 9 in Tris-HC1 buffer, and the activity is about half of that in the sodium phosphate buffer. Distribution. Homogenates of rat kidney, brain, lung, skeletal muscle, and heart muscle, in addition to liver, all catalyze a slow but significant hydrolysis of fl-aspartylglycine. Rat kidney enzyme, prepared under the same conditions as for fraction B of liver, showed a specificity similar to that of the liver enzyme. The specific activity of the kidney enzyme is about twice that of the liver enzyme at the same stage of purification? * See also the discussion of distribution of fl-aspartyl peptidase in Chapter [55] on the E. coli enzyme.
1 Ammoniawas determined by Nesslerization of an aliquot of the incubation mixture. The extent of hydrolysis was estimated by semiquantitative determination of hydrolysis products by paper chromatography in a desalted aliquot of the incubation mixture.
[57] Peptide Hydrolases in Mammalian Connective Tissue By CHRISTIAN SCHWABE Connective tissue constitutes about 20% of the body pro~ins of a macroorganism. Its function is as varied at its appearance in topographically different areas such as skin, bone, or as the supporting tissue of various organs. The cellular element of the connective tissue, the fibroblasts, are responsible for the production and maintenance of the intercellular components, collagen, and acid mucopolysaccharides. Peculiarly, in spite of its substantial contribution to the total body weight, large portions of homogeneous cellular connective tissue are rare. A notable exception is the pulp of teeth which consists of fibroblasts, collagen, and mucopolysaccharides with a negligible amount of endothelium (capillaries) and nerve fibers interwoven. This tissue provides an unambiguous source of fibroblasts needed for the study of connective tissue peptide hydrolases. The peptide bond-hydrolyzing enzymes found in connective tissue are divided into two groups, the neutral peptide hydrolases and the acid proteases, according to the hydrogen ion concentration at which they are
[57]
PEPTIDE HYDROLASES
741
metal ions. There was a slight activation by mercaptoethanol, while inhibition was caused by p-hydroxymercuribenzoate but not by iodoacetamide. pH Optimum and Buf]er Ef]ects. The pH optimum is 7.5-8.0 in sodium phosphate buffer. There is a broad pH optimum between 7 and 9 in Tris-HC1 buffer, and the activity is about half of that in the sodium phosphate buffer. Distribution. Homogenates of rat kidney, brain, lung, skeletal muscle, and heart muscle, in addition to liver, all catalyze a slow but significant hydrolysis of fl-aspartylglycine. Rat kidney enzyme, prepared under the same conditions as for fraction B of liver, showed a specificity similar to that of the liver enzyme. The specific activity of the kidney enzyme is about twice that of the liver enzyme at the same stage of purification? * See also the discussion of distribution of fl-aspartyl peptidase in Chapter [55] on the E. coli enzyme.
1 Ammoniawas determined by Nesslerization of an aliquot of the incubation mixture. The extent of hydrolysis was estimated by semiquantitative determination of hydrolysis products by paper chromatography in a desalted aliquot of the incubation mixture.
[57] Peptide Hydrolases in Mammalian Connective Tissue By CHRISTIAN SCHWABE Connective tissue constitutes about 20% of the body pro~ins of a macroorganism. Its function is as varied at its appearance in topographically different areas such as skin, bone, or as the supporting tissue of various organs. The cellular element of the connective tissue, the fibroblasts, are responsible for the production and maintenance of the intercellular components, collagen, and acid mucopolysaccharides. Peculiarly, in spite of its substantial contribution to the total body weight, large portions of homogeneous cellular connective tissue are rare. A notable exception is the pulp of teeth which consists of fibroblasts, collagen, and mucopolysaccharides with a negligible amount of endothelium (capillaries) and nerve fibers interwoven. This tissue provides an unambiguous source of fibroblasts needed for the study of connective tissue peptide hydrolases. The peptide bond-hydrolyzing enzymes found in connective tissue are divided into two groups, the neutral peptide hydrolases and the acid proteases, according to the hydrogen ion concentration at which they are
742
P~rIDASES
[57]
effective. With the exception of a leucine aminopeptidase, the neutral peptide hydrolases do not attack proteins but hydrolyze amino acid amides, dipeptides, and a variety of oligopeptides. The leucine aminopeptidases hydrolyze protein substrates, although very slowly. The enzymes which hydrolyze peptide bonds at low pH values all appear to be typical proteases, commonly referred to as "cathepsins." It should be emphasized that the name cathepsin is a collective term given to intracellular proteolytic enzymes and does not necessarily imply similarity within this group. Some eathepsins have been characterized, such as beef spleen, cathepsin A, B, C, 1 and D. 2 Hydrogen-Ion Activated Peptide Peptidohydrolases
Assay Method Principle. The main enzyme of this group in mammalian fibrobla.sts catalyzes the hydrolysis of internal peptide bonds of denatured proteins and some peptides (peptide peptidohydrolase 3.4.4). The activity is most conveniently assayed with hemoglobin as substrate but other proteins, such as oxidized ribonuelease, or peptides like the insulin A or B chain, are also hydrolyzed.
Reagents 4% Hemoglobin solution in pH 4.0 acetate buffer (0.1 M). [The hemoglobin substrate powder (Worthington) is stirred at pH 2.0 for ½ hour at room temperature, then readjusted to pH 4.0 and made up to volume.] 10% Triehloroacetic acid (TCA). This solution should be stored at 4 ° and prepared new every 2 weeks. 2.7% Solution of sodium potassium tartrate 1% Solution of copper sulfate Sodium carbonate-sodium bicarbonate buffer, 0.5M, pH 10.5, made up in 0.05 M in sodium hydroxide to provide base reserve. Phenol reagent (Folin-Ciocalteau), 8 1 N diluted from the commercially available 2 N reagent with water just prior to use.
Procedure. The total volume of the assay system should be adjusted according to the number of points desired to plot nOD vs. time. Usually 2 ml of 4% hemoglobin is placed into a test tube and incubated at 40 ° 1j. S. Fruton, in "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrb~ck, eds.), Yol. 4, p. 233. Academic Press, New York, 1960. 2E. M. Press, R. Porter, and J. Cebra, Biochem. J. 74, 501 (1960). a O. Folin and V. Ciocalteau, J. Biol. Chem. 73, 627 (1927).
[57]
PEPTIDE HYDROLASES
743
with 400--800 ~l of crude tissue extract and sufficient pH 4.0 acetate buffer (0.1 N) to bring the volume up to 4 ml. After 5 minutes, 500 ~l are pipetted into a tube containing 1.5 ml of 10% TCA. The TCA tube is vigorously shaken for a few seconds (test tube shaker) and after 15 minutes the precipitate is filtered through Whatman No. 4 paper. Plastic funnels, 35 mm in diameter, and 5-cm filter circles (Whatman No. 4) are most convenient, particularly when many assays are performed at the same time. The withdrawal of 500 ~l aliquots from the digestion mixture is repeated at any desired time interval. The color is developed using an adaptation of the Lowry method for proteins. 4 From every tube of filtrate 2 X 500 ~l are placed into a duplicate set of clean test tubes. Using a syringe pipette, 2 ml of a freshly prepared solution (1 ml of 2.7% sodium potassium tartrate and 1 ml of 1% copper sulfate made to 100 ml with the carbonate--bicarbonate buffer) are added to the 500 ~l of filtrate and the contents mixed. After 10 minutes at room temperature 0.2 ml of 1 N phenol reagent is added from a graduated pipette and every tube immediately and vigorously agitated on a test tube shaker. The test tubes may be left at room temperature for 1 hour, or at 40 ° for 10 minutes to fully develop the color. The readings are made on any suitable colorimeter or spectrophotometer at 700 nm. The units given in this description are defined for a 1-cm light path. The results are plotted as OD (700 nm) vs. time and the zero time is obtained by extrapolation from the linear portion of the plot. Since hemoglobin and extract do not precipitate completely immediately upon mixing, the direct zero time reading is not reliable when crude extracts are assayed. With purified samples this precaution is not necessary. Definition o] Activity Unit and Specific Activity. The enzyme unit is defined as the amount of enzyme giving rise to a change in optical density (700 nm) of 0.1 per minute. The reaction is essentially linear if the enzyme concentration is chosen to produce a AOD of 0.1 to 0.15 in 30 minutes. Slopes obtained during this period are used for computation. The specific activity is defined as units per milligram of protein. For protein determination it has been assumed that a concentration of 1 mg/ ml gives rise to an optical density of 1.0 at 280 nm in a 1-cm cell. Source and Sample Collection. Only the lower two molar teeth of 1 to 1½-year-old commercially slaughtered cattle (the usual age of animals used for meat production) are used since they are easy to recover and contain most of the desired tissue. The last tooth is usually buried under a thin plate of bone and the preceding tooth is about one-half to threefourths erupted. An unskilled laborer in a meat-packing plant can be O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
744
PEPTIDASES
[ST]
trained to chisel these teeth out of the demeated mandible and to crack the teeth and extract the pulp. The tissue (about 1 g per tooth) is washed briefly in running cold water to remove gross contamination and then immediately placed on dry ice for shipment. In about 2 months 100 kg of tissue can be obtained. In order to retain the protease activity indefinitely, the whole sample is lyophilized and stored in the dry state at --20 °. Lyophilization facilitates mechanical disruption of the tissue required for the extraction of enzymes. Collagenous tissue resists usual degradation methods in the hydrated state. While it is as yet unknown whether the enzymes described here occur in all fibroblasts, it is very likely that they do not occur in ectodermally derived tissues. 0nly cathepsin D is similar in molecular size but is less restricted in its activity. 2 (See [19], this volume.) Purification o] the Acid Protease. The dry lyophilized tissue is broken up in a Waring blendor and subsequently ground to a 60-mesh powder in a bench type Wiley mill. An extract is prepared by stirring 200 g of pulp powder in 3 liters of distilled water at room temperature. The suspension is centrifuged at 5000 rpm for 10 minutes and the supernatant collected. The pellet is resuspended in 1 liter of distilled water and centrifuged as 'before. The combined supernatants constitute about 4000 ml of original extract. Purification on DEAE-Cellulose. In order to effect a group separation of the acid and neutral peptide hydrolases, earlier purification methods were replaced by an initial chromatographic step on a DEAE-cellulose column2 A 9 X 100 cm jacketed column (made of acrylic resin) is packed with 700 g of DEAE-cellulose (a 1:1 mixture of Schleicher and Schuell type 40 and 70) suspended in 0.1 M N a 0 H , and subsequently washed with 16 liters of 5 X 10-2 M HC1 and 16 liters of pH 8.0 Tris buffer (5 X 10-2 M), containing 10-3 M calcium chloride, 10-2 M sucrose, and 10-8 M magnesium chloride. With an average liquid head of 120 cm this column should flow at 250 ml per hour, if maintained at 4 °. The extract (4000 ml) is permitted to run onto the column. The vessel containing the extract should be cooled in an ice bath since the application to a column takes approximately'16 hours. The first 2 liters of column effluent are discarded and subsequent effluent is collected in 25-ml fractions in a refrigerated fraction collector while the protein sample is still being applied to the top of the column. Following the application of the 4 liters of extract, elution is begun with 4 liters of the same buffer used to equilibrate the column initially, followed by the additional 4-liter portions of the same basic buffer but 0.1 M, 0.2 M, and 0.5 M in NaC1, respecs C. Schwabe and G. Kalnitsky, Arch. Biochem. Biophys. 109, 68 (1965).
[57]
PEPTIDE HYDROLASES
745
tively. This schedule leads to the elution of a protease (referred to as DEAE I) 6 in the first 4 liters of eluent. The next 2 liters contain no activity, followed by 2.5 liters containing a second protease (DEAE II) and an aminopeptidase activity. Purification o] DEAE I by Ammonium Sulfate Fractionation. The 4 liters of fraction 1 obtained from the DEAE column are 40% saturated with solid ammonium sulfate. 7 The heavy precipitate is centrifuged off and discarded, the remaining supernatant is saturated to 85% with ammonium sulfate and stirred for about 15 minutes. The second precipitate is collected by centrifugation at 5000 rpm and the supernatant discarded. This fractionation is performed at room temperature. Adsorption on Calcium Phosphate. The pellet obtained from the 85% ammonium sulfate fraction is redissolved in 250 ml of distilled water and dialyzed for 16 hours against a 10-2 M solution of CaCl~. Calcium phosphate slurry (Bio-Rad)s is added to the dialyzed solution in 20-g portions, followed by centrifugation at 5000 rpm between each addition. The protein contents of the original solution as well as each of the supernatants obtained has to be checked before the next addition of calcium phosphate. This procedure should be carried on until 70% of the protein has been adsorbed on the calcium phosphate gel. The remaining supernatant contains 70-80% of the original activity. Chromatography on Hydroxylapatite. About 45 g of wet hydroxyapatite slurry (prepared according to Jenkins) 9 is mechanically mixed with 50 g of a wet slurry of Whatman cellulose for-15 minutes. The adsorbent is then poured into a 2 X 25-cm jacketed column and permitted to settle under a gravity flow of 0.02 M phosphate buffer (pH 6.8). With a liquid head of 60 cm (between the outlet of the column and the buffer reservoir) a fairly constant flow rate of 16 ml per hour is attained. The total 260 ml supernatant from the calcium phosphate fractionation procedure is applied to the column in about 18 hours. A plateau of nonadsorbed protein is observed to elute from the column long before the protein sample has been applied in toto. The adsorbed protein is eluted by a simple gradient produced by 250 ml of 0.02M phosphate buffer (pH 6.8) in vessel I and 400 ml of 0.15 M phosphate buffer in vessel II (vessel I is a 250-ml Erlenmeyer flask, fitted with airtight inlet and outlet). The volume in vessel I (which is magnetically stirred) remains constant until depletion of the limiting buffer. After about 400"ml of e Naming of the connective tissue enzymes will be postponed until specificities are established beyond doubt. 7 See Vol, I, p. 76. 8 Bio-Rad Laboratories, Richmond, California. 9 W. T. Jenkins, Biochem. Prep. 9, 84 (1962).
746
PEPTIDASES
[57]
eluant (including the 250 ml from the protein peak) have been collected, a sharp peak of proteolytic activity should be observed, usually followed by a second, less pronounced, activity peak. The first larger fraction is pooled for further purification. These fractions are referred to as hydroxylapatite fractions I and II. Chromatography on Bio-Gel P-60. A 2 X 100 cm column with upward flow adapter is packed with Bio-Gel P-60 (Calbiochem, molecular exclusion value ~60,000) and equilibrated with an acetic acid triethylamine buffer. This buffer solution is prepared by the addition of 2 ml of trimethylamine to each liter of 0.1 N acetic acid. The flow rate through the column should be maintained at 4 ml/cm2/hour by means of a peristaltic pump. The inlet tubing leading to this pump is fitted with a Y-piece to permit the application of a sample through the buffer feeding line. Hydroxylapatite fraction I should be concentrated to 7 ml on an Amicon UM 11° filter and pumped into the Bio-Gel column. The activity elutes in a symmetrical peak in front of a larger inactive protein component. The enzyme, which should be better than 90% pure, is pooled and sucrose added to a final concentration of 0.05 M before lyophilization. The summary of the purification steps given in Table I represents one typical experiment. TABLE I PURIFICATION OF AN ACID PEPTIDE PEPTIDOHYDROLASEFROM BOVINE FIBROBLASTS
Specific activity (units/ m g X 10s)
Fraction
Volume (ml)
Protein (mg/ml)
Total protein (ml)
Crude extract DEAE Ammonium sulfate
4,000 4,200 230
19.3 2.0 6.7
77,200 8,400 1,540
318 185 109
4.4 22.0 71.0
-58 35
230 72 40
2.1 0.9 0.2
483 65 8
77 35 39
160.0 540.0 4,900.0
24 11 12
Total units
Percent ° yield
(4o-85%) GaPO~ Hydroxylapatite Bio-Gel P-60
Pertaining to the main fraction only.
In general the best results will be achieved if freezing and lyophilization can be avoided between procedures. Should the enzyme be kept in solution for a longer time CaCI2 (0.02M) must always be present. Sucrose (0.05 M ) protects the enzyme largely against denaturation during freezing. ioAmicon Corporation, Lexington, Massachusetts.
[ST]
PEPTIDE HYDROLASES
747
Properties of the Acid I-Iydrolase
Specificity. The highly purified preparation produces only very few peptides after 24 hours of incubation at pH 4.0 (40 °) with oxidized bovine ribonuclease A. The peptide containing residues 1-52 (N-terminal lysine, C-terminal alanine), 53-120 (N-terminal aspartic acid, C-terminal phenylalanine), 121-124. (N-terminal aspartic acid, C-terminal valine) have been positively identified. Several minor peptides have not yet been analyzed. The A-chain of oxidized insulin is hydrolyzed between residues 14 and 15 (=Tyr-Gln-) and 16 and 17 (-Leu-Glu-). At high enzyme concentration (100 /~g/mt) the tetrapeptide Leu-Phe-Glu-Ala is hydrolyzed between Phe and Glu. When the latter reaction is followed by thin-layer chromatography, some evidence for transamidation becomes apparent. It is not clear whether the transamidation at this unusual pH (4.0) is due to a trace impurity. The peptides (or derivatives) Tyr-Meth-AspPhe NH2, Gly-Phe-Phe, Z-Gly-Phe, N-Benz DL-Phe-fl naphthyl ester and Leu-Tyr NH~ were not hydrolyzed. Activators and Inhibitors. The enzyme is insensitive to diisopropylphosphofluoridate (DFP) and slightly inhibited by HgCl~. No activator other than hydrogen ions has been found. Purity. The enzyme obtained following the above procedure should show a single band upon discontinuous electrophoresis at pH 8.0 and 4.0.11 Occasionally a minor impurity above and below the active band may be observed which can be removed by repeating the Bio-Gel column step. Stability. The purified enzyme is unstable in dilute solutions unless 0.02 M CaCl~ is added. Denaturation during freezing and lyophilization is prevented by 0.05 M sucrose. About 50% of its activity is lost in 15 minutes if heated to 50 ° and total inactivation occurs after 15 minutes at 60 °. At pH values of 2.0 or less the enzyme will be irreversibly denaturated, while it retains activity well at higher pH values. The purification is performed at pH 8.0 without undue reduction of enzymatic activity. pH Optimum. The activity of the enzyme with hemoglobin as substrate is maximal at pH 3.5 to 4.0. At pH 2.0 the enzyme activity ceases abruptly. The catalytic activity slopes down more gently toward the neutral pH range with 50% activity remaining at pH 5.0. Molecular Weight. The molecular weight determined by exclusion chromatography is 40,000 ± 2000.11 According to preliminary data, the protein is formed by a single polypeptide chain. Distribution. The acid protease appears to be a lysosomal enzyme al1~C. Schwabe and G. Kalnitsky, Biochemistry 5, 158 (1966).
748
PEPTIDASES
[ST]
though it has not been established that this is its exclusive location i n the cell. Other Acid Proteases. The second active peaks obtained from the D E A E column and a second peak or shoulder observed during hydroxyl° apatite chromatography remain to be characterized. It seems certain that enzymes different from spleen cathepsins are present and that a carboxypeptidaselike activity does not occur in connective tissue cells. Neutral Peptide Hydrolases This group includes leucine aminopeptidase as a major enzyme. In addition an aminotripeptidase, glycylglycine dipeptidase, (gly)a tripep-
E
g
8.0
14 1 ~. , _. 2 ... . ~. .5 .= , ._ . .4 . . ~ . . 5. ~ , . . 6 _ ,
'- 2.0 [-
~
"~..-''-
8 o. I 0
.f.-~0.4 0.2 _~
0.5
0.1 I00
200
300
400
500
Tube number
FIG. 1. Distribution of various peptide hydrolases of mammalian fibroblasts after chromatography on DEAE-cellulose. The fractions as indicated above the protein line • • contain the following enzymes: (1) Acid peptide peptidohydrolase (cathepsin). (2) Acid peptide hydrolase, imidodipeptidase I. (3) Aminotripeptidase, iminodipeptidase, triglycine peptidase I, glycylalanine dipeptidase. (4) Glycylleucine dipeptidase, imidodipeptidase II, leucine aminopeptidase. (5) Formylleucine hydrolyzing enzyme. (6) Triglycine peptidase II.
tidase, glycylleueine dipeptidase, imino and imidodipeptidase, and a formylleucine hydrolyzing enzyme are found in a crude neutral extract of the fibroblasts of bovine dental connective tissue. Charging the large DEAE column described in the previous section with only about 50 g of protein and eluting by a buffer gradient rather than a stepwise procedure, the enzyme distribution as depicted in Fig. 1 is observed. 12 Fractions of the effluent of this column might be used to further purify a certain C. Schwabe, Biochemistry 8, 783 (1969).
[57]
PEP*rIDE HYDROLASES
749
enzyme of this group of neutral peptide hydrolases. Below the purification of the Mn 2÷ ion-dependent soluble leueine aminopeptidase (an a-aminopeptide aminoacidohydrolase 3.4.1) is described. Other enzymes will be considered briefly at the end of this chapter. Principle. The enzyme hydrolyzes leucinamide and a variety of peptides with N-terminal residues other than glycine, proline, or hydroxyproline. Denatured proteins with susceptible N-terminals are hydrolyzed much slower than dipeptides. Leucylleucine is a better substrate than leucinamide and is routinely used to follow purification steps. The activity is measured by a ninhydrin reaction essentially as described by Matheson and Tattri. TM The reagent reacts only slightly with dipeptides but yields normal color values with amino acids.
Reagents A 10 mM solution of leucylleucine in 0.1 M Tris buffer at pH 8.0 Acetic acid, 0.1 M MnC12 solution, 0.1 M Methyl Cellosolve, 50% in distilled water Stock ninhydrin solution: 50 ml 5% ninhydrin in Cellosolve, 250 ml methyl Cellosolve, 5 ml KCN 0.01 M. This reagent is stored at --20 ° . Working ninhydrin solution. Dilute stock ninhydrin 1:1 with 50% Cellosolve just prior to use. Tris buffer 0.1 M, pH 8.0 Citrate buffer, 0.25 M, pH 5.0
Procedure. An appropriate amount of crude extract (25 ~l) or partially purified enzyme preparation is made to 200 ~l with 0.1 M Tris HC1 buffer (pH 8.0, 2 X 10-8 M in MnC12) and placed into a water bath (40 °) for about 5 minutes. The prewarmed Leu-Leu solution is added (200 ~l) and three 25-~1 samples of the reaction mixture are immediately withdrawn and placed in three different tubes, each containing 0.5 ml of 0.1 N acetic acid. In 5, 10, 15, and 20-minute intervals again triplicate samples of 25 ~I each are withdrawn and pipetted into acetic acid-containing tubes (acid stops the reaction). All volume transfers, except the acetic acid, must be made with micropipettes to be sufficiently accurate. The whole contents of the acetic acid-containing tubes are used for color development as described bel~)w. To assay the column effluent for leucine aminopeptidase activity 2 X 25 ~l may be withdrawn from every third or fifth tube (depending upon the size of the column and volume collected) and placed into a duplicate is A. T. Matheson and B. L. Tattri, Can. J. Biochem. 42, 95 (1964).
750
PEPTIDASES
[57]
set of test tubes. Each member of one set receives 25 #1 of Tris buffer (pH 8.0) while the second set receives 25 ~l Leu-Leu substrate. Incubation for 15 minutes at 40 ° is usually sufficient to locate the enzyme in the effluent. The reaction is stopped by the addition of 0.5 ml of 0.1 N acetic acid from a pipette-syringe. Color Development. This procedure is identical for both the regular assay and the column assay. The tube racks are placed in an ice bath and 1 ml of citrate buffer (pH 5.0) is added (speedy addition of the reagents for color development is best achieved with pipette-syringes), followed by 1 ml of the working ninhydrin solution. Each test tube is removed from the rack, shaken vigorously, and placed back into the ice bath. After all tubes have been shaken, the whole rack is transferred to a vigorously boiling water bath for exactly 81/~ minutes. Thereafter the rack is rapidly transferred back into the ice bath and 2 ml of 50% aqueous isopropanol added. The test tubes are shaken again and left in the cold water for 5 minutes. Subsequently the tubes may be kept at room temperature for 30 minutes or may be warmed up briefly at 40 ° prior to color determination at 570 nm. The absorbance should be measured within 1 hour. The A0D is plotted as a function of time. Enzyme concentrations should be adjusted to give a linear reaction for at least 10 minutes. The column assay is evaluated by plotting the difference between blank and experimental sets of tubes as a function of tube number. Assay o] Tissue Slices or Whole Homogenates. Tissue slices may be prepared from either fresh or thawed connective tissue and suspended in Ringer's solution at pH 7.5 in double-sidearm Warburg flasks. 1~ A single 15-ml respirometer flask can be loaded with several grams of wet tissue slices. One sidearm of the flask receives 200 ~l .of a 0.2% solution of snake venom 15 or correspondingly less purified L-alpha amino acid oxidase, while the second sidearm is filled with a substrate peptide (about 1 millimole). At least one, preferably both amino acids of a dipeptide must be a substrate for the amino acid oxidase. The amino acid oxidase is added first to the tissue slices, and the immediate oxygen uptake, which is due to free amino acids in the tissue, is measured. When the rate drops to near zero the peptide substrate is added and the rate of peptide hydrolysis measured as 02 uptake. Appropriate controls can be used to compensate for respiratory activity. This interference will be low since no C02 absorbing base is included in the system. I'W. W. Umbreit, R. H. Burns, and J. F. Stauffer, "Manometric Techniques," Burgess, Minneapolis, 1964. Ross Allan's Snake Farm, Florida.
[57]
PEPTIDE HYDROLASES
751
The result should be expressed in micromoles oxygen used per minute per milligram of wet or dry weight of tissue. The oxidation of every mole of amino acid causes the uptake of 1 mole of 02 if no peroxidase is present. The efficiency of the system must be tested by the addition of standard amounts of leucine to the tissue slices. Definition o] Activity Unit and Specific Activity. The activity unit is defined as the amount of enzyme hydrolyzing 1 micromole of leucylleucine per minute in Tris-HC1 buffer at pH 8.0 (buffer effects are significant). Specific activity designates the number of units per milligram of protein, assuming that 1 mg of protein in 1 ml ~f water gives rise to an optical density of 1 in a 10-mm light path at OD = 280 rim. The conversion of ninhydrin color to micromoles is possible if the ~-values for products and reactants are determined in a system duplicating assay conditions. Considering the c~ is the molar extinction of reactants and cp represents the molar extinction of products and that 1 mole reactant (Leu-Leu) gives rise to 2 moles of product, the following relation is obtained. moles hydrolyzed liter minutes
AODsTo 2~ -- ~Train
AOD57o A~ rain
(In case of a mixed dipeptide the term 2cp is replaced by ~pl + c~2.) The ~-values will have to be determined by each investigator. The data may also be expressed as percent hydrolysis since the limiting AOD is the value of the denominator and any AOD obtained divided by the limiting value and multiplied by 100 gives percent hydrolysis. Knowing the assay conditions, it is possible to calculate Co values from specific activities obtained from initial velocities (zero order) for comparison. The ninhydrin method used here permits good measurements with as little as 2% hydrolysis if a 5 mM leucylleucine solution is used as substrate. In contrast to the discontinuous titration method of Grassman and Hyde16; the assay can always be restricted in time to observe the apparent zeroorder period of reaction. Purification. The purification of the leucine aminopeptidase of bovine fibroblasts begins with the DEAE column described under Purification of Acid Proteases. After 8.5 liters have been collected from the column, the leucine aminopeptidase begins to elute (this must be checked since batches of DEAE may vary). The following 6 liters of eluant are collected and lyophilized to provide a convenient, stable starting material for subsequent steps. Benzalkonium Chloride (Zephiran) Precipitation. This quaternary le W. Grassman and W. Hyde, Z. Physiol. Chem. 183, 32 (1929).
752
PEPTIDASES
[57]
ammonium compound has proven useful for the purification of the connective tissue LAP although its mechanism of action and limiting conditions are not well understood. The procedure should be followed as closely as possible as deviation might yield unpredictable results. The lyophitized LAP fraction (10 g) is redissolved in 500 ml of distilled water and the solution magnetically stirred in an ice bath. A 1.7% solution of Zephiran chloride in 0.1 M Tris at pH 8.0, 10.3 in MnCl~, is prepared and added to the enzyme solution in 20-ml increments. Slow addition is essential and is best achieved by siphoning the Zephiran chloride through an intravenous tubing into the gently stirred protein solution. Following each addition of 20 ml Zephiran, the solution is centrifuged at 5000 rpm, the pellet suspended in 100 ml of 0.1 M Tris buffer (pH 8.0, 10-3 M ,in MnC12, 10-2 M in MgCl~, and 10-1M in NaC12) and stirred gently at 4 °. Up to 60 ml of Zephiran chloride solution may be added without precipitating a significant portion of the activity, although it is advisable to retain all the pellets obtained until final tests have been made. The addition of the next 20 ml of Zephiran chloride precipitates the LAP activity. All pellets are stirred overnight at 4 ° and tested for activity. If a substantial number of enzyme units are missing after this procedure, the pellet of the enzyme-containing fraction is resuspended in 100 ml of Tris buffer. The combined supernatants should contain 7 0 ~ of the activity present prior to Zephiran frac'tionation. Generally, if the ionic strength is too high the enzyme will not precipitate at pH 8.0. The column effluent may be fractionated with Zephiran without prior lyophilizatkm,, in which case the buffers present should be removed by a short dialysis or the pooled effluent diluted with about 50% of its volume of distilled water. Acetone Fractionation. The enzyme solution ~)btained from the previous step is cooled to 0 ° in an ice bath and ice-cold acetone is added slowly. Any precipitate formed between 0 - 2 0 ~ acetone concentration is centrifuged off and discarded. The acetone concentration in the supernatant is then increased to 40%, the pellet collected by centrifugation at 4000 rpm and redissolved in 10 ml of 0.05M Tris buffer (pH 8.0, 10-3 M in MgC12). Ammonium Sul]ate Precipitation. In order to remove most of the solvent, the enzyme solution obtained from the acetone fractionation is diluted to 300 ml and reconcentrated to 100 ml with an Amicon UM I filter. This solution, containing about 1 mg protein per milliliter, is cooled in an ice bath and 50 ml of saturated ammonium sulfate containing 1 g of Tris base per liter, are added. No precipitate forms at this time. Adding solid ammonium sulfate the salt concentration is increased to 4 0 ~ saturation, and the precipitate centrifuged off at 5000 rpm and
[57]
PEPTIDE HYDROLASES
753
~x
Z Z
O
©
!
O
O
754
PE~mASZS
[ST]
discarded. The ammonium sulfate in the supernatant solution is increased to 6 0 ~ saturation and the precipitate, collected by centrifugation, is redissolved in a small amount of pH 8.0 Tris buffer. Agarose Chromatography. A 150 X 2.5 cm jacketed column maintained at 4 ° is used for exclusion chromatography with Agarose (BioGel A 1.5, exclusion limit 1.5 X 106 g/mole). The sample is concentrated to 5-10 ml, applied t~ the top of the column, and eluted with a 0.025 M Tris buffer (pH 8.0) containing 10-3 moles of MgC12 per liter. A broad protein peak elutes in tubes 100 to 300 (if 3-ml fractions are collected) which contain nearly all of the activity placed onto the column. The peak broadening is due to aggregates varying in size from 4 X 10~ to 5 X 10~ g/mole. An acrylamide gel run at pH 8.0 in a glycine buffer system 1~ of the pooled active fraction should show one band at the origin and four equally spaced bands traveling toward the anodeJ 2 The purification procedure is summarized in Table II. Properties of the Purified Connective Tissue Leucine Aminopeptidase
Specificity. The Specificity of the fibroblast enzyme is similar to that of other leucine aminopeptidases. ~s An absolute requirement exists for the L-configuration of the N-terminal residue possessing a free primary amine function. The penultimate residue must be :an L-amino acid other than proline or hydroxyproline but could also be glycine. Peptides with glycine or imino acids at Che N-terminal position are not hydrolyzed. The amides of leucine or phenylalanine are hydrolyzed one order of magnitude slower than dipeptides. In contrast to the kidney enzyme, no activity toward glycinamide has been detected. The rate of hydrolysis of dipeptides is greatest if norleucine, isoleucine, leucine, methionine, or phenylalanine occupies the N-terminal position. The activity decreases with increasing length of the peptide su.bstrate chain and becomes very slow with denatured proteins. N-Acetylated peptides, denatured cytochrome c (horse heart), and mercuripapain are not hydrolyzed. Activators and Inhibitors. In contrast to other leucine aminopeptidases, the connective tissue enzyme is activated solely by Mn 2÷ ions. EDTA removes the ions from the enzyme only at elevated temperatures (40°). The inhibition can be reversed by Mn ~÷ ions. In contrast, removal of Mn 2÷ ions by dialysis against distilled water inhibits the enzyme irreversibly. Copper ions reduce the rate of hydrolysis of Leu-Tyr (Ki 5 X 1 0 -SM) at pH 8.0 (the K~ for this substrate at pH 8.0 is 8 X 104 M). Some inhibition is also observed with Zn 2÷, Cd 2÷, and Ca 2÷ ions. Exposure to N-ethylmaleimide and p-mercuribenzoate abolishes the 1TL. Ornstein and B. J. Davis, Ann. N.Y. Acad. Sci. 121, 428 (1964). 18E. L. Smith, Advan. Enzymol. 12, 191 (1951); see also Vol. II, p. 88.
[57]
P~.P~D~. I~DROLASES
755
aminopeptidase activity. The enzyme can be reacted with iodoacetamide or 1-dimethylaminonaphthalene-5-sulfonyl chloride without influencing the activity or the rate of migration in an electric field at pH 8.0. Purity. Due to the multiple forms of this enzyme, i¢ is difficult to ascertain absolute purity of a preparation of connective tissue leueine aminopeptidase. Upon acrylamide electrophoresis in a discontinuous buffer system (pH 8.0), four bands are observed to travel toward the anode while a fifth band remains at the origin. Each 'band has been demonstrated to be active.12 Stability. The enzyme is relatively stable in solution and may be frozen and thawed repeatedly without causing inactivation. In frozen samples (--20 °) the activity is retained for at least 1 year. Lyophilization, in contrast, irreversibly inactivates the enzyme up to 80%. In 8.0 M urea (40 °) the LAP remains active for 16 hours while t`be addition of 0.1 M LiC1 to this solution destroys the activity as well as the banding pattern on acrylamide electrophoresis. The enzyme also remains active if kept at 55 ° for 30 minutes in 0.1 M Tris buffer (pH 8.0) or at 50 ° in the same buffer containing 30% acetone or ethanol. The presence of 30% dioxane in the Tris buffer deactivates the enzyme completely within 30 minutes. 19 The pH stability curve of connective LAP shows a maximum at pH 8.0 and a steep decline within 2 pH units above and below this value. At the higher pH the enzyme is protected by substrate. pH Optimum. The pH optimum of the connective tissue leucine aminopeptidase is substrate concentration dependent. At pH 7.5 the affinity for Leu-Leu is greatest but the maximal catalysis rate is obtained at pH 9.0 and high substrate concentration,s° Below pH 7.0 the activity disappears. Molecular Weight. In the crude extract aggregates of connective tissue leucine aminopeptidase as large as 1.5 X 106 g/mole have been observed during exclusion chromatography. The main form of the enzyme with a molecular weight of 4 X l0 s dissociates "into functional units, each 105 g/mole. Preliminary experiments suggest that smaller units (5 X 104 g/mole) exist and that these are possibly formed by two kinds of polypeptide chains. A leucine aminopeptidase isolated from bovine lenses by Kretschmer and Hansen 21 shows a similar pattern. Other Neutral Peptide Hydrolases ]rom Bovine Fibroblasts. The enzymes described here have not been purified. The fractions in which they are contained are shown in Fig. 1. Molecular weights were determined by exclusion chromatography. 1,C. Schwabe, Biochemistry 8, 795 (1969). ~*C. Schwabe, Biochemistry 8, 771 (1969). -'~K. Kretschmerand It. Hansen, Z. Physiol. Chem. 349, 831 (1968).
756
PEPTIDASES
[58]
Imidopeptidase. (Molecular weight ~150,000.) The existence of two forms of this enzyme is suggested by its appearance in two widely separated fractions of the DEAE column (2 and 4). The imidodipeptidase differs in some respects from the intestinal enzyme. In the presence of Tris buffer (pH 8.0) Mn 2÷ ions have no effect but stimulate in the presence of phosphate buffer. Zinc ion in Tris buffer inhibits the activity but stimulates in phosphate buffer. Glycylglycine Dipeptidase. The Co 2+ activated peptidase is observed in the crude extract but appears to denature upon DEAE chromatography. Differences in glyeylglycine dipeptidase from other tissues have not been observed. Aminotripeptidase. (Molecular weight ~100,000.) The activity is independent of Mn 2÷ ion, active in the presence of EDTA, and slightly depressed by Cd ~÷ions (DEAE fraction 2). Tripeptides of L-amino acids are hydrolyzed to a dipeptide and the free N-terminal amino acid. Triglycine is not a substrate. Triglycine Peptidase. (Molecular weight ~400,000, possibly 100,000 subunits.) The peptidase catalyzes the hydrolysis of triglycine to glycine and glyeylglyeine and does not require Co2÷ions (DEAE fraction 2). Imino Dipeptidase. (Molecular weight ~250,000.) Dipeptides possessing an N-terminal proline are hydrolyzed by this enzyme. The activity is stimulated by Mn 2÷ ions and inhibited by EDTA and inorganic pyrophosphate (DEAE fraction 2). Glycylalanine Dipeptidase. (Molecular weight ,-~50,000.) A small amount of 200,000 molecular weight activity was observed. The ion requirement is not known (DEAE fraction 2). Formylleucine Hydrolyzing Enzyme. This weak activity has not been further characterized (DEAE fraction 5). Triglycine Peptidase II. It is not known whether the enzyme differs from the triglyeine peptidase I (DEAE fraction 6).
[58] e - L y s i n e Acylas¢ f r o m Achromobacter pestiier
By ICHIRO CHIBATA, TSUTOMUISHIICAWA,and T~rsuYA TosA ¢-N-Acyl-L-lysine -{- H~O --* fatty acid ~ ~-lysine
Assay Method 1
Principle. The assay of ~-lysine acylase (¢-N-acyl-L-lysine amidohydrolase, EC 3.5.1.17) is based on the determination of liberated IT. Ishikawa, T. Tosa, and I. Chibata, Agr. Biol. Chem. 26, 581 (1962).
756
PEPTIDASES
[58]
Imidopeptidase. (Molecular weight ~150,000.) The existence of two forms of this enzyme is suggested by its appearance in two widely separated fractions of the DEAE column (2 and 4). The imidodipeptidase differs in some respects from the intestinal enzyme. In the presence of Tris buffer (pH 8.0) Mn 2÷ ions have no effect but stimulate in the presence of phosphate buffer. Zinc ion in Tris buffer inhibits the activity but stimulates in phosphate buffer. Glycylglycine Dipeptidase. The Co 2+ activated peptidase is observed in the crude extract but appears to denature upon DEAE chromatography. Differences in glyeylglycine dipeptidase from other tissues have not been observed. Aminotripeptidase. (Molecular weight ~100,000.) The activity is independent of Mn 2÷ ion, active in the presence of EDTA, and slightly depressed by Cd ~÷ions (DEAE fraction 2). Tripeptides of L-amino acids are hydrolyzed to a dipeptide and the free N-terminal amino acid. Triglycine is not a substrate. Triglycine Peptidase. (Molecular weight ~400,000, possibly 100,000 subunits.) The peptidase catalyzes the hydrolysis of triglycine to glycine and glyeylglyeine and does not require Co2÷ions (DEAE fraction 2). Imino Dipeptidase. (Molecular weight ~250,000.) Dipeptides possessing an N-terminal proline are hydrolyzed by this enzyme. The activity is stimulated by Mn 2÷ ions and inhibited by EDTA and inorganic pyrophosphate (DEAE fraction 2). Glycylalanine Dipeptidase. (Molecular weight ,-~50,000.) A small amount of 200,000 molecular weight activity was observed. The ion requirement is not known (DEAE fraction 2). Formylleucine Hydrolyzing Enzyme. This weak activity has not been further characterized (DEAE fraction 5). Triglycine Peptidase II. It is not known whether the enzyme differs from the triglyeine peptidase I (DEAE fraction 6).
[58] e - L y s i n e Acylas¢ f r o m Achromobacter pestiier
By ICHIRO CHIBATA, TSUTOMUISHIICAWA,and T~rsuYA TosA ¢-N-Acyl-L-lysine -{- H~O --* fatty acid ~ ~-lysine
Assay Method 1
Principle. The assay of ~-lysine acylase (¢-N-acyl-L-lysine amidohydrolase, EC 3.5.1.17) is based on the determination of liberated IT. Ishikawa, T. Tosa, and I. Chibata, Agr. Biol. Chem. 26, 581 (1962).
c-LYSINE ACYLASE FROM A. pestifer
[58]
757
T.-lysine from ~-N-acyl-L-lysines by the acidic ninhydrin colorimetric method. 2 Reagents Sodium acetate-acetic acid buffer solution, 1 X 10-1 M, pH 5.0 ~-N-benzoyl-L-lysine solution, 1.2 X 10-2 M, in water Enzyme. Prior to assay, dilute in 5 X 10-2 M sodium acetate solution (1 X 10-~ M) so that 1 ml will contain approximately 1-5 units. Acidic ninhydrin solution. One gram of ninhydrin is dissolved in 64 ml of acetic acid and 16 ml of 6 X 10-1 M phosphoric acid. 50% Ethanol-water solution (v/v) Procedure. A mixture of 0.5 ml of buffer solution, 0.5 mt of ~-Nbenzoyl-L-lysine solution, and 0.5 ml of enzyme solution is incubated at 42 ° for 10 minutes. After completion of the enzyme reaction, 0.5 ml of the above enzyme reaction mixture is placed into 1.0 ml of acidic ninhydrin solution. The mixture is heated at 100° in a boiling water bath for exactly 5 minutes, and cooled rapidly to room temperature. To the resulting mixture 3.5 ml of 50% ethanol-water solution (v/v) is added. The optical density of the solution is read at 340 nm. A reaction blank containing water instead of c-N-benzoyl-L-lysine solution is treated identically. Definition o[ Unit and Specific Activity. One unit of activity is the amount of enzyme liberating 1 mieromole of lysine per hour under the above conditions. Protein content is determined by the method of Lowry et al. 8 Specific activity is expressed as units per milligram of protein. Purification Procedure *,5 Preparation o] Cells 6 Five liters sterilized medium containing 1% peptone, 5% glucose, 0.2% KH2P04, and 0.1% MgS04-7 H20 in a 10-liter jar fermentor are inoculated with 150 ml of the seed culture of Achromobacter pesti]er EA ATCC 23584, which was incubated with reciprocal shaking (110 cycles/ minute, stroke 8 cm) at 30 ° for 48 hours in 500-ml flasks containing 100 ml of the same medium described above. Fermentation in the jar is I E. Work, Biochem. J. 67, 416 (1957). s 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4T. Ishikawa, T. Tosa, and I. Chibata, Agr. Biol. Chem. 26, 412 (1962). 5 I. Chibata, T. Ishikawa, and T. Tosa, Nature 195, 80 (1962). eT. Ishikawa, T. Tosa, and I. Chibata, Agr. Biol. Chem. 28, 193 (1962).
758
PEPTIDASES
IS6]
carried out at 30 ° for 24 hours with aeration (5 liters air/minute/5 liters medium) and agitation (400 rpm). The cells are harvested by centrifugation and washed with 0.9% sodium chloride solution. Step 1. Preparation o] Cell-Free Extract. One kilogram of wet cells from 75 liters of culture broth are mixed with 500 ml of toluene to initiate autolysis. After being liquefied, 12 liters of 6.67 X 10-2 M sodium sulfate solution is added, and the suspension is kept for 24 hours at 30 ° with occasional stirring. The suspension is centrifuged at 50,000 g and 0 ° for 30 minutes. A yellowish, clear, viscous solution (ca. 12 liters) is obtained. Step 2. Fractionation with Ammonium Sul]ate. The crude extract is brought to 40% saturation by addition of ammonium sulfate at 5 ° and adjusted to pH 6.5. After standing for 30-60 minutes at 5 °, the precipitate is removed by centrifugation at 20,000 g and 5 °. The resulting supernatant is brought to 70~ saturation by further addition of ammonium sulfate at 5 ° and is adjusted to pH 6.5. After standing for 3060 minutes at 5 °, the precipitate is collected by centrifugation at 20,000 g and 5 °. The precipitate obtained is dissolved in 350 ml of 5 X 10-~M sodium acetate solution and dialyzed against the same solution at 5 ° for 24 hours. After dialysis of the solution, any insoluble material is removed by centrifugation at 20,000 g and 5 °. Step 3. Rivanol Treatment. To the dialyzed solution (ca. 380 ml) a 15% volume of 2 ~ Rivanol solution is added dropwise at 5 °. After allowing the solution to stand at 5 ° for 1-2 hours, the precipitated acidic protein is removed by centrifugation at 20,000 g and 5 °. To remove excess Rivanol, the supernatant is passed through a column packed with Amberlite IRC-50 buffered with 5 X 10-1 M sodium acetate-acetic acid buffer solution, pH 6.0. The ettiuent containing c-lysine acylase activity is collected. Step ~. Fractionation with Acetone. To the efltuent (ca. 600 ml) cold acetone is added dropwise at --10 ° to the final concentration of 45%. Ti~e precipitate is immediately removed by centrifugation at 15,000 g and --10% The resulting supernatant is brought to 60% volume by further dropwise addition of cold acetone at --10% The precipitate is immediately collected by centrifugation at 15,000 g and --10% The preCipitate obtained is dissolved in a small volume (ca. 50 ml) of 5 X 10-3 M sodium acetate solution, and any insoluble material is removed by centrifugation at 20,000 g and 5 °. Step ,5. Fractionation with Ammonium Sulfate. The supernatant is brought to 48% saturation by dropwise addition of saturated ammonium sulfate solution at 5 ° and adjusted to pH 7.0. After standing for 60 minutes at 5 °, the precipitate is removed by centrifugation at 20,000 g and 5 °. The supernatant is brought to 60% saturation by further drop-.
[58]
C-LYSINE ACYLASE FROM A.
pestifer
759
wise addition of saturated ammonium sulfate solution at 5 ° and adjusted to pH 7.0. After standing for 60 minutes at 5 °, the precipitate is collected by centrifugation at 20,000 g and 5 °. The precipitate obtained is dissolved in a small quantity of Veronal buffer solution (pH 8.8, ~ : 0.0505) and dialyzed against the same buffer for 24 hours at 5 ° . Step 6. Vertical Zone Electrophoresis. The dialyzed solution is used for vertical zone electrophoresis to obtain the pure enzyme. The apparatus and technique are the same as those employed by H. Chiba et aU To allow the electrophoresis to be performed for a long time, the apparatus is furnished with Hg--Hg2Cl~ reversible electrodes, and is so designed to prevent electroosmosis and pH changes. The cellulose powder used as a supporting material is prepared by refluxing clean cotton of good quality in absolute methanol containing 1 M hydrogen chloride for 24 hours. The cellulose powder is packed in a column using a Veronal buffer solution (pH 8.8, /z ----0.0505). Nine milliliters of the dialyzed enzyme solution (step 5) containing 200 mg of protein (specific activity 10,800) is placed on the electrophoresis column (50 cm length) as described by H. Chiba et al. A potential difference of 300V (20 mA) between the two electrodes is applied for 160 hours in the Veronal buffer solution (pH 8.8, ~ ---- 0.0505) at 6 °. When the run is finished, the column is detached from the apparatus and mounted on a fraction collector. The elution is carried out with the same buffer solution at 6 ° and the elution rate is controlled to 12 ml per hour by a pump. Fractions of 3.2 ml are collected, and are assayed for protein content and enzyme activities. The fractions of higher specific aotivity (14,300-14,600 units/rag of protein) are collected. To the pure c-lysine acylase solution of higher specific activity, saturated ammonium sulfate solution is added to give 60% saturation; thus the enzyme is precipitated. The final preparation is stored in a refrigerator. The purification procedure is summarized in Table I. PadayatW and Van Kley 8,9 reported that the enzyme was effectively purified by the addition of gel filtration technique in 0.1 M sodium acetate buffer solution (pH 6.0) on Sephadex G-200 before column zone electrophoresis. Properties
Stability. A suspension of the pure enzyme in 60% ammonium sulfate may be stored at 0 ° for several months without decrease of enzyme activity. If. Chiba, E. Sugimoto, and M. Kito, Agr. Biol. Chem. 24, 428 (1960). "J. I). Padayatty and H. Van Kley, Biochemistry 5, 1394 (1966). 0j. D. Padayatty and H. Van Kley, Arch. Biochem. Biophys. 120, 296 (1967).
760
PEPTIDASES
[SS]
TABLE I PURIFICATION OF e-LYSINE ACYLASE
Step
Total Volume protein (ml) (mg)
Total enzyme activity (unit)
1. Cell-free 12,000 26,560 6,365 × 108 extract 2. Fractionation 380 6,570 5,130 X 10s with (NH,)2SO, 3. Rivanol 600 1,350 4,525 X 10s treatment 4. Fractionation 50 480 3,410 × 10~ with acetone 5. Fractionation 9 200 2,163 × 108 with (NH,hSO, 6. Vertical z o n e -130 1,885 × 10a electrophoresis
Enzyme CumulaC u m u l a - specific rive rive activity purificayield (unit/rag tion (%) of protein) (-fold) 100
240
1
80.6
780
3.3
71.1
3,360
14.0
53.6
7,140
29.8
34.0
10,800
45.0
29.6
14,500
60.4
Optimum pH. Optimum pH activity of the c-lysine acylase from A. pesti]er EA ATCC 23584 is found to be around 4.8-5.2. This value is different from those of the other reported E-lysine acylases, that is, the pH optima for the enzymes from rat kidney, Pseudomonas species and Aspergillus oryzae No. 10 are pH 7.0, 6.0, and 8.2-8.4, respectively. Purity and Physical Properties. In the vertical zone electrophoresis, specific activities of all fractions in the main component are apparently identical. Thus, the enzyme having a specific activity of 14,500 units per milligram of protein appears homogeneous on electrophoresis. In the absorption of ultraviolet light, the extinction coefficients of 1 ~ solution in a 1-cm cell at 280 nm is 12.00 and the ratio of optical density 280 nm/260 nm is 1.84. Inhibitors and Activators. ~-Lysine acylase activity is inhibited by both the heavy metal ions (Ag+, Hg ~ and Hg 2÷) and the sulfhydryl reagents (monoiodoacetic acid, p-chloromercuribenzoate and ~-chloroacetophenon). The inhibition by p-chloromercuribenzoate is completely recovered by the addition of eysteine. Among the chelating agents, the inhibitory effect of ethylenediaminetetraaeetate and citrate are slight, but oxalate and pyrophosphate inhibit markedly the enzyme activity. Potassium cyanide, azide, and o-phenanthroline show no inhibitory effect. The slight activation of the enzyme is observed by the addition of metal ions or salts in higher concentration (1 X 10-s M to 10-1 M). A specific and more effective activator is not found.
e'LYSINE ACYLASE FROM A. pestifer
[58]
761
TABLE II RATES OF HYDROLYSIS OF e-N-AcYLATED L-LYSINES BY e-LYSINE ACYLASE
Rate of hydrolysis, ~-Acyl-~lysines
at pH 5.0
at pH 7.2
Benzoyl Acetyl Monochloroaeetyl Dichloroacetyl Trichloroacetyl Monoiodoacetyl n-Butyryl /ao-Butyryl Methacrylyl n-Caproyl n-Caprylyl Glyeyl (HC1 salt) Carbobenzoxy Phenylacetyl p-Toluoyl a-Nitrobenzoyl m-Nitrobenzoyl p-Nitrobenzoyl a~-Naphthoyl Cyclohexanecarboxyl
15,000 2,700 5,300 34,800 7,500 14,500 16,000 6,400 4,500 15,700 12,500 4,100 5,200 1,500 8,700 0 3,100 5,900 1,200 4,200
6 100 1400 2 600 7 700 2 000 59oo 4 500 3 5o0 3 200 5,200 5,000 3,700 2,600 1,100 3,500 0 3,600 2,700 1,100 3,300
Liberated lysine mieromoles/hour/mg of the enzyme. Specificity. 1° Among the various N-acyl derivatives of lysine and its analogs, the enzyme is unsusceptible to the following compounds: c-Nbenzoyl-n-lysine, a-N-acyl, and a,~-N-diacyl-L-lysines, N-acetyl-DLmethionine, c-N-benzoyl-L-lysine ethyl ester, c-N-benzoyl-L-lysine amide, monobenzoyl cadaverine, c-N-benzoylaminocaproic acid, ~-N-acetylaminocaproic acid, a-chloro-c-N-benzoylamino-DL-caproic acid, a-bromoc-N-benzoylamino-DL-caproic acid, 8-N-benzoyl-L-ornithine, ~-N-benzoyl-DL-homolysine, c-N-benzoyl-c-N-me~yl-DL-lysine, and c-N-tosylL-lysine. From these results, i t is shown that the enzyme has a greater substrate specificity than a-aminoacylase, and susceptible substrates are limited to c-N-acyl-L-lysines. Table II shows the rates of hydrolysis of various c-N-acyl-L-lysines by the enzyme. Kinetics. Lineweaver-Burk plot yields a K , of 2.9 X 10-4 M for e-Nchloroacetyl-L-lysine and 5.0 X 10-" M for c-N-acetyl-L-lysine. The K,~ for e-N-benzoyl-L-lysine is below 1.0 X 10-4 M, but a correct measurement is practically impossible due to the low concentration of liberated lysine.
,oI. Chibata, T. Tosa, and T. Ishikawa, Arch. Biochem. Biophys. 104, 231 (1964).
762
P]~PTIDAS~.S
[58]
The activation energy of the enzyme on c-N-benzoyl-L-lysine is 9280 calories per mole by means of an Arrhenius' plot. Distribution. The enzyme is widely distributed in animal tissues 11-1s and in microorganisms? 4-~s Among the former, kidney (pigeon, chicken, pig, rat, etc.) is the richest source, but the enzyme is also found in brain, liver, spleen, pancreas, and heart of a few animals. TM Of the microorganisms, besides Achromobacter pesti]er EA, Pseudomonas species, ~4,~5 a number of soil bacteria, le,~7 Aspergillus oryzae, TM and mushrooms TM have been reported to contain the enzyme. c-Lysine acylase activities of various enzyme sources are summarized in Table III. TABLE III ¢-LYsINE ACYI,ASE ACTIVITY OF VARIOUS ENZYME SOURCES
Enzyme activity• Enzyme sources Rat kidney Hog kidney Pseudomanas (KT 83) Aspergillu8 oryzae No. 10 Achromobacter pestifer EA
Substrates ~-N-Acetyl-L-lysine e-N-Acetyl-L-lysine ¢--N-Benzoyl-L-lysine e-N-Benzoyl-L-lysine
Crude Purified Optimum extract preparation pH 0.023 0.05 0.32 2.90
e-N-Benzoyl-L-lysine 240
2.59 8.21 1,190 31.6 14,500
Ref.
7.0 8.0 6.0 8.2--8.4
11 13 15 18
5.0
4
Liberated lysine micromoles/hour/mg of the protein. T h e animal enzyme (rat kidney and hog kidney) cleaves the benzoyl derivative at a lower rate than the acetyl one, and is also susceptible to ~-N-~-N-disubstituted derivatives of L-lysine. T h e enzyme in Aspergillus oryzae is inducibly formed by ~,N-benzoyl-L-, -n-, or -DL-lysine, and does not have the optical specificity on t h e hydrolysis of c-N-acyl-DLlysine. These major differences indicate that the ~-lysine acylases from animal, mold, and bacterial sources are quite different enzymes. 1~W. K. Paik, L. Bloch-Frankenthal, S. M. Bimbaum, M. Winitz, and J. P. Greenstein, Arch. Biochem. Biophys. 69, 56 (1957). ~2W. K. Paik, Comp. Biochem. Physiol. 9, 13 (1963). is W. K. Paik and L. Benoiton, Can. J. Biochem. Physiol. 41, 1643 (1963). 1, y. Kameda ' E. Toyoura, Y. Kimura, and K. Matsui, Chem. Pharm. Bull. 6, 394 (1958). ~S. Wada, J. Biochem. (Tokyo) 46, 445 (1959). ~T. Ishikawa, T. Tosa, and I. Chibata, Agr. Biol. Chem. 26, 43 (1962). ~TI. Chibata, T. Ishikawa, and T. Tosa, Bull. Agr. Chem. 8oc. Japan 24, 31 (1960). 1~I. Chibata, T. Ishikawa, and T. Tosa, Bull. Agr. Chem. ,goc. Japan 24, 37 (1960).
[Sg]
LOBSTER MUSCLE TRANSPEPTIDASE
[59]
By
Lobster
Muscle
765
Transpeptidase
ROLF MYHRMAN and JOYCE BRUNF~-LORAND
Introduction Tissue coagulin, 1,2 an enzyme found in lobster muscle, induces clot formation when added to the plasma of the same species. The reaction is calcium dependent and m a y be inhibited by the same amines which prevent the cross-linking of vertebrate fibrin?,4 Sulfhydryl reagents such as mercurials and iodoaeetate s are also inhibitory. The enzyme catalyzes the incorporation of amines such as i~he fluorescent N-(5-aminopentyl)-5dimethylamino-l-naphthalene sulfonamide into a-casein.6 In this respect it is similar to guinea pig liver transglutaminase and the fibrinoligase (activated factor XIII) cross-linking enzyme of vertebrates (see this volume, [59a]).7-9 Guinea pig liver transglutaminase can substitute for the lobster enzyme in the clotting test described? ° W e thus consider the enzyme to be a sulfhydryl-dependent transpeptidase, and propose the name "lobster muscle transpeptidase." As with the other two related enzymes, it is also capable of hydrolyzing nitrophenyl esters. Assay Methods Activity is measured either by the ability of the enzyme to clot plasma or by its effect on the incorporation of N-(5-aminopentyl)-5dimethylamino-l-naphthalene sulfonamide into a-casein. Clotting Assay The standard assay is performed in a 10 X 75 mm glass tube at room temperature. The enzyme, 0.1 ml, is mixed with 0.1 ml of 0.2 M calcium 1j. Glavind, in "Studies on the Coagulation of Crustacean Blood" (A. Busuk, ed.). Nyt Nordisk Forlag, Kiobenhavn, 1948. 2G. Duehateau and M. Florkin, Bull. Soc. Chim. Biol. 36, 295 (1954). L. Lorand, R. F. Doolittle, K. Konishi, and S. K. Riggs, Arch. Biochem. Biophys. 1{}2, 171 (1963). 4L. Lorand, N. G. Rule, H. H. Ong, R. Furlanetto, A. Jacobsen, J. Downey, N. 05"er, and J. Bruner-Lorand, Biochemistry 7, 1214 (1968). 5R. F. Doolittle and L. Lorand, Biol. Bull. 123, 481 (1962). e R. V. Myhrman and L. Lorand, Biol. Bull. 135, 430 (1968). 7L. Lorand, T. Urayama, J. W. C. deKiewiet, and H. L. Nossel, J. Clin. Invest. 48,
1054 (1969). 80. Lockridge and L. Lorand, unpublished. *"Report of the Committee on Nomenclature of Blood Clotting lea~tors."Thromb. Diath. Haemorrhag. ~uppl. 13, 428 (1963). ~oj. Bruner-Lorand, T. Urayama, and L. Lorand, Biochem. Biophys. Res. Commun. 23, 828 (1966).
766
TRANSPEPTIDASES
[59]
chloride, and the reaction is initiated 45 seconds later by the addition of 0.2 ml of citrated lobster plasma containing approximately 12 mg of protein. Plasma is added rapidly and the tube is immediately shaken several times by hand to thoroughly mix the contents. Then the tube is gently tipped and inspected at short intervals to detect the first sign of gelation. The clotting time is taken as the interval between addition of plasma and first appearance of gel. Homarus plasma is obtained by entering the pericardial sinus with a siliconized Pasteur pipette through the first abdominal segment and it is collected by swirling into one fourth volume cold 0.1 M sodium citrate. The blood is then clarified by centrifuging at approximately 2000 g for 5 minutes. The supernatant plasma is decanted and may be frozen and stored in small aliquots (5 ml) at --30 ° for at least 10 months without loss of activity. Alternately, it may be freeze dried and stored at --30 °, without loss of activity, for a similar period of time. It is reconstituted by adding water. For duration of short-term experiments, the eitrated plasma is simply kept in ice where it retains its ability to clot for about 5 days. Definition o/Unit. A unit of activity is defined as that amount of enzyme which ~vill produce gelation in 100 seconds under the conditions described.
Incorporation o] N- (5-Aminopentyl) -5-dimethylamino-l-naphthalene Sul]onamide (or Monodansylcadaverine) into ,,-Casein Principle. As this fluorescent amine is incorporated enzymatically into casein, presumably by an amide exchange with some of the v-glutaminyl residues of the protein, a marked enhancement of fluorescence of the naphthalene residue is observed, as if the chromophore were placed in a more hydrophobie environment. The initial rate of enhancement is proportional to enzyme concentration over a wide range of dilutions for a given enzyme fraction. The simpler clotting assay is used to monitor the specific activity of preparations during purification of the enzyme. Reagents Tris-chloride, 0.05 M, 0.001 M EDTA, pI-I 7.5 (buffer I) Calcium chloride, 40 mM N- (5-Aminopentyl) -5-dimethylamino- 1-naphthalene sulfonamide, 1 mM, in 0.05 M Tris-chloride, pH 7.5 Substrate: a-casein (Mann) 7 mg dissolved in 1.0 ml 0.05 M Trischloride, pH 7.5 (care must be taken to see that all the casein is dissolved).
[59]
LOBSTER MUSCLE TRANSPEPTIDASE
767
Enzyme: Lobster muscle transpeptidase is contained in 0.05M Tris-chloride, 0.001 M EDTA, pH 7.5, and kept in ice until use.
Procedure. The Turner Model 111 fiuorometer is used with AA ---- 360 rim, XF = 546 nm. The reaction mixture contains: 1.0 ml Tris-chloride; 0.1 ml calcium chloride; 25X fluorescent amine; 0.1 ml a-casein; 0.1 ml enzyme. The reaction is initiated by the addition of enzyme, and run at 24°C. Control tubes, lacking enzyme, show no incorporation of the fluorescent amine into casein. Purification Procedure
Principle. The purification of lobster muscle transpeptidase is based on differential centrifugation and DEAE chromatography. Step 1. Homogenization. Fresh lobster tails (Homarus americanus) are cut into 5-g pieces and frozen at --30 ° for at least 2 weeks prior to use. To prepare the enzyme, frozen 5-g portions are minced in a small beaker with 2 ml cold 0.25 M sucrose, and homogenized for 2 minutes in a total of 10 ml cold sucrose, using a Teflon-glass homogenizer. (Kontes, size D, motorized.) Step 2. Differential CentriIugation. The homogenate is clarified at 9000 g for 20 minutes (Beckman Model L, No. 30 rotor, 10,000 rpm, 4°). The supernatant is respun at 90,000 g for 4 hours (No. 30 rotor, 30,000 rpm, 4°), and the resulting pelleted enzyme is suspended in cold 0.05M Tris-chloride, 0.001 M EDTA, pH 7.5 (buffer I) to an optical density of about 60 at 280 nm. Step 3. Sonication. Five-milliliter aliquots of the above enzyme solution are sonicated in three bursts of 15 seconds each, allowing 15 seconds between bursts for cooling. (Bronwill Biosonik III, with probe, 19 mm diameter, and an intensity setting of 60.) Step 4. DEAE-Cellulose Chromatography. The sonicate (about 12 ml) is applied to a 19 mm X 20 cm DEAE column (Whatman DE-52) previously equilibrated at 4 ° with buffer I, and the column is washed with 250 ml of 'buffer I at an average flow rate of 35 ml/hour. Eightmilliliter fractions are collected. The enzyme is eluted with a linear sodium chloride gradient to 0.4 M in 600 ml, which releases the activity at a sodium chloride concentration of about 0.03 M, as shown in Fig. 1. The fractions of highest specific activity are pooled and concentrated 4-fold at 0 °, by the addition of polyacrylamide gel (Lyphogel, Gelman Instrument Co.). The purification scheme is outlined in the table. Yields and purification factors vary from one preparation to another, but the concentrated DEAE pool represents a 200 to 400-fold purification relative to the 9000-g supernatant at a recovery of 20-40%. The DEAE pool shows two
768
TRANSPEPTIDASES I
I
[59]
I
I
I0 9
_1oo!
8-
7 I
i
II
78
,.~
0
ssssssSSSpSSSS
5-
0.2
200
4-
0.5 i
3-
2;I
0.1 ~
I00
-
0
20
40 60 Fraction number (8 ml each)
80
FIa. 1. DEAE-cellulose chromatography of lobster muscle transpeptidase.
PURIFICATION OF LOBSTER MUSCLE TRANSPEPTIDASE
Fraction 9000 g Supernatant 90,000 g Supernatant 90,000 g Ppt. in buffer I Sonicate DEAE pool DEAE pool concentrated
Volume Clotting (ml) (units/ml)
Total units
Protein (mg/ml)
PurifiSpecific cation % activity factor Yield
135.0
62.5
8430
53.4
1.17
121.0
27.8
3360
43.1
0.65
9.2
2000
16500
109.0
7.2 34.0 10.4
2000 133 315
14400 4520 3280
103.0 0.717 1.26
1
100
--
--
18.4
16
195
19.4 186 250
17 159 214
171 54 39
m a j o r bands in polyacrylamide disc gel electrophoresis at p H 9.3, one of which is essentially eliminated by subjecting the material to sedimentation in a cold sucrose gradient. T h e pool shows no loss of activity in 3 months if adjusted to 2 0 ~ glycerol and stored at - - 8 °. Polyacrylamide Disc Gel Electrophoresis. Gel solutions are made to 100 ml with deionized water and contain the following:
[59]
LOBSTER MUSCLE TRANSPEPTIDASE
A: Acrylamide
N,N'-MethylenebisaerylamJde B: Tris (hydroxymethylaminomethane) 1 N Hydrochloric acid
N,N,N',N'-TetramethylethylenediamJne C: Ammonium persulfate
769
30 0.8 18.15 24 0.24 0.14
g g g ml ml g
Gels 6 mm diameter X 60 mm are poured from a solution containing one part each of A and B and two parts of C. They are allowed to polymerize for 40 minutes at room temperature, and are then equilibrated with buffer III (28.8 g Tris, 6.0 g glycine in a total of 1 liter, pH 9.3) for at least 20 minutes at a current of 2.5 mA per gel. Samples are layered onto the gels, and a current of 2.5 mA per gel is applied for 80 minutes. Gels axe then stained in 1% amido black in 7% acetic acid for 30 minutes, and destained electrophoretically in 7% acetic acid. Protein concentration is estimated at 0D28o using bovine serum albumin as a standard. Physical Properties
Isoelectric Point. A gradient spanning pH 4 to 7 is established in the 110 ml LKB electrofocusing column, with the less dense solution in some of the middle fractions replaced by the concentrated DEAE pool. The voltage is raised stepwise from an initial 300 V to a final 700 V, and the column run for a total of 43 hours at 30-4 °. The resulting gradient is collected in 2-ml fractions for the determination of pH and clotting activity. The isoelectric point, as indicated by the peak in clotting activity, is 5.9. Molecular Weight. A linear sucrose gradient, 10% to 35% in 4.5 ml, is established in a 1/~ X 2 inch cellulose nitrate tube using a two-chamber Buchler mixer loaded with 2.6 ml of 8% sucrose in buffer I, and 2.4 ml of 40% sucrose in buffer I. The gradients are cooled to 4 °, and a mixture of 0.26 ml concentrated DEAE pool, 10)~ beef liver catalase [Mann, 16 mg/ml in 0.05 M Tris-chloride, pH 7.5 (buffer II) ], and 10X rabbit muscle lactic dehydrogenase (Calbiochem, 5 mg/ml in 2.2 M ammonium sulfate) is layered on top. The gradient is spun at 40,000 rpm )< 13 hours in the Beckman SW 50L rotor, and eluted in 28 fractions of 13 drops (approx. 0.17 ml) each. Clotting is assayed as above except that 0.05 ml enzyme fraction and 0.05 ml buffer I are substituted for the usual 0.1 ml enzyme. Catalase is assayed by the loss of 0D2,o of 2.0 ml of a 0.06~o solution of hydrogen peroxide in buffer II, in 6 minutes following the addition of 10X of gradient fraction. Lactic dehydrogenase is assayed by the loss of 0D34o in the minute following the addition of 0.05 ml of sodium pyruvate (2.5 mg/ml in buffer II) to a solution of 2.0 ml of buffer
770
TRANSPEPTIDASES
[59a]
II, 0.05 ml of reduced fl-diphosphopyridine nucleotide (1.25 mg/ml in buffer I I ) , and 10X of gradient fraction. For the further purification of tl~e concentrated D E A E pool by sucrose gradient sedimentation, 0.3 ml of the pool is substituted for the enzyme mix in the above procedure. A comparison of the distance migrated by lobster muscle transpeptidase and each of the other enzymes yields a sedimentation coefficient of about 10 and a molecular weight of about 200,000 assuming that all three proteins are spherical and have the same partial specific volume. Ester Hydrolysis Lobster muscle transpeptidase will catalyze the hydrolysis of p-nitrophenyl acetate in a mixture containing 0.2 ml of buffer I, 0.1 ml of 0 . 2 M calcium chloride, 0.4 ml of enzyme solution, and 0.1 ml of a 1 m M solution of the ester in acetone. Reactions are initiated by the addition of enzyme, and the increase in nitrophenol is followed at 400 nm. The reaction is inhibited by a 1-hour preineubation of the enzyme with iodoacetamide or iodoacetate at a concentration of 1 raM.
[59a]
Fibrinoligase 1
T h e Fibrin Stabilizing F a c t o r System B y L. LOm_~D and T. GOTOH
Introduction Fibrinoligase is a physiological transpeptidating (i.e., transamidating) enzyme TM which catalyzes the formation of selective ~,-glutamyl-~-lysine cross links between fibrin molecules. 2-T In terms of the (aflv)2 threechain fibrin structure 8,~ (a and fl contain N-termini of glycine, representThis work was aided by a USPHS Research Career Award and by grants from the National Institutes of Health (HE-02212) and the American Heart Association. ~l L. Lorand, K. Konishi, and A. Jacobsen, Nature 194, 1148 (1962). L. Lorand and H. H. Ong, Biochem. Biophys. Res. Commun. 23, 188 (1966). L. Lorand, H. H. Ong, B. Lipinstd, N. G. Rule, J. Downey, and A. Jacobsen, B/ochem. Biophys. Res. Commun. 25, 629 (1966). ' L. Lorand, J. Downey, T. :Gotoh, A. Jacobsen, and S. Tokum, Bioehem. Biophys. Res. Commun. 31, 222 (1968). L. Lorand, N. G. Rule, H. H. Ong, R. Furlanetto, A. Jacobsen, J. Downey, N. Offer, and J. Bruner-Lorand, Biochemislry 7, 1214 (1968). e S. Matacic and A. G. Loewy, Biochem. Biophys. Res. Commun. 30, 356 (1968). ~J. J. Pisano, J. S. Finlayson, and M. P. Peyton, ~cience 160, 892 (1968). 8L. Lorand and W. R. Middlebrook, Biochem. J. 52, 196 (1952). DB. Blomback and I. Yamashina, Arkiv Kemi, 12, 299 (1958).
770
TRANSPEPTIDASES
[59a]
II, 0.05 ml of reduced fl-diphosphopyridine nucleotide (1.25 mg/ml in buffer I I ) , and 10X of gradient fraction. For the further purification of tl~e concentrated D E A E pool by sucrose gradient sedimentation, 0.3 ml of the pool is substituted for the enzyme mix in the above procedure. A comparison of the distance migrated by lobster muscle transpeptidase and each of the other enzymes yields a sedimentation coefficient of about 10 and a molecular weight of about 200,000 assuming that all three proteins are spherical and have the same partial specific volume. Ester Hydrolysis Lobster muscle transpeptidase will catalyze the hydrolysis of p-nitrophenyl acetate in a mixture containing 0.2 ml of buffer I, 0.1 ml of 0 . 2 M calcium chloride, 0.4 ml of enzyme solution, and 0.1 ml of a 1 m M solution of the ester in acetone. Reactions are initiated by the addition of enzyme, and the increase in nitrophenol is followed at 400 nm. The reaction is inhibited by a 1-hour preineubation of the enzyme with iodoacetamide or iodoacetate at a concentration of 1 raM.
[59a]
Fibrinoligase 1
T h e Fibrin Stabilizing F a c t o r System B y L. LOm_~D and T. GOTOH
Introduction Fibrinoligase is a physiological transpeptidating (i.e., transamidating) enzyme TM which catalyzes the formation of selective ~,-glutamyl-~-lysine cross links between fibrin molecules. 2-T In terms of the (aflv)2 threechain fibrin structure 8,~ (a and fl contain N-termini of glycine, representThis work was aided by a USPHS Research Career Award and by grants from the National Institutes of Health (HE-02212) and the American Heart Association. ~l L. Lorand, K. Konishi, and A. Jacobsen, Nature 194, 1148 (1962). L. Lorand and H. H. Ong, Biochem. Biophys. Res. Commun. 23, 188 (1966). L. Lorand, H. H. Ong, B. Lipinstd, N. G. Rule, J. Downey, and A. Jacobsen, B/ochem. Biophys. Res. Commun. 25, 629 (1966). ' L. Lorand, J. Downey, T. :Gotoh, A. Jacobsen, and S. Tokum, Bioehem. Biophys. Res. Commun. 31, 222 (1968). L. Lorand, N. G. Rule, H. H. Ong, R. Furlanetto, A. Jacobsen, J. Downey, N. Offer, and J. Bruner-Lorand, Biochemislry 7, 1214 (1968). e S. Matacic and A. G. Loewy, Biochem. Biophys. Res. Commun. 30, 356 (1968). ~J. J. Pisano, J. S. Finlayson, and M. P. Peyton, ~cience 160, 892 (1968). 8L. Lorand and W. R. Middlebrook, Biochem. J. 52, 196 (1952). DB. Blomback and I. Yamashina, Arkiv Kemi, 12, 299 (1958).
[59a]
FIBRINOLIeAS~.
771
ing the end groups created by the hydrolytic removal of fibrinopeptides A and B from fibrinogen, ~, contains an N-terminal tyrosine), crosslinking consists essentially of the pairing of a and ~, chains, s,1°-18 The fl chains do not seem to be directly involved. The cross-linking reaction constitutes the last step in the enzymatic events of normal blood clotting. The enzyme itself appears only in a transient manner during the course of coagulation; no doubt, the continued functioning in the circulation of such an enzyme, which could secondarily act on proteins other than fibrin, cannot be safely tolerated. Normally only the zymogen form [fibrinoligase precursor; fibrin stabilizing factor (FSF) or, in relation to other coagulation components, also called factor X I I I 1~] is present in plasma. This constitutes a significant difference which distinguishes fibrinoligase from two other related and somewhat interchangeable enzymes,15 liver transglutaminase (Vol. XVIIA [127]) and the muscle transpeptidase (this volume [59]), both of which occur as fully active enzymes in their physiological states. In blood, conversion of fibrinoligase precursor to the active enzyme is brought about by a limited proteolysis with thrombin. 4,16,17 T'hus, the latter hydrolytic enzyme (it itself being only transiently produced in the cascading appearance of enzymes during coagulation) exercises a unique control function not only over the rate of production of the fibrin substrata which is to be cross-linked but also over the timing of the appearance of the enzyme which catalyzes cross-linking. Coordination of these biosynthetic steps, which represent the final enzymatic phase of clotting in vertebrates, can be summarized as follows: n fibrinopeptides (A + B) n fibrinogen J / : n fibrin " (fibrin), gel Thrombin 1% CICH2COOH Ca2+ fibrin stabilizing factor • l~ibrinoligase (FSF or factor XIII)
Ca- ~
" Amines inhibit cross-linked (fibrin), gel 1~L. Lorand and D. Chenoweth, Proc. Natl. Acad. Sci. U.S. 63, 1247 (1969). L. Lorand, D. Chenoweth, and R. A. Domanik, Biochem. Biophys. Res. Commun. 37, 219 (1969). R. Chen and R. F. Doolittle, Proc. Natl. Acad. Sci. U.S. 63, 420 (1969). ~aT. Takagi and S. Iwanaga, Biochem. Biophys. Res. Commun. 38, 129 (1970). ~4Thromb. Diath. Haemorrhag. Suppl. 13, p. 428 (1963).
772
TRANSPEFTIDASES
[59 a]
We have succeeded in separating the thrombin-catalyzed conversion thrombin
fbrin stabilizing factor Ca-~fibrinoligase
(1)
f
of the zymogen, from the later cross-linking reaction which takes place between fibrin (not fibrinogen !) and fibrinoligase: (fibrin)~ gel
fibrinoligase
=
cross-linked (fibrin), gel
(2)
Cal+
soluble in 1% C1CH=COOH
insoluble in 1% CICH2COOH
Both of these steps may now be studied individually in purified systems. Reaction (2) serves as the basis of bioassays for fibrinoligase activity. The "cross-linked" fibrin gel is characterized by a greatly increased elastic modulus is and it is also very much more resistant both to lytic enzymes 19-22 and to dispersion by added solutes, such as weak acids and bases23; urea24,25 or some neutral salts. 26,2~ Most routine assays make use of the latter property and 1% monochloroacetie acid proved to be the best choice ~s for the purpose. The "extent of cross-linking" of a given amount of fibrin (4-5 mg is used in most tests) is estimated by solubilization of the clot in monochloroacetic acid, following a fixed cross-linking period with (varying dilutions of) the enzyme. Complete or partial solubilization of the clot can be seen with the naked eye or m a y be readily quantitated either by measuring the protein content of the acid-insoluble clot residue or that of the acid-solubilized supernatant. T h e enzyme required to convert half of the fibrin substrate into a monochloroacetic acid-insoluble gel form is defined as possessing "one cross-linking unit = L. Lorand, J. Bruner-Lorand, and T. Urayama, Bioehem. Biophys. Res. Commun. 23, 828 (1966). 16L. Lorand and K. Konishi, Arch. Biochem. Biophys. 105, 58 (1964). 17K. Konishi and L. Lorand, Biochim. Biophys. Acta 121, 177 (1966). =W. Roberts, L. Mockros, and L. Lorand, Proc. Intern. Con]. Med. Biol. Eng. 8th No. 27-1 (1969). ~'L. Lorand and A. Jacobsen, Nature 195, 911 (1962). ~oL. Lorand, J. Bruner-Lorand, and T. R. E. Pilkington, Nature 210, 1273 (1966). 21L. Lorand, in "Dynamics of Thrombus Formation and Dissolution" (S. A. Johnson and M. M. Guest, eds.), pp. 212-244. Lippincott, Philadelphia, 1969. 1=L. Lorand, Thromb. Diath. Haemorrhag., Suppl. XXXIX, p. 75, 1970. ~ K. C. Robbins, Am. J. Physiol. 142, 581 (1944). 2~L. Lorand, Hung. Acta Physiol. 1, 192 (1948). ~L. Lomnd and K. Laki, Science 108, 280 (1948). S. Shulman, S. Katz, and J. D. Ferry, J. Gen. Physiol. 36, 759 (1953). ~7T. H. Donnelly, M. Laskowski, Jr., N. Notley, and H. A. Scheraga, Arch. Biochem. Biophys. 56, 369 (1955). L. Lorand, Nature 166, 694 (1950).
[59a]
FIBRINOLIGASE
773
(XLU)" of activity. 2sa Perhaps it should be pointed out that "crosslinking," as judged by the criterion of clot solubility has a rather complex meaning and that, quantification and reproducibility notwithstanding, bioassays based on this property must be regarded as empirical at best. A fibrin clot appears to be totally insoluble in monochloroaeetic acid when only about 60% of the protein is "cross-linked" and the rest is still monomeric. 4, 22 Furthermore, the cross-linked portion itself consists of a spectrum of olignmeric clusters of fibrin, in which the dimeric species predominates. Nevertheless, the clot solubility test proved to be of great use, particularly in the evaluation of specific inhibitors of fi'brin cross-linking? We have also developed (and are still in the process of developing) various rate assays for measuring the activity of fibrinoligase. These fall into two categories: (a) those based on the hydrolytic activity; and (b) those on the transpeptidating property of the enzyme, p-Nitrophenyl esters (e.g., acetate or trimethylacetate) have been used as hydrolytic substrates29; obviously, however, measurements have significance only in conjunction with the enzyme in its highest purity. The transpeptidating reaction utilizes synthetic amines of various types which were shown to be specific inhibitors in the enzyme-catalyzed cross-linking of fibrin and were also shown to act so by virtue of becoming incorporated into the acceptor function of fibrin itself. Various types of amine tracers could be used: hydroxylamine, hydrazine 8° as chemical markers; glyeine ethyl ester, ~°,31 histamine as radioisotopes82; N-(5-aminopentyl)-5-dimethylaminonaphthalene-l-sulfonamide (or "monodansylcadaverine") as a fluorescent label2 ,5 On account of the favorable Km, app (ca. 2 X 10-4 M) of the latter substrate, and because of the great sensitivity and the ease of fluorescent measurements, most quantitative data so far are based on the incorporation of monodansylcadaverine. This is particularly so in regard to the clinical diagnostic tests in plasma relating to the hereditary disease of fibrin stabilizing factor deficiency2 ~,3~The scope of the test for other clinical applications is being enlarged. "~ L. Lorand, in "Blood Coagulation, Hemorrhage and Thrombosis, Methods of Study" (L. M. Tocantins and L. A. Kazal, eds.), p. 239. Grune & Stratton, New York, 1964: ~'O. Lockridge, Ph.D. dissertation, Northwestern University, Evanston, Illinois, 1970. "L. Lorand and H. H. Ong, Biochemistry 5, 1747 (1966). L. Lorand and A. Jacobsen, Biochemistry 3, 1939 (1964). s~L. Lorand, T. Urayama, J. deKiewiet, and H. A. Nossel, J. Clin. Invest. 48, 1054 (1969). "L. Lorand, T. Urayama, A. C. Atencio, and D. Y. Y. Hsia, Am. J. Human Genetics o.2~89, 1970.
774
TRANSPEPTIDASES
[598]
Both fibrin and casein may be used as amine aeceptors; the test devised for clinical application makes use of casein22,8s Interestingly, fibrinogen is a much poorer acceptor substrate than fibrin. The hydrolytic removal of the small fibrinopeptide A fragment is the prerequisite for unmasking the incorporating sites of the protein?, 21,~2,so Assays for the precursor (i.e., fibrin stabilizing factor) are the same as for the ligase, except that the thrombin-catalyzed conversion step (reaction l) has to be carried out prior to measuring the generated enzyme activity either by the fibrin cross-linking, ester hydrolysis or the amine incorporation test. In the present description, two different methods are given for the assay of the zymogen. Its potency is expressed in the same units [i.e., "cross-linking units" (XLU) and "amine incorporating units" (AIU) ] as used for the fibrinoligase enzyme itself. If the latter alone is to be assayed, the activation step with thrombin is simply omitted. Assay Methods Bioassay, Based on Clot Solubility,/or the Fibrin Stabilizing Factor
The fibrin substrate is prepared essentially as given 'by Donnelly et al., 2~ an adaptation of the method of Lorand and Middlebrook2 The starting fibrinogen may be of bovine (e.g., Armour fraction I precipitated with 0.25 saturated ammonium sulfate, as prescribed by LakP') or of human origin (e.g., Kabi). A 0.1.% solution of fibrinogen in 0.05 M Tris0.1 M NaC1 solution at pH 7.5 (with HC1) is allowed to stand with 1 mM iodoacetic acid, and clotting is initiated by the addition of thrombin (ca. 20 NIH units per 100 ml of solution). The fibrin clot formed over a 30-minute period is collected on a glass rod, gently squeezed of excess liquid and is taken up in 1/30 of the volume of the original fi'brinogen in 2 M NaBr adjusted to pH 5.3 with acetic acid. When fully dissolved, water is added to lower the NaBr concentration to 1 M. The pro.tein solution is then poured into 20 times its volume of a phosphate buffer of pH 6.5 (0.03 M KH2PO,, 0.015 M Na2HP0,, and 0.09 M KC1) where the change in pH and ionic strength causes it to precipitate: The fibrin precipitating over a 2-hour period is wound out on a glass rod as before and is dissolved in 1/50 of the volume of starting fibrinogen in the 2 M NaBr (pH 5.3) solvent. It is then dialyzed overnight in the cold ( ~ 4 °) against 1 M NaBr of pH 5.3. (All the earlier steps are carried out at room temperature.) Any precipitate present after dialysis should be removed by centrifugation. The protein concentration is adjusted by dilution with the 1 M NaBr to about 1.5~ (absorbancy at 280 nm for u K. Laki, Arch,. Biochem.
32, 317' (1951).
[59a]
FIBRINOLIGASE
775
1% protein is 15.12~). The fibrin solution may be stored at 0 °. We arbitrarily set an expiration date of about 3 weeks. Different (chromatographically purified) thrombin preparations (bovine or human 36) may be used for preparing fibrin (as above) as well as for activating the fibrin stabilizing factor in the test. Thrombin is diluted to the required strength with the 0.05 M Tris-0.1 M NaC1 buffer of pH 7.5. (For definition of N I H units of thrombin activity see this volume, [8], [9], and [9a].) Cysteine and tosylarginine methyl ester (TAME) are each freshly made up to 0.1 M in the 0.05 M Tris-0.1 M NaC1 and the pH of the solutions is adjusted to pH 7.5 (with a few drops of 1 N NaOH if necessary). One millimolar aqueous CaCl~ is used. Purification of the fibrin stabilizing factor zymogen is described separately. It is made up in the Tris-NaC1 solution of pH 7.5. Monochloroacetic acid is prepared as a 2% aqueous solution. In its most useful version the multistage assay consists of the following main steps: (1) Activation of the zymogen by thrombin, in the presence of calcium and cysteine. (2) Quenching of thrombin by the addition of the competing substrate, TAME. (For less precise measurements, this step is often omitted.) (3) Cross-linking of fibrin by the enzyme generated in step (1). (4) Testing of the solubility of the fibrin, after the cross-linking period, in 1% monochloroacetie acid. A set of mixtures, containing various amounts of the fibrin stabilizing factor, is prepared in 18-mm wide test tubes at room temperature. Into each tube is pipetted 0.5 ml of CaCl~, 0.5 ml of cysteine, 0.6 ml of the fibrin stabilizing factor, and 0.1 ml of thrombin. After 10 minutes of activation, 0.5 ml of TAME is added and is followed by the admixing of 0.3 ml of fibrin solution, whereupon uniform gelation ensues. Half an hour later, 2.5 ml of 2% monochloroacetic acid is introduced in order to test the solubility of the fibrin clot. 28,2Sa Swirling of the tubes aids the penetration of the acid; however, breaking up of the clot is to be avoided if the extent of solubility is to be judged visually. Various concentrations of fibrin stabilizing factor are selected such that the test tubes would yield a range of clots from the fully soluble to the completely monochloroacetic acid-insoluble type. The "threshold" activity producing half an acid-insoluble clot represents 1 cross-linking unit (XLU). P. Johnson and E. Mihalyi~ Biochim. Biophys. Acta 102~ 476 (1965). a, p. S. Rasmussen, Biochim. Biophys. Acta 16, 157 (1955).
776
TRANSPEPTIDASES
[59a]
Tubes for residual clots are read 2 hours after the addition of monochloroaeetic acid and are checked again some 16 hours later. If quantitation is required, the remaining clot residues are sedimented by centrifugation. Optical density of the supernatants may be read at 280 nm and expressed in terms of either fibrin or standardized crystalline bovine serum albumin quantities. Alternately, the sediments are washed twice with 0.15 M NaC1, centrifuged, and digested in NaOH for colorimetric measurement by the Folin-Ciocalteau ~7 method.
Amine Incorporation Test ]or the Fibrin Stabilizing Factor An outline of the coupled rate assay may be given as: fibrin stabilizing factor (factor XIII) Ca2+ 1 Thrombin casein -{- H~.NR
fibrlnoligase ~ casein-NHR ~ (H) Ca2+
As mentioned above, the amine (H2NR) in the particular test to be described was chosen as the fluorescent monodansylcadaverine. When concentrations of Ca 2+, thrombin, casein, and that of the amine are optimized together with pH and time of conversion of the factor to the ligase enzyme, the rate of formation of casein-bound amine reflects the original concentration of fibrin stabilizing factor. If the activity of the preactivated fibrinoligase is to be measured, the simplified casein ~ H~NR
fibrinoligase , casein-NHR -~ (H) Ca2+
system is applicable. For highly purified fibrin stabilizing factor and fibrinoligase preparations, the continuous rate assay (described in this volume, [59]) may be used to good advantage. However, for measuring the fibrin stabilizing factor content of biological fluids, such as human plasma, a sampling procedure has been worked out with the following details22 Desensitization o] the Test Plasma. (This step is unnecessary when testing purified fibrin stabilizing factor.) Mix 0.2 ml of citrated plasma with 0.05 ml of 50~ (v/v) aqueous glycerol in a 16 X 100 mm (Sorvall Pyrex) test tube. Bring rapidly to 56 ° and keep at this temperature for 2.5 minutes. Then by immersion in ice, allow it to cool to room temperature (24°). Activation o] Fibrin Stabilizing Factor with Thrombin. Add 0.05 ml of 0.2 M glutathione solution [dissolved in 50~ (v/v) aqueous glycerol ~TA. Hirsch and C. Cattaneo, Z. Physiol. Chem. 304, 53 (1956).
[59a]
FIBRINOLIGASE
777
and adjusted to pH 7.5 with 3 N N a 0 H ] . Activation of factor X I I I is initiated by admixing 0.2 ml of thrombin [125 N I H units/ml; dissolved in 25% (v/v) aqueous glycerol and 0.02M CaC12; pH 7.5] and it is allowed to proceed for 20 minutes. Amine Incorporation. Add 0.5 ml of the synthetic amine substrate, 2 mM monodansylcadaverine ~ solution in 0.05 M Tris-HC1 of pH 7.5 and 3 mM CaC12. The incoporation reaction is started with the addition of 1.0 ml of 0.4% casein solution; made up in a mixture containing 10% glycerol, 3 mM CaCl~, 0.05 M Tris-HC1 at pH 7.5 [in preparing the casein (Hammarsten) solution, it is clarified after dialysis by centrifuging at 20,000 rpm for 60 minutes in a Spinco L No. 30 rotor]. Incorporation Measurement. Amine incorporation is stopped 30 minutes later by adding 2 ml of 10% trichloroacetic acid. The protein precipitate is washed successively with 6 X 10 ml of ethanol-ether (1:1) and dried. Finally, it is taken up in a 2 ml solution of 8 M urea, 0.5% sodium dodecylsulfate in 0.05 M Tris-HC1 at pH 8, for measuring the protein-bound fluorescent amine. Fluorescent intensities are obtained with excitation at approximately 355 nm; emission at approximately 525 nm. All fluorescence measurements are carried out in the mixture containing 8 M urea and 0.5% sodium dodecyl sulfate in 0.05M Tris buffer at pH 8. Monodansylcadaverine at a concentration of 1 micromole/liter in the same solvent system serves a~ reference. A given rate of amine incorporation is conveniently expressed in terms of units. It is of further advantage to normalize these unit values for 1 ml of citrated plasma. Using 0.2 ml of citrated plasma in a 2-ml test mixture, units are defined in the following manner: AIU per ml of eitrated plasma = 0-2 X ~ X
L ~ J
L~R - -
~sJ
The first term on the left accounts for the 10-fold dilution of plasma in the test; the second simply serves to reduce the numerical values into a convenient range and may be considered to express units in terms of a 10-minute incorporation instead of the measured 30 minutes; the third is to correct for the estimated 10% loss of factor during heat desensitization plasma. If units are to be expressed for original plasma and not the citrated one, another correction for dilution wth the anticoagulant should be included. The symbols within the brackets represent the measured fluorescent intensities (i) of the test sample (T) after 30 minutes of amine incorpo-
778
TRANSPEPTIDASES
[59a]
ration; of the blank (B) control mixture with only the monodansylcadaverine missing, but otherwise similarly treated; of a 1 t~M reference (R) solution of free monodansylcadaverine and finally of the mixed (urea-sodium dodecyl sulfate) solvent (S) system itself. Purification Procedure Fibrin Stabilizing Factor (Factor X I I I ) ]rom Plasma
Plasma, rather than serum serves as the source for isolating the fibrin stabilizing factor zymogen28, 89 (Again, it should be recalled that no trace of the active fibrinoligase enzyme is present in normal plasma.) Both oxalated bovine plasma (Pentex, Kankakee, Ill.) or citrated human plasma from a blood bank can be readily used as starting materials. The factor appears to be quite stable in freshly frozen plasma over extended periods (months) of storage. Purification procedures consist of two phases: (1) preparation of crude concentrates by one (or a combination) or several precipitation steps; and (2) chromatography on DEAE-cellulose. Precipitation procedures are based on the use of ether, 38,3~ alcohol,~° ammonium sulfate, '°,~1 polyethyleneglycoW or isoelectrie fractionation at pH 5.4. 88-40 Since the factor combines readily with fibrinogen,~s a mild heat treatment was developed~1 for the differential precipitation of the latter protein. This does not seem to affect the potency of the factor; however, it is not known whether its fine structure is modified. In any case, it is a useful step in preparing concentrates. The following is a well-proven procedure for production on a laboratory scale: To 8 liters of thawed plasma, 2 liters of saturated ammonium sulfate solution is added (4 °) and the precipitate formed in an huur is removed by centrifugation (International head No. 858; 8000 rpm X 20 minutes, 0°). The sediment is taken up in 0.15 M KC1 and the 1250 ml solution is adjusted to pH 5.4 with 1 N acetic acid. Then 240 ml of saturated ammonium sulfate is added again and the precipitate is removed as before. It is dissolved in 0.05 M Tris-0.1 M NaC1 buffer, with the total volume being 450 ml and the pH of the solution set to 7. (This concentrate may be stored for several days in the cold room which is particularly convemL. Lorand, Doetoral thesis, Leeds University, England, 1951. L. Lorand, Physiol. Rev. 34, 742 (1954). o L. Lorand and A. Jacobsen, J. Biol. Chem. 230, 421 (1958). ~IA. G. Loewy, C. Veneziale, and M. Forman, Biochim. Biophy8. Acta 26, 670 (1957). d M. Kazama and R. D. Langdell,Federation Proc. 28, 746, No. 2725 (1969). a L. Lorand, in "Anticoagulants and Fibrinolysins" (R. L. MacMillan and J. F. Mustard, eds.), p. 333. Macmillan,Canada, 1961.
[Sga]
FIBRINOLIGASE
779
nient if several batches of the above preparation are to be pooled for chromatography.) The solution is diluted 2-fold with the 0.05 M Tris-0.1 M NaC1 buffer and (in 225-ml aliquots) it is heated to 56 ° for 3 minutes (by rapid immersion in an 80 ° water bath). While at 56 °, the denatured fibrinogen is removed with the aid of a glass rod and the supernatant is squeezed out as completely as possible and is cooled to 15° . Small floating fragments are removed by centrifugation. The supernatant (ca. 460 ml) is brought to 4 ° and is mixed with 260 ml of saturated ammonium sulfate. The precipitate is removed by centrifuging, as in earlier steps, and it is dissolved by adding 40 ml of an 0.05 M Tris--0.001 M EDTA buffer set to pH 7.5 with HC1. The 42-ml solution is dialyzed against the buffer (2 X 1 liter) overnight in the cold room. Fibrin Stabilizing Factor Zymogen on DEAE-Cellulose
Both stepwise ~4 and gradient elution~,43 procedures have been reported. On account of the good reproducibility of eluting fibrin s~*abilizing factor with a salt gradient at pH 7.5 and because of our extensive experience with this methodology over the past 5 years, only this technique is recommended. DEAE-eellulose (Whatman DE-52, mierogranular, preswollen) is equilibrated with 0.05 M Tris-0.001 M EDTA set to pH 7.5 with HC1 and made into a column of approximately 3 X 24 em. Washing with the buffer is stopped when the pH and conductivity of the effluent is identical to that of the original. The chromatographic operations are carried out at 4 °. Flow rate is adjusted to about 32 ml per hour and approximately 8-ml fractions are to be collected. The fibrin stabilizing factor concentrate isolated earlier is then applied. Protein contents of these crude preparations vary appreciably. As much as 400 mg in 80 ml can be applied. (For larger quantities, we have successfully used a 4 X 60 cm column.) About 500 ml of buffer is allowed to pass so as to elute the protein impurities not retained by the DEAE-cellulose. Then a linear gradient is applied with the proximal chamber containing 600 ml of the 0.05M Tris-0.001 M EDTA at pH 7.5 and the distal one the same volume of 0.3 N NaC1 in the 0.05 M Tris-0.001 M EDTA buffer. (With the larger column 1300 ml solution is placed in each mixing chamber.) Figure 1 shows an elution pattern which is typical only in the position of the fibrin stabilizing factor activity on the gradient. The size of the impurities emerging before the gradient is applied as well as those eluting at higher salt concentration varies appreciably from one preparation to ~ A. G. Loewy, K. Dunathan, R. Kriel, and H. L. Wolfinger,Jr., J. Biol. Chem. 236, 2625 (1961).
780
TRANSPEPTIDASES
[Sga]
another. The active peak is pooled and concentrated with 0.36 saturation of ammonium sulfate. The precipitate is dissolved in minimal (2-3 ml) volume of 0.05 M Tris-0.1 M NaC1-0.001 M EDTA, pH 7.5 buffer (with HC1) and is dialyzed against the same. The solution is stored at w l 0 ° after adding glycerol (20%).
Zonal Centri]ugation and Disc Gel Electrophoresis o] Fibrin Stabilizing Factor Routinely, sucrose-density centrifugation 4,22 of the ehromatographed material (Fig. 2) reveals only minor impurities which sediment slower than the fibrin stabilizing factor activity. Tentatively these are identified as byproducts of the spontaneous activation of the factor to fibrinoligase. The latter sediments indistinguishably from the zymogen and does occasionally arise to some extent. The fractions with uniform specific activity obtained by zonal centrifugation give a single band in disc gel electrophoresis, ~ unless some of the spontaneously activated fibrinoligase is present. Sucrose density centrifugation proved to be so useful that it is often used as a final preparative step of purifying fibrin stabilizing factor. A linear gradient (4.5 ml) to 40% sucrose is formed in 0.05 M Tris-0.1 M NaC1-0.001 M EDTA buffer at pH 7.5 and 0.5 ml of chromatographed fibrin stabilizing factor is applied. Centrifugation (Spinco, SW 50 or 65 rotors) is carried out at 44,000 rpm for 18.5 hours at 0 °. Properties of Fibrin Stabilizing Factor In both chromatographic behavior on DEAE-eellulose and in sedimentation properties, the zymogen as well as the fibrinoligase enzyme itself are strikingly different from liver transglutaminase, an enzyme of related activity in m a n y respects.15 Transglutaminase elutes at much higher salt concentration ~5 and sediments slower in sucrose density centrifugation.29 With catalase and lactic dehydrogenase, fibrin stabilizing factor (and fibrinoligase, too), sediments between the two markers, whereas transglutaminase is considerably slower than lactic dehydrogenase. 22,s9 In chromatography and sedimentation, fibrinoligase and its precursor show great similarity, however, with the transpeptidase (see this volume [59]) isolated from lobster muscle. It must be recalled that the latter is not known to occur in the form of a zymogen, but only as the active enzyme. There are a number of kinetic dissimilarities29 among the three transpeptidating enzymes mentioned.
~J. E. Folk and P. W. Cole, J. Biol. Chem. 241, 5518 (1966).
[59a]
FIBRINOLIGASE l
T
"T"
781
T
T
0.20
1,0
9Z ,°~0. 5
250
500
750 IO00 Effluent volume,ml
1250
Fro. 1. Salt gradient-elution chromatography of fibrin stabilizing factor (bovine or human) from DEAE-cellulose. - - , Absorbancy at 280 nm; . . . . fibrin stabilizing factor activity in terms of cross-linklng units (XLU); ~ i , conductivity or NaCI concentration.
1.4
700 I-HI
A 1.2
600
i"
"7
sooi
1.0 g
08
..
g o
06 u.
04 (3_
f ,oo
02 Bottom
5
iO
15
20
25
Top
Fraction number
FIa. 2. Sucrose-density centrifugation of chromatographed fibrin stabilizing factor (bovine or human). Activity is given in terms of cross-linking units.
782
TRANSPEPTIDASES
[60]
Conversion of the Fibrin Stabilizing Factor Zymogen to the Fibrinoligase Enzyme Thrombin, as a hydrolytic enzyme, serves the purpose of physiologically converting the zymogen into the active transpeptidaseJ, le Disc gel electrophoresis ~ as well as electrofocusing 22 may be used to monitor the conversion and to analyze for fragments which seem to be released as byproducts of activation. The activating thrombin can be removed by gel filtration. ~ Trypsin ~e and reptilase 47 also seem capable of converting the zymogen to active fibrinoligase. Solid-phase enzymes such as water-insoluble trypsin have been used. 4,4~ 46K. Konishi and T. Takagi, Abstr. 7th Intern. Uongr. Biochem., Tokyo, 1967, J. 381. 4~M. Kopec, Z. S. Latallo, M. Stahl, and Z. Wegrzynowicz, Biochim. Biophys. Acta 181, 437 (1969).
[6o]
y-Glutamyl
Transpeptidase
By G. E. CONNELL and E. D. ADAMSON Glutathione + gly-gly --~ ~-glu-gly-gly + cySH-gly 2 Glutathione --* ~,-glu-~-glu-cySH-gly + cySH-gly Glutathione + H20 --* glutamic acid + cySH-gly
~-Glutamyl transpeptidase from most sources catalyzes the transfer of the T-glutamyl group from glutamine or a variety of substituted ~,amides of glutamic acid to many primary amines, most notably, the a-amino acids and small peptides. 1,2 ~-Glutamyl amides and peptides may be both acceptors and donors in the reaction: Assay
Principle. The method described below is a rapid spectrophotometric method devised by Orlowski and Meister2 ,~ When a preparation of ~glutamyl transpeptidase is incubated with T-L-glutamyl-p-nitroanilide, p-nitroaniline is liberated. The colorless substrate exhibits a maximum absorbancy at 315 nm and no absorbancy at 410 nm, while p-nitroaniline exhibits high absorbancy in the range 350 to 420 nm. The release of pnitroaniline in reaction mixtures may be followed directly by continuous photometric recording and is linear for several minutes at moderate enzyme concentrations. 1C. S. Hanes, F. J. R. Hird, and F. A. Isherwood, Nature 166, 288 (1950). i C. S. Hanes, F. J. It. Hird, and F. A. Isherwood, Biochem. J. 51, 25 (1952). s M. Orlowski and A. Meister, Biochlm. Biophys. Acta 73, 679 (1963). • M. Orlowsld and A. Meister, J. Biol. Chem. 240, 338 (1965).
782
TRANSPEPTIDASES
[60]
Conversion of the Fibrin Stabilizing Factor Zymogen to the Fibrinoligase Enzyme Thrombin, as a hydrolytic enzyme, serves the purpose of physiologically converting the zymogen into the active transpeptidaseJ, le Disc gel electrophoresis ~ as well as electrofocusing 22 may be used to monitor the conversion and to analyze for fragments which seem to be released as byproducts of activation. The activating thrombin can be removed by gel filtration. ~ Trypsin ~e and reptilase 47 also seem capable of converting the zymogen to active fibrinoligase. Solid-phase enzymes such as water-insoluble trypsin have been used. 4,4~ 46K. Konishi and T. Takagi, Abstr. 7th Intern. Uongr. Biochem., Tokyo, 1967, J. 381. 4~M. Kopec, Z. S. Latallo, M. Stahl, and Z. Wegrzynowicz, Biochim. Biophys. Acta 181, 437 (1969).
[6o]
y-Glutamyl
Transpeptidase
By G. E. CONNELL and E. D. ADAMSON Glutathione + gly-gly --~ ~-glu-gly-gly + cySH-gly 2 Glutathione --* ~,-glu-~-glu-cySH-gly + cySH-gly Glutathione + H20 --* glutamic acid + cySH-gly
~-Glutamyl transpeptidase from most sources catalyzes the transfer of the T-glutamyl group from glutamine or a variety of substituted ~,amides of glutamic acid to many primary amines, most notably, the a-amino acids and small peptides. 1,2 ~-Glutamyl amides and peptides may be both acceptors and donors in the reaction: Assay
Principle. The method described below is a rapid spectrophotometric method devised by Orlowski and Meister2 ,~ When a preparation of ~glutamyl transpeptidase is incubated with T-L-glutamyl-p-nitroanilide, p-nitroaniline is liberated. The colorless substrate exhibits a maximum absorbancy at 315 nm and no absorbancy at 410 nm, while p-nitroaniline exhibits high absorbancy in the range 350 to 420 nm. The release of pnitroaniline in reaction mixtures may be followed directly by continuous photometric recording and is linear for several minutes at moderate enzyme concentrations. 1C. S. Hanes, F. J. R. Hird, and F. A. Isherwood, Nature 166, 288 (1950). i C. S. Hanes, F. J. It. Hird, and F. A. Isherwood, Biochem. J. 51, 25 (1952). s M. Orlowski and A. Meister, Biochlm. Biophys. Acta 73, 679 (1963). • M. Orlowsld and A. Meister, J. Biol. Chem. 240, 338 (1965).
[60]
~/-GLUTAMYL T R A N S P E P T I D A S E
783
Reagents. T h e substrate is prepared so that 0.90 ml contains 5 micromoles ),-L-glutamyl-p-nitroanilide, 10 micromoles MgCl2, 100 micromoles Tris-HC1 buffer, pH 9.0. The substrate is sparingly soluble, and heating to 50°-60 ° may be required to achieve complete solution. Synthesis o] ~-~-glutamyl-p-nitroanilide. This compound is prepared by a modification of the general procedure of King and KiddS; 13.2 g (0.051 mole) phthaloyl-L-glutamic anhydride6,7 is suspended in 35 ml glacial acetic acid, and 6.9 g (0.05 mole) p-nitroaniline is added. The mixture is heated at 60o-65 ° for 30 minutes and then concentrated under reduced pressure at 25 ° to yield a crude product which is dried in a vacuum desiccator over CaC12 and NaOH. The product is ground to a fine powder and dissolved in 250 ml methanol. Hydrazine hydrate (80%, 0.1 mole) is added, and the solution filtered to remove a small amount of insoluble material. It is allowed to stand 48 hours at 26 ° while a crystalline product separates. The crystals are washed on a Biichner funnel with cold distilled water and with ethanol, then dried in a vacuum over CaCl2. The material is suspended with vigorous agitation in 10 parts of cold 0.5 N HC1 and the mixture filtered into a separatory funnel. Free p-nitroaniline is removed by extraction with 5 volumes of ethyl acetate, after which the aqueous layer is quickly filtered and brought to pH 7 by addition of 1 M Na2COa. A crystalline product forms immediately; this is washed with water and with ethanol and then dried in a vacuum over CaC12. The yield is 8.2 g (61%), m.p. 183°-186 °, c at 315 nm =- 13,000. Procedure. To 0.90 ml buffered substrate solution in a cuvette of 1-cm light path, held at 37 °, 0.10 ml enzyme solution is added with rapid stirring. Using a photometer with or without a recording device, the increase in absorbance (AA) at 410 nm is determined for 3-5 minutes and the average AA per minute calculated. This figure is converted to micromoles of p-nitroaniline/minute using the molar extinction coefficient of p-nitroaniline at 410 nm of 8800 M -1 cm-1. In an alternative procedure, the enzyme and buffered substrate may be incubated for a fixed time, e.g., 3-5 minutes; 2 ml 1.5 N acetic acid is added to terminate the reaction and precipitate the protein. The absorbance is then measured at 410 nm against a reference solution containing the same components, except that the enzyme is added after addition of acetic acid. This procedure may be useful in working with crude enzyme preparations which would give turbid reaction mixtures. Szasz 8 has suggested minor modifications of the p-nitroanilide method for use in clinical
F. E. King and D. A. A. Kidd, J. Chem. Soc. p. 3315 (1949). sj. C. Sheehan and W. A. Bolhofer, J. Am. Chem. Soc. 72, 2469 (1950). F. E. King, J. W. Clark-Lewis, and R. Wade, J. Chem. Soc. p. 886 (1957). s G. Szasz, Clin. Chem. 15, 124 (1969).
784
TRANSP~.PTIDASES
[50]
laboratories. The quantitative aspects of several assay procedures have been compared by Dimov and Kulhanek.' Definition o] Urdt and Specific Activity. One unit of y-glutamyl transpeptidase activity is defined as the amount of enzyme required to release 1 mieromole p-nitroaniline per minute under the conditions of the assay. Specific activity is expressed in units per milligram protein as determined by the method of Lowry et al. 1° Preparation of Enzyme n Beef kidneys (4 kg) free of connective tissue, which have been stored frozen at --15 °, are homogenized in a Waring blendor with 12 liters 0.9% NaC1 solution at 4 °. The homogenate is then centrifuged at 900 g and the supernatant retained. Step 1. Acetone and Sodium Deoxycholate Treatment. The supernatant is treated with half its volume of cold acetone at 4 °, the precipitate spun off at 900 g and washed with 2 volumes 0.9% NaC1 mixed with 1 volume acetone. The precipitate is suspended in 2 volumes 0.9% NaC1 and for each liter of suspension 50 ml 20% sodium deoxycholate solution is added. The mixture is homogenized for 5 minutes and left overnight at 4 °. After eentrifugation at 10,000 g the precipitate is discarded. Step ~. Ammonium Sul]ate Precipitation. To the clear supernatant, 472 g solid ammonium sulfate per liter is added to give 70% saturation. After standing overnight the precipitate is spun off, suspended in a small volume of water, and dialyzed for 48 hours against tap water. The residual precipitate is removed by centrifugation. Step 3. Butanol, Heat, and Acetone Treatment. To the supernatant 0.4 volume cold n-butanol is added slowly over 10 minutes and the mixture is warmed for 15 minutes at 38 °. The biphasic mixture is centrifuged and the lower water layer is collected and cooled. The protein dissolved in the water layer is precipitated by the addition of 1.5 volumes of cold acetone, the precipitate is spun off, suspended in a small volume of water and dialyzed overnight against distilled water. The solution is clarified by centrifugation and the precipitate discarded. Step 4. Second Arfimonium Sulfate Precipitation. The protein which salts out between 52 and 70% saturation in fractionation with solid ammonium sulfate is collected, suspended in a small amount of water, and dialyzed for 48 hours at 4 ° against 0.01 M Tris-citrie acid buffer, pH 8.9. ' D. M. Dimov and V. Kulhanek, Clin. Chim. Acta 16, 271 (1967). O. It. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). A. Szewczukand T. Baranowski, Biochem. Z. 338, 317 (1963).
[60]
~/-GLUTAMYL TRANSPEPTIDASE
785
Step 5. DEAE-Cellulose Treatment. The clear supernatant is applied, at 4 °, to a column (0.9 X 30 cm) of DEAE-cellulose (0.8 meq/g) previously equilibrated with 0.01 M Tris-citric acid, p H 8.~, at a flow rate of 5 ml per hour. The same buffer is used for elution, and the active fractions are combined and dialyzed against 0.01 M sodium acetate buffer, p H 5.5. Step 6. Second DEAE-Cellulose Column. The product of the previous column (55 rag) is applied to a DEAE-cellulose column (1.5 X 30 cm) previously equilibrated with 0.01 M sodium acetate buffer, p H 5.5. The column is eluted with a s tepwise gradient of (a) 0.01 M buffer, (b) 0.1 M buffer, (c) 0 . 2 M buffer, (d) 0.1 M NaC1 in 0 . 2 M buffer, (e) 0 . 2 M NaC1 in 0.2 M buffer, and (f) 0.5 M NaC1 in 0 . 2 M buffer. A flow rate of 30 m l / h o u r is used and fractions of 10 ml are collected. One active fraction of the enzyme A is eluted with 0.2 M buffer and a second active fraction B with 0.1 M NaC1 in 0.2 M buffer. Fraction A has 34% and fraction B has 52% of the activity of the preparation applied to the final column. The properties of the preparations at various stages of purification are summarized in the table. SUMMARY OF PURIFICATION OF ~7-GLUTAMYL TRANSPEPTIDASE
Step 1 2 3 4 5 6
Preparation Centrifuged homogenate Deoxycholate extract 1st Ammonium sulfate precipitation Butanol treatment 2nd Ammonium sulfate precipitation 1st DEAE-cellulose treatment 2nd DE&E-cellulose column Fraction A Fraction B
Volume (ml)
Total protein
Specific activity• (units/mg protein)
11,000 4,700 550
390 g 27.7 g 8.6 g
0.078 0.69 1.55
---
Yield (%) 100 63 44
53 37
1.22 g 470 mg
9.6 17.6
38 27.2
43
77 mg
66.5
16.8
21 mg 31 mg
81.5 85.5
14.3
a The specificactivities quoted in this table are based upon an assay using ~-glutamyla-naphthylamide as substrate rather than -y-L-glutamyl-p-nitroanilide.The values obtained are slightly lower than those which would be obtained using the standard procedure. Properties
Specificity. ~-Glutamyl transpeptidase has absolute specificity with respect to the donor substrate for the glutamyl moiety in v-linkage. It will not, for example, act upon L-asparagine, L-homoglutamine, or the
786
TRANSPEFTIDASES
[50]
p-nitroanilide of fl-aminoglutaric acid2 Among the ~-glutamyl peptides which have been shown to be donor substrates are glutathione, in both the oxidized and reduced form,2 and a number of dipeptides, for example, ~,-glutamylglycine, -phenylalanine, -tyrosine, -glutamic acid, -aspartic acid. Several ~,-giutamylarylamides also are donor substrates, namely, the derivatives of a- and fl-naphthylamine, aniline, and p-nitroaniline. s,1~-14 Glutamine has been shown to be a ~,-glutamyl donor using highly purified transpeptidase preparations from beef 11 or hog~ kidney. Glutamine is not a donor substrate for the sheep kidney enzyme in partly purified preparations. 2,15 Peptides containing D-glutamic acid in v-linkage are substrates but the reaction rates are much lower than with the analogous L-glutamyl peptides. The transpeptidase has broad specificity with respect to the nature of the acceptor amine. All of the naturally occurring L-amino acids which have been tested proved to be active. 2,16-~8 A substantial number of dipeptides and several tripeptides are also known to be active. 2,17 Glutathione itself will act as an aeceptor as well as a donor substrate. 19 Evidence has been obtained for the synthesis by the enzyme of tetra-, penta-, or higher peptides of glutamie acid in ~,-linkage beginning with a simple ~/-glutamyl substrate.4, ~10rlowski and Meister have shown that hydroxylamine is an acceptor substrate of transpeptidase and that the product is the ~,-hydroxamate of glutamic acid. 4 Most preparations of v-glutamyl transpeptidase catalyze hydrolytic as well as transfer reactions. 2,4,16,1~ After prolonged incubation all ~,-glutamyl peptides in a reaction mixture may be hydrolyzed completely to free glutamate. 1~ The pH optimum for the hydrolytic reaction is lower than that for the transfer reaction. The values for the pig kidney enzyme acting on v-glutamyl-p-nitroanilide are approximately pH 8.1 and pH 8.8 for hydrolysis and transfer, respectively. Leibach and Binkley have described a preparation from pig kidney which catalyzes ~,-glutamyl transpeptidation but not hydrolysis.2° The relationship of this enzyme to ~,-glutamyl transpeptidase is not clear; it differs from the transpeptidase with respect to specificity, heat lability, and carbohydrate content. 1~M. Orlowski and A. Szewczuk,Clin. Chim. Acta 7, 755 (1962). G. G. Glenner, J. E. Folk, and P. J. MeMillan, J. Histochem. I0, 481 (1962). 1,j. A. Goldbarg, E. P. Pineda, E. E. Smith, O. M. Friedman, and A. M. Rutenberg, Gastroenterology 44, 127 (1963). G. E. ConneU, Ph.D. Thesis, University of Toronto, Canada, 1955. ~P. J. Fodor, A. Miller, and H. Waelsch, J. Biol. Chem. 202, 551 (1953). 1~p. j. Fodor, A. Miller, and H. Waelsch,J. Biol. Chem. 203, 991 (1953). F. J. R. Hird and P. H. Springell, Biochem. J. 56, 417 (1954). 19j. p. Revel and E. G. Ball, J. Biol. Chem. 234, 577 (1959). F. H. Leibach and F. Binkley,Arch. Biochem. Biophys. 127, 292 (1968).
[60]
~-GLUTAMYL TRANSPEPTIDASE
787
Activators and Inhibitors Revel and Ball 19 demonstrated that ~-glutamyl transpeptidase is inhibited by serine in the presence of borate buffer. Kinetic analysis ~1 suggested that the mixture .of serine and borate forms an inhibitor competitire with the ~,-glutamyl substrate. Iodoacetamide (0.05 M) inactivates the enzyme rapidly and irreversibly. 21 Iodoacetate and other sulfhydryl reagents axe much less effective. Iodoacetamide may be reacting with a carboxyl group in the active center to form a glycolyl ester, in a reaction analogous to that of ribonuclease T1 with iodoacetate. 22 The enzyme is protected from iodoacetamide inhibition by serine in the presence of borate. 2~ Goldbarg ~3 demonstrated complete inhibition of ~/-glutamyl transpeptidase activity in rat kidney homogenate by sulfobromophthalein at 2 X 10.5 Mr Greenberg 24 also observed sulfobromophthalein inhibition of the enzyme in homogenates of human jejunal mucosa. Sulfobromophthalein inhibits weakly (about 50% at 2 X 10-~ M) the purified beef kidney enzyme. 11 Orlowski and Meister have reported slight activation of the pig kidney enzyme by Mg~÷.4 No activation by Mg 2÷ was observed with the beef kidney, 11 serum, 8,14 or bean 25 enzymes. The kidney enzyme of Leibach and Binkley ~° is absolutely dependent upon the presence of Mg 2÷. In general the activity of the enzyme is not significantly affected by the presence of traces of other metallic ions, nor of ethylenediaminetetraacetate.S, 11 pH Dependence. The pH optimum for transpeptidation is 8.8-9.0 for both the beef kidney 11 and the pig kidney 4 enzymes. A value of 8.2 has been reported for human serum transpeptidase by Szasz? The bean enzyme 28 has a higher pH optimum, 9.5 for transpeptidation, while for hydrolysis the optimum is between pH 6.0 and 6.5. Stability. ),-Glutamyl transpeptidase of beef kidney is stable for at least 10 months at --20 °, whether in the frozen or lyophilized state. 11 Physical and Chemical Properties. As highly purified preparations of ~-glutamyl transpeptidase from mammalian sources are heterogeneous, no meaningful physicochemical characterization has been possible. The heterogeneity may be explained by the membrane-bound state of the 21A. Szewczuk and G. E. ConneU, Biochim. Biophys. Acta 105, 352 (1965). K. Takahashi, W. H. Stein, and S. Moore, J. Biol. Chem. 242, 4682 (1967). 22j . A. Goldbarg, O. M. Friedman, E. P. Pineda, E. E. Smith, R. Chatterji, E. H. Stein, and A. M. Rutenberg, Arch. Biochem. Biophys. 91, 61 (1960). :4 E. Greenberg, E. E. Wollaeger, G. A. Fleisher, and G. W. Engstrom, Clin. Chim. Acta 16, 79 (1967). =J. F. Thompson, D. H. Turner, and R. K. Gering, Phytochemistry 3, 33 (1964). ~ M. Y. Goore and J. F. Thompson, Biochim. Biophys. Acta 132, 15 (1967).
788
TRANSPEPTIDASES
[50]
enzyme in tissues, and the solvation procedures which are required in the purification. Both the beef and pig kidney enzymes appear to be glycoproteins containing approximately 36% carbohydrate by weight. Enzymatic removal of the sialic acid results in no loss of activity. 2~ Indirect evidence suggests that a soluble form of the enzyme from mammalian tissues is also a glycoprotein. 28 The pig kidney enzyme preparation contains a small proportion of fatty acid." Distribution 7-Glutamyl transpeptidase was discovered by Hanes, Hird, and Isherwood 1,~ in sheep kidney and it has been highly purified both from beef 11 and pig' kidney. Although the enzyme is found in most tissues of man and other mammals, the level is far higher in kidney than in any other tissues. 1~,28 Histochemical analysis and centrifugal fractionation of tissue have shown that the transpeptidase is a membrane-bound enzyme, ls,1~,~8,29'3" Glenner, Folk, and McMillan demonstrated, a° by a specific histochemical method, that in the kidney the enzyme is located on the brush border of the proximal convoluted tubules and loops of Henle. This pattern of distribution has suggested the possibility that the enzyme may play a role in kidney function, for example in the tubhlar reabsorption of amino acids. Histochemical observations on the jejunal mucosa of man z' which in some respects parallel those of Glenner, Folk, and McMillan on kidney, have stimulated speculation regarding a general role for the enzyme in amino acid transport. Szewczuk 2s has shown that tissues of man contain, in addition to the membrane-bound enzyme, a small amount of 7-glutamyl transpeptidase which is recovered in the high-speed supernatant after centrifugal fractionation of homogenates. Evidence was presented that the serum enzyme was closely related in properties to the soluble enzyme of liver. The urinary enzyme differed from these in some respects but resembled, on the other hand, the soluble transpeptidase of kidney. Other workers 28,31-33 have provided evidence by electrophoresis and gel filtration for heterogeneity of the serum and urine enzymes. Thompson e t al. 2~ and Goore and Thompson 26 have purified a 7A. Szewczuk and G. E. Connell, Biochim. Biophys. Acta 83, 218 (1964). ~A. Szewczuk, Clin. Chim. Acta 14, 608 (1966). G. G. Glenner and J. E. Folk, Nature 192, 338 (1961). G. G. Glenner, J. E. Folk, and P. J. McMillan, J. Histochem. Cytochem. 19, 481 (1962). SIF. Kokot and J. Kuska, Clin. Chim. Acta I I , 118 (1965). = M. Orlowski and A. Szczeklik, Clin. Chim. Acta 15, 387 (1967). K. Jacyszyn and T. Laursen, Clin. Chim. Acta 19, 345 (1968).
[61]
789
~'-GLUTAMYL CYCLOTRANSFERASE
glutamyl transpeptidase from kidney bean fruit which resembles the mammalian enzyme in many respects. The plant enzyme is not membrane-bound but is recovered in the high-speed supernatant after centrifugal fractionation of homogenates, y-Glutamyl peptides accumulate in dormant 'bulbs and seeds in several species84 which suggests that the enzyme may have a special role in the metabolism of plants. An enzyme from Agaricus bisporus (mushroom) '~ differs from the bean enzyme; although it will catalyze hydrolysis and transfer of the y-glutamyl radical to hydroxylamine, it will not transfer to a-amino acids. y-Glutamyl transpeptidase has been found in several species of bacteria, for example, Escherichia coli, Proteus vulgaris, Proteus morganii, and Pseudomonas aeruginosa. A specialized form of transpeptidase from Bacillus subtilis has been characterized by Williams and Thorne? 6 This enzyme is involved in the synthesis of an extracellular soluble poly-yglutamyl peptide by the organism. A similar polypeptide appears to be present in a bound form as a component of the capsule of anthrax bacillus and other strains of the mesentericus group of bacilli? 7,as s, p. M. Dunnill and L. Fowden, Biochem. J. 86, 388 (1963). H. J. Gigliotti and B. Levenberg, J. Biol. Chem. 239, 2274 (1964). seW. J. Williams and C. B. Thome, J. Biol. Chem. 210, 203 (1954). 3rM. Bovarniek, J. Biol. Chem. 145, 415 (1942). V. Bruekner, J. Kovaes, and H. Nagy, J. Chem. Soe. p. 148 (1953).
[61] T-Glutamyl
Cyclotransferase
(y-Glutamyl
Lactamase)
By E. D. ADAMSON, O. E. CON~,LL, and A. Sz~,wczuK O II C--NH--R I
CH2 I
CH2 I
H2N--CH --COOH 7-L- GIu-NH- R
H2~ ~H2 .~.C C O~ \NJH"COOH
+ H2N--R
I H
2 Pyrrolidone-5-carboxylic acid + H2N--R
y-Glutamyl cyclotransferase catalyzes the transfer of the y-carboxyl group of terminal L-glutamyl residues from linkage with a-amino groups of amino acids and peptides to linkage with the amino group of the same glutamyl residue. The products are the cyclic derivative of glutamic acid, L-pyrrolidone earboxylie acid (PCA), and a free amino acid or peptide.
[61]
789
~'-GLUTAMYL CYCLOTRANSFERASE
glutamyl transpeptidase from kidney bean fruit which resembles the mammalian enzyme in many respects. The plant enzyme is not membrane-bound but is recovered in the high-speed supernatant after centrifugal fractionation of homogenates, y-Glutamyl peptides accumulate in dormant 'bulbs and seeds in several species84 which suggests that the enzyme may have a special role in the metabolism of plants. An enzyme from Agaricus bisporus (mushroom) '~ differs from the bean enzyme; although it will catalyze hydrolysis and transfer of the y-glutamyl radical to hydroxylamine, it will not transfer to a-amino acids. y-Glutamyl transpeptidase has been found in several species of bacteria, for example, Escherichia coli, Proteus vulgaris, Proteus morganii, and Pseudomonas aeruginosa. A specialized form of transpeptidase from Bacillus subtilis has been characterized by Williams and Thorne? 6 This enzyme is involved in the synthesis of an extracellular soluble poly-yglutamyl peptide by the organism. A similar polypeptide appears to be present in a bound form as a component of the capsule of anthrax bacillus and other strains of the mesentericus group of bacilli? 7,as s, p. M. Dunnill and L. Fowden, Biochem. J. 86, 388 (1963). H. J. Gigliotti and B. Levenberg, J. Biol. Chem. 239, 2274 (1964). seW. J. Williams and C. B. Thome, J. Biol. Chem. 210, 203 (1954). 3rM. Bovarniek, J. Biol. Chem. 145, 415 (1942). V. Bruekner, J. Kovaes, and H. Nagy, J. Chem. Soe. p. 148 (1953).
[61] T-Glutamyl
Cyclotransferase
(y-Glutamyl
Lactamase)
By E. D. ADAMSON, O. E. CON~,LL, and A. Sz~,wczuK O II C--NH--R I
CH2 I
CH2 I
H2N--CH --COOH 7-L- GIu-NH- R
H2~ ~H2 .~.C C O~ \NJH"COOH
+ H2N--R
I H
2 Pyrrolidone-5-carboxylic acid + H2N--R
y-Glutamyl cyclotransferase catalyzes the transfer of the y-carboxyl group of terminal L-glutamyl residues from linkage with a-amino groups of amino acids and peptides to linkage with the amino group of the same glutamyl residue. The products are the cyclic derivative of glutamic acid, L-pyrrolidone earboxylie acid (PCA), and a free amino acid or peptide.
790
TRANSPEPTIDASES
[61]
Assay
Principle. ~,-Glutamyl-7-glutamylnaphthylamide labeled with 1'C in the terminal glutamyl group yields 14C-labeled pyrrolidone carboxylic acid in the presence of the enzyme. The other product, 7-glutamylnaphthylamide is nut further degraded by the enzyme. The PCA which is formed is separated from other substances in the reaction mixture by paper electrophoresis, and the quantity is determined by liquid scintillaLion counting.
Reagents 7-L-~C-glutamyl-7-L-glutamyl-a-naphthylamide, 0.032 M, adjusted to pH 8.0 with 0.05 M Tris-HC1 buffer. Substrate solutions should be prepared freshly at least monthly.1 Tris-HC1 buffer, 0.80 M, pH 8.0
Synthesis of the Substrate y-L-GLuTAMYL-a-NAPHTHYLAMmE. Phthaloyl-L-glutamic anhydride (12.95 g, 0.05 mole) obtained by the method of Sheehan and Bolhofer,la is dissolved with heating in 30 ml dry dioxane, and after cooling, 7.15 g (0.05 mole) a-naphthylamine is added. The solution is heated to 45 ° for 30 minutes with occasional stirring and then evaporated at reduced pressure. The remaining oily product is dissolved in 50 ml 1 M sodium carbonate, filtered, diluted to 300 ml with water, and then acidified with 70 ml 2 N hydrochloric acid. The precipitate is filtered off, washed with distilled water, and dried in a vacuum desiccator over calcium chloride. The dried product is dissolved in 250 ml ethyl alcohol and 7.45 g (0.05 mole) triethanolamine, and 8.1 g (0.075 mole) pure phenylhydrazine is added. The mixture is boiled 2 hours with refluxing, cooled, and 3 g (0.05 mole) glacial acetic acid is added. The material is left overnight at 4 °, filtered on a Biichner funnel, washed three times with alcohol and once with ether, and then allowed to dry in air. The crude product is dissolved in 400 ml water with heating to 900-95 °, 0.5 g charcoal is stirred in, and t h e mixture filtered while hot. After 12 hours the crystals are collected on a funnel, washed with alcohol, and dried in a vacuum desiccator over calcium chloride. The yield is 6 g (44% of theoretical) pure ~,-L-glutamyl-a-naphthylamide (m.p. : 185 °, [a]n 2° -~ 28 °, 3% in 1 N HC1). 1If the substrate is radiochemicallyimpure and contains "C-PCA (i.e., if an aged solution is used) acceptable results may be obtained by running a blank reaction mixture containingno enzyme.The radioactivity of PCA in the blank is determined and the excess PCA formed in the presence of enzymeis calculated. J. C. Sheehan and W. A. Bolhofer, J. Am. Uhem. ,5oc. 72, 2469 (1950).
[61]
"y-GLUTAMYL CYCLOTRANSFERASE
791
"f-L-14C-GLuTAMYL-T-L-GLUTAMYL-a-NAPHTHYLAMIDE.This compound is synthesized 'by an adaptation of the method of Orlowski and Szewczuk2 from phthaloyl-L-1'-C-glutamic anhydride and unlabeled ~/-L-glutamyla-naphthylamide. For this synthesis I00 ~Ci L-14C-glutamic acid is diluted with 1.50 g unlabeled glutamic acid and phthaloyl-L-~'C-glutamic anhydride is prepared according to the method of Sheehan and Bolhofer. TM Phthaloyl-L-~4C-glutamic anhydride (780 rag) is dissolved in a hot solution of 816 mg 7-L-glutamyl-a-naphthylamide in 3 ml glacial acetic acid. The solution is heated for 30 minutes at 60 ° . Acetic acid is removed in vacuo, and the remaining powder is dissolved in 15 ml dry dioxane. After 3 hours the solution is filtered, concentrated at reduced pressure, and the residue is dissolved in 60 ml 0.1 M sodium carbonate. The material is precipitated by the addition of 30 ml 0.5 N hydrochloric acid. The precipitate is filtered, washed with water, and dried in a desiccator over calcium chloride. Of this material, 1.06 g is dissolved in 9 ml 0.5 M sodium carbonate and 1 ml 2 M hydrazine is added. After 2 days the mixture is acidified to pH 3 with fresh distilled hydroiodic acid, filtered, and concentrated under reduced pressure. Ethanol (50 ml, 99%) is added to the concentrate, the product is recrystallized from 99% ethanol and then dried (m.p. = 183°-184 ° with decomposition). Procedure. Incubation tubes are prepared by sealing one end of 2 mm inside diameter glass tubing (about 4 cm lengths). To 5 ~l 0.80M TrisHC1 buffer in an incubation tube, 10 ~l enzyme solution of appropriate dilution is added. Buffered substrate solution (25 #l) is then added with thorough mixing. The mixture is incubated at 37 ° for 60 minutes, and the reaction is stopped by heating to 100 ° for 3 minutes. The reaction mixture is transferred quantitatively to Whatman 3 MM paper for electrophoresis. The sample is applied in a band 1.2 cm long and is subjected to drying in a current of warm air during application. After high-voltage electrophoresis, pH 3.6, at 55 V/cm for 45 minutes, the paper is dried and a strip of the paper containing a marker spot of authentic PCA (0.1 micromole) is cut off. The strip is treated to locate the PCA by the method of Rydon and Smith, 3 as follows: The strip is placed in an atmosphere of chlorine for 7-10 minutes, then the excess gas is removed by placing the strip in a ventilated oven at 60 ° for about 1/2 to 1 hour. When the paper strip is sprayed lightly with 1% starch-l% potassium iodide solution, PCA is located immediately as a blue-black spot on a fainter colored background. The radioactive spots of PCA from the reaction mixtures are located by reference to the guide strip, cut out, and M. Orlowski and A. Szewczuk,Acta Biochim. Polon. 8, 189 (1961). *H. N. Rydon and P. W. G. Smith, Nature 196, 922 (1952).
792
TRANSPEPTIDASES
[51]
placed in scintillation fluid for counting. The scintillation fluid is prepared by dissolving 5 g 2,5-diphenyloxazole and 300 mg 1,4-bis-2-(5-phenyloxazolyl)-benzene in 1000 ml toluene. Counts are corrected for quenching by the channels ratio method. The yield of PCA is calculated as follows: Number of micromoles of PCA produced per minute PCA disintegrations/minute X 0.80 substrate disintegrations/minute X 60 The relationship between enzyme concentration and PCA formation is linear up to 3 0 ~ production of the theoretical amounts; with pure enzyme preparations this figure reaches 50%. Alternative assays have been used by Orlowski, Richman, and Meister. ~ Definition o] Unit and Specific Activity. One unit of enzyme activity is defined as that amount of enzyme which catalyzes the production of 1 mieromole of pyrrolidone carboxylie acid per minute under the conditions of the assay. Specific activity is expressed as the number of units of enzyme per milligram protein (determined by the method of Lowry
et al2). Adaptation o] the Assay ]or Crude Enzyme Preparations. The presence of ],-glutamyl transpeptidase in crude ],-glutamyl cyclotransferase preparations may deplete the theoretical amount of substrate added. The addition of L-serine and sodium tetraborate to the incubation mixture so that the final concentration of each is 0.05 M will completely inhibit transpeptidase activity. Rapid, Semiquantitative Estimation o] Enzyme Activity. Enzyme solution (10 ~l) is added to 10 ~l 0.80 M Tris-HC1 buffer, pH 8.0, in an incubation tube. The substrate, 20 ~l 0.05 M ~-glutamylalanine, is added rapidly so that thorough mixing is achieved. The mixture is incubated for a standard time (between 15 and 60 minutes may be chosen) at 37 ° and the reaction is stopped by heating to 100 ° for 3 minutes. A suitable aliquot (30/~l) is then transferred to Whatman 3 MM paper for electrophoresis. The paper is subjected to high-voltage electrophoresis at pH 3.6, 55 V/cm for 40 minutes, and dried and stained by the method of Rydon and Smith 8 (see above). Spots of PCA produced during incubation may then be visualized and the quantity estimated by the intensity and size of the colored spot by reference to a marker spot of PCA. In addition, the paper can then be sprayed with ninhydrin solution so as to con4 M. Orlowski, P. G. Richman, and A. Meister, Biochemistry 8, 1048 (1969). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
[51]
'y-GLUTAMYL CYCLOTRANSFERASE
793
firm these observations by estimation of the amount of alanine produced by enzymatic action.
Preparation of Enyme About 5 pig livers (7 kg) is a quantity conveniently handled in the preliminary steps. The tissue is obtained from freshly killed animals and processed immediately in the cold. The liver is first minced in a meat grinder. For every 100 g mince, 200 ml 0.05M Tris-HCl, pH 7.4, is added and the mixture is homogenized for 3 minutes in a Waring blendor. The homogenate is centrifuged at 12,000-13,000 g for 30 minutes; the supernatant is retained and diluted with an equal volume of 0.05 M Tris-HCl buffer, pH 7.4. Step 1. Ammonium Sul]ate Precipitation. The protein which precipitates at room temperature from the diluted supernatant between 60 and 90% saturation with solid ammonium sulfate is suspended in a small volume of 0.01 M potassium phosphate buffer, pH 6.5, and dialyzed in the cold against frequent changes of this buffer. The red solution is clarified by centrifugation. Step P. CM-CeIlulose Treatment. CM-Cellulose (microgranular) is equilibrated with 0.01 M potassium phosphate buffer, pH 6.5, and placed in a Biiehner funnel. For every 100 g CM-cellulose about 200 ml 10% protein solution may be processed. The solution is allowed to pass at room temperature through the CM-cellulose and is eluted with an equal volume of phosphate buffer. The eluate is conveniently concentrated to 1/3 its volume at this point, by pervaporation in dialysis tubing. The concentrated protein solution is brought to pH 5.4 by dialysis against 0.15 M sodium acetate buffer, and then clarified by centrifugation. Step 3. DEAE-Sephadex Chromatography. The protein solution (about 600 ml 10%) is passed at 4 °, through a column (10 cm X 45 cm) of DEAE-Sephadex previously equilibrated with 0.15 M sodium acetate buffer, pH 5.4, and is eluted with the same buffer. A great deal of colored inactive protein is eluted immediately from the column and when the protein concentration has dropped to a low level, the enzyme emerges. The active fractions are pooled and concentrated to about 2% protein concentration. To avoid assaying many fractions of eluate by the standard procedure, the more rapid, semiquantitative method may be employed (see above). Step 4. Preparative Polyacrylamide Gel Electrophoresis2 The concentrated product (500 mg protein) of the previous column is dialyzed against 0.1 M Tris-glycine, pH 9.5, and is subjected to electrophoresis eT. Jovin, A. Chrambach, and M. A. Naughton, Anal. Biochem. 9, 351 (1964).
794
TRANSPEPTIDASES
[51]
in 18% polyacrylamide gel (containing 1 g bisacrylamide per 100 g monomer) at pH 9.5. The gel height is 10 cm and the current is maintained at 40 mA during the run. The enzyme migrates ahead of other proteins emerging after 16 hours and is eluted with 0:1 M Tris-glycine buffer, pH 9.35. Active fractions are detected by the "rough assay," pooled, and immediately concentrated by ultrafiltration. Step 5. Preparative Isoelectric Focusing. 7-11 The product (100 mg protein) from electrophoresis is dialyzed overnight against 1% glycine solution and is applied in a sucrose gradient to an :~oelectric focusing column (440 ml) maintained at 15 ° with tap water cooling. Ampholine 11a of pH range 4.6 to 5.2 (previously prepared from Ampholine of pH range 4-6) at a final concentration of 1% is also incorporated into the gradient. A potential of 350 V is applied initially to the column, and as the ampholytes become focused this is raised gradually until a value of 600 V is reached after 48 hours. The column is emptied at a rate of 2.5 ml/min and fractions of 3-5 ml are collected. The pH and absorbance at 280 nm are measured, and fractions are assayed for activity. At least two proteins with different isoelectric points are obtained. Protein is separated from sucrose and Ampholine after prolonged dialysis against 0.05M Tris-HC1 buffer, pH 8.0, or by passage of the mixture through a column of Sephadex G-50. The purification process is summarized in Table I. Properties Specificity. The best substrates so far described for y-glutamyl cyelotransferase are those with two ],-glutamyl residues, namely ~-glutamyl-y-glutamyl-a-naphthylamide and the analogous p-nitroanilide. Neither ~,-glutamyl-a-naphthylamide nor ~-glutamyl-p-nitroanilide is a substrate. Di-y-glutamylnaphthylamide is acted upon much more rapidly than the triglutamyl analog. Among the compounds of glutamic acid with other amino acids, the best substrates are ~,-glutamylalanine, ~,glutamylglutamine, ~-glutamylglycine, and y-glutamyl-a-aminobutyric acid. The enzyme shows only slight activity toward the ~/-glutamyl derivatives of the hydrophobic amino acids phenylalanine, tyrosine, leucine, valine, and toward glutathione and ophthalmic acid. The enzyme is inactive toward glutamine.
•. Svensson, Acta Chem. Scand. 15, 325 (1961). 8H. Svensson, Aeta Chem. Scand. 16, 356 (1962). H. Svensson, Arch. Bioehem. Biophys. Suppl. 1, 132 (1962). I*O. Vesterberg and It. Svensson, Acta Chem. Seand. 20, 820 (1966). 110. Vesterberg, Acta Chem. Scand. 21, 206 (1967). 11,Ampholine from LKB-produkter AB, Stockholm-Bromma 1, Sweden.
[61]
"y-GLUTAMYL CYCLOTRANSFERASE
795
TABLE I SUMMARY OF THE PURIFICATION OF ~?'-GLUTAMYL CYCLOTRANSFERASE FROM 7 KG PIG LIVER
Step 1 2 3 4 5
Preparation
Volume
Total protein
Specific activity (units/mg protein)
Homogenate Ammonium sulfate extract CM-Cellulose treatment DEAE-Sephadex chromatography Preparative polyacrylamide gel electrophoresis Isoelectric focusing
20.33 liters 2 liters 670 ml 63 ml
1.31 kg 93.5 g 21.5 g 587 mg
0.0416 0.174 0.61 10.81
18 ml
56.8 rag
Yield 100 30 24 11.6
67
7
84-147
4.9
All substrates so far identified have a ~-glutamyl residue with free a-amino and a-carboxyl groups, linked to the a - a m i n o group of an adjacent amino acid residue with an ~-carboxyl group either free or bound in amide, ester, or peptide linkage. No compounds containing n-glutamyl residues have been identified as substrates. I n crude enzyme preparations containing ~-glutamyl transpeptidase TM in addition to cyclotransferase, some substances which are not substrates of cyclotransferase m a y be converted to pyrrolidone carboxylic acid by sequential action of the two enzymes. 4,1a pH Dependence. In the presence of Tris buffers the activity of the enzyme from h u m a n liver 13 and brain ~ and sheep brain 4 is practically independent of p H between p H 5 and 9. In phosphate and borate buffers the brain enzyme is less active over most of the range but a distinct o p t i m u m is observed in borate buffer near p H 8.0. Stability. Both the pig liver and h u m a n brain 4 enzymes are stable in solution near 0 ° for at least 1 month except in extremely dilute solutions. The enzyme is also stable in the frozen state at --20 ° . Physical and Chemical Properties. The sedimentation velocity of pig liver cyclotransferase in phosphate buffer-NaC1 (pH 6.5; ionic strength 0.20) is 2.1 Svedberg units, corrected to water at 20 °. The sedimentation velocity does not v a r y with concentration between 0.2 mg and 1.2 mg/ml. The h u m a n brain enzyme has a sedimentation coefficient of 1.2 Svedberg units ( s~0' w) in 0.05 M Tris-citrate, p H 8.8. G. E. Connell and E. D. Adamson, this volume [60]. 1~G. E. Connell and A. Szewczuk, Clin. Chim. Acta 17, 423 (1967).
796
TRANSPEPTIDASES
[61]
TABLE II AMINO ACID ANALYSISOF ~f-GLUTAMYLCYCLOTRANSFERASE
Amino acid Glyeine Alanine Leueine Isoleucine Valine Half-eystine Proline Methionine Serine Threonine Aspartic Glutamic Lysine Histidine Arginine Tyrosine Phenylalanine Tryptophan
Micromoles per 100 micromoles recovered
Number of residues per 22,400 MW
7.4
14.9
5.9
11.9
8.1 5.7 5.7 2.0 4.9 1.9 7.1 4.4 9.1 15.2 8.6 1.5 3.3 3.4 4.0 1.0
16.3 11.4 11.6 4.2 9.9 3.7 14.4 8.9 18.4 30.6 17.3 3.0 6.7 6.8 8.1 2.0
The amino acid composition of ~,-glutamyl cyclotransferase of pig liver is presented in Table II. The molecular weight of cyclotransferase from pig liver, determined by sedimentation equilibrium in phosphate buffer-NaC1 (pH 6.5, ionic strength 0.20) is 22,400. Dif]erent Forms o] the Enzyme. 7-Glutamyl cyclotransferase from pig liver occurs in at least two forms with isoelectric points between 4.82 and 5.05; the enzyme from human brain ~ is also reported to occur in at least two forms with isoelectric points at 4.06 and 4.25 while that of sheep brain 4 occurs in five forms with isoelectric points ranging from 6.20 to 4.65. The relationship between the different forms remains to be elucidated; they appear to have similar sedimentation coefficients and molecular weights but different isoelectrie points. Distribution The highest level of 7-glutamyl cyclotransferase so far known occurs in human brain is and the enzyme has been purified 1000-fold from this source b y Orlowski, Richman, and Meister.' Appreciable levels are found in other organs of mammals, namely, kidney, liver, spleen, pancreas, lung, and muscle of man, 18 mouse, * and guinea pig, 4 in liver of r a t / ' 14G. E. Connell and C. S. Hanes, Nature 177, 377 (1956).
[62]
RIBOSOMAL PEPTIDYL TRANSFERASE
797
rabbit, 1~ and pig,4 and in sheep brain, 4 A smaller amount occurs in calf and rabbit lens, 1~ and serum of man. 1~ In all cases so far recorded the enzyme has been recovered entirely in the supernatant fraction after high-speed centrifugation of homogenates or extracts of the above tissues. E. E. Cliffe and S. G. Waley, Biochem. J. 79, 118 (1961).
R i b o s o m a l P e p t i d y l T r a n s f e r a s e (E. coli)
[52]
By Rosin E. MONRO oR". ]
.
. OR.+1 . . I
.
.
. OR.+1 I
R'CHCO + NH2CHCO --~ R'CHCO--NHCHCO + HOR"'
I
R"~,
I
R "n + l
I
R"~.
I
R"n + l
where R' is an acylamido group; R ~' is the side chain of one of the 20 protein amino acids; HOR "~' is tRNA (HO is the 2' or 3' hydroxyl group of the terminal adenosine); and n corresponds to the number of amino acid residues in the growing peptide chain. Assay
Method
1, ~
Principle. Ribosomal peptidyl transferase, the catalyst of peptide bond formation in protein synthesis, is not a typical enzyme. It exists as a catalytic center on the 50 S ribosomal subunit, and its expression is interlinked (largely through the tRNA substrates) with that of other functional centers on the ribosome (Fig. 1). The peptidyl transferase activity of the 50S subunit normally requires, for its expression, the presence of the 30S subunit 3 and mRNA. 4, ~ However, it is possible to induce isolated 50 S subunits to catalyze peptidyl transfer by addition of methanol or ethanol. 1,2 Under such conditions the normal substrates can also be replaced by fragments thereof (or other small analogs such as puromycin), and by this means interactions with the ribosome in the immediate vicinity of the catalytic site can be investigated. This reaction R. E. Monro and K. A. Marker, J. Mol. Biol. 25, 347 (1967). 2 R. E. Monro, T. Staehelin, M. L. Celma, and D. Vazquez, Cold $pring Harbor Symp. Quant. Biol., in press, 1969. M. B. Hille, M. J. Miller, K. Iwasaki, and A. J. Wahba, Proc. Natl. Acad. Sci. U.S. 58, 1652 (1967). 4I. Rychilik, Biochim. Biophys. Acta 114, 425 (1966). M. S. Bretscher and K. A. Marcker, Nature 211, 380 (1966).
[62]
RIBOSOMAL PEPTIDYL TRANSFERASE
797
rabbit, 1~ and pig,4 and in sheep brain, 4 A smaller amount occurs in calf and rabbit lens, 1~ and serum of man. 1~ In all cases so far recorded the enzyme has been recovered entirely in the supernatant fraction after high-speed centrifugation of homogenates or extracts of the above tissues. E. E. Cliffe and S. G. Waley, Biochem. J. 79, 118 (1961).
R i b o s o m a l P e p t i d y l T r a n s f e r a s e (E. coli)
[52]
By Rosin E. MONRO oR". ]
.
. OR.+1 . . I
.
.
. OR.+1 I
R'CHCO + NH2CHCO --~ R'CHCO--NHCHCO + HOR"'
I
R"~,
I
R "n + l
I
R"~.
I
R"n + l
where R' is an acylamido group; R ~' is the side chain of one of the 20 protein amino acids; HOR "~' is tRNA (HO is the 2' or 3' hydroxyl group of the terminal adenosine); and n corresponds to the number of amino acid residues in the growing peptide chain. Assay
Method
1, ~
Principle. Ribosomal peptidyl transferase, the catalyst of peptide bond formation in protein synthesis, is not a typical enzyme. It exists as a catalytic center on the 50 S ribosomal subunit, and its expression is interlinked (largely through the tRNA substrates) with that of other functional centers on the ribosome (Fig. 1). The peptidyl transferase activity of the 50S subunit normally requires, for its expression, the presence of the 30S subunit 3 and mRNA. 4, ~ However, it is possible to induce isolated 50 S subunits to catalyze peptidyl transfer by addition of methanol or ethanol. 1,2 Under such conditions the normal substrates can also be replaced by fragments thereof (or other small analogs such as puromycin), and by this means interactions with the ribosome in the immediate vicinity of the catalytic site can be investigated. This reaction R. E. Monro and K. A. Marker, J. Mol. Biol. 25, 347 (1967). 2 R. E. Monro, T. Staehelin, M. L. Celma, and D. Vazquez, Cold $pring Harbor Symp. Quant. Biol., in press, 1969. M. B. Hille, M. J. Miller, K. Iwasaki, and A. J. Wahba, Proc. Natl. Acad. Sci. U.S. 58, 1652 (1967). 4I. Rychilik, Biochim. Biophys. Acta 114, 425 (1966). M. S. Bretscher and K. A. Marcker, Nature 211, 380 (1966).
798
[52]
TRANSPEPTIDASES "P" "A"
Peptidyl
site site ~'~"--~. A i A :
~..u.~.u..U...U..'AAAf--
/ b,," /
/ Fzo. 1. Diagrammatic representation of interactions occurring on the ribosome during two cycles of chain elongation. (a) Peptidyl transfer, (b) other reactions of protein synthesis. The "P-site" is defined as the collection of sites with which the peptidyl-tRNA interacts at the moment prior to peptide bond formation, and the "A-site" as the corresponding collection of sites at which the aminoacyl-tRNA interacts. The P- and A-sites of the peptidyl transferase center are depicted as interacting with the CCA-peptide and CCA-amino acid moieties of the two substrates. [Reproduced from Nature 223, 903 (1969) by kind permission of the editor.]
(Fig. 2) is termed the "fragment reaction." Other assay systems are also available 3-8 but are less resolved since they require the presence of the 30 S subunit, mRNA, and intact peptidyl donor substrate. e p. Leder and H. Bursztyn, Biochem. Biophys. Res. Commun. 25, 233 (1966).
[52]
RIBOSOMAL PEPTIDYL TRANSFERASE
799
Fro. 2. Diagrammatic representation of the "fragment reaction." Met-f is transferred from CCA to puromycin to give CCA and f-met-puromycin. CACCA-Leu-Ac and other related fragments can also serve as substrates.
The assay method is based on the reaction in Eq. (1) (footnotes 2 CACCA-(SH)leu-Ac ~- puromycin ---*Ac-(3H)leu-puromycin -Jr CACCA
(1)
and 7). The product, Ac-(SH)leu-puromycin, is selectively extracted into ethyl acetate, and the radioactivity estimated in a scintillation counter. The original ethyl acetate method e has been modified 8 by raising the ionic strength and lowering the volume of the aqueous phase, in order to increase the extent and rapidity of extraction. Alternative methods for estimation of the extent of reaction include alcohol precipitation or paper ionphoresis.1, 7
Reagents Buffered salts mix: Tris(hydroxymethyl) aminomethane (Tris)-HC1 buffer, pH 7.4, at 20 ° (giving pH 7.8 in the assay mixture at 0°), 0.25 M in Tris KC1, 2 M Mg acetate, 0.25 M Puromycin dihydrochloride, 10 mM CACCA- (3H) Leu-Ac (10-30 Ci/mM) ,7, 9 200-600 cpm/~l Ribosomes Methanol or ethanol (absolute) Buffered MgS04 solution: Na acetate, pH 5.5, 0.3 M in acetate saturated with solid MgS04 at room temperature ' R . E. Monro, J. ~ernh, and K. A. Marcker, Proc. Natl. Acad. Sci. U.S. 61, 1042 (1968). SB. E. H. Maden and R. E. Monro, European J. Biochem. 6, 309 (1968). R. E. Monro, Vol. XX [50].
800
WRANSPErrwAs~s
[52]
Scintillation fluid (modified from footnote 10) : Toluene, 1 liter 2-Methoxyethanol, 250 ml Butyl PBD (from Ciba), 5 g
Procedure2 The incubation mixture (150 /zl, assuming volumes of water and alcohol to be additive) is placed in small conical tubes at 0 °, and contains 20/zl of buffered salts mix, 10/~l of puromycin, water, ribosomes (1 0D26o unit), 50 ~l of methanol or ethanol (precooled to 0°), and 10/zl of CACCA-(3H)leu-Ac. The components are added in the order indicated, and the reaction is initiated by addition of CACCAleu-Ac. (If ethanol is used, a precipitate of ribosomes is formed, but this does not affect the kinetics of the reaction. If aliquots are taken from a common mix, care should be taken to disperse the precipitate before pipetting, in order to avoid uneven distribution of ribosomes.) After 2-30 minutes of incubation at 0 °, the reaction is stopped by addition of 0.1 ml of buffered MgSO4. Ethyl acetate, 1.5 ml, is then added, the mixture agitated for 5 seconds at room temperature, and centrifuged briefly at low speed. One milliliter of the top layer (ethyl acetate) is transferred to a 4-ml vial, mixed with 2.5 ml of scintillation fluid, and the radioactivity estimated in a scintillation counter (efficiency of counting about 20%). Over 95% of the Ac-leu-puromycin is recovered in the ethyl acetate phase after one extraction. Blank values obtained by addition of buffered MgSO~ before alcohol, or by incubation without ribosomes, puromycin, or alcohol amount to less than 3% of the added radioactivity. Estimates are reproducible to within ± 5 % . The extent of reaction is expressed as percentage of CACCA-leu-Ac converted to Ac-leu-puromycin. Values can be corrected for the presence of free Ac-(3H)leu in CACCA-(SH) leu-Ac preparations, as determined by paper ionphoresis. Initial rates are estimated by extrapolation of progress curves to zero time, or, less accurately, from single incubations in which less than 20% of the added CACCA-leu-Ac has reacted. Definition of Unit and Specific Activity. Because of technical difficulties in the preparation of large amounts of suitable substrate, 9 the CACCA-leu-Ac is labeled with very high specific activity (aH)leu, and is used at a very low concentration (in the order of 5 mM). This concentration is more than an order of magnitude less than the K,~ of CACCA-leu-Ac, and the other substrate, puromycin, is in large excess, so that (a) the rate of reaction is proportional to the concentration of CACCA-Ieu-Ac, (b) the reaction is first order with respect to CACCAleu-Ac, and (c) the time taken to convert a given percentage of the D. J. Prockop and P. S. Ebert, Anal. Biochem. 6~ 263 (1963).
[52]
RIBOSOMAL PEPTIDYL TRANSFERASE
801
added CACCA-leu-Ac to Ac-leu-puromycin is independent of the initial concentration of CACCA-leu-Ac. Until a substrate becomes available which can be used at saturating concentrations, it is proposed that a unit of peptidyl transferase activity be defined as that amount of ribosomes which catalyzes the conversion of CACCA-leu-Ac to Ac-leupuromycin at an initial rate of 10% per minute in the above assay. The unit defined in this way is independent of the concentration of CACCAleu-Ac (in the range under study), so that the concentration can be adjusted, with substrate preparations of various specific activities, to have the appropriate amount of radioactivity. The definition also obviates technical difficulties in obtaining CACCA-(~H)leu-Ac of accurately known specific activity2 The specific activity of peptidyl transferase is expressed as the number of units per milligram of ribosomes; ribosome concentration is determined by use of an ~eo ~1~ of 160. It is premature to define an expression for specific activity of components of the peptidyl transferase center derived from 50 S subunits. Range o] Application. The assay technique is applicable to ribosomes from a variety of species (see below), but not to preparations containing soluble proteins (due to the formation of bulky precipitates in the presence of alcohol). The inapplicability to crude extracts is not a serious handicap, since the purification of ribosomes is followed by physical criteria. The peptidyl transferase center is firmly integrated into the structure of the 50 S subunit and there is no tendency for it to be lost during the purification of ribosomes. 11,12,~3 Purification Procedure The purification of 70 S ribosomes and their 50 S subunits from crude cell extracts is effected by differential and zonal centrifugation under controlled ionic conditions. The method for Escherichia coli ribosomes is described elsewhere. 11,1" Active preparations of 70 S ribosomes and 50 S subunits from E. coli have specific activities of about 10 and 15, respectively. It is possible to remove about 30% of the proteins from the 50S subunit without loss of activity, thus giving a further purification of peptidyl transferase. 2,ix Removal of more proteins leads to inactivation. 11T. Staehelin, D. Maglott, and R. E. Monro, Cold Spring Harbor Syrup. Quan$. Biol. 34 (1969). B. E. H. Maden, R. R. Traut, and R. E. Monro, J. Mol. Biol. 35, 333 (1968). 1sR. E. Monro, J. Mol. Biol. 26, 147 (1967). 14T. Staehelin, Vol. XX [47].
802
Zr~Nsrzr'rmAsss
[62]
Recombination of the resultant fractions under suitable conditions leads to restoration of activity. Work is in progress to purify and identify components of the 50 S subunit which constitute the peptidyl transferase center. Properties
Stability. Ribosomes may be stored without loss of activity for several days at 0% or for several months at temperatures below --70 ° . Frozen ribosomes should be distributed in small aliquo~s and used only once, since repeated freezing and thawing leads to inactivation. Peptidyl transferase activity varies with the conformational state of the ribosome. Salt-washed ribosomes are more active than unwashed ribosomes. 12,13 Exposure to absence of monovalent cations leads to inactivation, which can be reversed by suitable treatment. 1~ Purity and Physical Properties. TM Subunits (50 S) prepared by the recommended procedure have less than 3% contamination with 30S subunits, and are free from other particulate components and supernatant proteins. It is possible that a small fraction of the ribosomal protein is lost during preparation, but evidence that the peptidyl transferase center is recovered intact is provided by the demonstration that typical 50 S preparations have approximately one such center per 50 S particle. 2 Subunits with 50 S have a sedimentation coefficient of 50 S in the presence of Mg 2÷ and monovalent cation. Their molecular weight is about 1.8 X 108. Each 50 S subunit is a highly organized structure containing 1 molecule of 23S RNA (molecular weight about 1.2 X 106) and 1 molecule of 5 S RNA (molecular weight 4.1 X 10~), and in the order of 30 different proteins, some of which are present as 1 mole per mole of ribosomes, others in lesser amounts. The proteins range in molecular weight from about 10,000 to 50,000. As already noted, work is in progress to determine which component (s) of the 50 S sUbunit is present at the peptidyl transferase center. 2,~ Specificity. Evidence from the use of various types of substrate analog~,7 suggests that effective interaction at the P-site of the peptidyl transferase center (Fig. 1) involves CpCpA, the 3' terminal grouping of nucleotides common to all species of tRNA. Interaction is also favored by acylation (at the a-amino group) of the aminoacyl residue attached to the adenosine, and is influenced by the nature of the amino acid side group. ~R. Miskin, A. Zamir, and D. Elson, Biochem. Biophys. Res. Commun. 33, 551 (1968).
For details and references see footnote 11.
[52]
RIBOSOMAL PEPTIDYL TRANSFERASE
803
Analogous studies with peptidyl acceptor substrates 17,18 indicate that there is specific interaction of the bases, " C " and "A," in the terminal dinucleotide of tRNA, at the A-site of the peptidyl transferase center. Evidence is not available as to whether the third base (C) also takes part in the interaction. The aminoacyl residue must be attached to the 3' position of the terminal adenosine, and must be of the L isomeric form. 19 Activity is influenced by the nature of the amino acid side group.iS, 19 Activators and Inhibitors. Catalysis of peptidyl transfer takes place most rapidly in the region of p H 8-9. s Below p H 8 the reaction is progressively inhibited• Above p H 8 it is difficult to assay the reaction, due to base-catalyzed hydrolysis of the substrates (for this reason the reaction is assayed at p H 7•8). The reaction shows an absolute dependence upon monovalent and divalent cations. Specificity toward cations, and effects of cation concentration have been reported, s The reaction is inhibited by Be ~*, Zn 2÷, and Hg2+,8 but sulfhydryl group reagents 13 and serine inhibitors (R. E. Monro and P. Siegler, unpublished data) are without effect• A number of antibiotic inhibitors of protein synthesis act specifically on the peptidyl transferase center, i° Distribution. Peptidyl transferase is integrated into the structure of the larger ribosomal subunit in all species which have so far been examined, i.e., E. coli, B. subtilis, 2~ Anacystis montana, ~ yeast, 22 protozoa, 23 rat liver, 24 and human tonsils. 2~ The ribosomal peptidyl transferases in these various species are similar to one another in many respects, but a difference between the fine structures at the catalytic centers of 70 S and 80 S ribosomes (from procaryotic and eucaryotic organisms, respectively) is indicated by differences in sensitivity to antibiotics. 2~
~' I. Rychlik, S. Chladek, and J. ZemliSka, Biochim. Biophys. Acta 138, 640 (1967). is R. H. Symons, R. J. Harris, L. P. Clarke, J. F. Wheldrake, and W. It. Elliott, Biochim. Biophys. Acta 179, 248 (1969). 19D. Nathans and A. Neidle, Nature 197, 1076 (1963). ~oR. E. Monro and D. Vazquez, J. Mol. Biol. 28, 161 (1967). ~1D. Vazquez, M. L. Celma, and R. E. Monro, unpublished data. "~D. Vazquez, E. Battaner, R. Neth, G. Heller, and R. E. Monro, Cold Spring Harbor Symp. Quant. Biol., in press, 1969. G. Cross, personal communication. 24T. Staehelin and A. Falvey, personal communication.
[•3]
STREPTOKINASE
807
[53] Strcptokinasc B y FLETCHER B. TAYLOR, JR., and RUSSELL H. TO~AR
Introduction Streptokinase (SK), an extracellular protein produced by various strains of streptococci, is capable of converting human plasminogen to plasmin. Its capacity to cause lysis of blood clots was first described by Tillet and Garner in 1933.I This effect was thought to be due to direct enzymatic'action on the fibrin of these clots. However, Milstone in 1941 demonstrated that streptokinase achieved its effect through activation of a plasma protein. 2 Thus, the proenzyme and enzyme forms of this plasma protein were given the names plasminogen (profibrinolysin), and plasmin (fibrinolysin), respectively, and the bacterial extract was given the name streptokinase2 As of now plasminogen and plasmin are the only substrates with which SK is known to to react. However, it is not known whether this interaction involves enzymatic hydrolysis of plasminogen by: (1) SK itself or (2) by an activator complex composed initially of SK and either plasminogen and/or plasmin or (3) by an activator complex composed of SK and a proactivator which is distinct from plasminogen. While there is little doubt that a complex of SK and plasminogen is formed, the first and third possibilities have not been excluded as being important in the conversion of plasminogen to plasmin by SK. In any case, however, it is still not known whether streptokinase is a proteolytic enzyme in the classic sense (hydrolysis of covalent bonds) or a physicochemical modifier of a preexisting enzyme. Since the interaction of SK with plasminogen is apparently unique, we will describe: (1) SK activity in terms of its effect on the activity of plasminogen and expand this description to include data on the nature of this interaction (under Assay Procedures), (2) purification of both SK and plasminogen (under Preparative Procedures) and (3) the known physical and chemical characteristics of SK (under Physical Properties). Assay of Streptokinase Activity and Discussion of Its Mode of Activity All of the many assays described detect the presence of streptokinase indirectly through its capacity to activate plasminogen to plasmin. lW. S. Tillet and R. L. Garner, J. Exptl. Med. 58, 485 (1933). 2H. Milstone,J. Immunol. 42, 109 (1941). 3At first streptokinase was called fibrinolysin by TiUet and Milstone until it was found that this material induced fibrinolysis indirectly through activation of a plasma protein. The term streptokinase was then coined by Christensen4 to describe
808
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[53]
Plasmin then hydrolyzes an indicator substrate such as a fibrin clot, casein, or synthetic substrate. The rate of hydrolysis of the substrate is a function of the amount of plasmin formed from the proenzyme, and, within the limits described, this plasmin formation in turn is directly related to the amount of SK present.
Clot Lysis Assay The most commonly used assay for SK is that originally described by Christensen. + It involves the determination of the smallest amount of streptokinase that will cause lysis of a standard fibrin clot in 10 minutes. One-tenth milliliter of serial dilutions of streptokinase in gelatin buffer, 0.8 ml of standard human "fibrinogen" solution (which also contains plasminogen), and 0.1 ml of thrombin solution (described below) are mixed and placed in a water bath at 37 °. The fibrinogen clots in 1 minute or less. The clots then lyse because the SK activates the plasminogen "contaminating" the original fibrinogen reagent. The lysis times of the tubes of the series are followed up to 20 minutes, and the lysis time of each dilution of SK is plotted against the reciprocal of the dilution on log-log paper (Fig. 1). A straight line can be drawn through the points beginning with a lysis time of 1.5 to 2 minutes and extending to about 20 to 30 minutes. Above and below these points the curve is not linear and therefore cannot be used for purposes of calculation. The upper limit 20 15 I0 4D
8 6
co
4
.J
0
l
I
I
1/2
114
118
I 1/12
I
J
1/20
1/40
Dilutions of streptokinase
Fro. 1. Plot of the logs of lysis time of the clot (minutes) vs. the reciprocal of the dilution of the SK solution being tested. the bacterial extract, and the terms profibrinolysin and fibrinolysin to describe the inactive (proenzyme) and active (enzyme) form of the plasma protein. 4L. R. Christensen, J. Clin. Invest. 28, 163 (1949).
[63]
STREPTOKINASE
809
of the curve is of course determined by the amount of substrate (fibrin) relative to active enzyme (plasmin). However, it also should be noted that the upper limit is influenced by the fact that high concentrations of SK combine with plasminogen or plasmin to form an activator complex (to be defined below) which has a lower affinity for fibrin than does plasmin. This assay of clot lysis can detect the presence of nanogram quantities of streptokinase. Under the above conditions, one unit is that amount of SK which will lyse a standard clot in 10 minutes as determined by interpolation of the lysis time curve. A sample assay and calculation is as follows: Onetenth milliliter aliquots of undiluted, 1 : 2, 1 : 4, 1:8, and 1 : 16, 1:32 dilutions of streptokinase are added to the system as described above and lysis time recorded. Figure 1 shows the log plot of the lysis time vs. the dilutions. Employing the above definition of 1 unit of SK activity together with interpolation of this plot, the units/ml or unit/mg sample can be calculated by recording the lysis time for a given dilution or concentration of SK and using the following equation: Lysis_time (minutes) 10 minutes / × (dilution factor) X (10, or correction to 1 ml of SK sample being assayed) = units/ml of sample being assayed
(1)
Interpolating along the plotted graph in Fig. 1,.one sees that a 1:12 dilution of the starting material should lyse a standard clot in 10 minutes. By definition then, 0.1 ml of a 1:12 dilution contains one SK unit. Therefore, the starting material contained 120 SK units/ml. 10 minutes × (12-fold dilution) (10) = 120 units/ml 10 minutes
(2)
The WHO Expert Committee on Biological Standardization used the Christensen assay to establish an international standard for SK based on assay of aliquots of a 6-g lot (¢~48035-154) of an impure but stable preparation of SK provided by Lederle Laboratories2 One international unit (that amount of the SK standard given above which would lyse a clot in 10 minutes) was found to be equivalent to 0.002090 mg of this material. Reagents
Fibrinogen (Containing Human Plasminogen). Cohn fraction I lyophilized and stored at 4 ° is made into a 0.25% solution w/v with borate saline buffer on the day of the study. 5Bull. World Health Organ. 33, 235 (1965).
810
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[53]
Thrombin. Lyophilized bovine thrombin (10,000 N I H units, ParkeDavis) is made into a solution containing I N I H unit thrombin activity per 0.1 ml with borate saline buffer on the day of the study. Lyophilized Standard and Test Sample. SK diluted in gelatin buffer on the day of the study. Buffers Borate Saline Sodium borate hydrated, 7 g Sodium chloride, 9 g Boric acid, 11.1 g Distilled water, 1000 ml HC1, 5 N, to pH 7.4 Gelatin Buffer Gelatin, 5 g dissolved in a small amount of warm water NaC1, 10 g KH2P04, 13.6 g make up to 1000 with distilled water NaOH, 10 N, to pH 7.4 Individual aliquots of these reagents can be prepared beforehand and stored at --60 °. However, neither the fibrinogen nor thrombin can be frozen and thawed more than once without affecting solubility and activity. Alternative reagents which are used less often are as follows: 0.7 ml of a 0.25% solution of highly purified fibrinogen (95% clottable) in phosphate buffer (pH 7.4, 0.1 M) 0.1 ml containing one Remmert and Cohen casein unit of highly purified plasminogen, 0.1 ml of 10 units/ml of bovine thrombin and 0.1 ml of the SK to be tested all in the same buffer. These reagents, though more difficult to prepare, can yield results which are more consistent than those obtained when fibrinogen (Cohn I) containing plasminogen is used. The Christensen assay is useful because it allows: (1) a comparatively simple determination of relative SK activity within a given laboratory; and (2) an approximation of specific activity of SK for biological purposes. However, the difficulty with such an assay for use in more rigorous biochemical studies is alluded to in the report on the establishment of the international unit of SK where a range of 2150 to 3886 Christensen units or 970 to 5093 British Units was recorded by different laboratories2 This wide range is due to the fact that this assay requires subjective determination of the end point and the use of three biological products,
[63]
STREPTOKINASE
811
fibrinogen, plasminogen, and SK. As with most biologicals, lot-to-lot variability, instability, and contamination by inhibitors multiply problems of standardization. This difficulty has been obviated in part by preparation of large batches of crude fibrinogen (plus plasminogen) for use solely in standardizing successive batches of SK. However, unless all laboratories have access to the same batch of SK or crude fibrinogen, more rigorous direct comparison of the various SK preparations is not possible. Other assays which have been employed for determinations of SK activity include the fibrin plate assay 6 and casein assay. 7 Synthetic Substrate (LMe) Assay
Synthetic esters instead of fibrinogen have been employed as substrates to define more rigorously the specific activity of SK2 '9 The following is a description of an assay for SK activity on lysine methyl ester (LMe) which utilizes plasminogen of known specific activity. It involves the addition of streptokinase to highly purified human plasmi~logen of known specific activity and assaying the rate of hydrolysis of a stable synthetic substrate (LMe). In this assay 0.200 mg of plasminogen (20 casein units/mg protein) in 0.25 ml 0.001 N HC1 is activated by 0.25 ml of the SK test sample in Tris buffer with 0.5 ml of 0.08M LMe (obtained from Mann Research Co., N.Y., N.Y.) in Tris buffer (final pH 7.4) at 37 °. At the end of the desired time interval (usually 30 minutes), 2 ml of a 1:1 mixture of reagents I and II (described below) are added to the above reaction mixture. The samples are then allowed to stand 2 minutes, followed by the addition of 1 ml reagent III. After at least 30 minutes and not more than 24 hours, the samples are centrifuged at 1000 q for 10 minutes to remove the precipitate. One milliliter of the supernatant is added to 4 ml of reagent IV and the color of the samples is read at 525 nm within 2 minutes. Control samples include LMe alone (0.5 ml) plus buffer (0.5 ml), and LMe (0.5 ml) plus plasminogen (0.25 ml) plus buffer (0.25 ml). In defining the activity of SK by esterolysis, it is necessary to do so indirectly in terms of the amount of plasmin activity generated by the SK conversion of plasminogen to plasmin. Therefore, for each batch or lot of SK a standard curve is drawn of the change in optical density T. Astrup and S. Mullertz, Arch. Biochem. Biophys. 40, 346 (1952). ~A. J. Johnson, W. R. McCarty, W. S. Tillet, A-O. Tse, L. Skoza, J. Newman, and M. Semar, in "Blood Coagulation Hemorrhage and Thrombosis," p. 449. Grune & Stratton, New York, 1964. 8F. B. Taylor and J. Botts, Biochemistry 7, 232 (1968). t p. R. Roberts, J. Biol. Chem. 235, 2262 (1960).
812
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[53]
0.500
A
0.4.00
U~ N
.-.q
E o
0.300
o 60
~
~o 20 I
2
,
I
5
i
i
i
i
50
*
i
100
Concentrotion (%)
FIG. 1. Assay curves for tissue plasminogen activator. Serial dilutions of stock solutions assayed as follows: (O O) reference standard containing 8.0 tissue activator units per milliliter, ( . m) solution prepared from pregnant hog ovaries (sample diluted 200 times), (O O) solution prepared from normal hog ovaries (sample diluted 100 times), (£ A) solution prepared from intestinal serosa from hogs (sample diluted 10 times). Abscissa (logarithmic): concentrations of serial dilutions in percentages of the test solution. Ordinate (logarithmic): diameter products in mm2 of lyzed zones (each the mean of triplicate). tion was (8 X 28 X 100 X 5000)/(100 X 447) ----2510 units per gram ovary. An extract (160 ml) made from 18 g of intestinal serosa from hog was diluted 10 times for assay (curve marked by triangles 'in Fig. 1). The test solution corresponded to 7 3 ~ of the standard solution. The activator concentration was (8 X 73 X 10 >( 160)/(100 X 18) = 520 units per gram fresh tissue. The accuracy of the fibrin plate assay of tissue activator is about -+-7% (coefficient of variation). In the assay of tissue samples this is an insignificant variation because of the large individual differences. Higher accuracy, about 1%, is provided by the lysis time method (see below) but this can be used only for concentrated activator solutions free from KSCN. The tissue activator unit was originally defined as the amount of activator in 1 mg of an arbitrarily selected pig heart preparation. 5 All data from our systematic studies of the content of plasminogen activator in tissues have been reported in these units2 One tissue activator unit (A and A unit) equals approximately 0.1 CTA unit of urokinase (as defined by the Committee on Thrombolytic Agents, advisory to the National
[64]
TISSUE PLASMINOGEN ACTIVATOR
829
Heart Institute) but, since tissue plasminogen activator differs chemically from urokinase, comparisons in the same unitage will be valid only with limitations. It is important to remember that the relationship between activator concentration and activity is linear only in a double logarithmic graph, so that, e.g., a 2-fold increase in activity might reflect a 5-f~ld increase in activator concentration. Purification of Tissue Activator
Source of Activator. The high specific activity of purified tissue plasminogen activator preparations, in terms of units of activator per milligram of protein, indicates that the molecular concentration in even the most active tissues is low. Hence, it is important, first, to select a highly active tissue for purification, and, second, to develop simple chemical procedures in the early steps of the purification amenable to the treatment of larger quantities of crude material. When the bulk of inert material has been removed, more sophisticated methods of purification can be applied. Unfortunately, nearly all animal tissues are low in activity and the few known exceptions are not easily available in large quantities. After trying several different tissues, we selected ovaries from pregnant hogs because of their extremely high activity. Normal hog ovaries contain from 200 to 5000 A and A units per gram fresh tissue. In late pregnancy they regularly reach 30,000 units, and occasionally even 50,000 units per gram, the highest concentrations yet observed in any organ or species. The selection of such potent material saves many preliminary steps aimed at concentrating the active material. The purification follows with minor modifications the procedure recently described. 16 Extraction o] Activator. One kilogram of frozen ovaries is partially thawed, passed twice through a meat grinder in the cold room, and disintegrated briefly in a rotating knife blender with 1.5 liters of cold 0.05 M KC1 adjusting the pH to 6.0 with 1 N HC1. After dilution with 8.5 liters of 0.05 M cold KC1, the suspension is stirred slowly for 2 hours in the cold room keeping pH at 6.0 by addition of HC1. The sediment is collected by centrifugation in the cold for 1/2 hour at about 1400 g, and the supernatant, containing only 3-5% of the total activity, is discarded. The sediment is then stirred overnight at room temperature with 10 liters of 2 M ammonium thiocyanate adjusting pH to 7.3-7.4 with 1 N KOH. Centrifugation at room temperature yields about 10 liters of an extract with a potency regularly reaching 3000 units per milliliter, corresponding to a total yield of 30 X 106 units per kilogram of ovaries. The protein nitrogen, determined by micro-Kjeldahl after removal of thiocyanate by 16p. Kok and T. Astrup, Biochemistry 8, 79 (1969).
830
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[64]
dialysis, amounts to about 0.6 mg/ml, yielding a specific activity of about 5000 units per milligram nitrogen. Separation o] Activator. The thiocyanate extract is diluted with 1 volume of water (10 liters) and pH adjusted to 4.5 with 1 N HC1. After standing for 15 minutes, addition of acid is continued until pH 2.5. The sediment is collected after ½ hour by centrifugation and kept overnight at --20 ° in order to destroy lipoproteins. The next day the material is thawed and extracted for 1 hour at room temperature with 1000 ml ] M NH4SCN, adjusting pH to 5.9 with 1 N KOH, followed, after centrifugation, by a second similar treatment with 500 ml NH~SCN. The combined extracts are acidified to pH 2.5 with 1 N HC1, left for ½ hour at room temperature and centrifuged. The sediment is suspended in 800 ml of cold ethyl ether and kept overnight at --20 ° or below. The next day the mixture is thawed and stirred briefly at 4 ° . The sediment is collected by centrifugation in the cold and again treated with 800 ml of ether followed by freezing overnight. The sediment is again collected by eentrifugation and then stirred for 1 hour with 1000 ml of 1 M NH~SCN adjusting pH to 5.9. After centrifugation the treatment is repeated and the two extracts combined, yielding approximately 2000 ml containing about 10,000 units per milliliter and 0.5-0.6 mg protein nitrogen per milliliter. The yield is about 20 X 106 units per kilogram of ovaries with a specific aetivity between 17,000 and 20,000 units per milligram protein nitrogen. 16a Fractionation with Zn 2+. A solution containing 0.2 M ZnC12 and 0.6 M glycine is prepared and adjusted to pH 8 with 7 M NH40H. Equal volumes (2000 ml) of this solution and the activator extract, similarly adjusted to pH 8, axe mixed and the pH carefully changed to 5.8 with 1 N HCI. After ½ hour the precipitate is separated by centrifugation, suspended in 1000 ml of a solution containing 0.02M ZnC12 and 0.06M glycine at pH 5.8 and again collected by centrifugation. It is then extracted for 30 minutes with 500 ml of a solution containing 0.01 M ZnCl2 and 0.03M glycine adjusting pH to 4.3. The solution is separated by centrifugation and the extraction is repeated. The combined extract (about 1000 ml) contains about 18,000 units/ml and 0.3-0.5 mg protein nitrogen per milliliter. The yield is about 18 X 10e units per kilogram ovaries with a specific activity between 35-60,000 units per /nilligram nitrogen. Activator Product I. The acidity is now adjusted to pH 5.8 with 0.5N ~ Utmost care must be applied in the ether treatment in the separation step. We have tried a number of other solvents in order to replace the ethyl ether with a less dangerous fluid. We have met with little success because of great losses of activity, except in the case of n-butanol which, preliminary experiments indicate, might be applicable in a modification of the method.
[64]
TISSUE PLASMINOGEN ACTIVATOR
831
KOH and the active precipitate collected by centrifugation after 1 hour in the cold room. After treatment in the cold for 1/2 hour at pH 5.8 with 200 ml 0.05 M glycine and centrifugation, followed by a similar treatment with 0.05 M KC1, the precipitate is treated with cold acetone and cold ether, each three times, and the product dried in the air. The resulting dry powder from 1 kg of ovaries weighs about 1.5 g. It contains about 10,000 units per milligram, equal to 70,000 units per milligram nitrogen. The yield is about 15 X 10~ units per kilogram of ovaries. The activity of product I is stable when kept in a dry place and is soluble at acid reaction in aqueous solutions of low ionic strength. Activator Product H. Four hundred milligrams of product I is stirred for 1 hour with 16 ml of 0.1 M glycine-HC1 buffer at pH 2.35 containing 0.1 M KC1 and the solution clarified by high-speed centrifugation (10,000 g, ½ hour). The solution, 15 ml, is applied to a Sephadex G-200 column, 90 X 2.5 cm, prepared with a 0.1 M glycine-HC1 buffer, pH 2.35, containing 0.2 M KC1. Effluents are collected at 30-minute intervals at a rate of 2-2.5 ml/cm 2 per hour using the same buffer and activities and ultraviolet absorbancies determined. Gel filtration is preferably performed in the cold room with glycine buffer half saturated with chloroform. (When processing a whole batch of product I, prepared from 1 kg of ovaries, a 100 X 5 cm column is used.) The most active fractions (Fig. 2) are combined (40-50 ml) and
252oC u
§ 15-
~ 10._~
g 0
~
5-
#
._~ ~ 0 30
40
50
60
?0
80
90
100 110
Froction number
FIG. 2. Collection of active fractions of product I by gel filtration on a Sephadex G-200 column (90 X 2~5 cm) by ascending flow in a 0.1 M glycine-ttC1 buffer at pH 2.35 and containing 09. M KC1. Effluents collected at intervals of 30 minutes. Abscissa: Fraction number. Ordinate: Activator concentration in units X 10~ per milliliter. For preparation of product II, fractions within the thin vertical lines are combined.
832
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[54]
dialyzed in the cold room against a 0.01 M glycine-HCl buffer at pH 2.35 containing 0.1 M KC1. The solution is adjusted to pH 7.1 with dilute KOH, left in the cold for 15 minutes, and the sediment collected by oentrifugation (in the cold). After treatment three times with cold acetone followed by anhydrous ethyl ether (3 times), it is dried in the air. The resulting powder (about 20 mg from 400 mg of product I) contains from about 100,000 to 175,000 units per milligram protein, and reaching about 1.2 X 106 units per milligram protein nitrogen. The yield is 'between 50 and 65% of product I corresponding to 8-10 X 108 units per kilogram of ovaries.
Properties The acid stability of the tissue activator greatly facilitates its isolation and purification. The products are stable for long periods when stored dry and cold. Crude extracts frequently contain additional amounts of activator activity of a heat-labile nature. Whether these labile activators originate in the blood or interstitial fluids or whether they represent a different type of tissue activator is not known. In the purification procedure described here they are destroyed by the acid treatment, leaving only acid-stable activator. Extraction of plasminogen activator from hog ovaries with 2 M thiocyanate gives higher yields than any of many other procedures tested. The purified activator is soluble in avid solutions at low ionic strength. At neutral pH a concentration about 30,000 units per milliliter can be obtained in saline :barbital buffer using product I. The yield and purity of activator preparations may vary in each step and from batch to batch. Both depend much upon the activity of the crude material. The data given represent those obtained in batches prepared from ovaries with activities around 30,000 units per gram. The preparations obtained are of uniform molecular size in gel filtration experiments, suggesting a molecular weight around 60,000. However, zone electrophoresis has shown them to be amenable to further purification, probably 3-5 times. The tissue activator preparations described here compare well in activity with those obtained from pig heart by Bachmann et alJ but the selection of a different source and method makes the purification procedure less cumbersome. The porcine tissue activator differs from human urokinase in molecular size 18 and in its interaction with ~-aminocaproic acid, 17 or with inhibitors in certain pathological blood samples28 Human tissue activator also differs from human urokinase in 1'S. Thorsen and T. Astrup, Proc. Soc. Exptl. Biol. Med. 130, 811 (1969). 18p. Brakman, E. R. Mohler, and T. Astrup, Scand. J. Haematol. 3, 389 (1966).
[64]
TISSUE PLASMINOGEN ACTIVATOR
833
its interaction with c-aminocaproic acid? 9 Tissue activator, though stable at acid reaction, is easily destroyed by pepsin. ~°, ~ Purified preparations of tissue activator from pig heart were found to activate plasminogen by an enzymatic reaction, 7,22 reportedly through the cleavage of an argininevaline bond. 2s Comments The fibrin plate method is applicable to the assay of crude tissue activator preparations and is little influenced by the presence of various salts (such as thioeyanate) and contaminants in the test samples introduced during purification procedures. I t often gives more reliable results than the otherwise more accurate lysis time method 17,24 which requires 100 times higher c~ncentrations of activator. The tissue activator acts specifically with plasminogen to form plasmin; it does not digest casein or plasminogen-free fibrin. The presence of enzymes, which can digest fibrin directly, can be tested on plasminogen-free fibrin plates. 1° The histochemical fibrin slide technique, 2~,26 useful for the cellular localization of plasminogen activator activity, is highly sensitive but does not yield quantitative results. The tissue plasminogen activator is widely distributed in the animal organism. 9'27 Its cellular localization in human venous endothelium was first reported by Todd. 2~ In animals, arteries may also be active, zs Budding capillaries in granulation tissue are particularly rich in tissue activator. ~9 Recently, additional sources of tissue plasminogen activator have been revealed, namely corneal epithelial cells, s° vaginal epithelial cells, s1,~2 endometrial epithelial cells, s~,~4 and serosal cellsY I t is quite l~p. Kok and T. Astrup, Federation Proc. 28, No. 2, 441 (1969). (Abstract ~1037). K. Alkjaer and T. Astrup, Trans. 6th Congr. European Soc. Haematol. Copenhagen, p. 42. Karger, Basel, 1957. ,1j. Rasmussen and O. K. Albrechtsen, Fertility Sterility 11, 264 (1960). ~D. C. McCall and D. L. Kline, Thromb. Diath. Haemorrhag. 14, 116 (1965). L. Summaria, B. Hsieh, and K. C. Robbins, J. Biol. Chem. 242, 4279 (1967). 24M. Lassen, Scand. J. Clin. Lab. Invest. 10, 384 (1958). A. S. Todd, J. Pathol. Bacteriol. 78, 281 (1959). H. C. Kwaan and T. Astrup, Lab. Invest. 17, 140 (1967). '~T. Astrup, in "Dynamics of Thrombus Formation and Dissolution" (S. A. Johnson and M. M. Guest, eds.), p. 275. Lippincott, Philadelphia, 1969. '~ B. A. Warren, Brit. J. Exptl. Pathol. 44, 365 (1963). :~H. C. Kwaan and T. Astrup, J. Pathol. Bacteriol. 87, 409 (1964). :~M. Pandolfi and T. Astrup, Arch. Ophthalmol. 77, 258 (1967). ~ J. Henrichsen and T. Astrup, J. Pathol. Bacteriol. 93, 706 (1967). ~:K. Tympanidis, A. E. King, and T. Astrup, Am. J. Obstet. Gynecol. 160, 185 (1968). asK. Tympanidis and T. Astrup, Acta Endocrinol. 60~ 69 (1969). K. Tympanidis and T. Astrup, J. Clin. Pathol, 22, 36 (1969). ~O. M. Jensen, S. B. Larsen, and T. Astrup, Arch. Pathol. 88, 623 (1969).
834
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[65]
possible that more than a single species of tissue activator exists in the organism. Since the activity of endometrial and vaginal epithelial cells is influenced by hormones, it could represent a type of activator different from that present in vascular endothelial cells.
[ 6 5 ] T i s s u e A c t i v a t o r of P l a s m i n o g e n ( C y t o k i n a s e )
By S. Y. ALI Mammalian tissues contain an activator which converts the blood proenzyme plasminogen to the active protease plasmin. 1 The purified tissue activator of plasminogen is referred to as cytokinase to be consistent with the terminology of plasminogen activators. Tissue activator has been purified from pig ovaries 2 and from pig heart, s The present paper will deal with the extraction and purification of tissue activator or cytokinase from rabbit kidney. 4 The preparation of plasminogen and casein and the fibrin-lysis assay method have been dealt with in earlier chapters and they will not be described here.
Assay Method
Principle. The activator is incubated with its substrate, the proenzyme plasminogen, in the first stage and the amount of plasmin so formed is measured by the extent of proteolysis of casein. The trichloroacetic acidsoluble aromatic amino acids and peptides released from casein are measured spectrophotometrically. Reagents 0.1 M Tris buffer, pH 7.4 4% (w/v) Casein (light white soluble casein, British Drug Houses Ltd.) 10% (w/v) Trichloroacetic acid Human plasminogen 5 Rabbit kidney cytokinase* 1T. Astrup, Federation Proc. 25, 42 (1966). 2p. Kok and T. Astrup, Biochemistry 8, 79 (1969). SF. Bachmann, A. P. Fletcher, N. Alkjaersig, and S. Sherry, Biochemistry 3, 1578
(1964). 4 S. Y. Ali and L. Evans, Biochem. J. 107, 293 (1968). 5D. L. Kline, J. Biol. Chem. 204, 949 (1953). Activity of plasminogen is expressed in milliunits as described by F. M. Davidson, Biochem. J. 76, 56 (1960).
834
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[65]
possible that more than a single species of tissue activator exists in the organism. Since the activity of endometrial and vaginal epithelial cells is influenced by hormones, it could represent a type of activator different from that present in vascular endothelial cells.
[ 6 5 ] T i s s u e A c t i v a t o r of P l a s m i n o g e n ( C y t o k i n a s e )
By S. Y. ALI Mammalian tissues contain an activator which converts the blood proenzyme plasminogen to the active protease plasmin. 1 The purified tissue activator of plasminogen is referred to as cytokinase to be consistent with the terminology of plasminogen activators. Tissue activator has been purified from pig ovaries 2 and from pig heart, s The present paper will deal with the extraction and purification of tissue activator or cytokinase from rabbit kidney. 4 The preparation of plasminogen and casein and the fibrin-lysis assay method have been dealt with in earlier chapters and they will not be described here.
Assay Method
Principle. The activator is incubated with its substrate, the proenzyme plasminogen, in the first stage and the amount of plasmin so formed is measured by the extent of proteolysis of casein. The trichloroacetic acidsoluble aromatic amino acids and peptides released from casein are measured spectrophotometrically. Reagents 0.1 M Tris buffer, pH 7.4 4% (w/v) Casein (light white soluble casein, British Drug Houses Ltd.) 10% (w/v) Trichloroacetic acid Human plasminogen 5 Rabbit kidney cytokinase* 1T. Astrup, Federation Proc. 25, 42 (1966). 2p. Kok and T. Astrup, Biochemistry 8, 79 (1969). SF. Bachmann, A. P. Fletcher, N. Alkjaersig, and S. Sherry, Biochemistry 3, 1578
(1964). 4 S. Y. Ali and L. Evans, Biochem. J. 107, 293 (1968). 5D. L. Kline, J. Biol. Chem. 204, 949 (1953). Activity of plasminogen is expressed in milliunits as described by F. M. Davidson, Biochem. J. 76, 56 (1960).
[55]
CYTOKINASE
835
"Procedure. Nine test tubes are placed in a water bath at 37 °. The first set of three test tubes contains (a) 0.2 ml of plasminogen (50 milliunits 5) the second three (b) 0.2 ml of cytokinase (about 200 units4). The third set (c) consists of 0.2 ml of cytokinase and 0.2 ml of ptasminogen. The volume in all the tubes is made up to 2 ml with Tris buffer and the tubes are incubated at 37 ° for exactly 10 minutes. Casein (1 ml) is then added to all the tubes. Triehloroacetie acid (3 ml) is added to one tube from each set. The remaining two test tubes from each set are incubated at 37 ° for another 30 minutes. Trichloroacetic acid is now added to these tubes as well. The precipitated protein is then removed by filtration through Whatman No. 3 filter paper. Extinction values of the filtrates are determined in a spectrophotometer at 280 nm. From the average extinction value obtained from the duplicate tubes in each set is subtracted the value for the first blank tube. The value thus obtained from set (a) provides the amount of plasmin contamination in the plasminogen preparation and when this is subtracted from (c) the difference indicates the plasmin formed by the action of cytokinase. Tbe value for set (b) is nil when dealing with purified preparations of cytokinase, but it is positive when crude tissue activator is used and this value should be subtracted from (c). Definition o[ Unit and Specific Activity. The E28o value, Obtained after subtraction of the plasmin and protease activity, is expressed in terms of an arbitrary unit of cytokinase activity. It is defined as the amount of cytokinase that, when allowed to activate 50 milliunits ~ of human plasminogen for 10 minutes and the resulting plasmJn is allowed to digest casein for 30 minutes at pH 7.4 and at 37 °, produces an E28o difference of 0.001. Specific activity is expressed in terms of this unit per milligram of protein 6 in the cytokinase preparation. Purification Procedure Kidneys, obtained from young rabbits, are weighed, minced with scissors, and introduced into a Potter-type homogenizer (smooth glass-walled tube and Teflon pestle) and homogenized at 4 ° at 5000 rpm in sufficient 0.25 M-sucrose to make a 10% (w/v) suspension. The homogenate is subjected to differential centrifugationJ The sediment obtained after centrifugation at 5000 g for 10 minutes is rejected and the supernatant fluid is then centrifuged at 140,000 g for 1 hour. The supernatant fluid is decanted and rejected. The sediment, which consists of the lysosome6 0. H. Lowry, N. J. Rosebrough, A. L. Fan:, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). ' S. Y. Ali and C. H. Lack, Biochem. J. 96, 6,3 (1965).
836
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[55]
microsome fraction, ~ is suspended in water to give a concentration equivalent to 0.5 g of the tissue/ml on a wet weight basis. The lysosome-microsome fraction, which contains about 90% of the cytokinase activity in the kidney homogenate, 7 is then adjusted to pH 10.5 with a few drops of 1 N NaOH, and centrifuged at 20,000 g for 10 minutes at 4 °. The supernatant fluid is neutralized with 1 N HC1 and then dialyzed against 5 mM phosphate buffer, pH 7.6, for 18 hours. The nondiffusate (12 ml) is then layered over a DEAE-cellulose column (2.2 X 35 cm) equilibrated with 5 mM phosphate buffer, pH 7.6. A linear gradient of the same buffer, 0.005 M (300 ml) to 0.4 M (300 ml), is then used for elution of cytokinase at room temperature. Cytokinase ~ and protein 8 assays are performed on those fractions which produce lysis when spotted on fibrin plates. 4,s Cytokinase appears in the eluate when the molarity of phosphate buffer reaches about 50 mM. The fractions that give the highest specific activity for cytokinase are pooled, dialyzed overnight against distilled water at 4 ° , and freeze dried. The freeze-dried material is stable at 4 ° almost indefinitely. The table shows a summary PURIFICATION OF RABBIT KIDNEY CYTOKINASE
Fraction Rabbit kidney homogenate Lysosome-microsome fraction Soluble supernatant fluid after alkaline extraction Column fractions 6, 7, and 8 Column fraction 7
Specific Total Total activity Volume protein activity (units/rag (ml) (rag) (units) of protein)
Yield (%)
Purification
66 13
416 74
27,300 21,800
66 295
100 80
1.0 4.5
12
53
11,700
222
43
3.4
99 33
3 1
7,750 3,070
2,600 2,910
28 11
40.0 44.3
of the purification steps. An 86-fold purification can be achieved by stepwise increase of phosphate buffer molarity but there is a consequent fall in percentage recovery of cytokinase activity.
Properties S t a b i l i t y . Cytokinase is quite stable in neutral buffer solution when stored at 4 °. When kept at 22 ° for 30 minutes cytokinase was stable over the range pH 3-11. It becomes unstable at acid pH values when the
s T. Astrup and S. Mullertz, Arch. Biochem. Biophys. 40, 346 (1952).
[65]
CYTOKINASE
837
temperature is raised. It retains all its activity at pH 8-9 even when treated at 37 ° or 56 ° for 30 minutes. It is inactivated completely when heated at 80 ° for 15 minutes. Purity and Physical Properties. From its behavior on free electrophoresis, and judging from ultracentrifugal sedimentation and immunological studies, it appeared that a cytokinase preparation with a specific activity of 3000 units/rag protein was about 70% pure. Cytokinase has /~o.1%--0.87 at 280 nm) and does an absorption maximum at 280 nm ~1o~ -not contain enough hexose or sialic acid to be a glycoprotein. It has a s°0.,, 2.9-3.1 S and a molecular weight of about 50,000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point at pH 8.6. ~ Activators and Inactivators. Although Triton X-100 appears to enhance the activity of crude tissue activator, presumably due to the disruption of subcellular membranes, 9 it does not have any enhancing effect on the activity of purified cytokinase. 4,7 Like urokinase, cytokinase is also inhibited in a competitive manner by ~-aminocaproic acid (0.2M) and aminocyclohexane-carboxylic acid (50 mM). At high inhibitor-tosubstrate ratio some nonspecific side effects are apparent and a partial competitive type of inhibition may be taking place. Cytokinase is also inhibited by arginine (50 raM) and by cysteine (10 mM) but is unaffected by p-chloromercuribenzoate (0.1 mM) and iodoacetamide (5 mM). Specificity. Cytokinase is specific for activation of plasminogen to plasmin although the mode of action is not completely understood. Like urokinase, and unlike streptokinase and staphylokinase, cytokinase can activate plasminogen from various mammalian species. It does not have any proteolytic activity against casein or when examined on a heatedfibrin plate in which the plasminogen has been inactivated. Its action on synthetic amino acid substrates has not been examined but, if its enzymatic nature is similar to urokinase, it should have an esterase activity toward lysine substrates. Kinetic Properties. Cytokinase activates plasminogen maximally at pH 8.5 and at 37 °. The kinetics of activation are those of a zero-order reaction. When the concentration of cytokinase is adjusted with streptokinase and urokinase, in terms of their individual units of activity, it appears to have the same reaction velocity. On this basis 1 Christensen unit of streptokinase was approximately equal to 4 Ploug units of urokinase or to 40 units of cytokinase. Cytokinase appears to follow typical Michaelis-Menten kinetics when activating plasminogen. Experimental points fit the Lineweaver and 9 C. H. Lack and S. Y. Ali, Nature 201, 1030 (1964).
838
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[55]
Burk plots and cytokinase and urokinase possessed the same Michaelis constant toward human plasminogen with a K~ ~ 3.1 X 10-~ M. D i s t r i b u t i o n . Tissue activator or cytokinase is present in most mammalian tissue 1,~ and the content in each varies from species to species. Kidney, lung, and spleen are found consistently to have a high tissue activator content. Its presence in the liver can be demonstrated only if the inhibitors present in a homogenate are removed in the supernatant fluid after differential centrifugation. Subcellular fractionation shows that tissue activator is distributed in the lysosome and microsome fractions and the proportion in either varies in different tissues. 4,lo The presence of a high content of cytokinase in the microsome fraction may imply that the tissue is synthesizing the activator. Its predominance in the lysosomes may indicate the storage capacity of that tissue and the release of activator can then be controlled by labilizers and stabilizers of lysosomes. It should be noted that cytokinase is a basic protein, with a dominant positive charge at physiological pH, and will therefore tend to form electrostatic complexes with negatively charged polymers and structural components such as ribonucteic acids, acid glycoproteins, and mucopolysaccharides. The relationship of plasminogen activators in tissues, in blood, and in other body fluids has not been determined satisfactorily. The vascular endothelium of veins and venules is now considered to be a major source of tissue activator as well as of blood activator. 1,11,12 Urokinase which was once considered to be filtered blood activator, has been shown to originate in the kidney :and urinary tract by tissue culture and other experiments.4,18,14 Rabbit kidney cytokinase has been shown to be similar to human urokinase 4 but its relationship to blood activator is not yet clear.
mE. L. Beard, M. H. Montuori, G. J. Danos, and R. W. Busuttfl, Proc. Soc. Exptl. Biol. Med. 129, 804 (1968). A. S. Todd, J. Pa$hol. Bacteriol. 78, 281 (1959). n R. Holemans, D. McConnell, and J. G. Johnstone, Thromb. Diath. Haemorrhag. 25, 192 (1966). n R. H. Painter and A. F. Charles, Am. J. Physiol. 202, 1125 (1962). 14K. Buluk and M. Malofiejew,Acta Physiol. Polon. 14, 351 (1963).
[66]
INHIBITORS OF PROTEOLYTIC ENZYMES
839
[ 6 6 ] N a t u r a l l y O c c u r r i n g I n h i b i t o r s of P r o t e o l y t i c E n z y m e s
By
BEATRICE KASSELL
Proteinase inhibitors are widely distributed in plants and animals, and a very large number of them have been reported. These chapters are restricted to some representative examples of well-characterized inhibitors from different sources. Other chapters in this volume cover inhibitors present in blood [68] and the secretory inhibitors of pancreas [67]. The book by Vogel, Trautschold, and Werle 1 is an excellent source of further information and includes data on all known inhibitors. Useful reviews are also available. 2,3 The assays and units in this series of articles are those used by the individual investigators whose purification procedures are described. Because so many different assays and variations of the same assay are used, it has not been possible to express t'he units of inhibiting activity in any uniform manner. This problem (previously discussed by Vogel et aU) is a serious one. Although methods and units have been recommended as international standards for trypsin and chymotrypsin, * it is unfortunate that these have not been generally adopted. Most of the inhibitors have been isolated as trypsin inhibitors. However, almost without exception, they inhibit other enzymes. Their function in pl:ant and animal tissues is a subject of considerable interest, although speculative at present. I am grateful to many colleagues in this field for personal communications that aided substantially in the preparation of t'hese articles. Thanks are due particularly to Drs. Y. Birk, D. Cechovh, J. W. Donovan, R. E. Feeney, H. Fritz, M. Laskowski, Jr., J. Mikola, S. Moore, R. J. Peanasky, P. Portmann, J. Pudles, J. J. Rackis, E. Sach, and E. Werle.
I R. Vogel, I. Trautschold, and E. Werle, "Natiirliche Proteinasen-lnhibitoren." Thieme, Stuttgart, 1966; also available updated in English translation, Academic Press, N e w York, 1968. 2 M. Laskowski and M. Laskowski, Jr.,Advan. Protein Chem. 9~ 203 (1954). 3 p. Desnuelle, in "The Enzymes" (P. D. Boyer, H. Lardy, and K. MyrbRck, eds.), Vol. 4, p. 128. Academic Press, N e w York, 1960. 4International Commission for the Standardization of Pharmaceutical Enzymes,
First Report, J. Mondial de Pharmacie 1, 6 (1965).
840
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66a] A Trypsin
[66a]
Inhibitor from Barley
B y BEATRICE KASSELL
Introduction Trypsin inhibitors have been isolated from several grains, e.g., whole wheat flour, 1,2 corn seed, s r y e and wheat germ, 4 and barley2 The inhibitor from barley, prepared by Mikola and Suolinna, 5 has been selected for detailed description. The presence of proteolytic inhibitors in barley had been noted previously2,7 Some properties of the other inhibitors are included in Table II. AssayS, 8
Principle. Inhibition of the tryptic hydrolysis of benzoylarginine pnitroanilide (BAPA) is measured by the change in absorbance at 410 nm. Reagents
Buffer 1: Tris chloride, 50 raM, pH 8.2. Just before use, add 1 ml of 2 M CaC12 to 100 ml of buffer. Substrate: Dissolve (by gently warming) 43.5 mg of DL-BAPA.HCI (Nutritional Biochemicals Corp., Cleveland, Ohio) in 1 ml of dimethyl sulfoxide. Be sure no crystals remain. Dilute to 100 ml witch warmed buffer 1. Use within 1 hour. Buffer 2: Sodium acetate, 50 mM, pH 5.4 containing 20 mM CaC12 Enzyme solution: 0.1 unit (about 100 #g) per milliliter in buffer 2 Inhibitor solution: Dilute with buffer 2 to a concentration to give about 50% inhibition of the trypsin (about 30 ~g/ml of purified inhibitor). Acetic acid: 3 0 ~ in water (v/v) Procedure. Pipette 5 ml of substrate and 0.5 ml of water into a series of 18 X 150 mm test tubes in a 25 ° bath. In a second series of small tubes in the bath, place 1 ml of trypsin plus 1 ml of buffer or 1 ml of
I G. Shyamala, B. M. Kennedy, and R. L. Lyman, Nature 192, 360 (1961). 2 G. Shyamala and R. L. Lyman, Can. J. Biochem. 4~, 1825 (1964).
' K. Hoehstrasser, M. Muss, and E. Werle, Z. Physiol. Chem. 348, 1337 (1967). 4 K. Hochstrasser and E. Wer]e, Z. Physiol. Chem. 350, 249 (1969). 5j. Mikola and E.-M. Suolinna, European J. Biochem. 9, 555 (1969). ej. Laporte and J. Tr~moli~res, CompL Rend. Soc. Biol. 156, 1261 (1962). TW. C. Burger and H. W. Siegelman, Plan~ Physiol. 19, 1089 (1966). SB. F. Erlanger, N. Kokowsky, and W. Cohen, Arch. Biochem. Biophys. 95, 271 (1961).
[66a]
A TRYPSIN INHIBITOR FROM BARLEY
841
trypsin plus 1 ml of inhibitor solution for standards and unknowns, respectively. Incubate 10 minutes. Timing with a stopwatch, add 0.5 ml of the enzyme (or enzyme and inhibitor) to the substrate and mix well. Exactly 10 minutes later, stop the reaction by adding 1 ml of 30% acetic acid. Measure the absorbance at 410 nm against a blank without enzyme or inhibitor. Subtract the absorbance of the unknowns from the absorbance of the trypsin standard. Definition of Unit2 One unit of enzyme is the amount that liberates 1 micromole of p-nitroaniline per minute. One unit of inhibitor decreases the reaction rate by one trypsin unit. Specific activity is expressed as inhibitor units per A2~. Purification
Procedure
5
The procedure is summarized in Table I. Operations are at room temperature except as indicated. Step I. Extraction. The starting material is Pirkka barley (a Finnish six-row variety, see Distribution). Suspend 1 kg of finely ground barley in 2 liters of water; acidify to p H 4.9 with 1 M acetic acid. Extract for 2 hours at 5 ° without agitation and centrifuge. Step 2. Heat Precipitation. Divide the extract into 250-ml portions in 500-ml conical flasks. H e a t 15 minutes in a boiling water bath. Cool to room temperature and centrifuge. Step 3. Ammonium Sul]ate Precipitation. Precipitate the active material by adding dry ammonium sulfate to 40% saturation at 5 ° . Stir for 1 hour at 5 ° and centrifuge. Resuspend the precipitate in 200 ml of water. TABLE I PURIFICATION OF BARLEY TRYPSIN INHIBITORa
Fraction
Volume (ml)
Total activity (units)
Extract After treatment at 100° Ammonium sulfate precipitate Same after dialysis DEAE-cellulose peak 20% Ethanol precipitate Sephadex G-75 peak 2nd Ethanol precipitate Final product
1,430 1,300 265 330 120 9.8 91 10.5 130 mg
738 650 597 525 412 400 355 345 333
Specific activity Recovery (units/A~so) (%) ---0.37 0.97 1.33 1.83 1.90 1.90
100 89 81 71 56 54 48 47 45
J. Mikola and E.-M. Suolinna, European J. Biochem.9, 555 (1969) (reproduced by permission of Springer-Verlag, Heidelberg and of the author).
842
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66a]
Stir for 30 minutes. Separate the inactive precipitate by centrifuging and wash it once with 70 ml of 0.1 M sodium acetate buffer of pH 4.9. Combine the two active supernatants, and dialyze against 2 X 10 liters of 10 mM Tris chloride buffer of pH 7.5 at 5 °. If a precipitate forms during dialysis, centrifuge and extract the solid (containing 5-15% of the activity) with 25 mM acetic acid. Adjust the extract to pH 7.5 and add it to the main solution. Step 4. Chromatography on DEAE-Cellulose. Prepare a 4 X 14 cm column of DEAE-cellulose, previously recycled with 0.5M HC1 and 0.5 M NaOH, adjusted to pH 7.5 and equilibrated with 10 mM Trischloride of pH 7.5, the starting buffer. Apply the solution of step 3. Elute inert proteins first with starting buffer and then elute the active material with 60 mM Tris chloride of pH 7.5. Pool the active peak and precipitate the inhibitor by adding 20 volumes percent of ethanol at --6 ° . Centrifuge after 30 minutes and dissolve the precipitate in 5-15 ml of 0.1 M sodium acetate buffer of pH 4.4. Step 5. Gel Filtration on Sephadex G-75. Prepare a 2.5 >( 95 cm column of the Sephadex equilibrated with sodium acetate buffer pH 4.9, ionic strength 0.1. Elute with the same buffer, collecting 5-ml fractions at the rate of 20 ml/hour. Some inactive material is eluted before the active peaks, which appears approximately in tubes 55 to 70. Pool the active peak and dialyze overnight at 5 ° against 1 liter of water. Adjust the dialyzed solution to pH 7.5 with N a 0 H and precipitate the inhibitor by adding 20 volumes percent of ethanol at --6 ° . Dissolve the precipitate in a few milliliters of 15 mM acetic acid and lyophilize. The yield is about 130 mg. Properties 5
Stability. The inhibitor is stable to exposure to 100 ° for 15 minutes at pH 4.9, and to 1 hour's exposure to pH 2.1 at 25 °. It is completely destroyed by incubation with pepsin under ~he latter conditions. Purity. The purified inhibitor is homogeneous on disc electrophoresis at pH 3.5 and on ultracentrifugation at p H 5.4. Physical Properties. The molecular weight is 14,400 by equilibrium eentrifugation. The s°s0.w is 1.79 S. The partial specific volume is 0.718 calculated from amino acid composition. The frictional ratio (]/]min) is 1.26. A solution of 1 mg of inhibitor per milliliter 'has an absorbance of 1.27 at 280 nm. Specificity. The barley inhibitor has equal effect on the tryptic digestion of casein, hemoglobin, and BAPA. The inhibitor is totally inactive against the proteolytic enzymes of
[66a]
A TRYPSIN INHIBITOR FROM BARLEY
843
g e r m i n a t e d b a r l e y : green m a l t e n d o p e p t i d a s e (gelatin s u b s t r a t e ) , green malt peptidase (BAPA substrate), and barley carboxypeptidase (Nc a r b o b e n z o x y p h e n y l a l a n y l - a l a n i n e s u b s t r a t e ) . I t does n o t affect the h y drolysis of casein b y c h y m o t r y p s i n , p a p a i n , s u b t i l o p e p t i d a s e A, Aspergillus oryzae proteinases, p H 6.5 a n d 10.3, Streptomyces griseus p r o t e i n a s e s or Pseudomonas fluorescens proteinases. I t does n o t i n h i b i t the h y d r o l y s i s of h e m o g l o b i n b y pepsin at p H 2. Kinetic Properties. I n h i b i t i o n of t r y p t i c digestion of casein or B A P A is l i n e a r u p to 65% i n h i b i t i o n . T h e m o l a r ratios of t r y p s i n to i n h i b i t o r are 0.94 a n d 1.12, respectively, for the two s u b s t r a t e s at 50% i n h i b i t i o n . TABLE II AMINO ACID COMPOSITION AND OTHER PROPERTIES OF TRYPSIN INHIBITORS FROM SEVERAL GRAINS
Barley~ Amino acid Alanine Arginine Aspartic acid Half-cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Amide groups Carbohydrate
Corn seedb
Rye germ~
Wheat germc
Residues per molecule 10 9 10 10 14 10 3 5 9 2 2 3 11 8 7 3 5 6 (12) 0
21 17 12 9 20 22 2 12 20 2 1 2 25 13 14 -3 9 -0
12 15 15 24 12 7 1 5 2 10 2 5 25~ 17 11 -3 7 --
9 15 15 24 10 9 0 5 3 10 2 5 23d 15 11 -3 9 --
Molecular weight
14,300
21,190
17,000 d
17,000 ~
Amino terminus Carbuxyl terminus
---
Serine Isoleucine
Serine --
Serine --
J. Mikola and E.-M. Suolinna, European d. Biochem. 9, 555 (1969). b K. Hochstrasser, M. Muss, and E. Werle, Z. Physiol. Chem. 348, 1337 (1967). * K. Hochstrasser and E. Werle, Z. Physiol. Chem. $50, 249 (1969). d Approximate value.
844
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[55b]
Distribution. Similar trypsin-inhibiting activity is present in 2-row varieties of ,barley tested (e.g., Balder, Ingrid, Zephyr). Trypsin inhibitor activity has been detected in several tissues of barley? The inhibitor of endospermal tissues (aleurone layer and starchy endosperm) is immunologically identical to the inhibitor obtained by the isolation procedure described. The inhibitors of embryos, coleoptiles, and rootlets are different from this inhibitor. A m i n o Acid Composition. A comparison of the barley inhibitor with some other grains is shown in Table II. The lack of resemblance of the barley inhibitor to the others may be explained by its derivation from a different tissue (see Distribution).
9j. Mikola and M. Kirsi, personal communication (1970).
[66b] Bovine Trypsin-Kallikrein Inhibitor (Kunitz Inhibitor, Basic Pancreatic Trypsin Inhibitor, Polyvalent Inhibitor from Bovine Organs) B y BEATRICE KASSELL
Introduction This protein was first isolated by Kunitz and Northrop 1 from bovine pancreas as a trypsin inhibitor. Later it was found that a kallikrein inactivator widely distributed in bovine tissues is identical in its properties 2 and in amino acid sequence 8 to the Hunitz trypsin inhibitor. ~,s It differs in structure and other properties from the specific trypsin inhibitor of the pancreatic juice 6 (this volume, [67]). The inhibitor is commercially available in highly purified form, e.g., as Iniprol (Laboratoire Choay, Paris), as Trasylol (Farbenfabriken Bayer AG, Elberfeld, Germany), as Basic Pancreatic Trypsin Inhibitor (Worthington Biochemical Corp., Freehold, N.J.). A related inhibitor has been isolated from bovine colostrum. 6a,6b A comparison of the two structures is included in this article. 1M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). 2H. Kraut, N. Bhargava, F. Schultz, and H. Zimmermann, Z. Physiol. Chem. 334, 230 (1964). F. A. Anderer, Z. Natur]orseh. 20b, 462 (1965). 4B. Kassell, M. Radicevic, S. Berlow, R. J. Peanasky, and M. Laskowski, Sr., J. Biol. Chem. 238, 3274 (1963). 5B, Kassell and M. Laskowski, Sr., Biochem. Biophys. Res. Commun. 202 463 (1965). eL. J. Greene and D. C. Bartelt, J. Biol. Chem. 244, 2646 (1969).
844
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[55b]
Distribution. Similar trypsin-inhibiting activity is present in 2-row varieties of ,barley tested (e.g., Balder, Ingrid, Zephyr). Trypsin inhibitor activity has been detected in several tissues of barley? The inhibitor of endospermal tissues (aleurone layer and starchy endosperm) is immunologically identical to the inhibitor obtained by the isolation procedure described. The inhibitors of embryos, coleoptiles, and rootlets are different from this inhibitor. A m i n o Acid Composition. A comparison of the barley inhibitor with some other grains is shown in Table II. The lack of resemblance of the barley inhibitor to the others may be explained by its derivation from a different tissue (see Distribution).
9j. Mikola and M. Kirsi, personal communication (1970).
[66b] Bovine Trypsin-Kallikrein Inhibitor (Kunitz Inhibitor, Basic Pancreatic Trypsin Inhibitor, Polyvalent Inhibitor from Bovine Organs) B y BEATRICE KASSELL
Introduction This protein was first isolated by Kunitz and Northrop 1 from bovine pancreas as a trypsin inhibitor. Later it was found that a kallikrein inactivator widely distributed in bovine tissues is identical in its properties 2 and in amino acid sequence 8 to the Hunitz trypsin inhibitor. ~,s It differs in structure and other properties from the specific trypsin inhibitor of the pancreatic juice 6 (this volume, [67]). The inhibitor is commercially available in highly purified form, e.g., as Iniprol (Laboratoire Choay, Paris), as Trasylol (Farbenfabriken Bayer AG, Elberfeld, Germany), as Basic Pancreatic Trypsin Inhibitor (Worthington Biochemical Corp., Freehold, N.J.). A related inhibitor has been isolated from bovine colostrum. 6a,6b A comparison of the two structures is included in this article. 1M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). 2H. Kraut, N. Bhargava, F. Schultz, and H. Zimmermann, Z. Physiol. Chem. 334, 230 (1964). F. A. Anderer, Z. Natur]orseh. 20b, 462 (1965). 4B. Kassell, M. Radicevic, S. Berlow, R. J. Peanasky, and M. Laskowski, Sr., J. Biol. Chem. 238, 3274 (1963). 5B, Kassell and M. Laskowski, Sr., Biochem. Biophys. Res. Commun. 202 463 (1965). eL. J. Greene and D. C. Bartelt, J. Biol. Chem. 244, 2646 (1969).
[55b]
BOVINE TRYPSIN-KALLIKREIN INHIBITOR
845
Assay Method 7
Principle. Inhibition of tryptic hydrolysis of N-a-benzoyl-DL-argininep-nitroanilide (BAPA) is measured by following the change in absorbonce at 405 nm. Reagents Substrate: Dissolve 100 mg of BAPA with warming in 100 ml of water. Buffer: 0.2 M Triethanolamine-HC1, pH 7.8 Trypsin: (Trypure, 8 Novo Industri A/S, Copenhagen). Dissolve 10 mg in 100 ml of 0.001 M HC1. Store in the cold. Diluted inhibitor solution in buffer: 0.2 ml approximately equivalent to 10 ~g of trypsin. Procedure. Place 3-ml, 1-cm cuvettes in a spectrophotometer connected to a circulating water bath at 25 °. Equilibrate the solutions in the water bath. Incubate 0.2 ml of trypsin solution, 0.2 ml of in~hibitor solution, and 1.6 ml of buffer for 3 minutes in the cuvette. Start the enzymatic reaction by adding 1 ml of substrate. Mix with a plastic spatula. Record the increase in absorbance at 405 nm for 5 minutes. Repeat the determination without the inhibitor. Definition of Unit and Specific Activity. One enzyme unit corresponds to the hydrolysis of 1 micromole of substrate per minute (AA4oJmin ---3.32 for 3 ml. One inhibitor unit decreases the activity of two enzyme units by 50%, thus decreasing the absorbance by 3.32/minute for 3 ml. The specific activity equals inhibitor units per milligram protein. Purification Procedure The purification is based on the selective adsorption of the inhibitor from a crude extract by an insoluble polyanionic derivative of trypsin (trypsin-resin) originated by Katchalski and co-workers. 9 The trypsininhibitor complex, attached to the resin, is washed free of contaminating material. The inhibitor is then removed by dissociating the complex under *aD. Pospi~ilovh, V. Dlouh~, and F. ~orm, Fifth Meeting, Federation European Biochem. Soc. Abstr. No. 971 (1968). 6bD. ~echovh-Pospi~ilovh,V. Svestkovh, and F. ~orm, Sixth Meeting, Federation European Biochem. Soc. Abstr. No. 225 (1969). H. Fritz, G. Hartwich, and E. Werle, Z. Physiol. Chem. 345, 150 (1966). SThis is a stabilized trypsin preparation. If another kind of trypsin is used, add 0.02 M CaC12to the buffer. 9y. Levin, M. Pecht, L. Goldstein, and E. Katchalski, Biochemistry 3, 1905 (1964).
846
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[55b]
acidic conditions. The procedure is that of Fritz et al. 1°-12 This method is particularly suited to the Kunitz inhibitor. Because of the extreme resistance of this protein to enzymatic hydrolysis, it is recovered entirely in its native form from the complex. 12a Lung has been selected as starting material, but liver or parotid gland may be used. The amount of tissue processed may be increased easily. All operations are carried out at 00-4 ° .
Preparation o] Trypsin-Resin Titration o] Maleic Anhydride-Ethylene Copolymer (EMA). ~ The product, obtained from Monsanto Chemical Co., Inorganic Chemicals Division, Des Plaines, Ill., varies from type to type and from lot to lot. Determine both the free carboxyl groups and the rate of saponification by titrating with a pH-stat as follows. Place a weighed amount of resin (about 1 rag) in a titration vessel of 2.5 ml volume. Introduce the electrodes and a magnetic stirrer. Add 1.2 ml of water. Record the uptake of 10 mM NaOH, added from a microburet. Calculate the content of free carboxyl groups from the initial rapid uptake of NaOH, i.e., from the beginning of the titration to the break in the curve. Resins with 1-5% of free carboxyl groups are suitable. To convert an excessive number of free carboxyl groups back to the anhydride, heat the resin at 105°-110 ° for 24 hours in vacuo, 9 then repeat the determination. The time for complete saponification, determined with different types and lots of EMA, varied from 0.17 to 43 hours. Binding Capacity and Binding Rate o] the Resin ]or Trypsin. ~ Add 20 mg of EMA to an ice-cooled (00-4 °) mixture of 2 ml of 0.1% hexamethylene diamine solution and 7.5 ml of 0.2 M triethanolamine buffer of pH 7.8. Homogenize the suspension briefly, then stir briskly with cooling. Two minutes after adding the resin, add a cold solution of 100 mg of trypsin in 7.5 ml of the same buffer. Remove aliquots every few minutes for rapidly saponifying resins and at longer intervals for slowly saponifying resins. Filter the aliquots at once and determine the trypsin in the filtrate. This is the amount not bound to the resin. For resins with 1-5% of free carboxyl groups, trypsin binding reaches 1oH. Fritz, H. Schult, M. Hutzel, M. Wiedemanu, and E. Werle, Z. Physiol. Chem. 348, 308 (1967). 11H. Fritz, M. Gebhardt, E. Fink, W. Schramm, and E. Werle, Z. Physiol. Chem. 350, 129 (1969). H. Fritz, personal communication (1969). ~a A simpler method for purification of the inhibitor by affinity chromatography is being developed in the author's laboratory, and will be ready for publication in the latter part of 1970.
[56b]
BOVINE TRYPSIN-KALLIKREIN INHIBITOR
847
its maximum of 80-95% within a few minutes. As the free carboxyl groups rise above 5%, the amount of trypsin bound falls rapidly. With resins of too low free carboxyl content, the binding of the enzyme is complete only after 6-10 hours. Trypsin-Resin Reaction. 1° Suspend 500 mg of copolymer in 50 ml of 0.2M potassium phosphate buffer, pH 7.5, homogenize briefly (cooling in ice), and dilute to 500 ml with the same buffer. Add 50 ml of 0.1% hexamethylene diamine in water and stir for 3 minutes. Add 2.5 g of trypsin (Novo Industries) dissolved in 250 ml of the phosphate buffer and stir for 12 hours with the pH maintained at 7.5. Dilute with 2 volumes of water and centrifuge. Calculate the trypsin content of the resin by determining the activity of the supernatant solution. To obtain a suitable flow rate, mix the solid enzyme-resin with a 2to 4-fold volume of cellulose powder (No. 123, Schleicher and Schull, Keene, N.H.), previously suspended and decanted several times. Pour the resin into a column of 4 cm diameter. Wash with 0.1 M triethanolamine buffer, pH 7.8, containing 0.3 M NaCl and 0.01 M CaCl~ until no more active trypsin can be detected in the effluent. The column may be stored for a long'period at 0 ° in the buffer, and may be reequilibrated and used at least 100 times without loss of activity. Each gram of trypsin attached to the resin will retain about 70 mg of inhibitor.
Inhibitor Purification Step 1. Crude Extract. 1°,12,~ Homogenize 1 kg of lung (containing approximately an amount of inhibitor equivalent to 600 mg of the Novo Trypure or 600 units) with 1 liter of 0.1 M triethanolamine buffer, pH 7.8, containing 0.3 M NaC1 and 0.01 M CaC12. Stir the mixture for 1 hour. Add 100 g of Celite 545 and centrifuge for 1 hour at 13,000 g in a refrigerated centrifuge. Resuspend the solid in an equivalent volume of the same buffer and centrifuge again. Combine the two supernatant solutions. Add 50% trichloroacetic acid (w/v) to a final concentration of 2.5%. Allow the solution to stand for 30 minutes at room temperature. Centrifuge as before and adjust the supernatant solution to pH 7.8 with 1 N NaOH. Determine the amount of inhibitor in the supernatant solution. Step 2. Affinity Chromatography. Pass the inhibitor solution through the column at a rate of 2 ml/min, collecting 15-ml fractions of effluent, until all the solution has passed through or until excess inhibitor appears in the effluent. Wash the column with the same buffer until the effluent is entirely protein-free. Elute the inhibitor with 0.25 M KC1-HC1 buffer ~*A few details have been added by the present author.
848
N A T U R A L L Y OCCURRING ACTIVATORS A N D INHIBITORS
[65b]
of p H 1.7-2.0 until ~he effluent is free from inhibitor. Reequilibrate the column with the p H 7.8 buffer. Step 3. Concentration and Desalting. Adjust the p H of the combined inhibitor fractions to 5--6 with 1 M K O H and concentrate the solution on a r o t a r y e v a p o r a t o r at 30 °. Remove the precipitated salts by centrifuging and wash the precipitate with a small amount of water. Desalt by passing through a column of Sephadex G-25, 2 X 50 cm, equilibrated with 0.1 M a m m o n i u m bicarbonate buffer of p H 7.0. Lyophilize the fractions containing inhibitor. At this stage, the inhibitor is 70-80% pure with 85% yield. Step 4. Final Purification. T h e final purification m a y be carried out by a second passage through the trypsin-resin. TM Alternate final purification steps are c h r o m a t o g r a p h y on CM-cellulose ~4'~ or on Amberlite IRC-50. * Properties
Stability. T h e inhibitor shows r e m a r k a b l e stability to high t e m p e r a ture, acid, alkali, and enzymes. I t m a y be heated in dilute acid TM at 100 ° and in 2.5% trichloroacetic acid 1 at 80 ° without loss of activity. I t m a y be k e p t for 18 months at room t e m p e r a t u r e in 0 . 1 4 M NaC1 without loss of activity. 14 The activity remains constant for 24 hours at room temperature up to p H 12.6, but begins to decrease at p H 12.8.1. The inhibitor is not inactivated by pepsin, ~8'1° trypsin, ~8'2° c h y m o t r y p s i n a ~8,2° or B, 2~ Pronase,lS, 2° kallikrein, 2° plasmin, ~° elastase, ~° collagenase, 2° carboxypeptidase A *~ or B, 2° cobra venom, 2° R u s s e l l ' s viper venom, 2° ficin, z° papain, 2° bromelain, *° Aspergillus oryzae protease, *° Bacillus subtilis proteases 2° A and B, Leucostoma peptidase A 2~ (from v e n o m of the western cottonmouth moccasin22), Streptomyces griseus proteases 2~ VI, V I I , and V I I I , silkworm digestive juice alkaline proteinase, 2~,22~ or crude 14E. Sach, M. Th6ly, and J. Choay, Compt. Rend. Aead. Sci., Paris 260, 3491 (1965). R. Avineri-Goldman, I. Snir, G. Blauer, and M. Rigbi, Arch. Biochem. Biophys. 121, 107 (1967). 16N. M. Green and E. Work, Biochem. J. 54, 257, 347 (1953). 1, M. P. Sherman and B. Kassell, Biochemistry 7, 3634 (1968). ~B. Kassell and M. Laskowski, Sr., J. Biol. Chem. 219, 203 (1956); Federation Proc. 24, 593 (1965). It. Kraut and N. Bhargava, Z. Physiol. Chem. 334, 236 (1963). H. Kraut and N. Bhargava, Z. Physiol. Chem. 348, 1498 (1967). 21B. Kassell and T.-W. Wang, unpublished experiments. F. W. Wagner, A. M. Spiekerman, and J. M. Prescott, J. Biol. Chem. 243, 4486 (1968). A gift of the enzyme is gratefully acknowledged. tl, We are grateful to Dr. J. I. Mukai, Kyushu University, Japan, for a gift of this enzyme.
[66b]
BOVINE TRYPSIN-KALLIKREIN INHIBITOR
849
Penicillium notatum proteinase. 21,22b It is, however, digested by thermolysin at 60°-80°. 2~c Purity. Inhibitor preparations purified by earlier chromatographic methods ~,14,15,2~ were homogeneous by electrophoresis, sedimentation, chromatography, amino acid analysis, and end group deLermination. Inhibitor purified as described in this article gives the correct amino acid analysis ~2 after step 4. Physical Properties. The molecular weight by osmotic pressure 1 is 6000 and by amino acid analysis ~ is 6513. In the ultracentrifuge, the inhibitor shows a monomer-dimer equilibrium, with the dimer predominating in neutral solution. The molecular weights are 11,600-11,700 in 0.9% NaC124 and 10,300-11,800 in 0.8% NaC1 at pH 7.3. 2~ At the extremes of the pH range, the monomer predominates. The molecular weights are 6500 in 8 M urea 25 at pH 10.5, 6700 in 0.1 M acetic acid, 25 6500 +__20026 at pH 1.1 and 10.5. The sedimentation constant (S2o,~) o is 0.9126 to 1.0. ~ The frictional coefficient2e (]/fo) is 1.387. The isoelectric point is at pH 10~e to 10.5Y The absorbance ~,~6 t,~xa"%1 e m at 280 nm) is 8.3. The inhibitor is soluble ~6 in water, 70% methanol, 70% ethanol, and 50% acetone. The optical rotation ~ ([a]D per milligram of nitrogen) is --65 °. The dispersion constant 2s of the one-term Drude equation (X~ = 234 nm) and the Moffitt constant 2s (bo = 169) are independent of pH and are not altered by the addition of urea or 2-chloroethanol. Specificity. The following enzymes are inhibited: Trypsins of cow, ~ pig, 2° man, 29 and turkey, 3° acetyl trypsin, 15 bovine chymotrypsins 3~ a and B (the latter only slightly), chicken chymotrypsin, 3° bovine 32 and porcine 2°,33 kallikreins, rabbit, 33~ human 29,~4,~4~ and porcine 2° plasmin, and =b W. E. Marshall and J. Porath, J. Biol. Chem. 240, 209 (1965). We are indebted to Dr. Marshall for a sample of this proteinase. 2~¢T.-W. Wang and B. Kassell, Biochem. Biophys. Res. Commun., in press. ~*V. Dlouh£, J. Neuwirthov£, B. Meloun, and F, ~orm, Collect. Czech. Chem. Commun. 30, 1705 (1965). ~H. Kraut, W. KSrbel, W. Scholtan, and F. Schultz, Z. Physiol. Chem. 321, 90 (1960). F. A. Anderer and S. HSrnle, Z. Natur]orsch. 20b, 457 (1965). W. Scholtan and S. Y. Lie, Makromol. Chem. 98, 204 (1966). J. Chauvet, G. Nouvel, and R. Acher, Biochim. Biophys. Acta 92, 200 (1964). ~sW. Scholtan and H. Rosenkranz, Makromol. Chem. 99, 254 (1966). R. E. Feeney, G. E. Means, andJ. C. Bigler, J. Biol. Chem. 244, 1957 (1969). ~C. A. Ryan, J. J. Clary, and Y. Tomimatsu, Arch. Biochem. Biophys. 110, 175 (1965). 31F. C. Wu and M. Laskowski, Sr., J. Biol. Chem. 213, 609 (1955). = H. Kraut, E. K. Frey, and E. Bauer, Z. Physiol. Chem. 175, 97 (1928). E. Sach and M. Thfily, Compt. Rend. Acad. Sci., Paris 266, 1200 (1968).
850
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[66b]
a trypsinlike component of Pronase. 35 Numerous other enzymes have been tested, but are not inhibited: pepsin, ~s elastase, ~5,2° liver esterase, a8 thrombin,2~,3~ carboxypeptidase A ~ and B, 2° renin, as angiotensin converting enzyme, 39 angiotensinase, 4° enterokinase, 4~ ribonuelease, ~ lyzozyme, ~ ribosomal polypeptide chain elongation factors, ~2 Bacillus subtilis proteases 2° A and B, Aspergillus oryzae protease, 2° clostripain 48 (from Clostridium h i s t o l y t i c u m ) , ficin, 2° papain, 2° or thermolysin. 22b I t does not react with diisopropyl phosphoryl trypsin, 44 but does form a complex with trypsinogen. 4~a K i n e t i c Properties. T h e inhibition of trypsin is stoichiometric ~ and is considered to be competitive 4~,45 or pseudononcompetitive. ~ Values for the dissociation constant (K~) between 10-9 M and 3 X 10-~ M have been reported.~6,,~, 44, ~e,4~ T h e latter value, '~ determined at p H 7.8, is p r o b a b l y the most accurate. T h e change in K~ with p H is shown in T a b l e I for complexes with trypsin and kallikrein. The values for trypsin are in good agreement with those obtained earlier by Green 86 below p H 4. The reaction with kallikrein is noncompetitive, and complete inhibition is not achieved even with a large excess of inhibitor2 s The reaction of the inhibitor with trypsin is not instantaneous. The rate of the reaction increases ~e,~ with p H and is at its m a x i m u m ~ (about 1 minute) between p H 7 and p H 10.5 at 25 °. T h e bimolecular reaction rate constant ~ at 25 ° and p H 7.8 is 3.05 X 10 ~ liters/mole/second.
ua E. G. Vairel and M. Th$1y, Ann. Biol. Clln. (Paris) 18, 363 (1960). •V. Mansfeld, M. Ryb£k, Z. Hor~kov~, and J. Hladovek, Z. Physiol. Chem. 318, 0 (1960). ~*A. DeBarbieri, M. E. Scevola, and M. Franchini, Boll. Chim. Farm. 103, 37 (1964). u M. Trop and Y. Birk, Biochem. J. 109, 475 (1968). u N. M. Green, Biochem. J. 66, 407 (1957). C. J. Amris, Scand. J. Haema~ol. 3, 19 (1965). mL. T. Skeggs, personal communication (1968). my.. S. Bakhle, Nature 220, 919 (1968). R. Ripa and P. Gilli, Boll. ~oc. Ital. Biol. 8per. 44, 1297 (1968). "~J. P[itter, Z. Physiol. Chem. 348, 1197 (1967). K. Moldave, personal communication (1968). eB. Labouesse and P. Gros, Bull. Soc. Chim. Biol. 42, 543 (1960), and personal communication (1963). N. M. Green, J. Biol. Chem. 205, 535 (1963). " V. Dlouh~ and B. Keil, Federation European Biochem. Soc. Letters 3, 137 (1969). P. Jeanteur and E. Fournier, Compt. Rend. Acad. 8ci., Paris 260, 722 (1965). d D. Grob, J. Gen. Physiol. 33, 103 (1949). 4~H. Kraut and R. KSrbel-Enkhardt, Z. Physiol. Chem. 312, 161 (1958). I. Trautschold and E. Werle, Z. Physiol. Chem. 325, 48 (1961).
[55b]
BOVINE TRYPS1N-KALLIKREIN I N H I B I T O R
851
TABLE I pH-DEPENDENCE OF THE DISSOCIATION CONSTANTS OF THE COMPLEXES OF TRYPSIN AND KALLIKREIN WITH BOVINE TRYPSIN-KALLIKREIN INHIBITORa
pH
Trypsin-inhibitor complex KI (mole/liter)
Kallikrein-inhibitorcomplex K1 (mole/liter)
2.0 3.0 4.0 5.0 6.0 7.8
4.5 × 10-4 2.4 × 10-6 2.6 × 10-9 -1.3 X 10-l° 2 X I0-rib
--Fully dissociated 1.9 X 10-~ 3 X 10-e 1.2 X 10-8
• From R. Vogel, I. Trautschold, and E. Werle, "Natural Proteinase Iahibitors," p. 91. Academic Press, New York, 1968. Interpolated value. T h e rate of r e a c t i o n with k a l t i k r e i n is also p H - d e p e n d e n t a n d n o t i n s t a n t a n e o u s .'9 Distribution. "9 T h e i n h i b i t o r occurs i n - n u m e r o u s organs of cattle a n d goats: p a n c r e a s , lung, p a r o t i d gland, l y m p h nodes, spleen, a n d liver. Amino Acid Composition and Sequence. T h e composition is shown in T a b l e I I . T h e s t r u c t u r e shown in Fig. 1, i n d e p e n d e n t l y d e t e r m i n e d for TABLE II AMINO ACID COMPOSITION OF THE BOVINE TRYPSIN-KALLIKREIN INHI]BITORa,b
Amino acid
Residues per molecule
Amino acid
Alanine Arginine Aspartic acid Half-cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine
6 6 5 6 3 6 0 2 2 4
Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Amide groups Total N
Residues per molecule 1 4 4 1 3 0 4 1 4 17.65%
" B. Kassell, M. Radicevic, S. Berlow, R. J. Peanasky, and M. Laskowski, Sr., J. Biol. Chem. 238, 3274 (1963); determined with trypsin inhibitor isolated from pancreas. F. A. Anderer and S. HSrnle, Z. Naturforschung 206, 457 (1965), found the same composition for the lung kallikrein inhibitor. *R. Vogel, I. Trautschold, and E Werle, "Natural Proteinase Inhibitors," p. 91. Academic Press, New York, 1968.
852
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66b]
19 ~"TYR-- THR-- GLY - -
PRO--CYS -- L Y S - - A L A - - A R G ~'
PROJ ~ R G - L Y S - A L A - A ~ - C Y S
~Ul
~
~
"YSI!~-~.(
-'k."- r . ~ -
] l/
TM
/v~
ILE l
, ~ ~o
51 J 1 | /Pe ~ , ~ - MET- c Y s - ,~s~ | I
TYR
JT~
PHE
PHEI, Asp -
G L Y ~ GLY
~ "~ PROt
1111111
(rL[U--- Gl.N --~' [GLYL. . . CYS . . k,.L ALA --
LYS -- ~ A
,
1¥~
- - ASN -J
I~G. I. A schematic representation of the structure of the trypsin inhibitor (based on footnotes 3 and 50). the proteins isolated as t r y p s i n '° and kallikrein 8 inhibitors, has been confirmed b y two o t h e r groups. 51,~2 Figure 2 c o m p a r e s the basic p a n c r e a t i c inhibitor with the bovine colostrum inhibitor3 s T h e location of at least 21 a m i n o acid residues is identical. I n c o n t r a s t to the p a n c r e a t i c inhibitor, the colostrum inhibitor is a glycoprotein. A
A r g ~ P h e ] r ~ L e u . G l y ~ P r o - Tyr-Thr',-Gly-Pro-Cy s-Lys-Ala.~Arg-lle-lle iArg-
[3
Phe-GIn-Thr-Pro ~Pro-Asp~Leu~L~y JGIwPr~Pr~ln-AlaoA,~ly-Pro-Cy s-Lys-A~Ala-Leu-LeuTA, -Tyr-Phe-Tyr-'Asn-Ala-Lys-Ala-Gly-Leu.~s-Gin-Tyr'-~Val ,'-Tyr-Gly-Gly-Cy1Arg-Ala-Lys-Arg-Asn-
B
-Tyr-Phe-Tyr!Asx-Ser-Thr-Ser-A -Ala
lu-l~ro[~[Thr[-Tyr-Gl¥-Gly-Cys(asx,asx,asx,asx~Ix,
A
-Asn-Phe-Lys-Ser-Ala-Gly-Asi~r-~Met]rA-~Tyr~-ls~31Y-Gly-Ala
B
,gix ,glx, thr,thr,fly, met, phe)~Cy~Leu~lle . . ~ A. s x . Pro-Pro-GIx-GIx . Thr-Glx Lys-Ser
Fro. 2. Comparison of the amino acid sequences of the basic pancreatic inhibitor (A) and of the main component of the cow colostrum inhibitor (B). Residues occupying identical positions in both proteins are outlined. (Reproduced with permission of North Holland Publishing Company, Amsterdam.) roB. Kassell and M. Laskowski, Sr., Biochem. Biophys. Res. Commun. 20, 463 (1065). a R. Aeher and J. Chauvet, Bull. 8oe. Chim. France p. 3954 (1967). a V. Dlouh£, D. Pospi§ilov£, B. Meloun, and F. ~orm, Collect. Czech. Chem. Commun. 33, 1363 (1968). = D. Oechovh, V. SvestkovK, B. Keil, and F. $orm, Federation European Biochem. Soc. Letters 4, 155 (1969).
[66c]
SOYBEAN TRYPSIN AND CHYMOTRYPSIN INHIBITORS
[66c] Trypsin
and
Chymotrypsin
Inhibitors
from
853
Soybeans
B y BEATRIC~ KASSr~L
Introduction In 1946, Kunitz 1 crystallized soybean trypsin inhibitor and Bowman 2 discovered a second inhibitor. Since then, other inhibitors have been purified from soybeans, s,4,4a-7 but these have not been studied as much as the first two inhibitors and are not included in this article. For purification of the Kunitz inhibitor, the procedure described is t h a t of Rackis et al., 3,s based on chromatography on D E A E cellulose. Several other purification methods have been used: similar D E A E chromatographies, 5-7 preparative eleetrophoresis, 8a and isoelectric focusing2, 9a For the second inhibitor, the purification procedure is derived mainly from the work of Birk and co-workers, 1°,11 but includes modifications made by F r a t t a l P 2 and in the author's laboratory. The name BowmanBirk inhibitor has been used previously. ~,~3 The 1.93 S inhibitor isolated by Yamamoto and Ikenaka e is the same or very similar. Both inhibitors are available commercially, the Kunitz inhibitor from several sources (e.g., California Corporation for Biochemical Research, Los Angeles, Calif.; Gallard-Schlesinger Chemical Manufacturing Co., Garden City, New York; Novo Industri A/S, Copenhagen, Denmark; xM. Kunitz, J. Gen. Physiol. 29, 149 (1946). J D. E. Bowman, Proc. Soc. Exptl. Biol. Med. 63, 547 (1946). s j. j. Rackis, H. A. Sasame, R. L. Anderson, and A. K. Smith, J. Am. Chem. Soc. 81, 6265 (1959). ~Y. Birk and A. Gertler, Bull. Res. Council Israel, Sect. A 11, 48 (1962). ~l y. Birk, A. Gertler, and S. Khalef, Biochlm. Biophys. Acta 67, 326 (1963). sj. j. Rackis and t L L . Anderson, Biochem. Biophys. Res. Commun. 15, 230 (1964). *M. Yamamoto and T. Ikenaka, J. Biochem. (Tokyo) 62, 141 (1967). V. Frattali and R. F. Steiner, Biochemistry 7, 521 (1968). s J. J. Rackis, H. A. Sasame, R. K. Mann, R. L. Anderson, and A. K. Smith, Arch. Biochem. Biophys. 98, 471 (1962). s, V. Frattali and R. F. Steiner, Anal. Biochem. 27, 285 (1969). DN. Catsimpoolas, C. Ekenstam, and E. W. Meyer, Biochim. Biophys. Acta 175, 76 (1969). u"N. Catsimpoolas, Separation Sei. 4, 483 (1969). Y. Birk, Biochim. Biophys. Acta 54, 378 (1961). 11Y. Birk, A. Gertler, and S. Khalef, Biochem. J. 87, 281 (1963). u V. Frattali, J. Biol. Chem. 244, 274 (1969). =R. Vogel, I. Trautschold, and E. Werle, "Natiirliche Proteinasen-Inhibitoren," pp. 8-21. Thieme, Stuttgart, 1966.
854
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66c]
Nutritional Biochemical Corp., Cleveland, Ohio; Sigma Chemical Corp., St. Louis, Mo.; Worthington Biochemical Corp., Freehold, N.J.; and the Bowman-Birk inhibitor from Miles Laboratories, Elkhart, Indiana). None of these commercial preparations is electrophoretically homogeneous.~2,14 Isoelectric focusing has been used 9~ for further purification of both inhibitors from commercial sources.
Assay Method The casein digestion method of Kunitz ~5 has already been described in a previous volume of this treatise. ~e Definition o] Unit. Kunitz ~ defined one tryptic unit as the enzyme activity which causes an increase of one unit of absorbance at 280 nm per minute of digestion of casein, under the standard conditions. The specific activity is expressed as units per microgram of trypsin protein. Trypsin-inhibiting activity is expressed as units of trypsin inhibited, and specific activity as units of trypsin inhibited per microgram of inhibitor. The inhibitor unit, defined by Rackis, s is the amount of trypsin inhibited in micrograms by 1 pg of a standard commercial preparation of the Kunitz soybean inhibitor. Birk ~1 uses the original Kunitz definition of the unit (see above). Kunitz Soybean Inhibitor (SBTIA2) Purification Procedure 8,s, 1~,is Step 1. Extraction. The starting material is 100 g of defatted soybean meal, a commercial product made from seed-grade dehulled beans of the Adams or Hawkeye variety. Extract the meal at pH 7.2 by stirring slowly, to avoid foaming, for 1 hour at room temperature with 1 liter of water. Centrifuge and reextract the solid with 500 ml of water for ½ hour. Combine the supernatant solutions and adjust to pH 4.4 with 1 M HC1. Discard the precipitate. Bring the solution (the whey) to pH 8.0 with 1 N NaOH and allow to stand at 4 ° for 1 hour to precipitate the phytate salts. Centrifuge and dialyze the whey at 4 ° against two changes of distilled water. Lyophilize. The product (4 to 5 g of crude whey protein,
14A. C. Eldridge, R. L. Anderson, and W. J. Wolf, Arch. Biochem. Biophys. 115, 495 (1966). M. Kunitz, J. Gen. Physiol. 30, 291 (1947). x M. Laskowski, Sr., Vol. II [3]. ~ J. J. Rackis, Proc. Seed Protein Con]erence, New Orleans, p. 98, Southern Utilization and Development Division, Agricultural Research Service, U~S. Department of Agriculture (1963). J. J. Rackis, personal communication (1969).
L56c]
S O Y B E A NTRYPSIN AND CHYMOTRYPSIN INHIBITORS
855
depending on the source of the beans) contains 0.8-1.0 g of total trypsin inhibitor, of which about half is the Kunitz inhibitor. 18 Step ~. Chromatography on DEAE-Cellulose. Cycle DEAE-eellulose19 (Type 40, Brown Co., Berlin, N.H.) with 0.5M HC1 and 0.5M KOH; neutralize with a concentrated solution of KH2P04. Wash several times on the Biichner funnel with 10 mM potassium phosphate buffer of pH 7.6, the starting buffer, and resuspend in the same buffer. Remove the fines by settling. Prepare a 2.25 X 39 cm column by allowing the 0.034M O,13M O . 1 7 M Noel N o C I NoCl
! F
0.25M NoCI
!
!
]I 28 rrr
2.4 E 2.0 ho
vr 1.6
c o
"~ 1.2 0.8 0.4 0
~tI 1
0 4
i
i
i
i
i
i
I
8 12 1 6 2 0 2 4 28 32 3 6 4 0 4 4 4 8
52
Effluent tube number
FIG. 1. Stepwise elution of whey proteins from DEAE-cellulose. Sample: 600800 mg of dialyzed and lyophilized protein in 30 ml of 0.01 M potassium phosphate buffer, pH 7.6. Column: 225 X 39 cm; fractions 10 ml. Absorbancy at 750 nm: optical density of 0.I ml aliquots with Folin-Lowry reagent. The arrows indicate points of change of sodium chloride concentration in buffer. [J. J. Rackis, Proc. Seed Protein Con]erence, p. 98 (1963). Reproduced with permission.1~]
DEAE to settle to a height of 43 cm and compressing to 39 cm. Dissolve about 600 mg of the whey protein in 30 ml of buffer. Clarify by centrifuging at 12,000 rpm for 5 minutes and apply to the column. Elute at room temperature with stepwise increments of NaC1 in starting buffer, as shown in Fig. 1. Inhibiting activity is present in peaks V and VI, the latter being inhibitor SBTIA2. Discard part of the leading edge of peak = E. A. Peterson and H. A. Sober, Vol. V [1].
856
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66c]
VI: pool the remainder. Dialyze and lyophilize. The yield is nearly 90% of the SBTIA2 in the whey. The purified inhibitor has 1.05 units of trypsin-inhibiting activity per microgram2 Properties
Stability. Below 30 ° the inhibitor retains its activity 1~,2o from pH 1 to 12, although the conformation shows some changes with pH. 21,22 It is reversibly 15 denatured by short heating to 80 °, and irreversibly 15 denatured by heating at 90 ° (50% in 33 minutes, using a 0.5% solution in 2.5 mM HC1). It is digested by pepsin at pH 2.0 TM or 1.5, 2s but not at pH 3.0.15 Purity. The inhibitor prepared by the method described gives a single peak 8 on rechromatography, on moving boundary electrophoresis at pH 7.6, and in the ultracentrifuge. However, polyacrylamide gel electrophoresis 14 at pH 9.2 shows several extra bands in very small amounts. Commercial preparations vary in the intensity of the extra bands 1~ appearing under these conditions. The inhibitor prepared by isoelectric focusing9 is 99% pure by microdensitometry of a disc electrophoresis gel, and shows only one component upon immunoelectrophoresis. Physical Properties. The molecular weight is 24,000 by osmotic pressure, TM 21,6008 and 21,50022 by ultracentrifugal analysis, 20,000 by light scattering, 2~ and 21,70022 and 22,0006 by amino acid analysis. The partial specific volume by density determinations is 0.735 s or 0.69822 (the latter in 1 M KC1) and by amino acid analysis is 0.74. 8 The sedimentation coefficient, s °, ~ is 2.29. 8 The eleetrophoretie mobility at pH 7.6, 2 ° and 0.1 ionic strength s is --8.0 X 10-~, and at pH 5.9 and 0.1 ionic strength s is --7.14 X 10-6. The isoelectrie point is, 21 is 4.5-4.6. The absorbance of a solution containing 1 mg/ml at pH 7.6 is 0.99481.013 s at 280 nm. Optical rotatory dispersion ~ values are ao = --552 ° and bo = --10 ° (Moffitt plots). The 0 R D and circular dichroism measurements indicate a nonhelical structure 25,2s in the native state. Some a-helix is formed in the presence of nopropanol, 28 anionic detergents, 28 or 2-chloroethanol. 28 The CD spectrum of the native inhibitor has a strong negative elliptieity M. Kunit~, J. Gen. Physiol. 32, 241 (1948). R. F. Steiner and H. Edelhoch, J. Biol. Chem. 238, 925 (1963). my. V. Wu and H. A. Scheraga, Biochemistry 1, 698 (1962). UB. Kassell and M. Laskowski, Sr., J. Biol. Chem. o.19, 203 (1956). H R. F. Steiner, Arch. Biochem. Biophy8. 49, 71 (1954). S K . Ikeda, K. Hamaguchi, M. Yamamoto, and T. Ikenaka, J. Biochem. (Tokyo) {}3, 521 (1968). " B . Jirgensons, J. Biol. Chem. 242, 912 (1967).
[66c]
SOYBEANTRYPSIN AND CHYMOTRYPSIN INHIBITORS
857
band at 200 nm and a weaker positive band at 226 nm. 26~ Changes in the C D spectrum with p H 26b are indicative of conformational changes below p H 7 and above p H 11.5. At p H 1.8-2.0, the amplitude of the positive Cotton effect at 226 nm decreases and the position shifts from 226 to 230 nm,26a Specificity. T h e trypsins of various species are inhibited, e.g., cow, ~ human, 27,2s salmon, 29 sting ray, ~° barracuda, 8° and t u r k e y ? ~ Of other enzymes, bovine c h y m o t r y p s i n 32 a and B, chicken chymotrypsin, 3~ h u m a n plasmin,27.33,a~ cocoonase,34,,34b plasma kallikrein, 35 and a serum trypsinlike enzyme 36 are inhibited. I t blocks the conversion of prothrombin to t h r o m b i n ? 7 Kallikreins from bovine organs, ~8 porcine kallikrein, as,s9 h u m a n 2s and bovine 37 thrombin, liver esterase, ~° and fl-subtilisin ~ B P N ' are not inhibited, although complexes are formed with both thrombins?; The Kunitz inhibitor does not form a complex with the tosyllysine chloromethyl ketone ~ia ( T L C K ) derivative of trypsin ~1~ or with diisopropylphosphoryl trypsin. ~1~ Kinetic Properties. T h e inhibition of trypsin is stoichiometric. With benzoylarginine ethyl ester as substrate, the inhibition is competitive. ~, ~ ~" B. Jirgensons, M. Kawabata, and S. Capetfllo, Makromol. Chem. 125, 126 (1969). ~ M . Baba, K. Hamaguchi, and T. Ikenaka, J. Biochem. (Tokyo) 65, 113 (1969). 27F. F. Buck, M. Bier, and F. F. Nord, Arch. Biochem. Biophys. 98, 528 (1962). R. E. Feeney, G. E. Means, and J. C. Bigler, J. Biol. Chem. 244, 1957 (1969). C. B. Croston, Arch. Biochem. Biophys. 112, 218 (1965). E. N. Zendzian and E. A. Barnard, Arch. Biochem. Biophys. 122, 714 (1967). 31C. A. Ryan, J. J. Clary, and Y. Tomimatsu, Arch. Biochem. Biophys. 110~ 175 (1965). "F. C. W u and M. Laskowski, Sr.,J. Biol. Chem. 213, 609 (1955). L. B. Nanning and M. M. Guest, Arch. Biochem. Biophys. 108, 542 (1964). "V. Mansfeld, M. Rybhk, Z. Hor~kov~, and J. Hladovec, Z. Physiol. Chem. 318, 6 (1960). u" F. C. Kafatos, J. H. Law, and A. M. Tarkakoff, J. Biol. Chem. 242, 1488 (1967). s,bH. F. Hixson, Jr. and M. Laskowski, Jr.,Biochemistry 9, 166 (1970). E. Werle and L. Mayer, Biochem. Z. 323, 279 (1952). A. M. Siegelman, A. S. Carlson, and T. Robert,son, Arch. Biochem. Biophys. 97,
159 (1962). z~G. F. Lanchantin, J. A. Friedrnann, and D. W. Hart, J. Biol. Chem. 244, 865 (1969). E. Werle and B. Kanfmann-Boetsch, Naturwi~senscha#en 19, 559 (1959). ~*E. Sach and M. Th~ly, Compt. Rend. Acad. Sci. Paris, Set D, 266, 1200 (1968). N. M. Green, Biochem. J. 60, 416 (1957). " H . Matsubara and S. Nishimura, J. Biochem. (Tokyo) 45, 413, 503 (1958). '1~E. Shaw, Vol. XI [80]. ~lbG. Feinstein and R. E. Feeney, J. Biol. Chem. 241, 5183 (1966). ,1~L. W. Cunningham, Jr., F. Tietze, N. M. Green, and H. Neurath, Faraday Soc. Symposium, Aug. 1952, p. 58. N. M. Green, J. Biol. Chem. 205, 535 (1953). "~P. M~tais, H. Schirardin, and J. Wafter, Compt. Rend. Soc. Biol. 57, 2307 (1963).
858
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[66¢]
TABLE I VARIATION OF THE ASSOCIATION CONSTANT a,b OF THE
KUNITZ SOYBEAN INHIBITORWITH pH
pH
q average number of protons released on association
3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 8.30
3.35 2.92 2.53 2.34 2.00 1.80 1.50 1.33 1.13 0h
L o g Kapp
(g~pp
[TI] ffi [ T ~ !
4. 578a 5. 349 6. 004~ 6.639 7. 182 7. 657 8. 070 8.424 8. 732 9. 832h
a From J. Lebowitz and M. Laskowski, Jr., Biochemistry I, 1044 (1962), determined by potentiometric measurement at 20 ° in 0.5 M KCI, 0.05 M CaCI~. (Copyright by the American Chemical Society. Reprinted by permission.) b The values are in good agreement with partial data obtained by an enzymatic method, ~ by light scattering, ~ by fluorescence polarization,d by sedimentation measurement,e and by gel filtrationJ c N. M. Green, J. Biol. Chem. 205, 535 (1953). a R. F. Steiner, Arch. Bioehem. Biophys. 49, 71 (1954). ° E. Sheppard and A. D. MeLaren, J. Am. Chem. Soe. 75, 2587 (1953). / G. A. Gilbert, Nature 210, 299 (1966). g Average of two methods of calculation. h Interpolated value.
With protein substrates, the type of inhibition is uncertain. "2 The association constants of the inhibitor-trypsin complex at different pH values are given in Table I. When the inhibitor reacts with trypsin at low pH (3.75), an arginyl-isoleucine bond in the inhibitor is hydrolyzed. ~4 Resynthesis of the cleaved bond by trypsin has been achieved, ~4a and an equilibrium mixture of 14% native and 86% cleaved form is established 4~b in the presence of trypsin at pH 4.0. Amino Acid Composition. The amino acid composition is shown in Table II. ~K. Ozawa and M. Laskowski, Jr., J. Biol. Chem. 241, 3955 (1966). ~l W. R. Finkenstadt and M. Laskowski, Jr., J. Biol. Chem. 242, 771 (1967). '~C. W. Niekamp, H. F. Hixson, Jr., and M. Laskowski, Jr., Biochemistry 8, 16 (1969).
[66c]
SOYBEAN TRYPSIN AND CHYMOTRYPSIN INHIBITORS
859
TABLE II AMINO ACID COMPOSITION OF TWO SOYBEAN INHIBITORS Residues per molecule Amino acid Alanine Arginine Aspartic acid Half-cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Amide groups Total N Molecular weight
Kunitz inhibitor~
Bowman-Birk b inhibitor
9 10 29 4 19 17 2 15 15 12 3 10 11 12 8 2 4 15 13
4 2 11-12 14 7 0 1 2 2 5 1 2 6 8-9 2 0 2 1 6
16.34% 22,000
15.9% 7,975
a M. Yamamoto and T. Ikenaka, J. Biochem. (Tokyo) 62, 141 (1967). b Based on the data from three laboratories: Y. Birk, Ann. N.Y. Acad. Sci. 146, 388 (1968); M. Yamamoto and T. Ikenaka, J. Biochem. (Tokyo) 62, 141 (1967); V. Fratteli, J. Biol. Chem. 244, 274 (1969). P a r t i a l Sequences. T h e a m i n o t e r m i n u s 45 is A s p - P h e - V a l - L e u - A s p . T h e c a r b o x y l t e r m i n u s is leucine. ~8 T h e sequences s u r r o u n d i n g t h e t w o disulfide b r i d g e s ~7 a r e : (1)
Glu-Arg-C~s-Pro-Leu-Thr Trp-Leu-Cys-Val-Gly-Ile-Pro-Thr-Glu
(2)
Val-Phe-C~s-Pro-Gln-Gln-~a
Gly-Ile-Asp-Gly-Cys-Lys-Asp-Asp-Glu ~T. Ikenaka, K. Shimada, and Y. Matsushima, J. Biochem. (Tokyo) 54, 193 (1963). a E. W. Davie and H. Neurath, J. Biol. Chem. 212, 507 (1955). ~7j . R. Brown, N. Lermaa, and Z. Bohak, Biochem. Biophys. Res. Commun. 23, 561 (19eS).
860
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66c]
Bowman-Birk Inhibitor from Soybeans (Inhibitor AA) Purification Procedure 11,12,~s
Starting Material. Defatted soybean flour is used, obtained by 48hour ether extraction (in a Soxhlet apparatus) of finely ground soybeans, Harasoy, 48 Lee, TM Hawkeye, ~2 or any other variety. "Hizyme" (Central Soya, Chicago, Ill.), a hexane-treated floui from mixed varieties of soybeans, proved satisfactory in the author's l~boratory without further extraction. Step 1. Preparation o] Crude Inhibitor. Warm 6 liters of 6 0 ~ ethanol to 60 ° and add 600 g of the starting material. Stir 1 hour at 55°-60 °, cool to room temperature, and filter through Whatman No. 50 paper on a 24-cm Biichner funnel, using a flattened circle of cloth of 21-cm diameter under the filter paper to prevent clogging. Discard the precipitate. Adjust the pH of the filtrate to 5.3 with 1 M HC1 (with some increase in cloudiness). Add 2 volumes of acetone with stirring and allow the precipitate to settle. Filter (as above) by decanting most of the liquid through the Biichner funnel before adding the rather sticky precipitate. Dissolve the air-dried precipitate in 500 ml of water and dialyze against water at 5 °, using Visking 18/100 cellulose casing. Step 2. Chromatography on CM-Cellulose. ~l,~s Cycle CM-cellulose TM with 0.5 N NaOH and 0.5 N HC1, adjust to pH 4.0 with acetic acid, and settle several times in the starting buffer (5 mM sodium acetate of pH 4.0). Prepare a 4 X 40 cm column and equili'brate with the starting buffer. Adjust the dialyzed inhibitor solution to pH 4.0 with 1 M acetic acid, centrifuge to remove a small amount of precipitate if present, and apply the clear supernatant solution to the column. Elute the column (a) with 1.5 liters of the starting buffer (including the volume of the applied sample); (b) a gradient of NaC1 concentration formed by passing 1 liter of O.12M N~C1 in starting buffer through 1 liter of the starting buffer in a constant volume mixing flask; (c) an increased gradient of NaC1 concentration formed by passing 1 liter of 0.25 M NaC1 in starting buffer into the mixing flask. A flow rate of 150 ml per hour with 8 ml fractions is satisfactory. Following elution of an inactive peak, the trypsin inhibitor appears in a peak extending from about tubes 250 to 350. Combine only the tubes of highest and constant activity (approximately the center half of ~he peak). Dialyze the solution against water at 5 ° and lyophilize. This gives an almost homogeneous inhibitor, but there is considerable sacrifice in yield. Almost the entire peak can be used if step 3 is to be a y. Birk and A. Gertler, in "Biochemical Preparations," (W. E. M. Lands, ed.) Vol. 12, p. 25. Wiley, New York, 1968. (Material from this article is reproduced by permission of the publisher.)
[66C]
SOYBEAN TRYPSIN AND CHYMOTRYPSIN INHIBITORS
861
carried out. In this case, concentrate the pooled peak (e.g., in an ultrafilter). Step 3. Chromatography on DEAE-Cellulose. 12 Adjust the solution to 140 ml with 0.05 M ammonium acetate solution, pH 6.5, the starting buffer, and dialyze at 5 ° against the same solution. Warm the protein solution to room temperature and apply to a column of DEAE-cellulose,19 2 X 28 cm, equilibrated with the same buffer. Elute with a salt and pH gradient, prepared in a rectangular Varigrad, by adding to compartments 1 through 9, respectively, (a) 0.20 M ammonium acetate, pH 5.0: 0, 5, 10, 20, 40, 60, 80, 100 and 100 ml; (b) 0.05 M ammonium acetate, pH 6.5, as needed to bring all compartments to 100 ml. A flow rate of 40 ml/hour and 5 ml fractions are satisfactory. The active peak appears approximately in tubes 175 to 195. Again, pool tubes of the highest and constant activity, dialyze against water in the cold, and lyophilize. The yield is about 800 mg (40%). The specific activity of the final product is 10 trypsin inhibitor units per milligram. Properties Stability. 1°,4s There is no loss in activity when the inhibitor is heated in the dry state at 105 ° or in 0.02% aqueous solution for 10 minutes at 100% Autoclaving for 20 minutes at 15 lb pressure destroys the activity. The inhibitor is stable to acid (pH 1.5, 2 hours, 37 °) and to peptic and Pronase digestion. Reduction with 2-mercaptoethanol in the presence of urea inactivates the inhibitor and the activity is not recovered upon reoxidation. Purity. The product of Birk et al. 1~,~8 (completed by rechromatography on CM-cellulose) behaves as a single substance by rechromatography on CM- and DEAE-cellulose, by sedimentation velocity, by paper electrophoresis at pH 4.9 and 6.9, and by acrylamide gel electrophoresis at pH 4.7 and 8.9. Amino acid analysis, however, shows a fraction of a residue of glycine (Table II). The preparation of Frattali ~2 (for which the final step is the DEAE chromatography above) is almost free of glycine, with ratios of amino acid residues very close to whole numbers. Polyacrylamide electrophoresis at pH 8.35 indicates a single component. Physical Properties. The molecular weight is 8,00049-16,4006 by ultracentrifugation (monomer-dimer-trimer equilibrium49). It is 7975 by amino acid analysis. 12 The s°20.wvalue is 1.96 to 2.3.11 The diffusion constant 11 (D2°0.,,) is 9.03 X 10-7 cm2/sec. The partial specific volume is 0.69 from amino acid composition,e,49 D. B. S. Millar, G. E. Willick, R. F. Steiner, and V. Frattali, J. Biol. Chem. 244,
281 (1969).
862
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[55d]
The isoelectric point is 4.06 to 4.2.11 The absorbance, --l~m~l~at 280 nm is 4.46,~9 to 4.8.11 The electrophoretie mobility s is 8.45 X 10-5 cm2/V/cm at pH 5.9. Specificity. s° The Bowman-Birk preparation inhibits both the proteolytic and esterolytic activities of trypsin and chymotrypsin, when assayed on casein, or on benzoylarginine ethyl ester (BAEE) and acetyltyrosine ethyl ester (ATEE), respectively. The inhibit~r-trypsin complex inhibits chymotrypsin (with casein or ATEE assay). The inhibitor° chymotrypsin complex inhibits trypsin (with BAEE, but not with casein assay). It also inhibits a trypsinlike component of Pronase21 It has a slight inhibiting effect on human trypsin and on human plasmin, but not on human thrombin. 2g Kinetic Properties. The inhibition of trypsin and chymotrypsin is noncompetitive 5° and nonstoichiometric. 1~ The KI for trypsin 5° (casein) is 5.6 X 10-s. The K~ for a-chymotrypsin 5° (casein) is 5.0 >( 10-7. When the inhibitor reacts with a catalytic amount of trypsin at pH 3.75, a Lys-X bond is hydrolyzed. 52,'53 The inhibitor retains activity against both trypsin and chymotrypsin, 52 but becomes a less efficient trypsin inhibitor22, ~4 When the inhibitor reacts with a small amount of chymotrypsin at pH 3.75, its activity toward chymotrypsin is markedly decreased, but that toward trypsin is unaffected22 Both modified inhibitors are reconverted in part to the native form by incubation with equimolar amounts of the original enzyme24 Distribution, The inhibitor is found in all varieties of soybeans. A m i n o Acid Composition. This is shown in Table II. There is no resemblance to the Kunitz inhibitor. Terminal group. T h e amino terminal group is aspartic acid. s, 5a my. Birk, Ann. N.Y. Acad. Sci. 146, 388 (1968). 6~M. Trop and Y. Birk, Biochem. J. 109, 457 (1968). Y. Birk, A. Gertler, and S. Khalef, Biochim. Biophys. Acta 147, 402 (1967). ~Y. Birk, A. Gertler, and S. Khalef, personal communication (1969). MV. Frattali and R. F. Steiner, Biochem. Biophys. Res. Commun. 34, 480 (1969).
[55d]
Trypsin
Inhibitors from Other Legumes
By BEATRICE KASSELL Introduction In the years following the isolation of inhibitors from soybeans, a large number of other vegetable food proteins have been shown to con-
862
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[55d]
The isoelectric point is 4.06 to 4.2.11 The absorbance, --l~m~l~at 280 nm is 4.46,~9 to 4.8.11 The electrophoretie mobility s is 8.45 X 10-5 cm2/V/cm at pH 5.9. Specificity. s° The Bowman-Birk preparation inhibits both the proteolytic and esterolytic activities of trypsin and chymotrypsin, when assayed on casein, or on benzoylarginine ethyl ester (BAEE) and acetyltyrosine ethyl ester (ATEE), respectively. The inhibit~r-trypsin complex inhibits chymotrypsin (with casein or ATEE assay). The inhibitor° chymotrypsin complex inhibits trypsin (with BAEE, but not with casein assay). It also inhibits a trypsinlike component of Pronase21 It has a slight inhibiting effect on human trypsin and on human plasmin, but not on human thrombin. 2g Kinetic Properties. The inhibition of trypsin and chymotrypsin is noncompetitive 5° and nonstoichiometric. 1~ The KI for trypsin 5° (casein) is 5.6 X 10-s. The K~ for a-chymotrypsin 5° (casein) is 5.0 >( 10-7. When the inhibitor reacts with a catalytic amount of trypsin at pH 3.75, a Lys-X bond is hydrolyzed. 52,'53 The inhibitor retains activity against both trypsin and chymotrypsin, 52 but becomes a less efficient trypsin inhibitor22, ~4 When the inhibitor reacts with a small amount of chymotrypsin at pH 3.75, its activity toward chymotrypsin is markedly decreased, but that toward trypsin is unaffected22 Both modified inhibitors are reconverted in part to the native form by incubation with equimolar amounts of the original enzyme24 Distribution, The inhibitor is found in all varieties of soybeans. A m i n o Acid Composition. This is shown in Table II. There is no resemblance to the Kunitz inhibitor. Terminal group. T h e amino terminal group is aspartic acid. s, 5a my. Birk, Ann. N.Y. Acad. Sci. 146, 388 (1968). 6~M. Trop and Y. Birk, Biochem. J. 109, 457 (1968). Y. Birk, A. Gertler, and S. Khalef, Biochim. Biophys. Acta 147, 402 (1967). ~Y. Birk, A. Gertler, and S. Khalef, personal communication (1969). MV. Frattali and R. F. Steiner, Biochem. Biophys. Res. Commun. 34, 480 (1969).
[55d]
Trypsin
Inhibitors from Other Legumes
By BEATRICE KASSELL Introduction In the years following the isolation of inhibitors from soybeans, a large number of other vegetable food proteins have been shown to con-
[55d]
TRYPSIN INHIBITORS FROM OTHER LEGUMES
863
tain trypsin inhibitors. This article describes the lima bean trypsin inhibitors in some detail, and includes some of the information available about inhibitors from other legumes. Lima bean (Phaseolus lunatus) inhibitors were purified and studied by T a u b e r and co-workers 1 and then by Fraenkel-Conrat et al. ~ Further purification and separation into several active fractions was later achieved using chromatographic methods by Jirgensons and co-workers, s by Jones, Moore, and Stein, 4 and by H a y n e s and Feeney.: The method of purification described by Jones et al. is used in the present article. The lima bean inhibitor is commercially available in partially purified form (e.g., Worthington Biochemical Corp., Freehold, N.J., and Sigma Chemical Co., St. Louis, Mo.). The purification procedure described below, starting with step 2, has also been used for further purification of commercial preparations2 Inhibitors have been highly purified from the following sources: red gram 6 (Cajanus cajan L.), mung bean ~ (Phaseolus aureus Roxb.), kidney bean s (Phaseolus vulgaris), navy bean 9 (Phaseolus vulgaris), black-eyed pea 1° (Vigna sinensis), peanut ~ (Arach~s hypogaea L.), field bean ~2 (Dolichos lablab), French bean ~s (Phaseolus coccineus), and sweet pea ~s (Lathyrus odoratus ) . Assay The casein digestion method of Kunitz has been described previously in this treatise. I~ Definition o] Unit." The specific inhibiting activity is expressed as milligrams of active trypsin (determined by the rate of hydrolysis of benzoyl-L-arginine ethyl ester) inhibited per milligram of inhibitor. 1H. Tauber, B. B. Kershaw, and R. D. Wright, J. Biol. Chem. 179, 1155 (1949). H. Fraenkel-Conrat, R. C. Bean, E. D. Ducay, and H. S. Olcott, Arch. Biochem. Biophys. 37, 393 (1952). B. Jirgensons, T. Ikenaka, and V. Gorguraki, Makromol. Chem. 39, 149 (1960). 4G. Jones, S. Moore, and W. H. Stein, Biochemistry 2, 66 (1963). R. Haynes and 1~. E. Feeney, J. Biol. Chem. 242, 5378 (1967). *S. Tawde, Ann. Bioehem. Exptl. Med (Calcutta) 21, 359 (1961). TH.-M. Chii and C.-W. Chi, Sei. Sinica (Peking) 14, 1441 (1965). *A. Pusztai, Biochem. J. 101, 379 (1966). oL. P. Wagner and J. P. Riehm, Arch. Biochem. Biophys. 121, 672 (1967). M. M. Ventura and J. X. Filho, Anales Acad. Brasil Cienc. 38, 553 (1967). ~1R. Tixier, Compt. Rend. Acad. Sci. Paris 266, 2498 (1968). A. P. Baner]i and K. Sohonie, Enzymologia 36, 137 (1969). "J. Weder and H.-D. Belitz, Deut. Lebensm. Rundschau 65, 78 (1969). ~' M. Laskowski, Sr., Vol. II [3].
864
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[65d]
P u r i f i c a t i o n P r o c e d u r e ~, ~5
Operations are at 4o-5 ° except as stated. Step 1. Extraction. The starting material is 100 g of finely ground lima beans (var. Fordhook, certified seed). Extract for 30 minutes with 500 ml of 80% ethanol. Filter by suction. Stir the semidry meal with 500 ml of 0.25 M H2SO~ for 1 hour. Centrifuge for 30 minutes at 1200 g. Reserve the supernatant solution and reextraet the solid in the same manner. Combine the extracts and adjust to pH 5.0-5.1 with 5 M NI-t4OH. Precipitate the inhibitor by adding solid ammonium sulfate with stirring to 50~ saturation. After 2 hours, remove the precipitated protein by centrifugation and take it up to 50 ml of 0.1 M ammonium formate buffer of pH 3.2. Remove insoluble material by centrifugation. The extract contains about 90% of the original inhibiting activity. Based upon the specific activities of the purified inhibitors, 100 g of beans contains about 0.5 g of inhibitors. The extract may be frozen at this stage. Step 2. Gel Filtration on Sephadex G-75. Prepare a 2 X 150-cm column of the Sephadex, after decanting fines and deaerating in the ammonium formate buffer. Apply 10 ml of the solution of step 1, and elute with the same buffer, at the rate of 16-20 ml per hour. Measure absorbance at 280 nm and inhibiting activity in the effluent. The peak containing the inhibiting activity appears when about 200 ml of effluent have been collected. Pool the active material and lyophilize. The specific activity is 1.6-1~, and the recovery is 80-90%. Repeat this step to bring all the material to this stage. Step 3. Chromatography on DEAE-Cellulose. Dissolve the material of step 2 in 50-100 ml of the starting buffer (0.01 M sodium phosphate of pH 7.60 __+0.02). Equilibrate the solution by passage through a 4 X 40 cm column of Sephadex G-25, medium, at a flow rate of 30--40 ml per hour, collecting 10-ml fractions. Pool the protein peak. Recycle DEAE-cellulose 18 with HC1 and NaOH and equilibrate with the starting buffer. Prepare a 2 X 55 cm column at 25 °. Apply up to 150 mg of the protein from the Sephadex column. Elute the column a~ 25 ° with a gradient solution prepared by passing 0.4 M NaCl in starting buffer through a constant-volume, l-liter mixing chamber containing the starting buffer. Collect 5-ml fractions at the rate of 20 to 25 ml per hour. Detect the protein in the effluent fractions by measuring the ninhydrin color of 0.1 ml aliquots after alkaline hydrolysis. 1~ Some inactive material S. Moore, personal communication, 1969. ~E. A. Peterson and H. A. Sober, Vol. V [1].
~' C. H. W. Hits, Vol. XI [35].
[66d]
TRYPSIN INHIBITORS FROM OTHER LEGUMES
865
is eluted first, then four or more peaks containing inhibitor appear between about 480 and 800 ml of effluent solution. Pool separately the main portion of each peak, sacrificing the material in the valleys between the peaks, and lyophilize. Step ~. Rechromatography on DEAE-Cellulose. Dissolve each fraction in 10-20 ml of water and equilibrate as before by passing through the Sephadex column. By carrying out the rechromatography under the same conditions as before, each peak appears in the expected position, with small amounts of contaminating neighboring peaks now separated. Lyophilize the pooled fractions from each peak, dissolve the product in 1020 ml of 0.1 M acetic acid, and desalt by passage through a 2 X 35 cm column of Sephadex G-25 equilibrated with 0.1 M acetic acid. Lyophilize the pooled fractions. The activities of components 1 to 4 were 2.69, 2.77, 2.28, and 2.44. Calculated on a molar basis (see below), this corresponds to 0.95, 0.99, 0.95, and 0.97 moles of trypsin inhibited by 1 mole of inhibitor. Variability with Dif]erent Batches o] Lima Beans. Different commercial preparations and different samples of fresh-frozen lima beans give peaks tha~ vary in amount, in position, and in amino acid composition. ~s As many as six active peaks have been found even with highly inbred varieties of lima beans, s Properties Stability. Most of the legume inhibitors show considerable stability to heat. There is no loss of activity when lima bean inhibitors 2 are heated for 15 minutes at 90 ° at pH 5 or pH 7. Kidney bean inhibitor ~9 does not lose activity in 90 minutes at 90 ° in 0.15 M NaC1. At 100° and pH 7.6, red gram inhibitor6 retains 50% of its activity after 80 minutes and guar meal inhibitoff° retains 50% after 10 minutes. At 100 ° and pH 7.2, field bean inhibitor 1~ loses 59% of its activity in 30 minutes and 86% in 1 hour; 30 minutes of autoclaving at 15 lb pressure destroys 93% of the activity. These inhibitors are also relatively stable to extremes of pH. Lima bean inhibitors 2 can be kept for 24 hours at 23 ° in 10 mM NaOH or for 24 hours at 40 ° in 10 mM HC1. Red gram inhibitor~ retains partial activity when exposed for 60 minutes to pH 2.5 and to pH 10. Field bean inhibitor 12 is stable for a week at 15° between pH 3 and pH 10. Kidney bean inhibitor 19 does not lose activity in 2 hours at pH 2 and 37 °. W. Ferdinand, S. Moore, and W. H. Stein, Biochim. Biophys. Acta 96, 524 (1965). 1,A. Pusztai, European J. Biochem. 5, 252 (1968). •J. R. Couch, C. R. Creger, and Y. K. Bakshi, Proc. Soc. Exptl. Biol. Med.
[email protected], 263 (1966).
866
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66d]
TABLE I PURITY OF SOME LEGUME INHIBITORS
Criteria of homogeneity
Inhibitors from Lima bean ~ Lima bean b M u n g bean, ~,d component B Kidney b e a n ' Navy bean/ Black-eyed peao Peanut h Field bean ~ Sweet pea ~'
Single peak Single component Single component on on in rechromatography electrophoresis ultracentrifuge Yes (peaks 1-4) -Yes -Yes Yes ----
~ No Yes Yes -Yes yes Yes Yes
Yes (fraction 6) Yes Yes Yes Yes ----
a G. Jones, S. Moore, a n d W. H. Stein, Biochemistry 2, 66 (1963). b R. Haynes and R. E. Feeney, J. Biol. Chem. 242, 5378 (1967). c H.-M. Chti a n d C.-W. Chi, ~gc/. Sinica (Peking) 14, 1441 (1965). d H.-M. Chii, S.-S. Lo, M.-H. Jen, C.-W. Chi, a n d T.-C. Tsao, Sc/. Sinica (Peking) 14, 1454 (1965). • A. Pusztai, European J. Biochem. 5, 252 (1968). / L. P. Wagner and J. P. Riehm, Arch. Biochem. Biophys. 121,672 (1967). g M. M. Ventura a n d J. X. Filho, Anales Acad. Brasil. Cienc. 38, 553 (1967). h R. Tixier, Compt. Rend. Acad. Sci. Paris 266, 2498 (1968). A. P. Banerji a n d K. Sohonie, Enzymologia 36, 137 (1969). J J. Weder a n d H.-D. Belitz, Deut. Lebensm. Rundsehau 65, 78 (1969).
Two of the inhibitors are known to resist inactivation by pepsin; they are the inhibitors from lima bean (24 hours at p H 2 ~ or p H 1.54) and kidney bean 1~ (2 hours at p H 2 and 37°). Lima bean inhibitor is not inactivated by papain. 2 Purity. From the criteria shown in Table I, several of the inhibitors have now been prepared in a high degree of purity. Physical Properties. The molecular weights of some inhibitors are shown in Table II. T h e lima bean inhibitors range from 8300 to 10,000, while the other inhibitors v a r y up to 24,000. Other physical constants are known for a few of the inhibitors. For lima bean inhibitors the s 20. o w is 1.5 for the mixture 2 and 1.81 for fraction 65; the calculated partial specific volume (~) for fraction 6 is 0.7252 For n a v y bean inhibitor, 9 ~ is 0.693. For black-eyed pea inhibitor, 1° s o20,w is 1.6, D2o,~ is 8.6 X 10-7 cm2/sec, ~ is 0.69 and the ratio ]/]o is ~ are: 8.23 for black-eyed pea, l° 7.58 for field 1.47. Some values of .~. % bean, 12 and 7.78 for red gram 8 inhibitors. The isoelectric points are: kidney bean ~ inhibitor, 5; field bean ~2 inhibitor, 4.7; and peanut ~ in-
[66d]
TRYPSIN INHIBITORS FROM OTHER LEGUMES
867
TABLE II MOLECULAR WEIGHTS
Inhibitors from Lima bean" Peak 1 Peak 2 Peak 3 Peak 4 Lima bean b Fraction 4 Fraction 6 Lima bean ~ Mung bean d Kidney bean" Navy beang Black-eyed peah Peanut ~ Field bean k
OF SEVERAL TRYPSIN INHIBITORS OF
LEGUbIES
Amino acid composition
1 : 1 Ratio to trypsin
Ultracentrifugal measurement
8,408 8,291 9,892 9,423
8,800 8,400 10,400 9,700
w
9,100 9,667 -8,113 --16,923 17, O00i --
8,950 9,050 --9,600 11,500 15,300 -23,700
Osmotic pressure
m
16,200 10,000 9,000 10,000I 23,000 17,10O
9,400 9,200
G. Jones, S. Moore, and W. H. Stein, Biochemistry 2, 66 (1963). b R. Haynes and R. E. Feeney, J. Biol. Chem. 242, 5378 (1967). c H. Fraenkel-Conrat, R. C. Bean, E. D. Ducay, and H. S. Olcott, Arch. Biochem. Biophys. 37, 393 (1952). d K. Wang, C.-W. Chi, and T.-C. Tsao, Aeta Biochim. Biophys. Sinica 5, 510 (1965) ; Chem. Abstr. 64, 13,000b (1966). * A. Pusstai, European J. Biochem. 5, 252 (1968). I The inhibitor shows a tendency to aggregate. o L. P. Wagner and J. P. Riehm, Arch. Biochem. Biophys. 121, 672 (1967). h M. M. Ventura and J. X. Filho, Anales Acad. Brasil. Cienc. 38, 553 (1967). R. Tixier, Compt. Rend. Acad. Sci. Paris 266, 2498 (1968). Calculated by the author. Two peanut inhibitors isolated by Hoehstrasser st al. ~ had molecular weights of 17.000 by gel filtration, but are believed to be made up of 4 subunits. A. P. Banerji and K. Sohonie, Enzymologia 36, 137 (1969). K. Hochstrasser, K. Illchmann, and E. Werle, Z. Physiol. Chem. 3.~, 929 (1969). h i b i t o r , 6.75. O p t i c a l r o t a t o r y d i s p e r s i o n m e a s u r e m e n t s h a v e been m a d e for m u n g b e a n i n h i b i t o r , 21 c o m p o n e n t A : t h e specific r o t a t i o n is - - 1 6 °, a n d X¢ is 298 n m ; for c o m p o n e n t s I I I , I V , V, a n d V I of l i m a b e a n inhibitors~: t h e v a l u e s are, r e s p e c t i v e l y , - - 3 2 . 0 °, - - 3 2 . 4 °, - - 2 0 . 5 °, a n d - - 2 0 . 3 ° for [a]548 a n d 258, 258, 280, a n d 274 for Xc. C i r c u l a r d i c h r o i s m s t u d i e s of t h r e e p u r i f i e d l i m a b e a n i n h i b i t o r s were m a d e b y I k e d a et al? 2 F r o m p H 7.4 to 11.8, t h e p o s i t i v e b a n d a t 248 n m increases, while t h e r e K. Wang, C.oW. Chi, and T.-C. Tsao, Acta Biochim. Biophys. Sinica 5, 510 (1965) ; Chem. Abstr. 64, 13,000 b (1966). ~ K . Ikeda, K. Hamaguchi, M. Yamamoto, and T. Ikenaka, J. Biochem. (Tokyo) 63, 521 (1968).
868
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
r~ O
r~
O
Q
O
[65d]
[66d]
TRYPSIN
INHIBITORS
FROM
OTHER
LEGUMES
~
~
~
~ ~o
O .
~4
.~3~
~
870
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[55d]
is little or no change in the negative band near 280 nm. These proteins are nonhelicah Specificity. Most of the purified inhibitors and many other unpurified extracts of legumesla,23 combine with both bovine trypsin and bovine ohymotrypsin. However, this double action has been shown to exist at different sites in the same molecule only for Bowman-Birk inhibitor from soybeans (this volume [66c]), for one of the lima bean inhibitors, 5 for black-eyed pea inhibitor, 1° and for kidney bean inhibitor. TM Bovine trypsin is inhibited stoichiometrically by lima bean, 4,5 blackeyed pea, ~° kidney bean, 19 mung bean, 24 green gram 2~ (Phaseolus mungo), red gram, ~ field bean, TM and double bean 26 (Faba vulgaris) inhibitors. Navy bean inhibitor9 appears to react with 2 molecules of trypsin; mung bean inhibitoff4 forms a second complex with 2 molecules of trypsin. Peanut inhibitor ~ reacts with different amounts of trypsin, depending on the substrate. Stoichiometric inhibition of bovine chymotrypsin occurs with kidney bean TM and field bean TM inhibitors, while black-eyed pea inhibitor1° reacts with 2 molecules of chymotrypsin. In the presence of high chymotrypsin concentrations, the kidney bean inhibitor TM combines with more than 1 mole in a nonstoichiometric manner. Other inhibitors show varying degrees of action against chymotrypsin, e.g., lima bean, 5 peanut, ~ mung bean, ~' double bean, 2~ and navy bean. 2s Human trypsin is inhibited by lima bean, navy bean, kidney bean, and black-eyed pea inhibitors. 2s Lima bean inhibitor is effective against turkey trypsin and chicken chymotrypsin.29 Bovine acetyltrypsin is inhibited by lima bean s and field bean TM inhibitors. A few examples of tests with other enzymes can be given, and the reader is referred to the book by Vogel, Trautschold, and Werle 3° for further data. Field bean inhibitor TM has antithrombin activity. Kidney bean inhibitor reacts stoichiometrically with elastase 19 and with human plasmin, ~,28 does not inhibit TM pepsin, papain, fl-subtilis protease, or 2~E. P. Abramova and M. P. Chernikov, Federation Proc., Transl. ~uppl. 24, 635 (1965). 24H.-M. Chii and C.-W. Chi, Acta Bioehim. Biophys. Sinica 6, 22 (1966); Chem. Abstr. 65, 4432h (1966). P. M. Honawar and K. Sohonie, J. Sci. Ind. Res. (India) 18C, 202 (1959). K. Sohonie and K. S. Ambe, Nature 175, 508 (1955). H.-M. Chu and C.-W. Chi, Acta Biochim. Biophys. Sinica 37 229 (1963); Chem. Abstr. 597 10405f (1963). s R. E. Feeney, G. E. Means, and J. C. Bigler,J. Biol. Chem. 2447 1957 (1969). C. A. Ryan and J. J. Clary, Arch. Biochem. Biophys. 108, 169 (1964). soR. Vogel, I. Trantschold, and E. Werle, "Natural Proteinase Inhibitors."Academic Press, N e w York, 1968.
[55d]
TRYPSIN INHIBITORS FROM OTHER LEGUMES
871
earboxypeptidase A, little 19 or no 28 action like component of Nagarse ~ (subtilisin
has weak 19 action on carboxypeptidase B, and has on thrombin. Lima bean inhibitor acts on a trypsinPronase (fraction C), 31 but does not inactivate B P N ' ) , Pronase ' (type VI from Streptomyces griseus), Aspergillus oryzae proteinase, 5 human thrombin, ~8 or Clostridium histolyticum collagenase22 N a v y bean and black-eyed pea inhibitots are weakly effective against human plasmin, 28 but are ineffective against human thrombin? 8 The peanut inhibitors act on chymotrypsin, plasmin, and serum kallikrein, but not on kallikreins from pancreas, submaxillary gland, or urine2 2a Kinetic Properties. The known trypsin inhibition constants for the legume inhibitors fall in a narrow range: lima bean, ss approximately 4 X 10-1°M; mung bean, ~7 10 -9 to 10-1°M; field bean, 12 3.3 X 101°M; green gram, ~5 6.1 X 10-l° M. The reaction with trypsin is instantaneous for field bean 12 and red gram ~ inhibitors. Distribution. The inhibitors are widely distributed in the seeds of leguminous plants. In double bean plants, ~4 the inhibitor is present in seeds, leaves, stems, and roots of the plant, with the highest concentration in the seeds and the lowest in the roots. Amino Acid Composition. The compositions of several inhibitors are shown in Table III. A few common features are: high cystine content, little or no methionine and tryptophan, and in most cases low tyrosine and phenylalanine content. Mung bean inhibitor has amino terminal s e r i n e / T h e two peanut inhibitors studied by Hochstrasser et al2 2~ differ in their amino terminal residues, one having serine and one valine.
sl M. Trop and Y. Birk, Biochem. J. 109, 475 (1968). • = I. Mandl, H. Zipper, and L. T. Ferguson, Arch. Biochem. Biophys. 74, 465 (1958). nl K. Hochstrasser, K. Illchmann, and E. Werle, Z. Physiol. Chem. 350, 929 (1969). D. Grob, J. Gen. Physiol. 33, 103 (1949). K. S. Ambe and K. Sohonie, Experientia 12, 302 (1956).
872
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[55e]
[66e] P r o t e o l y t i c E n z y m e I n h i b i t o r s f r o m Ascaris lumbricoides B y BEATRICe. K~S~T. Introduction
Early in this century, 1,2 it was discovered that Ascaris lumbricoides extracts inhibit trypsin, chymotrypsin, and pepsin. Several groups have since succeeded in purifying chymotrypsin and trypsin inhibitors from Ascaris.
The Ascaris trypsin inhibitor was partially purified by Collier3 and was further studied by Green. 4 Rhodes et al. 5 isolated two trypsin inhibitors, one from body walls and one from perienteric fluid. Portmann and Fraefel 6 obtained several trypsin inhibitors from whole ascarids. The inhibitor isolated by Pudles et al.7 which at first appeared to differ in amino acid composition, proved to be identical to the main inhibitor (CM-1 be'low) of Portmann and Fraefel when a Sephadex G-50 filtration was substituted for dialysis,s Recently, Kucich and Peanasky 9 reported two inhibitors from Ascaris body walls. It is clear that there are several inhibitors that show some resemblances in amino acid composition. The best characterized is the main inhibitor of Portmann and Fraefel2 The purification described is their procedure, but the properties of the other inhibitors are included. The chymotrypsin inhibitors were also studied by Collieff and by Green. 4 The predominant inhibitor of body walls was first obtained pure by Peanasky and co-workers 1°,~ and their procedure is described below. Rhodes et al2 found one inhibitor predominant in perienteric fluid and another (probably the same as Peanasky's) predominant in body walls. Rola and Pudles, ~2 using whole ascarids, obtained three inhibi¢ors. 1E. Weinland,Z. Biol. 44, 1 (1903). IL. B. Mendel and A. F. Blood,J. Biol. Chem. 8, 177 (1910). H. B. Collier, Can. J. Res. BIg, 90 (1941). N. M. Green, Bioehem. J. 66, 416 (1957). ~M. B. Rhodes, C. L. Marsh, and G. W. Kelley, Jr., Ezptl. Parasitol. 13, 266 (1963). e p. Portmann and W. Fraefel, Helv. Chim. Aeta 50, 2078 (1967).
J. Pudles, F. H. Rola, and A. K. Matida, Arch. Bioehem. Biophys. 120, 594 (1967). ' P. Portmann, personal communication,1969.
' U. Kucieh and R. J. Peanasky, Bioehim. Biophys. A~ta 200, 47 (1970). R. J. Peanasky and M. Laskowski,Sr., Bioehim. Biophys. Acta 37, 167 (1960). nl~. J. Peanasky and M. M. Szucs, J. Biol. Chem. 239, 2525 (1964). F. H. Rola and J. Pudles, Arch. Biochem. Biophys. 113, 134 (1966).
Ascaris INHIBITORS
[66e]
873
Trypsin Inhibitors Assay Method s, 13
Principle. Inhibition of tryptie hydrolysis of N-a-benzoyl-nL-argininep-nitroanilide (BAPA) is measured by the change in absorbance at 405 nm. Reagents Buffer: Tris chloride, 0.1 M, pH 8.0, containing 0.025 M CaC12 Substrate: Dissolve 47.7 mg of BAPA with warming in 100 ml of buffer. The final concentration in the reaction mixture is 0.844 raM. Trypsin: 10 mg/100 ml of 0.1 N HC1, containing 0.1 ml of chloroform and 3 drops of saturated CaC12. This solution keeps 2-3 weeks at 4 °. Before using, dilute to 40 ~g,/ml with water. Diluted inhibitor solution in buffer, 0.1 ml approximately equivalent to 10 ~g of trypsin
Procedure. Place 3-ml, 1-cm cuvettes in a spectrophotometer connected to a circulating water bath at 25 °. Equilibrate the solutions in the water bath. Add 2 ml of substrate, 0.5 ml of trypsin (20 ~g) and 0.1 ml of inhibitor solution in rapid succession. Mix with a plastic spatula. After 5 minutes, begin to record the increase in absorbance at 405 nm for an additional 5 minutes. Repeat the determination without the inhibitor to determine the total amount of trypsin present. Definition o] Unit.* One unit is equivalent to 0.26 micromole of p-nitroaniline not liberated by 20/~g of trypsin in 30 minutes. Specific activity = Units/mg N. Purification Procedure 6
Step I. Crude Extract. Collect 25 kg of swine ascarids and preserve in 90% ethanol until use. Grind the ascarids in a meat grinder and mix with 5-6 liters of 1% NaC1 solution. Homogenize the suspension in a blender in suitable portions. Add 1 g of Diastase and homogenize again until the particles are very fine. Cover with toluene and autolyze for 7 days. Add 25% trichloroacetie acid (TCA) (about 1.5 liters) until the pH of the mixture is 2.3-2.5. Heat rapidly to 80 ° and cool at once. Centrifuge, resuspend the precipitate in 3 liters of 2.5% TCA, and centrifuge again. The yellow, slightly turbid, supernatant solution contains almost 100% of the inhibitor originally present in the ascarids. ~A modification of the method of W. Nagel, F. WiUig, W. Peschke, and F. H. Schmidt, Z. Physiol. Chem. 340, 1 (1965).
874
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66e]
Step 2. Magnesium Sul]ate Precipitation. Adjust the crude extract (13-14 liters) to pH 3.0 with 1 N NaOH and heat to 70 °. Saturate with MgS04 and maintain at 70 ° until an oily, semisolid layer (containing the inhibitor) forms on the surface and can be easily skimmed off. Discard the lower layer, which contains little inhibitor. Step 3. Alcohol Fractionation (to reduce the sugar content). Dissolve the upper layer of step 2 in 3 liters of water and adjust to pH 5.0 with 1 N NaOH. Mix with an equal volume of absolute ethanol and cool to 4 °. After 24 hours, filter off the precipitated MgS04 and wash with 50% (v/v) alcohol until the solid is pure white. Adjust the filtrate, containing all the inhibitor, to pH 6.0 with NaOH and concentrate it to 200 ml in a rotary evaporator. Carefully adjust the very viscous solution to pH 7.5 with NaOH and add an equal volume of absolute ethanol. Centrifuge and discard the almost inactive precipitate. Raise the alcohol concentration to 70% by volume and again discard the precipitate, which contains only about 1% of the inhibiting activity. Add alcohol to 90% by volume. Separate the precipitate containing 90% of the inhibitor by centrifuging and dissolve it in 200 ml of water. Step ~. Ammonium Sul]ate Precipitation (]urther reduction o] sugar content). Allow the solution of step 3 to stand 24 hours at 4 °. Centrifuge and discard a small amount of inactive precipitate. Add solid ammonium sulfate to the clear supernatant solution to 80% saturation and centrifuge. Dissolve the sticky yellow precipitate in 200 ml water. Repeat the precipitation four times, and each time remove the precipitate by centrifuging. The precipitate formed in the fifth precipitation is yellowish and granular and contains 97% of the starting activity with 95% of the carbohydrate removed. Step 5. Picric Acid Precipitation. Dissolve the final precipitate of step 4 in 200 ml of water and add an equal volume of absolute alcohol. Keep overnight at 4 °. Filter off the precipitated ammonium sulfate on a sintered glass funnel. Evaporate the filtrate to 150 ml in a rotary evaporator. Add 450 ml of saturated (1.2%) picric acid. The inhibitor precipitates as the picrate. Centrifuge, wash the precipitate three times in the centrifuge bottles with 50 ml of absolute alcohol, and suspend it in 150 ml of water. Dissolve the precipitate by adding Dowex l-X8 (bicarbonate form) with stirring until C02 evolution stops. The pH of the solution should not go above 7.5 or at most 8.0. Filter off the yellow-colored Dowex 1 and wash it three times with 25-30 ml of water. Evaporate the combined filtrates in vacuo to 30 ml. Step 6. Gel Filtration on Sephadex G-50. Swell 60 g of Sephadex G-50 overnight in 5 liters of water and transfer it with water to a 3.5 X 55 cm column. Apply the solution of step 5 and make a quantitative transfer with 2 ml of water. Elute the column with water at the rate of 20 ml
[55e]
Ascaris INHIBITORS
875
per hour, collecting 5-ml fractions. Run ninhydrin determinations and trypsin-inhibiting activity on the fractions. The first three peaks contain relatively little activity, while the fourth (peak D) contains 70% of the inhibitor. Pool peak D (approximately tubes 87-103) and evaporate to dryness in a rotary evaporator. Dissolve in 15 ml of Tris acetate buffer [per liter: 1.21 g Tris (0.01 M) plus 0.525 g magnesium acetate (0.0025 M) adjusted to pH 8.0 with acetic acid]. Step 7. Chromatography on DEAE-Sephadex A-25. Swell 60 g of the resin in 3 liters of water, decant the water and cycle with 0.5 N HC1 and 0.5 N N a 0 H . Wash free of alkali with water and suspend in the Tris buffer above. Transfer to a 3.5 X 55 em chromatography tube and equilibrate with the buffer until the pH of the effluent is 8.0. Apply the inhibitor solution to the column and begin elution with the same buffer. After the first peak has appeared, elute with a gradient prepared by passing 0.1 M magnesium acetate in 0.01 M Tris acetate into 500 ml of the starting buffer in a constant volume mixing bottle. Collect 5-ml fractions at the rate of 20 ml per hour. Locate the peaks by ninhydrin assay and trypsin-inhibiting activity. The active material is distributed among three or four peaks. If the last peaks do not come off the column, change the gradient buffer to 0.2 M magnesium acetate and continue the elution. Pool each peak separately and evaporate to 5 ml on a rotary evaporator. Adjust each fraction to pH 7.5 and cool at 4 °. Add ethanol to a concentration of 95%. Centrifuge in the cold and wash the precipitated inhibitor with 20 ml of 95% ethanol. After centrifuging again, dissolve in doubly distilled water and lyophilize. The highest activity attained in the main (first) fraction is about 1200 units/mg N. The inhibitor should be kept a minimum time at pH 8 because there is some loss of activity. This pH is necessary, however, for sufficient retention of the inhibitor on the resin. At this stage, the hexose content is not greater than 31 ~g/mg N. Amino acid analysis and paper electrophoresis at pH 2 show that the fractions are not yet homogeneous. Step 8. Chromatography on CM-Cellulose. Dissolve the main fraction of step 7 in 3 to 5 ml of 0.01 M sodium acetate buffer, pH 5.5, and apply the solution to a 1.2 X 30 cm column of CM-ceUulose, 14 equilibrated with the same buffer. Elute the column with a sodium acetate gradient, prepared as above, 0.01 to 0.1 M, at pH 5.5. The activity appears in up to seven peaks, s CM-1, CM-2, etc. Evaporate each pooled fraction in vacuo to 5 ml, and precipitate the inhibitor by adding absolute ethanol to a concentration of 95%. Centrifuge in the cold and wash the precipitate twice with 95% alcohol. Dissolve in 0.1% acetic acid and lyophilize. The activities of fractions CM-1 and CM-2 are the highest, 260014E. A. Peterson and H. A. Sober, Vol. V [1].
876
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66e]
TABLE I TEMPERATURE AND p H DEPENDENCE a OF THE STABILITY OF INHIBITORb C M - 1
Percent activity remaining a t p H
N a t i v e inhibitor a t 25 ° (control) 10 minutes a t 60 ° 10 minutes a t 100 ° 24 hours a t room temperature
5
6
7
8
9
10
11
100
100
100
100
100
100
100
100 100 100
100 100 86
95 59 89
100 31 89
96 6 89
91 ~ 79
107 -56
U. R. H~inggi, thesis, University of Freiburg, Switzerland, 1969 (reprinted with permission of the author). b p. P o r t m a n n and W. Fraefel, Helv. Chim. Acta 50, 2078 (1967).
3200 units/rag N. s The other fractions are less active. CM-1 and CM-2 show only one component by paper electrophoresis at pH 2.0, but highvoltage eleetrophoresis at pH 7 shows that CM-1 still contains about 2% of CM-2. This does not affect the amino acid composition, since the two fractions are similar. If too large a sample is put on the column, incomplete separation results and this chromatography should be repeated. Properties
Stability. Inhibitor CM-18 is stable in acid and neutral solution (Table I). It is not affected by 8 M ureaY It is not hydrolyzed by TABLE II MOLECULAR WEIGHTS OF VARIOUS AscaP8 TRYPSIN INHIBITORS
Source
Inhibitor
Molecular weight
Whole ascarids a Whole ascarids ° Whole ascarids ° Body walls ~ Perienteric fluid d Body walls! Whole ascaridso
CM-1 CM-2 CM-3 --Peak 1 --
7,192,b 7,000' 6,500 b 15,000-20,000 ~ 4,650, 7,100, 5,520 b s °, ~ = 1.1
a p. P o r t m a n n a n d W. Fraefel, Helv. Chim. Acta 50, 2078 (1967). b F r o m amino acid analysis. By Sephadex chromatography. M. B. Rhodes, C. L. Marsh, and G. W. Kelley, Jr., Exptl. Parasitology 13, 266 (1963). • Derived from 1 : 1 ratio to trypsin. ] U. Kucich a n d R. J. Peanasky, Biochim. Biophys. Acta, 200, 47 (1970). g J. Pudles, F. H. Rola, a n d A. K. Matida, Arch. Biochem. Biophys. 120, 594 (1967).
Ascaris INHIBITORS
[56e]
877
chymotrypsin. 1~ I t is relatively stable to digestion b y pepsin; the activity falls to 80% of the control after 3 days of digestion and no further on longer digestion, s Physical Properties. Table I I summarizes the molecular weights of Ascaris inhibitors. Purity. Inhibitor C M - V is almost homogeneous (2% of C M - 2 ) , and is free from carbohydrate. Specificity. Inhibitor CM-1 of P o r t m a n n and FraefeP ,15 has a small amount of activity against chymotrypsin. I t does not inhibit pepsin, papain, or Pronase. The two inhibitors isolated by Kucich and P e a n a s k y 9 do not inhibit chymotrypsin. Kinetic Properties. Green ~ reported the rate constant for inhibitortrypsin reaction (0.007 ionic strength, Tris buffer, p H 7.8) to be 2.1 X TABLE III AMINO ACID COMPOSITION OF Ascaris TRYPSIN INHIBITORS Portmann and Fraefel ~ Amino acid (residues/mole) Lysine Histidine Arginine Aspartic acid Threonine
Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan NHa
CM-1
CM-2
Kucich and Peanasky ~ peak 1
7 0 3 5 4 1 11 6 6 5 10 2 0 3 0 0 2 1
6 O 3' 4 4 1 9-10 5 6 5 8 2 0 3 0 0 2 1
5 0 3 3 3 1 8 4 4 4 8 2 0 2 0 0 2 1
6-7 ~
--
P. Portmann and W. Fraefel, Helv. Chim. Acta 50, 2078 (1967)'. b U. Kucich and R. J. Peanasky, Biochim. Biophys. Acta 200, 47 (1970). c p. Portmann, personal communication. d W. Fraefel and R. Acher, Biochim. Biophys. Acta 154, 615 (1968). U, R. Hi~nggi, thesis, University of Freiburg, Switzerland, 1969.
9
878
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[66e]
10' liter/mole/second. Kucieh and Peanasky 8 report instantaneous reaction between their two inhibitors and trypsin. The K1 values found by Green, ~ using bovine trypsin at pH 7.8 with BAEE or casein substrate, are in the range of 0.5 to 3 X 10-8 M. The values found by Kucich and Peanasky, 8 using porcine trypsin at pH 7.5 with hemoglobin substrate, are: for peak 1, Kx = 9.0 X 10-8M; for peak 2, K1 = 1.3 X 10 -s M .
Amino Acid Composition. This is shown in Table III. The absence of histidine, leucine, methionine, and tyrosine from all of the inhibitors is notable. Terminal Groups. All of the Portmann and Fraefel e inhibitors have amino terminal glutamic acid. 15 Amino Acid Sequence.TM The sequence of inhibitor e CM-1 is: Glu-Ala-Glu-Lys-Cys-(Asx, Glx, Glx, Pro, Gly, Trp)-Thr-LysGly-Gly-Cys-Glu-Thr-Cys-Gly-Cys-Ala-Gln-Lys-Ileu-ValPro-Cys-Thr-Arg-Glu-Thr-Lys-Pro-Asn-Pro-Gln-Cys-ProArg-Lys-Gln-Cys-Cys-Ileu-Ala-Ser-Ala~Gly-Phe-Val-ArgAsp-Ala-Gln-Gly-Asn-Cys-Ileu-Lys-Phe-Glu-Asp-Cys-Pro-Lys The positions of the disulfide bridges are shown in Fig, 1. Chymotrypsin Inhibitors Assay Method 11'1~
Reagents Buffer: sodium phosphate, 0.1 M, pH 7.6 Globin TM substrate solution: Dissolve 3.6 g of globin in 116 ml of a 33% (w/v) aqueous solution of urea (recrystallized from 95% alcohol). Add 1 M NaOH with stirring and allow the globin to denature at 25 ° for 30-60 minutes. Adjust the pH to 7.5 with 18.5 ml of a solution containing 40 g of urea plus 100 ml of 1 M KH2P04. The solution may be kept for several weeks in the refrigerator. a-Chymotrypsin solution: Dissolve a few milligrams in 0.0025 M HC1. Measure the absorbance at 280 nm. A2so X 0.5 = ~g,/ml. W. Fraefel and R. Acher, Biochim. Biophys. Aeta 154, 615 (1968). 1, N. M. Green and E. Work, Biochem. J. 54, 257 (1953). a Peanasky's globin was prepared from hemoglobin crystallized from fresh, eitrated human blood.~ Globin may be obtained commercially from Pentex Bioehemieals, Kankakee, Illinois. ~A. Rossi-Fanelli, E. Antonini, and A. Caputo, Biochim. Biophys. Acta 30, 608 (1958) ; J. Biol. Chem. °.36, 391 (1961).
[55e]
Ascaris INHIBITORS
879
Fla. i. The complete structure of trypsin inhibitorCM-I, showing the position of the disulfidebridges. (From A. Induni, thesis,University of Freiburg, Switzerland, 1969; reproduced with permission of the author.) Adjust the solution to contain about 180 ~g of protein per milliliter. Chymotrypsin preparations are not 100% "active"; the chymotrypsin used by Peanasky 11 was 7 1 % active, determined by titration with N-trans-cinnamoyl imidazole.19a Inhibitor solution: Dilute the solution with water to about 20 ~g/ ml so that it gives about 5 0 % inhibition of the chymotrypsin. Trichloroacetic acid (TCA) ; 5 % solution Procedure. Place a series of polycarbonate centrifuge tubes (16)< 100 ram) in a bath at 37% Add 0.1 ml (2 /~g) of inhibitor solution, 0.8 ml of phosphate buffer and 0.1 ml (18 ~g) of chymotrypsin solution. Mix and equilibrate for 15 minutes. With timing, add 1 ml of preequilibrated globin solution, mix, and stop the reaction exactly 5 minutes later by adding 2 ml of TCA. The timed additions are best made with automatic pipettes. Place in ice for 30 minutes and centrifuge for 30 minutes at 20,000 g in a Sorvall centrifuge. Use cellulose nitrate tubes (17)< 95 m m ) as adapters for the polycarbonate tubes in the SM-24 rotor.
m" A. R. Schonbaurn, B. Zerner, and M. L. Bender, Y. Biol. Chem. @.36,2930 (1961).
880
NATURALLYOCCURRING ACTIVATORS &ND INHIBITORS
[65e]
Prepare a standard curve by using chymotrypsin without inhibitor in a range of concentrations. Include in each set of assays chymotrypsin standards and blanks prepared by adding equivalent amounts of inhibitor and chymotrypsin after the TCA. Definition o] Unit. 1~ One unit inhibits 1 ~g of "active" ehymotrypsin. Specific activity ----units per milligram of protein. Purification Procedure I°,~
Step 1. Crude Extract. Collect Ascaris lumbricoides, var. suis in the salt medium of Baldwin and Moyle. 2° Wash the ascarids and separate the body walls by dissection. Carry out subsequent operations at 5 °, except as indicated. Homogenize 100-g portions of body walls in 4 volumes of water in a blender for 2 minutes and extract for an additional 15 minutes. Centrifuge at 25,000 g for 20 minutes and then at 57,000 g for 3.5 hours. Step ~. Acid Precipitation. Adjust the clear red supernatant solution to pH 1.9 with 5 N H2S04 and incubate at 37 ° for 75 minutes. Cool to 5 ° and adjust to pH 5.7 with 5 M NaOH. Filter off the heavy precipitate with the aid of Celite. Step 3. Ammonium Sul]ate Fractionation. Adjust the filtrate to pH 4.7 and collect the fraction that precipitates between 50 and 80~ saturation with ammonium sulfate. Dissolve the precipitate in water (0.1 the volume of step 2) and adjust the concentration to 10 mK/ml (Biuret reaction, 21 As~o X 31.5 ----mg protein per 10 ml of Biuret solution). The yields in steps 1, 2, and 3 are almost quantitative. Step 4. TCA Precipitation. Add 90~ (w/v) TCA to bring the solution to 7.5% con,centration. After 5 minutes, centrifuge at 25,000 g for 5 minutes. Precipitat@ 2 the inhibitor from the supernatant solution by adding ammonium sulfate to 30% saturation. Centrifuge as before and suspend the precipitate in 0.2 M sodium phosphate buffer of pH "7.2 (0.2 the volume of step 3). Dialyze the suspension against 0.07 M sodium acetate buffer of pH 5.15 using acetylated2s 18/32 tubing. The yield at this step is about 50%. At this point repeat the procedure to accumulate about 500 mg of protein; this requires six to seven 100-g portions of ascarids. 24 Step 5. Continuous-Flow Electrophoresis. The protein concentration of N E. Baldwin and V. Moyle, J. Exptl. Biol. 23, 277 (1947). ~A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949). 2sModified procedure, R. J. Peanasky, personal communication, 1969. L. C. Craig, in "Methods of Protein Chemistry" (P. Alexander and R. J. Block, eds.) Vol. 1, p. 116. Macmillan (Pergamon), New York, 1960. R. J. Peanasky, personal communication, 1969.
[65e]
Ascaris INI~IBITORS
881
the dialyzed solution should be about 15 mg/ml. Equilibrate a continuous-flow electrophoresis apparatus (Beckman-Spinco, Palo Alto, Calif., model CP) with 0.07 M acetate buffer, pH 5.15. Introduce the solution onto the center tab at the rate of 1.5 ml per hour, with an applied potential of 500 V. Determine the absorbance at 280 nm and the ehymotrypsin and trypsin-inhibiting activity of each tube. The main chymotrypsin-inhibiting peak is located from tubes 2 to 4 and is free of trypsin-inhibiting activity. Collect fraction 3, containing about 20% of this peak. Lyophilize and dialyze against water, using the acetylated dialysis tubing. Step 6. Crystallization. Concentrate the dialyzed solution to a 10% solution by lyophilization. Add an equal volume of saturated ammonium sulfate solution. Adjust the pH to 7.2 with 5 M NH,OH plus a drop of 1 M phosphate, pH 7.2. Then add saturated ammonium sulfate dropwise to the first sign of silkiness. The inhibitor crystallizes in the form of fine needles after 8 hours at room temperature. The crystalline material has 100 times the activity of the solution of step 2. Step 7. Gel Filtration. Prepare two columns equilibrated with 0.05 M Tris chloride, pH 8.5. The first (2.4 X 34 cm) contains Sephadex G-75; the second (2.4 X 37 ~cm) contains Sephadex G-25. Apply about 75 mg of the inhibitor of step 6 to the first column and elute with the same buffer. Collecting 3.0 ml fractions, the most active material appears in fractions 25 to 33. Pool these tubes and lyophilize. Apply about 100 mg of the lyophilized material (from 2 to 3 runs) to the second column and again elute with the same buffer. Collecting 3-ml fractions, the most active material appears in fractions 22 to 29. Pool and lyophilize. Repeat these two gel filtrations in sequence. The product then gives a symmetrical peak with uniform specific activity (about 2000), when chromatographed once more on Sephadex G-75. Finally, dialyze against water in the acetylated bags and lyophilize. The final yield 2~ is about 10%. The losses occur mainly by selection of only the purest material in the last steps. If the less pure fractions are reworked, the yield is improved. Properties
Stability. The inhibitors of Rola and Pudles ~2 retain activity when kept at 70 ° and pH 7.2 for 2 hours, or in 7 M urea at room temperature and pH 7.2 for 1 hour. Peanasky's inhibitor retains full activity ~4 after digestion with leucine aminopeptidase, carboxypeptidases A and B, and trypsin, but is digested2~ by ficin and papain. Purity. Peanasky's inhibitor 11 appears pure by two criteria: constant
882
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66e]
TABLE IV MOLECULAR WEIGHTS OF CHYMOTRYPSIN INHIBITORS
Inhibitor
Source
Molecular weight
I II
Perienteric fluid~ Body walls" Body walls* Body walls' Whole ascarids/
12,400b 8,760 b 7,955 ~ 8,000~ 8,600o
Pooled
M. B. Rhodes, C. L. Marsh, and G. W. Kelley, Jr., Exptl. Parasitol. 13, 266 (1963). bDerived from 1:1 ratio to chymotrypsin. " R. J. Peanasky, personal communication, 1969. From amino acid analysis. *By sedimentation equilibrium, using p ffi 0.749 determined by pycnometry (p = 0.709 by amino acid analysis). / F. H. Rola and J. Pudles, Arch. Biochem. Biophys. ll3, 134 (1966). o By Sophadex chromatography. specific activity across the final chromatography peak and absence of more than traces of extra amino terminal groups. Rola and Pudles 1~ inhibitor I I behaves as a single component upon electrophoresis at p H 8.6; inhibitor I separates into two active components. Physical Properties. T h e isoelectric point ~5 is over 10.75. The al>sorbance at 280 nm (A~m) is 7.14.11 See Table IV for a summary of molecular weights of ehymotrypsin inhibitors. Specificity. Peanasky's preparation inhibits a-chymotrypsin, 11 chymotrypsin B, 1~ and subtilisin. 24 It does not inhibit 24 trypsin, leucine aminopeptidase, carboxypeptidase A or B, ficin, or papain. It also does not inhibit 2~ the chymotryptic activity of trypsin. Kinetic Properties. The inhibitor forms a 1:1 complex with a chymotrypsin and chymotrypsin B. The K1 values are as follows: complex with ~,-chymotrypsinll: 1.1 to 3 X 10-s M, at pH 5.0-9.0 and 31°; complex with chymotrypsin Bll: 2.1 to 3.2 X 10-SM at p H 5.0 to 9.0 and 31°; complex with subtilisin2~: 1.2 X 1 0 -6 M. The reactions ~ with chymotrypsin a and B are not instantaneous; both reactions require 12-15 minutes at p H 7.0 at 0 ° or 28 °. Distribution2 The inhibitors of Ascaris occur in body wall, perienteric fluid, intestines, ovaries, and uterus. Amino Acid Composition. The composition is given in Table V. The ~*R. J. Peanasky, Federation Proc. 22, 246 (1903).
[66f]
CHYMOTRYPSIN INHIBITOR I FROM POTATOES
883
TABLE V AMINO ACID COMPOSITION OF THE CHTMOTRYPSIN INHIBITOR 6
Amino acid
Residues/mole
Amino acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine
6 1 7 4 5 3 8 9 7
Half-eystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Amide groups
Residues/mole 10 4 3 1 2 0 0 1 4
1
Total N
16.59%
° R. J. Peanasky, personal communication, 1969.
absence of tyrosine and phenylalanine and the large amounts of eystine and proline are unusual. Terminal Groups. Peanasky's inhibitor has arginine in the amino 11 and carboxyl s" terminal positions.
[66f] Chymotrypsin Inhibitor I from Potatoes
By C. A. RYAN and BEATRICEKASSELL Introduction N u m e r o u s proteinase inhibitors h a v e been isolated from potatoes 1-8~ and sweet p o t a t o e s ? T h e present article describes c h y m o t r y p s i n inhibitor I, first isolated s b y R y a n and Balls in 1962 and crystallized' in 1963. 1E. Werle, L. Maier, and F. LStiier, Biochem. Z. 321, 372 (1951). s K. Sohonie and K. S. Ambe, Nature 176, 972 (1955). s C. A. Ryan and A. K. Balls, Proc. Natl. Acad. Sc/. U.S. 48, 1839 (1962). • A. K. Balls and C. A. Ryan, J. Biol. Chem. 238, 2976 (1963). M. Yoshikawa, T. Kiyohara, and K. Ito, Hyogo Noka Daigaku Kenkyu Hokoku, Nogei-kagaku Hen 6, 35 (1963). 6V. P~bek and V. Mandeld, Experientia 19, 151 (1963). ' F. H. Rola and C. Correia-Aguiar, Anale8 Acad. Brasil C/enc. 39, 521 (1967). 8j. M. Rancour and C. A. Ryan, Arch. Biochem. Biophys. 125, 380 (1968). s, K. Hochstrasser, E. Werle, R. Siegelmann, and S. Schwarz, Z. Physiol. Chem. 3,50, 897 (1969). , K. Sohonie and P. M. Honavar, Sd. Cult. (Calcutta) 21, 538 (1956).
[66f]
CHYMOTRYPSIN INHIBITOR I FROM POTATOES
883
TABLE V AMINO ACID COMPOSITION OF THE CHTMOTRYPSIN INHIBITOR 6
Amino acid
Residues/mole
Amino acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine
6 1 7 4 5 3 8 9 7
Half-eystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Amide groups
Residues/mole 10 4 3 1 2 0 0 1 4
1
Total N
16.59%
° R. J. Peanasky, personal communication, 1969.
absence of tyrosine and phenylalanine and the large amounts of eystine and proline are unusual. Terminal Groups. Peanasky's inhibitor has arginine in the amino 11 and carboxyl s" terminal positions.
[66f] Chymotrypsin Inhibitor I from Potatoes
By C. A. RYAN and BEATRICEKASSELL Introduction N u m e r o u s proteinase inhibitors h a v e been isolated from potatoes 1-8~ and sweet p o t a t o e s ? T h e present article describes c h y m o t r y p s i n inhibitor I, first isolated s b y R y a n and Balls in 1962 and crystallized' in 1963. 1E. Werle, L. Maier, and F. LStiier, Biochem. Z. 321, 372 (1951). s K. Sohonie and K. S. Ambe, Nature 176, 972 (1955). s C. A. Ryan and A. K. Balls, Proc. Natl. Acad. Sc/. U.S. 48, 1839 (1962). • A. K. Balls and C. A. Ryan, J. Biol. Chem. 238, 2976 (1963). M. Yoshikawa, T. Kiyohara, and K. Ito, Hyogo Noka Daigaku Kenkyu Hokoku, Nogei-kagaku Hen 6, 35 (1963). 6V. P~bek and V. Mandeld, Experientia 19, 151 (1963). ' F. H. Rola and C. Correia-Aguiar, Anale8 Acad. Brasil C/enc. 39, 521 (1967). 8j. M. Rancour and C. A. Ryan, Arch. Biochem. Biophys. 125, 380 (1968). s, K. Hochstrasser, E. Werle, R. Siegelmann, and S. Schwarz, Z. Physiol. Chem. 3,50, 897 (1969). , K. Sohonie and P. M. Honavar, Sd. Cult. (Calcutta) 21, 538 (1956).
884
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[66f]
A s s a y M e t h o d 3, ~o-~2
Principle. The hydrolysis of tyrosine ethyl ester (TEE) by ehymotrypsin is measured by potentiometrie titration at constant pH. Reagents Substrate: TEE (2.09 mg of tyrosine ethyl ester/ml), 10 raM, in 25 mM CaC12, adjusted to pH 6.3 Stock Enzyme: 1.00 mg,/ml o f a-chymotrypsin in 1 mM HG1. Determine and adjust the concentration by the absorbance, A2so X 500 : ~g/ml. Inhibitor: Prepare the inhibitor solution in 10 mM Tris buffer of pH 7.0. Mix suitable amounts of this solution with the chymotrypsin in small vials so that about 50% of the chymotrypsin is inhibited. (5 to 50-#1 aliquots of the mixture should contain 0 to 40 ~g of active chymotrypsin for accurate titrations.) Allow to stand 3-4 minutes. NaOH: Standard 0.100M, free from carbonate Procedure. The apparatus for manual titration consists of a small thermostated cup connected to a circulating water bath at 25 ° and mounted on a magnetic stirrer. Place the combination microelectrode of a pH meter having an expanded scale at pH 6--7, a fine tube to deliver nitrogen, and the tip of a microburet containing the NaOH in position so that they will be beneath the surface of the solution. Equilibrate the solutions in the bath. Pipette 2.0 ml of substrate into the cup. Adjust to pH 6.4 with NaOH from the buret. Start the reaction by adding 50 ~I of the solution prepared by mixing enzyme and inhibitor. When the pH reaches 6.3, start timing the reaction. Add 2 to 4 ~l of NaOH and note the time that the pH again reaches 6.3. Continue the addition of increments of NaOH and timing over a period of 3 to 5 minutes (5 to 6 readings). Repeat the entire procedure, substituting 50 ~l of a solution containing the same amount of enzyme, but with buffer in place of inhibitor. A pH-stat may be used. Calculate the activity of the chymotrypsin from the slope of the reaction curve without inhibitor and the inhibition from the difference between the two values. The reaction is first order, so the tangent to the initial rate ,curve is used. Definition of Unit and Specific Activity. An esterase unit is expressed 12 ~oG. W. Schwert, H. Neurath, S. Kaufman, and J. E. Snoke, J. Biol. Chem. 172, 221 (1948). ~IF. L. Aldrich, Jr. and A. K. Balls, J. Biol. Chem. 233, 1355 (1958). C. A. Ryan, Biochemistry 5, 1592 (I966).
[~f]
CHYMOTRYPSIN INHIBITOR I FROM POTATOES
885
as micromoles of substrate hydrolyzed per minute per milligram of enzyme in 2 ml. The reaction between chymotrypsin and inhibitor I is stoichiometric and therefore the milligrams of ehymotrypsin neutralized can be used to calculate the milligram of inhibitor in each preparation. The best values for milligrams of a-chymotrypsin neutralized per milligram inhibitor I approach 3.0.12 Purification Procedure la The procedure is based on the property of the inhibitor to dissociate into subunits in a solution at least 3 M in guanidine concentration. Inhibitor I is separated on Sephadex G-75, first as a molecule of 39,000 molecular weight, and then as subunits of 9000 to 10,000 molecular weight. Finally, the native protein is reformed and isolated. The starting material is 100 lb of new Russet Burbank potatoes (Solanum tuberosum). The procedure can be scaled up or down as desired. Gram quantities can be prepared in a relatively short time. Step 1. Extraction. Cut the potatoes into small pieces, with peels intact, and soak in 5-gallon buckets containing a sodium dithionite solution (7 g per liter) until the process of cutting is complete. Pour off the sodium dithionite solution and discard it. Homogenize the potato pieces to a mushy pulp in a 4-liter capacity Waring-type blendor. Add about 500 ml of the sodium dithionite solution to facilitate the blending. Collect the pulp in buckets and express the juice through style A nylon cloth (W. G. Runkles Machinery Co., Trenton, N.J.) using an apple-type press at approximately 1000 psi. For a laboratory scale, use four layers of cheesecloth and express the juice by hand. Adjust the expressed juice (about 15 liters) to pH 3.0, using about 22 ml of 6 N HC1 per liter, and then centrifuge for 15 minutes at 5 ° . Carefully pour off the clear supernatant solution and collect it in large glass cylinders. Step ~. Ammonium Sul]ate Precipitation. Add solid ammonium sulfate slowly at 5 ° to give 70% saturation (472 g/liter). Stir the mixture gently for 1 hour. Centrifuge as above and discard the supernatant solution. Suspend the precipitate in 2 liters of 70% ammonium sulfate. Filter at room temperature through Whatman No. 1 filter paper on a 25-cm Biichner funnel using a ¼-in. pad of Celite No. 545 filter aid. Wash the precipitate, while on the pad, with 1 liter of 70% ammonium sulfate. This removes residual sodium dithionite. Dissolve the precipitate in 2 liters of water by stirring in a beaker for 1 hour with a magnetic stirrer at room temperature. Remove the 11C. A. Ryan and J. C. Melville, manuscript in preparation.
886
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[55f]
Celite by filtering by suction through Whatman No. 1 filter paper. The yield of inhibitor I is 74~ based on the original potato juice. Step 3. Heat Treatment. Divide the clear filtrate into 500-ml portions and heat in 2-liter Erlenmeyer flasks in a steam bath, with stirring, to a temperature of 80 ° . The heating should not exceed 5-6 minutes. At approximately 60 ° precipitation begins and by 80 ° a voluminous precipitate results. Inhibitor I survives this step almost quantitatively. Filter the hot solution through Whatman No. 1 filter paper, using Celite No. 545 as a filter aid. Pool the clear filtrates from each heated fraction and lyophilize. Step ~. Dialysis. Disperse the dry lyophilized powder in 500 ml of water and place in dialysis tubing (4.5 cm flat width). Dialyze against distilled water for 48 hours using several changes. The mixture in the dialysis tubing first clears as salts are removed and then precipitates as salt-free globulins come out of solution. Remove the mixture from the tubing and filter as described above through Whatman No. 1 filter paper using Celite as a filter aid. Lyophilize the filtrate. The yield of inhibitor I is 90% from the ammonium sulfate precipitate. Step 5. Gel Filtration on Sephadex G-75 in Bullet. Dissolve 1-5 g of dry material from step 4 in 30 to 60 ml of 0.05 M Tris chloride, 0.1 M KC1, pH 8.2, and apply to a 100 X 10.0 cm column of Sephadex G-75 equilibrated with the same buffer. Apply the sample to the column from the bottom and use an upward flow of about 450 ml per hour ~or elution, regulating the flow with a peristaltic pump (e.g., Sigma Motor, Inc., Middleport, N.Y.). Monitor the effluent at 280 nm. Collect 50-ml fractions. Begin collection of the effluent just preceding the void volume of the column (previously determined with blue dextran). Figure 1 shows the elution pattern. The second peak to elute (V/Vo---1.33) contains inhibitor I. Pool fractions 27 through 50. Reduce the volume to 300 ml using a rotary vacuum evaporator and desalt the pooled solution by passage through a 100 X 5.0-cm column of Sephadex G-25 with distilled water as eluent. Collect the breakthrough peak (the only peak) in its entirety, and lyophilize. The resulting salt-free protein is approximately 90% inhibitor I and represents a 76~ yield of inhibitor I based on the heated, dialyzed, lyophilized material from step 4. Step 6. Gel Filtration on Sephadex G-75 in ~,M Guanidine. Dissolve 100 mg of the product of step 4 in 5 ml of 4 M guanidine, pH 8.0. Apply the solution to an upward flow Sephadex G-75 column (100 X 2.5 cm) equilibrated with 4 M guanidine, pH 8.5. Elute the column with 4 M guanidine, pH 8.5, collecting 3.5-ml fractions at a flow rate of 60 ml per hour. The major peak appears at V/Vo of 1.82 (Fig. 2). Pool fractions 36 through 59. Dilute the combined fractions 8-fold with water and dialyze overnight against 12 liters of distilled water. Reduce the volume
[55f]
CHYMOTRYPSIN INHIBITOR I FROM POTATOES
3]
I
I
I
I
1
I
t
I
I
I
887
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t
I
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20
~.5
~)
1.0
0.5
O0
20
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I
t
1(30
140
180
I
22(
Tube number
FIG. 1. Gel filtration of potato Inhibitor I on Sephadex G-75 in 0.05 M Tris chloride buffer, pH 82, containing 0.1 M KC1. Column 100 X 10 cm, flow rate 450 ml/hour, fractions 50 ml. See text, step 5. (From C. A. Ryan and J. C. Melville, manuscript in preparation.)
20[
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Fro. 2. Gel filtration of potato Inhibitor I on Sephadex G-75 in 4 M guanidine solution, pH 8~. Column 100 X 2~ cm, flow rate 60 ml/hour, fractions 35 ml. See text, step 6. (From C. A. Ryan and J. C. Melville, manuscript in preparation.)
888
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[55fJ
to 100 ml in a rotary evaporator and dialyze for 24 hours against several changes of distilled water. Filter through a medium sintered glass filter. Lyophilize and store at --20 °, although storage at room temperature is not detrimental. The yield of inhibitor I in this step is 8 2 ~ . The overall recovery of inhibitor I from the crude juice is 41%. Properties
Stability. Inhibitor I is stable at pH 1 to 10 at room temperature. Above pH 10 it is denatured; for example, its half-life of inhibitory activity toward chymotrypsin at pH 11.5 is 48 minutes and at 12.2 is 25 minutes. 14 It is stable at 80 ° for 10 minutes from pH 2 to 10. is Full activity can be restored after dissolving inhibitor 1 in 8 M urea or in 6 M guanidine and diluting 10-fold in a pH range of 4 to 9.14 It is stable in 5% TCA in the cold ~8 and in 2.5% TCA at room temperature. The denatured precipitate formed in 10~ TCA, when dissolved in 6 M urea and diluted 10-fold regains native characteristics. 13 Pepsin destroys the activity below pH 6.12 Purity. Inhibitor I gives a single peak upon gel filtration is and electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. It shows a single peak in the ultracentrifuge, both in buffer and in 4 M guanidine solutions (see the table and Fig. 2). MOLECULAR WEIGHT DETERMINATIONS OF INHIBITOR COMPLEX WITH CHYMOTRYPSINa
I
AND
ITs
Molecular weight or
Protein Inhibitor I
Method
equivalent weight
38,800 + 2,400 Ultracentrlfuge-Archibald (0.5-1.0% solution) Ultracentrifuge-Velocity (0.25-1.0% solution) 37,500 -b 2,800 Sephadex G-75 in buffer, pH 8.2 37,000 ~ 2,0O0 Sephadex G-75 in 4 M guanidine, pH 8.5 9,8O0 ~ 2O0 Sephadex G-75 in 0.1% sodium dodecyl sulfate • 9,300 ± 1,100 a-Chymotrypsin inhibition (synthetic substrate) b 9,500 -~ 500 a-Chymotrypsin inhibition (casein substrate) c 10,000
Ultracentrifuge-Velocity (1.0% solution) Inhibitor Ichymotrypsin complex
123,000
J. C. Melville and C. A. Ryan, Arch. Biochem. Biaphys. 138, 700 (1970). b Substrate: either L-tyrosine ethyl ester or benzoyl-L-tyrosine ethyl ester. C. A. Ryan, Biochemistry 5, 1592 (1966). u j-. C. Melville and C. A. Ryan, Arch. Biochem. Biophys. 138, 700 (1970). C. A. Ryan, unpublished observations.
[6fif]
CHYMOTRYPSIN INHIBITOR I FROM POTATOES
889
Physical Properties. T h e molecular weight is between 37,000 and 39,000 and the molecule consists of four subunits (see the table). The absorption maximum of the protein is at 280 nm with a second, sharp maximum at 290 nm and a shoulder at 275 nm. The optical factor (OD X optical factor ---- mg protein) is 1.27 ± 0.01 at p H 3.0 and 1.3 ~0.04 at p H 7.0. Specificity. T h e following enzymes are potently inhibited b y inhibitor I: Bovine a-chymotrypsin, ~ bovine chymotrypsin B, 1~ chicken chymotrypsin, 16 subtilisin, 12 and one of the pronase enzymesJ ~ Trypsin is inhibited, but only its proteinase activity and not esterolytie activity. ~2 Inhibitor I does not inhibit pepsin, ~2 rennin, 15 carboxypeptidases A and B, 1~,17 bromelain, 12 ficin, 12 and papain. 12 A milk-clotting enzyme from Endothia parasitica, called "Sure Curd" is also not inhibited. 15 H u m a n trypsin, plasmin, and thrombin are not inhibited, lg Inhibitor I forms a complex 19 with inactive, anhydrochymotrypsin =° (serine-195 converted to dehydroalanine) and also interacts ~ with T P C K - c h y m o t r y p s i n . 2~ Kinetic Properties. As shown in the table, 1 molecule of inhibitor I inactivates 4 molecules of chymotrypsin, i.e., each subunit is equivalent to a molecule of the enzyme. ~4 The association constant =~ (Ka) of the inhibitor a-chymotrypsin reaction at 30 ° is 2.9 ± 0.9 X 10l° M -~. The thermodynamic parameters for the reaction are: AF = - - 1 4 . 5 ± 0 . 2 kcal/mole enzyme; ~ - / = --18 ___4 kcal/mole and z~S = --11 -+- 4 entropy units. Distribution? ~ Inhibitor I has a transitory occurrence in all tissues of potato plants, except xylem and seeds, where it is absent.
16C. A. Ryan, J. J. Clary, and Y. Tomimatsu, Arch. Biochem. Biophys. 110, 175 (1965). t~Impure preparations of inhibitor I contain contaminants of potato carboxypeptidase B inhibitor. R. E. Feeney, G. E. Means, and J. C. Bigler, J. Biol. Chem. 244, 1957 (1969). R. J. Foster and C. A. Ryan, Federation Proc. 24, 473 (1965). D. H. Strumeyer, W. N. White, and D. E. Koshland, Jr., Proc. Natl. Acad. Sei. U.~g.50, 931 (1963). ~1G. Feinstein and R. E. Feeney, J. Biol. Chem. 241, 5183 (1966). = E. Shaw, Vol. XI [801. = J. De Moura and R. J. Foster, personal communication, 1970. 2'C. A. Ryan, O. C. Huisman, and R. W. Van Denburgh, Plant Physiol. 43, 589 (1968).
890
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[66g]
[66g] Proteinasc Inhibitors from Egg White B y BEATRICE KASSELL
Introduction Egg white was one of the earliest known '~rypsin inhibitors 1 and this inhibiting property was shown to be associated with the ovomucoid fraction by M e y e r et al. 2 and by Lineweaver and I,,urray2 This fraction consists of several closely related glycoproteins, *, 5 all with inhibiting activity, and of a second type of inhibitor, ovoinhibitor2 Trypsin a n d / o r chymotrypsin inhibitors have been purified from the egg whites of numerous avian species ~ and have been detected in still more species2 These homologous inhibitors a r e of great interest, since they differ in their reactions with trypsin and chymotrypsin, while resembling each other in chemical and physical properties. Chicken ovomucoid is commercially available in partially purified form from several sources (e.g., Worthington Biochemical Corp., Freehold, N.J.; Sigma Chemical Corp., St. Louis, Mo.; and L. Light and Co., Great Britain). The present article describes the chicken ovomucoids and ovoinhibitors in detail and includes properties of the trypsin and chymotrypsin inhibitors of other species. Several recent 9-1~ and older ~2-~ reviews are I C. Delezenne and E. Pozerski, Compt. Rend. ~oe. Biol. 55, 935 (1904). K. Meyer, J. W. Palmer, R. Thompson, and D. Khorazo, J. Biol. Chem. 113, 479 (1936). 8It. Lineweaver and C. W. Murray, J. Biol. Chem. 171, 565 (1947). ' R. E. Feeney, F. C. Stevens, and D. T. Osuga, Y. Biol. Chem. 238, 1415 (1963). 5R. E. Feeney, D. T. Osuga, and H. Maeda, Arch. Bioehem. Biophys. 119, 124 (1967). e K. Matsushima, Science 127, 1178 (1958). M. B. Rhodes, N. Bennett, and R. E. Feeney, J. Biol. Chem. 235, 1686 (1960). 8S. Nakamura, M. Nagao, and R. Suzuno, Comp. Biochem. Physiol. 18, 937 (1966). 'M. D. Melamed, in "Glycoproteins" (A. Gottschalk, ed.), Vol. V, Chap. 11, Section 2. BBA Library, Elsevier, Amsterdam, 1966. ~0R. Vogel, I. Truut~chold, and E. Werle, "Natural Proteinase Inhibitors," p. 45. Academic Press, New York, 1968. R. E. Feeney and R. G. Allison, "Evolutionary Biochemistry of Proteins," pp. 83, 208ff. Wiley, New York, 1969. K. Meyer, Advan. Protein Chem. 2, 264 (1945). It. L. Fevold, Advan. Protein Chem. 6, 188 (1951). 14R. C. Warner, in "The Proteins" (H. Neurath and K. Bailey, ecls.), Vol. IIA, p. 465. Academic Press, New York, 1954. M. Laskowski and M. Laskowski, Jr., Adva~. Protein Chem. 9, 203 (1954).
[~g]
PROTEINASE INHIBITORS FROM EGG WHITE
891
available. A ficin and papain inhibitor (a protein of 12,700 molecular weight) has also been purified from chicken egg white, le It does not inhibit trypsin or chymotrypsin. The term "ovomucoid" used below without mention of species refers to chicken ovomucoid. AssayT, 17, ~s
Principle. Change in the absorbance at 395 nm of the indicator, m-nitrophenol, is a measure of acid liberated during the hydrolysis of ester substrates by trypsin and chymotrypsin.
INHIBITION OF TRYPSIN Reagents
Substrate-buffer-indicator solution, p-toluenesulfonyl arginine methyl ester (TAME), 0.01 M, in 0.006M Tris-chloride buffer containing 0.02% m-nitrophenol, with a final pH of 8.2. Trypsin solution. 65-80 ~g/ml in 0.004 M acetic acid containing 0.02 M CaC12. Inhibitor solution. 15-20 ~g/ml in 0.006 M Tris-chloride buffer of pH 8.95. Procedure. A recording spectrophotometer connected to a thermostated bath at 37 ° is used. The speed of the chart is 4 in./minute. Equilibrate all the solutions in the bath. Pipette into the cuvette 0.3 ml of enzyme solution and 0.7 ml of inhibitor solution. The final pH is 8.2 ___0.05. Add 2 ml of substrate-buffer-indicator solution by blowing it into the cuvette from a fine-tipped pipette to insure mixing. Start the recorder within 2 to 3 seconds, and record the change in transmittance at 395 nm for 1 minute. Repeat the determination with 0.7 ml of 0.006 M buffer of pH 8.95 substituted for the inhibitor solution. The amount of trypsin inhibited is the difference between the two determinations. The slope has a linear relationship to the amount of trypsin. The conversion factor is predetermined with variable amounts of a trypsin preparation of known "active enzyme" content, e.g., titrated with p-nitrophenyl p-guanidinobenzoate. 19 Unit. Based on a standard preparation of chicken ovomucoid, 1 ~g of
K. Fossum and J. R. Whitaker, Arch. Biochem. Biophys. 125, 367 (1968). M. B. Rhodes, R. M. Hill, and R. E. Feeney, Anal. Chem. 29, 376 (1957). M. M. Simlot and R. E. Feeney, Arch. Biochem. Biophys. 113, 64 (1966). ~T. Chase, Jr., and E. Shaw, Biochem. Biophys. Res. Commun. 29, 508 (1967).
892
NATUPJ~LLY OCCURRING ACTIVATORS AND INHIBITORS
[55g]
inhibitor is equivalent to 0.86/~g of trypsin; 17 this is a 1:1 molar ratio. For ovoinhibitor, 2° equivalence was determined with three fractions of similar activity. The average value was 1 /~g of inhibitor equivalent to 0.85/~g of trypsin, or a molar enzyme to inhibitor ratio of 1.74. INHIBITION OF CHYMOTRYPSIN Reagents Substrate--buffer-inhibitor solution: ber.z,~yl-L-tyrosine ethyl ester ( B T E ) , 0.01 M, in 0.006 M Tris-chloride buffer containing 30% methanol and 0.02% m-nitrop.henol, with a final p H of 8.2. Acetyl-L-tyrosine ethyl ester may be substituted 21,~ for B T E ; in this case the methanol is omitted. Chymotrypsin solution. 65-80 ~g/ml in 0.004 M acetic acid, containing 0.02 M CaC12. Inhibitor solution. Same as for trypsin assay. Procedure. The method is the same as for the trypsin assay. The standardization should be carried out with an a-chymotrypsin preparation of known "active enzyme" content, e.g., titrated with N-transcinnamoyl imidazole. 2s Unit. Chicken ovomucoid does not inhibit chymotrypsin ;24,25 measurement of the inhibition of chymotrypsin is a measure of contamination with ovoinhibitor. ~5 For ovoinhibitor, 2° 1 /~g inhibits 0.84 /zg of chymotrypsin; this is a molar enzyme:inhibitor ratio of 1.65 (average value of three fractions). Chicken Ovomucoid Purification Procedure The ovomucoid is precipitated with trichloroaeetic acid ( T C A ) acetone by the method of Lineweaver and M u r r a y s and further purified by chromatography according to Feeney, Osuga, and Maeda2 Other ovomucoids are prepared by similar methods.' Step 1. Separation from Egg White2 Adjust 750 ml of fresh egg white J. G. Davis, J. C. Zahnley, and J. W. Donovan, Biochemistry 8, 2044 (1969). 21C. A. Ryan, J. J. CIary, and Y. Tomimatsu, Arch. Biochem. Biophys. 110, 175 (1965). =Y. Tomimatsu, J. J. Clary, and J. J. Bartulovich, Arch. Biochem. Biophys. 115, 536 (1966). *8G. R. Schonbaum, B. Zerner, and M. L. Bender, J. Biol. Chem. 236, 2930 (1961). 2, F. C. Wu and M. Laskowski, Sr., J. Biol. Chem. 213, 609 (1955). 2sR. E. Feeney, F. C. Stevens, and D. T. Osuga, J. Biol. Chem. 238, 1415 (1963).
[66g]
PROTEINASE INHIBITORS FROM EGG WHITE
893
at 25o-30 ° to pH 3.5 by the slow addition, with stirring, of about 750 ml of TCA-acetone solution (1 volume of aqueous 0.5M TCA plus 2 volumes of acetone). Continue stirring for 30 minutes, then store overnight at 4 °. Centrifuge for 30 minutes at 13,000 g in a refrigerated centrifuge. Discard the precipitate. A sample of the supernatant solution should remain almost clear when heated to 80 ° for 5 minutes. Precipitate the inhibitor at 4 ° by adding 3 to 3.5 liters of cold acetone. After settling, filter on a Biichner funnel, using Celite as a filter aid. Suspend the filter cake in about 400 ml of water. Remove the Celite by filtration and precipitate the inhibitor with 21/2 volumes of acetone. Filter or centrifuge the suspension and dissolve the precipitate in a minimum amount of water adjusted to pH 4.5. Dialyze the inhibitor solution against water to remove residual TCA, then against 0.02 M sodium phosphate buffer of pH 6.5. The yield is about 5 g, and the activity is increased 8.5-fold over the original egg white. Step ~. Chromatography on D E A E - C e l l u l o s e 2 Cycle DEAE-cellulose2e (Whatman DE-50, Reeve Angel and Co., Clifton, N.J.) with 0.5 M HCI and 0.5M NaOH. Adjust to pH 6.5 with a concentrated solution of NaH2P04. Wash several times on the Biichner funnel with O.02M sodium phosphate buffer, pH 6.5, the starting buffer, and resuspend in the same buffer. Remove the fines by settling. Prepare a 35 X 3.2 cm column by allowing the resin to settle to a height of 39 cm and compressing to 35 em. Equilibrate the column overnight. Apply the entire sample 27 of step 1 (or 5 g), and elute the column with two successive linear gradients of NaC1 in the starting buffer, as shown in Fig. 1. The details of the procedure are given in the legend. Table I indicates that the inhibiting activity is mainly in peaks II and III; peak I contains many constituents, including lysozyme and ovoinhibitor. Pool each peak, lyophilize, and dialyze against 0.1 M sodium acetate buffer of pH 4.2. Step 3. Chromatography on CM-Cellulose. ~, ~, 2~ Recycle CM-cellulose 26 (Whatman CM-70) with 0.5 M NaOH and 0.5 M HCI, and adjust to pH 4.2 with 4 M sodium acetate. Wash the resin on the Biichner funnel with 0.1 M sodium acetate pH 4.2, the starting buffer. Resuspend in this buffer and remove fines. Prepare two 35 X 1.8 cm columns as described for DEAE, and equilibrate with the starting buffer. Apply the material from peak II and peak III to the separate columns. Elute with 150 ml of starting buffer, collecting 10-ml fractions. Change to a linear gradient prepared from 500 ml of starting buffer and 500 ml of 1 M NaC1 in starting buffer in interconnected open bottles of the same size. **E. A. Peterson and H. A. Sober, Vol. V [1]. ~' The present author recommends applying a smaller sample and u~ng a slower flow rate to achieve greater resolution than that shown in Fig. 1.
894
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
8.0 c
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O. I I00
150
Tube number Fie. 1. Chromatography of trichloroacetic acid-acetone preparation of chicken ovomueoid on DEAE-cellulose. Sample 5 g, column 35 X 32 cm, flow rate 3.6 ml/minute, fractions 12.5 ml. ( ) Optical density; ( . . . . ) salt gradient; ( ..... ) 10-fold scale reduction of peak 1. Reproduced from Feeney, Osuga, and Maeda, Arch. Biochem. Biophys. 119, 124 (1967).
DEAE fractions II and III both give several peaks, ~ but in each case there is one predominant peak. Pool the most active fractions from the predominant peaks, lyophilize, dialyze extensively against water, and lyophilize again. TABLE I DISTRIBUTION OF CHICKEN OVOMUCOID FRACTIONS FROM DEAE-CELt.ULOSE~ (Fig. 1) Trypsin-inhibiting activity
Fraction no.
% (by weight)
Specific activity b
% of total activity
I tI III M~ IV V
11.0 21.6 48.0 14.4 2.2 3.2
0.3 1.0 1.0 1.0 0.58 0
4 24 54 16 1 0
Sialic acid content c (%)
0.50 0.63
a R. E. Feeney, D. T. Osuga, and H. Maeda, Arch. Biochem. Biophys. 119, 124 (1967). b With chromatographically pure turkey ovomucoid taken as 1.00. c After rechromatography on CM-cellulose (see text, step 3). Mixture of fractions between peaks II and I I I and between peaks I I I and IV.
[56g]
PROTEINASE INHIBITORS FROM EGG WHITE
895
Other Methods o] Purification. A procedure ~8 involving precipitation with alcohol instead of acetone m a y be used for the preliminary separation of the ovomucoid from egg white. Similar DEAE-cellulose 29 and T E A E cellulose 3°,31 methods have been suggested for further purification, as well as gel filtration, 23 chromatography on sulfoethyl cellulose, 32 chromatography on hydroxylapatite, s2 and electrophoresis2 s,33~ However, a thorough testing of these products 8~ by electrophoretic and immunoelectrophoretic methods has shown that chicken ovomucoid has not yet been prepared as a single entity.
Properties Stability. Chicken ovomucoid shows unusual stability to heat and to high concentrations of urea in neutral or acid solutions; activity is rapidly lost in alkaline solutions. The data have been summarized by Melamed? Between pH 3 and 7, after 30 minutes at 80 ° more than 90% of the activity remained~; at p H 9 only 6% was retained24 After 15 minutes 35 at 90 °, 80% of the activity remained at p H 5 and 62% at p H 7. At p H 6 and 100 °, 30% of the activity was still present after 15 minutes24 T u r k e y and pheasant ovomucoids are even more stable to heat than chicken ovomucoids24 Jirgensons and co-workers 8~ have shown that the denaturation in alkali, first detected by loss of a c t i v i t y y is also reflected in changes in viscosity and optical rotation (see Physical Properties). In 9 M urea at p H 4, more than 90% of the activity was retained ~ after 18 hours at 25 ° or after 30 minutes at 80°; at 100 °, 86% remained after 15 minutes and 66% after 30 minutes, s At p H 7.4, also in 9 M urea, 80% of the activity was still present 8~ after 90 minutes at 80 °. 0vomucoid in 8 M urea showed a small increase in viscosity. 28 In 9 M urea, turkey ovomucoid lost its activity against trypsin and
s E. Fredericq and H. F. Deutsch, J. Biol. Chem. 161, 499 (1949). A. K. Chatterjee and R. Montgomery, Arch. Biochem. Biophys. 99, 426 (1962). ,o F. R. Jevons, Biochim. Biophys. Acta 45, 384 (1960). sl j. G. Beeley and F. R. Jevons, Biochim. Biophys. Acta 101, 133 (1965). J. Montreuil, B. Castiglioni, A. Adam-Chosson, F. Caner, and J. Queval, J. BIOchem. (Tokyo) 57, 514 (1965). =M. Jutisz, M. Kaminaki, and J. Legault-D~mare, Biochim. Biophys. Acta 23, 173 (1957). =" M. D. Melamed, Biochem. J. 103, 805 (1967). 34F. C. Stevens and R. E. Feeney, Biochemistry 2, 1346 (1963). •~ H. Fraenkel-Conrat, R. C. Bean, E. D. Ducay, and H. S. Olcott, Arch. Biochem. Biophys. 37, 393 (1952). ~B. Jirgensons, T. Ikenaka, and V. Gorguraki, Makromol. Chem. 39, 149 (1960). '~ A. K. Balls and T. L. Swenson, J. Biol. Chem. 106, 409 (1934).
896
NATURALLY OCCURRING ACTIVATORS AND INttIBITORS
[65g]
chymotrypsin 8~ at different rates. After 90 minutes at pH 7.4 and 80 °, less than 5 ~ of the activity against trypsin remained, while 8 5 ~ of the chymotrypsin inhibiting ability was still present. Pheasant ovomucoid was stable under these conditions24 Chicken ovomucoid is readily digested by pepsin2 ~,a8 Purity. The electrophoretic heterogeneity of the earlier preparations 8,2s of ovomucoid has been recognized for a long time. 2s, sg,40 Present methods have reduced, but have not eliminated, the heterogeneity. Even ovomucoids from the eggs of single hens 5 give chromatographic patterns similar to Fig. 1. The distinct components do not differ significantly in antitryptic activity or in amino acid composition2 ,84 The isolation, 4°a from four components of ovomucoid, of four glycopeptides with the same amino acid composition, but with definite quantitative differences in carbohydrate content, makes it likely that differences in the size of the carbohydrate moieties account for most of the heterogeneity. However, the possibility 11 of microheterogeneity in amino acid composition or in amide content cannot yet be excluded. The ovomucoids of ten other avian species are also electrophoretically heterogeneous2 Physical Properties. Molecular weight determinations T M by osmotic pressure and by sedimentation and diffusion methods are in the range from 27,0007,28 to 31,5004~; 28,000 is usually taken as the average value. The sedimentation constants for chicken ovomucoid range from 2.3 to 2.8, with diffusion constants from 6.0 to 8.1 X 10-~ cm~/se e at 20 °. The partial specific volume is 0.68528 to 0.71. 7 The frictional ratio 28 from sedimentation and diffusion is 1.35, giving an axial ratio of 6.3 for the unhydrated particle. However, ovomucoid is probably not compact and highly asymmetric, but highly hydrated, with an axial ratio near unity. 42 Jirgensons 36 found the intrinsic kinematic viscosity in 0.25 M NaBr solution to be 5.1 ml/g. In 0.1 M NaBr ~ 0.1 M NaOH, it changed to 18. Donovan 42 reported 6.31 ml/g at pH 2.1, 5.51 at pH 3.8, and 5.46 at pH 4.6. Sedimentation constants of 2.1 to 2.3 have been found for other ovomucoids (Table IV), but a molecular weight (28,000) has been reported 7 only for duck ovomucoid. Presumably, they are all quite similar. *~B. Kassell and M. Laskowski, Sr., J. Biol. Chem. 219, 203 (1956). n L. G. Longsworth, R. K. Carman, and D. A. MacInnes, J. Am. Chem. Soc. 62,
258O (1940). M. Bier, L. Terminiello, J. A. Duke, P. J. Gibbs, and F. F. Nord, Arch. Biochem. Biophys. 47, 465 (1953). ~o~M. Kana Mori and M. Kawabata, Agr. Biol. Chem. 33, 75, 220 (1969). ~ If. F. Deutsch and J. I. Morton, Arch. Biochem. Biophys. 93, 654 (1961). d j . W. Donovan, Biochemistry 6, 3918 (1967).
[6{~g]
897
PROTEINASE INHIBITORS FROM EGG WHITE
The optical rotation [a]D was reported as - - 5 6 ° (5% in water) by Lineweaver and M u r r a y , ~ while Chatterjee and Montgomery 29 reported - - 7 8 ° (0.79% in water) ; the latter preparation was chromatographically purified. The optical rotatory dispersion has been studied in the visible ~6,'2 and in the ultraviolet '~,~ ranges and has led to the conclusion 4~ that ovomucoid has 22-27% of helical structure. The effect of p H on the rotatory TABLE II OPTICAL ROTATORYDISPERSION (ORD) AND CIRCVLARDICHROISM(CD) DATA FOR CHICKENOVOMUCOID [~]5,6
X~( + 3)
Type
pH
(degrees)
K X 10-s
(nm)
ORD ,
1.1 6.1 9.7 12.0
-84.1 -91.5 -83.6 -65.9
20.8 22.2 21.0 16.5
228.5 237.3 228.9 220.0
CD a,"
pH
(nm)
(degrees decimole-l cm2)
7.1 7.1 7.1 7.1 7.1 7.1
210 222 243 263 271 292
- 16,000 - 10,500 q-390 -210 - 190 - 100
° B. Jirgensons, T. Ikenaka, and V. Gorguraki, Makromol. Chem. 39, 149 (1960). (Reproduced with permission of Hiithig and Wepf Verlag, Basel.) b K. Ikeda, K. Hamaguchi, M. Yamamoto, and T. Ikenaka, J. Bioehem. (Tokyo) 63, 521 (1968). (Reproduced with permission of the Japanese Biochemical Society.) Ionic strength 0.1; see text for changes with pH. properties in visible light is shown in Table II. Denaturation in alkali decreases the dispersion constant, 44~ X¢, in the normal way, while the specific rotation becomes less negative than for the native protein. This is interpreted as indicating an unusual conformation26 In the ultraviolet, 44 in 0.1 M NaC1, there is a negative Cotton effect with a trough at 230 nm. This trough decreases when the p H is raised to 11.5 and is greatly diminished at p H 12.8. The data given by Tomimatsu and Gaffield 4a for ,s y. Tomimatsu and W. Gaffield, Biopolymers 3, 509 (1965). ~K. Ikeda, K. ttamaguchi, M. Yamamoto, and T. Ikenaka, J. Biochem. (Tokyo) 63, 521 (1968). ~" The dispersion constant is derived from the one-term Drude equation.
898
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[56g]
TABLE I I I REACTION OF ~NHIBITORS WITH VARIOUS TRYPSINS AND CHYMOTRYPSINSaob
Molar complexing ratio (enzyme/inhibitor)
Trypsins Ovomucojds Chicken (GaUus oallus) Peking duck (Anas
Chymotrypsins
Bovine
Human
Turkey
Bovine*a
Chicken
1 2
0
1
0 1
O
platyrhynckos) Khaki Campbell duck (Anas
platyrhynckos ) Turkey (Meleagris gaUopavo) Guinea fowl (Numida meleagris) Goose (Anser ans.er) Emu (Dromiceius novaehollandiae) GAX pheasant (a genetic cross between golden pheasant and Lady Amherst pheasant) Lady Amherst's pheasant
1
2 1 1
0
1
1 1
2 2
0 1
0
1
0
1
1
1
0
1
1
(Chrysolophus amherstiae) Ring-necked pheasant)
(Phasianus colchicus) Golden pheasant
(Chrysolophus pictus) Ostrich (,gtruthio camelus) Positive' Cassowary (Casuarius 1 easua~us) Red jungle fowl (Gallus aaUus) 1 California valley quail
Weak c 0 0 1
1-2
(Lophortyx californica ) Painted quail (Oreortyx picti) 1 Japanese quail (Coturnus Positive
0 0
Weak
coturnus japonica) Tinamou (Eudromia elegans) Penguin (Pygoscelis adeliac) Rhea (Rhea americana) Chicken ovoinhibitor
Weak Positive Positive 2
0 1
Positive Weak Weak 2
1
a The data are compiled from the following sources: F. C. Wu and M. Laskowski, Sr., J. Biol. Chem. 213, 609 (1955). M. B. Rhodes, N. Bennett, and R. E. ~'eeney, J. Biol. Chem. 235, 1686 (1960). A. J. Vithayathil, F. Buck, M. Bier, and F. F. Nord, Arch. Biochem. Biophys. 92, 532 (196]). C. A. Ryan, J. J. Clary, and Y. Tomimatsu, Arch. Biochem. Biophys. 110, 175 (1965).
[66g]
PROTEINASE INHIBITORS FROM EGG WHITE
899
p H 4.5 are as follows: Als~ 49 °, A~2s --503 °, bo --86 °, ao--524 °, Xc 231 nm. The circular dichroism spectrum .4 of ovomucoid at p H 7.1 shows minima at 292 and 264 nm and a maximum at 243 nm (Table I I ) . The helical content was estimated to be 26% from this data. At p H 11.6 the 243 maximum is greatly enlarged and the intensity of the band at 264 decreases; the band at 292 is almost unaltered. At p H 12.8, all the bands are greatly diminished, showing disorganization. Between p H 3.5 and 4.5, there is ~ a small decrease in specific rotation at both 578 and 313 nm accompanied by a decrease in solvent perturbation effect on the absorption spectrum; these observations indicate a limited conformational change. Spectrophotometric and spectrofluorometric methods 4~ associate the protonation of a carboxyl group of p K 2.8 with this change. Isoelectric points ranging from 3.9 to 4.5 have been reported ~s,Ss,~s for chicken ovomucoid. Bier et al./°,'~ in agreement with Fredericq and Deutsch, 2s separated five components with isoelectric points from 3.83 to 4.41; subsequent electrophoretic separations have confirmed the differences among the components. Osuga and Feeney 's have compared the isoionic points of ovomucoids of several species with the results shown in Table IV. The electrophoretic mobility '1 at p H 8.6 and 0.1 ionic strength is --3.5 X 10~. The absorbanee t.~l~mjrA1% 1 is 4.132s at 280 nm and 4.10' 2 at 277.5 nm. Specificity. F r o m the extensive work of Feeney and his co-workers with ovomucoids derived from the egg whites of many avian species, different patterns of specificity have emerged (Table I I I ) . Ovomucoids may T. T. Herskovits and M. Laskowski, Jr., J. Biol. Chem. 237, 3418 (1962). Z. Hesselvik, Z. Physiol. Chem. 254, 144 (1988). 4~M. Bier, J. A. Duke, R. J. Gibbs, and F. F. Nord, Arch. Biochem. Biophys. 37, 491 (1952). D. T. Osuga and R. E. Feeney, Arch. Biochem. Biophys. 124, 560 (1968).
Y. Tomimatsu, J. J. Clary, and J. J. Bartulovich, Arch. Biochem. Biophys. 115, 536 (1966). D. T. Osuga and R. E. Feeney, Arch. Biochem. Biophys. 118, 340 (1967). R. E. Feeney, G. E. Means, and J. C. Bigler, J. Biol. Chem. 244, 1957 (1969). R. E. Feeney and R. G. Allison, "Evolutionary Biochemistry of Proteins," p. 85. Wiley, New York, 1969. bFor reactions with other enzymes, see the text. The terms "weak" and "positive" are used when quantitative data were not available.
900
NATURALLY OCCURRING ACTIVATORS AND INHIBITORB
[56g]
specifically inhibit either trypsin or chymotrypsin or they may react with both enzymes, separately or at the same time ("double-headed" inhibitors). Some of the ovomucoids react with 2 molecules of trypsin. The reactions of chicken and turkey ovomucoids with turkey trypsin 21 and chicken chymotrypsin 2~ are the same as with the bovine enzymes (Table
III). H u m a n trypsin is not significantly inhibited by any of the ovomucoids tested29,~° Chicken ovomucoid inhibits porcine and ovine trypsin in a 1:1 molar ratio, 51 and also the acetyl derivative of bovine trypsin. 35,5~ Some of the ovomucoids show varying degrees of inhibition of subtilisin. ~ Several ovomucoids form complexes 52 with inactive anhydrochymotrypsin (active center serine replaced by dehydroalanine 5~) and with trypsin and chymotrypsin inactivated at the active center histidine residue ( T L C K - t r y p s i n 5~ and TPCK-,chymotrypsin~5), but not with tosylchymotrypsin 52 or D F P - t r y p s i n . 5e Numerous enzymes in addition to those in Table I I I are not significantly inhibited by chicken ovomucoid: bovine chymotrypsin B, 2~ Clostridium histolyticum collagenase, 57 kallikreins from several sources, ~8-s° human plasmin, z°, 5s human thrombin, 5° and liver esterase2 ~ Kinetic Properties. The dissociation constant (K~) of the complex of chicken ovomucoid with bovine trypsin is 5 X 10-9 M ~ to 5.8 X 10-g M. 5~ K~ values of 6 X 10-9 M were found for the complexes of chicken ovomucoid with porcine and ovine trypsins: ~ The inhibition has been characterized as noncompetitive e2,s8 by classical methods. Green 56 suggested that the usual criteria cannot be applied and regarded the inhibition as competitive. However, the formation of complexes between ovomucoids "F. F. Buck, M. Bier, and F. F. Nord, Arch. Biochem. Biophys. 98, 528 (1962). ~oR. E. Feeney, G. F. Means, and J. C. Bigler, J. Biol. Chem. 244, 1957 (1969). A. J. Vithayathil, F. Buck, M. Bier, and F. F. Nord, Arch. Biochem. Biophys. 92, 532 (1961). G. Feinstein and R. E. Feeney, J. Biol. Chem. 241, 5183 (1966). u D. H. Strumeyer, W. N. White, and D. E. Koshland, Jr., Proc. Natl. Acad. Sci. U.8. 50, 931 (1963). E. Shaw, M. Mares-Guia, and W. Cohen, Biochemistry 4, 2219 (1965). G. Schoellman and E. Shaw, Biochemistry 2, 252 (1963). N. M. Green, ]. Biol. Chem. 205, 535 (1953). ~7I. Mandl, H. Zipper, and L. T. Ferguson, Arch. Biochem. Biophys. 74, 465 (1958). E. Werle, J~rztliche Forschung 22, 41 (1968). N. Back and R. Steger, Federation Proc. 27, 96 (1968). ,o E. Sach and M. Th~ly, Compt. Rend. Acad. Sci., Paris, Ser. D 266, 1200 (1968). ~N. M. Green, Biochem. J. 66, 416 (1957). ~2H. Fraenkel-Conrat, R. S. Bean, and H. Lineweaver, J. Biol. Chem. 177, 385 (1949). ~sp. M6tais, H. Schirardin, and J. Warter, Compt. Rend. Soc. Biol. 157, 2307 (1963).
[66g]
PROTEINASE INHIBITORS FROM EGG WHITE
901
and inactive derivatives of trypsin and chymotrypsin (see Specificity) makes it doubtful that the inhibition is competitive. The rate of reaction of ovomucoids with trypsin and chymotrypsin has been studied 18 at pH 8.2 and 37 °. Turkey and duck ovomucoids react rapidly with trypsin (in less than 1 minute), but about 4 minutes are required to complete the reaction with chymotrypsin. 0vomucoid from the ring-necked pheasant reacts rapidly with both enzymes. Chicken ovomucoid reacts rapidly with trypsin and golden pheasant ovomucoid reacts rapidly with chymotrypsin. Chicken ovomucoid is a temporary 64 inhibitor of trypsin, i.e., it is gradually hydrolyzed in the presence of trypsin. 6s At pH 3.75, upon reaction with trypsin, an Arg-Ala bond is hydrolyzed.6~ Distribution. The occurrence of ovomueoid-type inhibitors in egg white appears to be common to all the avian .species tested. Composition and Terminal Groups. The data for nine ovomucoids are given in Table IV. There are some general similarities: large amounts of hydroxy, acidic, and branched chain amino acids, as well as lysine and cystine, and little or no methionine. Argjnine varies from zero to six residues. 0vomucoid resembles other trypsin inhibitors in its high cystine and low methionine content, and in the absence of tryptophan. The nitrogen content of chicken ovomucoid~,34 is 13.3%. Turkey and pheasant ovomucoids have 12.7 and 13.3%, respectively24 Chicken ovomucoid has amino terminal alanine e7 and carboxyl terminal phenylalanine2s,69 The other ovomucoids in Table IV have amino terminal valine.4s The ovomucoids are glycoproteins, unlike mos~ other trypsin inhibitors. Studies on the nature and linkage of the carbohydrate moiety of chicken ovomucoid have been reviewed by Melamed? Some data on carbohydrate composition are given in Table IV; the hexoses have been identified as D-mannose and D-galactose. Several investigations of the type of attachment of the carbohydrate to the protein have led to diverse conclusions (cf. footnote 9). However, the most recent work of Montreuil and co-workers7° appears to establish linkages between glucosamine and asparagine of the type N-(fl-aspartyl)-N-acetylglucosaminylamine. M. Laskowski,Sr., and F. C. Wu, J. Biol. Chem. 204, 797 (1953). L. Gorini and L. Audrain, Bioehim. Biophys. Acta 8, 702 (1952). mK. Ozawa and M. Laskowski,Jr., J. Biol. Chem. 241, 3955 (1966). e7 H . Fraenkel-Conrat and R. R. Porter, Bioehim. Biophys. Acta 9, 557 (1952). L. P~nasse, M. Jutisz, C. Fromageot, and H. Fraenkel-Conrat, Bioehim. Biophys. Aeta 9, 551 (1952). o R. A. Turner and G. Schmerzler,Biochim. Biophys. Acta 11, 586 (1953). ~oM. Monsigny, A. Adam-Chosson, and J. Montreuil, Bull. Soc. Chim. Biol. 50, 857 (1968).
902
[66~]
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
o
00 o0 o0
~.~
li
J
O
°~~
a~
©
~ [ , ~ O
Z
0 v
i! u
[65g]
PROTEINASE INHIBITORS FROM EGG WHITE
903
Chicken Ovoinhibitor
OvoinhibRor differs from chicken ovomucoid in several properties, including solubility, 6 molecular weight, 22 and ability to inhibit chymotrypsin and bacterial proteinases2 It is therefore considered a separate inhibitor. Purification Procedure ~'~ The starting material is 17 g of ovomucoid prepared by the Lineweaver-Murray method s (step 1, above) and dialyzed only against water. Commercially prepared ovomucoid may be used. Operations are carried out at 23 ° except for the dialysis step (4 °)/Oa Separation o] Ovoinhibitor ]rom Ovomucoid. Titrate a 5% solution of the ovomucoid in water to pH 6.0 with 1 N N a 0 H . Add solid ammonium sulfate to 50% saturation. Collect the precipitate by centrifugation, dissolve it in a minimum amount of water, and dialyze th¢ solution overnight against 0.25% NaC1. Centrifuge the bag contents. Bring the supernatant solution to 35% saturation with ammonium sulfate. Centrifuge and dissolve the precipitate in a minimum volume of water. Dialyze the solution against water, and lyophilize. The yield is 250 mg of ovoinhibitor. Other Methods o] Purification. There are several alternate methods.6,22 A more elaborate method has been developed by Davis et al.2°; it consists of separation from egg white by ammonium sulfate fracfionation, followed by gel filtration and DEAE-cellulose chromatography. This procedure results in the separation of five ovoinhibitor fractions, identical in amino acid composition, but differing in carbohydrate content (Table VI). These fractions, however, are still heterogeneous. Properties
Stability. ~1 In acid solution, ovoinhibitor shows considerable stability: 93-95% of the activity is re~ained when the inhibitor is kept for 15 minutes at 90 ° at pH 3 and 5, for 24 hours at 40 ° at pH 2, or for 3 hours at 40 ° at pH 1. In 0.01 N N a 0 H , the activity is stable at 23 ° for 24 hours, but the activity is lost in 3 hours at 40 ° in 0.1 N NaOH and in 15 minutes at 90 ° at pH 7 and 9. Pepsin at pH 2 destroys ~he activity, but papain at pH 5 does not. Purity. Ovoinhibitor preparations are electrophoretically 2°'22 and chromatographically ~° heterogeneous, but give a sihgle boundary in sedimentation experiments. S° Physical Properties. The data are summarized in Table V. The high 7oly. Tomimatsu, personal communication, 1969. 71K. Matsushima, J. Agr. Chem. Soc. Japan 32, 211 (1958).
904
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[66g]
TABLE V PHYSICAL PROPERTIES OF OVOINHIBITOR Property Molecular weight
Partial specific volume (~) Refractive incrementa dn/dc, ~ -- 436 nm Absorbance, ¢~1% t211emJ] at 278 nm
Method
Value
Light scatteringa Sedimentation (Archibald)° Sedimentation equilibriumb." Sedimentation equilibrium",d Composition'." Amino acid composition° Amino acid compositionc Density'
49,000 44,000 48,700 48,600 46,000-49,000 0. 693 0. 708-0. 712 0.707 0. 185 7.40 ~, 6.5--6.9c
" Y. Tomimatsu, J. J. Claw, and J. J. Bartulovich, Arch. Biochem. Biophys. 115, 536 (1966). b In. H~O and DzO. "J. G. Davis, J. C. Zahnley, and J. W. Donovan, Biochemistry 8, 2044 (1969). In 6 M guanidinium chloride plus 2 mM dithiothreitol, pH 8. • Molecular weight range for five fractions calculated as follows: amino acids 44,000 -b hexose 800-1800 ~ glueosamine 1500-3000.
molecular weight compared to most other inhibitors is not due to dimer formation, 2° since it remains unchanged in a denaturing, reducing solvent (Table V, footnote d). Specificity. 0voinhibitor reacts with trypsins and chymotrypsins of both bovine 7 and avian 21 origin (Table III). It also inhibits Aspergillus protease 6,71 and Bacillus subtilis s var. biotecus (Nagarse). With pronase, the esterase activity 2° and part of the protease activity ~2 are inhibited. Kinetic Properties. Enzyme-to-inhibitor molar combining ratios of 1.74 to 1.90 and 1.65 to 1.70 have been determined 2°,22 for the reactions with trypsin and ehymotrypsin, respectively. 0voinhibitor reacts with both trypsin and chymotrypsin simultaneously; the inhibition of one enzyme is unaffected by the presence of the other, indicating distinct inhibition sites for the two enzymes2 ,6,22 The two active sites differ in their stability toward heat and photooxidationY Subtilisin and trypsin are independently inhibited 22 in mixtures. With mixtures of chymotrypsin and subtilisin, the inhibition of one enzyme is affected 22 by the presence of the R. Haynes and R. E. Feeney, J. Biol. Chem. @-42,5378 (1967). 'ST.-S. Chu and C.-L. Hsu, Acta Bioehim. Biophys. ~inica 5, 434 (1965); Chem. Abstr. 64, 5373g.
[55g]
PROTEINASE INHIBITORS FROM EGG WHITE
905
o t h e r ; this suggests t h a t t h e t w o e n z y m e s c o m p e t e for the s a m e o r closely a d j a c e n t sites. I n h i b i t i o n c o n s t a n t s (KI) for b o t h e n z y m e s 2°,7~'~' a r e b e t w e e n I 0 -8 a n d 10-9 M . T h e i n h i b i t i o n of t r y p s i n a n d c h y m o t r y p s i n a p p e a r s t o be intermediate between competitive and noncompetitive inhibition, from L i n e w e a v e r - B u r k plots, s° W h e n t h e i n h i b i t i o n c o n s t a n t is c a l c u l a t e d 2° for 1:1 b i n d i n g for t h r e e s e p a r a t e o v o i n h i b i t o r f r a c t i o n s as c o m p e t i t i v e inhib i t i o n , K~ v a l u e s of 1.4 to 1.6 X 10 s M for t r y p s i n a n d 0.96 to 1.2 X 10 -8 M for c h y m o t r y p s i n a r e found. C a l c u l a t e d 2° for n o n c o m p e t i t i v e i n h i b i tion, K~ v a l u e s a r e 4.4 to 5.0 X 10-SM a n d 4.2 to 4.8 X 10-SM, respectively. Composition. 2° T h e a m i n o a c i d c o m p o s i t i o n is given in T a b l e V I . A1TABLE VI COMPOSITION OF OVOINIIIBITORa
Amino acid
Residues per mole b
Amino acid
Residues per moleb
Alanine Arginine Aspartic acid Half-cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine
20 21 47 34 39 32 14 17 22 23
Methionine Phenylalanine Proline Serine Threonine Tryptophan c Tyrosine Valine Amide groups Total N (%)
4 6 18 27 33
.Q E I. 0
,
0
0.1
012
,
,
0.3
0.4
Hirudin concentrotion in M.g/ml
Fro. 1. Inhibition of the clotting activity of thrombin by hirudin.
of fibrinogen to fibrin. Such an esterase--thrombin did not bind hirudin, and therefore, the esterolytie activity of this enzyme cannot be inhibited by hirudin. In contrast, diisopropylphosphoryl-thrombin (DIP-thrombin) in which the serin residue in the active enzyme center has been phosphorylated by diisopropylphosphofluoridate (DFP), is like the native form of thrombin bound by hirudin. When thrombin reacts with hirudin, which leads to the immediate neutralization of the enzymatic activity of thrombin, the active surface of the enzyme is apparently occupied by hirudin. It has recently been suggested~ that when thrombin reacts with fibrinogen, the fibrinopeptides, due to their polyanionic characteristics, are bound ,to positively charged sites on the surface of the enzyme. This brings the fibrinogen molecule into a position where its fibrinopeptides can be cleaved. From the finding that DIP-thrombin still binds hirudin, it can be concluded that the hydrolytically active groups of thrombin are not involved in the binding of hirudin. Apparently the binding sites for hirudin can be found in the cationic groups of the active surface of the enzyme which binds the fibrinopeptide portion of the fibrinogen substrate. Acetylation of these , L. Lorand, "Anticoagulants and Fibrinolysis" (R. L. MacMillan and J. F. Mustard, eds.), p. 333. Pitman, London, 1961.
[69]
H I R U D I N AS AN INHIBITOR OF THROMBIN
927
groups, as in esterase-thrombin, blocks the binding of hirudin and thrombin. Methods of Assay
For the standardization and control of hirudin activity, its inhibitory effect can be determined in biochemical test systems. A known amount of thrombin is added to a hirudin solution with unknown activity and the remaining thrombin activity is measured. Thrombin activity can be tested on fibrinogen (clotting activity) or on synthetic substrates (esterolytic activity) (Fig. 1). Since thrombin activity is standardized in National Institute of Health units, the activity of hirudin can easily be related to this international standard. The hirudin activity is expressed in antithrombin units (AT-U), whereby one antithrombin unit is the amount of hirudin which neutralizes one N I H unit of thrombin (fibrinogen assay). One antithrombin unit (AT-U) corresponds to 0.1 ~g of pure hirudin2 Hirudin Titration with Thrombin
Based on the specific, rapid, and stoichiometric reaction between hirudin and thrombin, hirudin activity can be quantitatively determined by titration with a standardized thrombin solution. 6 The following principle is involved: A fibrinogen solution to which hirudin has been added will not clot until enough thrombin is added to neutralize all of the hirudin present. An excess amount of thrombin can be noted within seconds by the appearance of a fibrin clot. Procedure. Aliquots of 0.01-0.1 ml of a hirudin solution with unknown activity are added to 0.2 ml of a 0.5% fibrinogen solution in Tris-NaC1 buffer, pH 7.4. After mixing, a standardized thrombin solution (100 N I H units/ml) is gradually added at room temperature. Since the formation of fibrin takes some time, the thrombin cannot be added as quickly as reagents are added when performing chemical titrations. Therefore, thrombin is added in 0.005-ml aliquots (corresponding to 0.5 N I H units) in minute intervals. The thrombin is added with micropipettes and immediately mixed with the fibrinogen-hirudin mixture. Only if the approximate amount of thrombin to be added is already known (repeat titration or after preliminary titration), can thrombin initially be added faster in larger quantities, but as the expected final concentration is approached, small amounts of thrombin are added in minute intervals. The end point of titration is reached when the fibrinogen coagulates within 1 minute. Instead of purified fibrinogen solutions, citrated plasma also can be used as the source of fibrinogen. The standard deviation for this method is ±0.5 AT-U.
928
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[59]
Use o/Hirudin in Coagulation Studies The effect which hirudin displays on thrombin can be employed for the determination of the activity of this enzyme. The practical application of this procedure has revealed certain advantages over other techniques. The determination of thrombin and those reactions which are closely related to the formation of thrombin can be easily handled in a rather simple procedure. The developed techniques require, however, pure or standardized hirudin which can either be obtained commercially (VEB Arzneimittelwerk, Dresden, German Democratic Republic) or purified by the technique outlined below. D e t e r m i n a t i o n of T h r o m b i n 1°
Principle. A~ter knowing that thrombin activity is neutralized by hirudin, hirudin can be employed for the determination of thrombin activity by titrating a thrombin solution of unknown activity against a known concentration of pure or standardized hirudin. In principle, the procedure is similar to 'the one already described for the determination of hirudin activity. Procedure. An aliquot of 0.1 ml hirudin solution (containing 10 AT-U hirudin) is mixed with 0.1 ml of a 0.5% fibrinogen solution or citrated plasma. Af'ter mixing, small quantities of thrombin (from 50 to 200 N I H units) are added at room temperature and the clotting of fibrinogen is recorded. The clotting of the substrate indicates that 10 N I H units of thrombin have been added. The standard deviation is ± 0.2 N I H units of thrombin. D e t e r m i n a t i o n of Prothrombin 11
Prothrombin can be assayed only after it is completely converted to thrombin. One unit of prothrombin is defined as the amount of prothrombin which gives rise to 1 N I H unit of thrombin. In order to correctly determine prothrombin, one must be certain that its conversion to thrombin is complete. Principle. Freshly drawn blood is mixed with an excess and known amount of hirudin. By adding tissue thromboplastin, a complete conversion of prothrombin to thrombin is achieved, but the generated thrombin will be immediately neutralized by the hirudin. The remaining amount of hirudin in the blood sample is next titrated by adding known quan.tities of standardized thrombin, until the blood sample clots. The ~oF. Markwardt, Arch. Pharm. 290/62, 281 (1957). 11F. Markwardt, Arch. Exptl. Pathol. Pharmakol. 232, 487 (1958).
[59]
HIRUDIN AS AN INHIBITOR OF THROMBIN
929
prothrombin content is calculated from the consumption of hirudin during the coagulation of the blood. Procedure. In a test tube, 0.05 ml hirudin (containing 30 AT-U) is mixed with exactly 0.2 ml freshly drawn blood and 0.1 ml thromboplastin solution. After 30 minutes of incubation, a thrombin solution of known activity is slowly added using a micropipette, as described for the assay of hirudin. The coagulation of the blood sample is recorded. The amount of thrombin that had to be added to facilitate clotting is substracted from the amount of hirudin added (30 AT-U), the difference being the amount of thrombin generated from the pmthrombin in the blood sample by tissue thromboplastin. Since one unit of prothrombin equals 1 NIH unit of thrombin, the exact amount of prothrombin can be calculated. The use of hirudin in other coagulation procedures, such as the hirudin tolerance test, has been described by MarkwardtY 12,13 Isolation of Hirudin TM Preparation o] the Animals and Extraction o] the Inhibitor The glands containing hirudin are located in the region of the body of the leeches which is next to the head region or suctorial disc or, more specifically, in body segments VII, VIII, and IX. These comprise the so-called neck region of the leeches. Since a clean anatomical dissection of the glands for the purpose of obtaining hirudin is not practical, the entire corresponding body parts are separated. For this purpose, leeches of more than 1.5 g body weight, who have not been leeching for at least 3 months, are sacrified by placing them in a 96% ethanol solution. After 24 hours, the fron'tal portion of the leeches is separated by dissecting this portion from the body approximately 5 mm before the anterior (male) genital orifice. The sections are once more placed into 96% ethanol and dehydrated for an additional 24 hours. The still-wet head sections are next chopped into small pieces and twice extracted for 30 minutes under stirring with a 10-fold volume of a 40% acetone-water solution. The extracts are combined and diluted with 1/~ volume of an 80% acetone-wa,ter solution. By adding glacial acetic acid, the pH is lowered to 4.3-4.5 and the resulting precipitate removed by centrifugation. The supernatant is mixed with a diluted ammonia solution until a pH of 6.0 is obtained. The volume is reduced to 1/10 of the original volume by placing the solution in a vacuum at 40 °. Next, the pH is adjusted to 1.8 by adding 10% trichloroacetic acid. The raw hirudin F. Markwardt, Kiln. Wochschr. 37, 1142 (1959). is R. Schmutzler and F. Markwardt, Klin. Wochschr. 40, 796 (1962). 14F. Maxkwardt, G. Schiifer, H. TSpfer, and P. Walsmann, Pharmazie 22, 239 (1967).
930
[69]
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS PURIFICATION OF HIRUDIN
Procedure 1. 1000 leech heads dehydrated in 96% ethanol, chopped to small pieces, extracted with 40% acetone, separation of impurities 2. Fractionation with ethanol 3. Adsorption on cation-exchange resin 4. Gel filtration 5. Chromatography on anion-exchange resin
Dry weight (mg)
Total activity (10a AT-U)
Specific activity (AT-U/rag)
900
450
500
270 80 35 20
405 360 295 208
1,500 4,500 8,400 10,400
product is precipitated from the 10-fold dilution with acetone, washed with acetone, and the solvents are removed in the vacuum. The raw hirudin preparations have a specific aotivity of approximately 500 AT-U/mg. (See the table.) Fractionation with Ethanol Approximately 1.0 g of raw hirudin is dissolved in 30 ml distilled water and chilled to 0°-5 °. Over a period of 30 minutes, 54 ml of 96% ethanol are slowly added. The resulting precipitate is removed by centrifuga~ion. The main bulk of hirudin remains in solution. In order to extract additional hirudin, the precipitate is extracted with ethanol two more times as described above. The combined supernatants are cooled to 0 ° and cold (--10 °) ethanol is slowly added until a concentration of 85% (v/v) is obtained. For more complete precipitation, 0.5% ammonium acetate was added to the ethanol. The precipitate is collected by centrifugation and after washing with ethanol, it is dried in a dessi.cator under vacuum. These hirudin preparations contain 10-15% pure hirudin. In addition, inert material and small amounts of a trypsin inhibitor are present. 1~ These preparations are useful for experimentally inhibiting coagulation and for certain blood clotting tests. Adsorption on Cation-Exchange Resins About 0.3 g of the ethanol-precipitated hirudin is dissolved in 90 ml of a 0.01 M ammonium acetate buffer, pH 4.6. Amberlite IRC-50 (80 ml) in its hydrogen form is added to this solution under stirring until the inhibitor is completely adsorbed into the resin. The resin is collected H. Fritz, K.-H. Oppitz, M. Gebhardt, I. Oppitz, and E. Werle, Hoppe-,.%ylers Z. Physiol. Chem. 350, 91 (1969).
[59]
H I R U D I N AS AN I N H I B I T O R OF THROMBIN
931
by' filtration and washed three times with 100 ml of distilled water. For elution of the hirudin, the resin is suspended in 50 ml of a 1 M ammonium acetate solution. The pH is adjusted to pH 7.0 by adding a 5% ammonia solution. The eluate is removed by filtration. The resin is once more suspended in 50 ml 1 M ammonium acetate solution and the pH adjusted to 8.0 by adding a 5% ammonia solution. The eluates are combined and concentrated to a volume of 50 ml by placing them in a vacuum at 20 °30 °. Next, the hirudin is precipitated in the cold from the eluate by adding a 10-fold volume of 95% ethanol containing 0.5% ammonium acetate. After storage for several hours, the hirudin-conta,ining precipitate is collected by centrifugation, washed with cold acetone, and dried in a vacuum dessicator. Instead of bulk adsorption column chromatography can be employed and Amberlite IRC-50, equilibrated with 0.05 M ammonium formate, pH 4.9, or CM-Sephadex C-50 can be used. First eluation is performed with the equilibration buffer, followed by a 1 M ammonium formate solution, pH 7.0. The obtained preparations contain 40-50% pure hirudin. Gel Filtration on Sephadex G-50 ApproximaCely 100 mg hirudin (40-50% pure) are dissolved in 1 ml of a 0.1 M NaC1 solution and placed on a Sephadex G-50 column (1 X 130 cm), previously equilibrated with 0.1 M NaC1, and elu*ated with 0.1 M NaC1. The eluate is collected in 3-ml aliquots. The tubes containing active material are combined, concentrated in a vacuum, desalted on Sephadex G-25 and lyophilized. These preparations contain about 1520% impurities. Chromatography on Anion-Exchange Resins About 40 mg hirudin (80-90% pure) are dissolved in water and placed on a DEAE-Sephadex A-25 column (0.7 X 20 cm) which has been equilibrated with a 0.05 M pyridine acetate solution, pH 7.4. Hirudin is eluted by establishing a linear gradient with 0.05 M pyridine acetate buffer, pH 7.4 and 1 M NaC1 in 0.5 M pyridine acetate buffer, pH 6.9. The tubes containing active material are combined, concentrated in a vacuum over P20~ and potassium hydroxide at 4 ° and desalted by filtering on Sephadex G-25. The salt-free solution contains pure hirudin and is dried from the frozen state. Criteria of Purity Hirudin is stable in dried form. In water (preservative added) it is stable for 6 months at room temperature. It is also stable when heated for 15 minutes at 80 °. The heat stability decreases with increasing pH
932
"I~ATURALLYOCCURRING ACTIVATORS AND INHIBITORB
[69]
values of the solvent, and in contrast, decreasing the pH increases the hea~ stability. Hirudin is also stable for 15 minutes at 20 ° in 0.1 N HC1 or 0.1 N Na0H. Trypsin and a-chymotrypsin do not destroy the hirudin activity. The resistance against these proteolytic enzymes is probably due to the tertiary structure of the peptide. This assumption is supported by the finding that oxidized hirudin is readily destroyed by trypsin. Papain, pepsin, and subtilopeptidase A destroy hirudin completely. Using paper electrophoresis, several ninhydrin-posi¢ive spots could be identified in the digestion products. Since hirudin does not contain arginine residues, the presence of arginine will identify proteins that contaminate hirudin. Also interesting is the absence of tryptophan and methionine residues in hirudin. The purity of the hirudin preparations was investigated by means of ultracentrifugation and electrophoresis, using a Tiselius electrophoretic apparatus. With both techniques hirudin was homogeneous. Homogeneity was also observed with other elect,rophoretic techniques. The staining properties of the spots with ninhydrin or amido black 10 b coincided with the antithrombin activi.ty.
[70]
INSOLUBLE ~.NZYM~.S
935
[70] Water-Insoluble Derivatives of Proteolytic Enzymes B y LEoN GOLDST~.IN
E n z y m e derivatives in which the biologically active protein is covalently bound to a water-insoluble polymeric carrier m a y serve as easily removable reagents of considerably improved shelf stability; they are well suited for repeated or continuous use and provide means for a more adequate control of enzyme reactions.1-3 Immobilized enzyme derivatives can be used as specific adsorbents for the isolation and purification of enzyme inh'ibitors.~-e Moreover, new properties m a y sometimes be imposed on the immobilized enzyme by the chemical nature of the polymeric carrier.I-3 The methods available for the immobilization of proteins have been recently summarized in several reviews. I-~ Some of the more important methods involving the covalent binding of proteins to water-insoluble polymeric carriers are listed below. The amino groups on proteins have been uti'lized to effect covalent linking to several carboxylic polymers via the corresponding azides,7-I° or by activation of the polymer carboxyls by carbodiimides ~-~3 or by Woodward's Reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate). ~' More recently, cellulose activated by 8-trichlorotriazine (cyanuric chloride) 15-" or by a dichloro-sym-triazinyl dyestuff (Procion brilliant IL. Goldstein and E. Katchalski,Z. Anal. Chem. 243, 375 (1968). 2I. H. Silman and E. Katchalski, Ann. Rev. Biochem. 35, 873 (1966). ~L. Goldstein, in "Fermentation Advances" (D. Perlman, ed.), p. 391. Academic Press, NSw York, 1969. ' H. Fritz, H. Schult, M. Hutzel, M. Wiedermann, and E. Werle, Z. Physiol. Chem. 348, 308 (1967). 5H. Fritz, M. Neudecker, and E. Werle, Angew. Chem. 78, 775 (1966); Angew. Chem. (Intern. Ed.) 5, 735 (1966). 6H. Fritz, K. Hochstrasser, E. Werle, E. Brey, and B. M. Gebhardt, Z. Anal. Chem. @.43, 452 (1968). F. Micheel and J. Evers, Makromol. Chem. 3, 200 (1949). 8 M. A. Mitz and L. J. Summaria, Nature 189, 576 (1961). 9 C. W. Wharton, E. M. Crook, and K. Brocklehurst, European J. Biochem. 6, 565
(1968). ~°W. E. l:Iornby,M. D. Lilly,and E. M. Crook, Biochem. J. 98, 420 (1966). 11N. Weliky and If. H. Weetall,Immunochemistry 2, 203 (1965). D. G. Hoave and D. E. Koshland, J. Biol.Chem. ~ 2447 (1967). N. Weliky, F. S. Brown, and E. C. Dale, Arch. Biochem. Biophys. 131, I (1969). ~4R. P. Patel, D. V. Lopiekes, S. R. Brown, and S. Price,Biopolymers 5, 577 (1967). B. P. Surinov and S. E. Manoilov, Biokhimiya 31, 387 (1966). 16G. Kay and E. M. Crook, Nature 216, 514 (1967).
936
WATER-INSOLUBI~ PROTEOLYTIC ENZYMES
[70]
orange MGS), is and Sephadex or Sepharose, activated by cyanogen bromide, 19-21 have been successfully applied to the immobilization of several enzymes. A polymeric acylating reagent, ethylene-maleic anhydride (1:1) copolymer (EMA), has been successfully used for the preparation of polyanionic water-insoluble derivatives of enzymes, antigens, and protein enzyme inhibitors2 -e, 22-24 In the case of acidic proteins containing large excess of carboxyl groups, immobilization could be achieved by coupling the protein to aminoethyl cellulose through soluble carbodiimide activation of the protein carboxyls. 25 Proteins conCaining relatively large amounts of aromatic amino acids have been coupled to the polydiazonium salts derived from p-aminobenzyl cellulose, 26,27 poly-p-aminostyrene, 2~,2s the m-aminobenzyloxymethyl ether of cellulose, ~,~5,29 a p-amino-DL-phenylalanine copolymer 36'31 and, more recently, a synthetic diazotizeable resin, S - M D A 2 ~ (See Scheme 2.) Kinetic Behavior of Immobilized E n z y m e s The kinetic b e h a ~ o r of immobilized enzyme systems is dominated by several factors not eneoun4ered in the kinetics of free enzymes: (a) effects of the chemical nature of the carrier, stemming from the modified environment within which the immobilized enzyme is located; (b) steric restrictions imposed by the carrier; and (e) diffusion control on the rate of substrate penetration. 1~G. Kay, M. D. Lilly, A. K. Sharp, and R. J. H. Wilson, Nature 217, 641 (1968). R. J. H. Wilson, G. Kay, and M. D. Lilly, Biochem. J. 108, 845 (1968). R. Axen, J. Porath, and S. Ernback, Nature 214, 1302 (1967). ~J. Porath, R. Axen, and S. Ernback, Nature 215, 1491 (1967). ~J. Porath, in "Gamma Globulins" (J. Killander, ed.), Nobel Syrup. No. 3 (June 1967), p. 287. Wiley (Interscience), New York, 1967. Y. Levin, M. Pecht, L. Goldstein, and E. Katchalski, Biochemistry 3, 1905 (1964). u B. Alexander, A. Rimon, and E. Katchalski, Biochemistry 5, 792 (1966). A. H. Sehon, in "International Symposium on Immunological Methods of Biological Standardization" Royaumont, 1965, Syrup. Series Immunobiol. Stand. 4, 51, Karger, Basel, 1967. = L. Goldstein, unpublished data. s D. H. Campbell, E. Leuscher, and L. S. Lerman, Proc. Natl. Acad. Sci. U.8. 37, 575 (1951). ~TL. Goldstein, M. Pecht, S. Blumberg, D. Atlas, and Y. Levin, Biochemistry 9, 2322 (1970). N. Grubhofer and L. Z. Schleith, Z. Physiol. Chem. 297, 108 (1954). ~gA. E. Gurvieh, Biokhimiya (Engl. transl.) 22, 977 (1957). mA. Bar Eli and E. Katchalski, J. Biol. Chem. 238, 1690 (1968). I. H. Silman, M. Albu-Weissenberg, and E. Katehalski, Biopolymers 4, 441 (1966).
[70]
INSOLUBLE ENZYMES
937
Low Molecular Weight Substrates The pH-activity profiles of the polyanionic derivatives of several proteolytic enzymes acting on their specific low molecular weight substrafes have been shown to be displaced toward more alkaline pH values by 1-2.5 pH units, at low ionic strength (r/2 ~ 0.01) as compared to the native enzymes.1,32 Polycationic derivatives of the same enzymes exhibit the reverse effect, i.e., displacement of the pH-activi~y profile toward more acidic pH values? These anomalies are abolished at high ionic strength (r/2 ~ 1). To illustrate this phenomenon, the pH-activity profiles of a polyanionic derivative of trypsin (EMA-~rypsin) and of the polyanionic and polycationic derivatives of chymotrypsin (EMA-chymotrypsin and polyornithyl chymotrypsin) are shown in Figs. 1 and 2. Charged derivatives of papain, 2s ficin,1° and subtilopeptidase A (subtilisin Carlsberg) =5 exhibit similar behavior. Furthermore, the apparent Michaelis constant (K~) of a polyanionic derivative of trypsin (EMAtrypsin), with the positively charged substrate benzoyl-L-arginine amide
LOC 90 8C =" 70 O
E 60
~ E 50 "S 4O
g ~. 3o 20 10
04
5
6
-f
8
9
IO
--I
pH
FIo. 1. pH-Activity curves for trypsin and a polyanionic, ethylene-maleic acid copolymer derivative of trypsin (EMA-trypsin), at different ionic strengths, using benzoyl-b-arginine ethyl ester as substrate (redrawn from the data of footnote 32). L. Goldstein, Y. Levin, and E. Katchalski, Biochemistry 3, 1913 (1964).
938
W A T E R - I N S O L U B L E PROTEOLYTIC E N Z Y M E S
[70]
?.
~5 8
o fL
.5
4
b
fO
f
B
~
I0
It
12
pH
FIG. 2. pH-Activity curves at low ionic strength (F/2 = 0.008) for chymotrypsin, a polyanionic derivative of chymotrypsin (EMA-chymotrypsin) and a polycationic, polyornithyl derivative of chymotrypsin, using acetyl-L-tyrosine ethyl ester as substrate.1'~ (O) chymotrypsin, ( 0 ) EMA-chymotrypsin, (A) polyornithyl-chymotrypsin.
(BAA) has been found to be markedly lower, at low ionic strength, than that of the native enzyme s2 (Figs. 3 and 4). Similar effects have been reported for the polyanionic derivatives of papain (EMA-papain), 25 fiein 1° (CM-cellulose-ficin) and bromelain 8~ (CM-eellulose-bromelain) using benzoyl-L-arginine ethyl ester and substrate. Again, the perturbation of the apparen¢ Michaelis constant is abolished at high ionic strength82, 88,s4 (see Fig. 4). K~ values closely similar to those of the native enzyme have been demonstrated with uncharged substrates for the polyu M. D. Lilly, W. E. Hornby, and E. M. Crook, Biochem. J. 100, 718 (1966). C. W. Wharton, E. M. Crook, and K. Brocldehurst, European J. Bioehem. 6, 572 (1968).
[70]
INSOLUBLE ENZYMES
939
EMA-Trypsin 1"/2=0.04
IO
•
"
Trypsin [ ' / 2 = 0 0 4 0.8
o
o
0.6 :>
04 0.2 0
I
I
I
I
I
2
3
4
[S] x 102 m o l e s / l i t e r
Fro. 3. Normalized Michaelis-Menten plots for trypsin and a polyanionic derivative of trypsin (EMA-trypsin), acting on benzoyl-L-arginineamide,=
anionic and polyeationic derivatives of ehymotrypsin and for a polyanionic derivative of papain?" These phenomena have been explained as resulting from the unequal distribution of ionic species between the charged "solid-phase," the pelyelectrolyte-enzyme particle, and the surrounding solution?2 I
>
K o E
|
I
, / E M A - trypsin, [ ' / 2 =0.5 K M =(5.2 ~ 0 . 5 0 ) x l 0 -3
E M A - trypsin, r / 2 = 0 . 0 4
•
..
K'M= (0.2 ± 0.05) x IO "s I I0
I 5
10-2/[S]
I 15
liter/mole
FIG. 4. Normalized Lineweaver-Burk plots for trypsin and a polyanionic derivative of trypsin (EMA-trypsin) acting on benzoyl-L-arginine amide.=
940
WATER-INSOLUBLE PRO'rEOLYTIC ENZYMES
[70]
Thus the local hydrogen ion concentration in the domain of the charged enzyme derivative could be described by Eq. (1) a~+ = a°÷e"'~/k~"
(1)
where ah+ and a% are the hydrogen ion activities in the polyelectrolyteenzyme derivative phase and the external solution, ~ = the electrostatic potential in the domain of the charged immobilized enzyme particle, = the positive electron charge, z = a positive or negative integer, of value unity in the case of hydrogen ions, k = the Boltzmann constant, and T = the absolute temperature. It follows from Eq. (1) that the local pH in the domain of a polyanionic enzyme derivative will be lower than that measured in the external solution. The reverse will be true for a polycationie enzyme derivative. Consequently the pH-a,ctivity profile of an enzyme immobilized onto a charged carrier will be displaced toward more alkaline or toward more acid pH values, for a negatively or positively charged carrier respectively, i.e., hpH = pH ~ - pH 0 = 0.43 ~-~
(2)
where ApH is the difference between the local pH within the polyelectrolyte-enzyme particle (pH i) and the pH of the external solution (pH°). pH i is deduced from the pH-activity profile of the native enzyme (see Figs. 1 and 2). The dependence of enzyma¢ic activity on p H is related to the dissociation of ionizing groups participating in the enzymatic catalysis mechanismS~, 86 (e.g., the acidic limb of the pH-activity curve of chymotrypsin associated with the ionization of histidine-5785,sT). The displaced pHactivity profiles of a polyelectr~lyte enzyme derivative can therefore be alternatively represented in terms of changes in the values of the apparent acidic dissociation constants [pK~ (app)] of the "active-site" ionizing group effected by the polyelectrolyte "microenvironment ''Ss of the enzyme derivative, i.e.,
z~
pg" = p g A- 0.43 k--T
(3)
or u H. Gutfreund, Trans. Faraday 8oc. 51, 441 (1955) ; M. L. Bender and F. J. K~zdy, Ann. Rev. Biochem. 34, 49 (1965). x M. Dixon and E. C. Webb, "Enzymes" (2rid ed.). Longmans, London, 1964. ,Tp. B. Sigler, D. M. Blow, B. W Matthews, and R. Henderson, J. Mol. Biol. 35, 143 (1968). =J. T. Edsall and J. Wyman, "Biophysical Chemistry," Vol. I, Chap. 9. Academic Press, New York, 1958; C. Tanford, "Physical Chemistry of Maeromolecules" Chaps. 7 and 8. Wiley, New York, 1961.
[70]
INSOLUBLE ENZYMES
941
hpK. = pK" - pK, = 0.43 zek k--T
(4)
pK, and pK" are the apparent dissociation constants for the native enzyme and polyelectrolyte enzyme derivative, calculated from the ~ppropriate pH-activity profile. Equations (1-2) ,and (3-4) are of course identical. The model summarized in Eqs. (1) to (4) explains satisfactorily most of the known data on the pH dependence of activity of chemically modified enzymes: The pH-activity profiles of both acetylated and succinylated, enzymatically active derivatives of trypsin and chymotrypsin are displaced toward higher pH's. 1,3, 22,38,29 A similar alkaline shift in the pH optimum has been reported for insoluble preparations of chymotrypsin coupled to Sepharose 19,2° (by means of cyanogen bromide activation of the polysaccharide carrier). These effects are most probably due to the increase in the net negative charge on the protein, resulting from the acylation of amino groups, and thus to an increase in the values of pK, (app) of the active-site histidines of these enzymes [see Eqs. (3) and (4) and footnotes 35-37]. Water-insoluble derivatives of enzymes bound to electrically neutral carriers such as a p-amino-DL-phenylalanine-leucine copolymer, 29,2° S-MDA resin (see Schemes 2 and 3), and p-aminobenzyl cellulose via the polydiazonium salts derived from these resins, often show pH-activity profiles displaced toward more alkaline pH's21,39a This anomaly is somewhat unexpected in view of the assumed chemistry of binding, i.e., coupling of the polymeric diazonium sal.t to the tyrosyl residues on the protein, via azo bonds. Amino acid analysis of acid hydrolyzates of several such derivatives has, however, revealed among the missing amino acids not only tyrosine but also lysine and arginine2 x It seems, therefore, that in this case as well, the increase in pK, (app) is due to an increase in the net negative charge on the protein, effected by the disappearance of lysyl-c-amino and guanido groups at some stage of the coupling reaction. In addition, the "acid-strengthening" effect of the azo-bond 4° on the hydroxyl ionization of "coupled" tyrosyl residues might also have contributed to the increase of net negative charge on the enzymes, and thus to the displacement of the pH-activity curves. s~j. Sri Ram, L. Terminiello, M. Bier, and F. F. Nord, Arch. Biochem. Biophys. 52, 464 (1954); J. Sri Ram, N. Bier, and P. H. Maurer, Advan. EnzymoI. 24, 105 (1962). ~ The water-insoluble derivatives of papain (with the above resins as carriers) are the only well-characterized case known to the author where both the pH-activity profiles and the values of K~ of the immobilized enzyme preparations (with BAEE as substrate) are closely similar to those of the crystalline enzyme. H. Zollinger, "Azo and Diazo Chemistry," Wiley (Interscience), New Yol'k, 1961; M. Sokolovsky and B. L. Vallee, Biochemistry ~, 3574 (1966) ; ibid., 6, 700 (1967).
942
WATER-XNSOLUBLV. PROTEOLY~C ENZYMES
[70]
The changes in the values of the apparent Michaelis constants (K~) of polyelectrolyte enzyme derivatives acting on charged low-molecular weight substrates (cf. Figs. 3 and 4) can be related to the unequal distribution [cf. Eq. (1)] of substrate between the charged enzyme particle and the external solution [Eq. (5) ].
[S]' = [~]°e"~l~T
(5)
[S]' and [S] o are the substrate concentrations in the domain of the polyelectrolyte enzyme particle and the external solution. It follows from Eq. (5) that [ S ] ' > [S] ° when polyelectrolyte enzyme derivative and substrate are of opposite charge; the enzyme derivative will attain the limiting rate, Vm,x, at lower bulk concentration of substrate as compared to the native enzyme, and the value of the Michaelis constant for the immobilized enzyme (K~) will be correspondingly lower. For substrate and polyelectrolyte enzyme particles of the same charge, the opposite will be true, i.e., [S]' < [S] ° and K~ of the enzyme derivative will be higher. This can be seen by introducing Eq. (5) into the MichaelisMenten rate equation
Vm.x[S] K~-b [S] to obtain Eq. (6).
v'=
gm~[S]°e"*lkr K~ q- [S]°e"~l~r
(6)
Primed symbols indicate quantities pertaining to the polyelectrolyte enzyme derivative. From Eq. (6) v ' - ~ 1/2V~,~ when [S] ° - - K m e - ~ l '/kT. Thus the value of external substrate concentration [S] °, at which halfmaximum velocity is attained, leads to an apparent Michaelis constant K'~ related to the Michaelis constant of the native enzyme by the expression K~ = K,e-,,~l~r
(7)
Eq. (7) can be rewritten as ApK,,, --- pK',,, -
K~
z~
pK,,, = log ~-~ -- 0.43 ~-~
(8)
High Molecular Weight Substrates The activities of immobilized proteases toward protein substrates, as determined by the standard procedures (Kunitz's casein digestion method, appearance of nonprotein nitrogen, potentiometric determination of the number of peptide bonds split, ninhydrin, etc.) are usually lower
[70]
INSOLUBLE ENZYMES
943
than those of the crystalline enzymes, when compared on a weight basis. Water-insoluble derivatives of polytyrosyl trypsin 29 and papain s° hydrolyzed casein at 15 and 30% of the rates which would have been expected from the amount of active bound enzyme (determined by a rate assay with low molecular weight substrate). Similar results have been obtained with carboxymethyl cellulose conjugates of ficin, 1°,3~ bromelain, 9 and trypsin, s and with poly.anionic polycations and polyalcohol derivatives of papain 2~ and subtilopeptidase A. 2~ In the majority of cases the lowering in proteolytie activity can be attributed to steric hindrance induced by the carrier. 1-~ An additional effect might become prominent in the case of polyelectrolyte derivatives of enzymes, i.e., the electrostatic interaction between charged carrier and charged, high molecular weight Substrate: In the digestion of casein by polyanionie derivatives of trypsin (see EMAtrypsin, p. 950) the rate of hydrolysis has been found to depend on the carrier-to-enzyme ratio. ~2 Preparations of high enzyme content (high enzyme :.carrier ratio) showed caseinolytic activities close to those of native trypsin while preparations of low enzyme content had about 20% of the expected proteolytic activity. 22 Raising the pH and thus increasing the charge density of the carrier led to a marked decrease in the caseinolyric activity of both EMA-trypsin preparations. 22 The data conveyed that the number of peptide bonds split could be controlled by varying the charge density on the polyelectrolyte enzyme derivative. Furthermore, indications that the sites of attack as well as the rates of cleavage of a high molecular weight substrate might be affected by the chemical nature of the polymeric carrier could be found in the literature? -3,~2,~3,4~-46 For example, both water-insoluble polytyrosyl trypsin and EMA-trypsin attacked prothrombin. The polytyrosyl trypsin derivative, like native trypsin, converted prothrombin to thrombin which was not digested any further. In contrast, EMA-trypsin rapidly degraded the newly formed thrombin. ~ It was also found that while the clotting factor VII was activated by insoluble polytyrosyl trypsin, it was not affected by EMA-trylasin. ~ These observations have been substantiated in studies on the EMAtrypsin digestion of myosin and the meromyosins. 4~,42 It was found that ,I S. Lowey, L. Goldstein, and S. Luck, Biochem. Z. 345, 248 (1966). " S . Lowey, L. Goldstein, C. Cohen, and S. M. Luck, J. Mol. Biol. 23, 287 (1967). ,3 It. S. Slayter and S. Lowey, Proc. Natl. Acad. Sci. U,$. 58, 1611 (1967). S. Lowey, in "Symposium on Fibrous Proteins, Australia 1967" (W. A. Crewther, ed.). Butterworth, London, 1,968; S. Lowey, H. S. Slayter, A. G. Weeds, and H. Baker, Y. Mol. Biol. 42, 1 (1969). E. B. Ong, Y. Tsang, and G. E. Perlmann, J. Biol. Chem. 241, 5661 (1966). T. L. Westman, Biochem. Biophys. Res. Commun. 35, 313 (1969).
944
V~ATER-INSOLUBLE PROTEOLYTIC ENYZMES
[70]
the first-order rate constants estimated for the EMA-trypsin digestion of myosin were about 50-fold lower as compared to those of native trypsin, and about half as many peptide bonds were split by the polyanionic trypsin derivative. Moreover, different protein fragments were obtained on limited EMA-trypsin digestion of both heavy-meromyosin (HMM) and myosin. In these investigations, a new helical subfragment (HMM-subfragment-2) was isolated from EMA-trypsin digests of heavy meromyosin.~l,4~ The separation of the globular part of the myosin molecule HMM-subfragment-1 as well as the isolation of its intact helical rod portion have been recently achieved by the controlled digestion of myosin with a water-insoluble papain derivative. '8, ~' It has been recently established "5 that whereas trypsin hydrolyzed the 15 lysyl peptide bonds in pepsinogen, the maximal number of bonds cleaved by EMA-trypsin never exceeded 10; the same difference was observed when reduced carboxymethylated pepsinogen was used as substate. These findings were confirmed by peptide mapping. The data indicate that the chemical nature of the carrier, at least in the case of polyelectrolyte enzyme derivatives, may impose additional restrictions on the specificity of the bound enzyme. These restrictions probably result from charge interactions between carrier and different regions, or different sequences on the high molecular weight substrate molecule. Hence, in studies of protein sequence, the restricted specificity displayed by polyelectrolyte enzyme derivatives could be utilized to obtain overlapping peptides by a series of derivatives of one enzyme only.~, Enzyme Columns Columns of immobilized enzymes have been employed for the continuous preparation of product, for regulation of the extent of conversion of substrate to product, and in automated analytical procedures.1-a,29,~3,~,'s A modified form of the integrated Michaelis-Menten rate equation has been used by Lilly et al.~ to describe the kinetics of substrate hydrolysis in an enzyme column. Defining the residence time of substrate in the enzyme column t, by
t = V~/Q
(9)
where Vz is the void volume of the column, Q the flow rate, and substituting Eq. (9) into the integrated Michaelis-Menten equation 4~R. Goldman, O. Kedem, I. H. Silman, S. R. Caplan, and E. Katchalski, Biochem/stry 7, 486 (1968). R. Goldman/O. Kedem, and E. Katchalski, Biochemistry 7, 4518 (1968).
[70]
INSOLUBLE ENZYMES
945
So - St = k3[E]t -t- K , , ln(SJSo)
Eq. (10) is obtained: PSo
-
-
g',, ln(1 - P ) = k~E~/O = C / Q
(10)
where fl = V ~ / V t is voidage of the column; P = ( S o - St)/So, fraction of substrate rea.cted in column; C = k3Efl, reaction capacity of column; Vt = total volume, and So, St, k3, E, and Km have their usual significance. Eq. (10) may be rearranged PSo = K',,, ln(1 - P ) -t- C / Q
(11) If values of P are measured when various initial concentrations of substrate are perfused through the same column at an identical flow rate (i.e., Q = const.), PSo plotted against (1 -- P) will give a straight line if K~ and C are constant at this flow rate. The slope of the line will be equal to Km and the intercept to C / Q . Thus Km and C can be determined for any flow rate through the column. Kinetic data on the hydrolysis of benzoyl-L-arginine ethyl ester in packed columns of carboxymethyl cellulose-ficin when "plotted according to Eq. (11) indicated that the apparent Michaelis constant (K'~) decreased with increasing flow rate and asymptoted toward a minimal value at high flow rates2 3 In general, the value of the Michaelis constant was higher than that observed under comparable conditions in stirred suspensions of the carboxymethyl cellulose ficin derivative. 1°,83 The data also suggested that at very low values of Q there was a tendency for C to increase. These deviations from the behavior expected on the basis of Eq. (11) could be qualitatively explained as due to diffusion-limited transport of substrate into the enzyme particles. A more general study of diffusion-controlled processes in immobilized enzyme systems has been carried out by Goldman et a I N ,4~ using papain-collodion membranes. Carriers The water-insoluble derivatives of trypsin, chymotrypsin, papain, and subtilopeptidase A (subtilisin Carlsberg), the preparation and properties of which are des.cribed below, can be divided into two groups according to the type of carrier used: 1. Polyanionic, water-insoluble derivatives, in which the enzyme is bound to a carboxylic polymer (an ethylene-maleic acid copolymer) via amide linkages. 2. Water-insoluble derivatives in which the enzyme is bound to a neutral, synthetic resin (see S-MDA, p. 946)mainly via azo linkages. The polyanionic, water-insoluble enzyme derivatives are prepared by coupling the enzyme protein to a polyfunctional acylating reagent, a
946
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[70]
N~ - C H 2 - C H 2 - C H - - C H -IC H 2 - C H 2 - C H - C H! I
OC ,,,o/CO
I
OC\ 0 ~CO
-+
N H2
NH2
.
-c.2-
C.oo_- c.2- c H2- ¢,.coo_-Vcoo_I
~ NH
NH 2
NH I OC CO0CO0" COOl I I I - C H 2 - CH2- CH -- C H - C H 2 - CH2- CH - - CH -
SCHEME 1. Coupling of proteins to an ethylene-maleic anhydride (1:1) copolymer (EMA).
1 :1
copolymer of ethylene and maleic anhydride, EMA (p. 948) 22,32 EMA reacts mainly with the ~-amino groups of the lysyl residues on the protein (Scheme 1). Additional cross-linking with hexamethylene diamine (1,6diaminoetha~le) enhances the insolubility of the materials. The unreacted maleic anhydride residues on the polymeric carrier subsequently undergo hydrolysis in the aqueous medium. The ratio of enzyme protein to carrier in the reaction mixture can be varied within fairly wide limits. Samples containing up to 8 0 ~ by weight of bound protein have been prepared by this procedure. 22 The physical properties of the EMA-protein derivatives are dependent on their protein-to-carrier ratio. 1,22,82 Preparations of low protein content are gel-like in appearance. The swollen EMA-protein gels contract significantly on increasing the ionic strength of the suspending medium. These materials can be separated from the medium by centrifugation. They cannot, however, be filtered except at very high ionic strength ( r / 2 / > 1.0). Preparations of high protein content (6080~o protein by weight) have flaky texture, are easily separated from the suspending medium by centrifugation, and in most cases by filtration , at moderate ionic strengths. High protein-content EMA derivatives can be used in columns. The second type of water-insoluble enzyme derivatives is obtained by coupling the protein, mainly through its aromatic amino acid residues, to the polydiazonium salt derived from a synthetic resin, S-MDA 48a (Schemes 2 and 3).al The S-MDA resin is prepared by condensation of ~l T h e name of the resin S - M D A is derived from the names of its chemical compon e n t s : (dialdehyde)-starch methylene-dianiline (see Scheme 2 ) . .
[70]
ISSOLVSL~, ~.~ZYM~ CH20H 0 H "CH II
.... 0
~(~ '---~O
H~
H II
o o
.... 0
n
BISHETHVLENE CH2OH
It
N
- -
O
O0
O
N
.....
66
d
NH2
'
DIANILINE
.
¢
Cd NH2
0 .....
It
STARCH
CH2OH
N
H
o I O.N.~Oc.~O=
DIALDEHVDE
H
947
N
..... O
~H
II H~ H
II
O CH2OH
CH2OH NaBH 4
"CH2OH -----0
H~
~I
0
0 .........
NH
OH
¢©
dd NH 2
H
NH 2
¢ d NH
NH I
.
.
.
.
.
.
o
66 NH
OH
,,~H,~c,, I(H
-o
NH I
~_ H2 H2C'~J
"i
'.\I---o4 CH2OH S_MDA
I
H!~.-'T~OH O~H
RESIN
SCHEME 2. S y n t h e s i s o f S - M D A
resin.
'-0 . . . . .
948
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[70]
o, HCI
~"CH2--NH- ' ~ S-MDA
CH2-~NH2 RESIN
POLYDIAZONIUM SALT
SCHEME 3. Prepsration of a polydiazonium salt derived from S-MDA resin.
dialdehyde starch (a commercially available periodate-oxidation product of starch) with bismethylene dianiline, and the subsequent reduction of the Schiff's base highly cross-linked polymeric product (Scheme 2). Preparations containing up to about 10% by weight of bound protein can be prepared using the S-MDA resin as carrier. 48b The water-insoluble, S-MDA enzyme conjugates have particulate form, are easily filterable, and can be conveniently used in columns.81 Ethylene-lYIaleic Anhydride (1 : 1) Copolymer (EMA) EMA samples of average molecular weight in the range of 20,000-100,000 are available commercially (Monsanto Chemical Co., Inorganic Chemical Division, St. Louis, Mo.). EMA undergoes, on standing, partial hydrolysis as a result of absorption of moisture. The carboxyl groups liberated in this process can be reconverted into the anhydride form on heating at 105°-110 ° for 24 hours over phosphorus pentoxide. Experience has shown, however, that when the coupling reaction is carried out in an aqueous medium, both the binding of protein and the recovery of enzymatic activity in the insoluble derivative are consistently higher with EMA samples in which only 60-80% of the carboxylic groups are in the anhydride form. When, on the other hand, the coupling reaction is carried out in a mixed solvent system, where a solution of the EMA polymer in an organic solvent (e.g., dimethylsulfoxide, dimethylformamide, acetone, etc.) is added to an aqueous solution of the protein, oven-dried EMA is to be preferred. ~bAs an alternative choice for a diazotizable resin, commercial p-aminobenzyl Cellulose (PAB-cellulose) could be used. This resin has, however, distinctly inferior properties as carrier, in comparison to the S-MDA resin. Both its protein binding capacity and the recovery of enzymatic activity in the insoluble form are considerably lower.~1 Moreover, insoluble enzyme preparations utilizing p-aminobenzyl cellulose as carrier have a tendency to liberate color into aqueous buffers. This deficiency of PAB cellulose can be partly overcome by refluxing the resin with methanol for about 30 minutes and air drying prior to diazotization.~
[70]
INSOLUBLE ENZYMES
949
Characterization. The anhydride content of an ethylene-maleic anhydride copolymer sample is determined as follows~2: The total amount of carboxyl plus anhydride groups in an EMA sample is determined after hydrolysis of the sample (50-100 rag) in boiling water for 10-15 minutes; the hydrolyzed copolymer in aqueous solution, 0.5 M in NaC1, is titrated with 0.1 N N a 0 H using phenolphthalein as indicator. Two moles of titrant are consumed per base mole of maleic acid or maleic anhydride. In a parallel experiment the intact copolymer is dissolved in anhydrous dimethylformamide and the solution is titrated with 0.1 N sodium methoxide in methanol-benzene, using thymol blue as indicator. ~9 One mole of titrant is consumed per base mole of anhydride, whereas 2 moles are consumed per base mole of maleic acid. The amount of anhydride and free carboxyl groups initially present in the ethylene-maleic anhydride copolymer sample is calculated from the two titration values. Preparation of S-MDA
R e s i n 31, 49a
Reagents. Dialdehyde starch, the periodate-oxidation product of starch (90% oxidized), can be obtained under the trade name Sumstar190, from Miles Chemical Co., Elkhart, Ind. Bismethylene dianiline (MDA, p,p'-diaminodiphenyl methane) and sodium borohydride are standard commercially available chemicals. Procedure. Dialdehyde starch, Sumstar-190 (10 g) is suspended in water (200 ml), stirred at room temperature for 10-15 minutes to obtain a fine slurry, and 2 M carbonate buffer, pH 10.5 (40 ml) is then added. The suspension is poured slowly into a vigorously stirred, 10% solution of bismethylene dianiline in methanol (300 ml). The reaction mixture is stirred at room temperature for 2-3 days. The insoluble, polymeric Schiff's base thus obtained is separated on a funnel, washed with methanol, and then suspended in water and reduced with sodium borohydride (40 g). The reaction mixture is brought to neutral pH with acetic acid and the resin is washed with water and methanol and then refluxed with methanol (3-4 changes of solvent, 300 ml each) to remove methanol-soluble aromatic amines. Refluxing is continued until a negative test is obtained with N-dimethylaminobenzaldehyde (Ehrlich's reagent) .50,~1 The material is then filtered and air dried. The total weight of dry material is 14-15 g. The nitrogen content of the S-MDA resin is 6.5-6.8%. The diazotization capacity is 0.24-0.26 meq/g, which comprises about 10% of the o A. Patchomik and S. Ehrlich-Rogozinki, Anal. Chem. 33, 803 (1961). 4°, S-MDA (dialdehyde)-starch-methylene-dianiline. C. Menzie, Anal. Chem. 28, 1321 (1956). ~1M. M. Sprung, Chem. Rev. 26, 297 (1940).
950
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[70]
total M D A content of the resin,sl The maximal protein binding capacity of the S - M D A resin (following diazotization) is 8-10 m g protein per 100 m g resin,sl Characterization o] the S - M D A Resin. The diazotization capacity of the S - M D A resins is estimated by coupling its polydiazonium salt with p-bromophenol and determination of the nitrogen and bromine contents of the reaction product: S - M D A resin (50 rag) is suspended in 50~o acetic acid (4 ml) and stirred for 1 hour over ice. An aqueous solution of sodium nitrite (10 m g in I ml) is then added dropwise to the chilled suspension. The diazotization mixture is stirred for I hour over ice and then brought to p H 8.5 by the dropwise addition of 5 N N a O H . The precipitate is separated on a funnel, washed with cold 0.2 M phosphate buffer, p H 7.8, and suspended in the same buffer (5 ml). A solution of p-bromophenol (50 rag, dissolved in water by the dropwise addition of 2 N N a O H ) is then added to the chilled suspension. The reaction mixture is stirred overnight at 4 ° . The precipitate is separated by filtration, washed with 0.1 M carbonate buffer, p H 10.5, water and methanol, and dried in vacuo over phosphorus pentoxide. The nitrogen and bromine contents of the dry resin are determined by the D u m a s and SehSniger combustion methods, 52 respectively. The protein binding capacity of the S - M D A resin can be estimated from the saturation curves obtained by coupling the polydiazonium salt, derived from S - M D A (50 rag) with varying amounts of protein (1-10 rag) and determining the enzymatic activity of the reaction mixture supernatants and the insoluble precipitates,8~ or by determining the amino acid composition of acid hydrolyzates of the latter,s~ Water-Insoluble Trypsin Derivatives
Trypsin (EC 3.4.4.4), X 2 crystallized, salt free and lyophilized is commercially available. Trypsin is assayed titrimetrically,~a,54 at 25 °, pH 8, using benzoyl-L-arginine ethyl ester (BAEE) as substrate (5 ml; 1.16 X 10-~ M BAEE, 0.002 M in phosphate) and 0.1 N N a 0 H as titrant. Polyanionic, Water-Insoluble Trypsin Derivative s2,s2
EMA- Trypsin Carrier. Ethylene-maleic anhydride (1 : 1) copolymer (EMA) (see p. 94s). a A. Steyermark, "Quantitative Organic Microanalysis" (2nd ed.).Academic Press, New York, 1961. B C. F. Jacobsen, J. Leonis, K. LindestrCm-Lang, and M. Ottesen,in "Methods of Biochemical Analysis" (D. Glick,ed.),Vol. 4, p. 171.Wiley (Interscience),1957. u M. Laskowski,Vol. H [3].
[70]
INSOLUBLE ENZYMES
951
Procedure. Ethylene-maleic anhydride copolymer, EMA (100 rag) is suspended in ice-cooled, 0.2 M potassium phosphate buffer, pH 7.5 (510 ml). The suspension is homogenized and the cross-linking agent, hexamethylene diamine (1 ml of a 1% aqueous solution), is added with stirring. The appropriate amount of trypsin (10-400 mg) dissolved in the same buffer (5-30 ml) is added and the reaction mixture left overnight at 4 ° with magnetic stirring. The precipitate is separated by centrifugation and washed by suspending in 1 M KC1 (100 ml) and recentrifuging (about twenty times) at 4 °, until no residual enzymatic activity toward benzoyl-L-arginine ethyl ester hydrochloride can be detected in the supernatant after filtration through a Millipore filter. The recovery of enzyme activity (tested with BAEE) in the waterinsoluble EMA-trypsin derivatives is 30-40%. 22'32 Notes. It should be stressed that the binding of enzymatically active protein is consistently higher when the EMA carrier is partially hydrolyzed. Best results are obtained when 60-~% only of the carboxyl groups are in the anhydride form. 22 In the preparation of high protein content samples (300-400 mg protein per 100 mg EMA in the coupling mixture), the cross-linking agent, hexamethylene diamine, can be omitted.2~ Determination o] Activity. The EMA-trypsin derivatives are assayed as described for trypsin, at pH 9.5 (the optimal pH of the EMA-trypsin derivatives is in the pH range 9.5-1022,a2). The amount of active enzyme is calculated from the rate of substrate hydrolysis. The protease activity is determined at pH 7.6 by the casein digestion method.54,55 Determination o] Bound Protein. The EMA-trypsin preparation is hydrolyzed in an evacuated, sealed tube using 6 N HC1 (48 hours, 110°). The acid is evaporated and the residue suspended in 0.2 M citrate buffer, pH 2.2 (4 ml); insoluble material is removed by centrifugation and amino acid analysis is carried out employing an automatic amino acid analyzer.58 The amount of protein is calculated from the amounts of alanine, leucine, glycine, and valine2 ~ In the case of EMA-trypsin preparations containing 60--80% protein by weight, an estimate of the protein content can be obtained from the nitrogen content (Kjeldahl) of the sample. Properties. EMA-trypsin derivatives are considerably more stable in the alkaline pH range (pH 7.0-10.7) than the crystalline enzyme.22 EMA-trypsin suspensions in distilled water or in 0.1 M phosphate buffer, M. Kunitz, J. Gen. Physiol. 30, 291 (1947). ~D. H. Spackmaa, Vol. XI [1]. 5, M. O. Dayhoff and R. V. Eck, "Atlas of Protein Sequence and Structure," National Biomedical Research Foundation, Silver Spring, Maryland, 1967-68.
952
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[70]
pH 7, can be stored in the cold (4 °) for several months without significant loss in enzyme activity, 22 They can be lyophilized without significant decrease in activity. The dried powders can be stored at room temperature or at 4 ° for long periods of time. 2. The pH-activity profiles of EMA-trypsin derivatives using benzoylL-arginine ethyl ester as substrate are displaced by about 2.5 pH units at low ionic strength (r/2 ~ 0.005) toward more alkaline pH values as compared with native trypsin under similar conditions. 22,32 The pH optimum for the polyanionic trypsin derivatives lies in the region of pH 9.5-10.022,32 (see Fig. 1). This effect is reversed on increasing the ionic strength. In the case of EMA-trypsin preparations of high protein content (60-80% by weight), the displaced pH-activity profile is essentially unaffected by increasing the ionic strength. 22,32 The apparent Michaelis constant (K~) of the EMA-trypsin preparations, with benzoyl-L-arginine amide (BAA) as substrate, at low ionic strength (K" = 0.2 X 10-3 M), is lower than that of the native enzyme (K~ = 6.85 X 10-3 M) 32 (see Figs. 3 and 4). The activity of EMA-trypsin preparations toward protein substrates is considerably lower than that of the native enzyme as calculated on the basis of the respective esterase activities. 22 Moreover, it has been shown in several cases1-8,.1,.2,.5-.8 that the sites of attack as well as the rates of cleavage of protein substrates such as myosin,41,.2 pepsinogen,.5 and oxidized ribonuclease,.6 are affected by the polyelectrolyte nature of the EMA-trypsin derivative (p. 944). Water-Insoluble Polytyrosyl Trypsin Derivative The direct coupling of trypsin to the polydiazonium salts derived from the S-MDA resin and other similar resins leads to excessive inactivation of the bound protein. 2~,3~ To circumvent this limitation, polytyrosyl side chains are grown onto the protein by initiating the polymerization of N-carboxyl-L-tyrosine anhydride5s with the enzyme in aqueous solution. Polytyrosyl trypsin, 59 subsequently coupled with the insoluble S-MDA polydiazonium salt, yields highly active water-insoluble derivatives2 ~ Polytyrosyl Trypsin 59 Materials. N-Carboxyl-L-tyrosine anhydridess is available commercially (Miles Chemical Co.). Procedure. Trypsin (1 g) is dissolved in 0.0025 N HC1 (36 ml) and 0.1 M phosphate buffer, pH 7.6 (36 ml) is then added. The final pH of u A. Berger, J. Kurtz, T. Sadeh, A. Yaron, 1~. Arnon, and Y. Lapidoth, Bull. Res. Council Israel 7A, 98 (1958). ~A. N. Glazer, A. Bar Eli, and E. Katchalski, J. Biol. Chem. 237, 1832 (1962).
[70]
INSOLUBLE ENZYMES
953
the solution is 7.2. The protein solution is chilled in an ice bath and a solution of N-carboxy-L-tyrosine anhydride (800 rag) in anhydrous dioxane (16 ml) is added dropwise with stirring. The milky reaction mixture is stirred magnetically at 4 ° for 16 hours and then exhaustively dialyzed against 0.0025 N HC1 (6 liters changed daily for about 7 days) until a clear solution is obtained. The solution is clarified by centrifugation, if traces of insoluble material are still present at the end of the dialysis, then lyophilized and stored at 4 °. The yield of protein and the recovery of enzymatic activity are almost quantitative. Characterization and Properties. Polytyrosyl trypsin is assayed as described for trypsin. The protease activity is determined by the casein digestion method? 4,~s The enrichment of the polytyrosyl trypsin preparations in tyrosine can be estimated spectrophotometrically by determining the absorbanee at 295 nm of a polytyrosyl trypsin solution in 0.1 N N a 0 H and correcting for the absorbance of the unmodified enzyme under the same conditions. The molar extinction coefficient of tyrosine at 295 rim, pH 13, is 2300..59 Polytyrosyl trypsin dissolves readily in aqueous solution of pH's lower than 5 or higher than 9.5.59 It shows limited solubility between pH 5 and 9.5.
Water-Insoluble PoIytyrosyl Trypsin Derivative 31 (S-MDA Polytyrosyl Trypsin Conjugate) Carrier. S-MDA resin (see p. 949). Procedure. S-MDA resin (100 rag) is suspended in 50% acetic acid (8 ml) and stirred for 1 hour over ice. An aqueous solution of sodium nitrite (20 mg in 1 ml) is then added dropwise to the chilled suspension. The diazotization mixture is stirred for 1 hour over ice and then brought to pH 8.5 by the dropwise addition of 5 N NaOH. Crushed ice is added, as needed, to keep the temperature down. The polydiazonium salt separates as a dark-brown lumpy precipitate. The precipitate is separated on a Biichner funnel, washed with cold 0.2M phosphate buffer, pH 7.8, and suspended in the same buffer (10 ml). A solution of polytyrosyl trypsin (30 rag) in 0.001 N HC1 (8 ml) is then added to the chilled suspension and the coupling reaction allowed to proceed overnight with stirring, in the cold room. The water-insoluble S-MDA-polytyrosyl trypsin conjugate is separated by filtration on a Biichner funnel, washed with 1 M KC1 (100-200 ml) and then with water. The precipitate is resuspended in water, or in 0.1 M phosphate buffer, ptI 7.0. The recovery of enzymatic activity (tested with BAEE) in the waterinsoluble polytyrosyl trypsin derivative is about 40%.
954
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[70]
Determination o] Activity. The S-MDA polytyrosyl trypsin derivatives are assayed as described for trypsin at pH 10 (the optimal pH of the S-MDA-polytyrosyl trypsin derivatives81). The amount of active enzyme is calculated from the rate of substrate hydrolysis. The protease activity is determined at pH 7.6 by the casein digestion method24, 55 Determination o] Bound Protein. The S-MDA-polytyrosyl trypsin preparation is hydrolyzed in an evacuated sealed tube using 6 N HCI (48 hours, 110°). The acid is evaporated and the : ~sidue suspended in 0.2 M citrate buffer, pH 2.2 (4 ml). Insoluble colored material is removed by strong eentrifugation (Sorval 12,000 rpm) and amino acid analysis is carried out employing an automatic amino acid analyzer. 5e The amount of protein is calculated from the amounts of alanine, leucine, glycine, and valine. 57 Note. After analysis of hydrolyzates of insoluble azo derivatives of proteins, the amino acid analyzer column is regenerated with five times the volume of 0.2N NaOH normally employed, in order to remove colored diazotization products adsorbed on the column. Properties. S-MDA-polytyrosyl trypsin samples can be stored in aqueous suspensions at 4 ° for long periods of time without significant loss of enzyme activity21 At room temperature, suspensions of these materials lose 15-20% of their activity after 1 week2 ~ S-MDA-polytyrosyl trypsin preparations are almost completely inactivated on lyophilization or air drying. 5~ The pH-activity profile of the S-MDA-polytyrosyl trypsin derivatives acting on benzoyl-L-arginine ethyl ester as substrate is displaced by 2 pH units toward more alkaline pH values as compared to the native enzyme under similar conditions. The pH optimum for this insoluble derivative lies in the region of pH 10-10.521 The easeinolytic activity of S-MDA-polytyrosyl trypsin is about 3 0 ~ that of crystalline trypsin or polytyrosyl trypsin21
Other Methods of Preparation Water-insoluble derivatives of trypsin have been obtained by coupling the enzyme with earboxymethyl cellulose azide. 8,6° Water-insoluble polytyrosyl trypsin derivatives have been prepared by coupling polytyrosyl trypsin 5s with the polydiazonium salt derived from a p-amino-nL-phenylalanine-leucine copolymer.29 Cross-linking trypsin with glutaraldehyde has led to an enzymatically active, insoluble preparation2 ~ The iraC. J. Epstein and C. B. Anfinsen, J. Biol. Chem. 237, 2175 (1962). ~A. F. S. A. Habeeb, Arch. Biochem. Biophys. 119, 264 (1967).
[?0]
INSOLUBLE ENZYMES
955
mobilization of trypsin adsorbed onto silica gel particles by glutaraldehyde cross-linking of the adsorbed protein has been recently described,s2 Water-Insoluble Chymotrypsin Derivatives
Chymotrypsin (EC 3.4.4.5) crystallized three times, lyophilized is available commercially {Worthington Bioehemicals). Chymotrypsin is assayed titrimetrically 53,5~ at 25 °, pH 8.2, using acetyl-L-tyrosine ethyl ester {ATEE) as substrate (5 ml; 0.018M ATEE, 0.01 M KCI) and 0.1 N NaOH as titrant. Polyanionic Water-Insoluble Chymotrypsin Derivative 1,25
E M A-C hymotrypsin Carrier. Ethylene-maleic anhydride (1:1) copolymer (EMA). Procedure. EMA-chymotrypsin derivatives are prepared by the procedure described for EMA-trypsin derivatives. The recovery of enzymatic activity {tested with ATEE) in the waterinsoluble EMA-chymotrypsin derivatives is about 30%.2~ Determination o] Activity. The EMA-chymotrypsin derivatives are assayed as described for chymotrypsin, at pH 9.5 (the optimal pH for these derivatives2~). The amount of active enzyme is calculated from the rate of substrate hydrolysis. The protease activity is determined at pH 7.6 by the casein digestion method2~, 5~ Determination of Bound Protein. The protein content of EMA-chymotrypsin derivatives is determined as described for EMA-trypsin. Properties. Aqueous suspensions of EMA-chymotrypsin preparations can be stored at 4 ° without significant loss of activity for long periods of time. The EMA-chymotrypsin preparations can be lyophilized, retaining most of their activity. The dried powders can be stored at 4 ° with no significant loss of activity. ~5 The pH-aetivity profile of the EMA-chymotrypsin derivatives, using acetyl-L-tyrosine ethyl ester (ATEE) as substrate is displaced at low ionic strength (F/2 ----0.01) by about 1.5 pH units toward more alkaline pH values as compared to crystalline chymotrypsin under similar conditions. 1,83 This effect is partially reversed on increasing the ionic strength. The pH optimum of the EMA-chymotrypsin derivatives lies in the region of pH 9.3-9.6. ~,2~ The apparent Michaelis constant of the EMA-chymo-
~R. Haynes and K. A. Walsh, Federation Proc. 28, 534 (1969); ibid., Biochem. Biophys. Res. Commun. 36, 235 (1969). *~E. L. Smith and M. J. Parker, J. Biol. Chem. 233, 1387 (1958).
956
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[70]
trypsin derivatives with ATEE as substrate (K~ = 1.2X 10-SM) is similar to that of the crystalline enzyme (K'~ z 0.8 X 10-3 M).1,25 The proteolytie activity of the EMA-chymotrypsin derivatives is considerably lower than that of the native enzyme as calculated on the basis of the respective esterase activitiesY 25 Other Methods of Preparation Sephadex and Sepharose conjugates of chymotrypsin have been prepared by cyanogen bromide activation of the polysaccharide carriers. 1~-21 Polyanionic, water-insoluble derivatives of chymotrypsin have been ob' tained by coupling the enzyme to CM-cellulose, polyglutamic acid, and polyacrylic acid through activation of the carrier carboxyls with Woodward's Reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate)1~ or to carboxymethyl cellulose azide2 ,e4 A water-insoluble derivative of polytyrosyl chymotrypsin (with a p-amino-DL-phenylalanine leucine copolymer as carrier) has been reported2 s,65a Water-Insoluble Papain Derivatives
It has been the experience of several investigators, as well as of the author, that in the preparation of immobilized derivatives of papain, the recoveries of enzyme activity in the insoluble form vary considerably with different batches of enzyme.~°,al In order to increase both the reproducibility of the methods as well as the recovery of "insolubilized" enzyme activity, the procedures described below employ the mercuryblocked enzyme, mercuripapain, e6,e~ as starting material. Papain (EC 3.4.4.10), crystallized twice, in suspension in aqueous acetate buffer pH 4.5 is available commercially (Worthington Biochemicals). Papain is assayed titrimetrically,53 at 25 °, pH 6.3 using benzoylL-arginine ethyl ester (BAEE) as substrate (5 ml; 0.05 M BAEE, 0.005 M cystei~e, 0.002 M EDTA), and 0.1 N NaOH as titrant25 One unit of esterase activity is defined as that amount of enzyme which catalyzes the hydrolysis of 1 micromole of substrate per minute in the above assay. Mercuripapain. Mercuripapain is prepared by a modification of the procedure of Kimmel and Smith. 68,67 A suspension of crystalline papain (1 ml; 20-30 mgfml; 13-18 esterase units per milligram) is added to a M. Lilly, C. Money, W. Horuby, and E. M. Crook, Biochem. J. 95, 45 P (1965). E. Katchalski, in "Polyamino Acids, Polypeptides and Proteins" (M. A. Stahmann, ed.), p. 283. University of WisconsinPress, Madison, Wisconsin, 1962; I. H. Silman, Ph.D. thesis, Hebrew University,Jerusalem, 1964. a, The direct coupling of chymotrypsin to the polydiazoniumsalt derived from the S-MDA resin leads to excessiveinactivation of the bound protein. ~J. R. Kimmel and E. L. Smith, Y. Biol. Chem. 207, 515 (1954) ; E. L. Smith, B. J. Finkle, and A. Stockell, Discussion Faraday Soc. 20, 96 (1955). ':: ~J. R. Kimmel and E. L. Smith, Biochem. Prep. 6, 61 (1958).
[70]
INSOLUBLE ENZYMES
957
solution 0.005M in cysteine, 0.002 M in EDTA, pH 7 (20 ml) and the mixture is incubated at 37 ° for 15 minutes. The solution of activated papain is brought to pH 5.5 with 0.1 N HC1 and a solution of mercuric chloride (33 mg HgC12, dissolved in 1 ml distilled water and adjusted to pH 5.5) is added dropwise with stirring. The reaction mixture is stirred for a few minutes, transferred to a dialysis tube (Visking Cellophane Tubing, size 23/32) and dialyzed exhaustively against distilled water (5 liters) at 4 ° . In the course of dialysis varying amounts of insoluble material at very low enzymatic activity separate out. The dialyzed mercuripapain solution is clarified by centrifugation and used directly in the binding experiments. Characterization and Properties. Mercuripapain is assayed as described under papain, es,eT,eTa The protein content of the mercuripapain solution is estimated from its absorbance at 280 nm using the value E1% -- 24 6 68.e9 The recovery of protein varies with the papain sample used and is in the range 60-85%. The recovery of enzymatic activity is 70-95%. The specific activity of the mercuripapain preparations after activation with cysteine 66,6~ is 18-24 esterase units per milligram of protein. ~5 280
n m
- -
•
•
Polyanionic Water-Insoluble Papain Derivative 25 E M A -Papain Carrier. Ethylene-maleic anhydride (1:1) copolymer. Procedure. The ethylene-maleic anhydride copolymer (EMA) is dried
before use, at 105°-110 ° for 24 hours, over phosphorus pentoxide. The mercuripapain solution is brought to pH 7.8 by the addition of 1 M phosphate buffer, pH 8; the final concentration of phosphate in the protein solution should be 0.1 M. A solution of dried EMA (50 mg) in redistilled dimethyl sulfoxide, D M S 0 (5 ml) is added dropwise to the ice-cooled, strongly stirred solution (0.1 M in phospha~, pH 7.8) of mercuripapain (10-200 rag). The cross-linking agent hexamethylene diamine (0.5 ml of a 1% aqueous solution) is then added and the reaction mixture stirred overnight at 4 ° . The insoluble precipitate is separated by centrifugation (Sorval; 12,000 rpm; 15 minutes) and washed by suspending and recentrifuging with 0.5M KC1, and then with water, until no residual enzymatic activity e'°The mercuripapain preparations are devoid of enzyme activity, as tested on BAEE, in the absence of reducing agent. The cysteine and EDTA present in the assay mixture restore their full activity. a A. N. Glazer and E. L. Smith, J. Biol. Chem. 236, 2948 (1961). aj. R. Whitaker and M. L. Bender, J. Am. Chem. Soc. 87, 2728 (1965).
958
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[70]
toward benzoyl-L-arginine ethyl ester can be detected in the supernatant, after filtration through a Millipore filter. The washed precipitate is then suspended in water. The recovery of enzymatic activity in the water-insoluble EMAmercuripapain derivative is 65-90%.25 When crystalline papain is coupled to EMA using the same procedure, the recovery of enzymatic activity in the EMA-papain derivative is about 30%.25
Notes 1. In the preparation of high protein content samples {300-400 mg protein per 100 mg EMA in the coupling mixture) the cross-linking agent, hexamethylene diamine, can be omitted. ~, ~ 2. The final concentration of dimethylsulfoxide in the coupling mixture should not exceed 20% (v/v). 3. Mixing dimethylsulfoxide with water is accompanied by evolution of heat. During the addition of the EMA-dimethylsulfoxide solution care should be taken to keep .down the temperature of the reaction mixture (e.g., by addition of crushed ice). Determination o] Activity. The EMA-mercuripapain derivatives are assayed as described for papain and mercuripapain at pH 7.52~ (the optimal pH for these derivatives). The amount of active enzyme is calculated from the rate of substrate hydrolysis. The protea~e activity is determined, at pH 7, by the casein digestion method.54, 55 Determination o] Bound Protein. The protein content of EMAmercuripapain derivative is determined as described for EMA trypsin. Note. After analysis of mercuripapain derivatives the amino acid analyzer column is washed with 50 ml of a 1% cysteine solution, to remove mercuric salts adsorbed on the column. Properties. Aqueous suspensions of EMA-mercuripapain preparations can be stored at 4 ° with no loss in activity for long periods of time. The EMA-mercuripapain derivatives can be lyophilized, retaining most of their enzymatic activity. The dried powders can be stored at 4 ° or at room temperature in a dessicator for extended periods of time with no significant loss of activity. ~5 EMA-mercuripapain derivatives are stable in the pH range 4-10. 25 The pH-activity profile of EMA-papain derivatives, using benzoyl-Larginine ethyl ester (BAEE) as substrate is displaced at low ionic strength (r/2 = 0.05), by about 1-1.5 pH units toward more alkaline pH values as compared to crystalline papain under similar conditions. ~5 The pH optimum of the polyanionic derivatives at F/2 = 0.05 lies in the region of pH 7.2-7.9. At high ionic strengths (r/2 : 1-2), this effect is partly reversed. The Michaelis constant (K~) of EMA-papain prepara-
[70]
INSOLUBLE ENYZMZS
959
tions acting on BAEE is considerably lower at low ionic strength than that of the crystalline enzyme?~ The activity of EMA-papain derivatives toward protein substrates is 40-50% of that of the native enzyme, as calculated on the basis of the respective esterase activities. 25 EMA-mercuripapain preparations of high protein content are well suited for the digestion of protein substrates. Because of their flaky texture they are easy to remove by centrifugation (and in certain cases by filtration through Millipore filters) from the reaction mixture.2~ They can also be preactivated, the activator (cysteine-EDTA) washed off, and the activated enzyme preparation added to the protein substrate solution without the added complication of reducing agent being present in the reaction mixture, v°,7~ W a t e r - I n s o l u b l e P a p a i n D e r i v a t i v e sl
S-M D A-M ercuripapain Conjugate Carrier. S-MDA resin. Procedure. S-MDA resin (100 rag), is diazotized as described in the procedure given for the preparation of water-insoluble polytyrosyl trypsin. The washed polydiazonium salt precipitate is suspended in 0.2 M phosphate buffer, pH 7.8 (6 ml). A solution of mercuripapain (8-10 rag) is added dropwise. The reaction mixture is stirred overnight at 4 °. The water-insoluble S-MDA-mercuripapain conjugate is separated by filtration on a Biichner funnel, washed with 1 M KC1 (200 ml), then with water (100 ml) and resuspended in water. The recovery of enzymatic activity in the water-insoluble mercuripapain derivative is about 60%. 31 When crystalline papain is coupled with diazotized S-MDA resin using the same procedure, the recovery of enzymatic activity in the insoluble precipitate is only 20-30%? 1 Determination o] Activity. The S-MDA-mercuripapain conjugates are assayed as described for papain and mercuripapain2 ~ The protease activity is determined at pH 7 by the casein digestion method.54, ~5 Determination o] Protein Content. The protein content of the SMDA mercuripapain conjugates is determined as described for S-MDApolytyrosyl trypsin. Properties. Aqueous suspensions of S-MDA-mercuripapain conjugates can be stored at 4 ° for long periods without significant loss of activity2 ~ On lyophilization, the S-MDA-mercuripapain conjugates retain about 7oj. j. Cebra, D. Givol, I. H. Silman, and E. Katchalski, J. Biol. Chem. 236, 1720 (1961). 71j. j. Cebra, D. Givol, and E. Katchalski, J. Biol. Chem. 237, 751 (1962).
960
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[70]
30% of their enzymatic activity21 Dry powders of S-MDA-mereuripapain can also be obtained by washing with 70% methanol and airdrying on a filter. The material thus obtained retains 50-60% of the enzymatic activity. Moreover, the methanol washing removes traces of physically adsorbed enzyme. The pH dependence of the esterase activity of the S-MDA-papain preparations with benzoyl-L-arginine ethyl ester (BAEE) resembles closely that of the native enzyme. The pH optimum is in the region of pH 6-6.5. These derivatives also have the same apparent Michaelis constant [K~(app) ---- 1.9 X 10-2 M] as crystalline papain21 The caseinolytic activity of S-MDA-mercuripapain derivatives is similar to that of the native enzyme, as calculated on the basis of the respective esterase activities. 3~ The S-MDA-papain derivatives can be easily removed from the digestion mixture by filtration or eentrifugation; they have good flow properties 81 and are suitable for column work (see footnotes 70 and 71). Other Methods of Preparation Water-insoluble derivatives of papain have been obtained by coupling the enzyme with the polydiazonium salts derived from a p-amino-DLphenylalanine-leucine copolymeff° or p-aminobenzyl cellulose81,'s," and by cross-linking papain with bisdiazobenzidine-2,2'-disulfonic acid2 ° A papain membrane has been prepared by adsorbing papain onto a synthetic collodion membrane and cross-linking the adsorbed protein with bisdiazobenzidine-2,2'-disulfonie acid. 4~ Water-Insoluble Derivatives of Subtilopeptidase A (Subtilisin Carlsberg) Subtilopeptidase A (Subtilisin Carlsberg, EC 3.4.4.16), crystalline, is available commercially (Novo Industries, Copenhagen, Denmark). Subtilopeptidase A is assayed titrimetrically/s at 25 °, pH 8.6, using acetyl-Ltyrosine ethyl ester (ATEE) as substrate (5 ml; 0.018M ATEE, 0.01 M in KCI) and 0.1 N NaOH as titrant/2''8 Polyanionic Water-Insoluble Subtilopeptidase A Derivative ~s
EMA-Subtilopeptidase A Carrier. Ethylene-maleic anhydride (1 : 1) eopolymer (EMA). Procedure. EMA-subtilopeptidase A derivatives are prepared by the procedure described for EMA-trypsin derivatives. " A . N. Glazer, J. Biol. Chem. 242, 433 (1967). A. O. Barel and A. N. Glazer, J. Biol. Chem. 243, 1344 (1968).
[70]
INSOLUBLE ENZYMES
961
The recovery of enzymatic activity (tested with ATEE) in the waterinsoluble EMA-subtilopeptidase A derivative is about 20%. 25 Determination of Activity. The EMA-subtilopeptidase A derivatives are assayed as described for subtilopeptidase A, at pH 9.4 (the optimal pH for these derivatives).25 The amount of active enzyme is estimated from the rate of substrate hydrolysis. The protease activity is determined, at pH 7.6, by the casein digestion method. 5~,~5 Determination of Bound Protein. The protein content of EMA-subtilopeptidase A derivatives is determined as described for EMA-trypsin. Properties. Aqueous suspensions of EMA-subtilopeptidase A preparations can be stored at 4 ° without significant loss of enzymatic activity for long periods of time. The EMA-subtilopeptidase A derivative can be lyophilized retaining 5(N60% of their activity. The dried powders can be stored at 4 ° with no significant loss of activity. The EMA-subtilopeptidase derivatives are stable in the pH range 5 to 10.25 The pH-activity profile of EMA-subtilopeptidase A derivatives, with acetyl-L-tyrosine ethyl ester (ATEE) as substrate, is displaced, by 1-1.5 pH units at low ionic strength (1"/2 = 0.01) toward more alkaline pH values as compared to crystalline subtilisin under similar conditions. The pH optimum of the polyanionic subtilopeptidase A derivatives, at low ionic strengths ( r / = 0.01), lies in the region of pH 9.5-9.6. This effect is partially reversed on increasing the ionic strength. The EMA-subtilopeptidase A derivatives have the same apparent Miehaelis constant (K,~ = 0.9 X 10-2 M), with ATEE as substrate, as the crystalline enzyme~ (see footnotes 72 and 73). The proteolytic activity of the EMA-subtilopeptidase A derivatives is considerably lower than that of the native enzyme as calculated on the basis of the respective esterase activities. The initial rates of hydrolysis have been found to vary considerably with the nature of the protein substrate used. 2s
Water-Insoluble Subtilopeptidasc A Derivative sl
S-MDA-Subtilopeptidase A Conjugate Carrier. S-MDA resin. Procedure. The carrier, S-MDA resin (100 mg), is diazotized as described in the procedure given for the preparation of water-insoluble polytyrosyl trypsin. The washed diazonium salt precipitate is suspended in 0.2 M phosphate, pH 7.8 (4 ml), and a solution of subtilopeptidase A (15 rag) in
962
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[70]
the same buffer (3 ml) is added dropwise. The reaction mixture is left overnight at 4 ° with magnetic stirring. The water-insoluble S-MDAsubtilopeptidase A conjugate is separated by filtration, washed with 1 M KC1 (200 ml), then with water (100 ml), and resuspended in water or in 0.1 M phosphate buffer, pH 7.0. The recovery of enzymatic activity (tested with ATEE) in the waterinsoluble subtilopeptidase A derivative is 15-20~. Determination o] Activity. The S-MDA-subtilopeptidase A derivatives are assayed as described for subtilopeptidase A, at pH 9.4 (the optimal pH of these derivatives)21 The amount of active enzyme is calculated from the rates of substrate hydrolysis. The protease activity is determined at pH 7.6, by the casein digestion method.5~, ~5 Determination of Bound Protein. The protein content of the S-MDA subtilopeptidase A conjugates is determined as described for S-MDApolytyrosyl trypsin. Properties. S-MDA-subtilopeptidase A samples can be stored in aqueous or dilute buffer (pH 7) suspensions at 4 ° for long periods of time without significant loss of enzymatic activity. S-MDA-subtilopeptidase A derivatives are almost completely inactivated on lyophilization or air drying21 The pH-activity profile of the S-MDA-subtilopeptidase A derivatives, with acetyl-L-tyrosine ethyl ester as substrate, is displaced by about one pH unit at low ionic strength ( r / 2 = 0.01) toward more alkaline pH values, as compared to the crystalline enzyme. The pH optimum of the S-MDA-subtilopeptidase k derivatives lies in the region of pH 9.2-9.6. 81 The apparent Miehaelis constant of the S-MDA-subtilopeptidase A derivatives, with ATEE as substrate [ K , ~ ( a p p ) - 1.7X10-2M] is slightly higher than that of the crystalline enzyme [K,~(app) = 0.9 X 10-2 M] .31 The proteolytic activity of S-MDA-subtilopeptidase A derivatives is slightly lower than that of the native enzyme21 Other Methods of Preparation A water-insoluble subtilisin Novo preparation has been prepared by cross-linking the enzyme with glutaraldehyde. ~4 More recently, subtilopeptidase A (subtilisin Carlsberg) has been coupled to Sepharose activated by cyanogen bromide. 75
,4 K. Ogata, M. Ottesen, and I. Svendsen, Biochim. Biophys. Acta 159, 403 (1968).
u C. B. Anfmsen,unpublished data.
[71]
CELLULOSE-INSOLUBILIZED ENZYMES
963
[71] Cellulose-Insolubilized Enzymes
By E. M. CROOK,K. BROCKLEHURST,and C. W. WHARTON In general, assay techniques applicable to soluble enzyme systems may be used for the estimation of the activity of cellulose-insolubilized (CI) enzymes. Direct spectrophotometric assays are not readily applicable, however, since the particulate nature of the support causes a high degree of scattering, particularly in the ultraviolet region of the speotrum. Diffuse reflectance spectroscopy has not been applied to CIenzyme systems, but it is this technique which holds the greatest promise. The titrimetric method has general applicability as a relatively simple continuous rate assay. Almost any available analytical technique may be used for discontinuous assays since the insoluble enzyme may be removed from the assay solution by either centrifugation or filtration. The catalytic behavior of 0-(carboxymethyl) cetlulose-insolubilized enzymes in columns has been examined (see p. 977) and this represents a possible method of assay. In this case the eluate is devoid of particulate material and continuous spectrophotometric measurement is possible. A number of parameters characteristic of the column and the CM-cellulose involved need to be determined before a meaningful estimate of the activity of the nreparation may be obtained. Continuous titrimetrie rate assays have been used exclusively in this laboratory for the determination of the specific activity of CI-enzymes in stirred suspensions. In order to assess the specific activity, it is necessary to obtain an estimate of the protein content of the CI-enzyme. Methods for determining both the enzyme protein content and in the case of the sulfhydryl enzymes, the quantity of attached enzyme possessing a reactive active center thiol group are given. Assay Methods
Determination o] the Dry Weight o] a Standard Suspension Procedure. A standard aliquot of suspension is placed in a preweighed Gooch filter, the water is removed by suction and the solid is washed with a little water. The Gooch filter is dried in the oven, allowed to cool to room temperature in a desiccator, and reweighed. Protein Content o] CI-Enzymes Method I. Mass Balance. A known mass of enzyme is used in the reaction in which the enzyme is coupled to the activated support material. The amount of enzyme attached is determined by difference after spec-
964
W A T E R - I N S O L U B LPROTEOLYTIC E ENZYMES
[71]
trophotometric measurement (A2~) of the total enzyme recovered from the coupling solution and washings. The spectrophotometric absorption parameters of the proteases which have been coupled to cellulose derivatives are given in Table I. TABLE I EXTINCTION COEFFICIENTS AND MOLECULAR WEIGHTS OF PROTEOLYTIC ENZYMES a
Enzyme
Molecular weight
10-~ ¢~ M -~ cm-1
E~
Bromelain Ficin Papain a-Chymotrypsin Trypsin
33,2001 25,0002 20,700 a 24,8005 23,8007
6.331 5.25 5.104 4.96 3.40
19 212 25 202 14.3*
Superscript numbers refer to text footnotes.
Method II. Amino Acid Analysis. This method is considerably more sensitive than method (I) and is used when only small quantities of enzyme and/or support are available. The method involves acid hydrolysis of the enzyme to amino acids followed by estimation of the amino acid amino groups using ninhydrin.9 Reagents Solid C02 Acetone Citrate buffer, 0.2 M, pH 5.0 5% w/v Ninhydrin in methyl Cellosolve Aq. KCN, 0.01 M Aq. KCN in methyl Cellosolve, 2 ~ v/v 0.01 M Procedure. CI-enzyme (1-10 rag) is placed in a thick-walled Pyrex tube with a neck prepared for vacuum sealing. A similar quantity of the CM-cellulose, from which the hydrazide was prepared, and a sample of enzyme (0.2-2.0 rag) in H20 (up to 1 ml) is placed in another tube. Constant boiling HC1 (5 ml) is added to both tubes which are then sealed IT. Murachi, M. Yasui, and Y. Yasuda, Biochemistry 3, 48 (1964). 2p. T. Englund, T. P. King, L. C. Craig, and A. Walti, Biochemistry 7, 163 (1968). SE. L. Smith, B. J. Finkle, and A. Stockell, Discussions Faraday Soy. 20, 96 (1955). ' A. N. Glazer and E. L. Smith, J. Biol. Chem. 236, 2948 (1961). 5 M. L. Bender, G. R. Schonbaum, and B. Zerner, J. Am. Chem. Soc. 84, 2540 (1962). ' M. Laskowski, Vol. II, p. 8. Tp. Desnuelle, in "The Enzymes" (P. D. Boyer, H. Lardy, and K. ,Myrbiick, eds.), 2nd ed., Vol. 4, p. 124. Academic Press, New York, 1960. s E. W. Davie and H. Neurath, J. Biol. Chem. 212, 507 (1955). ' E . C. Cocking and E. W. Yemm, Biochem. J. 58, xii (1958).
[71]
CELLULOSE-INSOLUBILIZED ENZYMES
965
in vacuo after repeatedly freezing in solid CO~/acetone, evacuating, and thawing to remove dissolved air. The tubes are incubated for 48-72 hours at 110 ° in an oven. The tubes are allowed to cool to room temperature and carefully opened. Solid material is removed by filtration and the illtrstes transferred to two 25-ml round-bottom flasks, the tubes and filters being washed with a little distilled water. The combined filtrates and washings in the flasks are evaporated to dryness in vacuo. The residues are transferred to 10 ml volumetric flasks and made up to the mark with w~,ter. Aliquots (0.01-1.0 ml) are taken for amino acid estimation, a standard curve being constructed for the hydrolyzate from the tube which contained uncoupled CM-cellulose and enzyme. The aliquots are placed in test tubes and made up to 1 ml with water. To each tube is added citrate buffer (0.5 ml) followed by ninhydrin solution (0.2 ml) and KCN solution (1 ml). The solutions are mixed well and heated in a boiling water bath for at least 15 minutes and then cooled for 15 minutes in running tap water. The resulting solution is made up to a convenient volume (10-100 ml) with 60% ethanol and the absorbance at 570 nm determined. The repeatability of the method is ± 0.2% and Methods I and II have been found to agree to within ± 0.25%, The quantity of enzyme attached t~ the support is calculated using the standard curve. Determination of Total Nitrogen Content. The total nitrogen content of the CI-enzyme preparations may be determined, e.g., by the Kjeldahl method. 1° Determination o] the Reactive Thiol Content o] CI-Sul]hydryl Proteases. The reactive thiol per mole attached enzyme is estimated by titration of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) according to the scheme: ES- ~ RSSR ~- ESSR + RSAt pH 8 RS- absorbs strongly at 412 nm (c,1~ = 1.36 X 10~) .11
Reagents Tris buffer, 0.2M, pH 8.0 (I = 0.1) DTNB, 0.05 M neutralized to pH 7.0
Procedure22 An aliquot of CI-thiol enzyme (ca. 50 mg in ca. 1 ml H20) is placed in a small beaker and stirred magnetically. Tris buffer (3 ml) is added, followed by DTNB solution (0.05 ml). The suspension 1oR. Ballentine,Vol. III, p. 964. ~1G.L. Ellman, Arch. Bioehem. Biophys. 82, 70 (1959). C. W. Wharton, E. M. Crook, and K. Brocklehurst, European. J. Biochem. 6, 565 (1968).
066
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[71]
is stirred for 30 minutes at room temperature. The solid material is removed by filtration, using a sintered glass filter under slight suction. The filtrate is made up to 5 ml with ca. 1 ml of buffer which is used to wash the filter. The absorbance of the solution at 412 nm (A1) is then measured. A similar quantity of CM-cellulose is treated as above and the absorbance at 412 nm (A2) subtracted from the value for the CI-thiol enzyme. The molarity of thiol (Mr), when distributed evenly throughout 5 ml is given by: A, - A~ M
M, =
1 . 3 6 X 1 0 -4
The thio] per moleof enzyme(TPM) is givenby: TPM =
WXPX2XIO
2
M W X Mt
where W is the weight of CI-enzyme present in the assay, P is the cent composition of the CI-enzyme [i.e., (wt. enzyme/total wt.) X and MW is the molecular weight of the enzyme (see Table I). Titrimetric Rate Assays. All the proteolytic enzymes which have attached to cellulose catalyze the hydrolysis of N-acyl-a-amino esters. At pH values near neutrality the products of the hydrolysis: RCOOR'
per100] been acid
H,O * RCO0- + R'OH + H +
are a carboxylate anion, a proton, and an alcohol. Accordingly, in order to maintain a constant pH value, a hydroxyl anion must be added to the assay system for each substrate molecule hydrolyzed. The reaction is followed using an automatic recording pH-stat, which maintains constant pH, recording the amount of alkali added as a function of time.
Reagents Na0H, 1 M ) NaOH, 0.1 M } volumetric KC1, 0.5 M ) EDTA (di-Na salt), 0.1 M
Substrates Bromelain Ficin 0.25 M a-N-Benzoyl-L-arginine ethyl ester (BAEE) Papain Trypsin a-Chymotrypsin 0.05 M N-acetyl-L-tyrosine ethyl ester (ATEE) in 50% aq. methanol
[71]
CELLULOSE-INSOLUBILIZED ENZYMES
967
Procedure. The reaction is followed using a Radiometer TTT1 automatic titrator, SBU1- 0.5 ml buret and SBR2 recorder. The micro reaction vessel (TTA 31) is maintained at 25 ° by means of a water jacket and water bath. A stream of O~-free nitrogen is passed through a wash bottle containing water at 25 ° and thence over the surface of the fluid in the reaction vessel to exclude COs and 02. The total reaction volume at the start of the reaction is 5 ml and the tRrant is 0.1 M Na0H. With the exception of the CI-bromelain catalyses, the substrate concentrations given above are sufficient to saturate the enzyme and thus approximate estimates of Vm~ may be obtained directly. AssAYs INVOLVING BAEE. One milliliter 0.5M KC1, 0.05 ml 0.1 M EDTA, x ml standard suspension (1 ml in this laboratory, containing ca. 50 mg CI-enzyme in the case of CI-bromelain, 5-10 mg in the case of CI-trypsin) and ( 1 . 9 5 - x) ml deionized water are placed in the reaction vessel and equilibrated to pH 7 using 1 M NaOH added from an Agla micrometer syringe. The equilibration takes about 5 minutes and must be complete before the substrate (2.0 ml) is added and the pH rapidly readjusted to 7.0 using the Agla syringe. The pH-stat is now switched on and the reaction followed for a time sufficient to obtain a zero-order trace (a degree of curvature may occur in the early stages of the reaction due to final reequilibration of the CI-enzyme, particularly in the case of CM-cellulose derivatives). AssAYs INVOLWNGATEE. The procedure is the same as that given for assays involving BAEE except that 1.0 ml ATEE and ( 2 . 9 5 - x) ml water are used. Convenient chart speeds for the titrimeter assays are 1-4 divs/minute and the rate of the reaction is given by: v~ = chart divisions per minute × 10-~ M min-1
Specific Activity. The unit of specific aotivity of CI-enzymes is defined as the number of micromoles of substrate hydrolyzed per minute per milligram CI-enzyme. The units of activity are derived from v~ by Units of activity --- 5 × 108 v~/W micromoles/minute/mg where W is the weight of CI-enzyme in the assay. It is important to note that in general the activity obtained by the above technique will not be strictly comparable with a similar estimate for the free enzyme catalysis. A number of factors which perturb the kinetic properties of CI-enzymes have to be taken into account {see p. 973).
Preparation of CI-Enzymes The principal methods which have been used to attach enzymes covalently to cellulose are summarized below. In all cases the attachment
968
[71]
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
depends upon the attack of a nucleophilic center in the enzyme on an electrophilic center in a cellulose derivative. Except in Method 4 the nucleophilie center in the enzyme is represented here as an amino group. While this group is the most probable attachment site on the enzyme, in no case has this been verified experimentally. 1. The Curtius Azide method was first used to attach enzymes to cellulose by Micheel and Ewers is and was modified subsequently by Mitz and Summaria. TM Cellulose--OH + CICHaCO2H
NaOH
CeUulose--O--CH= - - CO2H CH~£)H HCi
Cellulose- - 0 - - CH2~ CO --NHNH2
NH2NH2
Cellulose- - 0 --CH= --COACH3
I NaNO2 HCI Cellulose--O ~ CH2 -- CON3
mild alkali ~- Cellulose- - 0 - - CH=-- CONH-- Protein protein-NH2
2. Acylation of cellulose hydroxyl groups by bromoacetyl bromide followed by alkylation of the protein amino group I~ or presumably reactive thiol groups if they are available. Cellulose--OH + Br-- CO--CH2Br ~ Cellulose-- O--CO--CH2Br I Protein-- NH= Cellulose- - O - - CO-- CH=--NH--Protein
This method suffers from the lability, even at neutral pH, of the ester linkage produced in the first reaction. 3. Reaction of both cellulose and the protein with a reactive triazine. TM C I y N~T/C| Cellulose~OH
~ Cellulose--O~N~
C!
+
Cellulose--O.~ N ~ C 1
Nil
I
Protein F. Micheel and J. Ewers, Makromol. Chem. 3, 200 (1949).
/ P r o t e i n - - NH,
[71]
CELLULOSE-INSOLUBILIZED ENZYMES
969
This method has great potential; the third reactive chlorine atom may be replaced by a group whose function would be to perturb the environment of the matrix in a given manner, e.g., a charged group. 4. Attachment to cellulose of an aromatic amine, diazotization of the amino group and subsequent coupling with the protein, presumably with one of the activated positions on the ring of a tyrosine residue. ~7
Cellulose--OH
10% NaOH
+ CICH2- ~ N O 2
//"-'x\
Cellulose--O--CH2~
NO2
I Sn/HCl
_•+
Cellulose--O--CH2
NaNO2 N~'NCI~ ~" HCI
Cellulose --0
--CH2--'(/(~'~"- NH2
pH ~ 8.5 [ P r o t e i n - ~ O H
~ Cellulose - - O ~ C H 2 ~
otein
N= N
OH 5. Synthesis of amide bonds between CM-cellulose and the protein using isoxazolium salts, developed by Woodward, Olofson, and Mayer TM for peptide synthesis. This method has been used to attach proteases to a copolymer of alanine and glutamie acid. TM Details of Method I, the method used most extensively to date, are given below. A great deal of research in the authors' laboratory has led to adaptations and improvements of the original procedure of Micheel and Ewers and the CI-enzymes discussed in the section on properties have been prepared using this method.
1,M. A. Mitz and J. Sum_maria,Nature 189, 576 (1961). A. T. Jagendorf, A. Potchomik, and M. P. Sela, Biochim. Biophys. Acta 78, 516 (1963). 1~G. Kay and E. M. Crook, Nature 216, 514 (1967). 1~D. H. Campbell, E. Luescher, and L. S. Lerman, Proc. Natl. Acad. ~ci. U.S. 37, 575 (1951). T. Wagner, C. J. Hsu, and G. Kelleher, Biochem. J. 108, 892 (1968). ~R. B. Woodward, R. A. Olofson, and H. Mayer, J. Am. Chem. Soe. 83, 1Ol0 (1961).
970
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[71]
Preparation o] C M-C ellulose Bromelain (C M C B ) : Experimental Details This version of the Curtius azide method for attaching enzymes to CM-cellulose is illustrated by the preparation of CMCB. TM This procedure has been used also to attach papain, ficin, trypsin, and a-ehymotrypsin to CM-cellulose to produce CMCP, CMCF, CMCT, and CMCC, respectively.
Preparation of CM-Cellulose Hydrazide CM-11 (Whatman; exchange capacity 0.6 meet/g) (100 g) is suspended in dry methanol (1.5 liter) by stirring the mixture vigorously with ,a powerful magnetic stirrer in a 2-liter conical flask fitted with a reflux condenser. Concentrated HC1 (10 ml) is added and the mixture is heated carefully until the methanol is just refluxing. The mixture is stirred and heated under reflux for 4 hours. The CM-cellulose methyl ester is then collected by vacuum filtration and washed thoroughly on a Biichner funnel with dry methanol (3 X 500 ml). The conversion of the CM-cellulose methyl ester to the hydrazide is effected by stirring a mixture of the methyl ester (100 g), methanol (1 liter), and hydrazine hydrate (100 ml) in a 2-liter flask fibted with a mechanical stirrer and immersed in a water bath at 40 ° for 72 hours. The CM-cellulose hydra~ide is collected by vacuum filtration and washed successively with 2 X 250 ml of methanol, methanol saturated with CO2, 90% aqueous methanol, and dry methanol. This procedure removes unreacted hydrazine as hydrazine carbonate.
Preparation of C M-C ellulose Azide CM-cellulose hydrazide (5 g) is suspended in 0.6 N HC1 (200 ml) cooled to 0 ° in an ice bath. Aqueous sodium nitrite (25 ml of a 5% solution) is added slowly with vigorous magnetic stirring over 20 minutes. The suspension is stirred at 0 ° for a further 20 minutes and then the CM-cellulose azide is collected by vacuum filtration using a 250-ml sintered filter. The azide is washed with ice-cold water (2 X 200 ml) icecold 1 M NaC1 (200 ml) and again with ice-cold water (2 X 200 ml) and dried on the filter by suction.
Preparation of CMCB To a solution of purified bromelain (1 g) in water (100 ml) immersed in an ice bath and stirred magnetically is added the freshly prepared CM-cellulose azide (5 g) all at once. The pH is rapidly adjusted to 8.7 with saturated sodium tetraborate solution. The suspension is stirred in the ice bath for 20 minutes after which the pH is readjusted to 8.7 if necessary. The suspension is then stirred for a further 40 minutes. The
[71]
CELLULOSE-INSOLUBILIZED ENZYMES
971
solid is then collected on a 250-ml sintered filter by vacuum filtration and washed on the filter as follows: H20 (1 X 200 ml), 1 M NaC1 (2 X 200 ml), 0.05 M acetic acid (2 X 200 ml), 1 M NaC1 (2 X 200 ml), 0.5 M NaHC03 (2 X 200 ml), 1 M NaC1 (2 X 200 ml), and H20 (4 X 200 ml). The solid CMCB is then suspended in a known volume of water to give a standard suspension which is stored at 4 ° until required. For long periods of storage 1 drop of purified chloroform or sodium azide (0.1% w/v) is added to the suspension to prevent bacterial contamination. Reactivation o] C M C B ~2 A partly oxidized sample of CMCB may be reactivated by stirring a suspension of it in 5 mM aqueous dithiothreitol (DTT) at pH 7.0 and room temperature for 15 minutes. The CMCB is then filtered off, washed with water, 1 M NaC1, and 0.5 M NaHCO~ until the filtrate is free from DTT as shown by lack of reaction with DTNB. Properties Stability. CI-enzymes are a,t least as stable as the corresponding enzymes in free solution. In most cases stability to heat, extremes of pH, and oxidation is enhanced. The fact that attachment to CM-cellulose of ficin2° but not of bromelain TM stabilizes the enzyme toward heat denaturation may be a consequence of different pH-dependencies of the thermal stabilities of the two systems (see Levin et al?~). CI-proteases are not subject to self-digestion as are the corresponding enzymes in solution. The CM-cellulose enzymes may be s~ored for long periods at 4 ° either as suspensions in deionized water or as freeze-dried preparations. In general about 50% of the ,activity of a CM-cellulose enzyme is lost during the freeze-drying process, although CMCF has been freeze dried without loss of activity.2° 1. CMCB. CMCB prepared from purified bromelain (untreated with cysteine) stored at 4 ° in air as a suspension in. deionized water (50-100 mg/ml) maintains its activity toward BAEE for a period of 6 weeks. The activity toward BAEE in the absence of cysteine is about 50% of that obtained by preincubation of the CMCB with 5 mM cysteine. CMCB activated with D T T and washed free of activator has the same activity toward BAEE as the unactivated CMCB when this is assayed in the presence of 5 mM cysteine. The D T T activated CMCB maintains its activity also for about 6 weeks when stored at 4 °. After this time, in both cases, the activity may fall off rapidly. This is due to bacterial contamination and may be prevented by including 0.1% v/v chloroform or w. E. Hornby, M. D. Lilly, and E. M. Crook, Biochem. J. 98, 420 (1966). 21y. Levin, M. Pecht, L. Goldstein, and E. Katchalski, Biochemistry 3, 1905 (1964).
972
W A T E R - I N S O L U B LPROTEOLYTIC E ENZYMES
[71]
0.1~ w/v sodium azide in the stock suspension. Attachment of bromelain to CM-eellulose reduces both the rate of loss of activity and the rate of loss of the reactive thiol group when the enzyme preparations are stored at 4 °. The stabilities of bromelain and CMCB are comparable both at 60 ° and at 70 ° . At the latter temperature the rate of loss of activity is considerably higher than the rate of loss of the reactive thiol group. 2. CMCF. 2° A suspension of CMCF in water at pH 5.0 stored at 2 ° for 4 months retains > 85% of its activity toward BAEE. Attachment of ficin to CM-cellulose increases the stability of the enzyme toward thermal denaturation. Purity and Physical Properties. Most CI-enzyme preparations contain about 5-10% protein on a dry weight basis and most of their physical properties such as particle size, density, solubility, charge, etc. are thus determined by the nature of the cellulose matrix. Cellulose is stable over a wide range of pH, possesses great ease of wetting, is available in readily activatable forms, and can be prepared as powders and flocs with excellent filtration and flow characteristics. In the case of the CM-cellulose enzymes, the enzyme protein is attached to the cellulose backbone by stable, covalent linkages (probably amide bonds between the CM-cellulose carboxyl groups and protein amino groups) and cannot be removed by any process other than hydrolysis, e.g., by hot concentrated acid or alkali. The washing procedure described on p. 971 ensures that all protein adsorbed or ion-exchanged onto the cellulose is removed and only covalently linked enzyme remains. This enzyme remains firmly bound to the cellulose matrix even under conditions which lead to its complete denaturation. Inadequate washing of the preparations leads to products containing noncovalently linked protein, much of which may be brought into solution by buffer salts and other constituents of a reaction mixture with consequent loss of the advantages of an insolubilized enzyme. The magnitude of the apparent catalytic constant (see p. 977) for CI-enzymes shows that they are not homogeneous preparations; some enzyme molecules are attached to the cellulose in modes which are either unreactive or less reactive than the free solution enzyme. This loss of activity at least in the ease of CMCF, does not seem to be due to covalent attachment of the enzyme by its active center thiol group. 2° Activators and Inactivators. There is no reason to suppose that substances which either activate or inhibit catalyses in free solution by a given enzyme will not act in an analogous manner on CI-enzymes, although the rate of reaction of the modifier with the enzyme may be changed as a result of the attachment. For example, the rates of inactivation of both ficin2° and bromelain22 by reaction of their essential thiol C. W. Wharton, E. M. Crook, and K. Brocklehurst, unpublished results, 1968.
[71]
CELLULOSE-INSOLUBILIZED ENZYMES
973
groups with D T N B at low ionic strength are markedly decreased by attachment of the enzyme to CM-cellulose. This decrease in rate, which is due to the unfavorable electrostatic interactions of the CM-cellulose and the DTNB, is diminished at high ionic strength when the negative charges on the matrix and the reagent .are shielded from each other. Reactivation o] CMCB, CMCF, and CMCP. When these CI thiol enzymes are wholly or partly in a reversibly oxidized state, they may be reactivated by stirring a suspension of the CM-cellulose enzyme in an excess of a solution of a sulfhydryl compound, e.g., D T T at pH 6-7. The excess of reducing agent may be readily removed by filtration and washing the activated CM-cellulose enzyme with O~-free NaHC03 solution and then with O~-free water. The use of cysteine may be undesirable for reactivating CM-cellulose enzymes because although it gives full activation, its use sometimes results in preparations which possess abnormally high SH/protein ratios. This is due probably to reaction of the cysteine thiol group with residual CM-cellulose azide and subsequent intramolecular transfer of the CMcellulose acyl moiety to the cysteine amino group. Specificity. There has been no demonstration of a fundamental change in the specificity of an enzyme consequent upon its attachment to cellulose as a result of matrix-imposed conformational changes in the protein. Such specificity changes as have been observed are due to changes in the apparent kinetic parameters which control the specificity as a result of such factors as charged groups on the cellulose matrix, steric hindrance by the matrix, diffusion limitation of substrates in and of products out of the enzyme matrix system. Kinetic Properties. REACTIONS IN STIRREDSUSPENSIONS.The reactions catalyzed by CMCB, CMCC, CMCF, CMCP, and CMCT follow Michaelis-Mentcn kinetics (see, for example, footnotes 12 and 20). In addition to the factors which influence the rate of the free solution enzyme catalysis, the rates of the catalyses by CI-enzymes are determined also by (1) the nature of the support, particularly its charge when the substrate also is charged and (2) the rate of stirring of the suspension. As a result of these additional factors the ratios of reactivities of a series of substrates for a given free solution enzyme may not be the same for the corresponding CI-enzyme. For example, CMCF preparations exhibit, relative to free ficin, a greater loss in activity toward casein than toward BAEE. ~° This differential effect presumably reflects steric hindrance and diffusion limitation by the support. CI-enzymes may thus be used for the preferential removal of low molecular weight substrates in the presence of susceptible macromolecules. Michaelis Constants. The measured Michaelis constant, Kin, for a CI-enzyme catalysis is generally similar (within a factor of ca. 2) to
974
WATER-INSOLUBLE
o
Z
Z
z
c~ Z O
•
Z
Z
o
PROTEOLYTIC
ENZYMES
[~1]
~71]
CELLULOSE-INSOLUBILIZED ENZYMES
8
ddldddddddddl
d
I
dddddddddddddd
°! °~
°~
.
.
.
.
.
~
°~
.
.
.
.
.
~
a~
d
ddddddddddddd~
O
e
975
976
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[71]
that for the corresponding free enzyme catalysis when (1) the supporting matrix and/or the substrate is uncharged and (2) irrespective of {he charges on the components, the assays arc carried out at high ionic strength. When the matrix is negatively charged and the substrate is positively charged, the substrate concentration is higher in the interstices of the matrix than in the bulk medium and the K,~ for the CI-enzyme catalysis is considerably smaller at low ionic strength than the K.,~ for the free enzyme catalysis. When the substrate is negatively charged this effect is reversed and the K , for the CI-enzyme catalysis is larger at low ionic strength than the K , for the free enzyme cabalysis. This effect has not been demonstrated for a CM-'cellulose protease catalyzing the conversion of a negatively charged substrate but has been demonstrated with CM-cellulose-ATP-creatine phosphotransferase. 2s At high ionic strength the electrostatic interactions of the matrix and the substrate are shielded from each other and the K,,'s for the CI and free solution systems are similar (see Table II). The value of K,, for the CMCB-catalyzed hydrolysis of BAEE at pH 7.0 and 25.0 ° at a given ionic strength, I, is predicted satisfactorily 2~ by the equation: K, =
~,K,(lim)I ('yZmJ2 + I)
in which vK~ (lim) ----0.066 and ~,Zmc= 0.26; ~ is the ratio of the mean ion activity coefficients of the CM-cellulose matrix ~nd bulk phases, K , (lira) is the limiting value of K , when the net charge on the matrix is zero, Z is the modulus of the number of charges on the matrix and mc is the concentration of the matrix in its own hydrated volume. When appropriate values of ],K~(lim) and ~,Zm¢ are used, this equation should have general applicability for an enzyme attached to a negatively charged matrix catalyzing the conversion of a positively charged substrafe. Other equations 2°,25 have been derived to describe the equili.brium distribution of a charged substrate between the internal microenvironment of the enzyme on its matrix and the bulk solution, but they do not permit an assessment of the perturbing effect of the matrix on K~ in terms of readily measurable quantities. Catalytic Constants. A comparison in a meaningful way of the intrinsic activity of an enzyme attached to a solid matrix with that of an enzyme in free solution is difficult. A comparison of the reaction rate per W. E. Homby, M. D. Lilly, and E. M. Crook, Biochem. J. 107, 669 (1968). ~C. W. Wharton, E. M. Crook, and K. Brocklehur~, European. J. Biochem. 6, 572 (1968). L. Goldstein, Y. Levin, and E. Katchalski, Biochemi~$~ 3, 1913 (1964).
[71]
CELLULOSE-INSOLUBILIZED ENZYMES
977
milligram enzyme protein at a fixed substrate concentration can be misleading because of the marked change in K~ at a given pH which sometimes results from attachment of the enzyme to the matrix (see above) and also because of shifts in pit-activity curves consequent upon atbachment (see below). The latter may sometimes be obviated by working in a pH-independent region and the former by computing Vm~ for both the supported and free solution enzyme catalyses and comparing the values of the catalytic constant, kcat. The calculation of k c a t defined as Vm~x/ enzyme active site concentration, demands a knowledge of the concentration of oatalytically active enzyme both in solution and attached to the matrix. This constitutes one of the major problems besetting the study of enzymes attached to solid matrices. In the case of all the CI-enzymes examined to date, the apparent catalytic constant [kcat(app)] defined as Vma~/"enzyme concentration" is significantly lower than the value for the corresponding enzymatic catalysis ,in free solution. The usual method of measuring the amount of enzyme attached to the matrix, estimation of the total protein content of the preparation, does not distinguish active from inactive enzyme. When the total protein content of CMCB is used as a measure of the enzyme concentration, kcat(app) for the hydrolysis of BAEE at pH 7.0, I = 0.2 and 25 ° is 52% of the correspond,ing value for the catalysis by bromelain. TM A better estimate than the protein concentration o f active bromelain on the CM-cellulose matrix should be the concentration of thiol groups which are reactive toward a thiol reagent such as DTNB. When this is used as a measure of the concentration of active sites on CMCB, kcat(app) is 77% of the value for the bromelain catalysis. TM Thus some CMCB molecules which display a reactive thiol group are inactive or less active in catalyzing the hydrolysis of BAEE than are free bromelain molecules. The value of koat(app) for the CMCB-catalyzed hydrolysis of BAEE increases with increase in ionic strength as does kcat(app) for the corresponding bromelain catalysis 24 (see Table II). pH Characteristics. Interaction of charges on the cellulose matrix with hydrogen ions may cause a change in the rate-pH profile of the enzyme catalysis when the enzyme is attached to the matrix. In the case of CMCF the accumulation of hydrogen ions in the negatively charged matrix results in the pH in the matrix being lower than the pH in the bulk solution (in the range of pH where the CM-cellulose-carboxyl groups are ionized) with consequent shift of the alkaline limb of the ratepH profile to higher pH's. 2° Reactions in Packed Beds (Column Assays). Passage of a solution of substrate at a known rate through a column of a CI-enzyme leads to a readily measurable difference in the inflowing and emerging substrate
978
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[72]
concentrations. The characterizing kinetic parameters vary from column to column and must be established for each set of experimental conditions. The catalysis of the hydrolysis of both BAEE and casein by a column of CMCF is described by the modified Michaelis-Menten equation, ~e P[S0] - K,~ l n ( 1 - P) -- k~t(app)E~/Q where P is the fraction of the substrate reacted in the column ---- ([So] -[ S t ] ) / [ & ] ; [SO] -~ concentration of substrate entering the column; [St] -----concentration of substrate leaving the column; fl, the voidage of the column, is the ratio of the void volume and the total volume of the cylindrical packed bed; E =- total amount of enzyme (in moles) in the packed bed; Q -- flow rate. The kinetic parameters for the catalysis of the hydrolysis of BAEE by CMCF in packed beds are compared with the parameters for catalysis by CMCF in stirred suspensions and by free solution fiein in Table II. x M. D. Lilly, W. E. Homby, and E. M. Crook, Biochem. J. 100, 718 (1966). 2, C. Money and E. M. Crook, unpublished results.
[ 7 2 ] W a t e r - I n s o l u b l e T h r o m b i n 1'1a B y BENJAMIN ALEXANDER a n d ARACELI M . ENGEL
Water-insoluble forms of some enzymes can now be readily prepared from soluble, preferably highly purified material. The procedures are based upon two principles: The first involves the preparation of derivatives of certain enzymes or proenzymes, notably trypsin, chymotrypsin, papain, plasminogen, and prothrombin (factor II) which are supposedly linked to macromolecular inert synthetic backbone, thereby being converted into insoluble products? -7 The second consists of embedding the soluble enzyme in an insoluble, highly cross-linked synthetic polymer Supported by U.S_P.I=LS.Grants No. HE 09011 and No. HE 11447. ~'A preliminary report of this work was presented at the 49th Annual Meeting of t h e Federation of the American Societies for Experimental Biology?b 11'A. M. Engel and B. Alexander, Federation Proc. 24, 512 (1965). s J. J. Cebra, D. Givol, H. Silman, and E. Katchalski, J. Biol. Chem. 236, 1720 (1961). s A. Bar-Eli and E. Katchalski, J. Biol. Chem. 238, 1832 (1963). ' Y . Levin, M. Pecht, L, Goldstein, and E. Katchalski, Biochemistry 5, 1905 (1964). Q. Z. Hussain and T. F. Newcomb, Proc. Soc. Exptl. Biol. Med. 115, 301 (1964). dI. H. Silman and E. Katehalski, Ann. Rev. Biochem. 35, 873 (1966). ' S. Rimon, Y. Stupp, and A. Rimon, Can. J. Biochem. 44, 415 (1966).
978
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[72]
concentrations. The characterizing kinetic parameters vary from column to column and must be established for each set of experimental conditions. The catalysis of the hydrolysis of both BAEE and casein by a column of CMCF is described by the modified Michaelis-Menten equation, ~e P[S0] - K,~ l n ( 1 - P) -- k~t(app)E~/Q where P is the fraction of the substrate reacted in the column ---- ([So] -[ S t ] ) / [ & ] ; [SO] -~ concentration of substrate entering the column; [St] -----concentration of substrate leaving the column; fl, the voidage of the column, is the ratio of the void volume and the total volume of the cylindrical packed bed; E =- total amount of enzyme (in moles) in the packed bed; Q -- flow rate. The kinetic parameters for the catalysis of the hydrolysis of BAEE by CMCF in packed beds are compared with the parameters for catalysis by CMCF in stirred suspensions and by free solution fiein in Table II. x M. D. Lilly, W. E. Homby, and E. M. Crook, Biochem. J. 100, 718 (1966). 2, C. Money and E. M. Crook, unpublished results.
[ 7 2 ] W a t e r - I n s o l u b l e T h r o m b i n 1'1a B y BENJAMIN ALEXANDER a n d ARACELI M . ENGEL
Water-insoluble forms of some enzymes can now be readily prepared from soluble, preferably highly purified material. The procedures are based upon two principles: The first involves the preparation of derivatives of certain enzymes or proenzymes, notably trypsin, chymotrypsin, papain, plasminogen, and prothrombin (factor II) which are supposedly linked to macromolecular inert synthetic backbone, thereby being converted into insoluble products? -7 The second consists of embedding the soluble enzyme in an insoluble, highly cross-linked synthetic polymer Supported by U.S_P.I=LS.Grants No. HE 09011 and No. HE 11447. ~'A preliminary report of this work was presented at the 49th Annual Meeting of t h e Federation of the American Societies for Experimental Biology?b 11'A. M. Engel and B. Alexander, Federation Proc. 24, 512 (1965). s J. J. Cebra, D. Givol, H. Silman, and E. Katchalski, J. Biol. Chem. 236, 1720 (1961). s A. Bar-Eli and E. Katchalski, J. Biol. Chem. 238, 1832 (1963). ' Y . Levin, M. Pecht, L, Goldstein, and E. Katchalski, Biochemistry 5, 1905 (1964). Q. Z. Hussain and T. F. Newcomb, Proc. Soc. Exptl. Biol. Med. 115, 301 (1964). dI. H. Silman and E. Katehalski, Ann. Rev. Biochem. 35, 873 (1966). ' S. Rimon, Y. Stupp, and A. Rimon, Can. J. Biochem. 44, 415 (1966).
[72]
WATER-INSOLUBLE THROMBIN
979
without involving chemical bonding between enzyme and insoluble carrier2 In the procedure described below for the preparation of insoluble thrombin, the first principle was employed. M e t h o d of Preparation
The method consists in coupling purified thrombin (EC 3.4.4.13) to a diazotized copolymer of p-amino-DL-phenylalanine L-leueine, similar to the procedure used for the preparation of one form of water-insoluble trypsin. ~ Materials
"Citrate" bovine thrombin is derived from purified factor II obtained from oxalated plasma, according to the method of Goldstein et al2 Factor II is ~then converted to thrombin in 25% sodium citrate, 1° which is subsequently removed by dialysis. The specific activity of the thrombin is 800-1200 NIH units/rag protein. Some of this material is further purified by starch gel eleetrophoresis, according to Tishkoff et al. ~1 The material does not contain detectable factors VII (proconvertin) and X (Stuart factor). p-Amino-DL-phenylalanine L-leucine copolymer, MW ~ 5000 (Miles Laboratories, Elkhart, Ind.) p-Tosyl-L-arginyl methyl ester (TAME) and benzoyl arginyl ethyl ester (BAEE) (K and K Laboratories) Trypsinogen, 1 X crystallized and chymotrypsinogen A, chromatographically homogeneous (Worthington) Plasminogen (Lot No. 2259-164-28), derived from human Cohn plasma fraction 3, was kindly provided by Dr. A. J. Johnson of New York University Hammarsten casein (General Biochemical) was purified according to Norman ~2 Acetyl-L-tyrosine-ethyl ester (ATEE) (Calbiochem) Procedure. Water-insoluble thrombin is prepared as follows: the copolymer of p-amino-DL-phcnylalanine L-leucine (1:2 ratio) is diazotized by dissolving 50 mg of the copolymer in a mixture of 0.65 ml of
s p. Bernfeld and J. Wan, Science, 142, 578 (1963). oR. Goldstein, A. Le Bolloe'h, B. Alexander, and E. Zonderman, J. Biol. Chem. 234, 2857 (1959). W. H. Seegers,Proc. ~oe. Exptl. Biol. Med. 72, 677 (1949). 11G. H. Tishkoff,L. Pechet, and B. Alexander,Blood 15, 778 (1960). D. Norman, J. Exptl. Med. 106, 423 (1957).
980
WATER-INSOLUBLE PROTEOLYTIC ENZYMES
[72]
50% acetic acid and 0.65 ml 2 N HC1, cooling to 0°, and adding dropwise (while stirring) 0.30 ml of 0.5 N NaN0~. After 11/~ hours at 2 °, the pH is raised to 7.5 with 5 N Na0H. The evolved coarse brown-orange precipitate is centrifuged, and washed at least 4 times with 7 ml of phusphate buffer (0.1 M, pH 7.4) until the last wash, after separation by centrifugation and filtration from the precipitate, had a pH of 7.4. The precipitate, finely homogenized in an electric homogenizer (Tri-R Instrument, Inc.) and resuspended in 7.0 ml of the buffer, is mixed with a solution of purified thrombin in saline (6-7 mg/ml), and interaction is allowed to proceed for 40 hours at 4 ° with constant stirring. The mixture is then centrifuged, the supernatant is filtered through a Millipore (45 ~) pad (sieve appropriate for the size particle of the material under preparation), and the filtrate is tested for clotting activity. The filtered material is resuspended in the phosphate buffer, and washed (with the buffer) and separated by centrifugation, as many times as needed (usually around 20 times) until the supernatant of the last centrifugation, when Millipore filtered and tested, exhibits no clotting activity, as indicated beluw for a clotting test with the insoluble material. The precipitate, resuspended in the desired volume of buffer or saline, is kept at 20--4°. The p-amino-DL-phenylalanine L-leucine insoluble thrombin (PLITh) thus obtained is a reddish-brown fine suspension which constitutes the stock solution. Assay Methods The clotting activity of PLITh is determined with 0.2 ml of a suitable suspension-dilution in saline of the stock solution, to which 0.2 ml of a !,5% factor I (fibrinogen, 9 0 ~ clottable) is added. Conglomeration of the dispersed PLITh heralds the first appearance of fibrin, which is recorded as the clotting end point. The thrombic activity (in NIH units) is determined by interpolation of the observed clotting time on a standardization curve derived with dilutions of a preparation of NIH thrombin (Lot No. B-3), obtained from the Division of Biologic Standards of the National Institutes of Health. The esterase activity is measured by the catalyzed hydrolysis of 0.04M TAME or 0.01 M BAEE, calculated from the continuously recorded uptake of 0.10 N NaOH in the pH-stat with attached automatic buret (Radiometer, Copenhagen). The caseinolytic activity is determined with Hammarsten casein, according to the procedure of Engel e t al. 13 The protein of the insoluble thrombin is determined by measuring the valine content, as has been applied to water-insoluble trypsin? is A. M. Engel, B. Alexander, and L. Pechet, Biochemistry 5, 1543 (1966).
[72]
WATER-INSOLUBLE THROMBIN
981
Definition o] Units. One thrombin clotting unit of P L I T h is defined as t h a t amount which clots 0.2 ml of a 1.5% factor I solution in 15 seconds at room temperature. Units of thrombin esterase activity are defined as the number of micromoles H ÷ liberated from T A M E or B A E E by the sample in question at 22 °, titrated at p H 7.8 (___0.05) with 0 . 1 0 N N a O H / m i n u t e / m l of T A M E or B A E E / m l of sample. One caseinolytic unit is defined as the activity which increases the optical density, at 280 nm, b y 1 unit of the optical reading/minute, after 30 minutes of incubation at 37 ° of the enzyme with a 1% solution of H a m m a r s t e n casein. 13 Properties Some properties of P L I T h are summarized in T a b l e I. TABLE I SOME PROPERTIES OF PLITh
Thrombin (mg) ~ per 100 mg carrierb Bound protein (rag) c per 100 mg insoluble enzyme Clotting activity of bound protein (percent of.original native thrombin) TAME esterolytic activity of bound protein (percent of original native thrombin)
62 34 3 15
Specific activity, 300 NIH units/rag protein.
b p-Amino-nL-phenylalanine ~-leucine copolymer (MW ~ 5000). c Calculated from valine content, assuming valine/protein N ratio of 0.32 which was found for the parent native thrornbin. T h e o p t i m u m p H for esterolytic activity ( T A M E ) over a range of 6.5 to 10.0 is 9.0, as compared with 8.0 for the soluble parent thrombin. Stability. Stability of both esterase and clotting activities at 4 ° compared with soluble thrombin is shown in Fig. 1. No bacterial contamination was detected during the period of testing. In the frozen state the material maintained full activity for at least 6 months. Purity. P L I T h prepared as above does not contain measurable amounts of factors V I I and X as determined by the method of Owren 14 and B a c h m a n et al., ~ respectively. These procedures are capable of detecting as little as 1% factors V I I and X. Specific Clotting Activity. The average specific activity of seven ~ P. A. Owren, in "Transactions of the 5th Conference on Blood Clotting and Allied Problems" (J. E. Flynn, ed.), p. 98. Josiah Macy, Jr. Foundation, New York, 1952. a F. Bachman, F. Duckert, and F. Koller, Thromb. Diath. Haemorrhag. 2, 24 (1958).
982
W A T E R - I N S O L U B LPROTEOLYTIC E ENZYMES
[~2]
,oo ~_
I
Lo :~ a~
~ \
--.
".............................~...
/j\, y\ ,e,,. Thr.
........
...................
/ ............
Clot(Act\ ~ (Citr. The.) ~. \
20
0
I
5
....
/t
I
I
20
35
5
10
l
65
Weeks
Fro. 1. Comparative stability of PLITh and the parent soluble thrombin. The two preparations were kept at 4° (thrombin, 500 U/mg protein; PLITh, 10 U/rag protein), and their clotting and esterase activity measured at the intervals indicated. unselected preparations was 3.2 N I H units/mg of protein (range 0.?10.0) with an average yield of 3.0% of the original thrombin activity. Inhibitors. Clotting activity is markedly retarded by TAME, as has also been reForted for the native soluble enzyme. 16 Esterase activity was inhibited 'by 0.10 M to 0.20 M Na ÷, as we observed with the soluble thrombin, .and as has been confirmed more recently by Roberts and Burkat. 1~ Soybean trypsin inhibitor (Worthington) had no effect on the clotting or esterase activities of PLITh, in accord with its well-known inertness in these respects on soluble thrombin." Specificity. As is true with soluble thrombin, ",~9 besides clotting f~ctor I and catalyzing TAME .and BAEE hydrolysis, PLITh cleaves casein, and activates trypsinogen, plasmin.ogen, and chymotrypsinogen. Relevant comparative data with PLITh and parent thrombin are shown in Table II. Kinetic Properties. Kinetic enzyme d'ata on PLITh have not yet been obtained. Reversibility. As already indicated, during its preparation the waterinsoluble material had been washed extensively with buffer until the washings failed to exhibit thrombin-clotting activity. Over an extended interval ( ~ 6 months) samples o f the stock solution were Millipore filtered, and the filtrates were examined for clotting activity. None was ~S. Sherry, N. Alkjaersig, and A. Fletcher, Am. J. Physiol. 209, 577 (1965). 1Yp. S. Roberts and R. K. Burkat, Proc. 8oc. Exptl. Biol. Med. 127, 447 (1968). = G. F. Lanchantin, J. A. Friedmann, and D. W. Hart, J. Biol. Chem. 244, 865 (1969). mA. M. Engel and B. Alexander, Biochemistry 5, 3590 (1966).
[72]
WATER-INSOLUBLE THROMBIN
z
o
o~
N
0
~
N
N
~
983
984
W A T E R - I N S O L U B LPROTEOLYTIC E ENZYMES
[72]
detected, indicating that no freeing or "leaking" of soluble thrombin had occurred. Nevertheless, a cautionary note should be sounded. Extensive washing with buffer does not preclude the possibility that soluble parent material may not be released by washing with a protein acting as a possible substrate. In collaboration with Y. Shamash, "° we have found that insoluble trypsin previously washed extensively, suspended in buffer, and yielding a trypsin-free Millipore filtrate after long storage, when washed with a solution of albumin, exhibited trace esterase activity in the albumincontaining filtrate. Several albumin washings thereafter could remove the last traces ,of soluble tryptic activity. This has also been the experience of Ephraim Katchalski of the Weizmann Institute, Israel (personal communication). Similar observations 21 with PLITh are shown in Table III. The data indivate that the copresence of PLITh and albumin or fibrin monomer resulted in liberation of a trace amount of thrombic clotting activity into TABLE III THROMBIN C'LEAKAGE ~ FROM INSOLUBLE THROMBIN ~
Clotting time
PLITh suspended in
Nonfiltered suspension (minutes)
Millipore filtered (hours)
0.15 N NaC1 1.0 N NaCI 1.0 N NaBr 1.0% Albumin in 0.15 N NaC1 0.5% Fibrin monomerb in 1.0 N NaBr
11 12 12 12 14
24 hours 24 24 Firm clot in 2 hours Firm clot in 2 hours
"Procedure: To 0.1 ml of a 1:10 saline dilution of stock PLITh suspension was added 0.9 ml of each of the following: 0.15 N NaC1; 1.09% purified human albumin (Courtland) in 0.15 N NaCI; 1.0 N NaCl; 1.0 N NaBr; fibrin monomer. An aliquot of each solution was MiUipore filtered (0.45 ~), and 0.15 ml of the filtrate was added to 1.0 ml of a 0.5% solution of factor I (Pentex, bovine, 95% clottable) in 0.3 M phosphate-0.075 NNaC1 buffer, pH 6.8, followed by enough H~O or 0.15 N NaC1 to bring the salt concentration to 0.15 ionic strength, thereby obtaining the same ionic strength, volumes, and factor I concentrations throughout. The clottingtime (room temperature) was then recorded. b Prepared according to T. H. Donnelly, M. Laskowski, N. Notley, and H. A. Scheraga, Arch. Biochem. Biophys. 56, 369 (1955). Research Associate, New York Blood Center, N.Y. Present address : Marcus Center, Magen David Adorn, P.O. Box 15025, Jaffa, Israel. B. Alexander, Y. Shamash, and A. M. Engel, unpublished observations.
[72]
WATER-INSOLUBLE THROMBIN
985
the solution, albeit immeasurable, in contrast to suspensions of the insoluble thrombin in the respective electrolyte solutions alone. This could be progressively reduced by successive washing of the insoluble enzyme with albumin, as indicated by the following experiment: 1.5 ml of PLITh in 0.15 NaC1 (clotting activity, 0.5 NIH units/ml) was washed repeatedly with equal volumes of saline, and the washings set aside for clotting determination. The final centrifuged pellet was resuspended in 3.0 ml of the albumin solution, the mixture was ~centrifuged, the supernatant Millipore fltered, and the filtrate set aside for clotting test. This procedure was repeated five more times. The observed clotting times were as follows: all saline washes, ~24 hours; PLITh in albumin, 4 minutes; first albumin extract, 12 minutes; second albumin extract, 15 minutes; third extract, 24 minutes; fourth, 27 minutes; fif'th, 29 minutes; sixth, 29 minutes; PLITh resuspended in albumin after 6 albumin extractions, 61/~ minutes. Thus, although repeated washing with albumin substantially decreased thrombin "leakage," it could not be totally eliminated, in contrast to our experience with water-insoluble trypsin. PLITh, and a water-insoluble derivative of factor II (prothrombin) prepared by the same procedure,z2 have been particularly useful in our laboratory in the investigation of fibrinogen-fibrin conversion,2~ and in the activation of factor II ~ via several pathways.
:2A. M. Engel and B. Alexander, Intern. Congr. Biochem. (Tokyo) Abstract IV, F-177, 793 (1967). ~B. Alexander, "Symposium on Fluid Replacement in the Surgical Patient." Columbia Univ. Coll. of Physicians & Surgeons, Depts. of Surgery and Anaesthesiology, in association with Division of Medical Sciences of the National Research Council, May 26 and 27, 1969, in press.
986
AUTHOR
INDEX
Author Index A Aas, K., 158, 159(8) Abiko, Y., 197 Abflgaard, V., 915 Abita, J. P., 42, 49(7), 72, 81, 86(7), 87 (7), 92(7), 94(7), 406 Abramova, E. P., 870 Abramowitz, N., 502 Abramson, M., 621 Acher, R., 849, 852, 877, 878 Ackers, G. K., 566 Adam-Chosson, A., 895, 901 Adams, E., 312 Adamson, E. D., 795, 796(13), 797(13) Akabori, S., 455 Alais, C., 427 Albrechtsen, O. K., 822, 833 Album, I-I. E., 616(7), 619, 630(7) Albu-Weissenberg, M., 936, 941(31), 946 (31), 948(31), 949(31), 950(31), 952 (31), 953(31), 954(31), 950(31), 959 (31), 960(31), 961(31), 962(31) Alden, R. A., 208, 209(20), 210(20), 215. 220(3) Aldrich, Jr., F. L., 884 Alex, M., 132, 139 Alexander, B., 150, 163, 164(19), 936, 943 (23), 978, 979, 980, 981(13), 982(13), 984, 985 All, S. Y., 196, 296, 300, 302(28a), 312 (24a), 834, 835(4), 836(4, 7), 837(4, 7), 838(4) Alkjaer, K., 833 Alkjaersig, N., 190, 196, 198, 199(51), 272, 667, 671, 713, 714, 814, 822, 832(7), 833(7), 834, 982 Allan, B. J., 478, 479, 481, 487, 488, 906 Allison, A. C., 144 Allison, R. G., 890, 896(11), 898(a), 899, 900(11), 902 Ambe, K. S., 870, 871,883 Ambler, R. P., 498, 648, 649, 650(24) Amirkhayan, M. M., 333 Amris, C. J., 850 Anderer, F. A., 844, 849, 851
Anderson, R. L., 853, 854, 856(8, 14) Ando, T., 649 Andrews, E. B., 915 Andrews, J., 144 Andrews, P., 433 Anfinsen, r?. B., 63, 954, 962 Angelatti, P. U., 264 Anson, M. L., 54, 286, 317, 319, 337, 347, 349, 358, 364, 389, 406, 413, 438, 455, 460, 475, 479(33), 582 Antonini, E., 878 Anwar, R. A., 122, 133, 613 Aoi, F., 712 Appella, E., 566 Appelmans, F., 286 Applewhite, T. H., 75 Archer, R., 650 Archibald, W. T., 721 Arima, K., 373, 374, 375, 376, 399, 446, 447 (5), 448, 449(23), 450(36), 454(35, 36), 455(20, 21, 36, 39), 457(20, 21, 34, 35, 36, 37, 38, 39, 40, 41, 42), 458, 459, 460 Armentrout, R. W., 555, 500(4), 562(4), 504(4), 505(4), 566, 567, 508(4), 569 (4) Arnon, R., 34, 48, 49, 58(31), 226, 229, 233 (10), 234, 235(21), 235(21), 239(21), 242, 243, 330, 331, 341, 952 Astrup, T., 272, 706, 811, 821, 822. 823, 824 (6, 11), 825(5), 826, 827, 829, 832(9, 16), 833(9, 17), 834, 836, 383(1) Atencio, A. C., 773, 774(33) Atfield, G. N., 544, 545(4) Atlas, D., 936 Audrain, L., 901 Auld, D. S., 476, 495, 499, 500, 501, 503 (116) Auricchio, F., 517 Austen, K. F., 682 Avineri-Goldman, R., 848, 849(15), 850 (15) Avrameas, S., 121, 140 Awad, E., 139 Awaza, S., 494, 500
AUTHOR INDEX Axelrod, A. E., 39, 171, 183 Axen, R., 936, 941(19, 20) B
Baba, M., 857 Babel, F. J., 373, 446, 447(15), 448(15) Babin, D. R., 475, 499(36) Bachmann, F., 196, 822, 832, 833(7), 834, 981 Bachmayer, H., 63 Back, N., 900 Badley, R. A., 334 Bagdy, D., 120, 129(32) Bailey, K., 156 Baines, N. J., 60, 62 Baird, J. B., 21, 60, 62, 171, 183(54) Baker, H., 943, 944(44), 945(44), 950(44) Bakerman, S., 163(23), 164, 167(23), 168 Bakhle, Y. S., 850 Bakshi, Y. K., 865 Balasubramanin, K., 312 Baldwin, E., 880 Ball, E. G., 786, 787 Ballentine, R., 965 Balls, A. K., 45, 59, 61, 226, 231, 237(11), 243, 246, 252(10), 437, 883, 884, 889 (4), 895 Balb, J., 113, 135(5), 139 Bamann, E., 285 Banga, I., 113, 120, 121(33), 129(32), 135 (5), 139 Bang-Jensen, V., 435 Baranowski, T., 784, 786(11), 787(11), 788 (11) Baratti, J., 466(20), 467, 468(20), 473(20) Bard, It. C., 636 Bardawil, C. J., 880 Bardawil, W. A., 121, 122(42), 123(42) Barel, A. 0., 210, 212(33), 213, 235, 289 (26), 960, 961(73) Bar-Eli, A., 936, 941(30), 943(30), 952, 953(59), 956(30), 960(30), 978, 979 (3), 980(3) Bar-Eli, E., 358, 359(3), 364, 370(1) Barg, W. F., 815, 817(17) Bargetzi, J.-P., 477, 478, 479, 482(54), 484 (54, 55), 486(55), 487(54), 488, 489 (54, 55), 490(o, p), 491
987
Barlow G. H., 186(4), 187, 190(4, 15, 27), 192, 193, 194, 195, 196, 197(18), 198, 199(4), 665, 670(1), 672, 816, 820 Bamard, E. A., 32, 33(4), 36, 857 Barnerji, A. P., 863, 865(12), 866, 867, 870 (12), 871(12) Barnhart, M. I., 170, 199, 300 Barroso, J., 699 Bartelt, D. C., 844, 906(14), 907, 913 Bartulovich, J. J., 211, 898(a), 892, 899, 903(22), 904 Bartz, Q. R., 351, 357(8), 358, 363(4) Bates, R. G., 378, 389(41) Battaner, E., 863 Bauer, E., 849 Baughman, D. J., 145, 146(1), 148, 149(1), 152(1), 154, 155(1, 16), 172(61), 173 Baumstark, J. S., 121, 122(42), 123(42) Bavel, E. J., 457 Bayliss, It. S., 333, 334, 375, 435, 436 Bean, R. S., 863, 865(2), 866(2), 867, 870 (2), 895, 896(35), 900 Beard, E. L., 838 Beaven, G. H., 551 Bechet, J. J., 61 Becker, C. A., 427, 430(15) Beeley, J. G., 50, 895 Begue-Canton, M. L., 20, 21(2), 25(2), 26(2), 119, 129(26), 131(26), 136(26), 137(26), 227(7), 228, 234(7), 377 Behnke, W. D., 468, 473, 474 Belen~¢ii, B. G., 333 Belitz, H.-D., 863, 866, 870(13) Bell, P. H., 190(22), 196, 818, 819(25), 820 (25) BeUer, F. K., 826 Bender, M. L., 7, 9, 10, 15, 16, 18(19, 21, 22), 19, 20, 21, 25(2), 26(2), 41, 42, 60, 61, 74, 118, 119, 120(26, 27), 131 (26), 132(27), 135, 136, 137(23, 26), 138, 139(27), 210, 214, 215, 216, 217, 218, 219, 220(1, 4, 6), 221,227(7), 228, 232, 234, 236(12), 237(50), 242, 243, 377, 476, 477(44), 478, 490, 499, 500, 590, 879, 892, 895(23), 940, 941(35), 957, 964 Bennett, N., 211, 890, 891(7), 892(7), 893 (7), 895(7), 898, 004(7)
"988
AUTHOR INDEX
Bennich, H., 577 Benoiton, L., 762 Benson, A. M., 573, 649 Berger, A., 212, 236, 237, 241,317, 335, 502, 521, 523(3, 6), 531(3, 6), 952 Bergkvist, R., 386, 582, 584(4), 585(4), 588, 590(22), 591(22) Bergmann, M., 285, 292, 295(19), 300(19), 315(19), 476, 498(39) BergstrSm, K., 177 Berlow, S., 844, 851, 907 Bernfeld, P., 979 Bernhard, S. A., 61, 262, 268(4), 269, 272 (4) Bernik, M., 672 Berridge, N. J., 348, 416, 422, 423, 424, 482, 457 Bethge, P. H., 475, 491(38), 493(38), 495 (38), 497(38), 501(38), 502(38), 503 (38) Bethune, J. L., 486, 488, 490(n), 491, 495, 501 Bettleheim, F. R., 156, 181 Beyerman, H. C., 22, 23(10) Bhadrakom, S., 700 Bhargava, N., 844, 848, 849(20), 850(20) Bickford, A. F., 199 Bier, M., 57, 857, 896, 898, 899, 900, 941 Biggs, R., 145, 148(2), 156(2), 157(2) Bigler, J. C., 197, 849, 850(29), 857, 862 (28), 870, 871(28), 889, 898('a), 899, 900 Bilinski, T., 196 Binldey, F., 786, 787(20) Birk, Y., 117, 123(26), 131(20), 132(20), 135(20), 375, 651, 850, 853, 854, 859, 860, 861, 862, 871 Birktoft, J. J., 63, 68(d), 69, 129, 133, 134, 136(58), 208, 607, 648, 649 Bimbaum, S. M., 724, 762 Blaauw, J., 446(31), 448, 449 Bladen, H. A., 618(26), 619 Blgkeley, R. L., 20, 21(2), 25(2), 26(2), 119, 120(26), 131(26), 136(26), 137 (26), 227(7), 228, 234(7), 377 Blaquier, P., 704 Blatrix, C., 924 Blatt, W. F., 819
Blauer, G., 848, 849(16), 850(15) Blobel, H., 714 Bloch-Frankenthal, L., 762 Blomb~ck, B., 146, 156, 157, 161, 171, 180, 181, 196, 556, 770 Blomb~ck, M., 161, 181, 198 Blood, A. F., 872 Blow, D. M., 8, 49, 58(32), 63, 08(d), 69, 80, 127, 129, 131(52), 133, 134, 135 (52), ~36(68), 208, 607, 648, 649, 940, 941 (37) Blumberg, S., 936 Btumenfeld, O. 0., 330, 331 Bosrdman, N. K., 298 Boesman, M., 672 Boggiano, E., 815, 817(17) Bohak, Z., 181, 351, 352, 354(9), 356(9), 357(9, 11), 358, 363(6), 859 Bolhofer, W. A., 783, 790, 791(la) Bonfils, S., 349, 350(6) Bonner, J., 648, 649 Bontekoe, J. S., 22, 23(10) Bottazzi, V., 372 Botts, J., 196, 811, 816(8), 818(8)820(8) Boublik, B., 582, 589(3) Boucher, R., 700, 701(4) Boun~a, J. M. W., 300, 302(30), 309(30), 312(30) Bovard, F. C., 479, 484(55), 486(55), 488, 489(55), 490(p), 491 Bovamick, M., 789 Bovey, F. A., 316, 334(2), 335(2) Bowman, D. E., 853 Boyer, H. W., 201 Boyer, P. D., 219, 598, 724 Boyles, P. W., 708, 711(8) Bradshaw, R. A., 34, 48, 49, 56, 57(e), 58(31), 460, 471, 475, 479, 489, 490, 491, 493, 498(78), 499(36), 651 Brahinsky, R. A., 906 Brakman, P., 823, 824(10, 11), 832 Brandi, C. M. W., 583, 688 Brannen, Jr., W. T., 183 Braunitzer, G., 517 Braxton, H., 64 Brecher, A. S., 534 Bretscher, M. S., 797, 798(5) Brewer, J. M., 622
AUTHOR
Bricteux-Grdgoire, S., 47, 48, 49(24, 25), 52(c), 53 Brink, N. G., 113, 120(1), 121, 122(1), 124 (1), 127(1), 129(1), 131(1), 132(1), 133(1), 135(1), 139(1) Brinkhous, K. M., 158, 160(9), 181 Brocklehurst, K., 935, 938, 943(9), 965, 970(12), 971(12), 972, 973(12), 974 (12, 24), 976, 977 Brodthagen, H., 159 Broomfield, C. A., 178, 179 Brown, C. S., 74 Brown, D. M., 145, 163(24), 164, 167(24), 168(24), 207, 243, 489, 490 Brown, F. S., 935 Brown, J. J., 705 Brown, J. R., 112, 133, 178, 461, 462, 463 (12, 15), 465(13, 15), 466(13, 15), 467 (13), 468(12, 13), 469, 471, 472(12), 473(12), 474(10), 859 Brown, M. E., 687 Brown, R. K., 178, 179 Brown, R. S., 618(26), 619 Brown, S. R., 935, 956(14) Brnbacher, L. J., 20, 21(2), 25(2), 26(2), 119, 120(26), 131(26), 136(26), 137 (26), 227(7), 228, 232, ~234(7), 236 (12), 243, 377 Bruckner, V., 789 Bruner-Lorand, J., 765, 770, 771(15), 773 (5), 777(5), 780(15) Bruni, T. B., 517 Bryan, W. P., 61 Bryce, G. F.,. 723, 728(4) Buchanan, D. L., 736, 737, 740 Buck, F. F., 57, 815, 817(17), 857, 898, 900 Biicher, T., 109, 504 Bueker, E. D., 672 Buluk, K., 838 Bumpus, F. M., 700, 701(3), 702, 704. 705 Bundy, H., 906 Bundy, H. F., 427, 430(15) Burck, P. J., 906(12), 907(9), 908(9, 12), 912(10), 913(9, 12), 914(9) Burger, W. C., 840 Burges, 670 Burkat, R. K., 962 Burns, R. H., 750
989
I~DEX
Bursztyn, H., 798, 799(6) Burton, M. 0., 600 Busuttil, R. W., 838 Bynum, E., 63
C Camerman, N., 123, 125(47), 382 Cammarata, P. S., 303 Campbell, B. J., 722, 723(1), 724(2), 728 (1) Campbell, D. H., 936, 969 Campbell, L. L., 204 Caner, F., 895 Cannamela, D. A., 713 Cannan, R. K., 896, 899(39) Capetillo, S., 857 Caplan, S. R., 944, 945(47, 48), 960(47) Caputo, A., 878 Caputto, R., 410, 416(6, 7), 418(7) Carlson, A. S., 673, 857 Carlton, B. C., 201 Carroll, W. R., 504 Carson, F. W., 487, 494, 495, 500, 503(72) Carter, J. R., 163(23), 164, 167(23), 168 Casillas, G., 178, 179 Castiglioni, B., 895 Catravas, G. N., 536, 537(10) Catsimpoolas, N., 853, 854(9a), 856(9) Cattaneo, C., 776 Cayle, T., 246, 251 Cebra, J., 285, 292(4), 742, 744(2), 959, 960(70, 71), 978 (~echov{~-Pospi§ilov£, D., 844(6b), 845, 852 Celliers, P. G., 517, 519 Celma, M. L., 797, 799(2), 801(2), 802(2), 8O3 Cerna, J., 799, 802(7) Cerwinsky, E W., 906(12), 907(9), 908 (9, 12), 912(10), 913(9, 12), 914(9) Chambers, D. C., 598 Chan, J. Y. S., 199 Chaney, A. L., 274 Chao, L. P., 271, 279, 281(13), 282(13) Chapman, G. H., 714 Chapuis, R., 562 Charles, A. F., 838
990
AUTHOR INDEX
Charles, M., 47, 48, 49(26), 52(b), 53, 56, 59, 69, 88, 89(30) Chase, Jr., T., 21, 22(6), 25(6, 7), 26(7), 42, 44(9), 61, 171, 183, 891 Chatterjee, A. K., 895, 897(29), 899(29) Chatterji, R., 787, 788(23) Chauvet, J., 849, 852 Chavre, ~. J., 244 Cheeseman, G. C., 433 Chen, L. L., 583 Chen, R., 771 Chenoweth, D., 771 Cheret, A. M., 349, 350(0) Chemikov, 870 Chervenka, C. H., 80 Chevallier, J., 62 Chi, C.-W., 863, 866, 867, 868(i), 869, 870, 871(7, 27) Chiang, L., 407, 408, 409(2), 414(4), 416, 418(4, 10), 421(10) Chiba, H., 759 Chibata, I., 756, 757, 761, 762 Chibnall, A. C., 551 Chittenden, R. H., 273 Chladek, S., 803 Choay, J., 848, 849(14) Chow, R. B., 337, 338(2), 339(2), 347 Chrambach, A., 649, 793 Christensen, L. R., 190, 808, 817 Christie, R., 714 Christophe, J., 47, 52(o), 58(23) Chii, H. M., 863, 866, 808(i), 869, 870, 871 (7, 27) Chu, T.-S., 904, 905(73) Ciocalteau, V., 398, 742 Clarke, L. P., 803 Clark-Lewis, J. W., 783 Clary, J. J., 52(d), 53, 105, 211, 849, 857, 870, 889, 892, 898(a), 899, 900(21), 903(22), 904 Clegg, J. B., 478 Clement, G. E., 18(21), 19 Clement, M. T., 602 Cliffe, E. E., 797 Cliffton, E. E., 713 Close, J., 237 Coan, M. H., 52(m), 53 Cocking, E. C., 535, 655, 657(16), 730, 737, 964
Cohen, C., 943, 944(42), 945(42), 952(42) Cohen, E., 68(b), 69, 460, 461(2), 463(2), 468(2, 3), 469, 482(2) Cohen, G. H., 64 Cohen, It., 113 Cohen, I., 181 Cohen, P. O., 814 Cohen, P. P., 185, 186(2) Cohen, W., 44, 61, 62, 63, 229, 243, 244, 265, 268(18), 269, 270(18), 590, 664, 840, 900 Cohn, E. J., 129, 549(10), 550 Cole, E. R., 923 Cole, P. W., 100, 111, 112(4), 780 Cole, R. D., 50, 63 Coleman, J. E., 488, 490, 493(83), 495, 496, 497(100) Collier, H. B., 872 Condit, E. V., 20, 671 Cone, W. H., 268 Connell, G. E., 786, 787, 788(15), 795, 796 (13), 797(13) Cook, F. I)., 599, 600 Coombs, T. L., 478, 479(53), 490, 495, 496, 501 Cooper, A. G., 333, 358 Copeland, W. H., 153, 172 Coppola, T. C., 475, 491, 493(33), 495 (38), 497(38), 501(38), 502(38), 503 (38) Comish-Bowden, A. J., 335 Correia-Aguiar, C., 883 Couch, J. R., 865 Cox, D. J., 461, 463, 465(13), 466(13), 467 (13), 468(13), 469, 471, 477, 478, 479, 484(55), 486(55), 488, 489(55), 490(o, p), 491 Cox, J. M., 129, 131(55), 133(55) Craig, C. P., 120, 132(39) Craig, L. C., 262, 266(2), 268(2), 269, 272 (2), 565, 880, 964 Creger, C. R., 865 Crestfield, A. M., 260 Crewther, W. G., 584 Crook, E. M., 935, 937(10), 938(10), 943 (9, 10, 33), 945(10, 33), 956, 965, 968 (16), 969, 970(12), 971(12), 972(20), 973(12, 20), 974(12, 24), 975(26), 976 (20), 977(12, 20), 978
AUTHOR INDEX
991
(41), 59, 72, 81, 86(7), 87(7), 92(7), 94(7), 400 de la Haba, G., 303 de la Llosa, P., 924 DeLange, R. J., 34, 200, 205, 207, 210(17), 648, 649 Delezenne, C., 890 Delpierre, G. R., 316, 333, 334, 335(I0), 383, 418 D De Moura, J., 889 Dahl, E., 156, 181 Dempsey, E. W., 132 Dale, A., 393 Denburg, J. L, 335 Dale, E. C., 935 Denson, K. W. E., 715 Daniels, J. R., 618(20), 619 Derechin, M., 814 Danno, G., 582, 584(8), 586(8), 588(8), DeRenzo, E. C., 190(22), 196, 815, 817, 589, 590(8) 818, 819(25), 820 Danos, G. J:, 838 Dernby, K. G., 372, 376 Darling, S., 824 Deschamps, 421 Darzynkiewicz, Z., 32 Desnuelle, P., 34, 42, 47, 48, 49, 50, 52(b), Datta, R. K., 534, 541(1), 543(1) 53, 54, 56, 59, 68(], h, i), 69, 81, 82 David, M. M., 880 (24), 83, 84(24), 85(24), 87(22), 88, Davidson, F. M., 706, 707(2), 768(2), 713 89(30), 92, 95(33), 96(33), 374, 375 (30), 405, 471, 472, 497(28), 839, 964 (2), 714(2), 834, 835(5) Davie, E. W., 42, 47(2), 48, 49(2), 374, Desreux, V., 316 Determann, H., 517 498, 503(112), 859, 964 Deutsch, H. F., 895, 896, 899 Davies, D. L., 705 Davies, D. R., 64 deVries, A., 181 Davies, R. C., 476, 499, 500 DiCarlo, J. J., 906(14), 907 Davis, B. J., 143, 622, 754, 914 Dickie, N., 372 Davis, H. F., 113, 114(2), 120(2), 133(2), Dickinson, W. L., 421 135(2) Dillon, H. C., 819 Davis, J. G., 892, 903, 904, 905 Dimov, D. M., 784 Davis, N. C., 244, 533, 534(20), 642, 643, Diniz, C. R., 699 815 Dixon, G. H., 32, 34, 42, 49(3), 61, !07, Dayhoff, M. O., 951, 954(57) 463 DeBarbieri, A., 849(34a), 850 Dixon, M., 940, 941 (36) DeBellis, R., 636 Djurtoft, R., 432, 433 deChaing, L., 416, 418(10), 421(10) Dlouh~, V., 58, 844(6a), 845, 849, 850, 852 deChamplain, J., 700, 701(4) DSrfel, H., 544, 545(3) de Duve, C., 286 Deftos, L., 513 Doi, E., 372 De Groot, J., 145 Doi, S., 372 Dole, V. P., 252, 254,255(1) Dehm, P., 533 Domanik, R. A., 771 Deiss, Jr., W. P., 312 de Kiewiet, J. W. C., 773, 774(32), 765, Donnelly, T. H., 772, 774, 984 985 Donovan, J. W., 892, 896, 899(42), 903 (20), 904, 905(20) Dekker, C. A., 272 Donta, S. T., 358, 362, 363(1, 9) de Koning, J. J., 433 Delaage, M., 42, 49(7), 54, 55(41, 42), 58 Doolittle, R. F., 555, 556, 557, 560(4), 562
Cross, G., 803 Croston, C. B., 106, 857 Cunningham, L. W., 54, 59, 60(39), 74, 857 Curragh, E. F., 60, 171, 182(51) Czerkawski, J. W., 113, 120(10), 132 Czerwinska-Kossobudzka, B., 163(22), 164
992
AUTHOR INDEX
(4), 564(4), 565(4), 566, 567, 568(4), 569(4), 765, 771 Dopheide, T. A. A., 332, 333, 434 Doter, F. E., 736, 737, 740 Douglas, A. S., 712, 713(14), 714(14) Dowmont, Y. P., 301 Downey, J., 765, 770, 771(3, 4), 773(3, 4, 5), 774(3), 777(5), 779(4), 780(4), 782 (4) Drenth, J., 232, 234(13), 237, 238, 239(36), 240, 241(36) Dresden, C. M., 618(27), 619, 621(27) Dreyer, W. J., 65 Dr~ze, A., 260 Dubiski, S., 566 Ducay, E. D., 863, 865(2), 866(2), 867, 870(2), 895, 895(35), 900(35) Duchateau, G., 765 Duckert, F., 981 Dugas, H., 118, 119(22), 127(22), 131, 135, 136, 137(22, 61), 609,612 Duke, J. A., 896, 899 Dummel, B. M., 427, 430(15) Dunathan, K., 779 Dunn, F. W., 498 Dunnill, P. M., 789 Durell, J., 301 Dus, K., 648, 649 Duspiva, F., 221 Duthie, E. S., 708 Dyachenck, P. F., 446, 447(4), 450(4) Dyce, B., 906 Dzwiniel, M., 606 E Ebata, M., 243, 246, 251, 648(32), 649, 650 (32) Ebert, P. S., 800 Eck, R. V., 951, 954(57) Eckert, J., 697, 698(18), 699(18) Edelhoch, H., 856 Edelman, G. M., 579 Edmondson, H. A., 906 Edsall, J. T., 129, 549(10), 550, 940, 941 (38) Ehrenpreis, S., 157, 171 Ehrlich-Rogozinki, S., 949 Eisen, A. Z., 618(27, 32, 37a), 619, 621(27)
Ekenstam, C., 853, 856(9) Eklund, H., 605 Eldridge, A. C., 854, 856(14) Ei-Gharbawi, M., 276, 280(10) Elkins, E., 476 Elkins-Kaufman, E., 494, 495, 499 Elliott, S. D., 252, 253, 254, 255(1, 6), 256 (6, 7), 257(3, 7), 258, 261 Elliott, W. H., 803 Ellis, S., 302, 305(39), 306(39), 307(39), 308(39), 535, 543 Ellman, G. L., 529, 965 Elmore, D. T., 21, 42, 60, 61, 62, 171, 182 (51), 183(54) Elson, D., 802 Elwyn, D., 186(4), 187, 190(4), 192(4), 193(4), 194, 199(4), 820 Emannilof, I., 446, 447(8) Endo, S., 575, 646, 647(10), 648(10) Enenkel, A. G., 58(e), 69, 72, 82, 84(23), 86(23), 87 Engel, A., 156 Engel, A. M., 978, 980, 981(13), 982(13), 984, 985 Englund, P. T., 262, 266, 268(2), 269, 270, 272, 565, 964 Engstrom, G. W., 787 Ensign, J. C., 591, 592, 597(5) Epstein, C. J., 954 ErdSs, E. G., 508 Ericsson, L. H., 471, 475, 479, 489, 490, 491, 498(78), 499(36) Erlanger, B. F., 44, 62, 229, 244, 333, 358, 590, 840 Ernback, S., 936, 941(19, 20) Ernstrom, C. A., 427, 430(14), 431 Esnouf, M. P., 157(1), 158, 715, 718(1), 719(1), 720 Etherington, D. J., 316 Evans, H., 207 Evans, J. E., 446, 447(7) Evans, L., 196, 296, 300, 302(28a), 312 (24a), 834, 835(4), 836(4), 837(4), 838 (4) Evans, W. H., 34, 205, 210(17) Evanson, J. M., 618(29), 619, 621(29) Ewers, J., 935, 968 Ezawa, K., 386
AUTHOR INDEX F
Fabre, C., 42, 47(2) Fackre, D., 52(k), 53, 56(g), 57, 613 Fackre, D. S., 906, 910(8), 911(8), 912(8), 913(8), 914(8) Fahey, J. L., 168, 170(34) Fahrney, D., 317, 335(15), 590 Fahmey, D. E., 8 Falla, F., 335, 336(62) Falvey, A., 803 Fambrough, R. M., 048, 649 Fantes, K. H., 622 Farr, A. L., 387, 511,526(9), 528, 547, 583, 592, 596(6), 627(61, a), 671, 689(b), 691, 695(d), 706, 724, 735, 739, 743, 757, 784, 792, 835 Fasman, G. D., 79, 80(17) Fasold, H., 373, 386(11) Fawwal, I., 373, 375(16), 440, 442(9), 443 (9), 444(9) Feder, J., 20, 21, 25(2), 26(2), 61, 119, 120 (26), 131(26), 136(26), 137(26), 227 (7), 228, 234(7), 377, 573, 650, 651 Feeney, R. E., 197, 211, 849, 850(29), 857, 862(28), 863, 865(5), 866, 867, 868(a), 869, 870, 871(5, 28), 889, 890, 891, 892, 893(5, 7, 25), 894, 895, 896(5, 11, 34), 898(a), 899, 900, 901(18, 34, 48), 902, 9O4 Feinstein, G., 280, 281(14), 857, 889, 900 Felber, J.-P., 496 Feldsted, E. T., 163, 171(17) Femfert, U., 515, 518, 521 Ferdinand, W., 865, 869 Ferguson, J. H., 708, 711(8), 713, 714(20) Ferguson, L. T., 871,900 Ferold, H. L., 890 Ferrari, C., 139 Ferry, G., 446, 447(16) Ferry, J. D., 772 Fidlar, E., 163, 171(17) Fiedler, F., 697, 698(18), 699(18) Filho, J. X., 863, 866, 867, 868(h), 869, 870(10) Fink, E., 846 Finkenstadt, W. R., 858 Finkenstaedt, J. T., 300 Finkle, B. J., 233, 235(14), 236(14), 956, 957(66), 964
993
Finlayson, J. S., 770 Fischer, E. H., 482 Fischer, F. G., 544, 545(3) Fish, J. C., 435 Fisher, Jr., E., 616(14), 619 Fishman, J. B., 815 Fishman, L., 672 Fleisher, G. A., 787 Fletcher, A., 196, 198, 199(51), 671, 672, 713, 819, 822, 832(7), 833(7), 834, 982 Florkin, M., 47, 48, 49(24, 25), 52(c), 53, 765 Fodor, P. J., 786 Foldes, I., 139 Folin, O., 398, 742 Folk, J. E., 91, 94(32), 96(32), 97(32), 98 (32), 99, 100, 109, 110, 111, 112, 463, 476, 499, 500, 504, 505, 507, 598, 780, 786, 788(13) Foltmann, B., 422, 423, 424, 425, 427(8, 13), 429, 430, 431(4, 5, 8), 432, 433, 434, 435, 436, 455, 457 Forman, M., 778 Fortmann, B., 421 Fossum, K., 262, 271(7), 272(7), 891 Foster, R. J., 889 Fournier, E., 850 Fowden, L., 789 Fox, S. W., 272 Fraefel, W., 872, 873(6), 876, 877, 878 Fraenkel-Conrat, H., 405, 444, 049, 863, 865(2), 866(2), 867, 870(2), 895, 896 (35), 900, 901 France, E. S., 917 Franchini, M., 849(34a), 850 Frankel, S., 113, 114(13), 135(13), 609 Frater, R., 237, 239(37), 240(37), 241(37), 271, 510, 513(3), 533, 534(18) Frattali, V., 853, 854(12), 859, 860, 861, 862 Fredericq, E., 895, 896(28), 899 Freisheim, J. H., 460, 467, 498, 471,472(7) Frey, E. K., 683, 699, 849 Fried, M., 307, 308(42) Friedenson, B., 263, 265, 266, 271, 272(8) Friedman, L., 433 Friedman, O. M., 786, 787(14), 788(23) Friedmann, J. A., 145, 168, 169, 857, 982
994
AUTHOR INDEX
Fritz, H., 697, 698(18), 699(18), 845, 846, George, St. H., 714 847(10, 12), 848(12), 849(12), 906(13), Geratz, J. D., 60 907, 913(11), 930, 935, 936(4, 5, 6) Gerendas, M., 916 Fritze, I., 514 Gerheim, E. B., 708, 711(8), 713 Frolova, N. F., 383 Gering, R. K., 787, 788(25) Fromageot, C., 901 Gertler, A., 114, 115(16), 117, 122(16), 123 Fruton, J. S., 258, 272, 285, 286, 288, 290 (20), 127(16), 129(10), 131(20), 132 (14), 291(14), 292, 295(19, 22), 296, (20), 135(20), 375, 853, 854(11), 860, 299, 300(19), 301, 302(9), 303, 304(9), 861(11, 48), 862 305(9), 306, 307, 308, 309(8, 10), 315 Gervais, M., 57, 58 (19), 316, 333, 334, 335, 373, 383, 418, Gerwin, B. I., 252, 253(5), 258, 259 489, 742 Ghuysen, J. M., 591 Fry, K. T., 334 Gianetto, R., 286 Fu, T., 284 Gibbons, R. G., 619 Fuchs, H. J., 164 Gibbs, R. J., 896, 899 Fujii, S., 197, 301, 307(32), 308(32), 335 Gibson, D., 32, 107 Fujimoto, D., 635 Gibson, W., 618(35), 019 Fuke, I., 208, 209 Gigliotti, H. J., 789 Fukumoto, J., 201, 212(1), 214(I), 215, Gilbert, G. A., 858 373, 391, 395(13, 01), 396, 397, 406, Gillespie, D. C., 599, 600, 609, 611(25) 575 Gilli, P., 850 Fukushima, D., 404, 582, 586, 588(7), 589 Ginodman, L. M., 334, 418 (4) Givol, D., 959, 960(70, 71), 978 Fukushima, H., 694 Gjessing, E. C., 91, 94(32), 96(32), 97(32), FuUmer, H. M., 618(26, 28, 35, 36), 619, 98(32), 99(32b), 112 621 (28) Gladner, J. A., 155, 156(20), 178, 180, 498, Funabashi, M., 401, 403(16) 503(112), 504, 507(3), 508 Furehgott, R. F., 700 Glantz, R. R., 500, 501(114) Furka, A., 32, 33(5), 68(~, 69, 70(a), 71 Gla~ville, K. L. A., 708, 709, 712(11), 713 Furlanetto, R., 765, 770, 773(5), 777(5) (11), 714(11) Furminger, I. G. S., 622 Glas, P., 823, 826, 827 Glasser, D., 513 G Glatter, D., 520 Gabeloteau, C., 374, 375(30), 405 Glavind, J., 765 Gabison, D., 352, 357(11) Glazer, A. N., 61, 210, 211, 212(33, 37), Gaffield, W., 897 213, 215, 221, 227, 235, 237(6), 239 Gallop, P. M., 613(4, 6), 619, 623(4), (6, 26), 241, 952, 953(59), 957, 960, 626(4), 629(6), 631(6, 46), 632(6), 633 961(72, 73), 964 (6), 634(4), 635(4, 46) Glenner, G. G., 786, 788(13) Gan~ev, K., 582, 589(3) Goi, H., 386 Ganrot, P. 0., 158, 163(5), 168(5) Gold, A. M., 8, 590 Gans, H., 924 Goldbarg, J. A., 656, 786, 787(14), 788(23) Gardienaet, M. C., 61 Goldberger, R. F., 63 Gardiner, J. E., 120, 121(34), 135(34) Goldblatt, H., 704 Garner, R. L., 807 Goldfine, I. D., 191, 199 Gates, B. J., 52(l), 53 Goldman, R., 239, 241(38), 944, 945, 960 Gebhardt, M., 846, 930 (47), 979 Genest, J., 700, 701(4) Geneste, P., 118, 119(23), 135, 136, I37 Goldstein, J., 242 (23) Goldstein, L., 845, 846(9), 935, 936, 937(1,
995
AUTHOR INDEX
25), 939(25, 32), 941(1, 32), 943(1, 2, 22), 944(1, 3, 25, 41, 42), 945(41, 42), 946(1, 22, 32), 949(22), 950(22, 32), 951(22, 32), 952(1, 3, 22, 32, 41, 42), 955(1, 25), 956(1, 25), 957(25), 958 (22, 25), 959(25), 960(25), 961(25), 971, 976, 978 Goldstein, R., 163, 164(19) Golubow, J., 39, 171, 183 Gomi, Y., 400, 401(11), 406(il) Gomori, G., 583 Goodwin, T. L., 250 Goodwin, T. W., 260, 529 Goore, M. Y., 787, 788 Gorguraki, V., 863, 867(3), 868(a), 869, 895, 896(36), 897 Gorecki, M., 62 Gorini, L., 901 Gornall, A. G., 880 Gorter, J., 302, 308(38), 309(38) Gortner, W. A., 273, 276(2) Gotoh, T., 770, 771(4), 773(4), 779(4), 780(4), 782(4) Gouault, M., 158, 163(4) Gould, A. B., 704, 705 Gould, N. R., 270, 271 Gounaris, A., 209, 210(22), 214(22) Graae, J., 212 Granoth, R., 530, 531(13), 532(13) Grant, N. H., 111, 120, 131, 135(6), 616 (7), 619, 630(7) Grassman, W., 629, 751 Gratecos, D., 68(t, h, i), 69, 82(24), 83, 84(24), 85(24), 88, 89(30), 92, 95(33), 96(33), 466(20), 467, 468(20), 471, 472, 473(20), 497(28) Gray, J., 626 Gray, J. L., 819 Green, A. A., 585 Green, N. M., 196, 900 Greenbaum, L. M., 285, 286(7), 291(7), 295(7), 296, 299(7, 26), 300, 301(26), 315(7) Greenberg, D. M., 268 Greenberg, E., 787 Greenberg, J., 533 Green, N. M., 57, 59, 848, 849(16), 850, 857, 858, 872, 877, 878 Greene, L. J., 566, 567, 844, 906(14), 907,
910(8), 911(8, 14), 912(8), 913(8, 14), 914(8, 14) Greenshields, R. N., 461, 462(12), 463(12), 465(13), 465(13), 467(13), 468(12, 13), 469, 471, 472(12), 473(12) Greenstein, J. P., 729, 762 Greenwell, P., 335 Grillo, A. C., 618(33, 37), 619, 621(33, 37) Grimberg, M., 446(24), 448, 449 Grinnan, E. L., 906(12), 907(9), 908(9, 12), 912(10), 913(9, 12), 914(9) Grob, D., 850, 871 GrSndahl, N. J., 198 Gros, P., 636, 639(13), 641(14), 850 Groskopf, W. R., 52(i), 53, 193, 195, 196, 197(17, 18, 19), 198, 817 Gross, F., 705 Gross, J., 617(18, 19, 20, 21, 22), 618(27, 31, 33, 34, 37), 619, 620, 621(20, 27, 31, 33, 37), 622(20), 623(18, 20, 38), 624(19, 20), 625(2, 19) Grossman, M. I., 906 Gruber, M., 289, 300, 301(16), 302, 307 (37), 308(38), 309(30, 38), 312(30) Grubhofer, N., 936 Gubler, C. G., 585 Giientelberg, A. V., 200, 202, 203(13) Guest, M. M., 197, 199, 857 Guidoni, A., 47, 48, 49(26), 52(b), 53, 56, 59 Gunter, C. R., 20, 21(2), 25(2), 26(2), 74, 119, 120(26), 131(26), 136(26), 137 (26), 227(7), 228, 234(7), 377 Gupte, S. M., 264, 265(10), 266(10), 268 (10), 271(10), 273(10) Gurvich, A. E., 936, 941(29), 943(29), 944 (29), 952(29) Gutfreund, H., 16, 61, 138, 262, 265(3), 267, 268(3, 4), 269, 272(3, 4), 940, 941 (35) Gutmann, H. R., 299 Guy, O., 68(], i), 69, 81, 82(24), 83, 84 (24), 85(24), 87(22), 88(24), 92, 95 (33), 96(33), 471, 472, 497(28) H Haas, E., 699, 704 Habeeb, A. F. S. A., 954 Haber, E., 682
996
AUTHOR INDEX
Habermann, E., 682, 697 Hartley, Jr., R. W., 150 Hachimori, Y., 458 Hartley, S., 457 Hilnggi, U. R., 876, 877, 878(15) Hartmann, P. A,, 547 Hagemeyer, K., 373, 375(16), 440, 442(9), Hartnett, J. C., 91, 94(32), 96(32), 97(32), 443(9), 444(9) 98(32), 99(32b), 112 Hagihara, B., 200, 202, 204, 214, 274, 275 Hartree, E. F., 527 (4), 397, 398, 399(1), 569, 581(1), 582, Hartsuck, J. A., 475, 491, 493(38), 495 642, 648, 649, 651 (38), 497(38), 498, 501(38), 502(38), Haglund, H., 579 5o3(38) Hajnal, K. P., 272 Hartwick, G., 845, 906 Haley, E. E., 730, 736, 737, 740 Hata, T., 372 Hall, D. A., 113, 120, 121(8, 34, 35), 135 Hattori, A., 395(61), 396, 397 (34) Haughton, G., 708 Hall, P. L., 494, 560 Haupt, H., 197 Hamaguchi, K., 279, 856, 857, 867, 897, Hausmann, E., 619 899(44) Haverback, B. J., 906 Hamill, R. L., 906, 912(10) Hayashi, K., 404, 582, 586, 588(7), 589(4) Hamilton, G. A., 334 Hayashi, R., 372 Hamilton, M. P., 319, 324(20), 325(20), .Hayes, N., 121, 122(42), 123(42) 32?(20), 329(20), 330, 331 Haynes, R., 47, 863, 865(5), 866, 867, 868 Hamilton, P. B., 536 (a), 869, 870(5), 871(5), 904, 955 Hammond, B. R., 262, 265(3), 267, 268 Heam, W. R., 307, 308(42) (3), 2?2(3) Heberlein, P. J., 199 Han, M., 75 Heene, D. L., 178 Handa, Y., 400 Heidema, J. H., 6, 7(2), 9, 11, 13 Hanes, C. S., 782, 788(1), 796 Heidrieh, H.-G., 617(25), 619, 620 Hannig, K., 620" Heimburger, N., 197, 924 Hanson, H., 513, 515, 519(4), 520, 755 Hein, G. E., 62, 63, 118, 601 Hanson, H. T., 489, 490 Heinicke, R. M., 273, 276(2) Hara, T., 386 Harmison, C. R., 145, 155, 157(2a), 158, Heinrieh, C. P., 306, 308(40) Heller, G., 803 168, 178, 923 Harper, E., 616(9, 11), 619, 620, 625(2), Helmer, O. M., 700 626(11), 628(9), 629(9, 11), 630(9), Henderson, R., 8, 49, 58(32), 80, 127, 129, 131(52), 133(52), 134(58), 135(52), 631(9), 633, 635 940, 941(37) Harrington, W. F., 636, 639(16), 640(16), Henrichsen, J., 833 641(16), 642(16) Henschen, A., 156, 198 Harris, 374 Harris, C. I., 47, 48, 49(27), 56(h), 57, 59 Hentsehel, G., 714 Herriott, 1~. M., 41, 56(1), 202, 316, 317, Harris, Jr., E. D., 618(30), 619 320, 321, 326(1), 329(1), 331,333, 339, Harris, J. I., 54, 498 351, 357(8), 358, 363(4), 365, 582, 600 Harris, R. J., 803 Herskovits, T. T., 899 Hart, A., 191, 199 Hess, G. P., 44, 74, 75(11), 79, 80(17), 127 Hart, D. W., 145, 168, 169, 857, 982 Hartley, B. S., 15, 31, 63, 68(d), 69, 119, Hessel, B., 198 122, 123, 125(44, 47), 127, 129, 131 Hesseltine, C. W., 397 (63), 132, 133, 134, 136(50, 68), 168, Hesselvik, Z., 899 169, 178, 180(35), 208, 234, 332, 421, Hewson, K., 632 434, 599, 609, 648, 649 Hilderman, F. A., 59
AUTHOR
Hill, R. A., 533 Hill, R. J., 242 Hill, R. L., 235, 237(22), 241, 242(44), 649, 655 Hill, R. M., 891,892(17) Hille, M. B., 797, 798(3), 803(3) Hilmoe, A., 74, 75(11) Himmelhoch, S. R., 508, 509, 510, 513(2) Hinrichs, J. S., 272 Hiramatsu, A., 651 Hird, F. J. R., 782, 786(2), 788(2) Hirohata, R., 284 Hits, C. H. W., 167, 402, 403, 411, 557, 556, 692, 864 Hirsch, A., 776 Hirschke, H., 520 Hirshkowitz, A., 300 Hixson, Jr., H. F., 225, 857, 858 Hjert~n, S., 579 Hladovec, J., 849(34), 850, 857 Hoave, D. G., 935 Hochstrasser, H., 703, 764 Hoehstrasser, K., 840, 843, 848(4), 849 (4), 852(3), 867, 869, 871, 883, 935, 936(6) HSrnle, S., 849, 851 Hofmann, K., 476, 498(39) Hofmann, T., 42, 47, 48, 49(27), 52(g), 53, 56(h), 57, 59, 114, 115(16), 120, 122(16), 123, 125(47), 127, 128, 129 (16), 131(31), 134, 178, 179, 373, 374, 375, 376, 379(8, 24), 381(8, 35b), 382, 383, 385(8), 393, 396, 397(56), 405 Hofstee, B. H. J., 75 Hohnen, H. W., 181 Hol, W. G. J., 232, 234(13), 237(13), 240 (13) Holden, D. A., 201 Holemans, R., 838 Holey§ovsky, V., 56, 58, 68(d), 69 Holiday, E. R., 551 Hollands, T. R., 316 Hollaway, M. R., 270 Holter, H., 424 Honavar, P. M., 883 Honawar, P. M., 870, 871(25) Hoover, S. R., 437 Hoppe, H., 564
INDEX
997
Hor~kov~, Z., 849(34), 850, 857 Horbett, T., 64, 80 Horinishi, H., 458 Homby, W. E., 935, 937(10), 938(10), 943 (10, 33), 944(33), 945(10, 33), 956, 971, 972(20), 973(20), 975(26), 976 (20), 977(20), 978 Horvath, M., 113 Hospelhorn, V., 616(9), 619, 628(9), 629 (9), 630(9), 631(9) Houston, L. L., 58 Howes, E. L., 636 Hruska, J. F., 25, 27, 226(9) Hsia, D. Y. Y., 773, 774(33) Hsieh, B., 52(i), 53, 190, 193, 195, 196(8, 16, 18, 19), 197(17, 18, 19), 198, 672, 816, 817, 833 Hsu, C. J., 969 Hsu, C.-L., 904, 905(73) Huang, W. Y., 419, 420(15), 421 Hiitter, H. J., 515, 519(4) Huggins, C., 653 Hughes, W. L., 585 Huisman, O. C., 889 Huller, I., 906, 913(11) Hummel, B. C. W., 38, 42, 43(14), 654 Humphrey, J. J., 144 Hunt, J. A., 205 Hussain, Q. Z., 978 Hussain, S. S., 240, 271, 280, 281, 282 Hutchings, B. L., 190(22), 196, 818, 819 (25), 820(25) Hutzel, M., 846, 847(10), 935, 936(4) Hwang, K., 234 Hyde, W., 751 Hyman, R. W., 44 I
Ichishima, E., 400, 401, 402(18), 403, 404, 405, 405(9, 10, 11, 32), 581(2), 582 IgelstrSm, M., 243, 262, 263(6) Iizuka, H., 399, 400(5, 6) Ikeda, K., 279, 856, 867, 897, 899(44) Ikenaka, T., 853, 856, 857, 859, 861(6), 862 (6), 863, 867, 808(a), 869, 895, 896 (36), 897, 899(44) Ilie, 446, 447(11), 456(11), 457(11)
998
AUTHOR INDEX
Illchmann, K., 867, 869, 871 Inagami, T., 57(48), 58, 60, 61, 62, 244, 274, 275(5), 279, 282, 283(5) Induni, A., 879 Ingrain, V. M., 307 Ingwall, J. S., 163(25), 104, 167(25), 168 (25), 169 Inouye, K., 316, 333(10), 334(11), 335(10, 11), 383 Iodice, A. A., 292, 204(21), 295(21), 296, 309(21), 310, 311(21), 312(21), 313 (21)
Irving, Jr., G. W., 285, 292 Ishay, J., 32, 107 Isherwood, F. A., 782, 786(2), 788(1, 2) Ishii, S., 21 Ishikawa, T., 756, 757, 761, 762 Itano, H. A., 63, 564, 566 Ito, K., 883 Ito, T., 400 Ivanovie, N., 178 Ivy, A. C., 234 Iwamoto, M., 197 Iwanaga, S., 156, 198, 771 Iwasaki, K., 797, 798,(3), 803(3) Iwasaki, S., 373, 374, 375, 376, 446, 447(5), 448, 449(20), 450(36), 454(35, 36), 455 (20, 21, 36), 457(20, 21, 34, 35, 36) Izumiya, N., 306, 307(41) J Jaarverslag, 448, 449 Jackson, R. L., 591, 566 Jackson, W. T., 319, 335(21), 421 Jacobsen, A., 765,770, 771(3, 4), 772, 773 (3, 4, 5), 774(3), 777(5), 778, 779(4), 780(4), 782(la, 4) Jacobsen, C. F., 347(3), 348, 351(3), 673, 950, 955(53), 956(53), 960(53) Jacyszyn, K., 788 Jagendorf, A. T., 968(15), 969 Jatm, W. F., 582, 583(6), 589(6), 590(6) James, M. N. G., 609 Jang, R., 61 Jansen, E. F., 61, 243, 246, 252(10) Jansonius, J. N., 237, 239(36), 240, 241 (36) Janssen, F., 272
Jaques, L. B., 163, 171(17) Jeanteur, P., 850 Jeffrey, J. J., 618(27, 29, 31, 37a), 619, 621 (27, 29) Jen, M.-H., 866, 868(i), 869 Jenkins, W. T., 745 Jensen, O. M., 833 Jerstedt, S., 579 Jevons, F. R., 895 Jirgensons, B., 856, 857, 863, 867(3), 868 (a), 869, 895, 896, 897 JSnsson, A. G., 591 Johansen, A., 432, 433 Johansen, J. T., 207, 212, 214 Johnson, A. J., 190, 667, 811, 814, 819 Johnson, M. J., 598 Johnson, P., 775 Johnson, S. L., 44 Johnston, C. L., 708, 711(8) Johnston, R. B., 264, 272(13), 288, 290, 291(14) Johnstone, J. G., 838 Jones, G., 863, 804(4), 866, 867, 868(a), 869 Jones, G. M. T., 333 Jones, M. E., 285, 286(3), 307, 368(42) Jones, S., 382 Jones, W. M., 332 Jonsson, E. K., 252, 253(4), 258(4), 259 (4)
Jorpes, E., 146, 156, 171, 181 Josso, F., 158, 163(4), 924 Jovin, T., 549, 793 Judson, W. E., 700 Jukes, T. H., 648, 649 Jur~ek, L., 52(k), 58, 56(g), 57, 599, 607, 608(19), 609, 610, 611(25), 613 Jutisz, M., 895, 901, 924 Juvkam-Wold, C., 145, 146(1), 148(1), 149(1), 152(1), 155(1) g Kafatos, F. C., 52(j), 53, 221, 225, 857 Kaffka, A., 714 Kahn, J. R., 703, 704, 705 Kaiser, E. T., 6, 7(2), 11, 60, 334, 487, 404, 495, 50O, 5O3(72)
AUTHOR
INDEX
999
Keller, P. J., 460, 461(2), 463(2), 468(2, Kajihava, T., 648, 649 3), 469, 478, 479, 481(56), 482(2), 906 Kalnitsky, G., 376, 582, 585(5), 587(5), Keller, S., 616(8), 619, 629(8) 588(5), 590, 744, 747 Kelley, Jr., G. W., 872, 876, 882 Kameda, Y., 762 Kemp, E., 706 Kamen, M. D., 648, 649 Kennedy, B. M., 840, 848(1), 849(1), 850 Kaminski, M., 895 (1) Kana Mori, M., 896 Kenner, R. A., 57, 58 Kanaoka, Y., 21 Kershaw, B. B., 863 Kang, A. H., 617(21), 619, 624(21) Kaplan, H., 118, 119(22), 127, 131, 135, K6zdy, F. J., 15, 16, 18(19, 21), 19, 20, 21, 25, 26(2), 27, 41, 42, 60, 61, 74, 119, 136(50, 61), 137(22, 61), 140, 601, 608 120(26), 131(26), 136(26), 137(26), (1O), 609(10), 611(10), 612(10) 157, 171, 182(51), 183(52), 217, 224, Kasper, C. B., 34, 205, 207, 210(17) 227(7), 228, 234(7), 377, 590, 940, 941 Kassell, B., 337, 338(2), 339(2), 343, 344 (35) (12), 346, 347, 844, 848, 849, 850(18), Khalef, S., 853, 854(11), 860(11), 861(11), 851, 852, 856, 896, 907 862 Kataoka, T., 103 Katchalski, E., 181,317, 335, 352, 357(11), Khorazo, D., 890 521, 523(3, 6), 531(3, 6), 845, 846(9), Kidd, D. A. A., 783 935, 936, 937(1), 938(1), 939(1, 32), Kies, M. W., 312 941(1, 30, 31, 32), 943(1, 2, 22, 23, 30), Kikuchi, T., 446(33), 448, 449 944(1,' 2), 945(47, 48), 946(1, 22, 31, Kilby, B. A., 15, 119, 234 32), 948(31), 949(22, 31), 950(22, 31, Kilhetter, Jr., J. V., 29, 21, 25(2), 26(2), 32), 951(22, 32), 952(1, 2, 22, 31, 32), 119, 120(26), 131(26), 136(26), 137 953(31, 59), 954(31), 955(1), 956(1, (26), 227(7), 228, 234(7), 377 31), 958(22), 959(31), 960(31, 47, 70, Kim, O.-H., 334 71), 961(31), 962(31), 971, 976, 978, Kimmel, J. R., 226, 228(2), 230(2), 231, 979(3), 980(3) 235, 236, 237, 239(37), 240(37), 241, Katchburian, A. V., 688, 698 242, 243, 271, 272, 956, 957(66, 67) Kato, K., 282 Kimura, Y., 762 Katz, S., 636, 772 King, A. E., 833 Kauffman, D., 56(i), 57 King, F. E., 783 Kauffman, D. L., 61, 68(d), 69, 133 King, T. P., 262, 266(2), 268(2), 269, 272 (2), 565, 964 Kau£man, E., 619 Kaufman, S., 43, 476, 654, 884 Kinkade, J. M., 50 Kaufmann-Boetsch, B., 857 Kipfer, R. K., 168, 169, 178, 179, 182(67) Kawabata, M., 857, 895 Kira, H., 201, 212(1), 214(1), 215 Kitsch, J. B., 235 Kay, C. M., 59, 68(e), 69, 72 Kay, G., 935(17), 936, 968(10), 969 Kitsch, J. F., 243, 262, 263(6) Kirschke, H., 513 Kay, J., 334 Kayto, A., 694 Kirsi, M., 844 Kazal, L. A., 906 Kishida, T., 52(d), 53, 56, 591 Kitasoto, 712 Kazama, M., 778 Keay, L., 201 Kito, K., 281 Kedem, 0., 944, 945(47, 48), 960(47) Kito, M., 759 Keil, B., 58, 333, 850, 852 Kiyohara, T., 883 Keilin, D., 527 Kjeldgaard, N. O., 196 Kelleher, G., 969 Klein, H., 914
1000
AUTHOR INDEX
Klein, I. B., 235 Klein, L., 616(6), 619, 629(6), 631(6), 632 (6), 633(6) Kleinberg, W., 113 Klett, W., 682 Kline, D., 190, 667, 707, 814, 815, 817, 833, 834, 835(5) Klitgaaxd, H. M., 650 Kluh, I., 333 Knappenberger, M. H., 308, 335 Knight, S. G., 446, 447(17) Knowles, J. R., 333, 334, 335, 375, 435, 436 Koaze, Y., 386 Kobayshi, M., 648, 649 Kochalaty, W., 636 Koekoek, R., 237, 239(36), 240(36), 241 (36) KSrbel, W., 849 KSrbel-Enkhardt, R., 850 Kohlstaedt, K. G., 700 Koide, A., .103, 104 Kok, P., 823, 829, 832(16), 833, 834 Kokas, E., 139 Kokot, F., 788 Kokowsky, N., 44, 62, 229, 244, 840 Koller, F., 981 Komaki, T., 214 Kominz, D. R., 168, 169 Konigsberg, W., 242 Konishi, K., 765, 770, 771(16, 17), 782 (la, 16, 17) Kopec, M., 782 Koshland, Jr., D. E., 209, 210, 214, 215, 218(8), 219, 220(8), 221(8), 889, 900, 935 Kostka, V., 333 Kovacs, J., 789 Kowarzyk, H., 163(22), 164 Kowarzykowa, Z., 163(22), 164 Koyama, A., 400 Kozlov, L. V., 334, 418 Kraemer, E. O., 718 Kramer, C., 698 Kramer, D. E., 264, 265, 206(10), 268, 269, 271(10), 273(10, 16) Krane, S. M., 618(29, 30), 619, 621(29) Krauss, A., 650 Kraut, H., 683, 699, 844, 848, 849, 850
Kraut, J., 54, 65, 208, 209(20), 210(20), 215, 220(3) Krehbiel, A., 62, 63 Krejci, L. E., 636 Kress, L. F., 650 Kretschmer, K., 515, 519(4), 755 Kriel, R., 779 Krivtsov, V. F., 332(e), 333 Krook, H., 671 Krulwich, T. A., 591 Kubo, T., 401 Kuby, S. A., 585 Kucich, U., 872, 876, 877, 878 Kucinski, C. S., 672 Kulhanek, V., 784 Kunimitsu, D. K., 246, 249(12, 13), 251 (12, 13) Kunitz, M., 41, 42, 44, 49, 56(1), 197, 202, 220, 244, 245, 252, 262, 263, 274, 317, 348, 374, 376, 438, 569, 582, 642, 651, 844, 853, 854, 856, 857(1), 906, 951, 953(55), 954(55), 956(55), 961(55) Kurihara, K., 458 Kurtz, J., 952 Kuska, J., 788 Kuzuya, M., 280, 281 Kwa~n, H. C.. 833 L Labouesse, B., 57, 58, 127, 636, 639(13), 641(14), 850 Lack, C. H., 302(28a), 303, 708, 712, 714, 835, 836(7), 837(7) Lajtha, A., 296, 309(25), 312(25), 534, 535 (1), 541(1), 543(1, 12) Lak, M., 272 Laki, K., 149, 155, 156(20), 168, 169, 178, 180, 772, 744 Lamm, O., 718 Lamson, P. D., 273 Lamy, F., 120, 132(39), 145, 167, 168(32) Lanchantin, G. F., 145, 168, 169, 857, 982 Landaburu, R. H., 145, 155, 168, 171, 178 (33), 180 Landmann, H., 184 Landon, M., 34, 205, 207, 210(17) Lang, H. M., 346 Langdell, R. D., 778 Lansing, A. I., 132, 139
AUTHOR Lansing, W. D., 718 Lapiccirella, R., 139 Lapides, J., 653 Lapidoth, Y., 952 Lapiere, C. M., 617(18, 19, 20), 618(37), 619, 620, 621(20, 37), 622(20), 623(18, 20, 38), 624(19, 20), 625(19) Laporte, J., 840 La Presle, C., 285, 286(5), 309, 311(5, 6), 312(5, 6), 313, 314(6), 315(6) Larsen, 0. M., 833 Larson, M. K., 437, 442(3), 445 Laskowski, Jr., M., 43, 62, 63, 146(5), 147, 225, 582, 642, 654, 772, 774(27), 839, 857, 868, 890, 899, 901, 912, 914, 950, 951(54), 953(54), 955(54), 956(54), 961(54), 964, 984 Laskowski, Sr., M., 844, 848, 849, 850(18), 851, 852, 853, 856, 857, 863, 872, 880 (10), 892, 896, 898, 900(24), 901, 907 Lassen, M., 833 Latallo, Z. S., 782 Lather, A. L., 393 Laursen, T., 788 Lavergne, J. M., 158, 163(4) Law, J. H., 25, 27, 52(j), 53, 221, 225, 226 (9), 857 Layne, E., 443, 547 Lazarus, G. S., 618(26, 28, 36), 619, 621
(28) Lazdunski, M., 42, 49(7), 54, 55(41, 42), 58(41), 59, 72, 81, 86(7), 87(7), 92(7), 94(7), 406 Lea, A. S., 421 Le Belloc'h, A., 163, 164(19), 979 Lebowitz, J., 858 Leclerc, J., 335, 336(62) Leder, P., 798, 799(6) Lee, D., 316, 324, 326(30), 327(8), 328(8), 329(8, 30), 330, 334, 335(8), 336(8, 30) Lee, Y. C., 281 Legault-D~mare, J., 895 Leibach, F. H., 786, 787(20) Lenard, J., 44 Lenney, J. F., 372 Lennox, F. G., 584 Lentz, K. E., 703, 794 Leong, V., 292, 294(21), 295(21), 296(21),
INDEX
1001
309(21), 310(21), 311(21), 312(21), 313(21) IA~onis, J., 347(3), 348, 351(3), 673, 950, 955(53), 956(53), 960(53) Lepow, I. H., 636 Lerman, L. S., 936, 969 Lerman, N., 859 Lesuk, A., 196, 670 Leuscher, E., 936, 969 Levchuck, T. P., 352, 357(10), 358, 363(5) Levdikova, G. A., 629 Levenberg, B., 789 Lever, A. F., 702, 705, 706 Levin, E. D., 333 Levin, Y., 335, 352, 357(11), 845, 846(9), 936, 939(32), 941(32), 943(22), 946 (22, 32), 949(22), 950(22, 32), 951 (22, 32), 952(22, 32), 958(22), 971, 976, 978 Levine, L., 363, 370 Levine, W. G., 172(60), 173, 178 Levy, A. L., 468, 474(24) Levy, M., 672, 675, 677 Lewandowski, V., 197 Lewis, Jr., C., 650 Lewis, J. H., 713 Lewis, M. L., 163, 164(20) Lewis, O. J., 608' Lewis, S. H., 54 Lewis, U. J., 113, 120(1), 121, 122, 123(1, 45), 124(1), 127(1), 128(1), 120(1), 131(1), 132(1), 133(1), 135, 139, 914 Li, L. F., 145 Lichenstein, N. M., 292, 295(22), 296(22) Lie, S. Y., 849 Liener, I. E., 50, 52(b, d), 53, 54, 56, 59, 263, 265, 266, 268(17), 269, 270, 271, 272(8), 279, 281(13), 282(13), 321, 372 Light, A., 237, 239, 240(37), 241, 271, 510, 513(3), 533, 534(18) Lilly, M., 956 Lilly, M. D., 935(17), 936, 937(10), 938 (10), 943(10, 33), 944, 945(10, 33), 971, 972(20), 973(20), 975(26), 976 (20), 977(20), 978 Lin, Y. C., 722, 728(1), 728(1) LindestrCm-Lang, K., 347(3), 348, 351 (3), 673, 950, 955(53), 956(53), 960 (63)
1002
AUTHOR INDEX
Lindsley, J., 521 Lineweaver, H., 226, 231, 234, 237(11), 890, 892, 895(3), 896(3), 897, 900, 901 (3), 903 Ling, C.-M., 198 Ling, V., 122 Lipinsky, B., 770, 771(3), 773(3), 774(3) Lipmann, F., 290 Lipscomb, 493 Lipscomb, W. N., 475, 491, 493(38), 495 (38), 497(38), 498, 501(38), 502, 503 (38) Litwack, G., 636 Liu, T. Y., 252, 253(3, 4), 254, 250(7), 257 (3, 7), 258, 259, 261 Liu, W., 448, 457(41) Livingstone, B. J., 157, 182 Lloyd, A. M., 700, 701(3), 704, 705, 706 Lo, K. W., 14 Lo, S.-S., 866, 868(0,869 Lobareva, L. S., 383 Loch, T., 196 Lockhead, A. G., 600 Lockridge, O., 765, 773, 775(29), 780(29) L5ffler, F., 883 Loewy, A. G., 770, 778, 779 Lokshina, L. A., 335 Longsworth, L. G., 896, 899(39) Loomis, E. C., 167 Lopez-Ramos, B., 246 Lopiekes, D. V., 935, 956(14) Lorand, L., 20, 21, 156, 157, 168, 169, 171, 182(52), 183, 244, 671, 765, 770, 771 (3, 4, 5, 15, 16), 772, 773(3, 4, 5, 22), 774(3, 8, 21, 22, 30, 32, 33), 775(5, 28a), 778, 779(4, 43), 780(4, 15, 22, 29), 782(la, 4, 16, 17, 22), 926, 985 Lowe, G., 234, 240, 243, 262, 270, 271,280, 281, 282 Lowey, S., 943, 944(41, 42, 43, 44), 945 (41, 42, 43, 44), 952(41, 42), 960(43, 44) Lowry, O. H., 387, 511, 647, 526(9), 528, 583, 592, 596(6), 627(61, a), 671, 689(b), 691,695(d), 708, 724, 735, 739, 743, 757, 784, 792, 835 Lubnow, R. E., 375 Luck, S. M., 943, 944(41, 42), 945(41, 42), 952(41, 42)
Ludwig, M. L., 475, 491, 493(38), 495(38), 497(38), 498, 501(38), 502(38), 503 (38) Lumry, R., 75, 487, 494, 495, 499, 500, 501 (114) Lundblad, R. L., 334 Lux, S. E., 163(23), 164, 167(23) Lyman, R. L., 840, 848(1), 649(1), 850(1) M MacAulay, M., 168 McCall, D. C., 833 McCarty, W. R., 811 McClaughry, R. I., 168, 170(34) McClung, L. S., 636 McClure, W. 0., 476, 477(46), 495, 499, 500, 501(46) McConn, J., 79, 80(17) McConn, J. D., 571, 572(4), 573, 575(5, 6), 650 McConneU, B., 91, 94(32), 96(32), 97(32), 98(32), 99(32b) McConnell, D., 838 McCoy, L., 178, 179, 182(67) MacDonald, A. G., 163,171 (17) MacDonald, J. B., 619 McDonald, C. E., 583 McDonald, J. K., 302, 305(39), 306, 307, 308(39), 543 McDonald M. R., 49 Macfarlane, R. G., 145, 148(2), 156(2), 157(1, 2), 158, 715 Maclnnes, D. A., 896, 899(39) McIvor, B. C., 117, 131(21), 139 McLaren, A. D., 858 MacLennan, J. D., 636 McMillian, P. J., 786, 788(13) McNicol, G. P., 712, 713(14), 714(14) Maden, B. E. H., 799, 800(8), 801, 802(12) Maeda, H., 890, 892, 893(5), 894, 896(5) Maglott, D., 801, 802(11) Magnusson, B., 176, 177(63) Magnusson, S., 153, 155, 156, 158, 159, 160(12), 161, 162, 163(12), 164, 167(3. 12, 14), 168, 169, 170(16, 26), 171(16), 172, 174(59), 176, 177(3, 16, 59, 63), 178, 179, 180(35), 181 Maier, L., 883
AUTHOR INDEX Mains, G., 374, 375(28a), 383 Makino, S., 197 Malhotra, O. P., 163(23), 164, 167(23) Malofiejew, M., 838 Mammen, E. F., 157(2a), 158 Manahan, J., 616(8), 619, 629(8) Mandl, I., 113, 117(3), 120(4), 132(3, 4), 140(3), 616(8), 619, 629(8), 636, 871, 9OO Mangan, L. J., 551 Manion, R., 373, 581 Mann, R. K., 853, 854(8), 856(8) Manning N., 508 Mannsfeldt, H. G., 515, 519(4) Manoilov, S. E., 935, 936(15) Mansfeld, V., 849(34), 850, 857, 883 Marbach, E. P., 274 Marchis-Mouren, G., 47 Marciniak, E., 168, 169 Marcker, K. A., 797, 798(5), 799, 802(7) Mares-Guia, M., 60, 61, 62, 664, 699, 900 Marini-Bettolo, G. B., 264, 269, 270 Marker, K. A., 797, 799(1) Markland, F. S., 34, 205, 207, 210, 211, 213, 215 Marks, N., 296, 309(25), 312(25), 534, 535 (1), 541(1, 3, 5), 543(1, 12) Markwardt, F., 156, 180, 184, 924, 925, 927(6, 9), 928, 929(8) Maroux, S., 34, 49, 50 Marrama, P., 139 Marsh, C. L., 872, 876, 882 Marshall, T. H., 20, 21(2), 25(2), 26(2), 119, 120(26, 27), 131(26), 132(27), 136 (26, 27), 137(26), 138, 139(27), 227 (7), 228, 234(7), 377 Marshall, W. E., 373, 581, 849, 850(22b) Martin, C. J., 39, 171, 183 Martin, S. M., 591 Martinez, J., 170 Maschmann, E., 631, 636 Mashburn, L. T., 732 Mason, R. G., 181 Masuda, T., 235, 236(25) Matacic, S., 770 Matheson, A. T., 535, 723, 730, 737, 749 Mathias, A. P., 270 Matida, A. K., 872, 876
1003
Matsubara, H., 202, 204(12), 207, 208, 209, 210, 211, 214, 274, 275(4), 569, 573, 575, 642, 647, 648(17, 19, 31), 649, 650 (17, 23, 30, 31), 857 Matsueda, G. R., 597(7), 598 Matsui, K., 762 Matsuoka, Y., 103, 104 Matsushima, K., 211, 890, 903, 904(6, 71) Matsushima, Y., 859 Matthews, B. W., 8, 49, 58(32), 64, 80, 127, 129, 131(52), 133(5a), 135(52), 040, 941 (37) Matushima, K., 590 Matveeva, R. A., 333 Maurer, P. H., 941 Maxwell, R. E., 197 Maybury, R. H., 463 Maye, R. G., 566 Mayer, H., 969 Mayer, L., 857 Mayhew, S., 63 Meadway, R. J., 648, 649, 650(24) Means, G. E., 197, 849, 850(49), 857, 862 (28), 870, 871(28), 889, 898(a), 899, 9OO Megel, H., 113 Mehl, J. W., 145, 168, 169 Meilman, E., 616(6), 619, 623, 629(6), 631 (6, 46), 632(6), 633(6), 635(46) Meister, A., 782, 786(3, 4), 788(4), 792, 795(4), 796(4), 797(4) Meitner, P. A., 343, 344(12) Mejbaum-Katzenellenbogen, W., 163(22), 164 Melamed, M. D., 890, 895, 901 Mellanby, J., 164 Meloun, B., 849, 852 Melville, J. C., 885, 887, 888, 889(14) Mendel, L. B., 872 Mendes, J., 698 Menger, F., 476, 477(44), 478, 490, 499, 5OO Menzie, C., 949 Merler, E., 178 Merrett, T. G., 358, 359(3), 364, 370 Mertz, E. T., 199 Mesrobeanu, L., 196 Messing, R. A., 264 M6tais, P., 857, 900
1004
AUTHOR INDEX
Metrione, R. M., 264, 270, 272(13), 285, 302(9), 303, 304(9), 305(9), 306(9) Meyer, E. W., 853, 856(9) Meyer, K., 890 Micheel, F., 935, 968 Mickelsen, R., 431 Middlebrook, W. It., 156, 770 Mihalcu, F., 196 Mihalyi, E., 775, 915 Mihara, H., 197 Mike§, 0., 56, 68(d), 69, 582, 589(3) Miki, Y., 446 Mikola, J., 840, 841, 842(5), 843, 844 Millar, D. B. S., 861, 862(49) Miller, A., 786 Miller, C. G., 20, 21(2), 25(2), 26(2), 119, 120(26), 131(26), 136(26), 137(26), 227(7), 228, 234(7), 377 Miller, D. D., 74, 76 , Miller, K. I)., 21, 140, 143(1), 148, i53, 157, 163, 164(21), 166(21), 170, 171, 172, 176, 177(63), 178, 179, 180(41), 182(51), 183(52), 224, 915 Miller, L. L., 338 Miller, M. J., 797, 798(3), 803(3) Millet, J., 650 Mills, J., 408, 414(4), 416, 418, 421(10) ~Milojevic, S., 915 Milstone, H., 807 Mirsky, A. E., 54, 406, 413 Miskin, R., 802 Mitchell, W. M., 616(12), 619, 636, 638 (22a), 639(16), 640(16, 24), 641(16), 642(16, 25) Mitrica, N., 196 Mitsuda, It., 62 Mitsyasu, K., 923 Mitz, M. A., 494, 935, 943(8), 954(8), 956 (8), 968(14), 969 Miura, K., 372 Miyake, T., 282, 283 Mizuno, M., 282 Mizushima, S., 455 Mlynar, D., 521, 528(1) Mockros, L., 772 Mogi, K., 404, 582, 586, 588(7), 589(4) Mohler, E. R., 832 Moldave, K., 850
Money, C., 956, 975(27), 978 Monier, R., 636 Monkhouse, F. C., 180, 915, 917, 921, 922 (11), 923 Monro, R. E., 797, 799(1, 2), 800(8, 9), 801(2, 9), 802(2, 7, 11, 12, 13), 803 (13) Monsigny, M., 901 Montague, D., 705 Montgomery, It., 895, 897, 899(29) Montreuil, J., 895, 901 Montuori, M. H., 838 Moon, H. D., 117, 131(21), 139 Moore, 444 Moore, H., 168 Moore, H. C., 163(23), 164, 167(23) Moore, M. R., 52(n), 53 Moore, S., 252, 253(3, 5), 257(3), 258(3, 5), 259(5), 260, 261, 276, 277(9), 279 (9), 280, 281(9), 282(9), 283(9), 284 (9), 291, 292, 317, 319, 321(19), 326 (19), 328, 330, 331, 333, 334(11), 336 (19), 345, 346(11), 365, 383, 402, 403 (21), 411, 434, 439, 457, 476, 529, 557, 583, 643, 787, 863, 854 865, 866, 867, 868(a), 869 Moravek, L, 333 Morihara, K., 212, 213, 446, 447(14), 573, 648(32, 33, 34, 35), 649, 650(20, 32, 34, 35), 664 Morita, F., 633, 635(57) Moriwaki, C., 694 Moriya, H., 682, 691, 694, 697 Morris, J. 0. R., 544, 545(4) Morrison, W. L., 312 Morton, J. I., 896, 899(41) Morton, R. A., 251, 260, 529 Moser, P., 549, 550(9) Moser, P. W., 201 Movileanu, D., 196 Moyle, V., 880 Mozen, M. M., 190(27), 196, 665, 670(1) Miillertz, S., 706, 811, 815, 822, 824(6), 836 Muirhead, H., 129, 131(55), 133(55), 475, 491, 493(38), 495(38), 497(38), 501 (38), 502(38), 563(38) Mukai, J. I., 848 Mulczyk, M., 569
AUTHOR INDEX Muller, M., 372 Murachi, T., 244, 274, 275(5), 276, 277 (11), 278, 279, 280, 281, 282, 283, 964 Muramatu, M., 197 Murano, G., 178, 179, 182(61) Muratova, G. L., 333 Murphy, R. C., 915 Murray, C. W., 890, 892, 895(3), 896(3), 897, 901(3), 903 Murray, M. A., 383 Muss, M., 840, 843, 852(3) Mutt, V., 156, 181 Mycek, M. J., 258, 285, 288, 290(14), 291 (14), 301, 306(8), 307(8), 309(8) Myhrman, R. V., 765 Mzhel'skaya, T. I., 383 N Naci, A., 218(8), 219, 220(8), 221(8) Nagabhushan, N., 32, 33(5), 58(g), 69, 70 (a), 71 Nagabushnan, N., 606 Nagai, Y., 617(19, 20, 21), 619, 621(20), 622(20), 623(18, 20), 624(19, 20, 21), 625(19), 633, 635(37) Nagao, M., 890 Nagasawa, F., 713 Nagasawa, M., 373, 401, 402, 404, 406(17) Nagel, W., 873, 878(13) Nagy, H., 789 Nahas, L., 715 Nakai, M., 202, 204(12), 214, 274, 275(4), 569, 642 Nakamura, S., 890 Nakanishi, K., 373, 374, 375(29), 386, 406 Nanci, A., 209 Nanninga, L. B., 197, 199, 857 Narahashi, Y., 651, 655(7), 657(2, 7), 658 (7),'661(7), 663(2, 7) Narayanan, A. S., 613 Narayanan, A. Sampath, 133 Nathans, D., 803 Naughton, M. A., 113, 114(11), 115(11), 117(11), 121(11), 122(11), 123(11). 129(11), 131(11), 133, 134(11), 549. 611, 793 Neet, K. E., 209, 210, 214, 215, 218(8), 219, 220(8), 221(8) Neidle, A., 803
1005
Nelson, J. It., 375, 455, 457(46) Nelson, R., 335 Neth, R., 803 Neudecker, M., 906(13), 907, 935, 936(5) Neuhaus, O. W., 178 Neumann, H., 317, 333, 335 Neumann, N. P., 252, 253(3), 257(3), 258 (3) Neurath, H., 32, 33, 34, 42, 43, 47, 48, 49, 50, 52(a, b), 53, 56, 57, 58, 59, 60, 61, 63(29), 65, 58(j), 69, 100, 102(38), 112, 196, 276, 282(7), 283(7), 312, 315, 374, 460, 461, 462(12), 463, 465(13), 466(13), 467, 468, 469, 471, 472(7, 12), 473, 474(5, 10), 475, 476, 477, 478, 479, 481(56), 482(2, 54), 484(47, 54, 55), 485, 486(55), 487, 488, 489, 490 (o, p), 491, 493(35, 82), 494, 495, 496, 498, 499, 500, 501, 503(82, 112), 643, 654, 857, 859, 884, 964 Neuwirthov~, J., 849 Neves, A. G., 285, 302(9), 303(9), 304(9), 305(9), 306(9) Newcomb, T. F., 978 Newman, J., 811 Nichol, L. W., 383 Nickel, V. S., 197 Niekamp, C. W., 858 Niemann, C., 75, 118, 601 Nil~hn, J. E., 158, 163(5), 168(5) Nilsson, I. M., 181,671 Nilsson, K. K., 308 Nishimura, S., 210, 211,857 Nitschman, H., 455, 456 Nivard, R. J. F., 289, 300(16), 301(16) Noda, H., 616(10), 619, 629(10), 633 Noguchi, Y., 646 Nolan, C., 533, 534(19) Noltmann, E. A.. 585 Nomoto, M., 651 Nomura, N., 252, 253(4), 258(4), 259(4) Nord, F. F.. 57, 857, 896, 898, 899, 900, 941 Nordwig, A., 533, 582, 583(6), 589(6), 590 (6), 616(5), 617(17), 619, 634(5), 636, 641(11) Norman, D., 979 Norman, P. S., 706, 707(3) Norris, E., 63
1006
AUTHOR INDEX
Northrop, J. H., 41, 44, 49(19), 56(1), 197, 202, 316, 317, 326, 337, 351, 357 (8), 358, 363(4), 582, 844, 906 Nossel, H. A., 705, 773, 774(32), 985 Notley, N., 772, 774(27), 084 Nouvel, G., 849 Nuenke, J. M., 535 Nutting, M. D. F., 61 Nyburg, S. C., 123, 125(47), 382 O Oakley, C. L., 636 Oduro, K. K., 321, 329(26), 334 Ohman, B., 146, 171 Ogata, K., 962 Ogle, J. D., 635, 641(1) Ogura, Y., 633, 635(57), 645, 647(9), 648, 65O(9) Ohta, Y., 045, 047(9), 648, 650(9) Oka, T., 212, 573, 648(34, 35), 640, 050 (34, 35), 664 Okamoto, S., 197, 713 Okomoto, U., 197 Okunuki, K., 202, 204(12), 208, 209, 214, 274, 275(4), 569, 042 Olaitin, S. A., 205 Oleott, H. S., 863, 865(2), 866(2), 867, 870(2), 895, 896(35), 900(35) Olofson, R. A., 969 Olson, M. O. J., 606, 010 Olson, R. E., 145 Omote, Y., 478, 479(53) O~er, N., 765, 770, 773(5), 777(5) Ong, E. B., 332, 334, 943, 944(45), 045 (45), 952(45) Ong, H. H., 705, 770, 771(3, 5), 773(3, 5), 774(3, 30), 777(5) Onishi, T., 197 Oosterom, E., 263, 272(8) Oppenheimer, H. L., 127 Oppitz, I., 930 Oppitz, K.-H., 030 Orekhovich, V. N., 330, 334, 335, 352, 357 (10), 358, 363(5), 333, 418, 629 Oreshenko, L. I., 383 Orlowski, M., 782, 786(3, 4), 788(4), 791, 792, 795(4), 796(4), 797(4)
Omstein, L., 622, 754, 914 Oshiba, S., 197 Ostoslavskaya, V. I., 333 Osuga, D. T., 890, 892, 893(5, 25), 894, 896(5), 808(a), 899, 901(48), 904(4) Ota, S., 276, 277(9), 279(9), 280, 281, 282 (9), 283(9), 284 Ottesen, M., 95, 200, 202(3), 203(1), 205, 207, 208, 209, 210, 212, 214, 347(3), 348, 351(3), 450, 673, 950, 955(53), 956(53), 960(53), 962 Otto, K., 206, 299(26a), 301(20a) Ouchi, T., 651 OueUet, L., 16 Owren, P. A., 158, 159, 981 Ozawa, A., 446, 449(23) Ozawa, H., 448, 457(42), 458(42) Ozawa, K., 858, 901, 914 P
Padayatty, J. D., 759 Page, I. H., 700, 701(3), 704, 705 Paik, W. K., 762 Painter, R. H., 838 Paleva, N. S., 446, 447(3) Palmer, J. W., 890 Palmstierna, H., 693 Pandolfi, M., 833 Pansky, J., 636 Papiannou, S., 50 Parisoli,U., 139 Park, B. C., 74, 75(11) Parker, M. J., 228, 242(8), 244, 237(8), 955 Parkes, C. O., 32, 33(5), 08(g), 69, 70(a), 71, 86, 87 Partridge, S. M., 113, 114(2), 120, 132 (38), 133(2), 135(2), 140(38), 298 Patch, M. J., 182 Patchornik, A., 949, 968(15), 960 Patel, R. P., 935, 050(14) Paterson, G. M., 608 Payne, N., 706 Peanasky, R. J., 466(20), 467, 468(20). 473(20), 650, 844, 851, 872, 876, 877, 878, 879, 880, 881, 882, 883, 907 Peart, W. S., 706
AUTHOR INDEX
1007
Porter, M. C., 915 Peeh6re, J. F., 48, 463 Pechet, L., 156, 979, 980, 981(13), 982(13) Porter, R. R., 242, 285, 292(4), 309(4), 311(4), 312(4), 316, 317(7), 321(7), Pecht, M., 845, 846(9), 936, 943(22), 946 326(7), 329(7), 330, 335(7), 742, (22), 949(22), 950(22), 951(22), 952 744(2), 901, 915 (22), 968(22), 971, 978 Portmann, P., 872, 873(6, 8), 875(8), 876, Pedersen, K. O., 456 877, 878 Peel, J. L., 63 Pospi§ilov£, D., 844(6a), 845, 852 P6nasse, L., 901 Potts, Jr., J. T., 507, 513 Pentia, T. A., 446, 447(1) Poulik, M. D., 579 Pereira, A. A., 699 Povova, N. Y., 446, 447(3) Perez-Villasefior, J., 236 Powers, J. C., 32 Perham, R. N., 333 Perlmann, G. E., 330, 331, 332, 334, 341, Pozerski, E., 890 Prado, E. S., 683, 688, 698 943, 944(45), 945(45), 952(45) Prado, J. L., 683, 688, 698 Permin, P., 821 Prahl, 374 Peschke, W., 873, 878(13) Peterson, E. A., 322, 402, 508, 510, 690, Prahl, J. W., 33, 68(j), 69, 100, 102(38) 708, 726, 855, 860(19), 861(19), 864, Prescott, J. M., 848 Press, E. M., 285, 292(4), 309(4), 311, 312 875, 893 (4), 742, 744(2) P6tra, P. H., 63, 93, 95(34), 243, 460, 471, 475, 476, 477(47), 478, 479, 484(47), Pressman, B. C., 286 485, 486(57), 487(47, 57), 488, 489(47, Pressman, D., 498 Price, S., 935, 956(14) 57), 491(59), 499(36) Prockop, D. J., 800 Petrova, V. I., 330 Prokopov£, D., 620 Peyton, M. P., 770 Pfleiderer, G., 32, 107, 514, 517, 519, 521, Prou-Wartelle, 0., 158, 163(4) Provido, H. S., 673 650 Pudles, J., 872, 876, 881, 882 Phelan, A. W., 140, 143(1) Piitter, J., 850 Pickens, P. T., 700, 701(3), 794, 705 Pugacheva, I. B., 333 Pierce, J. V., 682, 691, 697 Puhan, Z., 446, 447(12) Piez, K. A., 594, 617(21), 619, 624(21) Purr, A., 475 Pilkington, T. R. E., 772 Pusztai, A., 197, 863, 865, 866, 867, 870 Pillemer, L., 636 (19), 871(19) Pineda, E. P., 786, 787(14), 788(23) Putnam. F. W., 489, 490 Piras, P., 460, 467(9), 468(9), 469, 473(9) Pisano, J. J., 770 Q Planta, R. J., 302, 307(37), 368(38), 309 (38) Queval, J., 895 Plapp, B. V., 63 Quick, A. J., 158 Ploug, J., 196 Quiocho, F. A., 475, 491(38), 493(38), 495 Pochon, J.. 236 (38), 497(38), 498, 501(38), 502(38), Polgar, L., 210, 214, 215, 216, 217(4), 218, 503(38) 219, 220(1, 4, 6), 221(4) Polgase, W. J., 494, 495 R Polglase, W. J., 513 Porath, J., 373. 577, 579, 581, 849, 850(22. Raaflaub, J., 530 R~bek, V., 883 b), 936, 941(19, 20) Rabin, B. R., 270 Porcelli, G., 264
1008
AUTHOR INDEX
Richmond, V., 410, 416(6) Rabin, R. B., 723, 728(4) Rick, W., 576, 583 Rabiner, S. F., 191, 199 Rickert, W., 459, 460 Rackis, J. J., 853, 854, 855, ~56(8) Riddiford, L., 222 Racz, M. M., 272 Riehm, J. P., 863, 866, 867, 868(g), 869, Rademaker, W., 302 870(9) Radhakrishnan, T. M., 42, 49(6) Rigbi, M., 848, 849(15), 850(15), 906, 910 Radicevic, M., 844, 851, 907 (8), 911(8), 912(8), 913(8), 914(8) Raftery, M. A., 63 Rajagopalan, T. G., 317, 321(19), 326 Riggs, S. K., 765 (19), 328(19), 330, 331, 334, 336(19), Riggsby, W. S., 201 343, 344(11)., 345, 346(11), 383, 583 Rimon, A., 197, 936, 943(23), 978 Rimon, S., 330, 978 Rancour, J. M., '883 Riniker, B., 705 Rand, A. G., 427, 430(14), 431 Randall, R. J., 387, 511, 526(9), 528, 547, Riordan, J. F., 472, 475, 476, 477, 487(37), 490, 495, 496, 497, 498(37), 499, 500, 583, 592, 596(6), 627(a, 61), 671, 501, 503(37, 101) 689(b), 691, 695(d), 768, 724, 735, Ripa, R., 850 739, 743, 757, 784, 792, 835 Rippon, J. W., 617(15, 16), 619 Rao, L., 123, 125(47) Roar, N. E., 113 Raper, K. B., 399 Robbins, B. H., 272, 273 Rappaport, H. P., 201 Robbins, K. C., 53, 113, 120, 131, 136(6), Rasmussen, J., 826, 833 185, 186(4, 5), 187, 190, 191, 192, 193, Rasmussen, P. S., 174, 775 194, 195, 196, 197(1, 17, 18, 19), 198, Ray, C., 32, 599, 600, 609(5), 610 199, 672, 772, 816, 817, 820, 833 Raymond, S., 622 Robert, L., 113, 135(12) Read, M. S., 181 Roberts, D. V., 62 Recke, Jr., G. N., 498 Reeke, G. N., 475, 491(38), 493(38), 495 Roberts, P. S., 687, 811, 982 (38), 497(38), 501(38), 502(38), 503 Roberts, R. C., 52(h), 53, 59 Roberts, W., 772 (38) Robertson J. I. S., 702, 705 Rees, M. W., 551 Robertson, P. S., 446(27), 448, 449 Reid, T. W., 317, 335(15) Robertson, T., 673, 857 Reidel, A., 244 Reilly, T. J., 302, 305(39), 306(39), 307 Robinson, D. S., 724 Robinson, N., 46 (39), 308(39), 543 Rocha e Silva, M., 686 Reimer, A. C., 118, 123(24) Rodman, N. F., 181 Reindel, F., 564 Roeske, R. W., 20, 21, 25(2), 26(2), 119, Reisfeld, R. A., 566, 608, 914 120(26), 131(26), 136(26), 137(26), Reisfield, R. A., 122, 123(45) 227(7), 228, 234(7), 377, 557 Remmert, L. F., 185, 186(2), 814 Rogers, H. J., 236 Remold, It., 373, 386(11) Roholt, O. A., 498 Ren~, A. M., 723, 724(2) Rola, F. H., 872, 876, 881, 882, 883 Revel, J. P., 786, 787 Rombauts, W., 435, 649 Rhodes, M. B., 211, 872, 876, 882, 890, Roncari, G., 544, 549(9), 550 891, 892(7, 17), 893(7), 896(7), 898, Rosebrough, N. J., 387, 511, 526(9), 528, 904(7) 547, 583, 592, 596(6), 627(a, 61), 671, Richardson, G. H., 375, 446(29), 448, 449, 689(b), 691, 695(d), 708, 724, 735, 455, 457(46) 739, 743, 757, 784, 792, 835 Richman, P. G., 792, 795(4), 796(4), 797 Rosen, H., 384, 408 Rosenberg, A., 75 (4)
AUTHOR INDEX Rosenberg, R. D., 154, 155 Rosenblum, E. D., 272 Rosenkranz, H., 849 Rosenthal, T. B., 132, 139 Rossi-Fanelli, A., 878 Rountree, P. M., 714 Rovery, M., 47, 48, 49, 50, 52(b), 53, 56, 59, 68(], h, i), 69, 81, 82(24), 83, 84 (24), 85(24), 87(22), 88, 89(30), 92, 95(33), 96(33), 466(20), 467, 468(20), 471, 472, 473(20), 497(28) Rubin, I., 706 Rule, N. G-, 183, 765, 770, 771(3), 773(3, 5), 774(3), 777(5) Rupley, J. A., 487, 489(70), 490, 491(70), 494, 495, 496 Ruscica, J., 210 Russell, K. E., 714 Rutenberg, A. M., 656, 786, 787(14), 788(23) Ruttle, D. I., 397 Ryan, C. A., 52(d), 53, 105, 211, 849, 857, 870, 883, 884, 885, 887, 888, 889, 892, 898, 900(21), 904(21) Rybhk, M., 849(34), 850, 857 Rychlik, I., 797, 798(4), 803 Rydon, H. N., 791,792 Ryle, A. P., 316, 317(7), 319, 320, 321(7, 25), 322, 324, 325(20), 326(7, 30), 327 (8, 20), 328(8), 329(7, 8, 20, 23, 25, 30), 330, 331, 333, 334, 335, 336, 358, 364
$ Saavedra, N. V., 168, 169 Sach, E., 848, 849, 857, 900 Sachar, L. A., 113, 114(13), 135(13), 609 Sachdev, G. P., 373, 383(29a) Sadeh, T., 952 Sakaguehi, K., 399, 400 Sakai, T., 279 Salvi, M. L., 264 Sampath Kumar, K. S. V., 477, 478, 479 (54), 482(54), 484(54), 487(54), 489 (54), 490(o), 491 Samsa, E. G., 54 Samuel, P., 113, 135(12) Sanb0m, B. M., 61, 62, 63
100~
Sanchez-Chiang, L., 407, 408, 409(2), 414 (4), 418(4) Sande, R., 485 Sanders, M., 46 Sanger, F., 113, 114(11), 115(11), 117(11), 121(11), 122(11), 123(11), 129(11), 131(11), 133, 134(11), 210, 589, 611 Sardinas, J. L., 373, 375(15), 439, 442, 444, 446, 447(16) Saresai, V. M., 673 Sarid, S., 521, 523(3, 6), 531(3, 6) Sasaki, R. M., 648(31), 649, 650(23, 31) Sasame, H. A., 853, 854(3, 8), 856(8) Savrda, J., 42, 49(7), 81, 406 Sawada, J., 373, 389(12), 390, 391, 406 Sbarra, A. J., 121, 122(42), 123(42) Scevola, M. E., 849(34a), 850 Schiifer, G., 929 Sehechter, I., 212, 236, 237, 241, 502 Seheidegger, J.-J., 717 Schellman, C. G., 210 Sehenkein, I., 672, 677 Scheraga, H. A., 146(5), 147, 157, 163(25), 164, 167(25), 108(25), 169, 171, 178, 179, 772, 774(27), 856, 984 Schirardin, H., 857, 900 Sehirmer, ]~. W., 91, 94(32), 96(32), 97 (32), 98(32), 109, 110, 111(1), 112(1), 476, 499, 500, 504, 505, 508(2) Schlamowitz, M., 316, 319, 335(21), 421 Schleith, L. Z., 936 Schmerzler, G., 901 Schmidt, F. H., 873, 878(13) Schmidt, J. J., 639 Sehmidt, W. R., 533 Schmutzler, R., 929 Schoellmann, G., 57(47), 58, 131, 616(14), 619, 664, 900 Scholtan, W., 849 Schonbaum, G. R., 7, 9(3), 10, 879, 892, 895(23), 964 Schram, E., 457 Schramm, W., 846 Schrier, E. E., 178, 179 Schroeder, D. D., 34, 50, 51, 62(35) Schroer, H., 923 Schult, H., 846, 847(10), 906(13), 907, 935, 936(4) Sehultz, F., 844, 849
1010
AUTHOR INDEX
Schulz, M. E., 446(30), 448, 449 Schwabe, C., 744, 747, 748, 754(12), 755 (12) Schwander, H., 433, 455, 456 Schwarberg, R. L., 375 Schwartz, H. C., 235, 237(22) Schwarz, S., 883 Schwert, G. W., 42, 43, 60, 477, 493(82), 495, 503(82), 636, 654, 655, 716, 884. 907 Schwick, H. G., 197, 199 Schwimmer, S., 234, 312 Schyns, R., 47, 48, 49(24, 25), 52(c), 53 Scocca, J., 281 Scrimager, S. T., 127 Searl, J., 491 Seegers, W. H., 145, 146(3), 148, 153, 155, 156(2), 157(2), 158, 160(10), 163, 164 (18), 167, 168, 169, 170, 171, 172(60), 173, 178, 179, 180, 182(67), 915, 917, 923, 979 Segal, H. L., 338, 724, 819 Sehon, A. H., 936 Seifter, S., 613, 616(4, 6, 9), 619, 620, 623 (4), 626(4), 628(9), 629(6, 9, 49), 630 (9), 631(6, 9, 46), 632(6, 49, 53), 633 (6, 49, 54), 634(4, 49), 635(57) Seijffers, M. J., 338 Sela, M. P., 317, 521, 968(15), 969 Seligman, M. A., 673 Seligson, D., 288 Seligson, H., 288 Semar, M., 811 Sen, S., 702 Seng, R., 264, 272(13) Senyutenkova, L. G., 333 Sgarbieri, V. C., 264, 265. 266, 268(10). 271(10), 273 Shah, R. J., 193, 195(16), 196(16), 672, 816 Shalitin, Y., 62 Shamash, Y., 197, 984 Shanberge, J. N., 915 Shapira, E., 226, 234, 235(21), 236(21), 239(21), 242, 243 Shapiro, B., 197 Shapiro, S. S., 170 Sharon, N., 317, 335 Sharp, A. K., 935(17), 936
Sharp, H., 3,35 Shaw, A., 319, 335(21), 421 Shaw, D. C., 133, 210 Shaw, E., 21, 22(6), 25(6, 7), 29(7), 32, 34, 35, 42, 44(9), 50, 51, 57(47), 58, 60, 61, 62, 131, 171, 183, 210, 211,444, 664, 857, 889, 891, 900 Shaw, R., 373, 374(8), 376, 379(8), 381 (8), 382(8), 383(8), 384, 385(8, 9). 406 Sheehan, J. C., 783, 790, 791(la) Sheena, R., 199 Shepard, R. S., 172(60), 173 Sheppard, E., 858 Sherman, M. P., 848 Sherman, R., 285, 286(7), 291(7), 295(7), 299(7), 315(7) Sherry, S., 157, 171, 180(46), 182(46), 196, 198, 199(51), 272, 671, 672, 713, 714, 822, 832(7). 833(7), 834, 982 Shiao, D. F., 75 Shibata, K., 458 Shibko, S., 296, 309(24) Shibuya, K., 651, 655(7), 657(7), 658(7), 681(7), 663(7) Shimada, K., 859 Shimazono. H., 373, 406 Shimizu, M., 197 Shimwell, J. L., 446, 447(7) Shinitzky, M., 239, 241(38) Shinowara, G. Y., 182 Shipley, R. E., 700 Shoichet, I., 300 Shotton, D. M., 58, 114, 118(15), 119(15), 122(15), 123, 125, 127, 128(15), 129, 131(15, 48, 55, 56), 132(15), 133(48, 54, 55, 56), 135(15, 48) Shpikiter, V. O., 330, 629 Shulman, S., 714, 772 Shyamala, G., 340, 848(1), 849(1), 850(1) Sicher, N., 113, 114(13), 135(13), 609 Siegelman, A. M., 857 Siegelman, H. W., 840 Siegelmann, R., 883 Sierra, G., 212 Sigler, P. B., 8, 49, 58(32), 80, 127, 129, 131(52), 133(52), 135(52), 940, 941 (37)
AUTHOR
Siiteri, P. K., 190(22), 196, 818, 819(25), 820(25) Silman, I. H., 521, 935, 936, 941(31), 943 (2), 944(2), 945(47), 946(31), 948 (31), 949(31), 950(31), 952(2, 31), 953(31), 954(31), 956(31), 959(31), 960(31, 47, 70), 961(31), 962(31), 978 Silver, M. S., 335 Silverton, E. W., 64 Simlot, M. M., 890, 901(18) Simpson, R. T., 477, 490, 496, 497(102), 498 Singer, A., 648(31), 649, 650(31) Singh, K., 591 Skeggs, L. T., 703, 764, 850 Skin, W. H., 260 Skinner, S. L., 702 Sklyankina, V. A., 335 Skoza, L., 811 Slavayanova, V. V., 446, 447(4), 450(4) Slayter, H. S., 943, 944(43, 44), 945(43, 44), 960(43, 44) Sletten, K., 648, 649 Sluyterman, L. A. iE., 232, 233, 234, 235, 236(24), 237(13, 20), 240(13, 20) Smeby, R. R., 700, 701(3), 702, 704. 705 Smillie, L. B., 32, 33(5), 52(k), 53, 56 (g), 57, 59, 67(e, g), 68, 69, 70(a), 71, 72, 82, 84(23), 86, 87, 122, 123 (44), 131(63), 132, 140, 600, 605, 606, 609, 610, 613 Smith, A. K., 853, 854(3, 8), 856(8) Smith, E. L., 34, 200, 205, 207, 210(17, 35), 211, 213, 215, 226, 227, 228, 230(2), 231,233, 235, 236, 237, 239(6, 37), 240 (37), 241, 242, 243, 244, 271, 272, 312, 475, 487, 489, 490, 493(34), 494, 495, 496, 498, 499, 500, 501(114), 510, 513, 517, 519, 533, 534(18, 19, 20), 642, 643, 648, 649, 655, 754, 786, 787(14). 788(23), 955, 956, 957(66, 67), 964 Smith, G. D., 383 Smith, H. P., 148, 153(7), 158, 160(9), 170 Smith, H. W., 714 Smith, L., 636 Smith, P. W. G., 791,792 Smith, R. A., 56
INDEX
1011
Smith, R. L., 35, 50 Smithies, 0., 920 Smyth, G. D., 66 Smyth, J. J., 21, 42, 61, 62, 171 Snellman, O., 301(35a), 392 Snir, I., 848, 849(15), 850(15) Snoke, J. E., 43, 476, 477, 487(41), 499, 500, 654, 884 Sobel, R. E., 534 Sober, H. A., 322, 402, 690, 708, 726, 855, 860(19), 861(19), 864, 875, 893 ~odek, J., 374, 375, 381(35b), 382(35, 35b), 383(25, 35), 393, 396, 397(56), 406 Soderstrom, N., 671 Soederberg, E., 671 Sohonie, K., 863, 865(12), 866, 867, 870, 871, 883 Sohr, C., 515, 519(14) Soko|ovsky, M., 495, 497, 501,941 Solov'eva, N. I., 629 Somkuti, G. A., 373, 446, 447(15), 448 (15) Somlo, M., 636 Sonneborn, H. H., 32, 107 Soons, J. B. J., 302 ~orm, F., 56, 58, 08(d), 69, 844(6a, 6b), 845, 849, 852 Soru, E., 196, 711,714 Soulier, J. P., 158, 163(4) Spackman, D. tt., 260, 513, 643, 951, 954 (56) Spadari, S., 590 Spector, A., 95, 200, 202(3), 207 Spicer, D. S., 906 Spiekerman, A. M., 848 Spies, J. R., 598 Spona, J., 334 Springell, P. H., 786 Springhorn, S., 61 Sprung, M. M., 949 Srinivasan, R. A., 446, 447(6) Sri Ram, J., 941 Staehelin, T., 797, 799(2), 801(2), 802(2, 11), 803 Stage, A., 822 Stahl, M., 782 Stahman, M. A., 498
1012
AUTHOR INDEX
Staib, F., 373, 386(11) Stainthorpe, E., 300, 302(28a) Stanulovic, M., 517 Staprans, I., 820 Stark, G. R., 66 Stauffer, J. F., 750 Steele, B., 178 Steger, R., 900 Stein, 444 Stein, E. A., 482 Stein, E. H., 787, 788(23) Stein, M., 271 Stein, W. H., 252, 253(3, 5), 257(3), 258 (3, 5), 269(5), 260, 261, 276, 277(9), 279(9), 280, 281(9), 282(9), 283(9). 284(9), 291, 292, 317, 319, 321(19), 326(19), 328(19), 330, 331, 333, 334, 336(19), 343, 344(11), 345, 346(11), 365, 383, 402, 403(21), 411, 434, 439. 476, 557, 583, 643, 787, 863, 864(4), 865, 866, 867, 898(a), 869 Steinbuch, M., 924 Steiner, R. F., 853, 856, 858, 861. 862 Steitz, T. A., 129, 134(58), 475, 491, 493 (38), 495(33), 497(38), 498, 501(38), 502(38), 503(38) Stepanov, V. M., 332, 3331 334, 383 Sternberg, M., 196, 711 Sternby, N. H., 671 Sterndorff, I., 822 Stevens, F. C., 890, 892, 893(25), 895, 896 (34), 901(34), 904(4) Stevenson, K. J., 32, 33(5), 63(g), 69, 70 (a), 71, 127, 136(50) Stewart, J. A., 16 Steyermark, A., 950 Stoekell, A., 235, 242(28), 489, 490, 956, 957(66), 964 Stone, N., 706 Stoops, J. K., 20, 21(2)~ 25(2), 26(2), 119. 129(26), 131(26), 136(26), 137(26), 227(7), 228, 234(7), 377 Straueh, L., 617(23, 24), 619, 629, 636, 641(11) Strominger, J. L., 591 Strumeyer, D. H., 889, 900 Stupp, Y., 978 Sturkie, P. D., 351 Sturtevant, J. M., 16, 57(48), 58, 62, 138
Subramanian, A. R., 376, 582, 585(5), 587 (5), 588(5), 59O Sugeno, K., 649 Sugimoto, E., 759 Summaria, L., 52(i), 53, 185, 186(4, 5), 187, 190, 191, 192, 193, 194, 195, 196, 197(1, 17, 18, 19), 198, 199, 672, 816, 817, 820, 833 Summaria, L. J., 935, 943(8), 954(8), 956 (8), 968(14), 969 Suolinna, E.-M., 840, 841, 842(5), 843 Surbeck, E., 66, 76, 77 Surinov, B. P., 935, 936(15) Sussman, A. J., 9O6(14), 907 Suzuki, A., 281 Suzuki, K., 649 Suzuno, R., 890 Svedberg, T., 456 Svendsen, I., 200, 210, 211(32), 212, 213, 214, 962 Svensson, H., 393, 581, 794 Svestkovh, V., 844(6b), 845, 852 Sweet, B., 712, 713(14), 714(14) Swen, H. M., 237, 239(36), 240(35), 241 (36) Swenson, T. L., 895 Symonds, V. B., 131, 136(61), 137(61), 6O9 Symons, P., 168, 169 Symons, R. H., 803 Szasz, G., 783, 787(8) Szewezuk, A., 569, 784, 786(11). 787(11), 788(11), 791 Szues, M. M., 872, 878(11), 879(11), 880 (11), 881(11), 882(11), 883(11) °
T Taehibana, A., 281 Tague, L. L., 508 Takagi, T., 771, 782 Takahashi, K., 787 Takahashi, N., 280, 281, 282(22), 283(22) Takahashi, S., 616(13), 619, 626, 639(49), 631, 632(49), 633(49, 54), 634(49) Takahashi, Y., 251 Takenaka, Y., 42, 477, 636, 655, 716, 907 Talamo, R. C., 082
AUTHOR INDEX
Tallan, H. H., 285, 286(3) Tamura, G., 373, 374, 375, 376, 446, 447 (5), 448, 449(20), 450(36), 454(35, 36), 455(20, 21, 36, 39), 457(20, 21, 34, 35, 36, 38, 39, 40, 41), 458, 459, 460(1) Tan, B. H., 924 Tan, W., 68(b), 69 Tanford, C., 940, 941(38) Tang, J., 332, 334, 407, 468, 409, 410, 414 (4, 5), 416, 418, 419, 420(15), 421 Tang, K., 409, 410(5), 414(5) Tang, L., 434 Taniguchi, K., 446, 449(23) Tanizawa, K., 21 Tanksley, T. D., 333 Tappel, A. L., 296, 309(24) Tartakoff, A. M., 52(j), 53, 221, 225, 857 Tattrie, B. L., 723, 730, 737, 749 Tauber, H., 863 Tauber, S., 120, 132(39) Tawde, S., 863, 865(6), 865(6), 870(6). 871(6) Taylor, F. B., 196, 811, 816(8), 818(8), 820(8) Taylor, Jr., F. B., 199 Taylor, Jr., S. P., 272 Taylor, W. H., 316 Teale, F. W. J., 334 Teller, D. C., 64, 74, 76, 80, 473 Tentori, L., 264 Terminiello, L., 196, 896, 899(40), 941 Tertrin, C., 924 Tesser, G. I., 289, 300(16), 301(16) Tetrault, P. A., 547 Thangamani, A., 405 Thatcher, G. N., 706 Th~ly, M., 848, 849(33a), 850, 857, 900 Thiele, E. H., 121,122(40) Thom, C., 399 Thomas, J., 120, 132(38), 140(38) Thompson, E. 0. P., 243, 469, 478, 479 (M), 482(M), 484(54), 487(54). 489 (54), 491(54) Thompson, J. F., 787, 788 Thompson, R. R., 226, 890 Thomson, A., 41 Thorne, C. B., 789 Thorsen, S., 832, 833(17)
1013
Tietze, F., 45, 59, 508, 857 Tillet, W. S., 807, 811 Tipograf, D. Ya., 446, 447(9) Tipograf, P. Y., 446, 447(1) Tipper, D. J., 591 Tiryakova, 0. Kh., 446, 447(10) Tiselius, A., 579 Tishkoff, G. H., 145, 163(24), 164, 167 (24), 168(24), 979 Tixier, R., 863, 866, 867, 868(g), 869, 870 (11) Tobita, T., 99, 112 Todd, A. S., 833, 838 T6pfer, H., 929 Toi, K., 63 Tokarsky, E., 672 Tokura, S., 770, 771(4), 773(4), 779(4), 780(4), 782(4) Tomar, R. H., 816, 819 Tomh~ek, V., 56, 68(d), 69 Tomimat~u, Y., 52(d), 53, 105, 211, 849, 857, 889, 892, 897, 898(a), 899, 900 (21), 903, 904 Tomodo, K., 373, 406 TorbjSrn, V., 146 Tosa, T., 756, 757, 761,762 Towers, A. G., 712 Toyoura, E., 762 Traut, R. R., 801,802(12) Trautschold, I., 197, 697, 839, 850, 851, 853, 870, 890 Traver, J. H., 196 Travis, B. L., 708, 711(8) Travis, J., 52(b, c, h, l, m, n), 53, 54. 56, 59 Tree, M., 702, 705 Tr~moli~res, J., 840 Triebel, H., 925 Trikojus, V. M., 383 Troll, W., 157, 171, 180(46), 182(46), 272, 521 Trop, M., 651,850, 862, 871 Trowbridge, C. G., 62, 63 Truceo, R. E., 410, 416(7), 418(7) Trujillo, R., 316 Tsai, C. S., 609, 610, 611(25) Tsang, Y., 943, 944(45), 945(45), 952(45) Ts'ao, C. H., 817 Tsao, T.-C., 866, 867, 868(i), 869
1014
AUTHOR INDEX
Tschesche, H., 914 Tse, A-O., 811 Tsugita, A., 648, 649 Tsugo, T., 446, 447(2), 448(2), 449 Tsuji, H., 395(61), 396, 397 Tsunoda, J. N., 244, 251, 649 Tsuru, D., 201, 212(1), 214(1), 2i5, 373, 391, 393, 395(13, 57, 61), 396, 397, 406, 571, 573, 575(5, 6, 12), 650 Tsuzuki, H., 212, 213, 573, 664 Tsuzuki, T., 648(34), 649, 650(20, 34) Tunnah, G. W., 715, 718(1), 719(1) Tuppy, H., 212, 514 Turkov~., J., 582, 589(3) Turner, D. H., 787, 788(25) Turner, R. A., 901 Tuttle, L. C., 290 Tye, R. W., 47, 52(e), 53, 56, 57(e), 59, 485 Tympanidis, K., 833 Tytell, A. A., 632, 635, 641(1) tJ Uchida, S., 401 Uchino, F., 372 Uliana, J. A., 557, 566, 567 Ulmer, D. D., 490 Umbreit, W. W., 750 Urayama, T., 765, 771(15), 772, 773, 774 (32, 33), 780(15), 985 Uren, J., 501 Uriel, J., 121, 140 Y Vaganova, T. I., 334 Vairel, E. G., 849(33a), 850 Vakhitova, E. A., 332(e), 333 Valiulis, R., 333 Vallee, B. L., 460, 467, 468(9), 469, 472, 473(9), 475, 476, 477, 478, 479(53), 487(37), 488, 490, 493(83), 494(91), 495, 496, 497, 498, 499, 500, 501, 503 (37, 101), 941 Vanaman, T. C., 649 Vandenbelt, J. M., 167 Van Denburgh, R. W., 889 Vandermeers, A., 47, 52(o), 53, 58(23) Van Heyningen, W. E., 636 Van Kley, H., 759 Van Melle, P. J., 54
Van Ness, W. P., 264 Van Vunakis, H., 170, 180(41), 331, 358, 362, 363, 364, 370 Vatier, J., 349, 350(6) Vaughan, R. J., 42, 61 Vazquez, D., 797, 799(2), 801(2), 802(2), 803 Velselov, I. Y., 446, 447(1) Vencelj, H., 617(23), 619 Veneziale, C., 778 Ventura, M. M., 863, 866, 867, 868(h), 869, 870(10) Vesterberg, O., 581, 794 Vestermark, A., 181 Veyrat, R., 700, 701(4) Vigyazo, L. V., 272 Villiard, P., 222 Vithayathil, A. J., 57, 898, 900 Vivaldi, G., 264 Vogel, R., 197, 839, 351, 353, 870, 890 Volgyesi, E. H., 272 Voynick, I. M., 285, 306(10), 307(10), 309(10), 316, 333(10), 335(10), 383 Vratsanos, S. M., 333, 358 Vrethammar, T., 171 W Wachsmuth, D., 514 Wachsmuth, E. D., 517, 521 Wada, A., 645, 647(9), 650(9) Wada, S., 762 Wade, R., 783 Wade, R. D., 65, 461, 465(13), 466(13), 467(13), 468(13), 469, 471, 473 Wiihlby, S., 651,662(5) Waelsch, H., 786 Wagner, F. W., 848 Wagner, L. P., 863, 866, 867, 868(g), 869, 870(9) Wagner, T., 969 Wahba, A. J., 797, 798(3), 803(3) Wahlby, S., 32 Wahlen, J. G., 446, 447(13) WaiUing, D. G., 714 Waite, H., 75 Wakil, S. J., 649 Waley, S. G., 797 Walker, D. G., 618(34), 619 Wallace, B. G., 252, 253(4), 258(4), 259 (4)
AUTHOR INDEX Wall~n, P., 198 Walschmidt-Leitz, E., 475 Walsh, K. A., 32, 33(5), 42, 46, 47, 48 (12), 49(6), 52(a), 53, 56, 57(e), 58, 59, 63(29), 460, 461, 468(7), 469(11), 471, 472(7), 475, 476, 477, 478, 479, 482(54), 484(54, 55), 486(55, 57), 487 (54), 488, 489, 490(o, p), 491, 498 (78), 499(36), 500, 501(46), 955 Walsmann, P., 184, 925, 929 Wan, J., 979 Wanamaker, L. W., 819 Wang, H. L., 397 Wang, K., 867 Wang, T.-W., 848, 849 Ware, A. G., 153, 158, 160(10), 163, 164 (20) Warner, E. D., 158, 160(9) Warner, R. C., 186, 890 Warrack, G. H., 636 Warren, B. A., 833 Warren, G. H., 626 Warter, J., 857, 900 Wasi, S., 120, 123(31), 127(31), 131(31) Wasserman, N., 333, 358 Watarai, T., 400, 401(11), 406(11) Watson, H. C., 58, 127, 129, 131(48, 55, 56), 133(48, 55, 56), 135(48) Watti, A., 264, 265, 565, 954 Wattiaux, R., 286 Waugh, D. F., 145, 146(1), 148, 149(1), 150, 152(1), 154, 155, 157, 167, 168 (32), 172(61), 173, 182 Webb, E. C., 940, 941(36) Webb, T., 285, 286(5), 309, 311(5, 6), 312 (5, 6), 313, 314(6), 315(6) Webster, M. E., 681(la), 682, 687, 691, 697 Weder, J., 863, 866, 870(13) Wedler, F. C., 16, 18(19), 19 Weeds, A. G., 943, 944(44), 945(44), 960 (44) Weetall, H. H., 935, 936(11) Wegrzynowicz, Z., 782 Well, L., 636 Weinland, E., 872 Weinstock, I. M., 292, 294(21), 295(21), 296(21), 309(21), 310(21), 311(21), 312(21), 313(21) Weis, P., 677
1015
Weliky, N., 935, 936(11) Welker, N. E., 204 Wellerson, R., 547 Werle, E., 197, 683, 697, 698(18), 099(18), 839, 840, 843, 845, 846, 847(10), 848 (4), 849(4), 850, 851, 852(3), 853, 857, 867, 869, 870, 871, 883, 890, 900, 906(13), 907, 930, 935, 936(4, 5, 6) Westall, F. C., 215, 220(3) Westberg, N. J., 427, 430(15) Westfall, R. J., 54 Westheimer, F. H., 42, 61 Westman, T. L., 943, 952(46) Wharton, C. W:, 935, 938, 943(9), 965, 970(12), 971(12), 972, 974(12, 24), 976, 977(12) Wheldrake, J. F., 803 Whitaker, D. R., 32, 131, 136(61), 137 (61), 140, 599, 600, 601(9), 602, 603 (13), 606, 607, 608(10, 19), 609(5, 10), 610, 611(10, 25), 612(10) Whitaker, J. R., 236, 237(50), 242, 244, 262, 264, 265, 266(10), 268, 269, 271 (7, 10), 272, 273, 276, 280, 281(14), 373, 375(16), 437, 440, 442(9), 443 (9), 444, 445(11), 476, 477(44), 478, 490, 499, 500, 890, 957 White, W. F., 190(27), 196, 665, 670(1), 672 White, W. N., 889, 900 Whiteley, H. R., 649 Wiedemann, M., 846, 847(10), 906, 913 (11), 935, 936(4) Wiesbauer, W., 514 Wiggans, D. S., 307 Wilcox, P. E., 32, 42, 48(12), 64, 65, 66, 68(b), 69, 76, 77 Williams A., 234, 243, 262, 270 Williams, C. M., 221 Williams, D. C., 273 Williams, D. E., 113, 120(1), 121, 122, 123 (1, 45), 124(1), 127(1), 128(1), 129 (1), 131(1), 132(1), 133(1), 135(1), 139(1), 608, 914 Williams, L. C., 145, 163(24), 164, 167 (24), 168(24) Williams, R. C., 330, 331 Williams, W. J., 720, 789 Willick, G. E., 861,862(49)
1016
AUTHOR INDEX
Willig, F., 873, 878(13) Willstiitter, R., 285 Wilson, H., 714 Wilson, J. B., 714 Wilson, R. J. H., 935(17), 936 Winitz, M., 272, 307, 762 Winnick, T., 268 Winter, K. K., 113, 114(13), 135(13), 609 Winter, W. P., 32, 33(5), 47, 52(1), 53, 56, 57(e), 58 Wintersberger, E., 463, 514 Winzler, R. J., 178 Wisser, J. W. E., 232, 234(13), 237(13), 240(13) Woitinas, F., 906 Wolf, S., 407, 468(2), 409(2), 410, 410(6, 7), 418(7) Wolf, W. J., 854, 856(14) Wolfe, R. S., 591, 592, 593(4), 595(4), 597(4), 598(4), 599(4) Wolff, E. G., 504, 508(2) Wolfmger, H. L., 779 Wollaeger, E. E., 787 Wolthers, B. G., 237, 239(36), 240, 241 (3e) Wong, R. C., 270, 271 Woodward, R. B., 969 Wootton, J., 178 Work, E., 757, 848, 849(16), 850(10), 878 Wright, C. S., 268, 209, 210(20), 215, 220 (3) Wright, H. T., 64 Wright, R. D., 863 Wriston, Jr., J. C., 732 Wu, F. C., 849, 857, 892, 898, 900(24), 901, 912 Wu, Y. V., 856 Wiinsch, E., 617(25), 619, 634 Wulf, R. J., 199 Wunsch, E., 244 Wybrandt, G. B., 212, 333, 334, 375, 435, 436 Wyman, J., 940, 941 (38) ¥
Yagisawa, S., 633, 635(57) Yamada, K., 400 Yamada, S., 564 Yamada, Y., 386
Yamamoto, M., 853, 856, 859, 861(6), 862 (6), 867, 897, 899(44) Yamamoto, T., 201, 212(1), 214(1), 215, 373, 391, 395(13), 396(13), 397, 408, 575 Yamamura, Y., 197 Yamasaki, M., 112, 461, 462(12), 463(12), 465(13), 465(13), 467(13), 468(12, 13), 469, 471, 472(12), 473(12), 474 (10) Yamashina, I., 181, 770 Yamauchi, K., 446, 447(z), 448(v) Yamazaki, K., 694 Yamazaki, M., 281 Yamazaki, S., 399 Yanagita, M., 651, 655(7), 657(2, 7), 658 (7), 661(7), 663(2, 7) Yanari, S., 316, 334(2), 335(2), 494 Yang, H. Y. T., 508 Yapel, A., 75 Yaron, A., 521, 528(1), 952 Yasuda, Y., 276, 277(11), 278, 279(11), 280, 281,282(11), 964 Yasui, M., 276, 277(11), 278, 279, 280(11), 281, 282, 446, 457(21), 964 Yasui, T., 374, 446, 455(21) Yasunaga, K., 168, 169 Yasunobu, K. T., 63, 243~ 244, 246, 249 (12), 251, 571, 573, 575(5, 6), 649, 650 Yatco-Manzo, E., 272 Yemm, E. W., 535, 655, 657(16), 730, 737, 964 Yon, J., 61, 62 Yonetani, T., 214 York, S. S., 60, 61 Yoshida, F,., 616(10), 619, 6'29(10) Yoshida, F., 373, 399, 400, 401, 402, 403, 404, 465, 406(9, 10, 11, 17, 32) Yoshikawa, M., 883 Yoshimura, S., 582, 584(8), 586(8), 588 ~ (8), 589, 590(8), 591 Yoshino, U., 446, 449(23) Youatt, G., 119 Yphantis, D. A., 456, 718 Yu, J., 373, 376, 446, 448, 450(36), 454 (35, 36), 455(36, 39), 457(34, 35, 36, 37, 38, 39, 40, 41, 42), 458, 459, 460(1) Yudkin, E. P., 156 Yulius, A. A., 446, 447(10)
AUTHOR INDEX
Zaborsky, O. R., 13 Zahler, P., 433, 455, 456 Zahnley, J. C., 892, 903(20), 904, 905 Zakrzewsi, K., 196 Zamir, A., 802 Zeffren, E., 334 Zehavi, U., 333 Zeitman, B. B., 302, 305(39), 306(39), 307 (39), 308(39), 543 Zemli~ka, J., 803 Zendzian, E. N., 857
1017
Zeppezauer, E. S., 605 Zeppezauer, M., 605 Zemer, B., 7, 9(3), 10, 221, 879, 892, 895 (23), 964 Zipper, H., 871, 900 Zollinger, H., 941 Zonderman, E., 163, 164(19), 979 Zendzian, E. N., 32, 33(4), 36 Zimmermann, H., 844 Zmrahl, Z., 68(c), 69 Zmrhal, Z., 69 Zuber, H., 544, 549(9), 550
1018
SUBJECT INDEX
Subject Index Agaricus bisporus, y-glutamyl transpeptidase from, 789 Absidia lichtheimi, milk-clotting enzyme AL-1 protease from Myxobacter, 591-599 from, 447 L-Alanine-p-nitroanilide, in aminopeptiN-Acetyl-L-alanine methyl ester, as dase assay, 514 elastase substrate, kinetics of, 137 Alcalase Novo, see Subtihsin Carlsberg a-N-Acetyl ,S-(fl-aminoethyl) cysteine Aldehyde reagents, as papain inaetivaethyl ester, as trypsin substrate, 62 tors, 236 N-Acetyl-L-diiodotyrosine, in pepsin Alkaline proteases, from Aspcrgillus spp., assay, 407-408 581-591 N-Acetylglycine ethyl ester, as trypsin Alternaria tenuissima, alkaline proteinsubstrate, 62 ase from, 591 a-N-Acetyl lysine ethyl ester, as trypsin 4-Amidinophenyl-pyruvic acid, as thromsubstrate, 62 b i n inhibitor, 184 Aeetyl-L-phenylalanyl-L-diiodotyrosine, 'Amino acids in pepsin assay, 316, 319-320 amino-terminal, enzymes releasing, N-Acetyl-b-tryptophan amide, in assay 508-569 of chymotrypsins A, 74, 75 carboxy-terminal, enzymes specific for N-Acetyl-L-tryptophan methyl ester, inreleasing, 461-508 activity of sulfonyl a-chymotrypsin L-a-Amino acids, derivatives of, hydrolto, 7, 8 ysis by aminopeptidase, 519 N-Acetyl-L-tyrosine ethyl ester (ATEE) Amino acyldipeptides, 534 in assay of chymotrypsins A, 73-74 in assay of chymotrypsinogens, 88, 92, Aminoacyloligopeptides, 534 p-Aminobenzamidine, as trypsin inhibi94-95, 100 tor, 61 as trypsin substrate, 62 7-Amino-l-chloro-3-toluene-p-sulfonaAchromobacter, pestifer, e4ysine acylase mide-2-butanone (TLCK), thrombin from, 756-762 reaction with, 183 Acidic proteases, 316-460 N-(5-Aminopentyl)-5-dimethylamino-1Endothia parasitica protease, 436-445 naphthalene sulfonamide, in assay gastricin and pepsin, 406-421 of lobster muscle transpeptidase, microbial acid proteinases, 372-397 765-770 of Mucor miehi, 459-460 Aminopeptidase, 534. (,See also Leucine mucor rennin, 446-459 aminopeptidase.) pepsins and pepsinogens, 316-372 activators and inactivators of, 517-518 prochymosin and chymosin, 421-436 amino acid composition of, 520 Activators of proteolytic enzymes, 805amino acid inhibition of, 518-519 932 assay of, 514 Active site titration chelating agents, 518 distribution of, 520 of proteolytic enzymes, 3-20 isolation of, 515~16 of trypsin, 44, 61 kinetic properties of, 520 e-N-Acyl-I,-lysine amidohydrolase, see from pig kidney, 514-521 •-Lysine acylase purification of, 514-515 Acyl-Lovaline esters, in chymotrypsin assay, 75 purity, and physical properties of, 517 A
SUBJECT INDEX
specificity of, 519 for leucylglycylglycine hydrolysis, 534543 activators and inhibitors of, 543 amino acid composition of, 540 assay methods for, 534-539 homogeneity and M. W. of, 539-540 microassay of, 540-541 purification of, 538-439 specificity of, 540 stability of, 540 thermophilic, from B. stearothermophilus, (API) 544-552 amino acid composition of, 551 assay of, 544-547 enzyme structure of, 550 kinetic properties of, 550, 562 purification of, 547-549 purity and physical properties of, 549-550 specificity of, 550 stability of, 550 thermophilic, from Talaromyces duponti, 552-555
assay of, 552 properties of, 554-555 purification of, 552-554 Aminopeptidase-P absorption spectrum of, 528 amino acid composition of, 528-529 assay of, 521-524 kinetic parameters of, 531-533 metal ion requirement for, 530 occurrence of, 533-534 pH profile of, 529 purification of, 524-527 purity and physical properties of, 528 sedimentation and diffusion coefficients of, 528 specificity of, 531 stability of, 527-528 p-Amino-DL-phenylalanineoleucine copolymer papain derivative of, 960 thrombin derivative of, 978-985 trypsin derivative of, 954 4- (4'-Aminophenylazo)phenylarsonic acid, as subtilisin inhibitor, 211 Aminotripeptidase, 534, 748 properties of, 756
1019
Anacystis montana, ribosomal peptidyl
transferase from, 803 Ananas comosus, see Pineapple Antherea pernyi, cocoonase from, 224-
226 Antherea polyphemus, cocoonase from,
222-224, 225 Anthrax bacillus capsule, T-glutamyl transpeptidase from, 789 Antihemophilic factor, thrombin and, 181 Antithrombin (s) action of, 156, 180 in plasma, see Plasma antithrombin AP I from Bacillus stearothermophilus, 5A.A. 552
from Talaromyces duponti, 522--555 Apocarboxypeptidase A, 495--496 Arthrobacter crystallopoietes, AL-1 protease hydrolysis of cell walls of, 591 Arthrobacter globi]ormis, in assay of Sorangium a-lytic protease, 600-601 Arthropoda, serine proteases in, 32 Ascaris lumbricoides proteolytic enzyme inhibitors, 872-883 chymotrypsin inhibitors, 872, 878-883 trypsin inhibitors, 872-878 Ascochyta vicae, milk-clotting enzyme from, 447 fl-Aspartylglycine, in assay of fl-aspartyl peptidase, 737-738 fl-Aspartylleucine, in fl-aspartyl peptidase assay, 730-732 fl-Aspartyl peptidase activators and inhibitors of, 735, 739741 assay methods for, 730-732, 737-738 distribution of, 736-737, 741 peptides hydrolyzed by, 740 pH optimum and buffer effects on, 735--736, 741 physical properties and purity of, 735 properties of, 735-737, 739-741 purification of, 732-735 from rat liver, 738-739 purity of, 739 specificity of, 735, 739 stability of, 735, 739
1~0
S~J~TIND~
substrates for, 736 substrate affinity of, 736 Aspergillopeptidase, assay and properties of, 617 Aspergillopeptidase A, 373, 386 activators and inactivators of, 405 amino acid composition of, 405 assay of, 397-399 distribution of, 406 nomenclature of, 373, 375 physical properties of, 404-405 purification of, 399-403 specificity of, 405-406 stability of, 403-404 trypsinogen activation by, 374-375 Aspergillopeptidase B definition of, 376 purification of, 586-588 Aspergillopeptidase C amino acid composition of, 589 inhibitor of, 590 molecular weight, 588 Aspergillus spp. alkaline proteinases from 581-591 activator and inhibitor, 589 amino acid composition of, 589 assay of, 582-583 chemical properties of, 589 distribution of, 591 purification of, 583-586 milk-clotting enzyme from, 447 trypsinogen-activating enzymes from, 374 Aspergillus flavus alkaline proteinase from, 582, 588, 591 amino acid composition from, 589 Aspergillus ]umigatus, alkaline proteinase from, 591 Aspergillus niger var. macrosporus, acid proteinases from, 386 Aspergillus oryzae alkaline proteinase from, 581-591 proteinase I I I from, 386 Takadiastase from, see Takadiatase Aspergillus saitoi acid proteinase, see Aspergillopeptidase A culture of, 399-401
Aspergillus sojae alkaline proteinase from, 582, 586, 588, 591 amino acid composition of, 589 Aspergillus sydowi, alkaline proteinase from, 582, 590, 591 Autoanalyzer, in aminopeptidase assay, 536-537, 541-542 Azocoll, m assay of AL-1 protease, 592593 AzoelastilL, in elastase assay, 113 B
Bacillus spp. milk-clotting enzyme from, 447 Bacillus amyloliqui]aciens neutral protease from, 575 subtilisin from, 204 Bacillus licheni]ormis, serine protease from, 201 Bacillus pumilis, serine protease from, 201 Bacillus stearothermophilus, AP I from, 544-552 Bacillus subtilis y-glutamyl transpeptidase from, 789 neutral protease from, 569-575 amino acid composition of, 574 assay of, 569-570 distribution of, 575 inhibitor specificity of, 573-575 metal derivatives of, 573 physicochemical properties of, 575 purification of, 570-572 purity of, 573 specificity of, 573 stability of, 575 pyrrolidonecarboxylyl peptidase from, 555-569 ribosomal peptidyl transferase from, 8O3 serine proteases from, see Subtilisins Bacillus subtilis var. amylosaccariticus, serine protease from, 201, 215 Bacteria ~/-glutamyl transpeptidases from, 789 serine proteases in, 32 , Bacterial acid proteinases, 372 Bacterial proteases, 569-613
s~J~.cT iNv~x
Bacterial proteinase Novo, see Subtilisin Novo Bacteroides melaninogenicus, collagenase from, properties, 616 Baker's yeast, proteinase A from, 372 Barley trypsin inhibitor, 840-844 amino acid composition of, 843, 844 assay of, 840-841 distribution of, 844 properties of, 842-844 purification of, 841-842 Basic pancreatic trypsin inhibitor, see Bovine trypsin-kallikrein inhibitor Benzamidine, as trypsin inhibitor, 61 N-Benzoyl-v-alanine methyl ester, as elastase substrate, kinetics of, 137 N-Benzoyl-L-alanine methyl ester, in assay, of elastase, 118-119 kinetics of, 135-137 Benzoyl-L-arginine in cathepsin B assay, 289-290 as trypsin inhibitor, 61 a-N-Benzoyl-L-arginine amide (BAA) in bromelain assay, 275-276, 283 as cathepsin B substrate, 285, 300-301 in assay methods, 288-289, 290-291 in papain assay, 228-229, 241,242 as trypsin substrate, 62 in assay methods, 43-44 a-N-Benzoyl-L-arginine ethyl ester (BAEE) in bromelain assay, 275, 283 in cellulose-insolubilized enzyme assay, 966-967 in cocoonase assay, 225 in ficin assay, 262 in papain assay, 227-228 reaction with trypsin derivatives, 50 as trypsin substrate, 62 in trypsin assay, 37-38, 42-43 a-N-Benzoyl-DL-arginine p-nitroanilide (BANA) in bromelain assay, 276 in ficin assay, 262 in papain assay, 229 reaction with trypsin derivatives, 50 as trypsin substrate, 62 N-a-Benzoyl-L-citrulline methyl ester, as trypsin substrate, 62
1021
N-Benzoyl-glycine methyl ester, as elastase substrate, kinetics, 137 N-Benzoyl-L-heptyline methyl ester, as trypsin substrate, 62 N-Benzoyl-L-leucine ethyl ester (BLEE), in assay of chymotrypsin C, 97, 109 of chymotrypsinogen C, 94-95 ~-N-Benzoyl-L-lysine, in ~-lysine acylase assay, 756-757 N-Benzoyl-L-tyrosine ethyl ester, in assay of chymotrypsin, 36, 38-39 Benzylamine, as trypsin inhibitor, 61 N-Benzylcarbonyl-a-L-glutamyl-iphenylalanine, as catheptic carboxypeptidase substrate, 285 N-Benzylcarbonyl-a-L-glutamyl-i-, tyrosine in assay of cathepsins, 291-292 as cathepsin A substrate, 285, 292, 295296 N-~-Benzyloxycarbonyl-L-arginine p-toluidide, as trypsin substrate, 62 Benzyloxycarbonylphenylalanine bromomethyl ketone (ZPBK), as subtilisin inhibitor, 210 N-Benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester in spectrophotometric assay of chymotrypsin, 39-41, 66 synthesis of, 39-40 4,4'-Biphenylenedisulfonyl chloride, as subtilisin inhibitor, 210 Bisdiazobenzidine-2,2'-disulfonic acid, papain derivative of, 960 Black-eyed pea trypsin inhibitor, 863 amino acid composition of, 868-869 properties of, 866, 867, 870-871 Blood coagulation, see Clotting Bombyx mori, cocoonase from, 224, 225 Bovine trypsin-kallikrein inhibitor, 844852 amino acid composition and sequence of, 851-852 assay method for, 845 distribution of. 851 kinetic properties of, 850-851 physical properties of, 849 properties of, 848-852, 913 purification of, 845-848
1022
SUBJECT INDEX
purity of, 849 specificity of, 849-850 stability of, 848-849 Bowman-Birk soybean inhibitor, 860-862 amino acid composition of, 862 properties of, 861-862 purification of, 860-861 BPN', see Subtilisin BPN' Brain aminopeptidase from, 534-543 Bromelain enzymes, 273-284 CM-cellulose derivative of preparation of, 970-971 properties, 971-972 from fruit, see Fruit bromelain from stem, see Stem.bromelain Bromelin, see Fruit bromelain p-Bromophenaeyl bromide, as pepsin inactivator, 333 m-Butylamine, as trypsin inhibitor, 61 B y s s o c h l a m y s ]ulva, milk-clotting enzyme from, 447 C Calcium ions, role in milk clotting, 457458 C a n d i d a albicans acid proteinase, 373 assay of, 387 purification and properties of, 386-389 N-Carbobenzoxy-L-alanine p-nitropbenyl ester, in assay of elastase, 119, 136, 137 Carbobenzoxy-L-glutamic acid, as papain inactivators, 236 N-Carbobenzoxy glycine p-nitrophenyl ester, reaction with elastin, 120, 134135 kinetics of, 137 N-Carbobenzoxy-L-isoleucine p-nitrophenyl ester, as elastase substrate, kinetics of, 137 N-Carbobenzoxy-L-norleucine p-nitrophenyl ester, as elastase substrate, kinetics of, 137 Carbobenzoxy-L-tyrosine-p-nitrophenyl ester, as thrombin substrate, 183 N-Carbobenzoxy-L-valine p-nitrophenyl ester, as elastase substrate, kinetics of, 137
Carboxymethyl cellulose chymotrypsin derivative of, 950 trypsin derivative of, 954 Carboxypeptidase A active site of, 502 assay methods for, 475-478 esterase activity, 477-478 peptidase activity, 475-477 chemical and crystal structure of, 491493 chemical modifications of, 496-498 esterase activity, kinetics, 502 forms of, kinetic parameters of, 501 inhibitors and activators of, 493-496 metal chelators, 495--496 structural organic type, 493-495 peptidase activity of, kinetic, 499 physical properties of, 489-491 preparation and purification of, 478487 carboxypeptidase A~, 484 carboxypeptidase Ap, 481 purity of, 489 solubility of, 486-487 stability of, 487-488 substrate specificity and mechanism of action, 498-503 Carboxypeptidase B, 504-508 activators and inhibitors of, 598 amino acid composition of, 507 assay of, 504 distribution of, 508 purification of, 505-507 purity and physicochemical properties of, 507 specificity of, 507-508 stability of, 507 Cariva p a p a y a
chymopapain from, 243 lysozyme from, 243 papain from, 226, 243 Casein chymopapain B assay, 245 in ficin assay, 261-262, 274-275 in papain assay, 226-227 in plasminogen assay, 184 in streptococcal proteinase assay, 262 in trypsin assay, 43 Cassowary, ovomucoid from, 898, 902
SUBJECT INDEX Cathepsins, 285-315 assay methods for, 286-292 for amidase activity, 288-289 for proteinase activity, 286-287 Cathepsin A, 292-296 activators and inhibitors of, 295 distribution of, 296 hydrolase assay of, 291-292 kinetic properties of, 296 proteinase assay of, 286 purification of, 292 specificity of, 285, 295 stability of, 295 Cathepsin B activators a n d inhibitors of, 300 amidase assay of, 288-289 comparison to papain, 300 distribution of, 301-302 esterase assay of, 289-290 kinetic properties of, 301 hydrolase assay of, 291-292 proteinase assay of, 286 purification of, 297-299 purity and physical properties of, 299300 specificity of, 285, 300-301 stability of, 299 transferase assay of, 290-291 Cathepsin C (dipeptidyl transferase), 302-309 activators and inhibitors of, 306 amidase assay of, 288-289 distribution of, 309 esterase assay of, 289-290 kinetic properties of, 309 hydrolase assay of, 291-292 physical properties and purity of, 305306 purification of, 303-305 specificity of, 285, 306-309 stability of, 305 transferase assay of, 290-291 Cathepsin D, 285, 309-313 activators and inhibitors of, 31 distribution of 312-313 hydrolase assay of, 291-292 kinetic properties of, 312 proteinase assay of, 286 purification of, 293-295, 309-311 purity and physical properties of, 311
1023
specificity of, 312 stability of, 311 Cathepsin E, 285, 313-315 activators and inhibitors of, 314 distribution of, 315 hydrolase assay of, 291-292 proteinase assay of, 286 purification of, 313 purity and physical properties of, 314 stability of, 314 specificity of, 314-315 Catheptic carboxypeptidase, 315 specificity of, 285 Cellulose-insolubilized enzymes, 963-978 assay methods for, 963-967 by amino acid analysis, 964-967 by protein content, 963-964 preparation of, 967-971 properties of, 971-978 Chaetomium brasiUieuse, milk-clotting enzyme from, 447 Cheese, acid proteinases' potential use to make, 375 Cheese rennet powder, chymosin purification of, 427-430 Chicken chymotrypsin from, 105-106 ovoinhibitor from, properties and pur.ification of, 903-906 ovomucoid from, properties and purification of, 892-895 pepsinogens and pepsin from, 347-363 Chloromethyl ketones, in test for serine proteases, 32, 36 1-Chloro-3-tosylamido-7-amino-2heptanone (TLCK) as active site titrant for trypsin, 61 as cocoonase inhibitor, 225 as trypsin derivative inhibitors, 50 Ch olecystokinin-pancreozymin, thrombin proteolysis of, 181 Chymopapain, comparison to papain, 243-244 Chymopapain A, physicochemical properties of, 250 Chymopapain B, 244-252 activation and inhibition of, 251 amino acid composition of, 250 assay method for, 244-245 homogeneity of, 249
1~4
S~J~TIND~
pH optimum of, 251 physicochemical properties of, 249 purification procedure for, 246-249 specificity of, 249-251 stability of, 251-252 Chymosin, 421-436 amino acid composition of, 431, 434, 456 assay of, 422--424 comparison with pepsin, 434, 435-436 molecular weights and structure of, 432-435 purification of, 427-430 Chymotrypsin(s), 64-103 autolyzed derivative of, 34 from chicken, 105-106 conformation related to elastase, 58 enzyme like, from hornet, 107 forms of, 64 inhibitor of, 131. (see also Chymotrypsin inhibitors) preparation of, 105, 106 properties of, 105-106 removal from trypsin, 57 from salmon, 106 water-insoluble derivatives of, 955--956 EMA-chymotrypsin, 955--956 miscellaneous types, 956 a-Chym0trypsin active site titration of, 3, 6-20 by 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone, 6-14 by p-nitrophenyl ester substrates, 14-20 by rapidly reversible, covalently bound substrates, 14-20 by slowly reversible, covalently bound inhibitors, 6-14 CM-cellulose derivative of, preparation and properties of, 970-978 elastase and, 129 Serl~ as site of sulfonylation, 7 stock solution preparation, 11 Chymotrypsins A assay methods for, 73-75 N-acetyl-L-tyrosine ethyl ester, 73 Caffi+effects on, 80 calcium in assay of, 75 from dogfish, 102-103 heavy metal inhibitors of, 80
N-terminal analyses of, 76 physical and chemical properties of, 79-80, 104 preparation and purification of, 102104 from sheep, 103-104 specific activity of, 73-74, 103 specificity of, 88 stability of, 79-80 Chymotrypsin Ao formation of, 64, 65 N-terminal analysis of, 76 physical properties of, 80 purification of, 75, 77 specific activity of, 74 spectrophotometric assay of, 37-40, 43 stability of, 79-80 Chymotrypsin A~ Ca z* effects on, 80 formation of, 64 purification of, 65, 66 Chymotrypsin Aj Ca s+ effects on, 80 formation of, 64 N-terminal analysis of, 76 phenylmethanesulfonyl derivative of, 78-79 purification of, 66 specific activity of, 74 trypsin-free, preparation of, 77-78 Chymotrypsin A~ phenylmethanesulfonyl derivative of, 79 purification of, 66 specific activity of, 74 Chymotrypsins B, 81--88 from fin whale, 104-105 specificity characterization of, 36, 8788 stability of, 87 Chymotrypsin B., 86 definition of, 81 formation of, 81 partial isolation of, 87 stability of, 87 Chymotrypsin B~, 81 crystallization and properties of, 86 stability of, 87 Chymotrypsin C activators and inhibitors of, 112
SUBJECT INDEX
amino acid composition of, 112 amino acid sequence of, 99 assay of, 97, 109 esteroproteolytic enzyme as, 91, 99, 112 molecular weight of, 98-99 from pig, 90-94, 97-100, 109-112 purification of, 97-98, 109-112 reaction with N-tosyl-L-phenylalanine chloromethyl ketone, 99-100 specificity of, 99-100, 112 stability of, 98, 111 Chymotrypsin inhibitors, 870 from Ascaris lumbricoides, 872, 878-883 from egg white, 892, 903-906 from potatoes, 883--889 Chymotrypsin inhibitor I, as subtilisin inhibitor, 211 Chymotrypsinogens, 64-108 activation of, 49 chain breaks in, 35 thrombin proteolysis of, 156 Chymotrypsinogens A activation of, 90, 102 amino acid composition of, 67-69, 102 amino acid sequence of, 67-71, 89 from various species, 90 assay of, 88, 100 calcium binding by, 72-73 chemical and physical properties of, 67-73 chromatographic purification of, 66-67 conversion to chymotrypsin A, 64 from dogfish, 100-102 electrophoresis of, 102 end group tests on, 66 from porcine pancreas, 88-90 physical properties of, 89-90 purification of, 88-89, 100-192 purity of, 65-66, 89 test for, 66 Chymotrypsinogen B, 81-88 activation of, 81, 87, 94 amino acid composition and sequences of, 68-71, 86, 94 assay of, 82-83, 92-94 Ca 2÷ binding by, 86 deoxyribonuclease in, estimation, 8384 isolation of, 81-82
1025
molecular weight of, 94 physical properties of, 72, 86 from pig, 90-94 purification of, 84-86, 92-94 purity of, 85--86, 94 specific activity of, 87 Chymotrypsinogen C activation of, 96-97 amino acid composition of, 96 assay of, 94-95 molecular weight, 96 from pig, 90--97 purification of, 95--96 purity of, 96 Cinnamoyl imidazole as a-chymotrypsin titrant comparison with nitrophenyl esters, 19 comparison with sultone titrant, 9-10 inactivity of sulfonyl a-chymotrypsin to, 7 as subtilisin titrant, 205 as thiosubtilisin substrate, 219, 220 Clostridia spp., acid proteinase of, 372 Clostridiopeptidase, see Clostripain Clostridiopeptidase A, see Collagenase A Clostridium histolyticum
clostripain from, 635, 637-638 collagenases from, 616, 620, 625-626 culture of, 626 Clostripain, 635-642 esterase and amidase activities of, 641 pH optimum of, 639 physical properties of, 639-640 properties of, 639-642 purification of, 637-638 standard assay of, 636--637 substrate specificity of, 640-642 sulfhydryl requirement of, 639 Clotting clot retraction in, 181 fibrinoligase in, 771-772 physical evidence of, 147 in thrombin assay, 145-157, 170-172 thrombin-dependent reactions in, 181 Coagulin, see Lobster muscle transpeptidase Cocoonases, 221-226 from A. polyphemus, 222-224 amino acid composition of, 52, 225
1026
SUBJECT INDEX
assay with BAEE and NPGB, 225 from B. mori, 224 function of, 221 inhibitors of, 225 properties of, 224-226 Coelenterata, serine proteases in, 32 Collagenases, 613-635 active center peptides of, 633-634 amino acid composition of, 630-631 assay of, 622-623 by release of glycine-containing peptides, 623 by viscosity, 623 binding to collagen, 634 from C. histolyticum, 616, 620, 625-626 calcium ions in function of, 631 cobalt and manganese effects on, 633 cysteine inhibition of, 631-632 definition of, 613-614 functions of, 613 inhibitors of, 631-633 kinetics of action, 635 list of, 616-618 preparation of, 620-622 properties of, 624-625 specificities of, 614 from tadpole, 616, 621-625 tissue culture method for, 620-622 Collagenase A amino acid composition of, 630-631 assay and properties of, 616 physical properties of, 628-629 possible of, subunits of, 629--630 preparation and purification of, 626628 specificity of, 634--635 Collagenase B amino acid composition of, 630-631 assay and properties of, 616 physical properties of, 628--629 preparation and purification of, 626628 specificity of, 634-635 CoUetorichum atramentarium, milk-clotting enzyme from, 447 Colletorichum lindenruthianum, milkclotting enzyme from, 447 Congo red-elastin, in elastase assay, 113115
Connective tissue, peptide hydrolases from, 741-756 Corn seed trypsin inhibitor, amino acid composition of, 843 Corynebacterium hoagi, milk-clotting enzyme from, 447 Cow chymotrypsins from, 64-88 ehymotrypsinogens from, 64-80 pepsins and pepsinogen from, 337--347 prothrombin from, 157-170 thrombin from, 170-184 trypsin amino acid composition of, 52 purification of, 49-51 Cyclohexylcarboxamidine, as trypsin inhibitor, 61 Cysteine as chymopapain activator, 251 as collagenase inhibitor, 631-632 as papain activator, 235 Cysteine proteases, 226-284 bromelain enzymes, 273-284 chymopapain B, 244-252 ficin, 261-273 papain, 226-244 streptococcal proteinase, 252-261 Cytokinase, 834-838 activators and inactivators of, 837 assay method for, 834-835 distribution of, 838 kinetic properties, 837-838 physical properties and purity of, 837 properties of, 836-838 purification of, 835-836 specificity of, 837 stability of, 836-837 O Dansyl chloride, as subtilisin inhibitor, 210 Deoxyribonuclease, in chymotrypsinogen B samples, estimation of, 83 Detergent, subtilisin use in, 201 Diazoacetyl amino acid esters, as pepsin inhibitors, 334, 345 a-Diazo-p-bromoacetophenone, as pepsin inactivator, 333 Diazonium-l-H-tetrazole, inactivation of aminopeptidase by, 520-521
SUBJECT INDEX 1,3-Dibromoacetone, in active site binding to papain, 240 Diisopropylphosphorofluoridate (DFP) as active site titrant, for trypsin, 61 as cocoonase inhibitor, 225 as elastase inhibitor, 131 as subtilisin inhibitor, 210 in test for serine proteases, 31, 36 as thrombin inactivator, 156, 180 as trypsin derivative inhibitor, 50 use in chymotrypsinogen analysis, 66 2,3-Dimercaptopropanol as cathepsin C inhibitor, 306 as chymopapain activator, 251 p-Dinitrophenol diethyl phosphate, as ' elastase inhibitor, 131 Dipeptides, hydrolysis by gastricsin and pepsin, 419 Dipeptidyl transferase, see Cathepsin C Diphenyldiazomethane, as pepsin inactivator, 333 Dithiothreitol, as cathepsin C inhibitor, 3O6 Dogfish chymotrypsin A from, 100-102 chymotrypsinogen amino acids of, 6869 chymotrypsinogen A from, 100-102 pepsinogens from, 364-372 trypsin amino acids of, 52 zymogens in, 33, 34 Double bean trypsin inhibitor, properties of, 870 Duck, ovomucoid from, 898, 902 E Echlnodermata, serine proteases in, 32 Egg white proteinase inhibitors chymotrypsin inhibition, 892 subtilisin inhibition, 211 trypsin inhibition, 890, 891-892 Elastase, 113-140 activators and inactivators of, 131-132 active center sequence of, 133 amino acid composition of, 128 amino acid sequences of, 130, 133 assay methods for, 113-120 Congo red-elastin procedure, 113-115 with synthetic substrates, 117-120 a-chymotrypsin and, 129
1027
conformation related to chymotrypsin, 58, 133-134 distribution of, 139-140 enzymatic activity and substrate specificity of, 132-135 kinetic properties of, 135--139 N-terminal analysis, 127 physical properties of, 128, 131 purification of, 120-125 summary, 126 specific activity of, 115-117 stability of, 125, 127-128 TNBS inactivation of, 128 Elastin, in assay of elastase, 113 Elastolytic e n z y m e (non-elastase)/ in pig pancreas, 140 EMA-chymotrypsin, preparation and properties of, 955-956 EMA derivatives of enzymes, 945-946 copolymer preparation of, 948-949 EMA-papain, preparation and properties of, 957-959 EMA-subtilopeptidase A derivatives, preparation and properties of, 960961 EMA-trypsin, preparation and properties of, 950-952 Emu, ovomucoid from, 898, 902 Endothia parasitica protease, 373, 447 activators and inhibitors of, 444 amino acid composition of, 444 assay of, 436-439 distribution of, 445 physical and chemical properties of, 442-446 potential use in cheese making, 375 production and purification of, 439-442 specificity of, 444-445 stability of, 442 Enterokinase, in activation of zymogens, 42 Enzyme, artificial, see Thiosubtilisin Escherichia coli
-~-glutamyl transpeptidase from, 789 ribosomal peptidyl transferase from, 797-803 Esterolytic assays, of serine proteases, 36 "Esteroproteolytic enzyme," as chymotrypsin C, 91, 99, 112
1028
SUBJECT INDEX
Ethylene-maleic acid (EMA) copolymer derivatives of enzymes, 945-946
Ethyl-p-guanidinobenzoate as active site titrant for trypsin, 61 reaction with thrombin, kinetics, 183 Ethyl-p-guanidinophenylacetate, as trypsin substrate, 62 Euglobulins precipitation for prothrombin isolation, 164 preparation for elastase isolation, 124 E v a s t e r i a s ~rochelii, see Starfish F
Factor V, see Proaccelerin Factor VIII, see Antihemophilic factor Factor X=, in proteolysis of prothrombin, 169-170 Factor XIII, see Plasma transglutaminase Fibrin stabilizing factor (factor XHI), purification of, 778--780 Fibrinogen assay of, 149-150 in assay of tissue plasminogen activator, ~4-8~5 standardization_ of, 152 stock solution of, 149 as thrombin substrste, 180-181 Fibrinoligase (fibrin stabilizing factor system), 770-782 action of, 770-771 assay of, 772-778 by amine incorporation, 776-778 by clot stability, 774-776 conversion of zymogen to fibrinoligase enzyme, 782 properties of, 780-781 purification procedure for, 778-780 Fibrinolysin, definition of, 807 Fibrinopeptidcs, formation of, 180, 181 Ficin, 261-273 activators and inhibitors of, 268, 270271 active site of, 271 amino acid composition of, 270 assay of, 261-263 CM-cellulose derivative of, preparation and properties of, 970-978 distribution of, 272-273
inhibitor from egg white, 891 pH effects on activity of, 268 physical properties of, 268 purification of, 264-265 purity of, 268 specificity of, 272 stability of, 265--268 synthetic substrates for, 261-262 F/cus spp., ficin distribution in, 272-273 Field bean trypsin inhibitor, 863 properties of, 865, 866, 867, 870-871 Fig, ficin isolation from, 264 Fin whale, see Whale Formylleucine hydrolyzing enzyme, 748 properties of, 756 French bean trypsin inhibitor, 863 Fruit bromelain amino acid composition of, 280 assay of, 283 properties of, 284 purification of, 283-284 Fungal acid proteinases, 373-374 N-3- (2-Furylacryloyl)-~alanine methyl ester, hydrolysis by elastase, kinetics, 135, 137 G Gastricsin activators and inactivators for, 418 amino acid composition of, 420 assay methods for (with pepsin), 4{}6410 chemical composition of, 421 dipeptide hydrolysis by, 419 distribution of, 421 enzymatic properties of, 416 kinetic properties of, 420--421 physical properties of, 418 purification of, 410-417 from pig, 414-417 specificity of, 418-426 stability of, 416--418 G l i o c l a d i u m r o s e u m , alkaline proteinase from, 591 Glucagon, in chymotrypsin specificity tests, 87-88 7-Glutamyl cyclotransferase, 789-797 amino acid analysis of, 796 assay of, 790-793 different forms of, 796
S~J~TIND~
distribution of, 796-797 function of, 789 pH dependence of, 795 physical and chemical properties of, 795 preparation of, 793-794, 795 properties of, 794-796 specificity of, 794-795 stability of, 795 "/-L-Glutamyl-T-L-glutamyl-a-naphthylamide in assay of T-glutamyl cyotransferase, 790-793 synthesis of, 791 T-Glutamyl lactamase, see T-Glutamyl cyclotransferase T-L-Glutamyl-p-nitroanilide in assay of "/-glutamyl transpeptidase, 782-784 synthesis of, 783 -/-Glutamyl transpeptidase, 782-789 activators and inhibitors for, 787 assay of, 782-784 distribution of, 788-789 function of, 782 pH dependence of, 787 physical and chemical properties of, 787-788 preparation of, 784-785 properties of, 785-788 specificity of 785-786 stability of, 787 Glutaraldehyde subtilopeptidase A derivative of, 962 trypsin cross-linking with, 954-955 Glutathione, as chymopapain activator, 251 Glycylalanine dipeptidase, properties of, 756 Glycyldehydrophenylalanine as substrate for renal dipeptidase assay, 722-724 synthesis of, 723-724 Glycylglycine dipeptidase, 748 properties of, 756 Glycylleucine dipeptidase, 748 Glycyl-L-phenylalaninamide, as cathepsin C substrate, 285 in assay methods, 288-289, 290-291
1~9
Glycyl-L-phenyalanyl ethyl ester, as cathepsin C substrate, 289-290 Green gram trypsin inhibitor, properties of, 870 p-Guanidinobenzoic acid HC1, synthesis of, 22-23 Guar trypsin inhibitor, properties of, 865 H Hemoglobin in bovine pepsin assay, 337-339, 406407 in bromelain assay, 276 in cathepsin assay, 287 in chicken pepsin assay, 348--350 in pepsin assay, 316, 317-319 Heparin, reaction with thrombin, 156, 180 Heparin cofactor, antithrombin I I I and, 156, 180 Hippurylamide, in ficin assay, 262 Hippuryl-L-leucinamide, in assay of B. sub$ilis protease Hirudin assay methods for, 927-928 in coagulation studies, 928 isolation of, 929-931 mode of action of, 925-926 purity criteria for, 931-932 terminal amino acid sequence of, 924 as thrombin inhibitor, 156, 180, 924-932 Histidine, as collagenase inhibitor, 632 Hornet protease, 32 preparation and properties of, 107 Horse, trypsin, amino acid composition of, 52 Horseshoe crab, trypsin, amino acid composition of, 52 Hydrocinnamate, as subtilisin inhibitor, 211 o-Hydroxybenzoic acid fatty acid esters, in chymotrypsin assay, 75 Hydroxylamine, as papain inactivator, 236 2-Hydroxy-5-nitro-~-toluenesulfonic acid sultone as a-chymotrypsin active site titrant, 6-26 advantages, 9-10 comparison with other titrants, 9-10
1~0
S~J~TIND~
kinetic aspects 8-9 site of reaction, 7-8 synthesis for, 13-14 titration procedure, 11-13 synthesis of, 14 2-Hydroxy-~-toluenesulfonic acid sultone, synthesis of, 13-14 I
Imidazole, as collagenase inhibitor, 632633 Imidopeptidase, 748 properties of, 756 Imino Dipeptase, 748 properties of, 756 Indole, as subtilisin inhibitor, 211 Inhibitors of proteolytic enzYmes, 839932
from Ascaris lumbricoides, 872-883 from egg white, 890-892 trypsin inhibitors, 839, 840-883 Insulin ehymopapain hydrolysis of, 251 fiein hydrolysis of, 272 Iodine, inactivation of aminopeptidase by, 521 K
Kallikreins (glandular), 681-699 activators and inhibitors of, 698 active center, 699 assay methods for, 682-688 chemical composition of, 699 distribution of, 699 kinetics of, 698--699 physical properties of, 697-698 purification of, 688-696 specificity of, 698 stability of, 697 Kidney, swine leueine aminopeptidase from, 508-513 particle-bound aminopeptidase from, 514--521 Kidney bean, ],-glutamyl transpeptidase from, 788-789 Kidney bean trypsin inhibitor, 883 properties of, 865, 866, 867, 870
Kunitz soybean inhibitor, 854--859. (See also Bovine trypsin-kallikrien inhibitor) amino acid composition of, 859 kinetic properties of, 857-858 physical properties and purity of, 856857 properties of, 856--859, 913 purification of, 854-856 specificity of, 857 stability of, 856 L Lactobacilli, acid proteinase from, 372 Legumes, trypsin inhibitors from, 862-871 Leucine amide, in leucine aminopeptidase assay, 508-509 Leucine aminopeptidase, 534 activators and inhibitors of, 754--755 assay of, 506-509, 748-750 molecular weight of, 755 pH optimum of, 755 properties of, 513 purification of, 509-513, 751-754 purity of, 755 specificity of, 754 stability of, 755 from swine kidney, 508--513 L-Leucine-p-nitroanilide in aminopeptidase assay, 514 in API assay, 544-547 Leueylglycylglycine, aminopeptidase which hydrolyzes, 534-543 Lima bean trypsin inhibitor, 862-863 amino acid composition of, 868-869 assay of, 863 properties of, 865-871 purification of, 864-865 specificity of, 870--871 Lobster muscle transpeptidase, 765-770 assay methods for, 765-767 by clotting, 765--766 by monodansylcadaverine incorporation into a-casein, 766--767 ester hydrolysis by, 770 isoelectric point of, 769 molecular weight of, 769-770 purification procedure for, 767-769
S~J~TIND~ e-Lysine acylase from Aehromobacter pesti]er, 756-762 assay method for, 756-757 distribution of, 762 inhibitors and activators of, 760 kinetics of, 761-762 pH optimum, 760 physical properties and purity of, 760 properties of, 759-762 purification procedure for, 757-759, 760 specificity of, 761 stability of, 759 from various sources, 762 Lysozyme, from Carica papaya, 243 ~-Lysyl-p-nitroanilide, as substrate for trypsin, 62 a-Lytic protease of Myxobacterium, 599613
M Malayan pit .viper venom protease, 715719 assay method, 715-716 properties of, 717-719 purification of, 716-717 Mast cells, serine proteases in, 32 fl-Mercaptoethylamine, as cathepsin C inhibitor, 306 p-Mercuribenzoate (PMB), in assay of thiolsubtilisin, 216-217, 220 Mercuripapain, formation of, 232 N-Methyl-L-alanine methyl ester, in 8orangium a-lytic protease assay, 601 N'-Methyl-N°-(p-toluenesulfonyl)-Llysine fl-naphthyl ester, as trypsin titrant, 21, 61 Metridium protease A, purification and properties of, 107-108 Metridium senile, see Sea anemone Microbial acid proteinases, 372--460 nomenclature of, 375 Milk clotting, calcium ion role in, 457458 Milk-clotting enzyme microorganisms producing, 447 from Mucor pusillus var. Lindt, see Mueor rennin Milk-clotting method for pepsin assay, 338-339, 365
1~1
Mold proteases, 569-613 in activation of zymogens, 42 Monascus anka, milk-clotting enzyme from, 447 Mouse submaxillary gland proteases, 672-681 amino acid composition of, 681 assay of, 673-676 chemical properties of, 680-681 enzymatic properties of, 680 preparation of, 676-680 Mucor spp., milk-clotting enzyme from, 447 Mucor miehei acid protease, purification and properties of, 459-460 Mucor pusillus var. Lindt, mucor rennin from, 446-459 Mucor rennin, 373 active site of, 457--459 amino acid composition, 456 assay of, 448-450 cheese-making experiments with, 449 clotting activity of, 454-455 physical properties of, 454, 457 potential use in cheese-making, 375 purification and crystallization of, 450454 specificity of, 458-459 stability of, 451 Mung bean trypsin inhibitor, 863 amino acid composition of, 868-869 properties of, 866, 867, 870-871 Mushroom, T-glutamyl transpeptidase from, 789 Mustelus cani~, see Dogfish Mycobacterium tuberculosis, collagenase, properties of, 616 Myxobacter AL-1 protease, 591-599 amino acid composition of, 598 assay of, 591-593 distribution of, 599 inactivators of, 597-59~ pH optimum of, 597 purification of, 593-595 purity and physical properties of, 595597 specificity of, 598-599 stability of, 597 Myxobacterium, a-lytic protease of, 599613
1~2
S~J~TIND~
N Nagarse, see Subtflisin BPN' Nagarse proteinase, see Subtilisin BPN' fl-Naphthamidine, as trypsin inhibitor, 61
Navy bean trypsin inhibitor, 863 amino acid composition of, 868-869 properties of, 866, 867, 870, 871 Neochymotrypsinogens, 35 Neochymotrypsinogens, B, formation of, 81 p-Nitroanilides, in aminopeptidase assay, 514-515 p-Nitrophenol diethyl phosphate (Paraoxon), in assay of elastase, 119 p-Nitrophenyl acetate (PNPA) in assay of thiolsubtilisin, 216-217, 219-221 synthesis of, 17 p-Nitrophenyl-N~-acetyl-Nl-benzylcar-
bazate, as trypsin titrant, 21, 61 p-Nitrophenyl N-acetyl-vL-trytophenate, as a-chymotrypsin titrant, 17 p-Nitrophenyl-p'-amidinobenzoate" HC1, as trypsin titrant, 21 p-Nitrophenyl N-benzyloxycarbonyl-Llysinate, as trypsin titrant, 21, 61 p-Nitrophenyl N-benzyloxycarbonyl-Ltyrosinate, as a-chymotrysin titrant, 17, 19 p-Nitrophenyl carbobenzoxyglycinate, in ficin assay, 261, 262-263 p-Nitrophenyl carbobenzoxy tyrosine (CTNP), in papain assay, 227 p-Nitrophenyl esters as active site titrants of ~-chymotrypsin, 14-20, 36 experimental methods, 17-20 limitations of, 20 in assay of esters, 119-120 elastase hydrolysis of, 136, 137 thiosubtilisin reaction with, 215 as thrombin substrates, 182-183 p-Nitrophenyl ethyl diazomalonate, as active site titrant, of trypsin, 61 p-Nitrophenyl-m'-guanidinobenzoate (m-NPGB), thrombin-reaction kinetics of, 183
p-Nitrophenyl p'-guanidinobenzoate HCI (p-NPGB) as active site titrant, 20-27 accuracy and specificity, 27 cocoonases, 225 method, 23-25 for trypsin, 20-27, 44, 60 reaction with thrombin, 171, 183 synthesis of, 22-23 as trypsin derivative inhibitor, 50 p-Nitrophenyl trimethyl acetate in elastase assay, 120 kinetics of hydrolysis, 138-139 synthesis of, 17 O Oncorhynchus tshawytscha, see Salmon
Orcein-elastin, in elastase assay, 113, 114 Ostrich, ovomucoid from, 898, 902 Ovoinhibitor, properties and purification of, 903-906 Ovomucoid(s) amino acid composition of, 901-902 from chicken, 892-902 from miscellaneous fowl, 898, 902 p Paecilomyces varioti acid proteinase, 373
assay of, 389 purification and properties of, 389-391 Pancreas serine protease isolation from, 33 trypsinogen purification of, 44-47 of various species, elastase in, 139--140 Pancreatic juice, enzymes in, trypsinogen activation and, 42 Pancreatic secretory trypsin inhibitors, 906-914 assay method for, 907-908 properties of, 912-914 purification procedures, 908-912 reactive site of, 914 Papain, 226-244 activation of, 235 active site of, 240-241, 243-244 size of, 241 amino acid sequence of, 238 assay methods for, 226-230 amidase activity, 228-229 esterase activity, 227-228
SUBJECT INDEX method comparison, 229-230 proteolytic activity, 226-227 biological properties of, 341-242 chymopapain compared to, 243-244 CM-cellulose derivative of, preparation of 970-978 enzymatic specificity of, 241-242 immunological properties of, 242 inactivation of, 235-236 inhibitor from egg white, 891 kinetic properties of, 242-243 membrane from, 960 mercuripapain from, 232, 956-957 physical properties of, 237, 239-241 properties of, 234-244 purification of, 230-234 for X-ray studies, 232-233 purity of, 233-234 stability of, 234-235 structure and conformation of, 239-240 water-insoluble derivatives of, 956-960 EMA-papain, 957-959 S-MDA-mercuripapain conjugate, 959-960 Paraoxon, see p-Nitrophenol diethyl phosphate Parapepsin I, see Pepsin B Parapepsin II, see Pepsin C Particle-bound azninopeptidase, 514-521 Peanut trypsin inhibitor, 863 amino acid composition of, 868-869 properties of, 866, 867, 870 Penguin, ovomucoid from, 898, 902 Penicillia, trypsinogen-activating enzymes in, 374 PeniciUium cyaneo-]ulvum, alkaline proteinase from, 591 Penicillium janthinellum, cultivation of, 379-380 Penicillium notatum, extracellular proteinase from 373, 576-581 assay method, 576-577 properties of, 579-581 purification of, 577-579, 580 Penicillium peptidase B, definition of, 376 Penicillo carboxypeptidase from Penicillium janthinellum
assay of, 384-385
1033
nomenclature, 373 properties of, 385 Penicillopepsin from Penicillium janthinellum
amino acid composition of, 382 assay methods, 376-379 enzymatic properties of, 382-384 molecular properties, 381-382 nomenclature, 373, 375-376 pepsin compared to, 375 purification of, 379-381 stability of, 381 trypsinogen activation by, 374-375, 376-377 Pepsins activators and inactivators of, 333-334 amino acid composition and sequences of, 331-333, 420 assay methods for, 316-321 in presence of gastricsin, 407-410 using hemoglobin, 4{}6-407 from chicken, 347-358 amino acid composition, 357 assay, 347-351 preparation, 352, 356-357 properties, 357-358 chymosin compared to, 435-436 from cow, 337-347 assay, 337-339 properties, 345 preparation, 344-345 dipeptide (synthetic) hydrolysis by, 419 distribution of, 336 pencillopepsin compared to, 375 from pig, 316-336 properties of, 329-336 purification of, 326-329 with gastricsin, 410-414 summary, 322 purity and physical properties of, 329331 specifity of, 334-336 Pepsin A (pepsin) amino acid composition and sequences of, 331-333 from chicken, 362, 363 definition of, 316 from dogfish, 371-372 inactivators of, 334
1034
SUBJECT INDEX
specificity of, 334-335 stability of, 329 Pepsin B assay of, 316, 319-320 from dogfish, 371-372 purification of, 326-327 purity and physical properties of, 329, 330 specificity of, 335 Pepsin C, 336 amino acid composition and sequences, 331-333 assay of, 316, 317-319, 320 from chicken, 362, 363 distribution of, 336 from dogfish, 371-372 inactivators of, 334 purification of, 327 purity and physical properties of, 329, 330 specificity of 335-336 stability of, 329 Pepsin D, 336 amino composition of, 333 assay of, 316, 317-321 from chicken, 362, 363 definition of, 316 from dogfish, 371-372 purification of, 327-328 purity and physicaI properties of, 329331 specificity of, 335 Pepsinogens amino acid composition and sequences of, 331-333 assay methods for, 316-321 from chicken, 347-363 amino acid composition, 357 isolation, 351-355, 358-361 properties, 357-358, 361-363 from cow, 337-347 amino acid composition, 347 properties, 345-347 purification, 339-344 from dogfish, 364-372 amino acid composition, 371 assay, 364-365 properties, 370-372 purification, 365-370 pepsin preparation from, 328--329
from pig, 316-336 purification of, 321-326 summary of, 322 Pepsinogen A amino acid composition and sequences of, 331-333 from chicken, 361, 362 from dogfish, 366-370 stability of, 329 Pepsinogen B from chicken, 363 chromatographic purification of, 324325 from dogfish, 369-370 purity and physical properties of, 329, 330 Pepsinogen C, 336 activation of, 334 amino acid composition and sequences, 331-333 from chicken, 361, 362 chromatographic purification of, 325326 from dogfish, 366-370 stability of, 329 purity and physical properties of, 329, 330 Pepsinogen D, 336 activation of, 334 amino acid composition of, 333 from chicken, 361, 362 chromatographic purification of, 326 from dogfish, 366-370 purity and physical properties of, 329331 stability of, 329 Peptidases, 722-762 fl-aspartyl peptidases, 730-741 e-lysine acylase from A. pesti]er, 756762 peptide hydrolases, 741-756 renal dipeptidase, 722-729 Peptidase A, see Penicillopepsin Peptides, for streptococcal proteinase assay, 252-253 Peptide hydrolases in connective tissue, 534, 741-756 hydrogen-ion activated peptide peptidohydrolases, 742-748 neutral type, 748-756
SUBJECT INDEX assay of, 748--750 individual enzyme properties, 754756 purification of, 751-754 Peptide peptidohydrolases (hydrogen-ion activated) activators and inhibitors of, 747 assay method for, 742-744 distribution of, 747-748 molecular weight of, 747 pH optimum of, 747 properties of, 747-748 purification of, 744-746 purity of, 747 source and sample collection for, 743744 specificity of, 747 stability of, 747 Peptide sequences, aminopeptidase in studies of, 519 pH-static assay of chicken pepsin, 350351 Phenole, as subtilisin inhibitor, 211 Phenylalanine diazoketones, as pepsin inactivators, 334 Phenylarsonates, as subtilisin inhibitors, 211 Phenylguanidine, as trypsin inhibitor, 61 Phenylhydrazine, as papsin inactivator, 236 Phenylmethanesulfonyl fluoride (PMSF) as subtilisin inhibitor, 210, 218 use in chymotrypsinogen analysis, 66 Phthaloyl glycyl derivatives, as cathepsin B substrates, 300, 301 Pig carboxypeptidase B from, 504-508 chymotrypsin C from, 90-94, 97-100, 109-112 chymotrypsinogens from, amino acids of, 68-69 chymotrypsinogen B from, 90-94 chymotrypsinogen C from, 90-92, 9497 elastase from, 113-140 gastricsin from, 414-417 pepsins and pepsinogens from, 316-336 trypsin, amino acid composition of, 52 Pineapple, proteolytic enzymes from, s e e Bromelain enzymes
1035
Plasma csrboxypeptidase B-line enzyme in, 508 prothrombin determination and isolation in, 141 Plasma antithrombin, 915--924 assay methods for, 915-917 properties of, 921-924 purification of, 917-921 Plasma transglutaminase, thrombin and, 181 Plasmin, 184-199 amino acid composition of, 193, 194 of SCM-light-chain derivative, 52, 195 assay of, 184-186 immunochemical properties of, 193 inhibitors of, 197 kinetic properties of, 199 origin as zymogen, 33 preparation of, 190-192 purity and physical properties, 192-193 as serine protease, 32 specificity of, 198 stability of, 192 terminal amino acid residues of, 195196 titration with p-nitrophenyl p'-guanidinobenzoate HCI, 20-27 Plasmin-streptokinase activator complex, isolation and properties of, 197-198 Plasminogen, 184-199 activation mechanism of, 193-195 activators of, 196-197 amino acid composition of, 193, 194 assay of, 184-186 distribution of, 199 immunochemical properties of, 193 plasmin from, 33 purification of, 186-190, 819-820 purity and physical properties of, 192193 stability of, 192 streptokinase activation of, 190, 196, 197, 807-808, 815-817 terminal amino acid residue of, 195196 tissue activator of, s e e Cytokinase; Tissue plasminogen activator
1036
SUBJECT INDEX
Plasminogen activator (urinary), see Urokinase Polyacrylic acid, trypsin derivative of, 956 Poly-L-glutamate, in pepsin assay, 317 Polyglutamin acid, trypsin derivative of, 956 Poly-L-proline, in aminopeptidase-P assay, 521-524 Polytyrosyl chymotrypsin, water-insoluble derivative of, 956 Polytyrosyl trypsin, characterization and properties of, 953 Polyvalent inhibitor from bovine organs, see Bovine trypsin-kallikrein inhibitor Potato chymotrypsin I inhibitor, 883--889 assay of, 884-885 properties of, 888-889 purification of, 885-888 Potentiometric assay, of trypsin, 37-38 P.P. reagent, 159, 161 Proaccelerin, thrombin and, 181 Procarboxypeptidases assay of, 93 from cow, 460-475 Procarboxypeptidase A from cow, 460-475 isolation and purification of, 461-467 Procarboxypeptidase A-S5 amino acid composition of, 471 properties of, 474-475 Procarboxypeptidase A-S6 amino acid composition of, 471 disaggregation of, 469-471 as metalloprotein, 467-468 purity and physical properties of, 468 stability and chemical properties of, 467-468 terminal analysis of, 468-469 Prochymosin, 421-436 amino acid composition of, 431, 456 assay of, 424 molecular weights and structure of, 432-435 purification of, 425-427 stability and activation of, 430-431 Profibrinolysin, definition of, 807 Proflavin, as trypsin inhibitor, 61
Pronase, 651-664 assay of, 651-658 aminopeptidase activity, 655-656 carboxypeptidase activity, 657-658 esterase activity, 653-655 proteinase activity, 651-653 properties of, 663--664 purification of, 658-663 trypsin and, 613 1-Propylguanidine, as trypsin inhibitor, 61 Prorennin, see ProchymOsin Proteases acidic type, see Acidic proteases from B. subtilis, 569-575 from bacteria and molds, 569-613 Protease A, from sea anemone, see M e t r i d i u m protease A Proteinase A, from baker's yeast, 372 Proteinase III, from A. oryzae, 386 Proteolytic enzymes, 29-762 active site titration of, 3-20 a-chymotrypsin, 6-20 principles of, 3-6 serine proteases, 31-226 water-insoluble type, 933-985 Proteolytic enzyme activators and inhibitors, 805-932 Proleus spp., 7-glutamyl transpeptidases from, 789 Prothrombin, 140-145 activators and inactivators of, 169-170 amino acid composition of, 168--169 assay methods for, 158-163 one-stage method, 159-160 principle of, 153 two-stage method, l~{)-lfi2 from cow, 157-170 crystallization of, 169 determination of, 928-929 distribution of, 170 from horse, 146-145 molecular weight of, 145 physical and chemical properties of, 144-145, 169-170 in plasma, determination, 162-163 preparation of, 140-143 purification of, 163-167 purity of, 144, 167-168 stability of, 143-144, 167
S~J~TIND~ thrombin from, 33 water-insoluble derivative of, 985 Protozoa, ribosomal peptidyl transferase from, 803 Pseudomonas spp., milk-clotting enzyme from, 447 Pseudomonas aeruginosa collagenase from, properties, 616 ~f-glutamyl transpeptidase from, 789 Pseudomonas fluorescens culture of, 558-559 pyrrolidone carboxylyl peptidase from, 555 Pseudotrypsin, definition and properties of, 50 L-Pyrrolidonecarboxylyl L-alanine, in pyrrolidonecarboxylyl peptidase assay, 556-557 Pyrrolidonecarboxylyl dipeptides, synthesis of, 557-558 Pyrrolidonecarboxylyl peptidase, 555-569 action of, 555 amino acids in, 566 distribution and function of, 568-569 PCA-peptides and PCA-proteins, 563565 physicochemical properties of, 565-566 preparation of, 560-563 specificity of, 566-568 stability of, 565 R
Rat, trypsin, amino acid composition of, 52 Rat liver, ribosomal peptidyl transferase from, 803 Red gram trypsin inhibitor, 863 properties of, 865, 866, 870, 871 Renal dipeptidase, 722-729 activators of, 728 amino acid composition of, 729 assay of, 722-724 distribution of, 729 inhibitors of, 728 kinetic properties of, 728 physical properties and purity of, 727728 purification of, 724-727 specificity of, 728 stability of, 727
i~7
Renin, 699-706 assay of, 699-702 Michaelis constants for, 704 pH optima of, 705 preparation of, 702-706 properties of, 706 Rennin (See also Chymosin) from Mucor species, see Mucor rennin Reptilase, thrombinlike activity of, 181 Rhea, ovomucoid from, 898, 902 Rhizopus spp., milk-clotting enzyme from, 447-448 Rhizopus chinensis acid proteinase, 373 amino acid composition of, 395-396 assay of, 391 enzymatic properties of, 396--397 molecular properties of, 395-396 purification of, 391-395 trypsinogen-activation by, 374 Ribonuclease, in pepsin assay, 317 Ribosomal peptidyl transferase (E. coli), 797-8O3 activators and inhibitors of, 803 assay methods of, 797-801 distribution of, 803 physical properties and purity of, 802 properties of, 802-803 purification of, 801-802 specificity of, 802-803 stability of, 802 Russell's viper venom protease, 719-722 activators and inhibitors of, 722 assay method, 719-720 properties of, 721 purification procedure, 720-721 Rye germ trypsin inhibitor, amino acid composition of, 843
Sea anemone protease A from, preparation and properties of, 107-108 serine proteases in, 32 Sea pansey, trypsin, amino acid composition of, 52 Secretin, thrombin proteolysis of, 156, 181 Sephadex, chymotrypsin conjugate of, 956
1038
SUBJECT INDEX
Sepharose ehymotrypsin conjugate of, 956 subtilopeptidase A derivative of, 962 Sericin, 221 Serine proteases, 31-226 assay methods for, 35-41 potentiometric, 36--38 principles of, 35-36 spectrophotometric, 36 characterization of, 31-32 by ehloromethyl ketones, 32 by DFP, 31 chymotrypsinogens, 64-108 chymotrypsins, 38--40, 64-108 cocoonases, 221-226 elastase, 113-140 general distribution and occurrence, 32-33 isolation of, 33 plasminogen and plasmin, 184-199 prothrombin (horse), 140-145 subtilisins, 199-215 thiolsubtilisin, 214, 215-221 trypsin, 37-38, 41--63 variants of, 33-35 as zymogens, 33 8erratia marcescens, milk-clotting enzyme from, 448 Serum albumin, as cathepsin E substrate, 315 Sheep chymotrypsin A from, 103-104 trypsin, amino acid composition of, 52 Shrimp, trypsin, amino acid composition of, 52 Silkworm pupae, source of, 221-222 S-MDA derivatives of enzymes, 945, 046-948 resin preparation of, 049-950 S-MDA-mercuripapain conjugate, preparation and properties of, 959-960 S-MDA polytyrosyl trypsin conjugate, preparation and properties of, 953954 S-MDA-subtilopeptidase A conjugate, preparation and properties of, 961962 Snake venom proteases, 715-722 of Malayan pit viper, 715-719 of Russell's viper, 719-722
8orangium sp., serine proteases from, 32 ~orangium a-lytic protease, 140, 599-613 activation and inactivation, 609 amino acid sequence and composition of, 606-607, 610 assay methods for, 600-601 crystallographic properties, 609 distribution, 612-613 kinetic properties of, 612 physical properties of, 608 production and purification of, 601-607 purity of, 608 specificity of, 609--612 stability of, 608 8orangium fl-lytic protease, AL-1 protease compared to, 599 Soybean trypsin inhibitors, 853-862 assay of, 854 Bowman-Birk type, 860-862 Kunitz type, 854--859 Speetrophotometric assay, of chymotrypsin Ao, 38--40 8qualus acanthias, see Dogfish 8taphylococcus aureus, cell walls of, AL-1 protease hydrolysis of, 591 Staphylokinase, 706-714 assay method, 706-708 distribution of, 714 inactivators of, 712-714 physical properties, 712 production and purification of, 708-711 properties of, 711-714 stability of, 711-712 Starfish serine proteases in, 32 trypsin, amino acid composition of, 52 Stem bromelain activators and inhibitors of, 282 amino acid composition of, 280 assay methods for, 274-276 amidase activity, 275-276 caseinolytic activity, 274-275 esterolytic activity, 275 chemical properties of, 281-282 physical properties of, 280-281 purification of, 276-279 purity of, 279-280 specificity and kinetic properties of, 282-283
SUBJECT INDEX Streptococcal proteinase, 252-261 activators and inactivators of, 258 active site of, 261 amino acid composition and sequence of, 259-201 assay methods for, 252-253 esterase activity, 253 peptidase activity, 252-253 proteolytic activity, 252 kinetic properties of, 259 preparation and purification of, 253257 culture medium for, 254-255 purity and physical properties of, 257258 serological reactivity of, 253 specificity of, 258-259 stability of, 257 zymogen of, amino acid composition of, 260 Streptokinase, 807-821 in activation of plasminogen, 190, 196, 197, 807-808, 815-817 amino acids of, 821 assay of, 807-817 by clot lysis, 808-811 reagents of, 817-820 by synthetic substrate, 811-815 mode of activity, 807-808, 815-817 physical properties of, 820 preparation of, 817-820 Streptomyces spp., milk-clotting enzyme from, 448 Streptomyces griseus, serine proteases from, 32 Streptomyces madurae, collagenase from, properties, 617 Submaxillary gland, proteases from, 672681 Subtilisins, 32, 199-215 activators of, 210 assay methods for, 201-202 esterolytic activity, 201-202 proteolytic activity, 202 chemical modifications of, 214-215 chemical and physical properties of, 205-215 commercial isolation of, 204-205 inhibition of, 210-211 isolation procedures of, 202-205
1039
nomenclature of, 199-201 optical rotation of, 208 physicochemical constants for, 207 primary structure of, 205, 207 secondary and tertiary structure of, 207-210 as serine proteases, 32 difference from other families of, 34 specificity of, 212-214 use in detergents, 201 Subtilisin A, see Subtilisin Carlsberg Subtilisin amylosaccariticus, isolation and structure of, 215 Subtilisin B, see Subtilisin Novo Subtilisin BPN' amino acid composition of, 205 amino acid sequences of, 205, 206 esterase kinetics of, 213 inhibitors of, 210-211 isolation of, 204 optical rotation of, 208 physicochemical constants of, 207 secondary and tertiary structure of, 206-210 similarity to subtilisin Novo, 200-201, 205 synonyms for, 200 thiol derivative of, see Thiolsubtilisin Subtilisin Carlsberg amino acid sequences of, 205-206 assay of, 202 chemical modification of, 214 estera.~ activity of, 212 kinetics of, 213 inhibitors of, 210-211 isolation of, 202-205 optical rotation of, 208 peptidase activity of, 212 physicochemical constants of, 207 similarity to subtilisin BPN', 208-209 synonyms for, 200 water-insoluble derivatives of, 960-962 EMA-subtilopeptidase A, 960-961 S-MDA-subtilopeptidase A conjugate, 961-962 Subtilisin Novo chemical modification of, 214 esterase kinetics of, 213 inhibitors of, 210-211 optical rotation of, 208
1040
SUBJECT INDEX
physicochemical constants of, 297 synonyms for, 200 Subtilopeptidase A, see Subtilisin Carlaberg Subtilopeptidase B, see Subtilisin Novo Subtilopeptidase C, see Subtilisin BPN' Succinyl-trypsin, preparation of, 102-103 Sulfhydryl reagents, as papain inhibitors, 236 Sulfite esters, in pepsin assay, 317 Sulfonyl fluorides, as elastsse inhibitors, 131 Sweet pea trypsin inhibitor, 863 properties of, 866 T Tadpole, collagenase from, 616 Takadiastase, 373 assay of, 386 purification and properties of, 386 Talaromyces" duponti, thermophilic aminopeptidase from, 552-555 Tetraethylpyrophosphate, reaction with serine proteases, 119 Tetrahymena pyri/ormis, acid proteinase of, 372 Tetranitromethane, inactivation of aminopeptidase by, 521 Thermolysin, 642-651 amino acid composition of, 647 assay of, 642--645, 651 distribution of, 650 inhibitors of, 648 kinetic properties of, 650 properties of, 647-650 purification of, 646-647 specificity of, 648-650 stability of, 647 Thioglycolie acid, as chymopapain activator, 251 Thiolsubtilisin, 215-221 activity of, 214-215 assay of, 216-217 kinetic properties of, 219 inhibitors of, 220 mechanism of action, 220-221 purification of, 218-219 specificity of, 219-220 stability of, 220 Thionine, as trypsin inhibitor, 61
Thrombin activators and inactivators of, 156, 180 amino acid composition of, 179 amino acid sequences of, 178-180 assay of, 145-157, 170-172 clotting time measurement, 147-148 conditions for, 148 method, 151-152 from cow, 170-184 determination of, 928 ester hydrolysis by, 157 hirudin as inhibitor of, 924-932 hirudin titration with, 927-928 kinetics of, 157, 181-i84 origin as zymogens, 33 physical properties of, 155-156, 177-180 preparation of, 154-155 purification of, 172-176 purity of, 155, 177" reaction with p-nitrophenyl p'-guanidinobenzoate, 171 as serine protease, 32 specificity of, 156, 180-181 stability of, 155, 177 standard solution of, 162 standardized, 152-153 synthetic substrates for, 157, 182 titration with p-nitrophenyl p'-guanidinobenzoate HCI, 20-27 units for, 153-154, 172 water-insoluble derivative of, 978-985 assay methods, 980-981 properties of, 981-985 preparation of, 979 Thrombocholecystokinin, biological activity of, 181 Thromboplastin, preparation of, 159 Thrombosthenin, function of, 181 Thrombosthenin M, as thrombin substrate, 181 Tinamou, ovomucoid from, 898, 902 Tissue plasminogen activator, 821-834 assay method for, 822-829 properties of, 832-833 purification of, 829-832 Titration methods for proteolytic enzymes, 1-27 active site methods, see Active site titration Toluamidines, as trypsin inhibitors, 61
S~J~TIND~ p-Toluenesulfonyl-L-arginine methyl ester (TAME) in pancreatic secretory trypsin inhibitor assay, 907-908 reaction with thrombin, 157, 182 with trypsin derivatives, 50 in subtilisin assay, ~01-202 in trypsin assay, 42--43 Tonsils, ribosomal peptidyl transferase from, 803 a-N-Tosyl-8-(fl-aminoethyl) eysteine ethyl ester, as trypsin substrate, 62 Tosyl-L-arginine methyl ester plasmin reaction with, 199 thrombin reaction with, 180 as trypsin substrate, 62 N -Tosyl-L-phenylalanine chloromethyl ketone (TPCK) as bromelain inhibitor, 282 as papain inactivator, 236 reaction with chymotrypsins, 99-100, 104, 131 in removal of chymotrypsin from trypsin, 57 Trametes sanguinea acid proteinase, 373 Transpeptidases, 763-803 fibrinoligase, 770-782 T-glutamyl transpeptidase, 782-789 ~,-glutamyl cyclotransferase, 789-797 from lobster muscle, 765-770 ribosomal peptidyl transferase, 797-803 Trichophyton schoenleinii, collagenase from, properties, 617 Triglycine peptidase, 748 properties of, 756 Triglycine peptidase II, properties of, 756 Trimethylacetyl elastase, deacylation of, 132 2,4,6-Trinitrobenzene sulfonic acid (TNBS), elastase inactivation by, 128 Tris buffer(s), for trypsin assay, 43 Trypsin e-N-acetylated, 57 in activation of zymogens, 33, 42, 6465 allotypes of, 34
1~1
amino acid composition of, from various species, 51-54, 57-58 amino acid sequences of, 56 assay methods for, 42--44 by active site titration with p-nitrophenyl p-guanidinobenzoate, 20-27, 44 titrants for, 60, 61 using amidase activity, 43--44 be nzoyl-L-argininamide, 43-44 benzoyl-L-arginine ethyl ester, 37--38 using esterase activity, 42--43 potentiometric, 37--38, 42 proteolytic activity to casein, 43 spectrophotometric, 42-43, 44 p-toluenesulfonyl-L-arginine methyl ester, 42--43 autolyzed derivative of, 34-35 minimization of, 57 products of, 50 chromatography of, 51 systems for, 50 chymotrypsin removal from, 57 CM-cellulose derivative of, preparation and properties of, 970-978 conformation of, 58 in enzyme evolution, 34 inhibitors of, 58-60 (See a/so Trypsin inhibitors) kinetic constants of, 60 in penicillopepsin assay, 376-377 physical properties of, 58, 59 properties of, 54-63 purification of, 49-51 in purification of streptococcal proteinase, 256-257 purity of, 57 role in conversion to serine proteinases, 33 specificity of, 60, 63 chymotrypsin and, 60 modification effects onl 63 stability of, 54-57 state diagram of, 55 succinyl derivative of, 102-103 synthetic substrates for, 62 from trypsinogen, 33 of various species, 41-63 water-insoluble derivatives of, 950--955 EMA-trypsin, 950-952
1042
SUBJECT INDEX
miscellaneous types, 954-955 polytyrosyl trypsin, 952-954 a-Trypsin chromatographic separation of, 51 definition and properties of, 35, 50, 51 t-Trypsin chromatographic separation of, 51 definition and properties of, 50, 51 substrates for, kinetic constants of, 62 Trypsin inhibitors amino acid composition of, 843, 868 from Ascaris lumbricoides, 872-878 from barley, 840-844 from egg white, 890-902 from miscellaneous legumes, 862-871 from pancreas, 906-914 from soybean, 853--862 Trypsin-kallikrein inhibitor, from bovine tissues, see Bovine trypsinkallikrein inhibitor Trypsin-resin, for purification of trypsinkallikrein inhibitor, 846-847 Trypsinogen activation of, 48 effects on pancreatic juice enzymes, 42 by fungal acid proteinase s, 374-375 nature of, 48-49 amino acid composition of, 52, 57-58 chromatography of, 46-47 physical properties of, 58, 59 properties of, 47-49 purification of, 44-47 state diagrams of, 55 thrombin proteolysis of, 156 of various species, 41-63 N-terminal sequences of, 48 Trypsinogen kinase from Aspergillus oryzae, see Takadiastase Turkey ovomucoid from, 898, 902 trypsin, amino acids in, 52
U Urokinase, 665-672, 838 in activation of plasminogen, 190 activators and inhibitors of, 671 assay methods for, 665-667 distribution of, 672 physical properties of, 670-671 purification of, 667--669 specificity of, 671-672 V Veronal buffer, for titration by p-NPGB, 23-24 Vespa orientalis, see Hornet Vitamin K, prothrombin and, 163 W Water-insoluble proteolytic enzymes, 933-985 carries for, 945-950 chymotrypsin derivatives, 955-956 kinetic behavior of immobilized enzymes, 936-962 enzyme columns, 944-945 on high molecular weight substrates, 942-944 on low molecular weight substrates, 937-942 papain derivatives, 956-960 subtilopeptidase A derivatives, 960-962 trypsin derivatives, 950-955 Whale, chymotrypsin B from, 104-105 Wheat germ trypsin inhibitor, amino acid composition of, 843 Woodward's Reagent K, 935 ¥ Yeast, ribosomyl pept, dyl transferase from, 803 Z Zymogens (See also Serine proteases) occurrence and distribution of, 32-33
