Methods in Enzymology, Volume 2: Preparation and Assay of Enzymes

METHODS IN ENZYMOLOGY Edited by SIDNEY P. COLOWICK and NATHAN O. KAPLAN McCoUum-Pratt Institute The Johns Hopkins Unive...

Author: Sidney P. Colowick | Nathan O. Kaplan

35 downloads 1266 Views 53MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

METHODS IN ENZYMOLOGY Edited by SIDNEY P. COLOWICK and NATHAN O. KAPLAN

McCoUum-Pratt Institute The Johns Hopkins University, Baltimore, Maryland

VOLUME

II

Advisory Board BRITTON CHANCE CARL F. CORI K. LINDERSTROM~ANG FRITZ LIPMANN

F. F. NORD SEVERO OCHOA JAMES B. SUMNER HUGO THEORELL

1955 ACADEMIC PRESS

New York

San Francisco

A Subsidiary of Harcourt Brace Jovanovich, Publishers

London

Contributors to Volume II Article numbers are s h o w n in parentheses following the n a m e s of contributors. Affiliations listed are current.

E. P. ABRAHAM (13), Sir William Dunn School of Pathology, Oxford University, Oxford, England N. J. BERRIDGE (7), National Institute for Dairy Research, University of Reading, Reading, England FRANCIS BINKLEY (40), Emory University, Atlanta, Georgia SANFORD M. BIRNBAUM (12, 55), National Institutes of Health, Bethesda, Maryland KOr~RAD BLOCH (45), Harvard University, Cambridge, Massachusetts ROGER K. BONNICHSEN (138), Nobel Medical Institute, Stockholm, Sweden ARNOLD F. BRODIE (121), Harvard Medical School, Boston, Massachusetts HARRr P. BROQUIST (104), Lederle Laboratories, Pearl River, New York KENNETH BURTON (23), Medical Research Council Unit for Cell Metabolism, Oxford, England G. C. B(:TLER (89), University of Toronto, Toronto, Canada P. S. CAMMARATA (6), Yale University, New Haven, Connecticut G. L. CANTONI (33, 34), National Institutes of Health, Bethesda, Maryland BRITTON CHANCE (136), University of Pennsylvania, Philadelphia, Pennsylvania H. CHANTRENNE (46), Facult$ des Sciences, Universit$ Libre de BruxeUes, Bruxelles, Belgium PHILIP P. COIIEN (19), University of Wisconsin, Madison, Wisconsin SIDNEY P. COLOWICK (68, 99), The Johns Hopkins University, Baltimore, Maryland BERNARD D. DAVIS (39), New York University, New York, N. Y. CHARLES R. DAWSON (145, 147), Columbia University, New York, N. Y.

ALEXANDER L. DOUNCE (137), University of Rochester, Rochester, New York S. W. EDWARDS(38, parts D,E,F), Harvard Medical School, Boston, Massachusetts NILS ELLFOLK (53), Biochemical Institute, Helsinki, Finland W. H. ELLIOTT (44), University of Oxford, Oxford, England HAROLD J. EVANS (59), North Carolina State College, Raleigh, N. C. CLAUDE FROMAGEOT (42), Universit$ de Paris. Paris, France J. S. FRUTON (6), Yale University, New Haven, Connecticut AKIJI FUJITA (103), Biochemical Institute, Kyoto Prefectural University, Kyoto, Japan COURTLAND C. GALEENER (31), University of Illinois, Urbana, Illinois ARTHUR W. GALSTON (140), California Institute of Technology, Pasadena, California DARCY GILMOUR (98), Commonwealth Scientific and Industrial Research Organization, Canberra, Australia CHARLES GILVARG (39), New York University, New York, N. Y. DAVID A. GOLDTHWAIT (78), Western Reserve University, Cleveland, Ohio ARDA A. GREEN (15, 151), The Johns Hopkins University, Baltimore, Maryland D. M. GREENBERG (5, 49), University of California, Berkeley, California a . ROBERT GREENBERG (78), Western Reserve University, Cleveland, Ohio JESSE P. GREENSTEIN (11), National Institutes of Health, Bethesda, Maryland SANTIAGO GRISOLIA (47), University of Kansas, Kansas City, Kansas I. C. GUNSALUS (18, 31, 109), University of Illinois, Urbana, Illinois

vi

CONTRIBUTORS TO VOLUME II

ERWIN HAAS (14, 122, 125), Mount Sinai Hospital, Cleveland, Ohio G. DE LA HADA (6), National Institutes of Health, Bethesda, Maryland OSAMU HAYAISHI (75), National Institutes of Health, Bethesda, Maryland LEON A. HEPPEL (57, 73, 80, 85, 90, 91), National Institutes of Health, Bethesda, Maryland DENIS HERBERT (139), Microbiology Research Department, Experimental Station, Porton, England ROGER M. HERRIOTT (1), The Johns Hopkins University, Baltimore, Maryland LEONARD A. HERZENBERG (17), California Institute of Technology, Pasadena, California R. J. HILMOE (85, 90), National Institutes of Health, Bethesda, Maryland B. L. HORECKER (73, 123), National Institutes of Health, Bethesda, Maryland f . EDMUND HUNTER, JR. (101), Washington University, St. Louis, Missouri MARGARET J. HUNTER (141), Harvard Medical School, Boston, Massachusetts R. M. JOHNSTONE (26), Montreal General Hospital, Montreal, Canada M. A. JOSLYN (149), University of California, Berkeley, California HERMAN M. KALCKAR (118), National Institutes of Health, Bethesda, Maryland NATHAN O. KAPLAN (69, 70, 86, 113, 114, 119, 135), The Johns Hopkins University, Baltimore, Maryland EDNA B. KEARNEY (108), Edsel B. Ford Institute for Medical Research, Detroit, Michigan W. WAYNE KIELLEY (95, 97), National Institutes of Health, Bethesda, Maryland DANIEL L. KLINE (16), Yale University, New Haven, Conn. W. E. KNox (32, 38), Harvard Medical School, Boston, Massachusetts SEYMOUR KORKES (129), Duke University, Durham, North Carolina ARTHUR KORNBERG (65, 76, 112, 116, 117), WasMngton University, St. Louis, Missouri

DANIEL E. KOSHLAND, JR. (87), Brookhaven National Laboratory, Upton, Long Island, N. Y. P. S. KRISHNAN (96), National Chemical Laboratory, Poona, India STEPHEN I~UBY (100), University of Wisconsin, Madison, Wisconsin HENRy LARDY (100), University of Wisconsin, Madison, Wisconsin M. LASKOWSKI (2, 3, 4), Marquette University, Milwaukee, Wisconsin HOWARD M. LEN~OFF (135), Loomis Laboratory, Greenwich, Connecticut ENzo LEONE (74), Istituto di Chimica Biologica dell' Universith, Naples, Italy AARON BUNSEN LERNER (146), University of Oregon, Portland, Oregon HENRY N. LITTLE (56), University of Massachusetts, Amherst, Massachusetts MARGARET R. MCDONALD (62, 63), Carnegie Institution, Cold Spring Harbor, New York WILLIAM D. MCELROr (150, 151), The Johns Hopkins University, Baltimore, Maryland R. W. McGILvERY (84), University of Wisconsin, Madison, Wisconsin WALTER S. McNuTT (67), Vanderbilt University, Nashville, Tennessee A. C. MAEHLY (136, 142, 143), University of Pennsylvania, Philadelphia, Pennsylvania RICHARD J. MAGEE (145, 147), Columbia University, New York, N. Y. HENRY R. MAHLER (120, 124), University of Indiana, Bloomington, Ind. INES MANDL (92, 93), Columbia University, New York, N. Y. A. H. MEHLER (29), National Institutes of Health, Bethesda, Maryland ALTON MEISTER (52), National Institutes of Health, Bethesda, Maryland HERSCHEL K. MITCHELL (17), California Institute of Technology, Pasadena, California SusuMu MITSUHASHI (39), New York University, New York, N. Y. ROBERT K. MORTON (81, 88), University, of Melbourne, Melbourne, Australia

vii

CONTRIBUTORS TO VOLUME II

AGNETE MUNCH-PETERSON (118), Uni-

versity of Copenhagen, Denmark

Copenhagen,

Institutes of Health, Bethesda, Maryland MURRAY SAFFRAN (77), McGiU Univer-

VICTOR A. NAJJAR (20, 61), The Johns

Hopkins University, Baltimore, Maryland ALVIN NASON (58, 59, 60, 128), 'The Johns Hopkins University, Baltimore, Maryland J. B. NEILANDS (134), University of California, Berkeley, California CARL NEUBERG (92, 93), New York Medical College, New York, N. Y. HANS NEURATH (8), University of Washington, Seattle, Washington GORDON NIKIFORUK (68), University of Toronto, Toronto, Canada LAFAYETTE NODA (100), University of Wisconsin, Madison, Wisconsin G. DAVID NOVELLI (102, 106,

SANFORD M. ROSENTHAL (54), National

115),

Western Reserve University, Cleveland, Ohio EVELYN L. OGINSKY (50), Merck Institute of Therapeutic Research, Rahway, New Jersey M. CLYDE OTEY (64), National Insgitutes of Health, Bethesda, Maryland A. M. PAPPENHEIMER, JR. (132), New York University, New York, N. Y. KARL-GusTAV PAUL (133), Nobel Medical Institute, Stockholm, Sweden S. V. PERRY (94), University of Cambridge, Cambridge, England PAUL PLESNER (64), University of Copenhagen, Copenhagen, Denmark B. DAVID POLIS (144), Naval Aviation Medical Research Laboratory, Johnsville, Pennsylvania VINCENT E. PRICE (64), National Institutes of Health, Bethesda, Maryland J. H. QUASTEL (26, 105), Montreal General Hospital, Montreal, Canada E. RACKER (127), Public Health Research Institute of the City of New York, New York, N. Y. S. RATNER (24, 28, 48), Public Health Research Institute of the City of New York, New York, N. Y. W. E. RAZZELL (109), University of Illinois, Urbana, Illinois

sity, Montreal, Canada ANTHONY SAN PIETRO (152), The Johns

Hopkins University, Baltimore, Maryland OTTO SCHALES (21, 22), Alton Ochsner

Medical Foundation, Louisiana

New

Orleans,

SELMA S. SCrIALES (21), Alton Ochsner

Medical Foundation, Louisiana

New

Orleans,

GERHARD SCHMIDT (79), Tufts Medical

School, Boston, Massachusetts H. W. SHMUKLER (144), Naval Aviation

Medical Research Laboratory, Johnsville, Pennsylvania Louis S~USTER (72, 86), National Institutes of Health, Bethesda, Maryland EMIL L. SMITH (9, 10), University of Utah, Salt Lake City, Utah LUCILE SMITH (130), University of Pennsylvania, Philadelphia, Pennsylvania C. V. SMYTHE (41), Rohm and Haas Company, Philadelphia, Pennsylvania JoHN E. SNOKE (45), University of Chicago, Chicago, Illinois B. H. SSRBO (43), Nobel Medical Institute, Stockholm, Sweden JOHN R. STAMER (18, 31), University of Illinois, Urbana, Illinois R. Y. STANIER (37), University of California, Berkeley, California ELMER STOTZ (131), University of Rochester, Rochester, New York HAROLD J. STRECKER(27), Columbia University, New York, N. Y. P. K. STUMPF (35), University of California, Berkeley, California JAMES B. SUMNER (51, 137), CorneU University, Ithaca, New York MARJORIE A. SWANSON (83), BowmanGray School of Medicine, WinstonSalem, North Carolina CELIA WHITE TABOR (54), National Institutes of Health, Bethesda, Maryland HERBERT TABOR (29, 54), National Institutes of Health, Bethesda, Maryland

viii

CONTRIBUTORS TO VOLUME I I

BIRGIT VENNESLAND (126), University of Chicago, Chicago, Illinois P. J. VIGNOS, JR. (34, part B), Western Reserve University, Cleveland, Ohio ARTTURI I. VIRTANEN (53), Biochemical Institute, Helsinki, Finland ELLIOT VO~KIN (82), Oak Ridge National Laboratories, Oak Ridge, Tennessee HEINRICH WAELSCH (36), Columbia University, New York, N. Y. T. P. WANG (66, 71, 110, 111), Institute of Physiology and Biochemistry, Shanghai, China

E. RoY WAYGOOD

(148), University of

Manitoba, Winnipeg, Canada H. G. K. WESTENBRINK (107), Laboratorture voor Physiologische Chemie, Utrecht, Holland W. A. WOOD (25), University of Illinois, Urbana, Illinois WALTER D. WOSILAIT (128), Western Reserve University, Cleveland, Ohio CHARLES YANOFSKY (30), Western Reserve University, Cleveland, Ohio MILTON ZVCEER (58, 60), Connecticut Agricultural Experiment Station, New Haven, Connecticut

Outline of Volumes I, HI, and IV VOLUME I P R E P A R A T I O N AND ASSAY OF E N Z Y M E S Section I. General Preparative Procedures A. Tissue Slice Technique. B. Tissue Homogenates. C. Fractionation of Cellular Components. D. Methods of Extraction of Enzymes. 1~. Protein Fractionation. F. Preparation of Buffers.

Section II. Enzymes of Carbohydrate Metabolism A. Polysaccharide Cleavage and Synthesis. B. Disaccharide, Hexoside and Glueuronide Metabolism. C. Metabolism of Hexoses. D. Metabolism of Pentoses. E. Metabolism of Three-Carbon Compounds. F. Reactions of Two-Carbon Compounds. G. Reactions of Formate.

Section III. Enzymes of Lipid Metabolism A. Fatty Acid Oxidation. B. Acyl Activation and Transfer. C. Lipases and Esterases. D. Phospholipid and Steroid Enzymes.

Section IV. Enzymes of Citric Acid Cycle

VOLUME

III

P R E P A R A T I O N AND ASSAY OF SUBSTRATES Section I. Carbohydrates A. Polysaccharide Analysis and Preparation. B. Substrates for Glucuronidases. C. Free Sugars. D. Sugar Phosphates and Related Compounds. E. Unphosphorylated Intermediates and Products of Fermentation and Respiration. F. Spectrophotometric Enzymatic Methods for Aldehydes and Ketoaldehydes.

Section II. Lipids and Steroids A. General Procedure for Separating Various Lipid Components of Tissues. B. Preparation and Determination of Higher Fatty Acids. C. Preparation and Analysis of Phospholipids. D. Phosphoric Monoester and Diester Derivatives of Phospholipids. E. Nitrogenous Phospholipid Constituents. F. Chromatographic Procedures for Deter... XVlll

OUTLINE OF VOLUMES I~ III~ AND IV

xi~x

mining Fatty Acids. G. General Procedures for Lower Fatty Acids. H. Molecular Distillation of Higher Fatty Acids and Other Lipids. I. Preparation and Assay of Cholesterol and Ergosterol.

Section I I I . Citric Acid Cycle Compounds A. Chromatographic Analyses of Organic Acids. B. Alpha-keto Acids. C. Beta-keto Acids. D. Tricarboxylic Acids. E. 4-Carbon Dicarboxylic Acids. F. Itaconic Acid and Related Compounds.

Section IV. Proteins and Derivatives A. Spectrophotometric Methods for Determination of Protein. B. Procedures for Measuring Amino Acids (Amino N). C. General Procedures for Preparation of Peptides. D. Resolution of DL Mixtures of Amino Acids. E. Determination and Preparation of Specific Amino Acids and Related Compounds.

Section V. Nucleic Acids and Derivatives A. Methods for Determination of Nucleic Acids in Tissues. B. Methods Nucleic Acids. C. Methods for Characterization of Nucleic Acids. D. Characterization and Isolation of Mono- and Oligonucleotides. E. Other Assay of Nucleotides and Nucleosides. F. Preparation and Assay of Cyclic G. Chemical Synthesis of Nucleosides and Nucleotides.

for Isolating Methods of Methods for Nucleotides.

Section VI. Coenzymes and Related Phosphate Compounds A. General Procedure for Isolating and Analyzing Tissue Organic Phosphates. B. Characterization of Phosphorus Compounds by Acid Lability. C. Determination and Preparation of N-Phosphates of Biological Origin. D. Methods for Preparation and Assay of ATP, ADP, and AMP. E. Methods of Assay and Preparation of Pyridine Nucleotides and Derivatives. Y. Assay and Preparation of Coenzyme A and Derivatives. G. Assay and Preparation of Lipoic Acid and Derivatives. H. Fluorometric Assay of Cocarboxylase and Derivatives. I. Assay and Preparation of Flavine Adenine Dinucleotide and Flavine Mononucleotide. J. Assay and Preparation of Pyridoxal Phosphate. K. Preparation and Analysis of UDPG and Related Compounds.

Section VII. Determination of Inorganic Compounds A. Nitrite and Nitrate. B. Total Nitrogen and Ammonia. C. Sulfur. D. Phosphorus. E. Metals.

XX

OUTLINE OF VOLUME I, III~ AND IV VOLUME IV

SPECIAL TECHNIQUES FOR THE ENZYMOLOGIST Section I. Techniques for Characterization of Proteins (Procedures and Interpretations) A. Electrophoresis; Macro and Micro. B. Ultracentrifugation and Related Techniques (Diffusion, Viscosity) for Molecular Size and Shape. C. Infra-red Spectrophotometry. D. X-ray Diffraction. E. Light Scattering Measurements. F. Flow Birefringence. G. Fluorescence Polarization and Other Fluorescence Techniques. H. The Solubility Method for Protein Purity. I. Determination of Amino Acid Sequence in Proteins. J. Determination of Essential Groups for Enzyme Activity.

Section II. Techniques for Metabolic Studies A. Measurement of Rapid Reaction Rates; Techniques and Applications, Including Determination of Spectra of Cytochromes and Other Electron Carriers in Respiring Cells. B. Use of Artificial Electron Aceeptors in the Study of Dehydrogenases. C. Use of Percolation Technique for the Study of the Metabolism of Soil Microorganisms. D. Methods for Study of the Hill Reaction. ~E. Methods for Measurement of Nitrogen Fixation. F. Cytochemistry.

Section III. Techniques for Isotope Studies A. The Measurement of Isotopes. B. The Synthesis and Degradation of Labeled Compounds (Including Application to Metabolic Studies).

Errata for Volume I Page xiii: for Milton F. Utter, read M e r t o n F. U t t e r Page xv: in Section I I B, for glucuronoside, read glucuronide. Page 63, 1. 7: for Ten Brock, read Tenbroeck Page 326: First sentence after Step 2. Acetone Precipitation should read: Dilute the yeast extract (170 ml.) with 940 ml. of cold water and adjust to p H 4.8 with about 20 ml. of 2 N acetic acid.

Outline of Organization VOLUME II PREPARATION

AND

ASSAY OF ENZYMES Article

Numbers

Pages

1-17

3-169

B. Enzymes in Amino Acid Metabolism (General)

18-26

170-220

C. Specific Amino Acid Enzymes

27-43

220-337

D. Peptide Bond Synthesis

44-46

337-350

E. Enzymes in Urea Synthesis

47-50

350-378

F. Ammonia Liberating Enzymes

51-55

378-400

G. Nitrate Metabolism

56-61

400-423

A. Nucleases

62,63

427-447

B. Nucleosidases

64-67

448-468

C. Deaminases

68-72

469-482

D. Oxidases

73-75

482-497

E. Nucleotide Synthesis

76-78

497-519

A. Phosphomonoesterases

79-88

523-561

B. Phosphodiesterases

89,90

561-570

C. Inorganic Pyro- and Poly-phosphatases

91-93

570-582

D. ATPases

94-98

582-598

1¢.. Phosphate-Transferring Systems

99-101

598-616

Section I. Enzymes of Protein Metabolism A. Protein Hydrolyzing Enzymes

Section II. Enzymes of Nucleic Acid Metabolism

Section III. Enzymes in Phosphate Metabolism

Section IV. Enzymes in Coenzyme and Vitamin Metabolism A. Synthesis and Degradation of Vitamins

102-105

619-632

B. Phosphorylation of Vitamins

166-109

633-649

110-118

649-677

C. Coenzyme Synthesis and Breakdown ix

x

OUTLINE OF ORGANIZATION Article Numbers

.Paoes

A. Pyridine Nucleotide-Linked, Including Flavoproteins

119-129

681-732

B." Iron-Porphyrins

130-144

732-817

C. Copper Enzymes

145-147

817-835

D. Unclassified

148-152

836-870

Section V. Respiratory Enzymes

Outlines of V o l u m e s I, III, and IV start on page xviii.

[1]

SWINE PEPSIN AND PEPSINOGEN

3

[1] Swine Pepsin and Pepsinogen By ROGER 71~/I. HERRIOTT Assay Method Pepsin Principle. This method, described by Anson, ~ involves digestion of denatured hemoglobin by dilute pepsin under standard conditions after which the undigested protein is precipitated with TCA and this is then removed by filtration. The extent of digestion which is the measure of protease action is determined on the filtrate with the aid of Folin's phenol reagent. A blue color is produced by a reaction with the tyrosine and tryptophan of the protein split products in the filtrate. Conversion of the color value to units of pepsin is readily made from a standard curve. Pepsinogen Principle. Swine pepsinogen may be determined in the presence of pepsin by destroying the latter at pH 8 where the precursor is stabile and then converting the pepsinogen into pepsin by acidification to pH 2. This pepsin is then assayed as described below. An evaluation of both components in a mixture of pepsin and pepsinogen entails two enzyme determinations, one after direct acidification which yields the sum of pepsin from pepsinogen and the initial pepsin, the other after first making the solution alkaline to pH 8 followed by acidification to pH 2 which measures the pepsinogen equivalent. The difference in the two values is the pepsin of the original mixture.

Reagents Substrate. The substrate solution contains 2% hemoglobin in 0.06 N HC1. An acidity of pH 1.8 is produced which denatures the hemoglobin. A satisfactory bovine hemoglobin may be purchased from Armour and Company or it may be prepared by plasmolysis of washed beef red cells followed by centrifugation of the stroma. Dry weight or colorimetry may be used to determine the concentration of hemoglobin. T C A - - 5 % (0.3 M). 0.5 N NaOH. Folin's phenol reagent 2 diluted 1:3 with water. Enzyme. Aqueous solutions containing 0.3 to 2 ~, of swine pepsin nitrogen per milliliter are optimal for this method. M. L. Anson, J. Ge~. Physiol. 22, 79 (1938). O. Folin and V. Ciocalt6u, J. Biol. Chem. 73, 627 (1927).

4

~NZYMES OF PROTEIN METABOLISM

[1]

Procedure. To 5 ml. of substrate in a test tube, equilibrated at 25 °, is added 1 ml. of enzyme solution. After 10 minutes' digestion, 10 ml. of 5% TCA is added and the precipitated protein removed by filtration. To 5 ml. of the filtrate in a 50-ml. Erlenmeyer flask is added 10 ml. of 0.5 N NaOH and 3 ml. of the diluted phenol reagent slowly with constant agitation. The blue color that develops is read after 5 minutes in a visible colorimeter against a standard or in a photoelectric colorimeter at 660 m~. The color value is expressed in milliequivalents of tyrosine. A color standard is readily produced with 8 X 10-4 meq. of tyrosine in 5 ml. of 0.2 M HC1 in place of the TCA filtrate. A hemoglobin enzyme blank is performed by adding the 1 ml. of enzyme to the TCA before the latter is added to the hemoglobin. Filtration and evaluation of the chromogenic equivalent of the filtrate completes this operation. An empirical standard curve in which the pepsin units plotted against the liberated tyrosine equivalents in 5 ml. of TCA filtrate permits a rapid conversion of color value to peptic units. Such a curve may be constructed from the following data taken from "Crystalline Enzymes": ~ 1, 2, 3, 4, 5, and 8 X 10-4 pepsin unit correspond to the release of 1.8, 3.5, 5.0, 6.3, 7.5, and 11 × 10-4 meq. of tyrosine, respectively, into 5 ml. of the TCA filtrate over and above the blank. Reproducibility. Duplicate assays agree within 5%, but analyses in different laboratories may not agree closer than 10%. Purification of Pepsin from Commercial Preparations Crystalline swine pepsin can be prepared readily from any of several crude products commercially available. A simple method which has considerable interest and merit employs ethanol fractionation and gives crystals low in nonprotein nitrogen. 4 The method described below uses salt fractionation and yields hexagonal bipyrimidal crystals which have been homogeneous in solubility studies. 4~ Step 1. One killogram of Cudahy U.S.P. 1:10,000 pepsin is dissolved in 2 1. of 0.5 M acetate buffer, pH 5.0. Step 2. To the above pepsin solution is added 4.5 1. of saturated magnesium sulfate and 50 g. of Hyflo Super-Cel (Johns Manville Co.), after which the suspension is filtered on large B~chner funnels. The residue is washed on the funnel with 1 1. of 0.6 saturated magnesium sulfate-0.2 M acetate, pH 5.0, hereafter termed "solvent." Step 3. The residue from step 2 is broken up in 14 1. of solvent and 3 j. H. Northrop, M. Kunitz, and R. M. Herriott, "Crystalline Enzymes," 2nd ed., Columbia University Press, New York, 1948. 4 j. H. Northrop, J. Gen. Physiol. 30~ 177 (1946). 4~ R. IV[. Herriott, V. Desreux, and J. H. Northrop, J. Gen. Physiol. 24, 213 (1940).

[1]


5

stirred for 20 hours at 20 ° , filtered, and the residue washed on the funnel with 1 1. of solvent. To the filtrate and washings is added 3 kg. of solid magnesium sulfate (crystalline, with 7 H20) and 100 g. of Filter-Cel. This is filtered and washed with 1 1. of solvent. Step 4. The residue from step 3 is stirred with 2.5 1. of solvent for 20 hours at 20 ° and filtered. Then 500 g. of magnesium sulfate is added to the filtrate, followed by 60 ml. of 5 N H2S04. The precipitate is filtered and washed with 100 ml. of cold N/50 H2S04. The cake is aspirated until no more mother-liquor can be withdrawn. Step 5. The filter cake is stirred with one-fourth its weight of water in a beaker. The beaker is placed in a 35 ° water bath, and the mixture is stirred to a smooth paste. 0.5 N NaOH is added very slowly through a capillary with continuous stirring until the precipitate just dissolves. The pH should be 3.9 to 4.0. The mixture is stirred slowly and allowed to cool. If hexagonal bipyramid crystals do not form, it should be reheated to 35 ° and the inside of the beaker scratched. Once the crystals form, the suspension should be stirred for 10 to 20 hours at 20 ° and then placed at 5° overnight. I t is then filtered. Purification of Pepsinogen from Swine Mucosa 3,5

Preparation of the Fundi. The stomachs from freshly killed swine are inverted and washed thoroughly with cold water. They can then be transported to the laboratory and the fundi cut out with heavy scissors. The fundus may be distinguished from the rest of the mucosa, since in general it appears slightly darker in color and usually occupies a 6- to 8-inch circular portion of the mucosa approximately midway between the entrance (cardiac) and exit (pyloric) of the stomach. Separation of the mucosa from the muscle tissue can best be performed with a scalpel or scissors. The separated mucosa should then have the slimy mucin removed by scraping gently with a straightedged piece of glass. The mucosa may now be used in step 1 or stored frozen. A fundus mucosa usually weighs about 100 g. and contains iust under a gram of pepsinogen. Step 1. One thousand grams of mucosa is minced twice through a meat grinder with 4-mm. holes and then mixed thoroughly with 4 1. of 0.45 saturated ammonium sulfate (SAS) in 0.1 M NaHC03 and stirred for an hour at room temperature. Step 2. F o r ty grams of Filter-Cel and 20 g. of Hyflo Super-Cel are added, and the suspension is filtered on 30-cm. B~tchner funnels. The filter cake is washed twice with 100 to 150 ml. of 0.42 SAS-0.1 M bicarbonate. The residue is discarded, and the filtrate and washings combined. 5 R. M. Herriott, J. Gen. Physiol. 21, 501 (1938).

6

ENZYMES OF PROTEIN METABOLISM

[1]

Step 3. The protein is precipitated from the above solution l~y the addition of 180 g. of solid ammonium sulfate per liter of solution. The addition of 15 to 30 g. of Hyflo Celite permits reasonably rapid filtration of this precipitate. The filtrate is discarded, and the residue suspended in water. The Celite is removed by filtration, but it should be washed thoroughly to recover all the pepsinogen. Step 4. The above pepsinogen solution is diluted to contain approximately 1 mg. of protein N per milliliter, titrated to pH 6.0 + 0.2 (yellow to methyl red and bromothymol blue) with 4 M acetate buffer, pH 4.7, and then mixed with an equal volume of " M / 1 " washed copper hydroxide. The mixture is stirred for 5 to 10 minutes, then filtered on large Bfichner funnels. If protein is found in the filtrate, more copper hydroxide may be added, filtered, and combined with the first residue. The combined residues are then broken up into a smooth creamy suspension in a volume of 0.1 M phosphate, pH 6.8, equal to the volume of the protein solution just prior to mixing with copper hydroxide. After being stirred for half an hour the suspension is filtered and the residue washed twice with 75 ml. of 0.1 M phosphate, pH 6.8. Step 5. The filtrate and washings from step 4 are combined and mixed with 50 g. of ~ilter-Cel per liter of solution. This is filtered with suction, and the cake washed twice with a volume of 0.1 M P Q , pH 6.8, equal to the weight of Filter-Ce] used, The filtrate is then brought to 0.7 saturation by the addition of 474 g. of solid ammonium sulfate per liter of filtrate. To this, 50 to 100 g. of Hyflo Super-Cel is added and the suspension is filtered on large Bfichner funnels, after which the protein in the residue is extracted as in step 3. Step 6. The protein solution is now diluted to contain about 1 mg. of PN per milliliter, after which steps 4 and 5 are repeated, except that no Hyflo Super-Cel is added to the 0.7 SAS suspension. The suspension is filtered slowly with suction on a hardened filter paper. Step 7. The residue from step 6 is stirred with 9 vol. of 0.4 saturated ammonium sulfate in 0.1 M P04, pH 6.25, filtered, and stirred slowly at 10° for a few hours. The solution~should become opalescent and a swirl of needle crystals develop. After two days the crystals may be filtered.

Properties Swine pepsin is unstable in solutions alkaline to pH 6, yet it is relatively stable to acid solutions, even to pH 1. The maximum protease action is observed at pH 1.8 to 2.0. Pepsinogen is unstable below pH 6, being autocatalytically converted to pepsin. In the absence of salts, pepsinogen is reversibly denatured at pH 7 above 50 °, or at room temperature and alkalinities of pH 8.5 to 11.

[1]


7

I n h i b i t o r s . A n i n h i b i t o r of p e p s i n h a s b e e n i s o l a t e d f r o m t h e a c t i v a t i o n m i x t u r e of p e p s i n o g e n 2 I t i n h i b i t s p e p t i c a c t i o n o n l y in t h e r e g i o n of p H 4 t o 6.

Other Species P u r i f i c a t i o n of p e p s i n a n d / o r p e p s i n o g e n f r o m species o t h e r t h a n swine h a v e b e e n d e s c r i b e d . T h u s , s a l m o n , 7 s h a r k , 8 b o v i n e , 9 a n d c h i c k e n 1° may be noted. TABLE I PREPARATION OF PEPSIN

Weight of cake

[PU]=~. ~b PN

500 g. (dry)

0.28 0.32 0.33-0.34 0.33-0.34

Crude pepsin Step 3 Step 4 Step 5

100 g. 40 g.

TABLE II PREPARATION OF PEPSINOGEN

Step

Volume, ml.

Protein N/ml.

Total protein

" [PU]ml" rfb,,,,

1 2 3 4 6 Crystals

4,700 3,800 1,000 1,470 135 37

0.67 0.37 1.3 0.33 2.1 3.5

3,150 1,400 1,300 485 280 130

0.042 0.048 0.15 0.075 0.43 0.74

1tl. . . . PN ,, [PU]mg.

0.06 0.13 0.12 0.23 b 0.21 0.21

Per cent of original activity 100 94 79 55 30 14

This is potential peptic activity, for it is determined only after active*ion of the pepsinogen. b This figure is probably not representative. In other preparations the activity per milligram of protein nitrogen was intermediate between this value and that of step 3. 6 R. M. Herriott, J. Gen. Physiol. 24, 325 (1941). 7 E. R. Norris and D. W. Elam, J. Biol. Chem. 134, 443 (1940). 8 G. P. Sprissler, "An Investigation of the Proteinase of the Gastric Mucosa of Shark," Dissertation, The Catholic University of America Press, Washington, D. C., 1942. 9 j. H. Northrop, J. Gen. Physiol. 16, 615 (1933). ~0R. M. Herriott, Q. M. Bartz, and J. H. Northrop, J. Gen. Physiol. 21, 575 (1938).

8


[9.]

[2] Chymotrypsinogens and Chymotrypsins B y ~I. LASKOWSKI

Two chymotrypsinogens and several chymotrypsins are known at present. The first, a-chymotrypsinogen, was crystallized b y Kunitz and Northrop. 1 Several modifications of the original procedure were later contributed b y Kunitz. 2-4 Small a m o u n t s of trypsin (0.5 mg. per 10 g., 5 °, 48 hours) slowly activate a - c h y m o t r y p s i n o g e n into a-chymotrypsin. 1 The latter undergoes a slow transformation into ~- and ~-chymotrypsins, presumably as a result of limited autolysis. C h y m o t r y p s i n s f~ and ~, were obtained in crystalline form. a Jacobsen, 5 using a fast activation of a - c h y m o t r y p s i n o g e n (35 rag. of trypsin per gram of a-chymotrypsinogen, 0 °, 1 to 2 hours), described two new forms of chymotrypsin, a very unstable form ~, and a fairly stable form 5, which were more active t h a n a - c h y m o t r y p s i n . The preparation of amorphous ~-chymotrypsin, was repeated b y Schwert and Kaufman. 6 The crystalline diisopropyl phosphate, 8" DP-~-chymotrypsin, was recently obtained b y Desnuelle and co-workers. 7 Considerable progress in elucidation of the activation process has been recently achieved. The activation process depends on rupturing of the cyclic molecule of a - c h y m o t r y p s i n o g e n b y trypsin, and b y chymotrypsin itself (autolysis). Schemes of the activation process have been suggested b y Gladner and N e u r a t h s and b y Rovery, Poilroux, and Desnuelle. 7 The latter scheme is reproduced in Fig. I sa. The mechanism of transformations leading to f~ and ~/forms is not yet clear. The terminal groups of a-, ~-, and ~,-chymotrypsins are the same, 9,1° but the rate of release of the C terminal groups b y carboxypeptidase differs. 9 M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). M. Kunitz, J. Gen. Physiol. 33, 349 (1950). 3 M. Kunitz, J. Gen. Physiol. 2, 207 (1938). 4 M. Kunitz, J. Gen. Physiol. 32, 263 (1948). 5 C. F. Jacobsen, Compt. rend. tray. lab. Carlsberg, sdr. chim. 25, 325 (1947). 6 G. W. Schwert and S. Kaufman, J. Biol. Chem. 180, 517 (1949). s~ The following abbreviations are used: diisopropyl fluorophosphate is called DFP; the enzyme which reacted with DFP is called DFP-treated enzyme or DP enzyme. 7 M. Rovery, M. Poilroux, and P. Desnuelle, Biochim. et Biophys Acta 14, 145 (1954). s j. A. Gladner and H. Neurath, J. Biol. Chem. 205, 345 (1953). s, F. R. Bettelheim and H. Neurath, g. Biol. Chem. 212, 241 (1955) on the basis of analysis of terminal groups postulated a new intermediate between ~ and a chymotrypsins, and concluded that it probably resulted from the action of chymotrypsin and not trypsin. 9j. A. Gladner and H. Neurath. J. Biol. Chem. 206, 911 (1954). i0 M. Rovery, C. Fabre, and P. Desnuelle, Biochim. et Biophys. Acta 10, 481 (1953).

9

CHYMOTRYPSINOGENS AND CHYMOTRYPSINS

[9.]

A second crystalline chymotrypsinogen, named B, which on activation leads to a crystalline c h y m o t r y p s i n B was obtained b y Laskowski and his co-workers, n-14 Proteins of the B series differ from those of the a series in electrophoretic mobility 15 and in the ease with which t h e y are retained on resins, ~6 b u t not in molecular weight. 17 C h y m o t r y p s i n B shows the same specificity as a - c h y m o t r y p s i n when tested on several synthetic substrates, is However, the rates at which the two enzymes hydrolyze various substrates 1~,~,~9,2° differ. The analysis of terminal groups ~-Chymotrypsin

~r-Chymotrypsin

a-Chyrnot rypsinogen [ q I t Tyr, Ileu I

',

Ala L

I

Base

trypsin >

T ~ e u I i Ala I Base

LLeu- ]

/ #

'

I Tyr

i

I

,

I

Ileu

(~)

Ala ~

trypsin /

+ x --Base Leu ( ~ II autolysis J

trypsio, autolysis

• a-Chy mot ry psin - -

~

Q

Tyr,

Ileu @ l

Ala ,

L

+ x - - Base

eu©

FIG. 1. Scheme of activation of o~-ehymotrypsinogen! confirmed the nonidentity of the B proteins with the c~ protein% b u t did not elucidate the mechanism of activation. 9 Preparation of a-Chymotrypsinogen 1 Ten to twelve average-size beef pancreases, removed immediately after slaughter, are immersed at once in enough 0.25 N ice-cold H~S04 to cover the glands. After removal of fat and connective tissue the glands are minced in a meat chopper. Three liters of minced pancreas is suspended in 6 1. of 0.25 N H2SO4 at 5 °, and the suspension is allowed to stand at this temperature for 18 to 24 hours. The suspension is strained n M. Laskowski, J. Biol. Chem. 166, 555 (1946). •~ M. Laskowski and A. Kazenko, J. Biol. Chem. 167, 617 (1947). ~3C. K. Keith, A. Kazenko, and M. Laskowski, J. Biol. Chem. 170, 227 (1947). 14K. D. Brown, R. E. Shupe, and M. Laskowski, J. Biol. Chem. 173, 99 (1948). l~ V. Kubacki, K. D. Brown, and M. Laskowski, J. Biol. Chem. 180, 73 (1949). i~ C. H. W. Hirs, J. Biol. Chem. 205, 93 (1953). ~7E. L. Smith, D. M. Brown, and M. Laskowski, J. Biol. Chem. 191, 639 (1951). ~sj. S. Fruton, J. Biol. Chem. 173, 109 (1948). 19j. A. Ambrose and M. Laskowski, Science 115, 358 (1952). ~0F. C. Wu and M. Laskowski, Federation Proc. 13, 326 (1954); J. Biol. Chem. 213, 609 (1955).

10


[2]

through two layers of gauze; the tissue is resuspended in 3 I. of cold 0.25 N H2S04 and immediately strained through gauze. The extracts are combined, and the residue is rejected. To each liter of extract 242 g. of solid ammonium sulfate is added to attain 0.4 saturation. The mixture is filtered through fluted paper (S. and S. No. 14501/~) in the cold room. ~ The precipitate is rejected. 22 The liquid is brought to 0.6 saturation with ammonium sulfate (205 g./1.). The copious precipitate which forms is allowed to settle at 5 ° and is filtered at that temperature on soft 22a filter paper on a large Bfichner funnel. ~3 The yield is about 100 g. of moist filter cake. The filtrate is saved for the preparation of ribonuclease (see Vol. II [62]). Each 100 g. of the precipitate is dissolved in 300 ml. of water, and 200 ml. of saturated solution of ammonium sulfate (20 to 25 °) is added. T M The precipitate which forms is filtered off with the aid of 5 g. of Celite and discarded. To each liter of filtrate 205 g. of solid ammonium sulfate is added slowly, and the precipitate which forms is filtered through a hardened paper. The yield is about 90 g. The filtrate is rejected. Crystallization of a-Chymotrypsinogen. Each 100 g. of the precipitate is dissolved in 150 ml. of water, treated with 50 ml. of saturated ammonium sulfate, and adjusted to pH 5.0 by dropwise addition of 5 N" NaOH (about 2 ml. per 100 g. of precipitate). The solution is allowed to stand for 2 days at 20 to 25 °. A heavy crop of chymotrypsinogen crystals (long needles) gradually forms. The suspension of crystals is filtered through hardened filter paper. The yield is about 25 g. The filtrate is saved for the preparation of trypsinogen (Vol. II [3]) or trypsin (Vol. II [3]), and afterward trypsin inhibitor (Vol. II [4]). 21 In this laboratory Sargent No. 500 filter paper is used and 10 g. per liter of SuperCel and 10 g. per liter of Celite No. 545 are added to facilitate filtration. 22 This precipitate contains deoxyribonuclease and chymotrypsinogen B. If the isolation of these two proteins from the same batch of pancreas is desired, it is recommended to follow the extraction procedure described for the preparation of deoxyribonuclease (see Vol. II [63]), using the less acid medium in order to protect deoxyribonuclease. The extract is then first brought to 0.2 saturation of ammonium sulfate (114 g./1.), and the precipitate is discarded. The liquid is brought to 0.4 saturation of ammonium sulfate (121 g./1.), and the precipitate (containing deoxyribonuclease and chymotrypsinogen B) is collected. The liquid is acidified by the addition of 6 ml. of concentrated sulfuric acid per liter of water used for the original extraction. From there on the procedure described in the text should be followed. 22a In this laboratory, for the soft paper Whatman No. 1 or 4 is used; for the hardened paper Whatman No. 50 or 52. 23 In this laboratory a stainless steel Btichner funnel, Model 503, is used, manufactured by American Biosynthetic Corp., Milwaukee, Wisconsin. ~ From there on all operations are carried at room temperature (20 to 25 °) unless specified otherwise.

[2]

CHYMOTRYPSINOGENS .A.ND CHYMOTRYPSINS

11

Recrystallization from A m m o n i u m Sulfate. 23bT h e crystalline filter cake is suspended in 3 vol. of water, and 5 N H2S04 is added from a b u r e t with stirring until the precipitate is dissolved. T h e solution is b r o u g h t to 0.25 saturation with a m m o n i u m sulfate b y addition of 1 vol. of s a t u r a t e d a m m o n i u m sulfate. An a m o u n t of 5 h r N a O H equivalent to the acid used is then added with stirring, and the solution is inoculated and allowed to stand at 20 ° . Crystallization should be practically complete in an hour. The yield is a b o u t 80%. Recrystallization from Alcohol. 4 After several (five or six) recrystallizations with a m m o n i u m sulfate, 10 g. of semidry filter cake of crystals of a - c h y m o t r y p s i n o g e n is stirred up with a b o u t 30 ml. of w a t e r are dissolved with the aid of several drops of 5 N H~SO~. T h e solution is dialyzed against slowly running distilled w a t e r for 24 hours at 5 °, preferably with stirring. T h e dialyzed solution of a - c h y m o t r y p s i n o g e n is filtered clear and then m a d e up with w a t e r to 50 ml. The p H of the solution is adiusted with dilute acid or alkali to a b o u t 4.0. T h e solution is cooled in an ice-salt b a t h to 1 to 3 °, and 12.5 ml. of ice-cold 95% ethanol is added slowly with stirring; the t e m p e r a t u r e of the solution is not allowed to rise a b o v e 5 ° during the addition of alcohol. T h e p H of the solution is then adjusted T M with 1 N N a O H to a b o u t 5.0 (0.01% m e t h y l red solution as an indicator on a test plate, and a 0.01 M acetate buffer as a standard). A h e a v y a m o r p h o u s precipitate forms at p H 5. T h e suspension is kept at 20 to 25 °. The precipitate gradually dissolves and is replaced within several hours b y a crop of large well-formed crystals. The crystallization is generally complete within 24 hours. T h e crystals are filtered with suction on hardened paper, washed with ice-cold acetone, and dried at room t e m p e r a t u r e for 24 hours. The yield is a b o u t 80%. T h e dried material is ground up in a m o r t a r and stored in refrigerator. 24 2~bIn the reviewer's laboratory, recrystallizations of a-chymotrypsinogen were repeatedly successful when the vriginal description of Kunitz was followed. Slight modifications of this method have been employed in other laboratories. E. F. Jansen (personal communication) suspends a-chymotrypsinogen in 5 vol. of water, adds H2S04 until pH 3.0 (glass electrode) is reached, then adds the required amount of ammonium sulfate and adjusts pH to 4.0 (instead to 5.0) with 5 N NaOH. Under these conditions recrystallization is essentially complete in about half an hour. G. W. Schwert (personal communication) similarly dissolves a-chymotrypsinogen at pH 3.0 (glass electrode), filters off the insoluble material, if any is present, but recrystallizes it at pH 5.0. 2acDuring the addition of alcohol the solution usually gels. On addition of NaOH the gel changes into a precipitate described below. ~4Neurath (personal communication) calls attention to a possible contamination of a-chymotrypsinogen with the active a-chymotrypsin. He noticed that when all necessary precautions in collecting glands were observed the first crystals of chymo-

12


[2]

Crystallization of a - C h y m o t r y p s i n 1

T e n g r a m s of the crystalline a - c h y m o t r y p s i n o g e n filter cake containing a m m o n i u m sulfate, or 5 g. of salt-free crystalline a - c h y m o t r y p s i n o gen, is suspended in 30 ml. of w a t e r and dissolved b y the addition of a few drops of 5 N H2S04. T e n milliliters of M / 2 p h o s p h a t e buffer, p H 7.6, and a q u a n t i t y of N N a O H equivalent to the acid used are added. T h e solution is inoculated with 0.5 mg. of crystalline trypsin and left for 48 hours at 5 °. T h e p H of the solution is then adjusted to 4.0 b y addition of a b o u t 5 ml. of N H2SO4. T w e n t y - f i v e g r a m s of solid a m m o n i u m sulfate is added, and the precipitate is filtered with suction. T h e filter cake is dissolved in 0.75 vol. of 0.01 N H2SO~ and filtered if the solution is not clear. T h e clear solution is inoculated with crystalline (rhombohedral) a - c h y m o t r y p s i n and allowed to stand at 20 ° for 24 hours. A b o u t 5 g. of crystalline filter cake should form. Recrystallization. ~4~ T h e crystalline filter cake is dissolved in 1.5 vol. of 0.01 N H2S04, and a b o u t 1 vol. of s a t u r a t e d a m m o n i u m sulfate is added cautiously until crystallization commences. After 1 hour at room t e m p e r a t u r e the crystallization is almost complete. Recrystallization from Alcohol. 4 a - c h y m o t r y p s i n is first recrystallized two to three times f r o m a m m o n i u m sulfate. T e n g r a m s of crystalline filter cake is dissolved in 30 ml. of w a t e r and dialyzed against slowly running 0.005 2V H2SO4 at 5 ° for 24 hours with stirring. T h e dialyzed solution is filtered clear, then m a d e up with w a t e r to a volume of 50 ml., and cooled in an ice-salt b a t h to 2 to 3 °. T h e p H of the solution is adjusted to a b o u t 4.8 with the aid of several drops of 1 hr N a O H . Ten milliliters of ice-cold 95 % alcohol is added slowly with stirring, while the t e m p e r a t u r e of the solution is m a i n t a i n e d at a b o u t 5 ° . T h e solution is then trypsinogen contained 0.2 % of the active enzyme. The best preparation of Gladner and Neurath 8 contained only 0.03% of chymotrypsin, and no tryptic activity could be detected. This preparation was recrystallized seven times from ammonium sulfate and twice from alcohol. On the other hand, it the precautions in collecting glands were not observed, the first crystals may contain as much as 7 to 10% of active enzyme, and subsequent recrystallizations cannot reduce this amount to less than 1%. Hirs 18 described a chromatographic separation of a-chymotrypsinogen from crude acid extracts of pancreas using the resin IRC 50 (XE-64) (see Vol. I [13]). Good separation was obtained after a single passage through the column. As yet no attempts have beeen made to obtain a-chymotrypsinogen free from a-chymotrypsin. However, chromatography was found useful in detection of impurities in crystalline preparations of a-chymotrypsinogen. See also Vol I [12]. 24, E. F. Jansen (personal communication) recommends that all the ammonium sulfate be added rather rapidly to an Erlenmeyer flask containing the enzyme with adequate stirring. By this procedure, a clear supersaturated solution is obtained, which, when seeded, results in fairly large crystals.

[2]

13


titrated with 1 N H2S04 to pH 4.0 (light green to bromocresol green on a test plate). A heavy amorphous precipitate is formed. The suspension is kept at 5°. The amorphous precipitate slowly changes into a paste of very fine needles and rosettes. Seeding with a drop of a suspension of the crystals assures prompt crystallization within 24 hours. The paste of crystals is filtered with suction on hardened paper at 5 ° . The filtration generally takes several hours. The filter cake is dried on a watch glass placed near the cooling coil of the refrigerator. The dry material is ground to a fine powder and stored in the refrigerator.

The first preparation of cL-chymotrypsin diisopropyl fluorophosphate derivative was accomplished by Jansen et al. 2~ The mechanism of the reaction has been elucidated as follows. ~8,27 O a-Ch--H +

OC3Hv

!!/ F--P \

O

OC3H~

H/ --+ a - C h - - P \ OC~H7

+ HF OC3H7

One mole of phosphorus 28 and two isopropyl groups 27 are introduced per mole of enzyme with the liberation of H F and the formation of diisopropyl phosphate a-chymotrypsin (DP-a-chymotrypsin). Thirty per cent of the protein-bound phosphorus of the DP-a-chymotrypsin, after complete hydrolysis, has been recovered as phosphoserine, ~8 suggesting that the diisopropyl phosphate radical is bound to chymotrypsin through the hydroxyl groups of serine. 28~ Balls and Jansen 29 critically reviewed the method of preparation. The method consists essentially in dissolving the several-times-recrystallized and salt-free enzyme in 0.2 M phosphate buffer, pH 7.7; the concentration of enzyme in solution is not critical2 ° Diisopropyl fluorophosphate is added as a 1 M solution in isopropanol; this is recommended as a safety precaution, 29 since D F P is highly toxic. When working with a-chymotrypsin (molecular weight ca. 22,500) only a small excess of D F P is recommended 3° (1.2 millimoles of D F P per millimole of enzyme). ~5 E. F. Jansen, M. D. F. Nutting, R. Jang, and A. K. Balls, J. Biol. Chem. 179, 189, (1949). ~6 E. F. Jansen, M. D. F. Nutting, and A. K. Balls, J. Biol. Chem. 179, 201 (1949). 27 E. F. Jansen, M. D. F. Nutting, R. Jang, and A. K. Balls, J. Biol. Chem. 185, 209

(1950). ~s N. K. Schaffer, S. C. May, Jr., and W. H. Summerson, J. Biol. Chem. 202, 67 (1953). 2s, Caution in accepting this evidence as final is suggested by R. M. Herriott, The Mechanism of Enzyme Action (W. D. McElroy and B. Glass, eds.), Johns Hopkins Press, Baltimore, 1954, pp. 24-49, who discusses other possible sites of binding of D F P by chymotrypsin. 2~ A. K. Balls and E. F. Jansen, Advances in Enzymol. 13, 321 (1952). 30 E. F. Jansen, personal communication.

14

ENZYMES

OF PROTEIN METXBOLIS~

[2]

With trypsin and other forms of chymotrypsins 31 a greater excess (2 millimoles per millimole of enzyme) of D F P is recommended. Desnuell~ and co-workers7 used 5 millimoles of D F P per millimole of 5-chymotrypsin. The reaction is fast but not instantaneous. Usually it is allowed to proceed overnight in the cold room or for 1 to 2 hours at room temperature. The reaction is stopped by adjusting the pH to 4.0 with the aid of 2 N H2SO4. After this, the amorphous enzyme derivative is precipitated with ammonium sulfate at 0.8 saturation. The amorphous DP-a-chymotrypsin is then recrystallized according to the method of Kunitz and Northrop 1 described for the native a-chymotrypsin. Crystals of DP-achymotrypsin have the same appearance as those of the native a-chymotrypsin but are virtually inactive. After two recrystallizations only traces of activity can be detected. Crystallization of ~- and ~,-Chymotrypsins 3 As a starting material, either a-chymotrypsin or the mother liquor from a-chymotrypsin crystallizations can be used. With the latter the protein is first salted out in 0.7 saturated ammonium sulfate, and the precipitate is then used in the following operations in the same manner as the crystal cake of a-chymotrypsin. One hundred grams of crystal cake of a-chymotrypsin (containing ammonium sulfate) is suspended in 100 ml. of water. Fifty milliliters of 0.5 M phosphate buffer, pH 8.0, is added, and the clear solution is allowed to stand at 5 ° for three weeks. Then 120 ml. of saturated ammonium sulfate is added, the pH is adjusted to 5.6 by means of 5 N H2SO4 added drop by drop, and the mixture is allowed to stand at 20 ° for 3 days. Large bipyramidal crystals of ~/-chymotrypsin are filtered with suction. The yield is about 30 g. of filter cake. The filtrate (first ~, mother liquor) is stored at 5°. The ~, crystals are recrystallized by dissolving 10 g. in 30 ml. of water and adding 20 ml. of saturated ammonium sulfate. After 24 hours the crop of second crystals of ~,-chymotrypsin is filtered with suction. The precipitate is stored at 5° . The filtrate is combined with the first ~, mother liquor, adjusted to pH 4.2 with 5 N H2SO4, and the protein is salted out by addition of 21 g. of solid ammonium sulfate to each 100 ml. of solution. The precipitate is filtered with suction and is dissolved in 0.75 vol. of 0.01 N H2SO4. I t is allowed to stand for several days at 20 to 25 ° until a heavy precipitate of fine needle crystals of crude f~ is formed. The solution frequently turns into a thick fibrous gel of crystals, which are filtered with suction. The filtrate on standing may yield another crop of needle crystals. The 31E. F. Jansen and A. K. Balls, J. Biol. Chem. 194, 721 (1952).

[2]


15

total yield is about 50 g.31~ of crude/~ filter cake per 100 g. of original a-chymotrypsin filter cake. Recrystallization of Crude ~ Crystals. Ten grams of crystal cake is dissolved in 30 ml. of water, and 30 ml. of saturated ammonium sulfate is added. The p H is adjusted to 5.6 b y means of a few drops of 5 N NaOH, and after inoculation with ~, crystals the solution is allowed to stand for several days at 20 °. The crystals of 7-chymotrypsin are filtered off, and the p H of the filtrate is adjusted to 4.2. Crude ~ crystals gradually appear. T h e y are filtered after several days and subjected to a second recrystallization for the crude f~ crystals. Isolation of Pure ~ Crystals. Ten grams of three-times-recrystallized crude crystal cake is dissolved in 250 ml. of water, and 10 ml. of 0.4 M borate buffer, 31b pH 9.0, is added. The solution is heated to 37 ° and allowed to stand at this temperature for 1 hour. I t is then cooled to 20 ° and adjusted to pH 4.2 by means of 5 N H2SO,. Sixty-five grams of ammonium sulfate is added, and if a precipitate forms it is filtered off with the aid of 5 g. of Super-Cel through 9-cm. W h a t m a n No. 3 filter paper. T o each 100 ml. of filtrate 21 g. of ammonium sulfate is added, and the precipitated protein is filtered on hardened paper. The filtrate is rejected. Each gram of the precipitate is dissolved in 3 ml. of water, and 2 ml. of saturated ammonium sulfate is added. The solution is adjusted to pH 4.2 and allowed to stand at 20 °. An amorphous precipitate forms which gradually changes into very fine crystals. After several days the crystals are filtered through a hardened paper. The yield is about 2 g. of pure ~ crystal cake per 10 g. of crude ~ filter cake. The procedure for the recrystallization of the " p u r e " fl crystals is the same as for the crude ~ crystals.

Preparation of ~-Chymotrypsin ~-Chymotrypsin was first prepared b y J a c o b s e n ) The procedure described here is essentially t h a t of Schwert and Kaufman. 6 First 1.1 g. of lyophilized, essentially salt-free, a-chymotrypsinogen (prepared from chymotrypsinogen recrystallized eight times) 32 is dissolved in 50 ml. of water. A small trace of insoluble material is removed by filtration, and the pH of the solution is adjusted to 7.3 with 0.1 N NaOH. The solution is placed in a refrigerated bath at 0 °. After 20 minutes 70 mg. of crystalline trypsin (containing 50% MgS04) is added. ~1~ H. Neurath (personal communication) states that in his laboratory the usual yield

is about 25 g. 31bStock borate solution contains 49.6 g. of boric acid and 80 ml. of 5 N NaOH per 1000 ml. of solution. Borate buffers (0.4 M), pH 8.0 and 9.0, are mixtures of 100 parts of stock borate and 78.6 and 17.6 parts of 0.4 N HC1, respectively. ~2Chymotrypsinogen recrystallized from alcohol would be equally good.

16


[2]

After a total of 98 minutes (half of this time interval would probably lead to the same result) the pH of the activation solution is rapidly adjusted to 4.2 with 2 N H~S04 and the solution is shell-frozen and lyophilized. The dry protein is stored at - 2 0 °. Over a period of two months no change in the activity of this preparation is observed. Crystalline DP-~-chymotrypsin was obtained 7 by following this activation procedure of Jacobsen, 5 and adding, at pH 7.6, 5 moles of D F P per mole of ~-chymotrypsin. The amorphous protein was then precipitated, and the needle-shaped crystals were obtained by following the procedure of Kunitz and Northrop 1 described for the crystallization of a-chymotrypsin.

Preparation of Chymotrypsinogen B The original extract from thirty average-size beef pancreases is prepared according to Kunitz and Northrop, 1 exactly as described for a-chymotrypsinogen. The whole operation is carried out in the cold room. The extract is brought to 0.2 saturation of ammonium sulfate (114 g. of solid salt per liter of extract). Ten grams of Celite No. 545 and 10 g. of Standard Super-Cel are added per liter, and the mixture is filtered through four large fluted filters (Sargent No. 500). The residue is rejected. The filtrate is brought to 0.4 saturation by addition of 121 g. of solid ammonium sulfate per liter. The precipitate which forms is filtered through a soft paper on a large Biichner funnel. The liquid may be used for the preparation of a-chymotrypsinogen, trypsinogen, trypsin inhibitor, and ribonuclease. The precipitate is dissolved in 5 vol. of water, and 20 ml. of a saturated solution of ammonium sulfate is added per 100 ml. of enzyme solution. A small precipitate is removed by filtration with the aid of 2 g. of Standard Super-Cel per 100 ml., using soft paper on a Btichner funnel, and is rejected. To each 100 ml. of filtrate 19 ml. of saturated ammonium sulfate is added. The precipitate which forms is collected on a hardened filter paper, and the filtrate is rejected. The precipitate is dissolved in 4 vol. of water plus 1 vol. of 1 M acetate buffer, pH 4.0, and the solution is adjusted to pH 4.0 (glass electrode). For each 100 ml. of solution 25 ml. of saturated ammonium sulfate is added. The precipitate is centrifuged off in the conical head of an International centrifuge at 5° and is washed twice, each time with one-half of the previous volume of 0.2 saturated solution of ammonium sulfate containing 20% of M acetate buffer, pH 4.0. The supernatant and washings are combined (the volume is measured), the pH is adjusted to 6.5 with 5 N NaOH (the volume of which is recorded), and a saturated solution of ammonium sulfate is added to at-

[2]


17

tain 0.4 saturation, a correction being made for the volume of NaOH used (33.3 ml. per 100 ml. of enzyme solution plus 0.67 ml. per each milliliter of NaOH). The precipitate is collected on a hardened filter paper on a Biichner funnel and is washed with 0.4 saturated ammonium sulfate containing 20% of M acetate buffer, pH 6.5. The washed precipitate is dissolved in a minimum amount of water kept at pH 4.0 by dropwise addition of 1 N HC1. The solution is centrifuged at high speed in a Servall SS-1 centrifuge in order to remove any of the extraneous material and is dialyzed in the cold, with stirring, against 0.01 M acetate buffer, pH 5.5, with frequent changes of buffer. Typical large plates of chymotrypsinogen B appear after several hours. The crystallization is usually complete after 2 to 3 days. Recrystallization is performed by dissolving crystals in a minimum amount of water which is kept at pH 4.0 by dropwise addition of 1 N HC1. After a complete solution is achieved, 0.5 N NaOH is added drop by drop until the first sign of "silkiness." The solution is allowed to stand for an hour at room temperature, after which it is transferred to a dialyzing bag and dialyzed for 24 hours against 0.01 M acetate buffer, pH 5.5, to complete crystallization. After three to five recrystallizations the crystals are centrifuged in a glass tube in a Servall centrifuge and are ]yophilized in the same tube. They may be kept in the refrigerator for several months without significant activation. If chymotrypsinogen B with a low content of chymotrypsin B is desired, this method should be followed. Even with this method partial activation occurs during the process of crystallization. 12 Fortunately, the activated enzyme remains in the mother liquor. In view of the lack of a better method, it is recommended that several (three to five) recrystallizations be made in rapid succession with a minimum exposure to room temperature. Preparations of chymotrypsinogen B containing only 0.15 % of the active chymotrypsin B have been occasionally obtained in the laboratory. An alternative method, which has the advantage of obtaining both crystalline deoxyribonuclease and chymotrypsinogen B, has been described. 17 This method, however, has two disadvantages: the yield is smaller, and considerable activation occurs during the process. This method can be used only when the ultimate goal is chymotrypsin B. Fresh pancreas is treated exactly as described by Kunitz 2 in the preparation of deoxyribonuclease (Vol. II [63]) up to the stage at which a precipitate with 0.7 saturation of ammonium sulfate was obtained. The 0.5 ammonium sulfate precipitate still containing Celite is suspended in 4 vol. of water, adjusted to pH 4.0 by addition of 5 N H2SO4, and stirred mechanically at low speed at 5° for 2 hours. The pH is checked several

18


[2]

times and readjusted to 4.0 when necessary. One volume of cold saturated ammonium sulfate is added slowly with stirring to produce a 0.2 saturated solution. A small precipitate together with Celite is removed by filtration on a Btichner funnel through a soft paper. The filtrate is brought to pH 5.3 by addition of 5 iV NaOH, and the protein is salted out by addition of 20.9 g. of solid ammonium sulfate per 100 ml. of filtrate. The resulting precipitate is collected as dry as possible on a Bfichner funnel with Whatman No. 50 filter paper. The precipitate is redissolved in a minimum amount of water kept at pH 4.0 by dropwise addition of 1 N HC1 and is dialyzed against 0.01 M acetate buffer, pH 5.5, exactly as described above. Preparation of Chymotrypsin B 1~,14 Five grams of five-times-reerystallized chymotrypsinogen B is dissolved in 50 ml. of 0.2 M borate buffer, pH 7.8, and adjusted to pH 7.8. Then 2.5 mg. of crystalline trypsin is added, and the solution is allowed to stand for 4 days at 5 °. It is then transferred to a dialyzing bag and is dialyzed against 0.01 M acetate buffer, pH 5.0, at 5 ° (buffer is changed frequently). A heavy amorphous precipitate forms, which is centrifuged down at high speed (Servall SS-1 centrifuge). The precipitate is dissolved in a minimum amount of water, kept at pH 4.0 by the addition of 1 N HCI, and a small amount of gelatinous insoluble material is removed by centrifugation. The clear liquid is dialyzed against 0.01 M acetate buffer, pH 5.5. The precipitate which forms on dialysis is composed of poorly shaped needles and prisms. The crystalline form improves considerably on recrystallization. The crystals are collected by either centrifugation or filtration through a hardened paper. Recrystallization is achieved by dissolving the semidry crystal cake in a minimum amount of water kept at pH 4 by dropwise addition of 1 N HC1, and subsequent dialysis against 0.01 M acetate buffer, pH 5.5. It was found convenient to include at least one recrystallization in which crystals were first dissolved in a minimum amount of 0.4 M borate buffer, pH 8.0, and dialyzed against 0.01 M acetate buffer, pH 5.0. Four recrystallizations are recommended, after which the crystal cake is lyophilized and stored in the refrigerator. A convenient nomogram for calculating required amounts of ammonium sulfate has been published by Dixon 32~ (see Vol. I [10]). The nomogram is based on a solubility value of 760 g./1., corresponding to room temperature (ca. 25°). The values used by Northrop and Kunitz are calculated for the cold-room temperature (ca. 5°) and differ considerably from the values of Dixon. 82~ M. Dixon, Biochem. J. 54, 457 (1953).

[2]

CHYMOTRYPSINOGENS AND CHYMOTRYPSIN8

19

Determination of Activity Activity of chymotrypsins can be determined b y a variety of methods which m a y be conveniently subdivided into two groups: (1) methods using natural substrates; (2) methods using synthetic substrates. B o t h types of method require dilute solutions of enzymes of accurately determined concentrations. Kunitz 33 introduced a very convenient method of expressing the concentration of pure proteins in dilute solutions b y measuring specific absorption at 280 mu. Kunitz's method is now a common practice in m a n y laboratories. I t was found convenient to express the specific absorption as the optical factor. The optical factor is defined as the reciprocal of the optical density at 280 m~, in a cell 1 cm. wide, when the concentration of protein is 1 mg./ml. The variation in absorption is comparatively small in the range of pH values from 2 to 6, and the factors are generally useful for this range. In establishing the optical factor, the purity of the preparation is of highest importance. Beside being homogeneous in regard to electrophoresis and sedimentation the preparation should be free from ultravioletabsorbing contaminants and should be corrected for the moisture content. The optical factors for chymotrypsins have been recently reinvestigated 2° and the following values were obtained. a-Chymotrypsinogen a-Chymotrypsin Chymotrypsinogen B Chymotrypsin B

0. 484 34determined at wavelength 282 mg, 0.500 34~ 0. 500,330.495,200.465 34~ 0.55(0. 546)30 0.54(0. 538)20

The solution of a desired enzyme concentration is prepared b y first making a solution in the range from 0.1 to 0.35 mg./ml. The exact concentration of this solution is then determined by its absorption at 280 mg, and the solution of a desired concentration is prepared b y an appropriate dilution. The majority of methods using the natural substrates are based on determination of the rate of proteolysis, and often the same m e t h o d can be applied to several proteolytic enzymes. The representative m e t h o d of this t y p e is the spectrophotometric method of Kunitz, 33 in which casein serves as substrate. T h e method was originally devised for the determination of trypsin and has been later applied to the determination of trypsin 33 M. Kunitz, J. Gen. Physiol. 30, 291 (1947). a~ M. A. Eisenberg and G. W. Schwert, J. Gen. Physiol. 34, 583 (1951). 34~p. E. Wilcox (unpublished), quoted from H. Neurath, J. A. Gladner, and E. W. Davie, "The Mechanism of Enzyme Action" (W. D. McElroy and B. Glass, eds.), pp. 50-69, Johns Hopkins Press, Baltimore, 1954.

20


[2]

inhibitors and chymotrypsins. The original method is described in detail in the next section on trypsin (see p. 32). This method is recommended for the purpose of following the purification of either an enzyme or an inhibitor. In respect to chymotrypsins, in the reviewer's laboratory 20 this method was modified in that 0.1 M borate buffer, pH 8.0, was substituted for the phosphate buffer, and CaC1236 was added to attain a final concentration of 0.005 M in the enzyme-substrate mixture. 1.2

1.0 O 0O

c~ 0.8

0.6 0.4 0.2

o

1'o

2'0

'

~o

'

45

'

50

Chymotrypsin, 3, per ml. 0.5% casein

FIG. 2. Standard activity curves for chymotrypsins ~ and B. ~°

Standard curves for chymotrypsins a and B obtained under the above conditions are illustrated in Fig. 2. Indicated concentrations of enzyme represent the final concentrations in the enzyme substrate mixture. Since the total volume in this procedure is 2 ml., the actual amounts of enzyme pipetted into the tube are twice the amounts indicated in the graph. Some of the methods utilizing the natural substrates are based on the ability of chymotrypsins to clot milk--the ability which was responsible for the original name of the enzyme and which distinguished it from trypsin. Several methods of the type have been described. Essentially the same methods are used for the determination of milk-clotting activity of rennin, pepsin, and chymotrypsin2 6 35 N. M. Green, J. A. Gladner, L. W. Cunningham, Jr., and H. Neurath, J. Am. Chem. Soc. 74~ 2122 (1952) found that calcium enhanced the activity of a-chymotrypsin. In unpublished work from this laboratory it was found that calcium accelerates also the rate of proteolysis by chymotrypsin B. With the casein method, a calcium concentration of 0.005 M is optimal for a-chymotrypsin and 0.0005 M is optimal for chymotrypsin B. With synthetic substrates or hemoglobin 0.1 M CaC12 is optimal for chymotrypsin B and 0.05 M for a-chymotrypsin. 8s See articles on pepsin and rennin, Vol. II [1, 7].

[2]


21

The introduction of simple synthetic substrates 37 for proteolytic enzymes must be credited to Bergmann and his co-workers2 8 The use of synthetic peptides made it possible to establish for each major proteolytic enzyme the peptide bonds susceptible to its action. F u r t h e r simplification of the substrate was achieved when it was found t h a t the amides 39 and esters t° of N-substituted aromatic amino acids are good substrates for chymotrypsins. The structural requirements for the specificity have been discussed in detail in the review of N e u r a t h and Schwert. 41 More recently Sprinson and Rittenberg 4~ have shown t h a t carbobenzoxy-L-phenylalanine, incubated in the presence of a-chymotrypsin in H20 Is, exchanges oxygen atoms of the carboxyl group with the medium, and that this reaction does not occur in the absence of a-chymotrypsin. D o h e r t y and T h o m a s 4~found t h a t a - c h y m o t r y p s i n is capable of hydrolyzing the C - - C bond in compounds of the type of ethyl 5-phenyl-3-ketovalerate with the formation of ethyl acetate and phenylpropionic acid. Only the methods employing synthetic substrates can be used when the specificity of the enzyme is to be determined. Similarly only these substrates can be used when the degree of contamination of c h y m o t r y p sin with trypsin is to be determined. 44 An additional advantage is t h a t zero-order kinetics persists for a longer period than with natural substrates. However, even with synthetic substrates the action of a-chymotrypsin m a y not be limited to the hydrolysis of a single susceptible bond, since transpeptidation (see Vol. I I [6]) has been shown to occur, a~-/ Numerous techniques, suggested for use with the synthetic substrates, were critically reviewed b y N e u r a t h and Schwert. 4° Two methods have 37 Methods of preparations of the typical substrates will be described by E. L. Smith in Vol. III [80]. 38See M. Bergmann, Advances in Enzymol. 2, 49 (1942). sq j. S. Fruton and M. Bergmann, J. Biol. Chem. 145, 253 (1942). 40G. W. Schwert, H. Neurath, S. Kaufman, and J. E. Snoke, J. Biol. Chem. 172, 221 (1948). 41 H. Neurath and G. W. Schwert~ Chem. Revs. 46, 69 (1950). 42D. B. Sprinson and D. Rittenberg, Nature 167, 484 (1951). 4a D. G. Doherty and L. Thomas, Federation Proc. 13, 200 (1954). a* Even with the synthetic substrates a careful selection must be made. Schwert et al.4O described a case of cross-reactivity with BAME (benzoyl-L-arginine methyl ester), a typical substrate for trypsin which was also susceptible to hydrolysis by a- and 7-chymotrypsins. See also J. A. Gladner and H. Neurath, Biochim. et Biophys. Acta 9, 335 (1952). ,4~ R. B. Johnston, M. J. Mycek, and J. S. Fruton, J. Biol. Chem. 187, 205 (1950). *4bj. S. Fruton, R. B. Johnston, and M. Fried, J. Biol. Chem. 190, 39 (1951). 44cM. Brenner, H. R. Mrtiler, and R. W. Pfister, Helv. Chim. Acta 33, 568 (1950). 44dM. Brenner, E. Sailer and K. Riifenacht, Helv. Chim. Acta 34, 2096 (1951). 44"H. Tauber, J. Am. Chem. Soc. 74, 847 (1952). 44! K. Blau and S. G. Waley, Biochem. J. 57, 538 (1954).

22


[2]

been added since t h a t time. Parks and Plaut 4s utilize the manometric technique (described in detail below), and Ravin et al. ~6 a colorimetric technique. The latter m e t h o d is based on the use of N-benzoyl-DL-phenylalanine-~-naphthyl ester. T h e hydrolyzed naphthol is coupled with tetrazotized diorthoanisidine; the resulting azo dye is extracted in ethyl acetate and determined colorimetrically. M a n y of the suggested techniques are applicable to the determination of b o t h c h y m o t r y p s i n and trypsin, provided t h a t appropriate substrates are used. Only a few of these methods have been employed in the reviewer's laboratory. T h e methods described below were chosen rather arbitrarily, and it is realized t h a t m a n y valuable methods have been omitted. Determination of the Amidase Activity of Chymotrypsins. 4° T h e reaction mixtures for determining amidase activity are made by mixing equal volumes of 0.1 M solution (or suspension) of substrate in 0.1 M phosphate buffer and of enzyme solution in phosphate buffer. 47 A stop watch is started, and the mixture is placed at 25 ° and shaken mechanically. At intervals 0.2-ml. samples are withdrawn for analysis and are introduced into the outer chambers of Conway plates, 48 which contain in the inner chamber 0.75 ml. of 2 % boric acid. One milliliter of saturated solution of K2C03 is added to the outer chamber to volatilize the ammonia. T h e m o m e n t at which the K 2 C Q solution touches the sample is considered as the end of the time interval for each withdrawn sample. The plates are allowed to stand for at least ] hour before being titrated with approximately 0.01 N HC1 and 1 drop of Tashiro's indicator. Thus, 0.01 ml. of acid corresponds to approximately 1% hydrolysis. Since the indicator color varies with the volume of the system at the end point, water is added to the plates in which the extent of hydrolysis is small in order to bring the final volume for all titrations close to a constant volume. The horizontal burets used for these titrations were made b y drawing out the ungraduated portions of Kimble Exax 1-ml. measuring pipets and fitting the other ends with Clay-Adams pipet suction units. Blank determinations are made b y placing 0.1 ml. each of substrate 45 R. E. Parks, Jr., and G. W. E. Plaut, J. Biol. Chem. 203, 755 (1953). 46 H. A. Ravin, P. Bernstein, and A. M. Seligman, J. Biol. Chem. 208, 1 (1954).

47If calcium is used in the system, borate buffer should replace the phosphate buffer. Since chymotrypsins are not very stable in dilute solutions at the pH of their optimal activity it is recommended to prepare the enzyme solution immediately before use, and to include 0.1 M CaCl2 in the borate buffer to increase the stability of chymotrypsins (unpublished). 48E. J. Conway, "Micro-Diffusion Analysis and Volumetric Error," Crosby, Lockwood and Son, London, 1939.

[2]


23

and enzyme solutions a short distance apart on Conway plates and by tipping the plates so that the solutions are mixed with the saturated K2C03 solution before they are mixed with each other. Typical substrates are glycyl-L-phenylalaninamide and acetyl-Ltyrosinamide. Potentiometric Determination of Esterase Activity of Chymotrypsin. This method was first described by Schwert et al. 4° The principle of the method, applicable to both trypsin and chymotrypsin, is a continuous titration of liberated carboxyl groups from an appropriate ester, at 25 °, using a potentiometer as a null instrument. This method is widely used with small modifications introduced in different laboratories. The following procedure is recommended by Balls and Jansen. 29 The reaction mixture of 20 ml., at pH 6.25, consists of sufficient L-tyrosine ethyl ester (TEE) to make it 0.025 M, sufficient NaC1 to make it 0.25 M, and routinely an amount of enzyme which would cause the liberation of 0.005 meq. of carboxyl groups per minute. The pH is maintained at 6.25 by addition of 0.02 N NaOH, and readings of the added alkali are recorded periodically. Under these conditions the reaction is of zero order. The potency of a chymotrypsin preparation is expressed in milliequivalents of carboxyl groups per milligram of enzyme or per milligram of enzyme nitrogen. When N-acetyl-L-tyrosine ethyl ester (ATEE) is the substrate, the procedure is essentially the same except that the final concentration of ATEE is 0.018 M and hydrolysis is allowed to proceed at pH 7.8 in the presence of 0.01 M phosphate buffer in place of NaC1. Since the hydrolysis of ATEE is of apparent first order, the results are calculated on the basis of the initial slope. In Desnuelle's laboratory 49 the assay for chymotrypsin is performed exactly as described for trypsin (see Vol. II [3]) except that the substrate is 0.1 M ATEE, and the solution of enzyme contains approximately 2 mg. of salt-free a-chymotrypsin per 100 ml. Since the reaction is of apparent first order, the concentration of substrate is critical. After the experimental points are plotted, the initial slope is used for calculations of activity and potency. Other typical substrates used for potentiometric titrations are carbobenzoxy-L-tyrosine ethyl ester, L-phenylalanine ethyl ester, and acetylL-phenylalanine ethyl ester. Manometric Assay of Esterase Activity. 4s This method has an advantage in that a series of determinations may be performed simultaneously, and that a zero-order reaction persists for a considerable period of time. ~9 M. Rovery, C. Fabre, and P. Desnuelle, Biochim. et Biophys. Acta 12, 547 (1953).

24

a-Chymotrypsinogen


[2]

TABLE I MOLECULAR WEIGHTS 36,000 Osmotic pressure a 22,000 Sedimentation-diffusion b 23,000 Sedimentation-diffusion c 22,500 Sedimentation-diffusion ~ 24,000 Osmotic pressure *

a-Chymotrypsin

40,000 32,000 17,500 21,500 22,500 27,000 23,000

Osmotic pressuref X-rayg Sedimentation-diffusion b Sedimentation-diffusion h Sedimentation-diffusion d Light scattering ~ Calculated from nitrophenol Released in preparation of E 600 derivativei

DP-a-Chymotrypsin

27,000 27,500 24,800

Light scattering k Osmotic pressure k From P content ~

DP-t~-Chymotrypsin DP-fl-Chymotrypsin

30,000 23,100

Osmotic pressures From P content ~

~-Chymotrypsin

DP-~,-Chymotrypsin

27,000 30,100 15,500 25,800

Osmotic pressurel X-rayo Sedimentation-diffusion b From P content k

Chymotrypsinogen B

22,500

Sedimentation-diffusion ~

Chymotrypsin B 22,500 Sedimentation-diffusion d a M. Kunitz and J. H. Northrop, J. Gen. Physiol. 18, 433 (1935). b G. W. Schwert, J. Biol. Chem. 179, 665 (1949). c G. W. Schwert, J. Biol. Chem. 190, 779 (1951). E. L. Smith, D. M. Brown, and M. Laskowski, J. Biol. Chem. 191, 639 (1951). e H. Gutfreund, Trans. Faraday Soc. 50, 624 (1954). s M. Kunitz, J. Gen. Physiol. 22, 207 (1938). a I. Fankuchen, in "Proteins, Amino Acids and Peptides as Ions and Dipolar I o n s " (E. J. Cohn and J. T. Edsall, eds.), Reinhold Publishing Corp., New York, 1943. h G. W. Schwert and S. Kaufman, J. Biol. Chem. 190, 807 (1951). K. J. Palmer, quoted from Balls and J a n s e n ) i B. S. Itartley and R. A. Kilby, Biochem. J. 56, 288 (1952). A. K. Balls and E. F. Jansen, Advances in Enzymol. 13, 321 (1952).

[2]


25

T h e a s s a y is p e r f o r m e d in 15-ml., 5° single side a r m W a r b u r g vesselh in a t o t a l fluid v o l u m e of 3 ml. a t 30 °. T h e e n z y m e s o l u t i o n is p l a c e d in t h e side a r m , a n d its final v o l u m e is b r o u g h t t o 0.3 ml. w i t h w a t e r . I t is a d v i s a b l e t o use c a r e f u l l y c a l i b r a t e d m i c r o p i p e t s t o d i s p e n s e t h e e n z y m e , since t h e v o l u m e is c r i t i c a l in o b t a i n i n g t h e m o s t a c c u r a t e results. I f t h e e n z y m e s o l u t i o n t o b e a s s a y e d c o n t a i n s a p p r e c i a b l e a m o u n t s of acid, i t is n e c e s s a r y t o b r i n g i t t o p H 6.5. TABLE II ISOELECTRIC POINTS

a-Chymotrypsinogen

5.0 6.3 9.5 9.1

Cataphoresis a Donnan equilibrium b Electrophoresis in 0.01-t` buffers ~ Electrophoresis in 0.1-# buffers d

a-Chymotrypsin

5.4 8.6 8.1 8.3 8.5

Cataphoresis a Electrophoresis in Electrophoresis in Electrophoresis in Electrophoresis in

~-Chymotrypsin ~,-Chymotrypsin

8.6 8.5

Electrophoresis in 0.05-t` buffers' Electrophoresis in 0.05-t~ buffers"

Chymotrypsinogen B

5.2

Electrophoresis in 0.1-• buffers d

0.01-t` buffers c 0. l-t` buffers c 0.l-t, buffers e 0.05-t` buffers'

Chymotrypsin B 4.7 Elcctrophoresis in 0.l-t` buffers d M. Kunitz and J. H. Northrop, J. Gen. Physiol. 18, 433 (1935). b V. M. Ingrain, Nature 170, 250 (1952). c A. E. Anderson and R. A. Alberty, J. Phys. and Colloid Chem. 52, 1345 (1948). V. Kubacki, K. D. Brown, and M. Laskowski, J. Biol. Chem. 180, 73 (1949). R. Egan, personal communication. The isoelectrie points of a- and ~-chymotrypsins are apparently identical, and that of fl form is very similar. The mobilities in more acid or more alkaline regions are different for each of these three chymotrypsins, a-Chymotrypsin on prolonged electrophoresis exhibits some heterogeneity. T h e m a i n c o m p a r t m e n t c o n t a i n s t h e s u b s t r a t e a n d b i c a r b o n a t e solution. L - P h e n y l a l a n i n e e t h y l e s t e r h y d r o c h l o r i d e ( P h E E ) , t h e m o s t useful of t h e s u b s t r a t e s t e s t e d , is a d d e d in 2.0 ml. of 0.0375 M s o l u t i o n (prep a r e d a n d a d j u s t e d t o p H 6.5). T h e n 0.7 ml. of a n 0.18 M s o l u t i o n of s o d i u m b i c a r b o n a t e is a d d e d . F i n a l c o n c e n t r a t i o n s of 0.025 M for P h E E a n d 0.042 M for s o d i u m b i c a r b o n a t e a r e t h u s o b t a i n e d . T h e p H of t h i s c o n c e n t r a t i o n of b i c a r b o n a t e w i t h an a t m o s p h e r e of 1 0 0 % c a r b o n d i o x i d e is 6.5. A c o n t r o l vessel c o n t a i n i n g no e n z y m e m u s t b e i n c l u d e d in t h e 50 In this laboratory 12-ml. vessels with a side arm blown up to contain 1 ml. of fluid are used.

26


[3]

set, since the s u b s t r a t e alone shows a slight b u t measurable rate of hydrolysis. After a 10-minute period of gassing with 100% carbon dioxide followed b y a 5-minute period of t e m p e r a t u r e equilibration in the w a t e r bath, the e n z y m e is tipped into the m a i n c o m p a r t m e n t . I t has been found advisable to t a k e the zero readings after 2 or 3 minutes. T h e reaction is linear with t i m e until a b o u t 6 0 % of the ester is h y d r o lyzed. T h e rate of COs evolution is proportional to the concentration of e n z y m e over a range 10 to 100 ~,/ml. (best 20 to 70 ~,/ml.).

[3] Trypsinogen and Trypsin B y M. LASKOWSKI

T r y p s i n ~,~ and trypsinogen 3 were obtained in crystalline form from beef pancreas b y N o r t h r o p and Kunitz. F u r t h e r work 4 has i m p r o v e d m e t h o d s of p r e p a r a t i o n and established t h a t the t r a n s f o r m a t i o n of trypsinogen i n t o trypsin is a proteolytic process and m a y be accomplished 5 either b y autocatalysis or b y enterokinase, or b y the kinase f r o m a mold of the genus P e n i c i l l i u m . ~ T h e m e c h a n i s m of activation of trypsinogen has been studied in detail. 7-9 T h e t r a n s f o r m a t i o n is not quantitative, since in addition to trypsin an " i n e r t p r o t e i n " is formed. T h e relative a m o u n t of " i n e r t p r o t e i n " depends on the p H and the presence of other ions in the m e d i u m in which a c t i v a t i o n occurs. ~° The f o r m a t i o n of the " i n e r t prot e i n " is suppressed b y calcium ions. 1° T h e latter finding led to an improved m e t h o d of crystallization of trypsin. ~1 Evidence has been presented ~,~3 t h a t during the a c t i v a t i o n of t r y p sinogen a peptide is split off the N terminal end of the trypsinogen mole1j. H. Northrop and M. Kunltz, Science 73, 262 (1931). J. H. Northrop and M. Kunitz, J. Gen. Physiol. 16, 267 (1932). a j. H. Northrop and M. Kunitz, Science 80, 505 (1934). M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). 6 M. Kunitz and J. H. Northrop, Science 80, 190 (1934). 8 M. Kunitz, J. Gen. Physiol. 21, 601 (1938). 7 M. Kunitz, J. Gcn. Physiol. 22, 293 (1939). s M. Kunitz, J. Gen. Physiol. 22, 429 (1939). g M. Kunitz, Enzymologia 7, 1 (1939). 10 M. R. McDonald and M. Kunitz, J. Gen. Physiol. 26, 53 (1941). 11 M. R. McDonald and M. Kunitz, J. Gen. Physiol. 29, 155 (1946). ~ E. W. Davie and H. Neurath, Biochim. ct Biophys. Acta 11, 442 (1953). 1~ M. Rovery, C. Fabre, and P. Desnuelle, Biochim. et Biophys. Acta 12, 547 (1953).

[3]

TRYPSINOGEN AND TRYPSIN

27

cule. A tentatively suggested '2 composition of the peptide is H2N Val. (Asp) 5ore-Lys. COOH. 13~ Crystallization of Trypsinogen 4 Beef pancreas is treated exactly as described in Vol. II [2] up to the crystallization of a-chymotrypsinogen.13b The mother liquor and the washings from the crystallization of a-chymotrypsinogen are adjusted to pH 3.0 (pink with 0.01% methyl orange on a test plate) with about 1 ml. of 5 N H2S04 per 100 ml. of filtrate. Solid ammonium sulfate is then added (30.4 g. per 100 ml.), the precipitate which forms is collected on a hardened paper, and the filtrate is rejected. Each 10 g. of the precipitate is dissolved in 30 ml. of water and is treated with 20 ml. of saturated ammonium sulfate and 2 g. of Filter-Cel. The mixture is filtered with suction through a soft paper, and the precipitate is washed with 0.4 saturated ammonium sulfate and is discarded. The volume of the filtrate is measured, and an equal volume of saturated ammonium sulfate solution is added. The mixture is filtered with suction through a hardened paper on a large Bfichner funnel (18.5 cm.) until the precipitate is quite hard (the cracks which appear are worked out with a spatula). A saturated solution of magnesium sulfate in 0.02 N H2S04 is then poured over the precipitate to form a layer of about 5 mm. and is allowed to remain on the filter for 1 to 2 minutes. After this, the funnel is removed, and the magnesium sulfate solution is poured off by tipping the funnel. The filtration is continued until the rest of the washing solution is removed. This precipitate is referred to as "crude trypsinogen" and may be used for either the purification of trypsinogen or for the activation to trypsin, followed by the isolation of the trypsin inhibitor-trypsin complex. Crude trypsinogcn (30 g.) is dissolved in 30 ml. of 0.4 M borate 14 buffer, pH 9.0, at 2 to 5° (in an ice-water bath), and saturated solution of potassium carbonate is added dropwise until the pH is brought to 8.0. The volume of the solution is measured, and an equal volume of a saturated solution of magnesium sulfate is added. The mixture is allowed to stand in the cold room. Short triangular prisms of trypsinogen appear in the course of 2 to 3 days. If the solution is inoculated with crystals of 13~ I n a personal communication to the reviewer Dr. N e u r a t h stated t h a t E. W. Davie and H. N e u r a t h [J. Biol. Chem. 212, 515 (1955)] established the n u m b e r of aspartie acid residues as four. 13b Unless otherwise specified all operations are carried out a t room t e m p e r a t u r e (20 to 25°). 14 Stock borate solution contains 49.6 g. of boric acid a n d 80 ml. of 5 N sodium hydroxide per 1000 ml. of solution. Borate buffers, 0.4 M, p H 8.0 a n d 9.0, are mixtures of 100 p a r t s of stock borate a n d 78.6 a n d 17.6 parts of 0.4 M hydrochloric acid, respectively.

28


[3]

trypsinogen, crystallization is much more rapid but the crystals are not so well formed. If the crystallization is delayed for more than 4 to 5 days, crystals of trypsin may appear. The crystals are filtered at 5° . The precipitate is washed on the funnel several times with cold 0.5 saturated magnesium sulfate made up in 0.1 M borate buffer and finally with saturated magnesium sulfate made up in 0.1 N H2S04 at room temperature. The crystals are then dried in an electric refrigerator at 5 ° and stored in the icebox. The dried material generally contains about 40% of trypsinogen protein and 60% of magnesium sulfate. No safe method for the recrystallization of trypsinogen can be recommended. Direct recrystallization leads to a mixture of trypsinogen and trypsin. Recrystallization has been accomplished in the presence of an excess of the pancreatic trypsin inhibitor. 4 This method is expensive owing to the high price (or low yield) of the pancreatic trypsin inhibitor (for the method of preparation, see Vol. II [4]). If this method is used the trypsin content of crystalline trypsinogen should be determined and an equal weight of the inhibitor (equivalent to a twofold excess) should be added. The recrystallized trypsinogen is then treated as follows, s Ten grams of trypsinogen crystals is dissolved in 200 ml. of N/400 HC1, and 200 ml. of 5 % trichloroacetic acid is added. The solution is left at 20° for 1 hour and then filtered with suction and washed several times with small amounts of 2.5% trichloroacetic acid and finally with water. The semidry precipitate is dissolved in twenty-five times its weight of N/50 HC1 and allowed to stand for about 30 minutes. Ammonium sulfate (242 g./1.) is added to attain 0.4 saturation. The precipitate is filtered off and rejected. The filtrate is brought to 0.7 saturation with solid ammonium sulfate (205 g./1.) and filtered with suction. Yield is about 30%. Tietze 15 recently described a method which led to a preparation of recrystallized trypsinogen, almost free from trypsin (0.016%). However, the author does not recommend his procedure for the routine preparations, since failures have been encountered. In Tietze's procedure 0.25 ml. of pure diisopropyl fluorophosphate (DFP) is added to a solution of 96 g. of "crude trypsinogen" in 96 ml. of 0.4 M borate buffer, pH 9, in order to inhibit any of the free trypsin present or forming. As in the procedure of Kunitz and Northrop 4 the pH is adjusted to 8.0 with saturated potassium carbonate, an equal volume of saturated magnesium sulfate is added, and the mixture is allowed to stand for 48 hours in the cold room. The crystalline trypsinogen (7.8 g.) is collected and then dissolved in 70 ml. of water, containing 0.003 ml. of DFP per milliliter. The pH is adjusted to 3.0, and a small amount of insoluble matter is removed by filtration through hardened filter paper. 15 F. Tietze, J. Biol. Chem. 204, 1 (1953).

[3]


29

The solution is dialyzed against 0.001 N HC1 and lyophilized. Trypsinogen is recrystallized in the following manner. Each gram of the lyophilized protein is dissolved in 4 ml. of ice-cold 0.4 M borate buffer, pH 9.3, and filtered in the cold. The pH is adjusted to 8.0 by a dropwise addition of 5/V H2S04, and an equal volume of saturated magnesium sulfate solution is added. The mixture is exposed to room temperature for a period of 24 hours, which results in formation of a copious crystalline precipitate. Crystallization of Trypsin In the method of Kunitz and Northrop 4 trypsin was obtained by activation of the crystalline trypsiuogen. The yield was small, however, owing to a side reaction leading to the formation of "inert protein." 1V[cDonald and Kunitz ~1 improved the yield by introducing calcium ions into the activating mixture. If trypsin, and not trypsinogen, is the desired product, the best yields are obtained in a minimum time by following the method of Kunitz and Northrop up to the stage of "crude trypsinogen" and activating the crude trypsinogen according to McDonald and Kunitz, as described below. Fifty grams of "crude trypsinogen" (or 30 g. of crystalline trypsinogen) is dissolved in 200 ml. of 0.005 N HC1. This solution is added to a previously prepared ice-cold mixture of 100 ml. of 1 M calcium chloride, 250 ml. of 0.4 borate buffer, ~4 pH 8, and 400 ml. of distilled water. 15~ The volume of the mixture is adjusted to 1000 ml. with cold water, and the mixture is allowed to stand for 24 hours in the cold room. The solution is treated with 2 g. of Standard Super-Cel and is filtered in the cold room through soft paper. The precipitate is rejected. The filtrate is adjusted to pH 3.0 (tested with methyl orange on a spot plate) with 5 N H2SO4 (about 4 ml.). Solid ammonium sulfate is added (242 g./1.) to 0.4 saturation, and the mixture is left in the cold room for 2 days. A heavy sediment of calcium sulfate crystals is formed. They are removed by filtration through a soft (Whatman No. 3) filter paper. The precipitate is rejected. The filtrate is brought to 0.7 saturation by addition of 205 g. of ammonium sulfate per liter, and the mixture is filtered with suction through the hardened filter paper. The filtrate is rejected. The filter cake (about 50 g.) is dissolved in 150 ml. of distilled water, and trypsin is reprecipils~ McDonald and Kunitz 11 used crystalline trypsinogen which always contains sufficient amounts of active trypsin to start the autocatalytic reaction. If the "crude trypsinogen" containing an excess of the trypsin inhibitor is the starting material, an activating agent (excess of trypsin, or enterokinase) should be added, otherwise trypsinogen, and not trypsin, would be crystallized. In the reviewer's laboratory 200 E.K.U. of enterokinase (5 mg. of the purified preparation) per 100 g. of crude trypsinogen is used.

30


[3]

tated b y a slow addition of 350 ml. of saturated ammonium sulfate from a dropping funnel, while the mixture is mechanically stirred. The mixture is filtered with suction through a piece of hardened paper 18.5 cm. in diameter or larger, and the precipitate is washed as described for the "crude t r y p s i n o g e n " with saturated magnesium sulfate in 0.02 N H2S04 in order to remove the excess of ammonium sulfate. The semidry filter cake is dissolved in ice-cold 0.4 M borate buffer, p H 9.0 (10 ml. per 10 g. of filter cake), ~6 in an ice-water bath. Crystals of fine needles appear almost immediately. The mixture is left in the cold room for 24 hours and is filtered in the cold on hardened paper with suction. The crystals are washed with cold 0.5 saturated magnesium sulfate in 0.1 M borate buffer, pH 8.0, in the cold room and then at room temperature with saturated magnesium sulfate in 0.1 N H~S04. The yield is 15 to 20 g. The m o t h e r liquor (filtrate E) and the washings from the crystallization of trypsin are combined and saved for the preparation of pancreatic trypsin inhibitor-trypsin complex (see Vol. I I [4]). RecrystaUization of Trypsin. 4 T h e semidry filter cake is dissolved in 0.02 N H2S04 (10 ml. per 10 g. of filter cake) b y mixing it gradually into the acid to avoid foam. I t is filtered on a small W h a t m a n No. 3 fluted paper, which is washed with several milliliters of 0.02 N H2S04. The filtrate is cooled to a b o u t 5 ° and is adjusted to p H 8.0 (pink to 0.01% phenol red, b u t not to 0.01% cresol red on a spot plate) with cold 0.4 M borate buffer, p H 9.0 (about 8 ml. per 15 ml. of solution). Crystallization begins almost immediately. The mixture is left in the cold room for 24 hours. T h e precipitate is collected and is washed as described for the first crystallization. T h e yield is about 10 to 12 g. The filter cake is allowed to d r y in the refrigerator, after which it is ground to a fine powder and stored in the cold room. Purification of Trypsin by Trichloroacetic Acid. 4 When first crystallized, trypsin sometimes has a low specific activity, owing p a r t l y to the presence of some inhibitor; the activity m a y be raised to the m a x i m u m value b y repeated recrystallizations or b y precipitation with trichloroacetic acid followed b y crystallization. Ten grams of crystalline filter cake of trypsin is dissolved in 100 ml. of water, and 100 ml. of 5 % solution of trichloroacetic acid is added. T h e 16In this laboratory, occasionally, such proportions resulted in a pH of the final solution lower than 8.0, owing to the acid retained from the wash solution. If that occurs, saturated potassium carbonate is added until pH 8.0 is reached, followed by solid magnesium sulfate to start crystallization (not more than 0.36 g./ml, of solution). A preferred alternative is to thoroughly stir the precipitate with one-half of the required volume, adjust the pH to 8 with potassium carbonate, and bring up to volume with pH 8.0 buffer.

[3]


31

mixture is allowed to stand at room temperature for 30 minutes, until precipitation is about complete. It is filtered with suction; the precipitate contains trypsin, and the filtrate contains trypsin inhibitor. The precipitate is washed with water to remove the free acid. Each gram of the precipitate is dissolved in 20 ml. of 0.02 2V HC1 and allowed to stand at room temperature for 30 minutes, after which 5 g. of solid ammonium sulfate is added (per each 20 ml. of 0.02 ~V HC1 used). The mixture is filtered through Whatman No. 42 fluted filter paper until clear. The precipitate is rejected. An additional 5 g. of ammonium sulfate is dissolved (per each 20 ml. of solvent used), and the precipitate which forms is collected with suction on hardened filter paper. The filtrate is rejected. The precipitate is washed on the paper with saturated magnesium sulfate in 0.02 N H2SO4 (as described for the "crude trypsinogen"). Each gram of the precipitate is dissolved in 0.5 ml. of water, cooled to about 5°, and about 0.5 ml. of 0.4 M borate buffer, pH 9.0, is added to bring the pH of the solution to 8.0 (pink to 0.01% phenol red but not to 0.01% cresol red on a test plate). The volume is measured, and an equal volume of saturated solution of magnesium sulfate is added. The mixture is allowed to stand at 5° for 24 hours. Typical crystals of trypsin are collected and washed as previously described. The yield is low, about 0.3 g./g. o'f the original trypsin. Preparation of Purified Enterokinase 17

The contents of duodena of freshly killed swine are collected by gentle squeezing. Then 2.5 1. of duodenal contents is diluted with 7.5 1. of tap water at room temperature. Next 450 g. of Hyflo Super-Cel is added, and the whole mass is filtered with suction through filter cloth on a large Btichner funnel. The first extract is saved. The residue is resuspended in 3 1. of tap water and refiltered through cloth. The second extract is combined with the first, and the residue is discarded. The combined extracts are cooled to 5° and are adiusted with 5 iV H~SO4 to pH about 4.0 (tested with methyl orange). The precipitate formed is filtered off rapidly with suction with the aid of 20 g. of Standard Super-Cel per liter of solution. The filtrate is brought immediately to pH 8.0 with 5 iV NaOH. Solid ammonium sulfate (530 g./1.) is added to bring the filtrate to 0.8 saturation. The pH of the solution is again adiusted to pH 8.0 with 5 N NaOH. Four milliliters of 0.4 M borate buffer, pH 9.0, is then added to every liter of solution. The formed flocculant precipitate is allowed to rise to the surface and is then easily collected into a doughlike mass and removed from solution. The weight of the precipitate is about 20 g. 17M. Kunitz, J. Gen. Physiol. 22, 447 (1939).

32


[3]

The precipitate is dissolved in about 5 vol. of cold water, and solid ammonium sulfate (24.2 g. per 100 ml.) is added to 0.4 saturation. The mixture is filtered with suction with the aid of 5 g. per 100 ml. of Standard Super-Cel. The residue is rejected. The filtrate from 0.4 saturated ammonium sulfate is brought to 0.8 saturation with solid ammonium sulfate and filtered with suction. The filtrate is discarded, and the precipitate is once more fractionated between 0.4 and 0.8 saturation as described above. The yield is about 10 g. Activity. The activity of enterokinase is determined as follows. 8 Into a 50-ml. volumetric flask are pipetted 5 ml. of 0.065% solution of trypsinogen in 0.005 N HC1 and 10 ml. of 0.1 M phosphate buffer, pH 5.8. The solution is left at 5 ° to equilibrate. One milliliter of enterokinase solution and precooled water to make up the volume are added. The flask is allowed to stand at 5 °. Aliquots are withdrawn at hourly intervals, and the amount of activated trypsin is determined by any suitable method. The reaction is of apparent first order. One enterokinase unit (1 E.K.U.) is defined as that amount of kinase that brings about the activation of 0.065 mg. of crystalline trypsinogen in 0.02 M S6rensen's phosphate buffer, pH 5.8, per hour at 5°.

Determination of Tryptic Activity Methods for the determination of trypsin are similar to those used for the determination of chymotrypsins (see Vol. II [2]), with the exception that synthetic substrates are amides and esters of basic amino acids. The range of optimal activity, similar to that of the chymotrypsins, lies between pH 7.0 and 8.0, but the activity can be measured over a wider range. At the range of the optimal activity trypsin is unstable. Gorini TM and Bier and Nord 19 independently found that the stability of trypsin is enhanced in the presence of calcium ions. These findings were extended to several other cations. ~°,21 The effect is attributed to the stabilization of the active form of trypsin. The enhanced stability is manifested as an increased activity in a majority of the systems investigated. However, no increase was found in the rate of proteolysis of protamine or in the rate of activation of a-ehymotrypsinogen.2° Recently the nitrogen content of trypsin was reported 2° as 15.0%. A correction for the optical factor (see section on chymotrypsins, Vol. II [2]), which was originally reported 22 as 0.585, was also introduced 2° as 18 L. Gorini, Biochim. el Biophys. Acta 7, 318 (1951). 19 M. Bier a n d F. F. Nord, Arch. Biochem. and Biophys. 33, 320 (1951). s0 N. M. Green a n d H. N e u r a t h , J. Biol. Chem. 204, 379 (1953). 2i W. G. Crewther, Australian J. Biol. Sci. 6, 597 (1953). ~2 M. :Kunitz, J. Gen. Physiol. 30, 291 (1947).

[3]


33

0.695. In this laboratory the optical factor was determined on a preparation of DP-trypsin, kindly sent to us b y Dr. E. F. Jansen, and was found to be 0.67, in fairly good agreement with the value of Green and Neurath. 20

Determination of Activity by the Casein Digestion Method (Kunitz22). A stock solution of casein is made b y suspending 1 g. of casein (preferably " H a m m a r s t e n " ) 23 in 100 ml. of 0.1 M S6rensen phosphate buffer, pH 7.6. 24 The suspension is heated for 15 minutes in boiling water, 25 thus bringing about a complete solution of the casein. This 1% casein solution IO-~(T.UJ.... 0.6 0

20

40

I

60

i

80 i

'

0.5 0

~ 0.4

~'0.3 o

~

~

-0.2

,y "

c,,s

0.25

-3

IlK, I I It

0

n

I

I

a

I

I

0.004 0.008 0.012 Trypsin protein, mg. per rnl. 0.5% casein

FIG. 1. Standard activity curve for trypsin according to Kunitz. 22

is stored in a refrigerator and is stable for at least a week. Prior to use, the casein solution is placed in a water bath at 35 ° for at least 5 minutes. A solution of crystalline trypsin (or a solution in which tryptic activity is to be determined, or a mixture of trypsin and inhibitor) is pipetted into 15-ml. pyrex centrifuge tubes. 2~ The volume of the enzyme solution in each tube is brought to 1 ml. with an appropriate buffer, and the tubes are placed in the water bath. One milliliter of casein solution is pipetted into the first tube, and a stop watch is started. Each subsequent tube receives 1 ml. of casein at 30-second intervals. Three milliliters of 5 % trichloroacetic acid (TCA) is added in the same order to each test tube ex~3 Casein prepared according to M. S. Dunn [Biochem. Preparations 1, 22 (1949)] is recommended. 24 If calcium is to be used in the system, 0.2 M borate buffer containing 0.005 M CaCl~ is recommended. 25 With casein prepared according to Dunn, 8-minute heating was found sufficient. ~8 I t was found convenient to use 15-ml. Lucite tubes for the Servall SS-1 centrifuge. The time of standing after addition of TCA can be reduced to 20 minutes, and the time of centrifugation to 7 minutes a t 8000 r.p.m.

34


[3]

actly 20 minutes after the addition of casein. The content of the tubes is mixed well. The tubes are removed from the bath, allowed to stand for an hour, and centrifuged for 20 minutes. The optical density of the supernatants is read at 280 m~. The readings are corrected for the values of blanks. The blanks are prepared by first mixing 1 ml. of casein solution with 3 ml. of TCA solution, and then adding 1 ml. of the highest concentration of enzyme used, or 1 ml. of the buffer used in making up the trypsin dilutions. The corrections for blanks for the intermediate concentrations of trypsin are calculated by interpolation. The standard curve for trypsin is shown in Fig. 1. The abscissa is expressed in two scales: as concentration (milligrams per milliliter, calculated on the basis of the optical factor of 0.585) of the standard trypsin as prepared by Kunitz, 22 and in Kunitz's tryptic units. Determination of Trypsin by the Hemoglobin Digestion Method of Anson. ~ In this method denatured hemoglobin is the substrate. It is digested for 5 or 10 minutes (at 37 ° or 25°), the reaction is stopped by the addition of trichloroaeetic acid, and the nondigested hemoglobin is removed by filtration. The amount of split products remaining in solution is determined colorimetrically by means of the phenol reagent, or spectrophotometrically. Either a crystalline hemoglobin, or ~ hemoglobin prepared according to Anson (see Vol. II [1]), is used. First 2.2 g. of hemoglobin is placed in a 100-ml. volumetric flask, half filled with water, 36 g. of urea and 8 ml. of 1 ~V NaOH are added, and the solution is made up to volume with water. The alkaline solution is kept at room temperature for 30 to 60 minutes to denature the hemoglobin and is then mixed with 10 ml. of 1 M potassium dihydrogen phosphate ~8 and 4 g. of urea. The final pH is 7.5. Two milligrams of Merthiolate (Lilly) is added as a preservative, and the solution is stored at 5° . The procedure for the determination of trypsin is identical with that for pepsin (see Vol. II [1]), except that after addition of trichloroacetie acid the solution is allowed to stand for 30 minutes before filtration. Methods of determination of tryptic activity with synthetic substrates are based on determining either the amidase or the esterase activity. The amidase activity of trypsin is conveniently determined by the method of Schwert et al., 29 the details of which have been described in ~7 M. L. Anson, J. Gen. Physiol. 22, 79 (1938). ~8 If calcium is to be used in the system, borate buffer should be used plus enough HCI to bring p H of the mixture to 7.5. ~9 G. W. Schwert, H. Neurath, S. Kaufman, and J. E. Snoke, J. Biol. Chem. 172, 221 (1948).

[3]


35

TABLE I ISOELECTRIC POINT OF TRYPSIN

ca. 7.0 ca. 11.0 10.8

Cataphoresis a Electrophoresis b Electrophoresis in the presence of Ca, two components at p H range 3-7 c

M. Kunitz and J. H. Northrop, J. Gen. Physiol. 16, 295 (1935). b M. Bier and F. F. Nord, Arch. Biochem. and Biophys. 33, 320 (1951). c F. F. Nord and M. Bier, Biochim. et Biophys. Acta 12~ 56 (1953).

TABLE II MOLECULAR WEIGHT OF TRYPSINOGEN AND TRYPSIN

Trypsinogen

23,700

Sedimentation-diffusion a

Trypsin

36,500 35,000 41,000 (dimer) 15,100 30,600-34,000 24,000 17,000 24,000

Osmotic pressure b Spread monolayers c Spread monolayers d Sedimentation-diffusion, By deutron and electron bombardments Light scatteringg Osmotic pressure h Calculated from 1 : 1 ratio with soybean inhibitov

DP-trypsin

20,700 21,400-24,800 24,000 23,800

Calculated from P contentJ Calculated from P content ~ Sedimentation-diffusion ~.~ Sedimentation-diffusion"*

a F. Tietze, J. Biol. Chem. 204, 1 (1953). b M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). c H. B. Bull, J. Biol. Chem. 185, 27 (1950). d E. Mishuck and F. Eirich, J. Polymer Sci. 7, 341 (1951). • V. G. Bergold, Z. Naturforsch. 1, 100 (1946). I E. Pillard, A. Buzzell, C. Jeffreys, and F. Forro, Jr., Arch. Biochem. and Biophys. 33, 9 (1951). R. F. Steiner, Arch. Biochem. and Biophys. 49, 71 (1954). h H. Gutfreund, Trans. Faraday Soc. 50, 624 (1954). ' M. Kunitz, J. Gen. Physiol. 30~ 291 (1947). J E. F. Jansen and A. K. Balls, J. Biol. Chem. 194, 721 (1952). k L. W. Cunningham, Jr., F. Tietze, N. M. Green, and H. Neurath, Discussions Faraday Soc. 13, 58 (1953). z F. F. Nord and M. Bier, Biochim. et Biophys. Acta 12, 56 (1953). "~L. W. Cunningham, Jr., J. Biol. Chem. 211, 13 (1954).

36


[4]

Vol. II [2]. For the determination of trypsin the authors used arginine derivatives: a-benzoylargininamide (BAA) and a-p-toluensulfonyl-L-argininamide (TSAA). Other substrates which have been used are a-benzoyl-L-lysinamide (BLA), a-p-toluensulfonyl-L-lysinamide (TSLA), and a-hippuryl-L-lysinamide (HLA). The potentiometric determination of esterase activity of trypsin is carried out by the method of Schwert et al. 2" according to Rovery et al. 13 The buffer solution of pH 7.9 is 0.005 M in respect to tris(hydroxymethyl)aminomethane, 0.04 M in respect to NaC1, and 0.02 M in respect to CaC12. Eight milliliters of this solution is placed in a small beaker, and 1 ml. of 0.1 M benzoyl-L-arginine ethyl ester (BAEE) is added. The beaker is placed in a 25 ° water bath, and the electrodes of a (Beckman Model G) potentiometer and a small mechanical stirrer are introduced into the liquid. With the aid of a horizontal buret, 30 0.1 N NaOH is added to adjust the pH to 8.0. One milliliter (100 7) of trypsin solution (approximately 10 mg. of trypsin containing 50 % of magnesium sulfate per 50 mh of 0.001 iV HC1) is added. The pH of the reaction mixture decreases. The stop watch is started when the pH of 7.9 is reached. At that time approximately 0.01 ml. of 0.1 N NaOH (free from carbonate) is added, and the time at which the pH 7.9 is again reached is recorded. After five or six repetitions the volume of NaOH added is plotted versus time. A straight line is obtained. The slope of this line represents the activity. The potency of the preparation of trypsin could be expressed by dividing the activity by either the micrograms of enzyme protein (which can be calculated from the optical density) or the micrograms of enzyme nitrogen. Other commonly used substrates are a-p-toluensulfonylL-arginine methyl ester (TSAME), a-benzoyl-L-arginine methyl ester (BAME), and L-lysine ethyl ester (LEE). 80See section on chymotrypsin, Vol. II [2].

[4] N a t u r a l l y O c c u r r i n g T r y p s i n I n h i b i t o r s B y M. LASKOWSKI

The following naturally occurring trypsin inhibitors will be discussed: (1) pancreatic inhibitor of Kunitz and Northrop, 1 (2) a second inhibitor from pancreas crystallized by Kazal, Spicer, and Brahinsky, 2 (3) soybean 1 M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19~ 991 (1936). L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, J. Am. Chem. Soc. 70, 3034 (1948).

[4]

NATURALLY OCCURRING TRYPSIN INHIBITORS

37

inhibitor, 3,4 (4) c o l o s t r u m inhibitor, s,6 (5) lima b e a n inhibitor, 7,8 (6) ovomucoid, 9-11 (7) blood p l a s m a inhibitor, 12 and (8) inhibitor f r o m A s c a r i s 2 3 T h e n a t u r a l l y occurring t r y p s i n inhibitors h a v e been r e c e n t l y reviewed 14 in regard to their m o d e of action and properties. All inhibitors isolated so far a p p e a r to be proteins. ~5 M o s t of t h e m (Nos. 1, 2, 4, 5, 6, and 8) are r e m a r k a b l y stable t o w a r d acid and heat, whereas the others (Nos. 3 and 7) are relatively labile. T h e original conclusion of K u n i t z a n d N o r t h r o p ~ t h a t one molecule of the inhibitor reacts with one molecule of trypsin, and t h a t the complex is an addition c o m p o u n d , has been essentially confirmed b y the recent evidence. T h e first four of the a b o v e listed inhibitors are obtainable in crystalline form, a n d Nos. 1, 3, a n d 4 are also obtainable as crystalline complexes with trypsin. These complexes are v i r t u a l l y devoid of either t r y p t i c or t r y p s i n i n h i b i t o r y activity. W i t h i n a fairly wide p H range these complexes fulfill several criteria of h o m o g e n e i t y (electrophoresis, u l t r a c e n t r i f u g e ) ; outside this range complexes dissociate into their active c o m p o n e n t s . I t has been r e c e n t l y shown t h a t complexes of t r y p s i n with o v o m u c o i d ~s,~7 a n d with the K a z a l ' s inhibitor ~s are unstable, the inhibitor being digested with t h e s i m u l t a n e o u s liberation of the free trypsin. This p h e n o m e n o n was called t e m p o r a r y inhibition2 ~ T h e a c t i v i t y of the inhibitors is m e a s u r e d in t e r m s of the t r y p s i n inhibited, a n d therefore all m e t h o d s used for t h e d e t e r m i n a t i o n of t r y p t i e s 3~[. Kunitz, Science 101, 668 (1945). 4 M. Kunitz, J. Gen. Physiol. 29, 149 (1946). 5 M. Laskowski, Jr., and M. Laskowski, Federation Proc. 9, 194 (1950). 6 M. Laskowski, Jr., and M. Laskowski, J. Biol. Chem. 190, 563 (1951). 7 H. Tauber, B. B. Kreshaw, and R. D. Wright, J. Biol. Chem. 197, 1155 (1949). s H. L. Fraenkel-Conrat, R. C. Bean, E. D. Ducay, and H. S. Olcott, Arch. Biochem. and Biophys. 37, 393 (1952). 9 A. K. Balls and T. L. Swenson, J. Biol. Chem. 106, 409 (1934). 10 H. Lineweaver and C. W. Murray, J. Biol. Chem. 171, 565 (1947). 11 E. Fredericq and H. F. Deutsch, J. Biol. Chem. 181, 499 (1949). 12 R. J. Peanasky and M. Laskowski, J. Biol. Chem. 204, 153 (1953). is H. B. Collier, Can. J. Research 19B, 91 (1941). 14 M. Laskowski and M. Laskowski, Jr., Advances in Protein Chem. 9, 203 (1954). 15 No distinction is being made between proteins and large polypeptides. 16L. Gorini and L. Audrain, Biochim. et Biophys. Acta 8, 702 (1952). 17L. Gorini and L. Audrain, Biochim. et Biophys. Acta 10, 570 (1953). 18 M. Laskowski and F. C. Wu, J. Biol. Chem. 204, 797 (1953). is, In order to explain the mechanism of temporary inhibition it was postulated 18from the kinetic data that one molecule of inhibitor combines with 2 molecules of trypsin to form an intermediate trypsin-inhibitor-trypsin (TIT) complex, which subsequently breaks into products +2 T. Recently, J. Sri Ram, L. Terminiello, M. Bier, and F. F. Nord [Arch. Biochem. and Biophys. 52, 451 (1954)] supplied additional evidence for the existence of TIT.

38


[4]

activity (see section on trypsin, Vol. II [3]) are applicable to the determination of inhibitors. The solution of inhibitor (in a buffer to be used) and the solution of trypsin (in 0.0025 N HC1) are mixed, allowed to react, and the remaining trypsin activity is determined. With the exception of the pancreatic trypsin inhibitor, which requires up to 5 minutes for the completion of the reaction with trypsin, all other inhibitors react almost instantaneously over the range of pH values from 6 to 8. In the majority of cases within a proper pH range the inhibition is a linear function of the inhibitor concentration and does not depend on the purity of the preparation used. Two exceptions are noted: inhibition by the crude blood plasma 19 and by the inhibitor from Ascaris. 13 The former exhibits the linear relationship only when the ionic strength of the medium is close to physiological range; the latter in many respects behaves abnormally. Since the activity of inhibitors is expressed in terms of the inhibited trypsin, it is important that the standard preparation of trypsin be used. In the reviewer's laboratory it was found convenient to accept the activity curve of trypsin published by Kunitz ~° as a standard (see section on trypsin, Vol. II [3], Fig. 1). Preparation of Pancreatic Inhibitor of Kunitz and Northrop 1 Fresh pancreas is treated by the procedure of Kunitz and Northrop, as described in the article on chymotrypsinogens and chymotrypsins (Vol. II [2]), through the stage of crystallization of the ~-chymotrypsinogem The mother liquor is then treated as described in the chapter on trypsinogen and trypsin through the stage of crystallization of trypsin (Vol. II [3]). The mother liquor from the crystallization of trypsin is referred to as filtrate E and is treated as follows. 2°" Crystallization of Inhibitor-Trypsin Compound. ~ Filtrate E is adiusted to pH 3.0 with 5 N H2S04, saturated with crystals of magnesium sulfate at 25 ° , and filtered with suction through hardened paper. The filtrate is rejected. The precipitate (10 g.) is dissolved in 50 ml. of N/16 HCI and poured with stirring into a large beaker containing 250 ml. of N / 1 6 HC1 at 90°. After 1 minute it is cooled in running cold water to 25 °. Then 24.2 g. of solid ammonium sulfate is dissolved in each 100 ml. of solution, and the suspension is filtered through fluted paper; next 20.5 g. of solid ammonium sulfate is dissolved in each 100 ml. of filtrate, and the suspension is refiltered with suction. The last filter cake (3 g.) is dissolved in 19 S. F. M c C a n n a n d M. Laskowski, J. Biol. Chem. 204, 147 (1953). 20 M. Kunitz, J. Gen. Physiol. 30, 291 (1947). 20~ All m a n i p u l a t i o n s are carried out a t room t e m p e r a t u r e (20 to 25 °) unless otherwise

specified.

[4]-


39

12 ml. of water and cooled in ice water. About 3 ml. of 0.4 M borate, 21 pH 9.0, is added in order to bring the solution to pH 8.0, and the mixture is poured with stirring into a large beaker containing 75 ml. of boiling distilled water. A heavy precipitate forms. After 1 minute the suspension is cooled to 25 ° in running cold water, and 24.2 g. of solid ammonium sulfate per 100 ml. is added. The mixture is filtered with suction through hardened paper, and the precipitate is rejected. The filtrate is adjusted to pH 3.0 by addition of several drops of 5 N H2SO4, and then 20.5 g. of solid ammonium sulfate is dissolved in each 100 ml. of solution. The suspension is filtered with suction on a large funnel, the filtrate is rejected, and the precipitate is washed with saturated magnesium sulfate. The precipitate (1 g.) is dissolved in 5 ml. of M/IO acetate buffer, pH 5.5, and the pH is adjusted to 5.5 with about 1 ml. of'0.4 M borate buffer, pH 9.0. The suspension is filtered through Whatman No. 42 paper into a flask containing enough crystals of magnesium sulfate to saturate the solution. The filter paper is washed with 4 ml. of M/IO acetate buffer, pH 5.5. The solution is stirred after completion of filtration. Hexagonal crystals of inhibitor-trypsin compound rapidly appear. The suspension is allowed to stand for 1 day at 20 ° to complete crystallization. The yield is about 0.25 g. of filter cake. Recrystallization. The filter cake of crystals (accumulated from several preparations) is dissolved in 10 vol. of M/IO acetate buffer, pH 5.5, and filtered through Whatman No. 42 fluted paper. Crystals of magnesium sulfate are added to saturate the solution. 2~ Hexagonal crystals of the inhibitor-trypsin compound rapidly appear. After 1 day at 20 to 25 ° they are.filtered off; the yield is about 60%. Crystallization of the Free Trypsin Inhibitor. To a solution of 1 g. of crystalline filter cake of three-times-recrystallized inhibitor-trypsin compound in 10 ml. of water, 10 ml. of 5% trichloroacetic acid is added, and the mixture is allowed to stand at 20 ° for 30 minutes, until precipitation is about complete. The mixture is filtered with suction; the precipitate may be used for crystallization of trypsin. The filtrate is heated for 5 minutes at 80 °, cooled to 25 °, and filtered through fluted Whatman No. 42 paper. The precipitate is rejected. The pH of the filtrate is adjusted to 3.0 with 5 N NaOH. Solid ammonium sulfate (5.6 g. per 10 ml.) is added, the suspension is filtered with suc31 Stock borate solution contains 49.6 g. of boric acid a n d 80 ml. of 5 N sodium hydroxide per 1000 ml. of solution; 0.4 M borate buffers, p H 8.0 a n d 9.0, are mixtures of 100 parts of stock borate a n d 78.6 a n d 17.6 parts of 0.4 M hydrochloric acid, respectively. ~ Green a n d Work 24 recommend 0.7 s a t u r a t e d MgS04 for recrystallization. T h e reviewer has successfully used 0.75 saturated.

40

ENZYMES

OF PROTEIN

METABOLISM

[4]

tion, and the filtrate is rejected. The precipitate (0.25 g.) is dissolved in 2.5 ml. of water, and the pH is adjusted to 5.5 with 0.4 M borate buffer, pH 9.0. Saturated ammonium sulfate is added to slight turbidity, and the suspension is filtered through Whatman No. 42 filter paper. The paper is washed with 0.5 saturated ammonium sulfate. More saturated ammonium sulfate is added to the combined filtrate and washings until a slight precipitate forms. The amorphous precipitate gradually changes into long hexagonal prisms. The suspension is allowed to stand for 2 days at 20 ° and is then filtered with suction. The yield is 0.15 g. of inhibitor crystals. If it is desired to have crystals free from ammonium salt, the filter cake should be washed with saturated magnesium sulfate and then recrystallized from a magnesium sulfate solution. Recrystallization. The crystals (0.15 g.) are dissolved in 1.5 ml. of M/IO acetate buffer, pH 5.5. Then 7.5 ml. of saturated ammonium sulfate or 7.5 ml. of saturated magnesium sulfate plus a few crystals of solid magnesium sulfate are added. The mixture is allowed to stand at 20 ° for 1 day. Crystals of inhibitor gradually appear. The yield is about 0.1 g. of filter cake. 23 A different method for the preparation of the crystalline trypsintrypsin inhibitor complex and crystalline inhibitor has been recently described by Green and Work. 24 In this method the residue remaining after the extraction of pancreas for insulin serves as starting material. Preparation of the Second Crystalline Trypsin Inhibitor from Pancreas,

According to Kazal, Spicer, and Brahinsky ~ This inhibitor is extracted from pancreas together with crude insulin and remains in solution after insulin is precipitated from an acid 15% NaC1 brine. In order to obtain sufficient quantities of the starting material, several tons of pancreas must be extracted. Very few laboratories have such facilities; therefore the details of this preparation are omitted and the reader is referred to the original paper. 2

Preparation of the Crystalline Soybean Trypsin Inhibitor 4 Step 1. Washing with 80 % Alcohol. One thousand grams of cold-processed, defatted soybean meal 2s is added to a mixture of 2400 ml. of 95% 3a Green and Work 24 recommend the following procedure for recrystallization: 0.6 g. of the inhibitor is dissolved in 0.1 M acetate buffer, p H 6.5 (6 ml.), and the solution is saturated with MgS04. A little of the partially crystalline material which precipitates is filtered off. The filtrate is acidified to p H 3. A precipitate which forms rapidly changes into crystals. It is filtered off after standing overnight. Yield, 0.3 to 0.4 g. after two recrystallizations. 24 N. M. Green and E. Work, Biochem. J. 54, 257 (1953). 2s Soybean meal, Nutrisoy X X X , in the form of flakes, supplied by the ArcherDaniels-Midland Co., Chicago, Illinois.

[4]


41

alcohol cooled to 5° and 450 ml. of distilled water. The suspension is stirred well and left at 20 to 25 ° for 30 minutes. It is then filtered with suction on a 32-cm. Biichner funnel through filter cloth. The filtrate is reiected. Step 2. Extraction in 0.25 N H2S04. The semidry meal is resuspended in 5000 ml. of 0.25 h r H2SO4 (7 ml. of concentrated H~SO4 per liter of water) at 20 to 25 ° and left for 1 hour at room temperature with occasional stirring. The suspension is refiltered with suction on the same filter cloth. The meal residue is rejected. Step 3. Removal of Inert Protein by Means of Bentonite. Twenty grams of stock mixture of 1 part by weight of Bentonite (U.S.P. powder, Amend Drug and Chemical Co., New York) and an equal part of Hyflo SuperCel (Johns-Manville Corp.) are added to the acid filtrate, and then it is stirred for 10 minutes. The suspension is filtered with suction on 32-cm. E-D No. 303 filter paper (The Eaton-Dikeman Co., Mr. Holly Springs, Pennsylvania). The residue on the paper is washed twice with portions of 125 ml. of water. The residue is rejected. Step 4. Adsorption of the Inhibitor on Bentonite. One hundred grams of the stock of Bentonite-Super-Cel mixture is added gradually to the combined filtrate and washings from step No. 3. The solution is stirred gently while the Bentonite mixture is added, and the stirring is continued for 10 minutes. The suspension is filtered and the residue is washed, as described in step 3. The filtrate and washings are rejected. Step 5. Elution with Pyridine and Dialysis. The Bentonite residue is stirred up with 270 ml. of water. At this stage the suspension can be stored in the refrigerator overnight. The suspension is warmed to 25 °, and 30 ml. of pyridine (Stock No. 214, Eastman Kodak Co.) is added with stirring. The thick suspension is filtered with suction on 24-cm. E-D No. 303 filter paper in a hood. The filtration generally requires several hours. The residue on the funnel is washed once with 200 ml. of 5 % pyridine in water. The combined filtrate and washing is dialyzed overnight in 12-inch-long cellophane tubings, 28 placed in a tall jar with running tap water, in the hood, if possible. Step 6. Removal of Inert Material at pH 5.3. The dialyzed solution, free of any gummy residue adhering to the dialysis tubes, is adjusted to pH 5.3 with the aid of about 2 ml. of 1 N HC1. (The pH is tested by the drop method on a plate with 0.1 M acetate buffers as standards and 0.01% methyl red as indicator.) Four grams of Bentonite-Super-Cel mixture is stirred into the solution which is then filtered with suction on 15-cm. E-D No. 303 filter paper, and the residue is washed several times NuJax Visking Cellulose Casing manufactured by the Visking Corporation, Chicago, Illinois.

2~ 27/~2_inch

42


[4]

with 15-ml. portions of water. The washings, if not clear, are refiltered. The residue is rejected. Step 7. First Precipitation of the Inhibitor at pH 4.65. The combined filtrate and washings of step 6 is cooled to 5 ° and then titrated with 1 N HCI to pH 4.65 (tested carefully with 0.05% bromocresol green on a drop plate). A heavy precipitate is formed which is filtered off at 5 to 8 ° on 15-cm. E-D No. 303 filter paper on a Bfichner funnel without suction. The filtration is completed with a very light suction. The weight of filter cake is from 10 to 12 g. The filtrate is rejected. Step 8. Second Precipitation at pH 4.65. The filter cake is suspended in 100 ml. of water cooled to 5 °, the water being added gradually to the precipitate and incorporated thoroughly with a porcelain spatula. Then 1 N NaOH is added dropwise with stirring until the precipitate is dissolved. Care should be taken, however, not to raise the pH of the solution above 6.4. The clear solution is warmed to 25 ° and titrated slowly with 1 N HC1 until a slight permanent precipitate is formed. Two grams of Standard Super-Cel is stirred into the solution which is then filtered with suction on 7- to ll-cm. E-D No. 303 filter paper. The residue on the paper is washed with several milliliters of water. The combined filtrate and washings is cooled to 5°, titrated to pH 4.65, and then filtered with light suction on 15- to 18-cm. E-D No. 612 filter paper at 5 to 8 °. The yield is about 8 to 10 g. of filter cake which is stored in refrigerator. The filtrate is rejected. Step 9. Crystallization. 27 The filter cake of step 8 (about 10 g.) is ground up to a uniform suspension with 10 ml. of cold water and then warmed to about 35 °. Next 0.5 N NaOH is added dropwise, with careful stirring, until the precipitate is almost completely dissolved and the pH of the solution is about 5.2. The clear solution is decanted into a 50-ml. centrifuge tube. Any residue in the beaker is stirred with 1 to 2 ml. of cold water, dissolved with the aid of a dro p of 0.1 N NaOH, and added to the main bulk of the solution in the centrifuge tube which is then placed at 35 to 37 ° for crystallization. A heavy sediment of crystals is obtained within 5 to 6 hours. Inoculation with a few crystals greatly facilitates the process of crystallization. The suspension is centrifuged for 10 minutes at about 3000 r.p.m. The residue is stored in the refrigerator, whereas the supernatant liquid is either stored or, if time permits, titrated with a few drops of 0.2/V HC1 to pH 5.1 at 36 to 37 °, inoculated, and left at that temperature. Another crop of crystals is gradually formed, which is centrifuged off after several hours and added to the first crop of crystals. The supernatant liquid is rejected. ~ I t is advisable to begin the crystallization (step 9) with at least 50 g. of amorphous precipitate collected from several preparations.

[4]


43

It is preferable to begin step 9 in the morning, so as to be able to centrifuge before the end of the day. The crystals, as well as the supernatant solution from the first crystallization, should be stored overnight in the refrigerator. Step 10. Recrystallization. The combined crystal residues of step 9 (about 7 ml.) are stirred up with twice the volume of cold water and titrated with 0.5 N NaOH to clearing, the final pH being about 6.0. The clear solution is warmed to 35 ° and titrated with 0.5 N HC1 to pH 5.1 when a slight permanent precipitate is formed. The solution is mixed with 2 g. of Standard Super-Cel and filtered clear with suction on a small E-D No. 303 filter paper. The filtrate is inoculated and left at 36 to 37 °. A heavy suspension of crystals forms gradually and is centrifuged after 5 to 6 hours. The residue is stored at 5°. The pH of the supernatant liquid is readiusted with 1 to 2 drops of 0.2 N HC1 to 5.1 and left for several hours longer at 36 to 37 °, when another crop of crystals is formed which is centrifuged off and added to the first crop. The final supernatant solution may yield still more crystals by cooling it to 5 ° and then adding one-quarter of its volume of cold 95 % alcohol as described in the following paragraph. The crystallization is repeated three times. Step 11. Crystallization in Dilute Alcohol. The centrifuged crystals are stirred up with five times the volume of cold water, and 0.5 N NaOH is added drop by drop until the crystals are all dissolved. The pH of the solution is not allowed, however, to rise above 6.6. The clear solution is titrated with 0.2 N HC1 to pH 5.2. Any precipitate formed is filtered off with suction on No. 303 filter paper with the aid of 4 g. of Standard Super-Cel per 100 ml. of solution. The residue on the funnel is washed with several milliliters of water. The volume of the filtrate and washings is measured, and the solution is then cooled in an ice-water bath to about 5° . One-quarter of its volume of 95% alcohol, cooled to 5 °, is added slowly to the cold solution. A heavy precipitate is formed. The pH of the mixture is adjusted with 0.2 N HC1 to 5.0, and the mixture is left at 30 °. The amorphous precipitate changes within 2 hours into well-formed hexagonal and rhomboid crystals and plates which settle rapidly to the bottom of the vessel. The supernatant solution is decanted every hour, adjusted with 0.2 N or more dilute HCI to pH 5.0, and returned to the original vessel containing the settled crystals. This is continued for several hours until no formation of precipitate is noticed when the pH of the supernatant solution is adjusted to 5.0. The crystallization mixture is allowed to stand at 30 ° for 30 minutes longer and then filtered with suction on hardened paper, washed on the funnel several times with cold acetone, and allowed to dry in the room for 24 hours. It is stored in the refrigerator.

44


[4]

Step 12. Recrystallization in Alcohol. The dry crystals are suspended in thirty times their weight of cold water, allowed to soak for 5 to 10 minutes, and then treated exactly as in step 11. Preparation of Crystalline Trypsin-Soy Inhibitor Complex 28

Step 1. Preliminary Step. One gram of dry soy inhibitor crystals is suspended in 40 ml. of distilled water at 5°. The mixture is titrated with 0.2 iV NaOH to pH 7.5. This brings about complete solution of the inhibitor crystals. One gram of a preparation of dry crystalline trypsin (containing about 50% anhydrous MgS04) is then added slowly with stirring. The mixture thus contains an excess of inhibitor in order to avoid any proteolysis by trypsin. If necessary, the pH is readjusted to 7.5 with several drops of 0.1 M borate buffer, pH 9.0. The solution, if turbid, is filtered with suction with the aid of 1 g. of Super-Cel on a small Bflchner funnel. The residue on the funnel is washed with about 5 ml. of cold H20. The washings, if clear, are added to the main bulk of the filtrate which is titrated with 0.1 N HC1 to about pH 6.0 and dialyzed overnight against slowly running distilled water at 5 to 10°, preferably with stirring. A granular precipitate gradually forms in the dialysis bag. The dialyzed suspension is titrated with a few drops of 0.1 N HC1 to pH about 5.4 (tested on a drop plate with 0.01% solution of methyl red). The suspension is centrifuged. The residue yields the crystalline compound; the supernatant solution (designated as "first supernatant solution") contains the excess of soy inhibitor which can be partly recovered. Step 2. Crystallization of the Compound. The residue is suspended in about 20 ml. of cold H20 and recentrifuged. The washed residue is resuspended in 40 ml. of H20 at about 5° and titrated dropwise with 0.2 or 0.5 N NaOH to pH 9.0 (pink to 0.1% phenolphthalein on a test plate), when complete solution generally occurs. The solution is then titrated with a few drops of 0.2 N HC1 to very slight opalescence and stored at about 20 ° . Fine crystals in the form of small rosettes or bundles of needles and plates gradually appear. The suspension of crystals is centrifuged after a day or so; several drops of 0.1 N HC1 are added to the supernarant solution until a slight turbidity appears. The solution is stored for several hours at 20 °. A second crop of fine crystals generally appears, which is centrifuged on the top of the first crop of crystals. 5iore acid is added to the supernatant solution, and the process is repeated until pH 5.8 is reached or until the final acidified supernatant solution no longer yields crystals. It is rejected or is combined with the "first supernatant solution" to be worked up for soy :inhibitor, as described in step 7. 2a M. Kunitz, J. Gen. Physiol. 30, 311 (1947).

[~]


45

Step 3. Recrystallization of the Compound. The crystals are suspended in about 20 vol. of cold water and titrated with several drops of 0.5 N NaOH to incipient clearing. The solution is allowed to stand for 5 to 10 minutes at 5 ° and then filtered, if turbid, on fluted W h a t m a n No. 3 paper moistened with cold water, pH 9.0. The filter paper is washed once with cold water. The combined clear filtrate and washing is titrated with several drops of 0.2 N HC1 to very slight opalescence, seeded, and left at 20 °. Crystallization is generally complete within 24 hours. The crystals are centrifuged. The supernatant solution is titrated with 0.2 N HC1 to slight turbidity and left at 20 ° for several hours. A second crop of crystals is obtained and collected by centrifugation in the same tube used to collect the first crop of crystals. The operation is repeated several times until no further yield of crystals is obtainable. The supernatant solution is treated as described in step 5. Step 4. Drying of Crystals. The combined crystals are resuspended in a small amount of distilled water and filtered with suction on hardened paper. The crystals are dried for 24 hours in a refrigerator at about 5 ° and then in a desiccator over anhydrous CaS04 (Drierite) at 20 °. The dried material is ground fine in a mortar and stored in a refrigerator. Step 5. Crystallization in Dilute Alcohol. The final supernatant solution in step 3 m a y further yield crystals if it is cooled to 5 °, one-fourth of its volume of cold 95 % alcohol is added, and the pH of the solution is adjusted with 0.2 N HC1 to 5.8. A precipitate forms which, when left at 20 ° , changes gradually into rosettes of fine plates. The crystals are filtered on hardened paper and dried first in a refrigerator and then in a desiccator over anhydrous CaSO4 (Drierite). Step 6. Recrystallization in Dilute Alcohol. The dry crystalline powder is suspended in about fifty times its weight of water. (The centrifuged residue of crystals, not dried, is suspended in twenty-five times its volume of water.) The suspension is titrated with several drops of 0.5 N N a O H to pH 9.0. The crystals gradually dissolve when left for about 10 minutes at 5° . The solution, if turbid, is filtered, and then one-fourth of its volume of cold 95% alcohol is added. The pH of the solution is adjusted to about 5.8. A heavy precipitate is formed which changes into crystals on storing for a day or two at 20 °. The crystallization in alcohol is more rapid and the yield of crystals is greater than in the absence of alcohol. There is also the advantage t h a t the alcohol keeps the solution sterile. There is, however, the possibility t h a t alcohol causes slight denaturation of the protein. Step 7. Partial Recovery of Excess of Inhibitor. The first supernatant solution of step 1 is titrated with 0.5 N HC1 to pH 4.65 at 5 ° and centrifuged at about the same temperature. The supernatant solution is re-

46


[4]

jected. The residue is suspended in about 5 vol. of cold water and is titrated with 0.2 N NaOH to pH 5.2. Any precipitate left undissolved is centrifuged off and is rejected (or worked up for compound as described in step 2). The supernatant solution is cooled to 5 °, and one-fourth of its volume of 95% alcohol precooled to 5° is added. The solution is adjusted with several drops of 0.1 N HC1 to pH 5.0, seeded with soy inhibitor crystals, and left at 30 °. Crystals of inhibitor gradually form. The crystals are filtered after several hours, washed with cold acetone, and dried in the room. The yield is 0.1 to 0.2 g. of dry soy inhibitor crystals.

Preparation of the Crystalline Trypsin Inhibitor from Colostrum 6

Step 1. To each liter of bovine colostrum, 1 1. of water and 1 1. of 7.5% trichloroacetic acid are added (final concentration 2.5% with respect to trichloroacetic acid). The mixture is heated to 80 ° with constant stirring and allowed to stand at that temperature for 5 minutes. It is then cooled to 25 ° and filtered on a large stainless steel Biichner funnel 29 with Whatman filter paper No. 1. The heavy cheeselike precipitate is discarded. The filtrate is brought to 80% saturation by addition of solid ammonium sulfate (603 g./1.) a° and allowed to stand overnight at room temperature. The slight precipitate which floats on the surface is removed by filtration with suction through Whatman filter paper No. 4. The filtrate is discarded. Step 2. The precipitate (plus crude fractions from previous preparations) is dissolved in 7 vol. of water with the aid of a Waring blendor, and enough trichloroacetic acid is added to attain a final concentration of 2.5%. The mixture is heated to 80 ° for 5 minutes, cooled to 25 °, and filtered with suction through Whatman No. 4 filter paper. The precipitate is washed with 2.5% trichloroacetic acid and discarded. The combined filtrate and washings is brought to 80% saturation of ammonium sulfate, and the mixture is filtered with suction through Whatman filter paper No. 4. The filtrate is discarded. Step 3. After the precipitate has been dissolved in 5 vol. of water (Waring blendor), the solution is adjusted to pH 6.5 with 1 N NaOH (glass electrode) and brought to 30% saturation of ammonium sulfate (22.6 g. per 100 ml.). After addition of 5 g. of Celite No. 545 per 100 ml., the mixture is filtered with suction through Whatman filter paper No. 4. The filtration is slow. The dark precipitate is discarded. The filtrate is 20 I n this l a b o r a t o r y a stainless steel Biichner funnel, Model 503, is used, m a n u f a c t u r e d b y American Biosynthetic Corp., Milwaukee, Wisconsin. 30 These figures are higher t h a n t h e figures used b y K u n i t z a n d Northrop, 1 who refer t o a s a t u r a t e d solution a t 5 °, whereas these figures refer to 25 °. See Vol. I [10].

[4]


47

brought to 70% saturation of ammonium sulfate (26.7 g. per 100 ml.), which results in the formation of a rubberlike precipitgte. The latter is filtered with suction through Whatman filter paper No. 4 and kept as precipitate 3. Some inhibitor can be saved by adjusting the filtrate to pH 2 and 80% saturation, filtering, and adding it to the next preparation (step 2). Step 4. Precipitate 3 is dissolved in 5 vol. of water, trichloroacetic acid is added to attain a concentration of 2.5%, and the solution is shaken thoroughly in a separatory funnel with an equal volume of ether. It is then allowed to stand for 2 hours. The water layer is separated and kept. The ether layer is washed with one-half of the previous amount of water, the washings are added to the next preparation (step 2), and the rest is discarded. The liquid, which is still saturated with ether, is brought to 30% saturation of ammonium sulfate (22.6 g. per 100 ml.). The rubberlike precipitate which forms is filtered through filter paper Whatman No. 4 and the filtrate is discarded. Step 5. The precipitate is dissolved by stirring in a minimum amount of water, and the solution is dialyzed against distilled water overnight in the cold room21 The dialyzed liquid is adjusted to pH 5.5 with 1 h r NaOH and is treated with an equal volume of methanol, precooled to - 1 8 °. The solution is allowed to stand in a deep-freeze ( - 1 8 °) for an hour and is centrifuged in the conical head of a refrigerated centrifuge at - 1 0 ° for 15 minutes at 3500 r.p.m. The precipitate may be used in the next preparation (step 2). The slightly cloudy supernatant is treated with four times the previous volume of methanol (a total of 5 vol.). It is set in a deep-freeze for an hour and is centrifuged at - 1 0 ° for 20 minutes at 3500 r.p.m. The supernatant is discarded. Crystallization of Trypsin-Colostrum Trypsin Inhibitor Complex. Precipitate 5 is dissolved in a minimum amount of water, and the solution is adjusted to pH 3. The amount of trypsin required to neutralize the inhibitor completely is determined on an aliquot. The calculated amount of recrystallized trypsin is added, the solution is adjusted to pH 5.5, and dialyzed in the cold against 0.01 M acetate buffer, pH 5.5, for a period of 10 to 14 days, ~2 with frequent (every 12 hours) changes of buffer. The small precipitate is centrifuged off and discarded. To the solution an equal volume of saturated ammonium sulfate is added, followed by dropwise addition until the first sign of turbidity. After seeding with crystals, the solution is allowed to stand for 3 to 4 days at room temperature. The ~ Sizable amounts of inhibitor are lost during dialysis, since the inhibitor slowly passes through the cellophane membrane. The dialysis is, however, necessary for

the succeeding precipitation with methanol[ 3~M. Laskowski, Jr., P. H. Mars, and M. Laskowski, J. Biol. Chem. 198t 745 (1952).

48


[4]

mixture first becomes gelatinous; then needles of the crystalline complex slowly form. Crystallization of the Free T r y p s i n Inhibitor f r o m Colostrum. The twicerecrystallized trypsin-trypsin inhibitor complex is dissolved in a small a m o u n t of water. An equal volume of 5 % trichloroacetic acid is then added, and the solution is allowed to stand for an hour. The precipitated trypsin is centrifuged off and saved for recrystallization. The solution containing the inhibitor is heated at 80 ° for 5 minutes, cooled to 25 °, and filtered through a small fluted filter to remove a slight precipitate, which is discarded. T h e filtrate is brought to 80% saturation with ammonium sulfate and centrifuged in the high-speed head of a refrigerated International centrifuge at about 25,000 r.p.m. The precipitate is dissolved in a TABLE I BALANCE SHEET OF EXPERIMENT WITH

4

GALLONS OF COLOSTRUMa

Step

Total amount of trypsin inhibited,b g.

Potency, trypsin inhibited (~,) E~800 m"

1 2 3 4 5

1.7 1.1 1.0 0.6 0.4

43 130 220

550 730

M. Laskowski, Jr., and M. Laskowski, J. Biol. Chem. 190, 563 (1951). b Calculated on the basis of 0.585 as an optical factor for trypsin. minimum a m o u n t of 0.05 M acetate buffer, p H 5.5, an equal volume of saturated a m m o n i u m sulfate is added, and then, v e r y carefully, an excess of a few drops until the minute t h a t t u r b i d i t y appears. Crystals (long needles) form almost immediately. The solution is allowed to stand overnight, and the crystals are collected b y centrifugation in a highspeed a t t a c h m e n t . Recrystallization is carried out in the same m a n n e r as the first crystallization. Table I summarizes the purification procedure.

Preparation of the Lima Bean Trypsin Inhibitor T a u b e r et al. ~ described a m e t h o d for crystallization of the trypsin inhibitor from lima beans. Fraenkel-Conrat et al. s repeated the method of T a u b e r et al. 7 and introduced a modification of the recrystallization procedure which resulted in the removal of inhibitory activity from the crystals and concentrating it in the m o t h e r liquor. Fraenkel-Conrat el al. s described a m e t h o d for the preparation of the amorphous inhibitor, the reported potency of which is about four and one-half times t h a t of the crystalline material of T a u b e r et al. 7 and about two and one-half times

[4]


49

that of the crystalline soybean trypsin inhibitor. 4 The procedure is similar to the procedure of Kunitz 4 for the crystallization of soybean trypsin inhibitor. Preparation of Ovomucoid The simplest and probably the most widely used method is that of Fredericq and Deutsch. 11 It leads to an amorphous product with ninefold higher potency than the original egg white. The two steps are: (1) precipitation of the inactive proteins of the egg white by addition of an equal volume of 10% trichloroacetic acid previously adjusted to pH 3.0, and readjustment of the pH of the mixture to 3.5; (2) precipitation of ovomucoid from the filtrate by addition of 2 vol. of 95% alcohol at pH 6.0, at - 8 °. This precipitate represents crude ovomucoid, which is electrophoretically heterogeneous. Further purification improves the electrophoretic pattern but does not improve the potency of the preparation. Preparation of Partially Purified Blood Plasma Inhibitor 12 Indirect evidence suggests the presence of more than one trypsin inhibitor in blood plasma. 14 The preparation presented here refers to the quantitatively predominant trypsin inhibitor. Partial purification of trypsin inhibitor from blood plasma with properties different from the inhibitor described here has been described, 3~ confirmed, 34 and denied. 35 Step 1. Bovine blood is collected in the slaughterhouse, oxalated, and centrifuged. Plasma, usually about 6 1., is diluted with an equal volume of physiological saline. The mixture is acidified with 5/V H2SO4 to pH 4.0 and cooled to 5 ° . It is brought up to 40% saturation (for that temperature) by a slow addition of solid ammonium sulfate (250 g./1.) and allowed to stand overnight. It is filtered in the cold with the aid of Celite No. 545 (20 g./l.) on a large stainless steel Biichner funnel 29 through one sheet of Whatman No. 1 filter paper (32 cm.) with gentle suction. The precipitate is reiected. The filtrate is brought to 90% saturation with ammonium sulfate (375 g./1.). The mixture is filtered through the same Bilchner funnel at room temperature with the aid of 5 g. of Celite per liter. The filtrate is reiected. Step 2. The precipitate is suspended in 20 vol. of water, and the Celite is filtered off. The clear filtrate is adjusted to pH 4.7 with 5 N NaOH and brought to 50% saturation with solid ammonium sulfate (377 g./1.). It is allowed to stand at room temperature overnight. The precipitate is filtered off with the aid of Celite (10 g./1.) and is discarded. The filtrate 33 A. Schmitz, Z. physiol. Chem. 255, 234 (1938). 84 D. Grob, J. Gen. Physiol. 26, 405 (1943). 35 E. S. Duthie and L. Lorenz, Biochem. J. 44, 167 (1949).

50


[4]

is b r o u g h t t o 6 5 % s a t u r a t i o n w i t h a m m o n i u m s u l f a t e (100 g./1.) a n d a l l o w e d t o s t a n d for s e v e r a l h o u r s . T h e p r e c i p i t a t e is c o l l e c t e d on a 18.5-cm. B i i c h n e r f u n n e l w i t h t h e a i d of 2 g. of C e l i t e p e r liter. TABLE I I EXTENT OF THE PURIFICATION OF BLOOD PLASMA TRYPSIN INHIBITORa

Fraction

trypsin inhibited (~)b E~0m'

Potency,

Whole plasma Step 1 Step 2 Step 3 Step 4

12 65 160 300 500

(11-13) (60-70) (140-180) (280-330) (480-520)

Yield, % 100 45 20 10 5

" R. J. Peanasky and M. Laskowski, J. Biol. Chem. 204, 153 (1953). b Calculated on the basis of 0.585 as an optical factor for trypsin. TABLE I I I OPTICAL PROPERTIES

Substance Pancreatic inhibitor

Factor a

Remarks

Kazal's inhibitor Soybean inhibitor Colostrum inhibitor

1.26 1.22 1.54 1.10 2.00

Acid solutionb pH 7 buffer c,d pH 5.7 ° Acid solutionl Acid solutiong

Pancreatic complex Soybean complex Colostrum complex

0. 810 0. 765 0. 840

Acid solution b Calculated from Kunitz's datal Acid solution ~

a After multiplying the observed value E ~ " by this factor, the concentration of protein would be expressed in milligrams per milliliter. b M. Laskowski, Jr., P. H. Mars, and M. Laskowski, J. Biol. Chem. 198, 745 (1952). c N. M. Green and E. Work, Biochem. J. 64, 257 (1953). N. M. Green, J. Biol. Chem. 205, 535 (1953). L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, J. Am. Chem. Soc. 70, 3034 (1948). f M. Kunitz, J. Gen. Physiol. 30, 291 (1947). g M. Laskowski, Jr., and M. Laskowski, J. Biol. Chem. 190, 563 (1951). Step 3. T h e p r e c i p i t a t e is s u s p e n d e d in 2 vol. of w a t e r , a n d t h e C e l i t e is f i l t e r e d off. A n a d d i t i o n a l v o l u m e of w a t e r is u s e d t o w a s h t h e C e l i t e . T h e f i l t r a t e a n d w a s h i n g a r e c o m b i n e d a n d a r e b r o u g h t t o 30 % s a t u r a t i o n w i t h solid a m m o n i u m s u l f a t e (22.6 g. p e r 100 ml.). T h e c l e a r s o l u t i o n is carefully a d j u s t e d t o p H 3.6 a n d is a l l o w e d t o s t a n d u n t i l a h e a v y p r e c i p i t a t e f o r m s . T h i s u s u a l l y r e q u i r e s n o t m o r e t h a n 15 m i n u t e s . L o n g e r

[4]


51

exposure results in a partial loss of activity. The mixture is centrifuged at full speed in a Servall type SS-la centrifuge for 8 minutes. The centrifuge is stopped b y applying mechanical brake. The clear supernatant is decanted and quickly adjusted to p H 6.5. An equal volume of 30% TABLE IV ISOELECTRIC POINTS

Substance

pH

Remarks

Pancreatic inhibitor

ca. 10.0 > 8,7 10.1 4.8, 5.2, 5.9 4.5 5.0 4.2 7.2 Higher than 3.6 4.5 4.3 3.9

Electrodialysis a Electrophoresis b Electrophoresis b Three separate peaks, electrophoresis ~ Cataphoresis d Cataphoresis a Electrophoresis b Electrophoresis b Electrophoresis e Electrophoresis/ Electrophoresis~ In 0.1-u buffers; five peaks in 0.01-~ buffersh Five separate peaks in 0.01-~ buffers ~,i

Pancreatic complex Kazal's inhibitor Soybean inhibitor Soybean complex Colostrum inhibitor Colostrum complex Lima bean inhibitor Ovomucoid

4.41, 4.28, 4.17, 4.01, 3.83

N. NI. Green and E. Work, Biochem. J. 54, 257 (1953). b M. Laskowski, Jr., P. H. Mars, and M. Laskowski, J. Biol. Chem. 198, 745 (1952). c L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, J. Am. Chem. Soc. 70, 3034 (1948). d M. Kunitz, J. Gen. Physiol. 30, 291 (1947). o H. L. Fraenkel-Conrat, R. C. Bean, E. D. Ducay, and H. S. Olcott, Arch. Biochem. and Biophys. 37, 393 (1952). f L. Hesselvik, Z. physiol. Chem. 254, 144 (1938). o L. G. Longsworth, R. K. Cannan, and D. A. McInnes, J. Am. Chem. Soc. 62i 2580 (1940). E. Fredericq and H. F. Deutsch, J. Biol. Chem. 181,499 (1949). M. Bier, A. J. Duke, R. J. Gibbs, and F. F. Nord, Arch. Biochem. and Biophys. 37, 491 (1952). i lV[. Bier, L. Terminiello, A. J. Duke, R. J. Gibbs, and F. F. Nord, Arch. Biochem. and Biophys. 47, 465 (1953). saturated a m m o n i u m sulfate is added, followed b y addition of 15.1 g. of solid a m m o n i u m sulfate per 100 ml. of mixture. The precipitate which forms is filtered off with the aid of Celite (1 g. per 100 ml.) and is rejected. To the clear filtrate 10 g. of a m m o n i u m sulfate per 100 ml. is added, and the precipitate containing most of the activity is collected on a small Btichner funnel with the aid of Celite. Step 4. The precipitate is suspended in a small volume of water; the Celite is removed and washed. The liquid is dialyzed against running t a p

52

ENZYMES

OF

PROTEIN

METABOLISM

.~

"~.~ ~

0

~~

[4]

°~

°r~

~

~

©

¢4

~ ~

O ~q C~

~2

~o o

0

0

O~q

I

Cq

~9

% o

°~

°~

O

¢D O

[4]

NATURALLY

0CCUR~ING

TRYPSIN

INHIBITORS

53

~.~ ~~

"~

e~

,.4

o~.

~

~

'~

~o

Lea v

h- ~" .,o~,'~

o

~

.~

~

~

O~

~0

oo

0

•- ~ •

~

"~'~ ~ " ~

.....

m

0

©

~'~

"~.~

h,

~. ~

•

~

•

~

~'~'~ ~ '~ ~0"~- ~

~

~

~

.

, ~. ' ~ ~~o ~. ~- ~ . ~ - ~ ~~. ~ ~- ~

-

m ~.~

.=~

I

~,~

~ ~

.~

~I~,i 0 .~

m~ ~

-~_~ ~.~-o.~o.~ ~ ...o o . ~ " ~ ~ ~ ~ ~- ~

~

.

~

~

.~

~..

,.~ ,

.

~

~'~ ~

~.,~ ~..~

~ ~..~ ~

~

=

=

~

=

.

~..~ . . . .

~

,.~

r/i~

~.~

.

.

o

~~~

. . .~ . . ' ~0 ~

~.~

~ ~

.

..~

~-~ ~ ~

.~ ~ . . ~~.; ~. ~. 0,~ •

" ~ 0 ~ ' ~

~

~

,"~.'~

~.~

• h-~

54


[5]

water for 60 hours. It is diluted with distilled water to produce a solution having an optical density of 2.0 at 280 m~. The pH is carefully adjusted to 3.6 with 0.5 N H~SO~. Twenty milligrams of bentonite is added per each milliliter, and the mixture is allowed to stand overnight at 5 ° . The Bentonite is centrifuged off, and the supernatant, containing most of the activity, is lyophilized. Table II summarizes the purification procedure. Preparation of the Inhibitor from Ascaris 13 The worms are homogenized in 1% NaC1 in Waring blendor. Diastase is added, and the suspension is allowed to autolyze under toluene for several days. Trichloroacetic acid is added to attain a concentration of 2.5%. The mixture is heated to 80 ° for 5 minutes, cooled, and filtered. The precipitate is rejected. The filtrate is treated with charcoal to remove the color and odor, and after adjustment to pH 3 it is saturated with MgSO4. The precipitate which contains the activity is collected on hardened filter paper. The inhibitor from Ascaris appears to be the only trypsin inhibitor which forms with trypsin a dissociable complex.13 Furthermore, trypsin treated with the inhibitor was reportedly no longer precipitable with trichloroacetic acid. Some properties of the trypsin inhibitors are summarized in Tables III, IV, and V, taken from Advances in Protein Chemistry. 14 The potencies of different inhibitors and the substrate on which they were determined are specified in the last column of Table V.

[5] P l a n t Proteolytic E n z y m e s B y D. M. GREENBERG

Distribution and Properties Proteolytic enzymes have been found in both dicotyledonous and monocotyledonous plants. Representative of the former are the fig, papaya, milkweed, and euphorbia; of the latter, the pineapple and cereals. There appears to be two major groups of these proteinases, one requiring free SH groups for activity, having their optimum activity at pH ~ 7 for the digestion of hemoglobin, casein, egg albumin, etc., and strong milk-clotting activity; the other group lack active SH groups, have a more alkaline optimum pH, and an inferior milk-clotting power. Both groups of plant proteinases are unusually heat resistant, often maintaining their activity at temperatures of 60 to 70 ° .

[5]

PLANT PROTEOLYTIC ENZYMES

55

The physiological function of the proteinases in plants is unknown. Certain of the plant proteinases have a number of technological and medical usages. I Probably commercially the most important is the tenderizing of meat and other protein material by papain and bromelin. Other technological uses are in the preparation of leather and in the brewing industry. They are used medically to some extent in the treatment of digestive disturbances involving the proteolytic enzymes and in the treatment of sloughing wounds. Most of the plant proteinases are able to digest living parasitic worms, such as ascaris and trichuris, but it has not been found safe to employ them as vermifuges. The characteristic properties of these enzymes are summarized in Table I.

Determination of Activity The most reliable and convenient procedures are those which determine the split products, soluble in trichloroacetic acid (TCA), of a standard protein, and the clotting activity against milk. Phenol Color Method3 Five milliliters of a solution of denatured hemoglobin at pH 7.4 (or other well-defined protein) is digested by i ml. of a suitably diluted proteinase solution for 5 minutes at 25 °. The reaction is terminated by the addition of 10 ml. of 3 M TCA, the mixture is filtered through Whatman No. 3 paper, and the tyrosine and tryptophan in an aliquot of the filtrate is determined by the blue color given with the FolinCiocalteau phenol reagent 3 in alkaline solution. The phenol reagent is diluted with twice its volume of H20 before use. This color is compared against a standard tyrosine solution dissolved in 1 M HC1. To 5 ml. of the digestion filtrate in a 50-ml. Erlenmeyer flask are added 10 ml. of 0.5 M NaOH and 3 ml. of the phenol reagent. The solution is kept agitated during addition of the phenol reagent. The color is read against a standard prepared in the same manner after 2 to 10 minutes of standing. The standard tyrosine solution contains 8 X 10-4 meq. of tyrosine (0.0112 mg. of tyrosine N) in 5 ml. of 0.2 M HC1, with 0.5% formaldehyde as a preservative. The tyrosine standard can be prepared by accurately weighing out 289.7 mg. of pure, dry tyrosine, dissolving it in 1 1. of 0.2 M HC1, and diluting this solution ten-fold in 0.2 M HC1 for use. Alternatively, the tyrosine nitrogen can be determined by micro-Kjeldahl. A blank is to be run with each series of determinations. The SIt protein1 See H. Tauber, "The Chemistry and Technology of Enzymes," Johla Wiley & Sons, New York, 1949. M. L. Anson, J. Gen. Physiol. 20, 561 (1937); 22, 79 (1938), 30. Folin and V. Cioealt6u, J. Biol. Chem, 78~ 627 (1929),

56

ENZYMES

OF

PROTEIN

METABOLISM

[~]

ND

Nil

~ r~

~.~

=

N

m ©

m

~ o"~'~

"g o

( 10 -5 and 1.4 >( 10-4 in moles per liter, respectively. With the soybean enzyme, however, T P N H and D P N H are almost equally effective as elec6 H. J. Evans and A. Nason, Plant Physiol. 28, 233 (1953). 7 D. J. D. Nicholas and A. Nason, J. Biol. Chem. 207, 353 (1954).

[59]

NITRATE REDUCTASE FROM NEUROSPORA

415

tron donors for nitrate reduction. The Km for the enzyme-nitrate complex is 1.4 × 10-3 mole per liter. Activators and Inhibitors. Nitrate reductase has a definite flavin requirement which can be fulfilled by boiled pig heart extract, 2 FAD, or in part by FMN, resulting in a 2- to 5-fold increase in enzyme activity. Although FAD has been established as the prosthetic group of the enzyme, FMN, which gives only half as much reactivation, serves as a cheap and convenient flavin for the assay. The dissociation constants (Kin) for the FAD- and FMN-enzyme complex are 3.2 × 10-7 and 30 × 10-7 in moles per liter, respectively. The sulfhydryl nature of the enzyme is shown by its complete inhibition by 5 × 10-8 M p-chloromercuribenzoate; this is almost completely restored by glutathione or cysteine. Iodoacetate at 10-2 M final concentration has no effect. Various metal-binding agents such as cyanide, azide, thiourea, 8-hydroxyquinoline, potassium ethyl xanthate, and o-phenanthroline are inhibitory, indicating a heavy metal constituent. The latter has been identified as molybdenum. 7 The addition of molybdate to the reaction mixture does not stimulate activity unless the enzyme has been freed of its metal by previous dialysis against cyanide. Nor does the addition of Fe ++, Fe +++, Mn ++, Zn++, Mg ++, BOa--, or Cu ++ to the reaction mixture stimulate activity. Cu ++ is markedly inhibitory (65%) at 10-4 M final concentration. Adaptive Nature of the Enzyme. Nitrate reductase activity is present in mycelia grown in the presence of nitrate or nitrite. There is no activity in mycelia grown in ammonia or alanine as a sole nitrogen source. Equilibrium Constant. From the E0' at pH 7.0 for T P N H : T P N +, which is probably about -0.28 volt on the assumption that it is close to that of D P N H : D P N +8 and E0' at pH 7.0 for NO3-:N02- of +O. 54, 9 the free energy change and equilibrium constant of the nitrate reductase reaction are calculated to be -30,000 calories and 1027, respectively. Mechanism of Action. With purified nitrate reductase from Neurospora it has been shown 1° that during the enzymatic transfer of electrons from T P N H to nitrate both FAD (or FMN) and molybdenum function as electron carriers. The reduction sequence mediated by the enzyme in the absence or presence of added indophenol dye is as follows: T P N H --* FAD (or FMN) --* Mo --* NO~2,3',6-trichloroindophenol s H. Borsook, J. Biol. Chem. 133, 629 (1940). gL. Anderson and G. W. E. Plaut, in "Respiratory Enzymes" (H. A. Lardy, ed.), rev. ed., p. 84, Burgess Publishing Co., Minneapolis, 1949. 1o D. J. D. Nicholas and A. Nason, J. Biol. Chem. 211, 183 (1954).

416

ENZYMES OF PROTEIN ~ETABOLIS~

[60] H y d r o x y l a m i n e R e d u c t a s e f r o m Neurospora

[60]

crassa

NH20H + D P N H + H +--~ NH~ + DPN + -~ H~O

By MILTON ZUCKER and ALVIN NASON Assay Method Principle. The method is based on the fact that the absorption of D P N H at 340 mt~ disappears on oxidation. The reaction may be followed by measuring the rate of D P N H oxidation spectrophotometrically.

Reagents NH~OH.HC1 (0.4 M). Dissolve 69.5 mg. in 2.5 ml. of H20. The solution is prepared daily. No nitrite is present in the Eastman Kodak product. D P N H (approximately 6 X 10-3 M), enzymatically reduced. 1 Boiled pig heart extract. Grind 10 g. of acetone-dried pig heart in 70 ml. of cold 0.1 M phosphate buffer, pH 7.5. Centrifuge, and boil the supernatant in a water bath for 5 minutes in the dark. Recentrifuge, and store the supernatant in the cold, protected from light. Not a reliable source of flavin after two to three weeks of storage. FAD, 2 X 10-5 M (Sigma Chemical Company, 40% pure). Dissolve enough FAD in distilled water to give an extinction of approximately 0.200 at 455 m~. The extinction coefficient at this wavelength is 1.13 X 10-7 sq. cm./mole. 2 Store frozen and protected from light. 0.1 M sodium pyrophosphate-HC1 buffer, pH 8.0. Enzyme. When necessary, dilute stock enzyme with 0.5 M K~HPO4 to obtain 1000 units or less per milliliter. (See definition below.)

Procedure. Mix 2.60 ml. of p i t 8.0 buffer, 0.05 ml. of DPNH, and 0.05 ml. of boiled pig heart extract or FAD in a cuvette having a 1-cm. light path. Take readings at 340 m~ at 30-second intervals after addition of 0.2 ml. of enzyme for 2 to 3 minutes to obtain an endogenous rate of D P N H oxidation. Start the reaction by addition of 0.1 ml. of NH~OH.HC1, and continue readings at 15- to 30-second intervals for 2 minutes, beginning 45 seconds after addition of substrate. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which causes an initial rate of change (AE~40) of 0.001 per 1See Vol. III [127] for enzymatic reduction of DPNH. O. Warburg and W. Christian, Biochem. Z. 298, 150 (1938).

[60]

ttYDROXYLAMINE REDUCTASE FROM NEUROSPORA CRASSA

417

minute after correction for the endogenous rate of oxidation. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al2 Application of Assay Method to Crude Tissue Preparations. In other wild-type Neurospora strains, i.e., strain 146, the enzyme is detected only in the purified fraction, presumably as a result of inhibitors in the crude preparation. T P N H must be used as the electron donor. Hydroxylamine reductase has also been detected in crude extracts of bacillus organisms by measuring the disappearance of NH2OH 4 or by measuring D P N H oxidation. 5 NH2OH is assayed for according to Czaky. 6 In Neurospora the enzyme is found only in extracts of mycelia grown in the presence of nitrate or, to a lesser extent, nitrite.

Purification Procedure

Culture Conditions. Mycelial mats of Neurospora crassa (wild type, macroconidial strain Em5297a) are grown from spore innoculums on 125 ml. of a modified Fries minimal medium 7 in 500-ml. Erlenmeyer flasks at 30 ° . Four- and five-day-old mats are used as the source of enzyme. The procedures described below have been repeated successfully a number of times in this laboratory. All steps were carried out at 0 to 4 °, and precipitates were centrifuged at 3000 × g. Step 1. Preparation of Crude Extract. Mycelial mats are collected on a Biichner funnel, washed with distilled water, and frozen at - 1 5 ° for 1 to 3 hours. The frozen mats are coarsely powdered in a cold mortar and pestle, homogenized in four times their weight of cold 0.1 M K2HP04 with a Tenbrock glass homogenizer, and centrifuged at 3000 × g at 0 ° for 20 minutes. The resulting supernatant solution, designated as the crude cell-free extract, contained 85 % of the total protein. Step 2. Ammonium Sulfate Fractionation. Eighty-three milliliters of saturated ammonium sulfate solution adjusted to pH 7.0 to 7.3 with concentrated ammonium hydroxide is added to 100 ml. of crude cell-free 30. H. Lowry, N. J. Rosebrough, ~. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4 S. Taniguchi, H. Mitsui, J. Toyoda, T. Yamada, and F. Egami, Japan. J. Biochem. 40, 175 (1953). R. Klausmeyer and R. Bard, J. Bacteriol. 68, 129 (1954). T. Z. Czaky, Acta Chem. Scand. 2, 450 (1948). 7 In units per liter: sodium tartrate, 5 g. ; NH4NO3, 3 g. ; I4H2PO~, 3 g. ; MgSO4-7H~O, 0.5 g. ; NaC1, 0.1 g. ; CaCl2, 0.1 g. ; sucrose, 20 g. ; biotin, 5 tLg.; 1 ml. of a complete race element solution containing sodium tetraborate 8.8 × 10-5 g. ; (NH4)4 MoTO24.6H20, 6.4 × 10 -5 g.; FeCI~-6H~O, 9.6 × 10 -4 g.; ZnSO4.7HsO, 8.8 × 10 -3 g.; CuC12, 2.7 X 10 -5 g.; M n C l r 4 H : O , 7.2 X 10 -8 g.

418


[60]

extract making the mixture 45% saturated. After standing for 5 minutes the precipitate is removed by centrifugation, and the supernatant solution is brought to 65% saturation by addition of 104 ml. more of ammonium sulfate solution. After 10 minutes the resulting precipitate is collected by centrifugation and dissolved in 40 ml. of cold distilled water (fraction 2). Step 3. Absorption of Reductase by Ca3(P04)2 Gel. Twenty milliliters of Caa(PO4)2 gel 8 containing 20 mg./ml, dry weight is added to the enzyme solution (fraction 2). After intermittent stirring for 15 minutes, the gel precipitate is collected by centrifugation and eluted by suspending in 20 ml. of cold 0.5 M K~HPO4 buffer for 15 minutes. The supernatant solution obtained from subsequent centrifugation is used as the purified enzyme fraction (fraction 3). Fraction 3 also contains a pyridine-nucleotide nitrite reductase which parallels hydroxylamine reductase activity during fractionation as well as under various conditions and treatments. As yet no separation of the two activities has been obtained. The ratio of the two activities varies in different extracts, and addition of hydroxylamine to a system saturated with nitrite usually produces a small stimulation in the rate of D P N oxidation. However, when extracts of strain 146 are carried through the above procedure, hydroxylamine reductase preparations free of nitrite reductase activity are obtained. SUMMARY OF PURIFICATION PROCEDURE

Fraction

Total Total Specific volume, Units/ml., units, Protein, activity, Recovery, ml. thousands thousands mg./ml, units/mg. %

1. Crude extract 100 2. (NH4)~SO4 fraction 45 3. Ca~.(PO4)~geleluate 20

0.20 0.40 0.50

20 18 10

3.4 2.1 1.4

56 190 360

-90 50

Properties Specificity. The purified enzyme is specific for NH2OH and will not catalyze the reduction of O-methyl hydroxylamine, hydrazine, or nitrophenylhydroxylamine. Both D P N H and T P N H serve as electron donors in the reaction. The Km's calculated from Lineweaver-Burke plots are 0.7 X 10-4 M for D P N H and 1.0 × 10-4 M for T P N H . The maximum velocity obtained with D P N H is one-half that obtained with T P N H . The turnover number of hydroxylamine reductase is approximately 250 moles of T P N H oxidized per minute per mole of enzyme, assuming a molecular 8 See Vol. I [11] for preparation of Caa(PO,), gel.

[60]

HYDROXYLAMINE REDUCTASE FROM NEUROSPORA CRASS&

419

weight of 100,000 for the enzyme. The K~ for NH20H is 3.8 × 10-3 M. Activators and Inhibitors. FAD (3 X 10-7 M, final concentration) but not F M N stimulates the rate of DPNH oxidation two- to threefold and completely replaces the stimulation obtained with boiled pig heart extracts. A number of chelating agents inhibit the oxidation of DPNH, KCN being the most effective (50% at 5 X 10-6 M KCN). Other inhibitors at a final concentration of 5 X 10-3 M are salicylaldoxime, potassium ethyl xanthate, diethyldithiocarbamate, o-phenanthroline, a,a-dipyridyl, and sodium azide. Cysteine and glutathione have no effect alone, nor are they effective in reversing the inhibition obtained with p-chloromercuribenzoate. Iodoacetate at 10-2 M has no effect on the enzyme. Bisulfite (1.6 X 10-3 M, final concentration) completely inhibits the enzyme. Stability of Enzyme. The enzyme is most stable between pH 7.5 and 9.0, losing all its activity after a week at - 1 5 °. There is a complete loss of activity on standing overnight at 4 °. The enzyme loses all activity after 5 minutes at 50 °. Hydroxylamine reductase can be dialyzed against 3 X 10-3 M sodium pyrophosphate and 10-3 M cysteine for at least 2 hours, losing less than half its activity during this time. However, there is a complete loss of activity after 1 hour if 10-2 M phosphate, pH 8.0, is the dialyzing solution. This loss of activity on dialysis cannot be restored by addition of boiled enzyme, boiled pig heart extract, FAD, or FMN (10-6 M final concentration). At a final concentration of 10-~ M, ferrous, ferric, zinc, calcium, molybdate, cupric, manganous, borate, and magnesium ions did not restore activity either. Effect of pH. The effect of pH on enzymatic activity was determined by use of various buffers of suitable pH values. There is maximum enzyme activity between pH 8.0 and 9.0 when pyrophosphate or tris(hydroxymethyl)aminomethane is the buffer. Little or no activity is obtained with phosphate buffer unless 10-3 M Versene is included in the reaction mixture, suggesting the removal of a metal inhibitor.

420


[61]

[61] Nitrite M e t a b o l i s m Enzymatic Formation of Nitrogen Gas (N~) from Nitrite and Nitric Oxide Gas (NO) 2 N O ~ - ~ N~ + 4 0 2 NO--~ N2 + 2 0

Formation of Nitric Oxide Gas from Nitrite N02 --~ NO + 0 By VICTOR A. NAJJAR

Assay Method

Principle. The method of measuring nitrogen and nitric oxide formation from nitrite is based on the fact that gas (N~ and NO) is formed which can be measured manometrically by the increase in pressure and that NO can be absorbed from the gas phase by alkaline sulfite. However, the formation of nitrogen from nitric oxide results in a decrease in pressure, as NO is also in the gas phase and two molecules of NO form one molecule of N2. Reagents NaNO2, 0.1 M. Hydrogen donor system. 0.3 % Difco yeast extract, glucose-6-phosphate 0.1 M, malate 0.1 M, and cofactors (see below). KOH, 20%. Nitric oxide gas, oxygen-free nitrogen gas. Phosphate buffer, pH 6.8, 0.2 M; tris(hydroxymethyl)-aminomethane buffer, pH 8.0, 0.2 M.

Procedure. Buffer (0.7 ml.), nitrite (0.1 ml.), and hydrogen donor (0.1 ml.) are placed in the main compartment, and the enzyme (0.5 ml.) in the side bulb. Alkali (0.2 ml. of 20 % KOH) is pipetted into the center well to absorb COs that might be formed. The manometers, while shaking, are flushed adequately for 15 to 20 minutes with oxygen-free nitrogen gas. After a period of equilibration the reaction is started by tipping in the enzyme. The increase in pressure as a function of time is recorded, and the amount of gas evolved is calculated. The bath is kept at a convenient temperature, 25 to 37% For the measurement of nitrogen (N2) formation from nitric oxide (NO) gas, NO is introduced into the gas phase after the system is thoroughly flushed with oxygen-free N2. It is essential that oxygen be rigor-

[61]

NITRITE METABOLISM

421

ously excluded from the whole system, as nitric oxide reacts readily ~Tith oxygen to form nitrogen dioxide gas (2 NO + 02 --* 2 NO2 ~ N204) which can be recognized by the red color of NO~ gas only if the latter is present in considerable amounts. The nitrogen dioxide formed reacts with water to produce nitrous and nitric acids (2 NO2 + H20--~ H N Q + HN02). This can occur to such an extent as to tax the capacity of the buffering system. The pH may drop to such a level as to precipitate and inactivate the enzyme. The decrease in pressure resulting from the conversion of NO to N2 is recorded as a function of time, and the amount of NO uptake can thus be calculated. Since 1 micromole of N2 gas is formed from 2 micromoles of NO gas, the resulting decrease in volume is a direct measure of the nitrogen formed. Nitric oxide gas is commercially available and can be generated readily in the laboratory in one of two reactions. I~NO~ + H I -~ ~/~ I~ + NO + H~O 3 C u + 8 H + + 2 N O 3 - - ~ 3 C u ++ + 4 H 2 0 + 2 N O

(1) (2)

In both reactions oxygen should be excluded rigorously to avoid the formation of nitrogen dioxide. The latter reaction is conveniently carried out by dropping 6 N HNO~ over copper wire in a closed system filled with N2 gas. The NO generated is allowed to bubble through water over which it is collected in order to remove traces of NO2 gas that is also formed. The reaction is preferably carried out in an ice bath.

Preparation of the Enzymes The enzymes are obtained from denitrifying bacteria, ~ Pseudomonas stutzeri, and a thermophilic strain of Bacillus subtilis (strain 115). These organisms are grown anaerobically in 0.3% (Difco) yeast extract and 0.5% KNO3. The latter is incubated at 50 to 55 ° for 8 hours, and the former at 25 ° for 24 to 38 hours or at 37 ° for 16 to 20 hours. A standard inoculum consisting of a 16-hour culture is used for inoculating fresh cultures (10 to 15 ml./1.). Cells are harvested by centrifugation and washed once with water. The enzymes are prepared by grinding P. stutzeri cell paste with alumina powder in the cold and extracting with 1 vol. of water at 4 to 6 °. This is then centrifuged for 30 minutes at 2100 × g to remove alumina, intact cells, and large cell fragments. The turbid supernatant is the source of enzyme. B. subtilis is lysed by adding a few crystals of lysozyme (Nutritional Biochemicals) to the cell paste and incubating at room temperature until complete lysis takes place. A volume of water is then added, and the nonlysed cells are centrifuged down. This supernatant is i V. A. Najjar and M. B. Allen, J. Biol. Chem. 206, 209 (1954).

422


[61]

also a source of enzyme. Crude extracts so prepared produce 1 to 2 micromoles of N2 per milliliter of extract per hour at 25 ° in a complete system containing yeast extract as hydrogen donor and nitrite in phosphate buffer, pH 6.8. When the extracts are centrifuged at 15,000 X g for 30 to 60 minutes at 6 ° to separate the small particles, the resulting supernatant shows little or no activity. The activity is restored, however, by the addition of the particles. 1In this system DPN and TPN have no stimulating effect. Nitrogen formation from nitric oxide is similarly dependent on the particles which presumably contain the reducing system. Crude extract of P. stutzeri produces NO gas from nitrite in the complete system described above. The NO formed is trapped in alkaline sulfite to form Na~N~O2SO3 (5% Na2SOa in 0.1 N NaOH). The extent of NO formation is appreciated by the difference in the amount of gas formed in duplicate vessels, one of which contains 0.2 ml. of the sulfite in one side arm. The amount of NO trapped in the sulfite can be obtained by liberating N20 upon the addition of 0.1 ml. of 5% H2S04. Purification Procedure

The enzymes from P. stutzeri have been purified to some extent by ammonium sulfate fractionation. ~,3 Nitrogen formation from nitrite can be obtained with the highest activity in an ammonium sulfate fraction obtained at 0.4 to 0.55 saturation. After 48 hours of dialysis against water this fraction shows little activity with the complete system. However, with the addition of excess T P N H or glucose-6-phosphate and catalytic amounts of TPN, the activity is regenerated. That the enzyme has glucose-6-phosphate dehydrogenase can readily be shown in the Beckman spectrophotometer. The enzyme activity is also stimulated by the addition of D P N H or malate and catalytic amounts of DPN, but to a lesser extent than that exhibited by the TPN system. In these extracts the reduction of DPN by malate can also be demonstrated. The reduction of NO to N2 by an ammonium sulfate fraction obtained at 0.4 to 0.7 saturation is likewise poorly active after a 48-hour dialysis against distilled water. 2,3 The activity is regenerated by TPN and glucose-6-phosphate and to a lesser extent by DPN and malate. Properties 3

The reduction of nitrite to nitrogen has a pH optimum around 6.8 in phosphate buffer. However, the pH optimum for the formation of nitrogen from nitric oxide is at 8.0 in tris(hydroxymethyl)-aminomethane 2 C. W. C h u n g a n d V. A. Najjar, Federation Proc. 15 t 192 (1954). a C. W. C h u n g a n d V. A. Najjar, u n p u b l i s h e d observations.

[61]

NITRITE METABOLISM

423

buffer. At a concentration of 1 × 10-3 M, Ca ++, Mg ++, Mo, and Co ++ have no effect on nitrite reduction. Cyanide at a concentration of 1 X 10-3 M is slightly inhibitory. Further supplementation with FAD or FMN produces many-fold stimulation of NO to N2 conversion. The host of enzymes described above belong to the class of adaptive enzymes. It is necessary to have nitrate or nitrite in the medium for the development of these enzymes. Nitrate has proved consistently superior, since nitrite is inhibitory to cell growth when present in necessarily large amounts.

[62]

RIBONUCLEASES

427

[62] Ribonucleases By

MARGARET R. McDoNALD

Enzymes capable of hydrolyzing R N A are present in a great variety of cells. Only the ribonuclease (RNase) of bovine pancreas 1 has been purified and crystallized. 2 Its properties and mode of action have been extensively studied. There is no basis for assuming t h a t the RNases of other tissues have the same properties or mode of action, although their gross effect on R N A m a y be similar. In fact, studies on crude tissue extracts show m a n y dissimilarities between crystalline pancreatic RNase and RNases from other sources. The hydrolysis of R N A by pancreatic RNase is accompanied b y the gradual formation of free acid groups without any significant liberation of free phosphoric acid. 3,~ The split products, unlike the undigested RNA, are not precipitable b y acetic acid, hydrochloric acid, 4 or a solution of uranium salt in trichloroacetic acid. 2 T h e y readily diffuse through collodion or cellophane membranes t h a t are impermeable to the undigested RNA. 2 Digestion of R N A b y RNase is accompanied b y a shift in the ultraviolet absorption spectrum of the substrate toward the shorter wavelengths. This shift is most distinct in the region of 290 to 305 m~. 5 Most of these phenomena have been utilized in developing methods of RNase assay. 6 The one described here has been found b y the author to be the most convenient for the routine assay of pancreatic RNase. I t is essentially Kunitz's modification 2 of the "acid-soluble phosphorus" method of Dubos and T h o m p s o n J

Assay Method P r i n c i p l e . The method is based on the fact that, during digestion of I~NA b y RNase, 40% of the total nucleic acid phosphorus is converted into a form soluble in acid uranium acetate.

1Partially purified RNase has been prepared from sprouted soybeans [M. Schlamowitz and R. L. Garner, J. Biol. Chem. 163, 487 (1946)], but its properties have not been studied. M. Kunitz, J. Gen. Physiol. 24, 15 (1940). 8 W. Jones, Am. J. Physiol. 52, 203 (1920). 4 R. J. Dubos, Science 85, 549 (1937). 5 M. Kunitz, J. Biol. Chem. 164, 563 (1946). a See J. S. Roth and S. W. Milstein [J. Biol. Chem. 196, 489 (1952)] for detailed literature references. 7 R. J. Dubos and R. H. S. Thompson, J. Biol. Chem. 124, 501 (1938).

428

ENZYMES OF NUCLEIC ACID METABOLISM

[62]

Reagents Substrate. RNA, 8 dissolved in 0.1 M acetate buffer, p H 5.0, to a concentration of 0.5 rag. of total P per milliliter. The solution should be fresh and the p i t carefully adjusted in order to obtain reproducible results. M a c F a d y e n ' s reagent2 0.25% uranium acetate in 2.5% trichloroacetic acid. Procedure. One milliliter of RNase is mixed with 1 ml. of substrate, the mixture is left for 10 minutes at 25 °, then 2 ml. of M a c F a d y e n ' s reagent is added with thorough mixing. T h e suspension is left at 25 ° for 30 minutes, then filtered through 7-cm. Whatm.an No. 42 filter paper, and 2 ml. of the filtrate is analyzed for total phosphorus, l° This is designated as soluble phosphorus. With partially purified RNase preparations, ultraviolet absorption measurements at 260 m~ of a fivefold aqueous dilution of the filtrate, with a tenfold dilution of M a c F a d y e n ' s reagent as the blank solution, can be satisfactorily substituted for the more time-consuming phosphorus determinations. Definition of Unit and Specific Activity. The R N a s e activity unit is defined as the activity which gives rise under the standard conditions described above to t h e formation of 1 X 10-4 mg. of soluble phosphorus per milliliter of digestion mixture per minute in a range of concentrations of enzyme where the a m o u n t of soluble phosphorus formed is proportional to the concentration of enzyme used. For convenience, a standard curve is plotted--soluble phosphorus vs. activity u n i t s - - f r o m data obtained b y measuring the activity of a series of dilutions of ribonuclease of a known enzyme content. T h e activity of any unknown solution of RNase is then determined from a single measurement b y means of the standard curve. Specific activity is expressed as RNase units per milligram of protein. Protein is determined b y the m e t h o d of L o w r y et al. 11 Application of Assay Method to Crude Preparations. Of the various procedures 6 for the assay of RNase in crude tissue homogenates, the one described above is probably most applicable, provided t h a t control determinations are simultaneously made without added substrate. The press If commercial RNA is used, it should be purified by the procedure of G. E. Woodward [J. Biol. Chem. 156, 143 (1944)] to remove inhibitory mononucleotides. 9 D. A. MacFadyen, J. Biol. Chem. 107~ 297 (1934). 10By the method of E. J. King [Biochem. J. 26, 292 (1932)] or a similar procedure; also see Vol. III [114]. 11O. H. Lowry, N. J. Rosebrough, A. L. Farr. and R. J. Randall, J. Biol. Chem. 193, 265 (1951); also see Vol. III [73].

[62]

RIBONUCLEASES

429

ence of phosphatases which, by removing the formed products, would tend to increase the rate of the reaction, can be detected by assaying the filtrate for inorganic phosphorus. I°

Purification of Pancreatic RNase The procedure described below is essentially t h a t of Kunitz 2 as modified b y McDonald. 12 The method has proved to be very reproducible, both in the hands of the author and in m a n y other laboratories. I t has been used for the preparation of radioactive RNase. 1~ The saturated (NH4)2S04 is prepared at 20 to 25 ° (760 g. of salt per liter of H20). Determinations of p H are made on a test plate by mixing 1 drop of the appropriate 0.01% indicator with 1 drop of the solution to be tested and comparing the color with t h a t found by mixing 1 drop of the same indicator with 1 drop of 0.1 M standard buffer of the desired pH. This gives only apparent p H values but is adequate for reproducing the necessary conditions. All filtrations, unless otherwise specified, are done with suction on Bfichner funnels. Step 1. Preliminary Purification. About 20 pounds of fresh TM beef pancreas is collected in ice-cold 0.25 N H2S04. The glands are drained, freed of fat and connective tissue, and minced in a meat grinder. The ground pancreas is suspended in 2 vol. of ice-cold 0.25 N H2S04 and left at 0 to 5 ° for 18 to 24 hours, with occasional stirring. The suspension is then strained through cheesecloth, and the strained fluid is saved. The residue is resuspended in an equal volume of cold 0.25 N H2SO4 and restrained after 1 hour. The residue is discarded. The combined extracts are brought to 0.65 saturation of (NH,)2S04 by the addition of 430 g. of salt per liter of strained fluid. The suspension is filtered by gravity through 50-cm. fluted filter papers (Eaton-Dikeman No. 612 or W h a t m a n No. 12), at 0 to 5 °, and the clear filtrate is saved. The residue is suspended in a volume of cold H20 equal to that of the original minced pancreas, and 430 g. of (NH4)2SQ is added per liter of H20 used. The mixture is refiltered through fluted paper. This filtrate 15 is combined with the first one, and 105 g. of (NH4)~SO, is added per liter of filtrate (final concentration of (NH4)2S04, 0.8 saturation). The resulting precipitate is all3 M. R. McDonald, J. Gen. Physiol. 32, 39 (1948). 13C. B. Anfinsen, J. Biol. Chem. 185, 827 (1950). 14Frozen pancreases, obtainable from any of the large slaughterhouses, can also be used if only ribonuclease (or deoxyribonuclease) is to be prepared. They should be thawed by leaving them immersed in 0.25 N H2SO, at 5 °. 15The residue on the paper can be used for the isolation of deoxyribonuclease (M. Kunitz, J. Gen. Physiol. 33, 349 (1950); see also Vol. II [63]), chymotrypsinogen, trypsinogen, trypsin, and trypsin-inhibitor compound (M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 1002 (1936) ; see also Vol. II [2, 3, 4]).

430


[62]

lowed to settle for 2 days at 0 to 5°; the settling is greatly facilitated by occasional stirring of the mixture and removal of foam during the first day of standing. The clear supernatant fluid is siphoned off and discarded; the remaining suspension is filtered through hardened paper (Schleicher and Schuell No. 576). The yield is about 4 g. per liter of ground pancreas used. Step 2. Removal of Proteolytic and Potential Proteolytic Activity. Each gram 18 of filter cake is dissolved in 5 ml. of H~O, and the resulting solution is poured into 20 ml. of boiling 0.2 saturated (NH4)2SQ previously adjusted with H2SO4 to pH 3.0 (methyl orange). The mixture is stirred for 5 minutes at 95 to 100 °, cooled quickly to 25 °, and left at 20 to 25 ° for approximately 1 hour. The suspension is filtered through soft paper (Eaton-Dikeman No. 617) with the aid of 10 g. of Standard Super-Ce117 per liter, and the filter cake is washed three times with small quantities of 0.2 saturated (NH4)2SO4. The residue is discarded. The filtrate is brought to 0.5 saturation of (NH4)2SO4 by the addition of 188 g. of salt per liter of filtrate; 10 g. of Standard Super-Cel is then added per liter, and the suspension is filtered with suction through soft paper. The residue is again discarded. The filtrate is brought to 0.8 saturation of (NH4)2SQ by the addition of 210 g. of salt per liter, and the resulting suspension is filtered on hardened paper; the yield is approximately 3 g./1. of ground pancreas used. The filtrate is discarded. Each gram 16 of filter cake is dissolved in 5 ml. of H~O, the pH of the solution is adjusted to 4.8 (methyl red or bromocresol green) with a few drops of 5 N NaOH, and 5 ml. of saturated (NH4)2S04 is added. The mixture is filtered through soft paper with the aid of 1 g. of Standard Super-Cel per 100 ml. of suspension. The residue is discarded. The filtrate is adjusted to pH 4.2 (bromocresol green) with 1 N H2SO4, after which 67 ml. of saturated (NH4)2SO4 is added slowly with constant stirring for each 100 ml. of solution (final concentration of (NH4)2SO4, 0.7 saturation). The suspension is filtered with suction on hardened paper. The yield is approximately 2 g./1. of minced pancreas originally used. The filtrate is discarded. TM Step 3. Crystallization. Each gram of filter cake is dissolved in 1 ml. of H20. The solution is filtered through soft paper with the aid of 5 g. of Standard Super-Cel per 100 ml. of solution; the residue on the paper 16 This expression denotes the relative a m o u n t s of material used. I t does not m e a n t h a t each gram of m a t e r i a l is processed separately. 17 Supplied b y Johns-Manville, 22 E a s t 40th Street, New York. 18 The yield of ribonuclease can be increased b y adding (NH4)2SO, to this filtrate to 0.8 saturation, filtering on h a r d e n e d paper, a n d reworking t h e filter cake with the next b a t c h of material being processed for the removal of proteolytic c o n t a m i n a n t s according to t h e second p a r a g r a p h of step 2.

[62]

RIBONUCLEASES

431

is washed several times with small quantities of H20 and then discarded. The combined filtrate and washings are brought with H20 to a final volume of 2 ml. Saturated (NH4)~S04 is then added slowly, with stirring, until a v e r y faint t u r b i d i t y appears (approximately 40 ml. per 100 ml. of solution is required), and the p H of the mixture is adjusted immediately to 4.6 (methyl red or bromocresol green) with a few drops of 1 N N a O H . T h e solution clears rapidly and is left at 20 to 25 °. Crystals of ribonuclease gradually form. 19 T h e y are filtered on hardened paper after 3 days; the yield is approximately 1.2 g./1. of minced pancreas originally used. The filtrate is adjusted to p H 4.2 (bromocresol green) with 1 N H2SO4 and brought to 0.8 saturation of (NH4)2SO4 by the slow addition, with constant stirring, of saturated (NH4)2SO4. The suspension is filtered on hardened paper, and the filtrate is discarded. The yield of filter cake is about 0.4 g./1. of minced pancreas. Additional ribonuclease crystals can be obtained b y reprocessing this filter cake according to the second paragraph of step 2. Step 4. Recrystallization. Each gram of filter cake of crystals is dissolved in 2 ml. of H20. The solution is filtered through soft paper with the aid of 0.1 g. of Standard Super-Cel. The residue is washed several times with small amounts of H~O. T h e combined filtrate and washings are brought to 3 ml. with H20. Saturated (NH4)2SO4 is added slowly, with stirring, until the solution becomes very faintly turbid (about 4 ml. per 10 ml. of solution is required). The mixture is left at 20 to 25°; crystals form rapidly. T h e y are filtered on hardened paper after 2 days; the yield is about 0.6 g./g. of filter cake. The filtrate is adjusted to p H 4.2, and saturated (NH4)2SO~ is added slowly to 0.8 saturation. The suspension is filtered on hardened paper, and the filtrate is discarded. The yield is about 0.3 g./g. of filter cake. Additional ribonuclease crystals can be obtained from this filter cake by processing it as described in step 3. Step 5. Recrystallization in Ethanol. Ribonuclease is recrystallized twice b y means of (NH4)~SO4, as described in the preceding section. Each gram of filter cake from the third crystallization is dissolved in 1.5 ml. of H20, and the solution is dialyzed 2° in collodion or viscose din19If too much (NH4),S04 has been added, an amorphous precipitate will form rapidly. This may change within 1 or 2 days into a mass of fine crystals; if not, water should be added dropwise until the amorphous material dissolves and typical "silkiness" is seen when the solution is stirred. Almost complete crystallization should then occur within the next 3 days. If no "silkiness" is seen the solution shouId be adjusted to pH 4.2, brought to 0.8 saturation with saturated (NH4)2SO,, and filtered with suction through hardened paper; the filter cake should again be processed as described in step 3. 90 M. Kunitz and It. Simms, J. Gen. Physiol. 11, 641 (1927-28). If the dialyzer of Kunitz and Simms is not available, it is advantageous to dialyze with slow mechani-

432


[62]

lyzing tubing at 0 to 5 ° against cold H 2 0 for 24 hours. The dialyzed solution is diluted with H : O to 5 ml. and cooled to 5 °, and 60 ml. of 95% ethanol of the same t e m p e r a t u r e is added with stirring. A h e a v y a m o r phous precipitate i~ formed, which, on standing at 10 to 20 °, changes within several hours into a mass of fine fan-shaped rosettes of rectangular or needle-shaped crystals. T h e crystals are filtered on hardened p a p e r after 2 days and washed several times with cold 9 5 % ethanol. T h e y are then dried for 24 to 72 hours Over CaC12 in a desiccator. T h e d r y powder can be stored in a cool place indefinitely; the yield is a b o u t 0.3 g. of d r y crystals per g r a m of filter cake. SUMMARY OF PURIFICATION OF BOVINE PANCREATIC R I B O N U C L E A S E a

Fraction 1. Preliminary purification Acid extract 0.65-0.8 sat. (NH4)2S04 2. Removal of proteolytic contaminants 0.5-0.7 sat. (NH4)2S04 3. Crystallization Crystals Mother liquor 4. Recrystallization 2 X crystallized 3 X crystallized 5. Crystallization from ethanol Dialyzed ribonuclease Crystals

Total units, b )< l0 s

Specific activity, units/mg. protein

Yield, %

2000 1200

5 82

100 60

886

119

44

585 289

135 92

29 14

472 315

151 166

23 16

267 255

167 166

13 12

M. R. McDonald, J. Gen. Physiol. 32, 39 (1948). Based on 12.5 1. of ground pancreas. P r o p e r t i e s of P a n c r e a t i c R N a s e Specificity. Crystalline pancreatic R N a s e hydrolyzes R N A b u t not polymerized or depolymerized D N A . 4,21,22 D e a m i n a t e d R N A is degraded b y RNase, showing t h a t NH2 groups are not essential for its action. 2~ T h e action of R N a s e appears to involve specifically pyrimidine nucleo-

cal stirring for 48 hours against 2 1. of cold distilled H20 which is changed twice daily. 21L. M. Gilbert, W. G. Overend, and M. Webb, Exptl. Cell Research 2, 138 (1951). 22 M. C. Durand and R. Thomas, Biochim. et Biophys. Acta 12, 416 (1953). ,8 L. Vandendriessche, Compt. rend. tray. lab. Carlsberg. Sdr. chim. 27, 342 (1951).

[6 9.]

RIBONUCLEASES

433

tides. ~4 Cyclic pyrimidine ribose nucleotides (2',3'-monohydrogen phosphate esters of nucleosides) are digested by RNase to give 3'-riboside phosphate, whereas the analogous purine derivatives are not. 2~ RNase appears to be a specific phosphodiesterase which hydrolyzes only secondary phosphate esters of pyrimidine riboside 3'-phosphates. 26 Recent publications state that it degrades polyribophosphate 27 and thymic acid. 22 Kinetics. A mathematical analysis of the kinetics of the hydrolysis of RNA by RNase is complicated by the complexity of the reaction and by the fact that the enzymatic reaction is always accompanied by a significant amount of spontaneous hydrolysis of the substrate. The time required for any amount of digestion is inversely proportional to the concentration of enzyme in solution, whereas the ultimate amount of digestion is independent of the amount of enzyme used. 2 A marked rate reduction of enzyme activity is observed when the substrate concentration is increased, with a definite lag in attainment of the maximal rate of digestion. 28 The rate of formation of titratable acid groups is much slower than the rate of formation of acid-soluble split products. 2 Activators and Inhibitors. No specific activators are required for the enzymatic activity of RNase; no specific inhibitors are known. Its activity, as determined spectrophotometrically5 or turbidimetrically, 29 is inhibited by Mg ++, Ca ++, and Mn ++, the minimal inhibitory concentration being less than 0.0005 M. This inhibition is not reduced by F1- or citrate ions; it is not suppressed by Na + or NH4 +, which, in the concentration range of 0.0005 to 0.1 M, stimulate ~'ibonuclease activity. Higher concentrations of Na + and NH4 + have an inhibitory action, 0.33 M NaC1 decreasing the initial rate of reaction about 40 %.2s The liberation of acid groups and the formation of acid-soluble split products by RNase is enhanced by 0.1 M NaC1 or 0.1 M MgC12, the latter being the more effec~4 R. A. Bolomey and F. W. Allen, J. Biol. Chem. 144, 113 (1942); H. S. Loring, F. H. Carpenter, and P. M. Roll, J. Biol. Chem. 169, 601 (1947); G. Schmidt, R. Cubiles, and S. J. Thannhauser, Cold Spring Harbor Symposia Quant. Biol. 12, 161 (1947); C. E. Carter and W. E. Cohn, J. Am. Chem. Soc. 72, 2604 (1950); G. Schmidt, R. Cubiles, N. Z611ner, L. Hecht, N. Strickler, K. Seraidarian, M. Seraidarian, and S. J. Thannhauser, J. Biol. Chem. 192, 715 (1951); B. Magasanik and E. Chargaff, Biochim. et Biophys. Acta 7, 396 (1951). 25 R. Markham and J. D. Smith, Biochem. J. 52, 552 (1952); D. M. Brown, C. A. Dekker, and A. R. Todd, J. Chem. Soc. 1952, 2715. ~6R. Markham and J. D. Smith, Biochem. J. 52, 558 (1952); E. Volkin and W. E. Cohn, J. Biol. Chem. 205, 767 (1953). 2TS. Zamenhof, G. Leidy, P. L. FitzGerald, H. E. Alexander, and E. Chargaff, J. Biol. Chem. 203, 695 (1953). 2s C. Lamanna and M. F. Mallette, Arch. Biochem. 24, 451 (1949). ~* M. McCarty, J. Exptl. Med. 88, 181 (1948).

434


[62]

tive. 8°,31 NaC1 or MgC12 does not liberate t i t r a t a b l e acid groups in the absence of the enzyme. 31 R N a s e - c a t a l y z e d liberation of acid groups is m a r k e d l y inhibited b y 2 X 10 -8 M Cu ++ or Zn ++ and slightly inhibited b y Ni ++ and Ag + of the same concentration; Co ++, Cd ++, Fe +++, and H g ++ h a v e a negligible effect. 3~ Higher concentrations (1 X 10 -3 M) of Co ++ and H g ++ inhibit RNase. 33 S t r e p t o m y c i n has been reported as an a c t i v a t o r s° and an inhibitor a4 of R N a s e ; penicillin 35 and basic dyes such as acridine 34 are also inhibitors. T h e enzyme is inhibited b y t r e b u r o n a6 (a synthetic sulfated polygalacturonic acid) and b y heparin 36,37 b u t not b y chondroitin-sulfuric acid, hyaluronic acid, or alginic acid. as This inhibition is reversible, the a c t i v i t y of the inhibited R N a s e being restored b y acid hydrolysis at 80°. a7 Mononucleotides 39 and D N A 4° inhibit RNase. Benzimidazole, 2-aminobenzimidazole, and 5,6-dimethylbenzimidazole accelerate R N a s e action, s~ I n c u b a t i o n of R N a s e with N a p-chloromercuribenzoate first increases then decreases its enzymatic activity; 33 incubation with iodoacetate, iodoacetamide, 32 periodic acid, 41 formaldehyde, ninhydrin, and phenylisocyahate 42 inactivates RNase. I t is also i n a c t i v a t e d b y X - r a y s 43 and b y O H radicals. 44 R N a s e is readily i n a c t i v a t e d b y digestion with pepsin. 2,4 Physicochemical Properties. Crystalline pancreatic R N a s e is a protein of the albumin t y p e with the following e l e m e n t a r y composition in per cent d r y weight: C, 48.2; H, 6.2; N, 16.1; S, 3.6 (partly inorganic); P, trace; residue, 0.1. 2 I t s amino acid composition, expressed as grams per 100 g. of protein (ash-, sulfate-, and moisture-free), is as follows: arginine, 5.2; aspartic acid, 14.2; cysteine, 0.6; cystine (half), 6.5; glutamic acid, 30 G. Ceriotti, Nature 163, 874 (1949). 3z C. E. Carter and J. P. Greenstein, J. Natl. Cancer Inst. 7, 29 (1946-47). Electrolytes, in the absence of RNase, do degrade RNA to dialyzable components. 32 C. A. Zittle, J. Biol. Chem. 163, 111 (1946). 33y. Miura and Y. Nakamura, Compt. rend. 232, 1874 (1951). a4L. Massart, G. Peeters, and A. Lagrain, Arch. intern, pharmacodynamie 76, 72 (1948) [Chem. Abstr. 42, 6383 (1948)]. a3L. Massart, G. Peeters, and A. Vanhoucke, Experientia 3, 494 (1947). 33j. S. Roth, Arch. Biochem. and Biophys. 44, 265 (1953). 37 N. ZSllner and J. Fellig, Am. J. Physiol. 173, 223 (1953). 33L. Ledoux, Biochim. et Biophys. Acta 10, 190 (1953). a9 C. A. Zittle, J. Biol. Chem. 160, 527 (1945). 40 M. R. McDonald in Carnegie Inst. Wash. Year Book No. 47, 148 (1948). 4i W. F. Goebel, P. K. Olitsky, and A. C. Saenz, J. Exptl. Med. 87, 445 (1948). 4~C. A. Zittle, J. Franklin Inst. 246, 266 (1948). 43D. Lea, K. M. Smith, B. Holmes, and R. Markham, Parasitology 36, 110 (1944); E. S. G. Barron, S. Dickman, J. A. Muntz, and T. P. Singer, J. Gen. Physiol. 32, 537 (1949); B. Holmes, Nature 165, 266 (1950). 44E. Collinson, F. S. Dainton, and B. Holmes, Nature 166, 267 (1950).

[69.]

RIBONUCLEASES

435

13.0; glycine, 1.3; histidine, 4.2; hydroxyproline, 0; isoleucine, 3.1; leucine, 0; lysine, 10.4; methionine, 4.4; phenylalanine, 3.6; proline, 3.6; serine, 12.0; threonine, 9.0; tryptophan, 0; tyrosine, 7.9; valine, 7.3; amide NH~, 2.5. 45 No indication of a special prosthetic group is evident from its ultraviolet absorption spectrum which shows a maximum molecular extinction coefficient of 11,540 near 280 m~ and a minimum of 6160 (5000) at 252 m~. 46 The following physical constants have been obtained for the enzyme: isoelectric point (by electrophoresis), ca. pH 7.8; 47 diffusion coefficient 2 at 20 ° in 0.5 M (NH4)RSQ, 0.092 sq. cm./day; diffusion coefficient47 at 25 ° in 0.5 M (NH4)2S04, 0.117 sq. cm./day; sedimentation constant 47 at 25 ° in 0.5 M (NH4)2SO4, 1.85 X 10-13; specific volume 47 at 25 °, 0.709; molecular volume 2 (calculated from diffusion coefficient), 14,850; molecular weight 47 (calculated from sedimentation and diffusion data), 13,000; molecular weight 2 (by osmotic pressure measurements), 15,000 + 1000; molecular weight (by X-ray analysis), 15,700 ± 300, 4s 13,400; 49 optical rotation per milligram of N at 25 ° (5 % aqueous solution), -0.470. 2 Ribonuclease is a good antigen despite its low molecular weight. 5° Aqueous solutions of crystalline pancreatic ribonuclease are quite stable 2 over a wide range of pH when kept at temperatures below 25 °. Heating to higher temperatures causes gradual loss in enzymatic activity. The rate of inactivation varies, however, with the pH of the solution, the concentration of the enzyme, and the concentration of electrolytes present. 51,52 The region of maximum stability is between pH 2 and 4.5. Effect of pH and Temperature. 2 The optimum pH for the action of RNase is ca. 7.7 (7.2 to 8.2); the optimum temperature is ca. 60 °. Crystalline RNase appears to be homogeneous from electrophoretic and ultracentrifugal studies. 47 Solubility studies 2,1~ indicate the possible presence of small amounts of impurities. Chromatographic fractionation has revealed the presence of two enzymically active components. 63,54 4~ E. Brand, as cited in J. H. Northrop, M. Kunitz, and R. M. Herriott, "Crystalline Enzymes," 2rid ed., p. 26, Columbia University Press, New York, 1948. 4~ F. M. Uber and V. R. Ells, J. Biol. Chem. 141, 229 (1941); D. Shugar, Biochem. J. 52, 142 (1952). 47 A. Rothen, J. Gen. Physiol. 24, 203 (1940). 4s I. Fankuchen, J. Gen. Physiol. 24, 315 (1940); not corrected for solvent of crystallization. 49 C. H. Carlisle and H. Scouloudi, Proc. Roy. Soc. (London) A207, 496 (1951) [Chem. Abstr. 46, 316 (1952)]. 50 j. Smolens and M. G. Sevag, J. Gen. Physiol. 26, 11 (1942). 5~ M. R. McDonald, J. Gen. Physiol. 32, 33 (1948). 52 A. Kleczkowski, Biochem. J. 42, 523 (1948). 53 A. J. P. Martin and R. R. Porter, Biochem. J. 49, 215 (1951). ~4 C. H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 200, 493 (1953).

436


[62]

Whether the two components possess the same enzymatic specificity and properties has not been determined. The degree of inhomogeneity of a given preparation probably depends on the exact experimental conditions employed in the isolation and crystallization of the enzyme, sinc~ some preparations are more nearly homogeneous than others. Chromatographically homogeneous samples of the predominant fraction can be isolated either from crystalline preparations or directly from acid extracts of pancreas utilizing preparative scale chromatography. 54

Properties of Other RNases The following observations, made with tissue homogenates or crude extracts, indicate that RNases differing markedly in their properties from the digestive one of pancreas and from each other occur in various cells. Polynucleotide fractions, obtained by exhaustive digestion of RNA with crystalline RNase and resistant to further incubation with it, are hydrolyzed with phosphatase-free RNase preparations from beef spleen. 55 The pH optima for the action of diverse tissue RNases vary, values of 4.5, 6.0, 7.8, and 7.0 having been obtained for calf thymus, s6,s7 calf spleen, 57 rat liver, rat kidney, 58 and chick erythrocytes. 59 The heat lability of the various preparations also vary, as do their reaction to electrolytes. 57,58 More than one RNase may be present in the same tissue. 5s Hirs et al. 54 were unable to prepare, by the chromatographic procedure successfully used for pancreas, RNase from acid extracts of beef liver, spleen, and thymus. These findings, together with the always possible and almost probable fact of zymogens and inhibitors being present in tissues, make the comparison of the RNase content of various tissues, when assayed under the same conditions, extremely unreliable. It would appear that each tissue RNase (as with all enzymes) should be studied as an individual entity and that generalizations based on one isolated example are extremely hazardous. ~ G. Schmidt, R. Cubfles, and S. J. Thannhauser, J. Cellular Comp. Physiol. 38, Suppl. 1, 61 (1951). 56K. D. Brown, G. Jacobs, and M. Laskowski, J. Biol. Chem. 194, 445 (1952). 57M. E. Mayer and A. E. Greco, J. Biol. Chem. 181, 861 (1949). 58j. S. Roth, Biol. Bull. 103, 288 (1952). 69Z. B. Miller and L. M. Kozloff,J. Biol. Chem. 170, 105 (1947).

[63]

DEOXYRIBONUCLEASES

437

[63] D e o x y r i b o n u c l e a s e s B y MARGARET R. MCDONALD

Several enzymes (deoxyribonucleases) capable of hydrolyzing highly polymerized DNA occur in various cells. Although some of their properties are similar, others are markedly dissimilar. Only pancreatic deoxyribonuclease (DNase) has been highly purified and crystallized. Its preparation and properties will be discussed first. Methods for the partial purification of thymus, spleen, yeast, and streptococcal DNase will then be given, followed by a comparison of their properties.

Assay Method When solutions of DNA are hydrolyzed by DNase, their viscosity decreases and their specific absorption of ultraviolet light increases. Titratable acid groups are liberated without the formation of free phosphoric acid. The split products are not precipitable by mineral acids, proteins, or alcohol; they diffuse through collodion or cellophane membranes. Methods of assaying DNase based on all these phenomena have been extensively used; their relative merits have been discussed. L,2 Kunitz's spectrophotometric procedure 3 is probably the most convenient for routine measurements of purified DNase. The procedure described here has been found by the author to be the most generally useful in studies on DNase, applicable to both tissue homogenates and purified preparations. It is essentially Allfrey and Mirsky's modification2 of Laskowski's acidsoluble method. 4 Principle. The method is based on the colorimetric determination of the acid-soluble deoxypentose compounds released in the course of enzyme action. Reagents

Substrate. 200 mg. of Na-DNA 5 in 100 ml. of H20 or 0.05 M MgSO4, depending on the Mg ++ requirement of the DNase being assayed. 1N. B. Kurnick, Arch. Biochem. 29, 41 (1950). 2V. Allfrey and A. E. Mirsky, J. Gen. Physiol. 36, 227 (1952). 3 M. Kunitz, J. Gen. Physiol. 33, 349 (1950). 4 M. Laskowski, Arch. Biochem. 11, 41 (1946). 5Highly polymerized Na-DNA is obtainable from the Worthington Biochemical Sales Co., Freehold, New Jersey. For methods of isolation and purification of this compound, see Vol. III [103].

438


[63]

0.2 M buffer. The composition and pH of the buffer is determined by the DNase being assayed. 3.0 M trichloroacetic acid.

Procedure. One milliliter of substrate plus 1 ml. of buffer is incubated with 1 ml. of enzyme solution at 35 ° for various times, after which 1 ml. of 3.0 M trichloroacetic acid is added. The mixtures are left in an icewater bath for 15 minutes, then filtered through 7-cm. Whatman No. 42 paper. Aliquots of the filtrate are analyzed by Dische's diphenylamine procedure, 6 and the optical densities obtained are converted to deoxypentose-P equivalents by comparison with those obtained from a standard solution of DNA. Definition of Unit and Specific Activity. The DNase activity unit is defined as that quantity of enzyme which catalyzes the formation of 1 "~ of acid-soluble deoxypentose-P per hour under the conditions described above. Specific activity is expressed as DNase units per milligram of protein. Protein is determined by the method of Lowry et alJ I. Crystalline Pancreatic Deoxyribonuclease Purification Prccedure The procedure described here is essentially that of Kunitz2 The yield of crystalline DNase is low, owing partly to the fact that at pH 2.8, which is most favorable for crystallization, the enzyme is gradually denatured. From 3 to 5 mg. of dry first crystals are usually obtained for each kilogram of ground pancreas extracted. The method has been found to be reproducible in several laboratories. The saturated (NH4)2S04 solution is prepared at 20 to 25 ° (760 g. of salt per liter of H20). All filtrations, unless otherwise specified, are done with suction. Step 1. Preliminary Purification. 8 Fresh *~beef pancreases are collected in ice-cold 0.25 N H2SO4. The glands are drained, cleaned of fat and connective tissue, then minced in a meat grinder. The minced pancreas is suspended in an equal volume of ice-cold H20, and ice-cold 0.25 N H2SO4 is added with stirring until the pH of the suspension is approximately 3.0 (tested with 0.01% methyl orange on a test plate); a volume of acid equal to half that of the H20 added is generally required. The suspension e Z. Dische, Mikrochemie 8, 4 (1930); see also Vol. I I I [99]. 7 O. H. Lowry, N. J. Rosebrough, A. L. Farr, a n d R. J. Randall, J. Biol. Chem. 193, 265 (1951); see Vol. I I I [73]. 8 Based on the procedure of M. M c C a r t y , J. Gen. Physiol. 29, 123 (1946). 9 Frozen pancreas, obtainable from a n y of the large slaughter-houses, can also be used if only deoxyribonuclease (or ribonuclease) is to be prepared. T h e y should be t h a w e d b y leaving t h e m immersed in 0.25 N H~S04 a t 5 °.

[63]

DEOXYRIBONUCLEASES

439

is left at 2 to 5 ° for 18 to 20 hours. I t is then strained through cheesecloth. The residue is resuspended in 1 vol. of ice-cold H20 and again strained. The residue is then discarded, and the combined filtrates are brought to 0.2 saturation of (NH4)2SO4 b y the addition of 114 g. of salt per liter of filtrate. The precipitate formed is filtered through a rapid filtering paper (such as E a t o n - D i k e m a n No. 617) with the aid of 10 g. of Celite No. 503 10 and 10 g. of Standard Super-Cel ~° per liter of solution. The filter cake is discarded. The clear filtrate is brought to 0.4 saturation of (NH4)2SO4 b y the addition of 121 g. of salt per liter and refiltered with the aid of 3 g. of Celite No. 503 per liter through double paper, E-D No. 612 on top of No. 617. The residue H is suspended in five times its weight of water, the suspension is brought to 0.3 saturation of (NH4)2S04 by the addition of 176 g. of salt per liter of H~O used and refiltered on E-D No. 617 paper; the filtrate is discarded. Step 2. Incubation at 37 ° Followed by Fraetionation with A m m o n i u m Sulfate. The residue is suspended in ten times its weight of H20, and the suspension is brought to 0.15 saturation of (NH4)2SO4 b y the addition of 83.7 g. of salt per liter of H20. The solution is titrated to p H 3.2 (glass electrode) with about 2 ml. of 5 N H2SO4 per liter. It is heated to 37 ° and left for 1 hour at t h a t temperature. I t is then cooled to 20 ° and filtered through E-D No. 617 paper with the aid of an additional 5 g. of Celite No. 503 per liter of suspension. The residue is discarded. The filtrate is titrated to p H 5.3 (glass electrode) with 5 N N a O H (about 2 ml./1.) and brought to 0.5 saturation of (NH4)2S04 by the addition of 220 g. of salt per liter. The precipitate formed, designated 0.5 precipitate, is filtered on E-D No. 617 paper with the aid of 5 g. of Celite No. 503 per liter of solution. The clear filtrate is titrated with a few drops of 5 N H2SO4 to p H 4.0 (tested with bromocresol green on a spot plate) and brought to 0.7 saturation of (NH4)2S04 by the addition of 135 g. of salt per liter. The scant precipitate formed, designated 0.7 precipitate, is filtered on E-D No. 612 paper with the aid of 2 g. of Standard Super-Cel per liter and stored. The filtrate is discarded. The 0.5 precipitate is resuspended in ten times its weight of water and step 2, including the incubation at 37 °, is repeated several times until no appreciable 0.7 precipitate is formed. lo Supplied by Johns-Manville, 22 East 40th Street, New York. 11The filtrate, when adjusted to 0.25 N H2SO4 by the addition of 7 ml. of concentrated H2SO~ per liter of H20 used in the extraction and washing of the ground pancreas, can be utilized for the preparation of chymotrypsinogen, trypsinogen, trypsin, trypsin-inhibitor compound (M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 1002 (1936) ; see also Vol. II [2, 3, 4]) and for ribonuclease (M. Kunitz, J. Gen. Physiol. 24, 15 (1940); see also Vol. II [62]).

440


[63]

The 0.7 precipitates are combined and suspended in ten times their weight of H~O and filtered through E-D No. 612 paper. The residue is washed with H20 until the washing is water clear. Step 3. Fractionation with Ethanol. The combined filtrate and washings are diluted with H~O to a concentration of approximately 1% protein (the approximate concentration of protein can be determined spectrophotometrically at 280 m~, the optical density being 1.2 per milligram of protein per milliliter). The pH of the solution is adjusted with 5 N H2SO4 to pH 3.8 (tested with methyl orange on a spot plate), and 2 ml. of saturated (NH4)2SQ is added per 100 ml. of solution. The mixture is cooled in an ice-salt bath to 2 °, and one-quarter of its volume of ice-cold 95% ethanol is added slowly, with stirring, keeping the temperature of the solution between 2 and 5 °. The mixture is stored for 24 hours at 2 to 5 ° and is then centrifuged at the same temperature. The residue is discarded, and the clear supernatant is left at - 10° for 24 hours, after which it is centrifuged at the same temperature. The supernatant is discarded. Step 4. Crystallization. The precipitate is dissolved in approximately ten times its volume of ice-cold H20, after which it is brought to 0.38 saturation by the addition of 60 ml. of saturated (NH4)2S04 per 100 ml. of solution. The precipitate formed is filtered with suction on hardened paper (such as Schleicher and Schuell No. 576) at 5 to 10 °. It is then suspended in three times its weight of ice-cold H20 and dissolved by the slow addition of several drops of 0.25 N NaOH, keeping the pH of the solution below 4.8. If the solution is turbid it is centrifuged clear at about 5 °, then adjusted to pH 2.8 (glass electrode) with several drops of 0.2 N H2SOt. The heavy precipitate, which usually forms at approximately pH 3.5, dissolves readily as the pH of the solution reaches 3.0 or lower. The clear solution is left at 5 ° overnight and then at approximately 20 ° for 6 to 8 hours. Crystals appear during the latter step. Step 5. Recrystallization. The suspension of crystals is centrifuged. The residue is snspended in approximately 3 vol. of 0.02 saturated (NH4)2SO4 and dissolved with the aid of a few drops of 0.2 N NaOH at a pH of about 4.6. The solution is centrifuged if turbid, titrated to pH 2.8 (glass electrode), and left at 20 °. Crystals of DNase form within an hour. They are filtered on hardened paper at 5 °, then washed, first with ice-cold acidified 30% ethanol (1 drop of 5 N H2SO4 per 100 ml.), then with ice-cold acetone, and dried at room temperature for several hours. The mother liquors in steps 4 and 5 yield additional crystals when treated as follows: The solution is diluted threefold with ice-cold H20 and titrated with 0.2 N NaOH to pH 4.6 (tested with bromocresol green on a spot plate). Any insoluble material formed is removed by centrifugation. The clear supernatant is titrated with 0.2 N H2SO~ to ptI 4.0

[63]

DEOXYRIBONUCLEASES

441

and t h e n b r o u g h t to 0.38 s a t u r a t i o n of (NH4)~S04, as described in step 4, which is t h e n followed t h r o u g h in e v e r y detail. TABLE I SUMMARY OF PURIFICATIONPROCEDUREa OF PANCREATICDEOXYRIBONUCLEASE Fraction 1. 2. 3. 4.

Precipitate from 0.3 saturated (NH4)~S04 Filtrate, after 1 hour of incubation at 37 ° Precipitate from 20% alcohol at - 1 0 ° First crystals First mother liquor 5. Second crystals Second mother liquor

Specific activity b 0.2 3-5 5-6 8-10 5-6 8-10 8-10

Yield' 100 30 15 5 10

M. Kunitz~ J. Gen. Physiol. 33, 349 (1950). b The specific activity, i.e., activity per milligram of protein, is expressed in terms of that of the best preparations, which is taken as equal to 10. c The yield is given in per cent of the activity of the first fraction, precipitated in 0.3 saturated (NH4)~SO4.

Properties Specificity. Crystalline pancreatic D N a s e h y d r o l y z e s h i g h l y polymerized D N A and D N A which has been d e n a t u r e d so t h a t its physical properties, b u t n o t its chemical composition, are altered. 12 D e t a c h m e n t of a p o r t i o n of the purines 12,13 inhibits the h y d r o l y s i s ; apurinic acid 12 is n o t h y d r o l y z e d b y D N a s e . it ( M g ++ alone causes disintegration of apurinic acid. 12) D N a s e does n o t h y d r o l y z e R N A . 8,15 Analysis of the p y r i m i d i n e a n d purine c o n t e n t of the p r o d u c t s of D N a s e hydrolysis of D N A shows the p y r i m i d i n e - p u r i n e ratio of the dialyzable p r o d u c t s to be higher t h a n the p a r e n t s u b s t r a t e ; t h a t of the nondialyzable " c o r e , " lower. ~6,17 This does n o t necessarily m e a n t h a t D N a s e preferentially h y d r o l y z e s p y r i m i d i n e nucleotide groupings, ~6 and until m o r e is k n o w n a b o u t the composition a n d s t r u c t u r e of the nucleotides formed, little can be said a b o u t the b o n d specificity of D N a s e . Kinetics. T h e various changes in the physical and chemical properties

1~C. Tamm, H. S. Shapiro, and E. Chargaff, J. Biol. Chem. 199, 313 (1952). 18 C. A. Zittle, J. Franklin Inst. 243, 334 (1947). 14This has been confirmed cytochemically by A. Howard and S. R. Pelc, working in London, and by H. Gay in the author's laboratory. 1~L. M. Gilbert, W. G. Overend, and M. Webb, Exptl. Cell Research 2, 138 (1951). 16S. Zamenhof and E. Chargaff, J. Biol. Chem. 178, 531 (1949); 187, 1 (1950). 17 M. G. Overend and M. Webb, J. Chem. Soc. 1950, 2746.

442


[63]

of D N A catalyzed b y D N a s e occur at unequal rates. 17-19 T h e change in viscosity and ultraviolet absorption generally precedes a n y noticeable change in the precipitability of the nucleate with strong acids or the liberation of acid groups. W h e n the concentration of s u b s t r a t e is low, the hydrolysis approxim a t e s closely a reaction of the first order, 18,2° the unimolecular constant being independent of the concentration of enzyme. At relatively higher concentrations of substrate, the initial rate of reaction decreases rapidly with increase in s u b s t r a t e concentration. 18,2°,2~ T h e products of the reaction are also inhibitory. 21 Activators and Inhibitors. M g ++ 4.8.22 (or other divalent ions ~3) are obligatory for the enzymic action of pancreatic DNase. T h e concentration of M g ++ required increases with increasing concentration of s u b s t r a t e and is practically independent of the concentration of enzyme. ~s,2° T h e relative concentrations of M g ++ and of D N A for the optimal rate of activation are such t h a t there is always a considerable excess of M g ++ over the a m o u n t necessary to change N a - D N A into M g - D N A stoichiometrically. T h e concentration-activation function for M g ++ on D N a s e passes through a m a x i m u m ; at concentrations of M g ++ a b o v e 0.02 M there is a decrease in the rate of digestion. ~s This inhibitory effect is also shown b y NaC1. 4,~8 Arginine, lysine, and histidine 24,25 h a v e been reported as activators ~-6 of D N a s e when used in concentrations ranging from 0.001 to 0.01 M ; at concentrations greater t h a n 0.01 M the effect decreases and lysine becomes inhibitory. 24 Fluoride, citrate, s arsenate, 2~ borate, and selenite ions 27 inhibit the action of D N a s e , owing p r o b a b l y to their ability to remove the a c t i v a t ing M g ++. Thioglycolic acid, Na2S, Cu ++, Zn ++, Fe ++, Fe +++, Cr ++, and Ni ++ are inhibitory 27,28 in the presence of M g ++. N a - u s n a t e 28 inhibits 18 M. Kunitz, J. Gen. Physiol. 33, 363 (1950). 19G. Jungner, I. Jungner, and L. G. Allg4n, Nature 164, 1009 (1949); R. Vercauteren, Nature 165, 603 (1950). 20 j. Gr4goire, Compt. rend. 231, 384 (1950). 2, L. F. Cavalieri and B. Hatch, J. Am. Chem. Soc. 75, 1110 (1953). 22F. G. Fischer, I. BSttger, and H. Lehmann-Echternacht, Z. physiol. Chem. 271, 246 (1941). 23 C. E. Carter and J. P. Greenstein, J. Natl. Cancer Inst. 7, 29 (1946); T. Miyaji and J. P. Greenstein, Arch. Biochem. and Biophys. 32, 414 (1951). 24 W. Frisch-Niggemeyer and O. Hoffmann-Ostenhof, Monatsh. 81, 607 (1950) [Chem. Abstr. 44, 9497 (1950)]. 2~V. L. Nemchinskaya and V. S. Shapot, Biokhimiya 18, 210 (1953) [Chem. Abstr. 47, 8132 (1953)]. 26This activation may be due to impurities, since a commercial sample of arginine which activated DNase did not do so after purification. ~ 27L. M. Gilbert, W. G. Overend, and M. Webb, Exptl. Cell Research 2, 349 (1951). 2s A. Marshak and J. Fager, J. Cellular Comp. Physiol. 85, 317 (1950).

[63]

DEOXYRIBONUCLEASES

443

DNase in the presence, but not the absence, of Co ++. Some tissues contain a protein(s) which markedly inhibits pancreatic DNase. 29 Physicochemical Properties. Crystalline DNase is a protein of the albumin type with the following elementary composition in per cent dry weight: C, 50.16; H, 6.91; N, 14.88; S, 1.09; P, 0; ash, 0.47. 3 No indication of a special prosthetic group is evident from its ultraviolet absorption spectrum which shows a maximum molecular extinction of 70,000 at 280 m~ and a minimum of 26,000 at 250 m~. 3 I t contains about 8 % tyrosine and 2 % tryptophan. 3 Its molecular weight calculated from diffusion measurer~.ents (assumed specific gravity, 1.33) is 63,000;3 from inactivation by deuteron and electron bombardment, 62,000. 30 The isoelectric point of DNase is in the region of p H 4.7 to 5.0. 3 The stability of solutions of DNase depends markedly on their concentration. Solutions containing > 0.1 mg. of protein per milliliter in dilute buffer in the p H range of 4.0 to 9.0 are stable for at least a week at 50. 3 Solutions containing < g. 3~Over 90% of the activity present in the minced (or homogenized) spleen is found in the extract. s5S. Zamenhofand E. Chargaff,J. Biol. Chem. 180, 727 (1949).

446


[63]

The residue 86 is suspended in 300 ml. of 1 M NaC1, and the viscous mixture is left at 4 ° for four months, during which period its viscosity disappears completely and its DNase activity increases markedly (0.6, 0.6, 17, 25, 30, and 30 viscosimetric unitsS/ml, after 0, 14, 19, 52, 90, and 120 days, respectively). Step 2. Concentration and Purification. The mixture (total units, 15,200) is then centrifuged at 1900 X g for 1 hour, dialyzed with rocking against ice-cold H20 for 4 hours, and dried in vacuo from the frozen state. The residue (total units, 15,000; units/rag, protein, 14) is suspended in 30 ml. of H20 and centrifuged at 31,000 × g for 2 hours. The residue is discarded, and (NH4)2SO4 is added to the yellow supernatant to 0.6 saturation. The resulting precipitate is collected by centrifugation at 31,000 X g, drained with suction on Whatman paper No. 50, suspended in 45 ml. of H20, and dialyzed with rocking against ice-cold H20 for 7 hours. The dialyzed mixture (total units, 10,200) is centrifuged at 31,000 × g for 1 hour, and the supernatant is discarded. The sediment is washed with H20, then extracted, first with 30 ml. and then again with 12 ml. of 1 M NaC1. The combined extracts (total units, 6200) are centrifuged at 31,000 X g for 1 hour, and the supernatant is dialyzed for 6 hours against ice-cold H20. The dialyzate is then dried in vacuo from the frozen state, yielding 27 rag. of pale yellow fluff, insoluble in H20 and soluble in salt solutions (total units, 4300; units/rag, protein, 1250; yield, 35%). V. Purification of Streptococcal Deoxyribonuclease 37

Group A hemolytic streptococci produce, during growth, appreciable amounts of extracellular DNase. It is readily precipitated from the culture medium by (NH~)2SO4. Step 1. Elaboration. Fifteen liters of neopeptone broth 38 is inoculated with a strain of group A hemolytic streptococcus (H105) and incubated at 37 ° for 20 hours. The cells are then removed by centrifugation in a Sharples centrifuge. Step 2. Concentration and Purification. The slightly turbid superuatant is brought to 0.4 saturation of (NH4)2SO4 by the addition of 243 g. of salt per liter of supernatant. The suspension is filtered with suction with the aid of 1 g. of Filter-Cel and 1 g. of Hyflo Super-Cel per liter. The residue is discarded, and the filtrate is brought to 0.8 saturation by the addition of 281 g. of (NH4)2SO4 per liter. The resulting precipitate is recovered by filtration and dissolved in 100 ml. of H20. (This solution 3s The s u p e r n a t a n t can be used for the preparation of yeast D N a s e inhibitor. 35 ~7 M. McCarty, J. Exptl. Med. 90, 543 (1949). as V. P. Dole, Proc. Soc. Exptl. Biol. Med. 63, 122 (1946).

[63]

DI~OXYRIBONUCLEASES

447

contains a l m o s t all the original D N a s e activity.) T h e solution is b r o u g h t to 0.4 s a t u r a t i o n of (NH4)~S04, filtered, a n d the residue discarded. T h e filtrate is b r o u g h t to 0.5 s a t u r a t i o n of (NH4)~S04, a n d the p r e c i p i t a t e is recovered b y filtration. I t is dissolved in a small v o l u m e of H~O, dialyzed against H20, a n d dried i n vacuo f r o m the frozen state. T h e dried m a t e rial (170 mg.) contains the bulk of the original D N a s e a c t i v i t y a n d has 25,000 viscosity s u n i t s / m g . Properties

& c o m p a r i s o n of some of the properties verse sources is p r e s e n t e d in :Fable I I . T h e fied D N a s e f r o m spleen a n d m o u s e leukemic e x t r a c t s of m a n y o t h e r m a m m a l i a n tissues, t h y m u s D N a s e . 2,~'I.s9,4°

of several D N a s e s f r o m diproperties of partially puritissues, a n d of crude D N a s c a p p e a r to resemble those of

TABLE II SUMMARY OF PROPERTIES OF VARIOUS DEOXYRIBONUCLEASES

Deoxyribonuclease from StreptoPancreas ~ Thymus b Serum c Yeast ~ coccus* 1. pit optimum 2. Mg ++ requirement 3. Inhibition by: F1 or citrate ions Bacterial RNAI Yeast protein s Pancreatic DNase antisera" Streptococcal DNase antisera'

7.0 -F

5.2 --

7.5 +

-{-

--

+

-+

--

6.0 +

7.5 + + +

4+

M. McCarty, J. Gen. Physiol. 29, 123 (1946). b M. Webb, Exptl. Cell Research 5, 27 (1953). c F. Wroblewski and 0. Bodansky, Proc. Soc. Exptl. Biol. Med. 74, 443 (1950). S. Zamenhof and E. Chargaff, J. Biol. Chem. 180, 727 (1949). M. McCarty, J. Expll. Med. 90, 543 (1949). s A. W. Bernheimer, Trans. N. Y. Acad. Sci. [II] 14, 137 (1952). 89 M. E. Mayer and A. E. Greco, J. Biol. Chem. 181, 861 (1949). 40 K. D. Brown, G. Jacobs, and M. Laskowski, J. Biol. Chem. 194, 445 (1952).

448


[64]

[64] Preparation of Nucleoside Phosphorylase from Calf Spleen I By

VINCENT E . PRICE, M . CLYDE OTEY, a n d PAUL PLESNER

Purine nucleoside phosphorylase was first described by Kalckar 2,3 in 1945 and shown to catalyze the equilibrium Inosine -~ phosphate ~- Hypoxanthine ~- ribose-l-phosphate The equilibrium was shown to greatly favor synthesis of the nucleoside. 4 Both ribosides and deoxyribosides of hypoxanthine and guanine are rapidly attacked by the phosphorylase. Xanthosine and deoxyxanthosine are phosphorolyzed at a much slower rate. 5 Pyrimidine nucleosides are attacked by a different enzyme, pyrimidine nucleoside phosphorylase. 6,7 Nucleoside phosphorylase has been very useful in the synthesis of new intermediates. Friedkin 8 has used nucleoside phosphorylase in the synthesis of the ribosides and deoxyribosides of 8-azaguanine. Korn et al. 9 have recently used a purified preparation of beef liver nucleoside phosphorylase in the synthesis of 4-amino-5-imidazolecarboxamide riboside. Rowen and Kornberg have presented evidence 1° indicating that purine nucleoside phosphorylase is active in the phosphorolysis and synthesis of nicotinamide riboside (pyridinium N + riboside). With this substrate the equilibrium is far toward free nicotinamide and ribose-l-phosphate formation. If hypoxanthine is removed from the above phosphorylase reaction by its oxidation to uric acid with xanthine oxidase, ribose-l-phosphate can be isolated. This is the basis for an excellent preparative method for 1 This is a hitherto unpublished method based on work initiated by Dr. Vincent E. Price while working with Dr. Herman Kalckar at the University of Copenhagen in 1951 and completed with Mr. M. Clyde Otey at the National Cancer Institute. The method has been checked by Dr. Paul Plesner and Dr. Hans Klenow of the University of Copenhagen. H. M. Kalckar, J. Biol. Chem. 158, 723 (1945). It. M. :Kalckar, Federation Proc. 4, 248 (1945). 4 H. M. Kalckar, J. Biol. Chem. 167, 477 (1947). M. Friedkin, J. Am. Chem. Soc. 74, 112 (1952). 6L. A. Manson and J. O. Lampen, J. Biol. Chem. 193, 539 (1951). M. Friedkin and D. Roberts, J. Biol. Chem. 207, 245 (1954). 8 M. Friedkin, J. Biol. Chem. 209, 295 (1954). 9 E. D. Korn, F. C. Charalampous, and J. M. Buchanan, J. Am. Chem. Soc. 75, 3611 (1953). 10 j. W. Rowen and A. Kornberg, J. Biol. Chem. 198, 497 (1951).

[64]

NUCLEOSIDE PHOSPHORYLASE FROM CALF SPLEEN

449

this acid-labile ester. 4 Friedkin H has more recently used the phosphorolysis of guanine deoxyriboside in the p r e p a r a t i o n of the extremely labile deoxyribose-l-phosphate. I n this case guanine was removed b y its conversion to xanthine b y the guanase in the phosphorylase preparation. Purine nucleoside phosphorylase is also an i m p o r t a n t enzyme in the methods for the differential s p e c t r o p h o t o m e t r y of purine compounds developed b y Kalckar. 12 For these analytical techniques a highly purified phosphorylase preparation is invaluable. Assay Method

P r i n c i p l e . The assay method is based on the determination of h y p o xanthine formed during the phosphorolysis of inosine b y nucleoside phosphorylase. The hypoxanthine is oxidized to uric acid b y xanthine oxidase and m a y be followed b y differential s p e c t r o p h o t o m e t r y as described b y Kalckar. 12 E n z y m e . X a n t h i n e oxidase is prepared according to Ball ~ with the modification of K a l c k a r et al., TM or according to Horecker and Heppel. 1~ One enzyme unit is defined as the a m o u n t of enzyme which causes an increase in optical density of 0.001 per minute at 293 mt~ in a cuvette with a light p a t h of 1 cm. under the following standard conditions:

Phosphate buffer, 0.05 M, pH 7.4 Hypoxanthine, 0.0075 M Enzyme T water to a volume of

2.7 ml. 0.2 ml. 3.0 ml.

Procedure. The assay is done under the following conditions:

Phosphate buffer, 0.05 M, pH 7.4 Inosine, 0.0075 M Xanthine oxidase, seven- to tenfold excess in units as determined using hypoxanthine as substrate Enzyme sample -{- water to

2.7 ml. 0.2 ml. 3.0 ml.

Unit. One unit of nucleoside phosphorylase is defined as the a m o u n t of enzyme which under the above conditions causes an increase in optical density of 0.001 per minute at 293 m~ in the initial rate when read in a cuvette with a light p a t h of 1 cm.

11 M. Friedkin, J. Biol. Chem. 184, 449 (1950). 1~H. M. Kalckar, J. Biol. Chem. 167, 429, 445, 461 (1947). la E. G. Ball, J. Biol. Chem. 128, 51 (1939). 14H. IV[. Kalckar, N. O. Kjeldgaard, and H. Klenow, Biochim. et Biophys. Aeta 5, 575 (1950). 1~B. L. Horecker and L. A. Heppel, J. Biol. Chem. 178, 683 (1949); see Vol. II [73].

450


[64]

Preparation

Reagents

Acetate buffer, pH 4.0:0.02 mole of Na acetate and 0.1 mole of acetic acid made up to 1000 ml. Acetate buffer, pH 5.2:0.02 mole of Na acetate adjusted to pH 5.2 with acetic acid and made up to 1000 ml. Ethanol-acetate buffer solution, 60 %: 600 ml. of absolute ethanol made up to 1000 ml. with the acetate buffer, pH 5.2. Ethanol-acetate buffer solutions, 12%, 9%, and 6%: 120 ml., 90 ml., and 60 ml. of absolute ethanol, respectively, made up to 1000 ml. with the acetate buffer, pH 5.2. Glycine-acetate buffer: 0.5 mole of glycine and 0.02 mole of Na acetate in 800 ml. of H20, adjusted to pH 5.3 with acetic acid and taken to 1000 ml. Procedure. 2600 g. of calf spleen is homogenized in a Waring blendor with 2.5 vol. of cold distilled water and filtered through gauze; the residue is washed with 0.5 vol. of water. The pH is adjusted to 5.2 with 1300 ml. of the acetate buffer, pH 4.0, and the homogenate is allowed to stand for 1 hour in an ice bath. After centrifugation in a refrigerated centrifuge at 0 °, the active supernatant, $1, is taken to 20% ethanol by addition of 0.5 vol. of the 60% ethanol-acetate buffer solution, which is cooled to - 1 0 ° before addition, and then allowed to stand overnight at - 5 °. In the morning, most of the supernatant, $2, above the precipitate formed is siphoned off and discarded after assaying for phosphorylase. The precipitate, P~, is centrifuged from the remainder of the supernatant and collected in eight 100-ml. plastic tubes. The precipitate of each tube is submitted to fractional extraction 16 as described below. A plastic Potter-Elvehjem homogenizer plunger made to fit the 100-ml. tubes is recommended for suspension of the precipitate at each step. Each tube of P2 is homogenized with 65 ml. of the 12% ethanol-acetate buffer solution, allowed to extract for 30 minutes at - 5 °, and centrifuged, giving $3 and P3. Each tube of Pa is homogenized with 65 ml. of the 9% ethanol-acetate buffer solution and after 30 minutes at 0 ° centrifuged, giving $4 and P4. Each tube of P4 is homogenized with 33 ml. of the 6% ethanol-acerate buffer solution and after 30 minutes at 0 ° centrifuged, giving $5 and Ps. 10E. J. Cohn, F. R. N. Gurd, D. M. Surgenor, B. A. Barnes, R. K. Brown, G. Derouaux, J. M. Gillespie, F. W. Kahnt, W. F. Lever, C. H. Liu, D. Mittelman, R. F. Mouton, K. Sehmid, and E. Uroma, J. Am. Chem. Soc. 72, 465 (1950).

[64]

NUCLEOSIDE

PHOSPHORYLASE

$:x, ¢~ ¢q

~9 O

Z O

° °

~9

Q ¢g

c~ o O

FROM

CALF

SPLEEN

451

452


[64]

To extract the phosphorylase the precipitate, Ps, in each tube is homogenized with 33 ml. of glycine-acetate buffer, and after 30 minutes at 0 ° centrifuged, giving $6 and Pe. To each 100 ml. of $6 is added 20 g. of ammonium sulfate (182 g./1. final concentration) and after 30 minutes at 0 ° centrifuged, giving $7 and PT. The supernatant, $7, is the active enzyme preparation, and gives 133-fold purification based on the extinction at 280 m~, and about 65-fold purification based on nitrogen. A summary of the fractionation is presented in the table. Notes and Comments on the Preparation

1. The solutions used should always be made up at room temperature and then cooled down to the desired temperature. The hydrogen ion concentration and the temperatures of the buffers given must be strictly followed; a difference of a few degrees of temperature or of 0.1 unit of pH from those stated will considerably alter the fractions obtained. 2. The fractional extractions with ethanol-acetate buffers remove the more soluble contaminating proteins. The phosphorylase is then extracted with glycine-acetate buffer, leaving behind a dense fatty pellet which is nearly immiscible even in 0.02 M phosphate buffer, at pH 5.2, and only slowly goes into solution. From its solubility properties 18 it is thought to be largely fl-lipoprotein. Fractional extraction 1~ permits more rapid equilibration than fractional precipitation, and by keeping the enzyme in a precipitated state while extracting away other proteins denaturation of the phosphorylase is minimized. In fractional extraction the volume of the eluting solution used in a given extraction is very important. If the volume is too small the concentration of protein in the eluate will be high and will cause the phosphorylase to be extracted at the ethanol-acetate buffer steps, resulting in a low yield of enzyme. 3. Phosphate, 0.02 M, at pH 5.2, may be used in place of the glycineacetate buffer to extract the phosphorylase from the lipoprotein precipitate. T h e use of phosphate gives a somewhat higher yield but extracts some lipoprotein as well which requires further ammonium sulfate fractionation for its removal. 4. Only the early steps of the fractionation procedure are described. These provide a simple reproducible method of obtaining a very active preparation. Further fractionation steps (unchecked) are: (1) Precipitate the activity of $7 by adding 20 g. more of ammonium sulfate to each 100 ml. of volume. (2) Centrifuge and wash the pellet twice with the same volume of 30% ethanol in 0.02 M succinate at pH 5.8 and - 5 ° to remove ammonium sulfate. (3) Extract the 30% ethanol pellet twice with

[64]

NUCLEOSIDE PHOSPHORYLASE FROM CALF SPLEEN

453

the same volume of 10 % ethanol in 0.02 M succinate at pH 5.8. (4) Centrifuge and treat the combined active supernatants with 0.1 M Zn acetate in 10 % ethanol-succinate buffer. A large portion of the phosphorylase precipitates at 0.002 M Zn ++ concentration. A smaller fraction precipitating between 0.002 M and 0.010 M Zn ++ is very highly active with an activity about 800 times the original homogenate based on absorption and about 400 times based on nitrogen. Alternative Methods of Preparation. An alternative preparation of purine nucleoside phosphorylase from beef liver has been carried out by Korn in Buchanan's laboratory. 17 An acetone powder extract of the liver is subjected to three successive ethanol precipitations followed by fractionation with ammonium sulfate and silica gel. The purity of the preparation is about 200-fold based on the absorption of the various fractions at 280 mu. Recently this preparation has been used in the synthesis of 4-amino-5-imidazolecarboxamide riboside2 Suggestive evidence was obtained that adenine could also be utilized by their preparation. The preparation of nicotinamide (N +) riboside phosphorylase from beef liver acetone powder has been described by Rowen and Kornberg. 1° The powder was extracted with 0.1 M Na:HP04 and carried through successive steps using ammonium sulfate, calcium phosphate gel, and alumina gel/C~, with 60-fold purification. This preparation was also active in the phosphorolysis of inosine, aud ino.sine markedly inhibited the phosphorolysis of nicotinamide riboside. It was suggested that the same enzyme may be active in the phosphorolysis of both inosine and nicotinamide riboside. Purine nucleoside phosphorylase from yeast has been purified and separated from hydrolase activity by tteppel and Hilmoe, TM using ammonium sulfate and calcium phosphate gel with some 55-fold purification. This enzyme resembles Kalckar's phosphorylase from animal sources with a pH optimum near 7.0 and having activity against inosine, guanosine, and nicotinamide riboside only in the presence of free phosphate or arsenate.

Eaitor's note: For the preparation of pyrimidine nucleoside phosphorylase (thymidine phosphorylase from horse liver), see Vol. III [26]. ~7j. M. Buchanan, in "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 2, p. 419, Johns Hopkins Press, Baltimore, 1952. ~8L. A. Heppel and R. J. Hilmoe, J . Biol. Chem. 198, 683 (1952); see Vol. II [66].

454


[65]

[65] Nicotinamide Riboside Phosphorylase 1 Nicotinamide Riboside + + Phosphate ~ Nicotinamide Ribose-l-phosphate W H +

By ARTHUR KORNBERG Assay Method Principle. Nicotinamide riboside but not nicotinamide yields a fluorescent condensation product with acetone;2 the cleavage of nicotinamide riboside is measured b y this fluorometric method. Reagents

Nicotinamide riboside (NR), 0.003 M. See Vol. I I I [129]. KH2PO4-K2HPO4 buffer, 0.1 M, p H 7.4. Procedure. The incubation mixture (0.50 ml.) contained 0.05 ml. of NR, 0.10 ml. of phosphate buffer, enzyme (0.5 to 0.7 unit), and water. After 10 minutes at 38 °, the reaction was stopped b y a twentyfold dilution with ice-cold water and an aliquot of 0.20 ml. was immediately assayed fluorometrically. 2 Definition of Unit and Specific Activity. One unit of enzyme is defined as t h a t a m o u n t which causes the cleavage of 1 micromole of N R in 1 hour. Specific activity is expressed as units per milligram of protein. Protein was determined b y the m e t h o d of Lowry et al.3

Purification Procedure Ten grams of beef liver acetone powder 4 was extracted with 100 ml. of 0.1 M Na2HPO4 for 10 minutes with gentle shaking. This and subsequent operations were carried out at 2 °. T o 80 ml. of extract (see the table) were added an equal volume of 0.1 M acetate buffer, p H 5.0, and 48 g. of ammonium sulfate. After 10 minutes the precipitate was removed b y centrifugation and 12 g. of ammonium sulfate was added to the supernarant. The resulting precipitate was collected in the centrifuge and dissolved in 13 ml. of water (fraction 2). 1j. W. Rowen and A. Kornberg, J. Biol. Chem. 193, 497 (1951). 2j. W. Huff and W. A. Perlzweig, J. Biol. Chem. 167, 157 (1947); for a description of fluorometric assay, see Vol. III [128]. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193~ 265 (1951). 4 A. Kornberg, J. Biol. Chem. 182, 779 (1950).

[65]

NICOTINAMIDE RIBOSIDE PHOSPHORYLASE

455

This fraction was diluted with water to 45 ml. and treated with 52 ml. of aged calcium phosphate gel 6 (10 mg./ml., dry weight). The gel was collected b y centrifugation and washed once with 65 ml. of water, and the enzyme was eluted twice with 32.5-ml. portions of phosphate buffer (0.005 M, p H 7.5) (fraction 3). The addition to 65 ml. of fraction 3 of 6 ml. of 0.1 M acetate buffer, p H 5.0, reduced the p H to 6.2. Then 14.2 ml. of alumina gel C~ 6 (1.6 m g . / ml., dry weight) was added. The gel was collected in the centrifuge and washed with 71 ml. of water; the enzyme was eluted with two 35.5-ml. portions of 0.02 M phosphate buffer, p H 7.5. This final fraction represents a sixtyfold purification over the crude extract, with a yield of 17 %. Fractions 2 and 3 lose no activity after storage for several weeks at - - 1 0 % SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. 2. 3. 4.

Crude extract Ammonium sulfate Calcium phosphate gel eluate Alumina gel eluate

NR splitting activity, units

Protein, mg./ml,

Specificactivity, units/rag, protein

6912 3159 1890 1140

20.2 21.0 0.17 0. 067

4.3 11.5 162.0 ~ 255.0

a In repeated preparations the specific activity of this fraction varied between 144 and 180 units.

Properties pH Optimum. The p H optimum of the enzyme is approximately 8; the reaction rates at p H 7 and 9 are about 80% of the value at p H 8. The Michaelis constant for N R i s 1.1 × 10-3 M. The constant for phosphate is 2.8 × 10-4 M. Specificity. The purified preparation did not a t t a c k N M N , D P N , T P N , or methylnicotinamide, nor were there any inhibitory effects of these compounds at equimolar concentration on the rate of N R splitting. The purified enzyme fractions are active in the phosphorolysis of inosine, and, in addition, inosine markedly inhibited N R phosphorolysis. Although the question as to whether one enzyme is responsible for both activities has not been settled, available evidence does suggest identity. The ratio of specific activities of N R and inosine splitting for fractions 1, 2, and 3 were 1.3, 1.4, and 1.8, respectively. 5 D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124, 397 (1938). R. Willst~ttter and H. Kraut, Ber. 56, 1117 (1923).

456


[66]

The Michaelis constant (Ks) for inosine was shown to be 1.3 X 10-~ M, as compared with a value of 1.7 × 10-5 M obtained by Friedkin and KalckarJ The affÉnity of inosine as an inhibitor of N R phosphorolysis was determined at several concentrations of inosine and also at various levels of N R and was found to be approximately 3.5 X 10-5 M. Since the experimental conditions under which the K8 and KI values were determined, were for technical reasons, different, it is difficult to evaluate properly the disparity between them. 7 y[. Friedkin and H. M. Kalckar, J. Biol. Chem. 184, 437 (1950).

[66] H y d r o l y t i c N u c l e o s i d a s e s PuR 1 + H~O ~ Pu 1 + R 1 P y R I + H20--~ pyl + R 1

By T. P. WANG I. LactobaciUus pentosus Assay Method Principle. The products of action of the hydrolytic nucleosidase from Lactobacillus pentosus are the nitrogenous base, purine or pyrimidine, and the free ribose. Demonstration of the appearance of one of these products constitutes the basis of assay of this enzyme. However, in view of the fact that the nitrogenous base is also one of the products of phosphorolytic cleavage of nucleosides, the appearance of free ribose, as determined by the reducing sugar method, would be the more reliable method for assay of hydrolytic nucleosidase.

Reagents a. PuR. Any of the PuR such as AR, GR, HxR, UAR, or X R 1 can be used for this purpose. Tris buffer (0.05 M), pH 7.5. Extract, 1 ml., equivalent to 200 mg. of wet cells. b. PyR. Either CR or UR. 1 Arsenate or phosphate buffer (0.05 M), pH 7.5. Extract. ~The following abbreviations are used in this article: PuR, purine riboside; PyR, pyrimidine riboside; Pu, purine base; Py, pyrimidine base; AR, adenosine; GR, guanosine; H×R, inoglne; UAR, uric acid riboside; XR, xanthosine; CR, cytidine; UR, uridine; A, adenine; G, guanine; Hx, hypoxanthine; UA, uric acid; X, xanthine; C, cytosine; U, uracil; R, ribose; R-l-P, ribose-l-phosphate; and R-5-P, ribose-5phosphate.

[66]

HYDROLYTIC NUCLEOSIDASES

457

Procedure. Six micromoles of P u R (or P y R ) is incubated at 37 ° wit5 3 ml. of a cell-free extract of L. pentosus and 2 ml. of Tris buffer (or arsenate or phosphate buffer in the case of P y R ) . At the end of 2 hours, two 1-ml. samples are taken from the mixture, one deproteinized by an equal volume of 4 % HC104 (ice-cold), and the other b y 1 ml. of Ba(OH)~ (0.3 N) and 1 ml. of 5 % ZnSO4'6H20 2 in the order mentioned. The precipitate is removed b y filtration or centrifugation, and the protein-free filtrates are analyzed for reducing sugar a n d / o r the nitrogenous base. The perchloric acid filtrate should be kept in an ice bath and neutralized immediately with solid K H C Q before any analysis is made. Any K C 1 Q fo~med can be removed by centrifugation. 1. Reducing Sugar. This determination is made with 0.2 to 0.5 ml. of the perchlorie acid filtrate or 0.3 to 0.75 ml. of the Ba-Zn filtrate according to Nelson's colorimetric method. 3 The perchloric acid filtrate would contain free ribose and ribose phosphate, if any is formed, whereas the Ba-Zn filtrate would contain only the free ribose. Since the nucleoside is split hydrolytically by the L. pentosus extract, no phosphate ester of ribose should be formed. The reducing value obtained from the perchloric acid filtrate should thus be the same as that obtained in the Ba-Zn filtrate. The activity of the extract is as follows: for Pyr, 6 micromoles of substrate will be completely cleaved by 0.3 ml. of extract (equivalent to 60 mg. of wet cells) in 2 hours at 37 °. For PuR, 70 to 80 % of the 6 micromoles of nucleoside (in the case of XR, about 40 %) will be split. 2. Nitrogenous Base. (.~) CHROMATOGRAPHIC OR IONOPHORETIC METHOD.4 All the Pu or P y bases can be distinguished from their nucleosides and from each other b y either chromatographic or ionophoretic methods. For instance, when a reaction mixture containing G R is applied to a strip of W h a t m a n No. 1 filter paper which is then developed by the 5 % Na~HPO4-isoamyl alcohol system of Carter, the newly formed G will stay around the origin with an Rs value of 0.02, differing quite distinctively from the remaining G R which has an Rs value of 0.62 in the same system. (B) SPECTROPHOTOMETRICMETHODS. In the case of PuR, the formation of free Pu base can be demonstrated with suitable enzyme or a mixture of enzymes in a Beckman spectrophotometer. See following table:

2 Method of M. Somogyi, described in footnote 3. N. Nelson, J. Biol. Chem. 153, 375 (1944). 4Paper chromatography: R. J. Block, E. L. Durrum, and G. Zweig, "A Manual of Paper Chromatography and Paper Eleetrophoresis," Chapter 9, Academic Press, New York, 1955; Ionophoresis: W. C. Werkheiser and R. J. Winzler, J. Biol. Chem. 204, 971 (1953).

458


Base Adenine Guanine Hypoxanthine Xanthine Uric acid

[66]

Change in millimolar extinction for complete reaction

Enzyme X a n t h i n e oxidase Guanase a n d x a n t h i n e oxidase X a n t h i n e oxidase X a n t h i n e oxidase Uricase

+15.53 + 7.25 +10.88 +10.03 - 12.17

at at at at at

305 290 290 290 290

mt~ 5 mt~ ~ m~ 6 m~ ~ m~ 6

Since the substrate specificity of xanthine oxidase is rather broad, it is essential to use other independent methods to determine the exact nature of the base formed if such a determination is necessary. In addition to the chromatographic methods mentioned in the previous section, such procedure as taking a general spectrum in the ultraviolet region of the reaction mixture is frequently used, since all the Pu bases have their characteristic absorption spectra and can be distinguished from each other by such. For instance, in a reaction mixture containing AR, if the solution gives a maximum absorption at 260 m~ in addition to a positive reaction with xanthine oxidase, it can be assumed that one of the products is A instead of Hx. The Py bases differ from their nucleosides by having higher absorption at 300 m~ in alkaline solution. The following values are calculated from the absorption data given by Hotchkiss. 7 MILLIMOLAR EXTINCTION CHANGES AT 300 m ~ WHEN THE REACTION SOLUTION I s CHANGED FROM NEUTRAL TO ALKALINE

C +0.81

CR -0.28

U + i . 93

UR Almost no change 8

In addition, the appearance of free Py bases can also be demonstrated by enzymatic methods. Base Cytosine Uracil

Enzyme

Assay M e t h o d

Cytosine deaminase Uracil oxidase

After a decrease in absorption a t 280 m ~ 9 Measuring oxygen u p t a k e manometrically 1°

H. Klenow, Biochem. J. 50, 404 (1952). H. M. Kalekar, J. Biol. Chem. 167, 429 (1947). R. D. Hotchkiss, J. Biol. Chem. 175, 315 (1948). 8 j . M. Ploeser and H. S. Loring, J. Biol. Chem. 178, 431 (1949). 9 A decrease in absorption of 58% at 280 m~ will be observed when cytosine is deamiHated to uracil, according to the d a t a of Hotchkiss. ~ lo T. P. Wang a n d J. O. Lampen, J. Biol. Chem. 194, 785 (19527.

[66]


459

Preparation of Enzyme Preparation of L. pentosus Cells. L. pentosus 124-2 is the source of the hydrolytic nucleosidase. Stock of this organism is kept in agar stabs containing 5 g. of Difco yeast extract, 5 g. of Difco peptone, 10 g. of glucose, 10 g. of NaAc, 0.5 g. of KH~P04, 0.2 g. of MgSO4.7H20, 0.01 g. each of FeSO4"7H20, MnSO4.4H~O, and NaC1, and 20 g. of Difco agar per liter of distilled water. Stabs are stored at 5 ° and transferred monthly. Liquid medium of the same composition as listed above, with the omission of agar, is used for preparing resting cells. The inoculum is made by transferring cells from the stab to 10 ml. of the liquid medium. After incubation at 37 ° for 20 hours, the inoculum is added to a liter of medium and the culture is again incubated at 37 ° for 20 hours. The cells are then harvested by centrifugation at 4500 r.p.m, for 15 minutes and washed once with 1% KC1. The yield of wet packed cells is around 5 g./1. of medium. Preparation of Cell-Free Extracts. Cell-free extracts are prepared by grinding the wet cells with either powdered glass 11 or alumina A-303 12 or by breaking the cells with sonic oscillations. With the alumina grinding method, the resultant paste is extracted with 5 ml. of water per gram of initial wet cells. In the case of sonic disintegration, the cells are suspended to a concentration of 1 g. per 5 ml. of water and disrupted for 30 minutes in a magnetostriction sonic oscillator (type R-22-3, Raytheon Manufacturing Company). The insoluble residue is removed by centrifugation at 20,000 r.p.m, for 20 minutes. All operations involved in the preparation of cell-free extracts are made at a temperature between 0 and 5 °. The clear extracts are stored around - 2 0 ° in a deep-freeze. Properties

Specificity. Regarding substrate specificity, the hydrolytic nucleosidase(s) from L. pentosus differs from the phosphorolytic nucleosidase in (1) its ability to act on AR, CR, UAR, and XR in addition to GR, HxR, and UR, and (2) its inability to act on deoxyribosides of C, Hx, and U. Thymidine also is not attacked. UAR, a riboside found in beef erythrocytes, is split by the L. pentosus nucleosidase, whereas thymine riboside, a synthetic compound, is not cleaved. Stability. The rate of splitting PyR by the extract is faster in phosphate, arsenate, sulfate, and succinate than in Tris, KC1, or KNO3. This apparent stimulation of phosphate, arsenate, etc., is due to the fact that these anions have the ability to stabilize the pyrimidine nucleosidase. On the other hand, the monovalent anions do not possess this ability. Thus, ~1G. Kalnitsky, M. F. Utter, and C. H. Werkman, J. Bacteriol. 4.9, 595 (1945). ~2H. McIlwain, J. Gen. Microbiol. 2, 288 (1948).

460

ENZYMES

OF NUCLEIC

ACID METABOLISM

[66]

the activity of the enzyme is retained if a 10-minute incubation in arsenate precedes addition of the P y R substrate. Almost complete loss of activity results if the enzyme is preincubated with Tris under similar conditions. The possibility of inhibition by the Tris is unlikely in view of the fact that the enzyme is fully active if it is incubated in a mixture of arsenate and Tris buffers. Contrary to the behavior of the pyrimidine nucleosidase, the purine nucleosidase is stabilized by the monovalent anions and rapidly loses its activity in arsenate or phosphate buffers. The inactivation of purine nucleosidase in the presence of phosphate ion cannot be prevented by the addition of bovine serum, cysteine, Versene at pH 7.0, pyrophosphate at pH 8.0, or a boiled extract of an active nucleosidase preparation. The striking difference between the purine and the pyrimidine nucleosidases toward inorganic anions suggests that two types of enzyme are present in the L. pentosus extract, one specific for PuR and the other for PyR. Attempts toward purification of these enzymes have been made but with little success. The crude extract is rather stable. No significant loss of activity occurs when the extract is stored at - 20 ° for a year. Repeated freezing and thawing also do not appreciably affect the activity. Mechanism of Action. Because of the wide distribution of the phosphorolytic nucleosidase in animal tissues and in microorganisms, the existance of a hydrolytic enzyme is, in a way, not expected. Efforts therefore have been made to study the actual mechanism of action of L. pentosus nucleosidase. Evidence for a hydrolytic cleavage is briefly presented as follows. 1. As mentioned in the assay section, the reducing sugar value obtained from either the perchloric acid filtrate or the Ba-Zn filtrate is the same. This indicates that no phosphate ester is formed or at least that it does not accumulate. 2. Neither R-1-P nor R-5-P is dephosphorylated by the extract. Addition of a phosphatase inhibitor, such as NaF (final concentration, 0.1 M) to the reaction mixture does not affect the reducing sugar value obtained in the perchloric acid filtrate. Therefore, the lack of accumulation of phosphate ester in the reaction mixture cannot be a result of the presence of phosphatase in the extract. 3. The extract does not catalyze the arsenolysis of R-1-P. If R-1-P is the product, as in the case of phosphorolytic nucleosidase, R-1-P should remain as such. However, all efforts indicate that free ribose instead of R-1-P is the product found in the reaction mixture. 4. R-5-P is degraded only slowly in extracts of cells grown on glucose but is metabolized rapidly in extracts prepared from cells grown on xy-

[66]


461

lose. 13 Free ribose is not degraded b y either type of extract. When U R or H x R is incubated with the extracts from cells grown on xylose, quantitative recovery of free ribose is obtained at the end of incubation. Thus, free ribose rather than the phosphate ester of ribose must be the p. ~uct of action of L. pentosus nucleosidase. 5. When a reaction mixture containing R-1-P is deproteinized by the cold perchloric acid method, no significant a m o u n t of reducing sugar can be demonstrated in the protein-free filtrate. The recovery of free ribose from nucleoside cleavage in perchloric acid filtrate, therefore, cannot be due to a secondary splitting of the sugar phosphate b y the acidic deproteinizing agent.

II. Yeast A. Uridine Nucleosidase 14

URWH~O--*U~-R

Assay Method Principle. Two methods, both based on the differential spectrophot o m e t r y of U R and U, are employed. The first method consists in measuring the absorption increase at 290 m~ in alkaline solution (0.01 N N a O H ) when U R (Era = 30) 1~ is split to U (Era = 5.4 X 103). The second m e t h o d measures the decrease of absorption at 280 m~ and p H 7.0, since U R has an Em of 3.5 X 103, and U has an Em of 1.4 X 103 at these conditions. Reagents

UR, 20 mg./ml. Buffer (phosphate, borate, glycine, or Veronal), 0.1 M, p H 7.0. Enzyme. Procedure. M e t h o d 1. In the first method, 0.025 ml. of U R is incubated with 0.1 ml. of enzyme and 0.2 ml. of buffer at 38 ° for 30 minutes. F o u r milliliters of 0.01 N N a O H is then added, and the solution is read at 290 m~ in the Beckman spectrophotometer. The increment in absorption at this wavelength read against a zero time blank is a measure of the U formed. This method, rapid and sensitive, is employed during purification of the enzyme. M e t h o d 2. Incubation is conducted in a 3-ml. cuvette at 26 °. The incubation mixture consists of 0.05 to 0.2 ml. of enzyme, 0.01 to 0.03 ml. of UR, and 3 ml. of buffer. Readings at 280 m~ are t a k e n at various time intervals to follow the cleavage of U R to U. This method is used for kinetic study of enzyme activity. i~ j. O. Lampen and H. R. Peterjohn, J. Bacteriol. 62, 281 (1951). 14C. E. Carter, J. Am. Chem. Soc. 73, 1508 (1951). 15E~ = molar extinction.

462


[66]


Steps 2 to 4 are conducted at 4 to 10 °. Step 1. Three pounds of Fleischmann baker's yeast is plasmolyzed in toluene according to Kunitz. 16 Step 2. To the clear plasmolyzate, solid (NH4)2SO4 is added to the concentration of 445 g./1. After standing for 1 hour, the precipitate is collected by centrifugation, dissolved in distilled water, and dialyzed against distilled water. Step 3. To 350 ml. of this solution, 385 ml. of saturated (NH4)2SO4 is added. After standing for 1 hour, the precipitate is centrifuged, dissolved, and dialyzed as above. The dialysis runs for 48 hours with frequent change of water. Step 4. The fraction is adjusted to pH 4.7, and the precipitate is discarded. The supernatant solution, adjusted to pH 7.0 with dilute NaOH, contains about 60 % of the original activity and represents a purification of ten- to fifteenfold. Properties

Specificity. The purified enzyme is specific for UR and has no activity on AR, GR, HxR, CR, or TDR. 17 Uridylic acid also is not attacked. Kinetics. When 227 ~, of U R is incubated with 0.2 ml. of enzyme (containing 400 ~/of protein) and 3 ml. of 0.1 M buffer, pH 7, at 26 °, the reaction follows first-order kinetics up to 83 % of hydrolysis of the substrafe. Addition of 200 ~ of U to the reaction mixture produces 27 % inhibition, whereas addition of 3000 ~ of R produces only 30 % inhibition. pH Optimum. There is a well-defined optimum at pH 7.0 in either phosphate, glycine, or Veronal buffers. B. Purine Nucleosidase P u R + H 2 0 - ~ Pu + R Assay Method

Principle. Spectrophotometric methods are generally used. Reducing sugar determinations are also made on occasion. Reagents and Procedures. See hydrolytic nucleosidase from L. pentosus section. Purification Procedure 18

Step 1. Fresh baker's yeast (Fleischmann) is rapidly dried in a thin layer (1/~ inch) at 22 to 25 ° overnight with the aid of a fan. Slow drying 16 M. S. Kunitz, J. Gen. Physiol. 29, 393 (1947). 17 T O R - T h y m i d i n e deoxyriboside or thymidine. 18 L. A. Heppel and R. J. Hilmoe, J. Biol. Chem. 198, 683 (1952).

[66]


463

destroys the hydrolytic nucleosidase. Autolyzates are prepared by mixing 500-g. portions of yeast with 1500 ml. of 0.2 M acetate buffer, pH 5.1, incubating for 6 hours at 37 °, and centrifuging. Step 2. 140 ml. of autolyzate is mixed with 27.2 g. of (NH4)~SO4, and the pH is adjusted to 4.6 with 17.2 ml. of 2 M acetic acid. Another 13.3 g. of (NH4)2SO4 is then added (0.45 saturation). After 15 minutes, the mixture is centrifuged for 8 minutes at 13,000 X g. The supernatant (164 ml.) is brought to 0.55 saturation by the addition of 9.7 g. of (NH4)2SO4. The precipitate is collected by centrifugation dissolved in 0.1 M acetate buffer, pH 6.0, and dialyzed for 6 hours against flowing 0.01 M acetate buffer, pH 6.0. This fraction is then centrifuged to remove any precipitate formed during dialysis. Step 3. To 25 ml. of the clear solution, adjusted to pH 7.3 with 1 ml. of 0.2 M NH4OH, is added 10.1 g. of (NH4)2SO4 (0.6 saturation). The precipitate is removed by centrifugation, and the supernatant (27.5 ml.) is brought to 0.7 saturation with 1.7 g. of (NH4)~SO4. The second precipitate is collected by centrifugation and dissolved in 0.02 M NaAc to give a volume of 15.5 ml. Step 3- A 1-ml. portion of the last fraction is treated with 2.5 ml. of distilled water and 0.2 ml. of aged calcium phosphate gel 19 (10.2 mg. of solid per milliliter). After the gel is removed by centrifugation, the supernatant shows a 55-fold purification compared with the original autolyzate. The over-all yield is 21 to 22%. The purity of the enzyme can be doubled by adsorbing the enzyme on calcium phosphate gel and eluting successfully with water M/600 and M/300 phosphate buffers, pH 7.4. But the yield for this step is low (14%) and considered "not useful" by the investigators of this method. Is Steps 2 to 4 are carried out at 2 °. The activity of the enzyme is expressed in a unit which represents the amount of enzyme causing the cleavage of 1 micromole of substrate per hour.

Properties Specificity. The enzyme splits AR, GR, HxR, XR, and a number of synthetic PuR. In addition, nicotinamide riboside is cleaved to nicotinamide ribose. CR, UR, and uridylic acid are not attacked. The hydrolysis of HxR is competitively inhibited by AR and GR. The same enzyme for all these three ribosides is suggested. Other Properties. Phosphate and arsenate are not required. Phosphate buffers (0.02 M) with a pH value below 6.7 have no effect on the rate; ~9For preparation of calcium phosphate gel, see Vol. I [11].

464


[67]

more alkaline phosphate buffers are inhibitory when compared with glycine and glycylglycine buffers. The purified enzyme is relatively unstable, b u t the (NH4)~S04 fractions can be stored for several m o n t h s at - 1 0 °. T h e p H of the optimal activity of enzyme is around 7 to 8. With C14-adenine, it has been demonstrated t h a t the hydrolytic nucleosidase from yeast does not catalyze either the synthesis of nucleoside from free ribose and a purine base or the exchange reaction between a nucleoside and a purine base. PURIFICATION OF YEAST PURINE, NUCLEOSIDASEHYDROLITE

Step 1. 2. 3. 4.

Autolyzate (NH4)2SO4fraction (NH4)2SO4fraction Calcium phosphate gel supernatant

[67] N u c l e o s i d e

Volume, ml.

Total units

140 27.5 15.5

2800 1730 700

54.5

595

Over-all yield, % 62 25

1.7 16.2 31

21.2

94

Transdeoxyribosidase

+R t

,.

Specific activity against HxR, units/mg, protein

from Bacteria

~

+R

OH OH R and R r represent certain of the naturally occurring purines and pyrimidines.

By WALTER S. MCNUTT Assay Method

Principle. The m e t h o d employed consists in the quantitative estimation of deoxyribosides b y their specific growth-promoting effect upon the microorganism, Thermobacterium acidophilus R26. This bacterium responds equally in growth to equimolecular amounts of the deoxyribosides of adenine, guanine, hypoxanthine, thymine, uracil, and cytosine. T h e microbiological process, described b y Hoff-JCrgensen, 1 is very sensitive, estimating 1 ~, of deoxyriboside with an error of about 10%. T h e micro1 E. Hoff-JCrgensen, Biochem. J. 50, 400 (1951); see Vol. III [110].

[67]

NUCLEOSIDE TRANSDEOXYRIBOSIDASE FROM BACTERIA

465

biological analysis is more reproducible than are most microbiological procedures. To separate the individual deoxyribosides in a mixture, one-dimensional, ascending chromatography on paper may be carried out in glass jars 11 X 48 cm. The compounds are placed along Whatman No. 4 filter paper in quantities of 10 to 20 ~/of purine or pyrimidine per centimeter. The solvent (25 to 30 ml.) is added to the dry vessel; the paper is suspended above the solvent in the closed system, and the system is equilibrated at room temperature. The distance transversed by the solvent is 45 cm. No special precautions were taken to ensure a constant temperature (usually about 23°), and Rf values varied somewhat on independent runs. The following systems were employed: (1) n-Butanol-water-ammonia: 1 ml. of 15 N NH40H was added to 100 ml. of n-butanol saturated with water. After thorough shaking the clear supernatant was used. Equilibrated for 11 hours. Time to traverse 45 cm., 1.5 to 2 days. (2) n-Butanolwater-acetic acid: 25 ml. of n-butanol saturated with water -4- 5 ml. of glacial acetic acid. Equilibrated for 8 hours. Time to traverse 45 cm., 1.5 to 2 days. (3) n-Butanol-water-ethyl acetate-morpholine: 5 ml. of n-butanol saturated with water A- 5 ml. of ethyl acetate saturated with water -4- 10 ml. of morpholine. Equilibrated for 24 hours. Time to traverse 45 cm., 2 to 2.5 days. (4) n-Butanol-water-morpholine-methylglycol: 10 ml. of n-butanol saturated with water -4- 10 ml. of morpholine + 5 ml. of methylglycol -4- 2 ml. of water. Equilibrated for 24 hours. Time to traverse 45 cm., 3 to 3.5 days. The Rf values of the deoxyribosides and certain related compounds in the several systems are shown in Table I. The purines, pyrimidines, and their deoxyribosides, if present in sufficient quantity (about 5 ~, of purine or pyrimidine per 1-cm. spot), show up as dark areas on the dry chromatogram when examined beneath ultraviolet light (h = 247 mg). The appropriate areas are cut from the sheet and eluted with warm water, the filtered solution being assayed microbiologically. The difference in microbiological activity between the corresponding areas in the control and in the experimental samples may be used to calculate the amount of a given deoxyriboside which has formed or disappeared. Procedure. (Example: Uracil deoxyribosido -t- adenine ~ adenine deoxyriboside -4- uracil.) Uracil deoxyriboside (0.44 gM. in 10 gl.), adenine (500 3'), 0.2 M phosphate buffer (500 gl., pH 8.04), and Lactobacillus helvetieus enzyme (2.0 mg. of dry bacterial substance, dialyzed for 3 days, 500 pl.) were incubated under toluene for 9 hours at 37 °. Ethanol (7ml., 97 %) was added to stop the action of the enzyme. The control tube differed from the experimental in having the uracil deoxyriboside added

466

[67]


a f t e r t h e a d d i t i o n of t h e e t h a n o l . T h e v o l u m e s w e r e r e d u c e d t o a b o u t 500 ~l. a t 55 °, a n d t h e walls of t h e t u b e w e r e w a s h e d w i t h t h e liquid. T h e p r o t e i n was r e m o v e d b y p r e c i p i t a t i o n t h r o u g h t h e a d d i t i o n of 7 ml. of e t h a n o l . T h e s u p e r n a t a n t w a s c o l l e c t e d , a n d t h e p r e c i p i t a t e was w a s h e d t w i c e m o r e w i t h 2 ml. e a c h t i m e of w a r m 9 5 % e t h a n o l . T h e c o m b i n e d s u p e r n a t a n t w a s r e d u c e d t o d r y n e s s a t 55 ° a n d t a k e n up in 500 ~l. of w a t e r , 100 ~l. of t h i s s o l u t i o n b e i n g p l a c e d a l o n g a 4-cm. d i s t a n c e on t h e p a p e r c h r o m a t o g r a m . T h e c h r o m a t o g r a m w a s d e v e l o p e d in t h e n - b u t a n o l w a t e r - a m m o n i a s y s t e m . S p o t s of u r a c i l d e o x y r i b o s i d e , a d e n i n e d e o x y r i boside, a n d h y p o x a n t h i n e d e o x y r i b o s i d e w e r e p l a c e d a l o n g s i d e t h e s a m ples ( c o n t r o l a n d e x p e r i m e n t a l ) . A s s a y of t h e e l u t e d a r e a s s h o w e d t h a t 0.37 ~IV[. of a d e n i n e d e o x y r i b o s i d e ( R / = 0.40) w as f o r m e d ; 0.45 ~ M . of u r a c i l d e o x y r i b o s i d e ( R / = 0.34) d i s a p p e a r e d ; a n d less t h a n 0.01 ~ M . of h y p o x a n t h i n e d e o x y r i b o s i d e ( R / = 0.15) w a s f o r m e d . TABLE I R$ VALUES OF DEOXYRIBOSIDES AND RELATED COMPOUNDS

Rf values in system

Compound Adenine Guanine Hypoxanthine Cytosine Uracil Thymine 4 (5) -Amino-5 (4) -imidazole carboxamide Adenine deoxyriboside Guanine deoxyriboside Hypoxanthine deoxyriboside Cytosine deoxyriboside Uracil deoxyriboside Thymine deoxyriboside Cytidine 5-Methylcytosine Inosine Uric acid

n-Butanolwaterammonia 0.40 0.15 . 0.19 0.28 0.33 0.50 0.32 0.41 0.18 0.17 0.26 0.34 0.48 0.15 0.36 O. 08 0.02

n-Butanolwateracetic acid --

n-Butanol- n-Butanolwater-ethyl wateracetatemorpholinemorpholine methyl glycol 0.45

-----

0.48 . 0.36 ----

----0.35 0.44 0.56 -----

0.44 0.70 0.57 0.54 0.70 0.88 0.94 -----

-0.66 0.58 0.55 0.68 0.77 0.85 -----

.

.

-----

It has been pointed out by R. Markham and J. D. Smith [Biochem. J. 45, 294 (1949)] that the capacity of the system for guanine is very limited.

[67]

NUCLEOSIDE TRANSDEOXYRIBOSIDASE

FROM BACTERIA

467

Preparation of the Enzyme Enzymes capable of catalyzing the above reaction have been prepared from Lactobacillus helveticus S, Lactobacillus delbriickii ATCC 9649, and Thermobacterium acidophilus R26. All these organisms respond to one or more of the deoxyribosides as growth factors. The cells from which the TABLE II COMPOSITION OF THE MEDIA USED IN THE CULTIVATION OF Lactobacillus helveticus S AND Thermobacterium acidophilus R26

Component Glucose H C l - h y d r o l y z e d casein E n z y m a t i c a l l y digested casein Sodium acetate S o d i u m citrate, 5 H 2 0 KH:PO4 K2HPO4 NaC1 MgSO4-7H~O MnSO4.4H20 FeSO4.7H20 L-Asparagine L-Cystine DL-Tryptophan Dl~-Cysteine T w e e n 80 Adenine Guanine Uracil Thymine Cytidylic acid Oleic acid Riboflavin Calcium p a n t o t h e n a t e Niacin Nicotinic acid p - A m i n o b e n z o i c acid T h i a m i n e chloride.HC1 Pyridoxal.HC1 Pyridoxamine.2HC1 P t e r o y l g l u t a m i c acid Biotin

Lactobacillus helveticus S,

Thermobacterium acidophilus R26,

g./1.

g./1.

10 5 0.6 10 10 3 3 5 2.8 0.56 0.14 0.1 0.2 0.1 -1 mg./1, 10 10 10 --10 0.4 0.4 0.4 -0.2 0.2 0.2 0.2 0.01 0. 002

15 12.5 2.5 15 -1 1 -0.1 0.02 0.01 --0.1 0.25 0.5 mg./1, 10 10 -10 25 -0.5 0.5 -0.5 0.5 ---0. 025 --

468


[67]

enzymes were prepared were obtained from growth in so-called" synthetic media" supplemented with thymidine. 2 The enzyme from Lactobacillus helveticus S is prepared by growing the organism in 250-ml. lots of complete basal medium, 3 supplemented with thymidine at a level of 1 mg. per 250 ml. (see Table II for composition of the medium). After incubation for 24 hours at 37 ° the cells from 500-ml. portions of medium are collected by centrifugation and washed twice with 75 ml. of 0.05 M citrate buffer at pH 6.0. The washed cells are suspended in 15 ml. of the same buffer and.are broken up by agitation with glass beads for 30 minutes in the Mickle electric shaker. During this operation the cell suspension is kept cool by intermittent cooling in ice water. The turbid solution is transferred to a dialyzing membrane and dialyzed against distilled water at 3 ° for several days. The insoluble material which settles out is removed by centrifugation at 3000 r.p.m., and the slightly turbid supernatant solution is used. No further purification of the enzyme is required in order to obtain essentially uncomplicated results. The enzyme preparation does, however, split the deoxyribosides to a slight extent (17 to 20%).

Properties These bacterial extracts retain their transdeoxyribosidase activity over a period of a month or more when preserved in the frozen state. The enzyme functions over a wide range of pH values (5 to 9). Specificity. Extracts from Lactobacillus helveticus S, prepared as described above, catalyze the exchange with the following purine acceptors: adenine, guanine, hypoxanthine, and xanthine. The pyrimidines, thymine, uracil, cytosine, and 5-methylcytosine act as acceptors in this system as does, also, 4-amino-5-imidazole carboxamide, although only a low yield of microbiologically active compound is obtained. Uric acid, 2,6diaminopurine, and dihydrothymine are inactive. The transfer of the deoxyribosyl group from one purine or pyrimidine to another does not proceed through deoxyribose-l-phosphate or through hydrolysis followed by resynthesis, since the enzyme preparation is incapable of catalyzing the synthesis of deoxyribosides when incubated with deoxyribose-1phosphate or deoxyribose in the presence of an appropriate purine or pyrimidine. 2 W. S. M c N u t t , Biochem. J. 50, 384 (1951). 3 W. S. M c N u t t a n d E. E. Snell, J. Biol. Chem. 182, 557 (1950).

[68]

5-ADENYLIC ACID DEAMINASE FROM MUSCLE

469

[68] 5'-Adenylic Acid Deaminase from Muscle 5-AMP -t- H20--, 5-IMP W N H ,

By GORDON NIKIFORUK and SIDNEY P. COLOWICK

Assay Method Principle. The method originally used by Schmidt 1 was based on the measurement of ammonia liberation. The method described below was developed by Kalckar 2 and is based on the fact that deamination of adenine compounds is accompanied by a shift in the ultraviolet absorption spectrum. At 265 m~ the molecular extinction of 5-IMP is only 40 % of that of 5-AMP. Reagents 5'-AMP stock solution (0.04 M). Dissolve 139 mg. of the free acid 8 in 10 ml. of 0.08 M NaHC03. This solution may be stored at - 1 5 ° for many months without change. 0.01 M citric acid--NaOH buffer, pH 6.5. 0.01 M succinic acid--NaOH buffer, pH 5.9. Enzyme. Dilute the stock enzyme with succinate or citrate buffer to obtain 200 to 1000 units of enzyme per milliliter. (See definition below.)

Procedure. Dilute the stock solution of 5'-AMP 1 : 1000 in the succinate buffer, and place 3 ml. of the resulting solution in a quartz cell having a 1-cm. light path. Take readings at 265 m~ before and at 30-second intervals after mixing with 0.1 ml. of enzyme. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which causes an initial rate of change in optical density (AE265) of 0.001 per minute under the above conditions. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. 4 The values reported in the table are for activities determined in succinate buffer, pH 5.9. The activity in citrate buffer, pH 6.5, is about three times as high. (See section on "Properties. ") 1 G. Schmidt, Z. physiol. Chem. 179, 2~3 (1928); 208, 185 (1932); 219, 191 (1933). 2 H. M. Kalckar, J. Biol. Chem. 167, 429, 461 (1947). 3 Crystalline 5-adenylic acid is obtainable commercially from various sources. For methods of isolation and purification of this compound, see Vol. I I I [119]. 4 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see Vol. I I I [73].

470


[68]

Application of Assay Method to Crude Tissue Preparations. With extracts or homogenates of most tissues, the a m o u n t of material required is such t h a t the light absorption b y the enzyme preparation itself is too high to p e r m i t direct spectrophotometric measurements. F o r such preparations, it is recommended t h a t a high concentration of substrate (1 : 10 dilution of stock A M P ) be used and t h a t the reaction mixtures be fixed at appropriate intervals b y addition of an equal volume of 10 % perchloric acid. The filtrates, after appropriate dilution, m a y be analyzed either spectrophotometrically at 265 mu, or b y ammonia determination. Certain precautions are necessary in the interpretation of results with crude tissue preparations. The presence of phosphatase plus adenosine deaminase m a y readily simulate 5-adenylie deaminase activity. Purification Procedure Steps 1 and 2 of the following procedure are based largely on the m e t h o d originally described b y Schmidt. ~ Step 3 is adapted from the m e t h o d of Kalckar, ~ and step 4 is based in principle on the reports of Tiselius 5 and Mitchell et al. ~ The procedure through step 3 has been carried out successfully m a n y times in several laboratories. Step 4 has not been explored as thoroughly and is not recommended as a routine procedure. Step 1. Preparalion of Crude Extract. Rabbit muscle (from hind limbs and back) is chilled, passed through a meat grinder, and washed four times b y occasional stirring with 4 vol. of cold 0.85% NaC1 for 20 minutes and squeezing through a cheesecloth3 The deaminase is extracted from the colorless residue b y occasional stirring with 1 vol. of cold 2 % NaHCO3 for 1 hour and filtering through a folded W h a t m a n No. 12 filter paper. Longer periods of extraction with N a H C 0 3 lead to a loss of activity. Step 2. Adsorption of Deaminase by Alumina. a Alumina C~ (dry weight 0.025 g./ml.) is added slowly to fraction 1 with continuous stirring. F i f t y milliliters of alumina per liter of fraction 1 is usually sufficient for complete adsorption, b u t occasionally as much as five times this q u a n t i t y is required. T h e suspension is k e p t at 8 ° for 15 minutes, with occasional stirring. I t is then centrifuged in the cold, the supernatant fluid dis5 A. Tiselius, Arkiv Kemi, Mineral. Geol. 26B, No. 1 (1948-49). e H. K. Mitchell, M. Gordon, and F. A. Haskins, J. Biol. Chem. 180, 1071 (1949). 7The ground muscle may be extracted once with 1 vol. of 0.03 N KOH prior to the four washings with 0.85 % NaCl. This step does not decrease the yield of deaminase and has the advantage that the KOH extract may be used as starting material for the preparation of other muscle enzymes. See Vol. I [39, 60]. 8 For the preparation of alumina C~, see Vol. I [11].

[68]

5 - A D E N Y L I C ACID DEAMINASE FROM MUSCLE

471

carded, and the deaminase eluted at 25 to 30 ° with 1.0 M Na2HP04, 9 using 50 ml./1, of fraction 1. The eluate is stored in the cold to permit crystallization of the bulk of the Na~HP04, which is discarded. Step 3. Fractionation with Ammoniacal Ammonium Sulfate. A solution of ammonium sulfate, saturated at 0 ° and adjusted to p H 7.6 with 0.01 vol. of 18% ammonia, is added slowly to the eluate at 0 ° to give 0.27 saturation. After 10 minutes the precipitate is removed b y centrifugation in the cold at 15,000 r.p.m. The supernatant fluid is brought to 0.45 saturation, and, after centrifugation, the 0.27 to 0.45 fraction is dissolved in the least possible volume of 0.1 M Na2HPO4 (approximately 0.5 ml./1, of fraction 1). This preparation can be kept for at least two months at 4 ° without loss of activity. Step ~. Separation on Filter Paper. W h a t m a n No. 1 filter paper (50 X 40 cm.) is rolled into a cylinder, and the edges are stapled together. The enzyme solution (fraction 3) is deposited on the paper around the cylinder at a distance of 2 inches from the bottom. The paper is placed in a glass vessel containing 0.4 saturated ammonium sulfate at p H 7.6 (depth of solution about 1 inch). I t is i m p o r t a n t to perform this procedure rapidly, as even the slightest drying imparts lyophobic properties to the area containing the deposited enzyme. About 7 to 9 hours is required for ascension of the solvent. The cylinder is then unfolded and cut horizontally into four equal strips. These are extracted with 2 % N a H C Q , using ten times the volume of fraction 3 originally deposited. The extracts, although varying in total activity, are combined, since each shows higher specific activity than t h a t in fraction 3. The purification achieved here appears to be due to selective elution of deaminase from the paper by NaHCO3. T h e resulting material is unstable and cannot be further purified b y repetition of steps 2, 3, or 4. SUMMARY OF PURIFICATION PROCEDURE a

Fraction

Total Total Specific volume, Units/ml., units, Protein, activity, Recovery, ml. thousands thousands mg./ml, units/mg. %

1. NattCO3 extract 5300 2. Eluate from alumina 200 3. (NH4)~SO4fraction, 0.27-0.45 2.3 4. Eluate from paper 14

0.38

2000

1.46

260

--

7.6

1520

9.2

820

76

60.0 3.0

200 42

22.0 0.30

2,700 10,000

10 2.1

G. Nikiforuk and S. P. Colowiek, J. Biol. Chem. in press. g As reported earlier by Schmidt, 1lower phosphate concentrations (0.1 M) are in effective in eluting deaminase and, when followed by elution at higher phosphate concentrations, result in a low recovery of enzyme.

472


[68]

The table summarizes the results of the purification procedure. The yields were in most cases better than those reported here.

Properties Specificity. The purified enzyme is absolutely specific for 5'-AMP, having no action on adenine, adenosine, 2'-AMP, 3'-AMP, ADP, ATP, DPN, or TPN. Activators and Inhibitors. The activity of the enzyme, when tested in succinate or malonate buffer at pH 5.9, can be increased by addition of certain anions, including citrate, chloride, acetate, and lactate. The concentrations of citrate and chloride required to double the rate are 8 )< 10-4 M and 3 X 10-2 M, respectively, but the maximum degree of stimulation by either citrate or chloride is about 2.5-fold. A system maximally stimulated by either anion is not further activated by addition of the other. The nature of the cation is immaterial in these experiments; the chlorides of magnesium, sodium, and potassium show practically identical stimulatory effects, whereas the sulfates of these three cations are uniformly without effect, even at concentrations as high as 0.1 M. Certain anions, including fluoride, phosphate, and pyrophosphate, are strongly inhibitory. The inhibition by fluoride is increased by raising the substrate concentration, whereas the inhibition by phosphate is decreased at higher substrate concentration. Thus, at low substrate concentration (4 X 10-~ M AMP) the concentrations of fluoride and phosphate required to produce 50% inhibition are 5 × 10-3 M and 3 )< 10-3 M, respectively; at high substrate concentration (4)< 10-3 M AMP), the concentrations required for 50% inhibition are 0.2 X 10-3 M and 8 X 10-3 M, respectively. The per cent inhibition by fluoride or phosphate is approximately the same in the absence or presence of stimulatory anions. The competitive nature of inhibition by phosphate has also been noted by Conway and Cooke. 1° Although inhibition by fluoride suggests participation of a metal in the reaction, no evidence for a metal requirement could be obtained. 4 Various metal-binding agents, including ethylenediaminetetraacetate, 8-hydroxyquinoline, cyanide, and thiocyanate (as well as citrate), failed to inhibit the enzyme. Addition of Mg or Mn ions failed to increase the activity. Certain heavy metal ions inhibit the enzyme. Mercuric ions at 1 >( 10-~ M cause 50 % inhibition. This inhibition is reversible by cysteine. Iodoacetate, even at 0.01 M, causes no detectable inactivation after incubation with the enzyme for 30 minutes at 25 ° in succinate buffer o.f pH 5.9. 10 E. J. Conway a n d R. Cooke. B~:oehem, J. 38, 479 (1939),

[691

SFECIFIC ADENOSINE' DE AMINASE FROM INTESTINE

473

Effect of pH. The enzyme exhibits a sharp optimum for activity at pH 5.9 in succinat~ and other 1 buffers, the activity falling to one-half of optimal at pH 5.5 or 6.4. In citrate buffer (0.1 M), no.t only is the activity increased markedly (see above), but the optimum pH is shifted to pH 6.5, the activity falling to one-half of optimal at pH 5.6 or 7.3. The degree of inhibition of the enzyme by fluoride is also dependent on pH. Thus, 0.01 M fluoride, which produces 96% inhibition of the enzyme in succinate buffer at pH 5.6, causes no inhibition of the enzyme at pH 6.7. Similarly, in citrate buffer, 0.01 M fluoride causes 83% inhibition at pH 5.6, but no inhibition at pH 7.3. Effect of Substrate Concentration. The Michaelis-Menten constant, Kin, determined from a Lineweaver-Burk plot which gave a straight line over the concentration range 4 X 10-~ to 4 X 10-3 M AMP, was 6.0 X 10-6. Degree of Purity. The absolute percentage purity of the most active fraction is not known, since none of the criteria for homogeneity have been applied. The turnover number under favorable conditions (fraction 4 in citrate buffer, pH 6.5, 4 X 10-3 M AMP, 25 °) may be calculated to be about 2500 moles of substrate per 10~ g. of protein per minute. The purity of fraction 3 or 4 in terms of separation from interfering enzymes is satisfactory. None of the following activities is detectable: adenosine deaminase, hexokinase, myokinase, ATPase, or nucleotide pyrophosphatase. These fractions are therefore suitable for use in spectrophotometric analysis H of compounds which can give rise to 5'-AMP, such as ATP, ADP, and DPN. 11 See Vol. I I I [111].

[69] Specific Adenosine Deaminase from Intestine By

NATHAN O. KAPLAN

Adenosine + H20 --~ Inosine + NH3 The deaminase from intestine, unlike the enzyme from takadiastase (see Vol. II [70]), is specific for adenosine and will not deaminate other adenine derivatives. Kalckar ~ has separated the deaminase from the potent phosphatase present in the intestine. Kornberg and Pricer, ~ however, have described a method in which separation of the phosphatase is not essential for the assay of adenosine, the phosphatase being inhibited by addition of phosphate. Both of these procedures will be outlined. 1 H. M. Kalckar, J. Biol. Chem. 167, 445 (1947). A. Kornberg a n d W. E. Pricer, Jr., J. Biol. Chem. 193, 481 (1951).

474


[69]

Principle of Assay. Deamination of adenosine is followed by measurement of the decrease at 265 m~ and is based on the same principle as that for 5'-AMP deaminase (see Vol. II [68]). Assay Procedure. Thirty-six micrograms of adenosine is introduced into 0.05 M glycylglycine or phosphate, pH 7.6 (see below), in a total volume of 3 ml. The deaminase is then added, and the change at 265 m~ followed. No specific units have been defined for the enzyme. Separation of Deaminase from Phosphatase by the Method of Kalckar.1 The first steps are identical with those outlined by Schmidt and Thannhauser for the preparation of intestinal phosphatase. 3 The mucosa from calf intestine is digested with trypsin in the presence of toluene for 36 hours and then filtered through a cake of Hyflo. The filtrate is then treated with 600 g. of ammonium sulfate per liter; the precipitate is collected on a film of Hyflo and dissolved in 0.1 M ammonium acetate, pH 8.5. Solid ammonium sulfate is then added to the solution to bring the concentration to 500 g./1. The precipitate is redissolved in the ammonium acetate and dialyzed overnight at 0 ° against 0.025 M ammonium acetate, pH 8. The adenosine deaminase is separated from phosphatase activity by addition of 0.1 vol. of alumina (containing 25 mg. of aluminum hydroxide per milliliter) to the above dialyzed fraction. After 10 minutes of stirring at room temperature the mixture is centrifuged, and to the supernatant 0.05 vol. of alumina is added. Both precipitates are combined and eluted with 0.2 M phosphate (pH 8) and dialyzed against 0.02 M ammonium acetate, pH 8. The supernatant from the alumina mixture contains all the phosphatase and about 35 to 40 % of the adenosine deaminase. The eluates contain almost no phosphatase and about one-half of the deaminase activity. 5'-Adenylic acid is deaminated at about 2 % of the rate of adenosine by the eluates; hence the eluates can be used for adenosine determination. About 0.4 to 1.0 ~' of the purified protein per milliliter can be used for a spectrophotometric determination. Adenosine Assay in the Presence of Phosphate. Kornberg and Pricer 2 describe the following procedure for obtaining the intestinal deaminase without the necessity for separating the phosphatase from the deaminase. One hundred milligrams of Armour intestinal phosphatase (stated to contain 15 Schmidt-Thannhauser units/mg.) is dissolved in 10 ml. of ammonium acetate buffer (0.02 M, pH 8.0). The solution is dialyzed against 0.04 M sodium acetate at 2 ° for 3 hours. This solution can be stored in the deep-freeze without loss in activity. Adenosine is deaminated very rapidly when the reaction is carried out in 0.05 M phosphate a G. Schmidt and S. J. Thannhauser, J. Biol. Chem. 149, 369 (1943).

[70]

NONSPECIFIC ADENOSINE DEAMINASE FROM TAKADIASTASE

475

(pH 7.4). Under identical conditions, no detectable deamination of 5'-AMP, 3'-AMP, or 2'-AMP takes place. pH Optimum. The intestinal deaminase is active over a very wide pH range. Although the pH optimum is near the neutral point, the activities at pH 9 and pH 6 are about two-thirds of that at the optimal pH. Use of Deaminase to Assay for Adenosine Derivatives. When the assay with the preparation of Kornberg and Pricer is carried out in phosphate, an unknown can be assayed specifically for adenosine. However, when the reaction is carried out in a nonphosphate medium, the total bound adenosine can be determined (i.e., coenzyme A, TPN, DPN, etc.). This is due to the fact that the crude fraction contains a pyrophosphatase as well as a monoesterase. Therefore, it is possible to determine total bound adenosine by means of the nonphosphate medium. 4,~ 4 T. P. Wang, L. Shuster, and N. O. Kaplan, J. Biol. Chem. 206, 299 (1954). 5 L. Shuster, N. O. Kaplan, and F. E. Stolzenbach, J. Biol. Chem. 215, 195 (1951).

[70] Nonspecific Adenosine Deaminase from Takadiastase Adenosine compound -~ H20 --* Inosine compound ~ NH3

By NATHAN O. KAPLAN Assay Method 1

Principle. The method is based on the change in absorption at 265 mp which manifests the conversion of adenosine to inosine. The principle of the procedure is identical to that used for following the specific 5'-AMP deaminase (see Vol. II [68]). Reagents 0.1 M phosphate, pH 6.8. 0.004 M adenosine. Enzyme. Diluted in 0.1 M phosphate, pH 6.8, if necessary.

Procedure. To 3 ml. of phosphate, add 0.05 ml. of 0.004 M adenosine. After observing the absorption at 265 m~, start the reaction by the addition of approximately 20 units of enzyme. Take readings at 15 and 120 seconds after addition of enzyme. Definition of Unit and Specific Activity. A unit represents the change in optical density of 0.01 in the 15- to 120-second interval. Specific act N. O. Kaplan, S. P. Colowick,and M. M. Ciotti, J. Biol. Chem.194, 579 (1952).

476


[70]

tivity is expressed as units per milligram of protein, determined by the procedure of Lowry et al. 2 Application of Assay Method to Crude Extracts. Since the crude takadiastase extracts are quite pigmented, it is difficult to determine the activity by the procedure outlined above. However, some estimation of the enzyme content can be determined by ammonia release. This method has been used in assaying the crude extract in the purification procedure outlined below. Purification Procedure The first step in the purification procedure is taken principally from the method of Mitchell and McElroy2 The further steps are essentially from the procedure of Kaplan et al., 1 with some modifications introduced by Astrachan (personal communication). Step 1. One hundred grams of takadiastase 4 is added to 2000 ml. of H20 and 200 g. of Permutit. The precipitate is centrifuged and washed with 300 ml. of water. The supernatant and wash are combined. Alcohol is then added at 0 ° to 33%. After centrifugation, the resulting precipitate is discarded. Alcohol is then added to 65%, and, after standing for 20 minutes, the precipitate is collected by centrifugation at 0 ° and dissolved in 500 ml. of H20. The solution is passed through a charcoal Permutit pad (60 g. of charcoal plus 60 g. of Permutit); this removes a considerable amount of pigmented material. The pad is washed with about 250 ml. of H20. The washings are combined to give a total volume of approximately 600 ml. This solution is the product of step 1 as outlined in Table I and has an activity of 33 units/rag, of protein. Step 2. Precipitation with Acetone. Acetone is added at 0 ° to the above solution (dissolved 65% alcohol precipitate) to 23 %. The resulting precipitate is removed by centrifugation at 0°; this precipitate contains only a negligible amount of activity. The solution is then brought to 40 % acetone; the resulting precipitate contains most of the activity, although some activity is present in a further fraction with 50 % acetone. The 40 % acetone precipitate is dissolved in a 100 ml. of water, and the specific activity is 219 units/mg, of protein. Step 3. Fractionation with Ethanol. Ethanol is added to the solution from step 2 to 10 % at 0 °. The resulting precipitate contains only a slight amount of activity. The supernatant is then chilled to - 1 2 °, and as a result a second precipitate ensues. The precipitate is dissolved in 25 ml. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. I I I [73]. 3 H. K. Mitchell and W. D. McElroy, Arch. Biochem. 10, 351 (1946). 4 Takadiastase powder can be obtained from Parke, Davis and Company.

[70]

NONSPECIFIC A D E N O S I N E DEAMINASE FROM TAKADIASTASE

477

of phosphate (0.1 M, p H 6.8) and contains the deaminase. This step gives approximately a threefold purification. Step 4. Ammonium Sulfate Fractionation. Some further purification is achieved b y bringing the active fraction to 70 % ammonium sulfate. The precipitate is colored and discarded. A fraction obtained between 70 and 100% ammonium sulfate contains the enzyme with a considerable increase in purity. However, the yield is quite low, and it has been our general practice to stop the purification at step 3. The advantage of the ammonium sulfate fractionation is t h a t this procedure removes phosphatase which is still present in small amounts in the ethanol precipitate. TABLE I SUMMARY OF PURIFICATION

Step Crude water extractsa of takadiastase (I00 g.) I. 66 % alcohol precipitate II. 40 % acetone precipitate III. - 12° ethanol precipitate IV. 100% ammonium sulfate fraction

Total units Units/mg. Recovery, % 522,000 227,000 70,800 32,420 8,100

4 33 219 614 1110

-43.0 13.5 6.2 1.5

The activity of crude preparations varies considerably. The, activity of the crude extract was determined by ammonia release from adenosine. Properties

Stability. The enzyme is quite stable and can be kept in the deepfreeze for over six months without loss in activity. The dissolved precipitate of step 3 has been lyophilized and kept as a dry powder for over a year with no decrease in activity. pH Optimum. Mitchel and M c E l r o y a report a broad p H optimum (5 to 8) in phosphate. This occurs with high and low levels of adenosine. However, with low substrate levels in succinate, a sharp p H optimum of 6.3 is found. Specificity. The enzyme, unlike the adenosine deaminase from intestine, is not specific. I t will deaminatc 5'-AMP, 3'-AMP, ATP, ADP, D P N and adenosine diphosphate ribose (ADPR), as well as adenosine. I I t does not deaminate 2'-AMP, T P N , or adenine. 3',5'-Diphosphoadenosine is deaminated, but 2',5'-diphosphoadcnosine is not. 5 I t is of interest to note t h a t the " s y n t h e t i c " 3'-isomer of T P N is deaminated at a slow rate. 6 The takadiastase enzyme deaminates adenosine twice as fast as 5'-AMP and approximately four times as fast as 3'-AMP. D P N , ADP, and A T P are deaminated at considerably slower rates. 5 T. P. Wang and N. O. Kaplan, J. Biol. Chem. 206, 311 (1954). e L. Shuster and N. O. Kaplan, J. Biol. Chem. 215, 181 (1955).

478

[71]


K,~. A s u m m a r y of the affinities of various substrates for the deaminase is given in T a b l e I I . Distribution. As yet, the nonspecific deaminase described a b o v e has been found only in takadiastase. TABLE II AFFINITIES OF VARIOUS SUBSTRATES FOR DEAMINASE

Substance

Approximate Km

ATP DPN 5'-Adenylic acid ADP 3'-Adenylic acid ADPR Adenosine

M X 10-~ 1.2 1.8 0.8 0.7 1.7 1.5 0.6

[71] Cytosine Nucleoside Deaminase from Escherichia

coli

C R 1 ~ H20--~ U R 1 -~ NH3 C D R i ~ H 2 0 --* U D R 1 -t- NH3

(1) (2)

By T. P. WANG

Assay Method Principle. S p e c t r o p h o t o m e t r i c m e t h o d s are used in following the act i v i t y of this enzyme, since the deamination of the cytosine c o m p o u n d s is accompanied b y a decrease in absorption of 55 % at 282 m~. T h e molecular extinctions of C R and U R at 282 m~ are 6000 and 2700, respectively. 2 T h e absorption spectra of the corresponding deoxyribosides are not m a t e rially different from those of the ribosides. Reagents C R or C D R , a n y suitable concentration. E. coli extract, 1 ml., equivalent to 50 to 100 mg. of wet cells. Tris buffer (0.1 M), p H 7.5.

Procedure. Place in a 3-ml. silica B e c k m a n cuvette 1.5 ml. of Tris buffer and a solution of C R or C D R containing a b o u t 0.3 micromole of the 1 CR, UR, CDR, and UDR stand for cytidine, uridine, cytosine deoxyriboside, and uracil deoxyriboside, respectively. 2 T. P. Wang, H. Z. Sable, and J. O. Lampen, J. Biol. Chem. 184, 17 (1950).

[71]

CYTOSINE NUCLEOSIDE DEAMINASE FROM ESCHERICHI~_ COLI

479

nucleoside. Make up to 2.9 ml. with water, and take an initial reading at 282 m~. Then add 0.1 ml. of the enzyme. After a quick stirring, take readings at 30-second intervals. The reaction will be finished in about 30 minutes.

Preparation of Enzyme 2

Preparation of E. coli Cells. E. coli strain 15 (9723 of the American Type Culture Collection) is the source of this enzyme. Stock culture of E. coli is kept on agar slants containing 0.3% Difeo beef extract, 0.2% Difco yeast extract, 0.7 % Difco peptone, 0.4% glucose, and 1.5 % Difco agar. An inoculum is made by transferring a loopful of bacteria from the slant to 10 ml. of medium of the same composition as listed above except the agar. The inoculum is then incubated for 24 hours at 37 °. The cells are collected by centrifugation at 4500 r.p.m, for 15 minutes. Preparation of Cell-Free Extract. The packed wet cells of E. coli, washed once with 0.9 % NaC1, are ground in an ice-chilled mortar with two and one-half times their weight of alumina powder (A-303 or A-301 of the Aluminum Company of America) according to McIlwain. 3 The paste is then mixed with 10 to 20 vol. (with respect to the original cells) of cold 0.05 M Tris buffer, pH 7.5, allowed to stand at 2 ° for 30 minutes, and centrifuged at 20,000 X g for 15 minutes in a Servall centrifuge. The supernatant is slightly opaque and light yellow in color. Attempts have been made to purify the enzyme by alcohol and (NH4):SO4 fractionations. The efforts were unsuccessful. Properties

Specificity. The cell-free extract is specific for the cytosine nucleosides. No action is observed when adenine, adenosine, cytosine, isocytosine, cytidylic acid, guanine, and guanosine are tested. The deamination is faster with CDR than with CR. The Km for CR is 1.74 X 10-4 M, and that for CDR, 8.9 X 10-5 M. General Properties. The enzyme is not inactivated by prolonged dialysis or by freezing and thawing. Preparations kept for several months at - 2 0 ° retain their original activity. The enzyme has a broad pH oPtimum between 6.5 and 8.5. Products of the Reaction. The products of the reaction are uracil nucleosides and ammonia. The former can be identified by their absorption spectra in ultraviolet region and by paper chromatographic or ionophoretic methods. Ammonia can be easily demonstrated by any of the standard methods such as by use of Nessler's reagent. H. McIlwain, J. Gen. Microbiol. 2, 288 (1948).

480


[72]

When a demonstration of the f o r m a t i ~ of uracil nucleosides is desired, it is preferable to use a thoroughly dialyzed extract. Because of the presence of a pyrimidine nucleoside phosphorylase which requires inorganic phosphate for its activity in the extract, 2 it is essential to remove any inorganic phosphate present in the extract to prevent any splitting of the uracil nucleosides formed from the deamination of cytosine nucleosides.

[72] Guanase Guanine ~ H~O --* Xanthine ~ NH3

By Louis SHUSTER Assay Method Principle. When guanine is deaminated to xanthine, there is a shift in the ultraviolet absorption spectrum, the greatest change being a decrease of about 50% in the extinction at 245 m~. This change is the basis for the method of Roush and Norris. 1 The method of Kalckar, 2 which is more commonly used, involves measurement of the xanthine produced in the reaction by oxidation with xanthine oxidase. This oxidation is followed spectrophotometrically by measuring the increase in optical density at 290 m~ due to the formation of uric acid. The increase obtained is roughly sixfold, which makes this method more sensitive than that of Roush and Norris.

Reagents Guanine. A stock solution of 0.001 M can be made up by dissolving 15 rag. of free guanine in a few milliliters of 1 N N a O H and diluting up to 100 ml. 0.1 M glycylglycine or tris (hydroxymethyl)aminomethane buffer, pH 8.0. Xanthine oxidase, prepared from milk (see Vol. II [73]). An aliquot of 0.1 ml. should contain enough enzyme to oxidize 50 ~/of hypoxanthine per milliliter per hour. Guanase. The enzyme is diluted with glycylglycine or Tris buffer to contain 200 to 1000 units of enzyme per milliliter (see definitions below). i A. R o u s h a n d E. R. Norris, Arch. Biochem. 29, 124 (1950). z H. M. Kalckar, J. Biol. Chem. 167, 461 (1947).

[72]

GUANASE

481

Procedure. Dilute the stock solution of guanine 1 : 15 with buffer, and place 2.9 ml. of the resulting solution in a quartz cell having a 1-cm. light path. By the method of Roush and Norris, add 0.1 ml. of guanase solution and follow the decrease in optical density at 245 mr*. By Kalckar's method add 0.05 to 0.10 ml. of xanthine oxidase, and read the optical density at 290 mu. Then add 0.1 ml. of guanase solution, and read at 30-second intervals after mixing. Application to Crude Tissue Preparations. Changes in turbidity during the course of the reaction may be checked at 320 mtL where no change ascribable to uric acid occurs. If uricase is present (this can be checked by adding 10 y of uric acid and noting any decrease at 290 m#), the reaction should be followed at 270 m,, the xanthine peak, where the optical density decreases about 80% during oxidation to uric acid, and this is unaffected by the action of uricase. Purification Procedure The following procedure is given by Kalckar, 2 who presents no data on specific activities or yields. Step 1. Preparation of Crude Extract. Rat liver freed of blood is blended with 2 vol. of cold distilled water and extracted for 15 minutes in a mechanical shaker. The homogenate is centrifuged at low speed (2000 r.p.m, for 5 minutes), and the precipitate is discarded. The supernatant is then centrifuged at high speed (15,000 r.p.m, for 20 minutes at 0°), and the precipitate is again discarded. The clear supernatant is used for the succeeding steps. Step 2. Fractionation with Ammonium Sulfate. Saturated ammonium sulfate is added to fraction 1 to give 0.4 saturation. The resulting precipitate is removed by centrifugation and discarded. The supernatant is brought to 0.6 saturation with more saturated (NH4)~SO4: The precipitate is centrifuged down and dissolved in a small volume of water. Step 3. Fractionation with Ethanol. Fraction 2 contains both guanase and nucleoside phosphorylase. Almost complete separation of these activities can be achieved by fractionation with alcohol at - 5 °. Fraction 2 is adjusted to pH 5.5 with 0.3 vol. of 0.1 M succinate-acetate buffer. Ethanol is added to a concentration of 15%. The resulting precipitate, which contains both guanase and nucleoside phosphorylase, is discarded. More ethanol is added to the supernatant to give a concentration of 40%. The precipitate is centrifuged down and extracted with 0.1 M glycine buffer, pH 9.1. Any insoluble residue is discarded. The clear glycine extract still shows slight nucleoside phosphorylase activity, but in the ,absence of large amounts of inorganic phosphate this preparation can be

482


[73]

used to measure guanine in the presence of guanosine. N o unit is available for the enzyme.

Properties Specificity. B o t h guanine and 8-azaguanine are d e a m i n a t e d b y this enzyme. 1 Guanosine and guanylic acid are not attacked. When acting on guanine the e n z y m e exhibits a broad p H o p t i m u m in the range of p H 6 to 10. When acting on 8-azaguanine the p H o p t i m u m is 6.3, and points of 5 0 % a c t i v i t y are a t p H values 5.5 and 7.5. T h e Km values for guanine and 8-azaguanine at p H 6.5 in 0.05 M p h o s p h a t e buffer are 5 X 10 -e and 7 X 10 -~, respectively.

[73] Xanthine Oxidase from Milk H y p o x a n t h i n e + 2 02 ~ - Uric Acid + 2 H202 B y B. L. HORECKER and L. A. HEPPEL

Assay Method P r i n c i p l e . X a n t h i n e oxidase m a y be assayed b y m e t h y l e n e blue reduction, 1 oxygen uptake, ~ uric acid formation, 3 and c y t o c h r o m e c reduction. 4 T h e m e a s u r e m e n t of uric acid f o r m a t i o n b y the absorption at 290 mg, as originally introduced b y Kalckar, is p e r h a p s the m o s t convenient of these methods. However, since the purification procedure described here was developed with the aid of the c y t o c h r o m e reduction test, the results are given in t e r m s of this assay. T h e rate of a p p e a r a n c e of the reduced band of c y t o c h r o m e c at 550 mg is expressed as the firstorder velocity constant for the reaction. Reagents

C y t o c h r o m e c (2.5 X 10 -4 M). P r e p a r e b y the m e t h o d of Keilin and Hartree, 5,6 and analyze b y the m e t h o d of Theorell. 7 1 M. Dixon, Biochem. J. 20, 703 (1926). 2 E. G. Ball, J. Biol. Chem. 128, 51 (1939). 8 H. M. Kalckar, J. Biol. Chem. 167, 429 (1947). 4 B. L. Horecker and L. A. Heppel, J. Biol. Chem. 178, 683 (1949). 5 D. Keilin and E. F. Hartree, Biochem. J. 39, 289 (1945); see Vol. II [133]. 6 Commercial preparations are available which are satisfactory for the assay. 7 H. TheoreI1, Biochem. Z. 285, 207 (1936).

[73]

XANTHINE OXIDASE FROM MILK

483

Catalase, 5.0 units/ml. Prepare by the method of Sumner and Dounce. 8.9 Hypoxanthine (0.05 M). Dissolve 68 mg. in 10 ml. of 0.05 M NaOH. Albumin. Dissolve 60 rag. of crystalline bovine serum albumin (Armour Laboratories) in 10 ml. of water. 0.1 M phosphate buffer, pH 7.4. Enzyme. Dilute in water to obtain 0.25 to 2.5 units of enzyme per milliliter. (See definition below.)

Procedure. To 1.0 ml. of buffer in a 1.0-cm. absorption cell add 0.1 ml. of cytochrome c, 0.1 ml. of catalase, 0.1 ml. of albumin, 0.2 ml. of water, and 0.04 ml. of diluted enzyme. Take readings at 550 mu at 1-minute intervals after mixing with 0.01 ml. of hypoxanthine. After 7 minutes add about 1 mg. of solid Na2S204 and take a final reading. Definition of Unit and Specific Activity. Calculate the concentration of oxidized cytochrome c (ferricytochrome) from the equation dR -Ferricytochrome - 1.96 × dt 104 moles per liter where d~ and dt are the density readings after addition of Na2S204 and at any time t during the rate determination, respectively. A unit of enzyme is that quantity which will give a value of 1.0 for (A log ferricytochrome)/At, the first-order velocity, where t is expressed in minutes. The specific activity is the number of units per milligram of protein in the test. Protein is determined by the turbidimetric method of Bficher. 1° Purification Procedure Steps 1 and 2 are based on the procedure of Ball. 2

Step 1. Preparation of Buttermilk. Churn 2 quarts of fresh raw cream (40 to 42 % butter fat) in a mechanical mixer at 2 ° until the butter separates as fine hard particles. Strain through several layers of cheesecloth. Step 2. Trypsin Digestion and Heating. To the strained buttermilk (780 ml.) add 0.2 M Na2HP04 (470 ml.) to bring the pH to 7.5 and 2.9 g. of trypsin (Wilson 1:300), dissolved in 50 ml. of water. Incubate at 37 ° for 31/~ hours. Cool the incubation mixture to 20 °, and test aliquots t o determine the minimum amount of 0.5 M CaC12 required to produce a nearly clear supernatant solution. Generally 0.12 to 0.15 vol. is sufficient; further addition will greatly reduce the yield of enzyme. To the bulk of the digestion mixture (1280 ml.) add 190 ml. of 0.5 M CaCI~, s j . B. Sumner and A. L. Dounce, J. Biol. Chem. 19.7, 439 (1939). 9 Commercial catalase preparations may be used. i0 T. Bficher, Biochim. et Biophys. Acta 1, 292 (1947); see also Vol. I I I [73].

484


[73]

incubate for 15 minutes at 20 °, and centrifuge. Warm the slightly opalescent supernatant solution to 60 ° in 3 minutes, keep at this temperature for 5 minutes, and cool as rapidly as possible. Step 3. Ammonium Sulfate Fractionation. To the heated fraction (1200 ml.) add 271 g. of ammonium sulfate, and filter overnight. Add 72 g. of ammonium sulfate to the filtrate, collect the precipitate by centrifugation, and dissolve in water. Step 4. Aluminum .Hydroxide Gel Adsorption and Elution. Add the ammonium sulfate fraction (273 ml.) to 153 mg. of aluminum hydroxide gel C~/11 which has been previously centrifuged. Stir to suspend the gel, centrifuge, and discard the supernatant solution. Elute the gel with three 12-ml. portions of 0.5 M phosphate buffer, pH 7.5. Step 5. Calcium Phosphate Gel Adsorption and Elution. Combine the eluates, and add 54 ml. of ammonium sulfate solution, saturated at 0 °. Collect the precipitate, and dissolve in 60 ml. of water. Add 99 mg. of calcium phosphate gel (aged about 3 months and centrifuged before use). Suspend the gel thoroughly, centrifuge, and discard the supernatant solution. Wash the gel twice with 12-ml. portions of 0.1 M phosphate buffer, p i t 6.2, and elute the enzyme with two 12-ml. portions of 0.5 M phosphate buffer, pH 6.2. To the combined eluates add 36 ml. of saturated ammonium sulfate solution, collect the precipitate by centrifugation, and dissolve in 30 ml. of water. SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. 2. 3. 4. 5.

Buttermilk Heated fraction (NH4)~SO4fraction AI(OH)3eluate Ca3(PO4)2eluate

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 780 1200 267 36 41

1.67 0.67 2.33 11.1 5.40

1300 49.0 800 1.73 624 2.58 400 2.85 221 0.98

0.034 0.39 0.90 3.89 5.51

-62 48 31 17

Properties Specificity. The purified enzyme will oxidize hypoxanthine, xanthine, and aldehydes. 1 The ratio of rate of oxygen uptake to cytochrome c reduction remains constant during the purification procedure. ~ Activators and Inhibitors. Xanthine oxidase is slowly and irreversibly inactivated by cyanide, 12 and this effect is now attributed to its metal 11R. Willstatter and H. Kraut, Ber. 66, 1117 (1923). 1~A. Szent-GySrgyi,Biochem. Z. 173, 275 (1926)~

[74]

URICASE

485

content. 13 The enzyme contains FAD. The inactivation on dialysis reported by BalF can be reversed by sulfhydryl compounds. TM The enzyme is inhibited by buffers and salts such as phosphate, imidazole, and sodium and potassium chloride. 15 Stability. The heated fraction m a y be stored at 2 ° for several weeks with little loss in activity. I t can be kept in the frozen state indefinitely. The final solution slowly loses activity when frozen and stored at - 1 6 °. i8 D. E. Green and H. Beinert, Biochim. et Biophys. Acta 11, 599 (1953). 14H. M. Kalckar, N. O. Kjeldgaard, and H. Klenow, Biochim. et Biophys. Acta 5, 575 (1950). is D. B. Morell, Biochem. J. 51, 666 (1952).

[74] U r i c a s e Uric Acid + 02 + 2 H20 --~ Allantoin + H202 + CO2 B y ENZO LEONE

Assay Method Principle. The rate of 02 uptake b y the enzyme is measured manometrically, in the presence of uric acid. The method was originally developed by Keilin and Hartree, ~ who used Barcroft differential manometers; a dangling cup, containing uric acid, is tipped in after equilibration. The procedure described below applies to Warburg manometers. Reagents

Uric acid, lithium salt. Dissolve 500 mg. of uric acid in 31.25 ml. of boiling 0,1 N LiOH; cool; bring to 100 ml. with water. This solution, of which 0.4 ml. contains 2 mg. of uric acid, must be prepared daily. 0.1 M boric acid-KC1/NaOH buffer, p H 9.0. Enzyme. Amounts of enzyme are chosen so as not to exceed an uptake of 60 to 65 pl. of 02 during the first 15 minutes. Procedure. In Warburg manometer flasks, place 1 ml. of borate buffer, enzyme, and water up to a final volume of 2.6 ml. In the center well place 0.3 ml. of 20% K O H adsorbed on a filter paper roll; in the side arm, 0.4 ml. of uric acid solution. T e m p e r a t u r e 39°; gas phase, air. After equilibration, tip in uric acid, and take readings every 5 minutes for the first 15 minutes. F r o m the O~ uptake during the first 15 minutes calculate Che corresponding u p t a k e per hour. i D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) Bllg~ 114 (1936).

486


[74]

Definition of Unit and Specific Activity. One unit of enzyme is defined as the amount which takes up 1 ~1. of O2 per hour, under the above conditions; the same value, divided by the volume, in milliliters, of the preparation used, represents the concentration of enzyme. The specific activity is expressed as Qo, i.e., units, calculated as above, per milligram dry weight of nondialyzable material. Application of Assay Method to Crude Tissue Preparations. The manometric assay is the method of choice, especially when examining unknown biological material for uricase activity. Allowance must be made for the 02 uptake of the control, in the absence of uric acid. It is also advisable, when dealing with tissues in which the presence of uricase is uncertain, to perform a colorimetric determination of residual uric acid, after an incubation period of at least 1 hour. Brown's 2 phosphotungstic acid or Fearon's 3 dichloroquinone chlorimide color reactions are suitable for this purpose. In crude tissue preparations or in fractions prepared by differential centrifugation, uricase can also conveniently be studied by the spectrophotometric method developed by Kalckar 4 (as described by Schneider and Hogeboom~) ; this method is based on the decrease in the ultraviolet absorption spectrum which takes place during uric acid oxidation; although in this procedure the 02 tension and the temperature are less rigorously controlled, it is of value owing to its simplicity and the relatively small amounts of enzyme required. Purification Procedure

Highly purified preparations of uricase (Qo2 -- 2000 to 6000) can be obtained by the procedure originally described by Davidson 6 and later modified and improved by Holmberg, 7 but the final yield is small and not always reproducible. Thus, although the above procedure may be followed to obtain a highly purified enzyme, the following method (Leone s) is recommended when a Qo, value not exceeding 700 to 800 is aimed at and a good yield of enzyme is required. Step 1. Borate-Butanol Extraction of Ox Kidneys. Ox kidneys, which can be stored for several weeks in the frozen state without much loss of activity, are freed from adhering fat and tissue and passed through a H. Brown, J. Biol. Chem. 158, 601 (1945). 3W. R. Fearon, Biochem. J. 38, 399 (1944). 4H. M. Kalckar, J. Biol. Chem. 167, 461 (1947). 5 W. C. Schneider and G. H. Hogeboom, J. Biol. Chem. 195, 161 (1952). J. N. Davidson, Bioehem. J. $2, 1386 (1938); 36, 252 (1942). C. G. Holmberg, Biochem. J. 83, 1901 (1939). 8 E. Leone, Biochem. J. 54, 393 (1953).

[74]

URICASE

487

mechanical meat mincer. The mince is then homogenized for 5 minutes in a Waring blendor with 0.1 M borate buffer, pH 10, and n-butanol (300 ml. of borate buffer and 10 ml. of n-butanol to every 100 g. of mince)2 The borate is preheated to about 40 ° before homogenization, and the suspension is brought to pH 10 by the addition of 0.1 N NaOH. The whole suspension is incubated at 37 ° for 16 to 18 hours, cooled down, and centrifuged for 30 minutes at 3000 r.p.m.; a yellow, cloudy supernarant is obtained, which is decanted and preserved; more 0.1 M borate buffer, pH 10, is added to the residue to bring the mixture to the original volume; the suspension is well stirred and centrifuged for 30 minutes at 3000 r.p.m. This is repeated twice, and the four supernatants are combined. See the table for a summary of yield and increase in purity. Step 2. Calcium Phosphate Gel Treatment (Alkaline). To fraction 1, about one-fifth of its volume of calcium phosphate gel is added; the gel (prepared according to Keilin and Hartree 1°) should have a dry weight content of about 25 mg./ml. The mixture is centrifuged after 10 to 15 minutes. The supernatant is preserved, and the residual calcium phosphate cake is thrice extracted with small amounts of a one-fifth-saturated solution of (NH4)2SO4. The three (NH4)2SO4 extracts are added to the supernatant from the first centrifugation.

Step 3. (NH4)2S04 Precipitation and Further Purification by Dialysis. The concentration of (NH4)~SQ in fraction 2 is increased to 50% saturation by the addition of solid salt. The mixture, after some time in the cold, is centrifuged. The bulk of the supernatant fluid is siphoned off; a further period at 0 ° and repeated centrifugation may be needed to separate the (NH~)~SO4 precipitate. This is next suspended in distilled water, made up to a volume corresponding to about one-fifth of the original extract from step 2, and dialyzed for 12 to 20 hours at room temperature against running tap water, until a brown precipitate settles out in the cellophane bag, while the fluid portion becomes clear. The dialyzed preparation is centrifuged at about 5000 r.p.m. The precipitate is ground in a precooled mortar with ice-cold 0.1 M phosphate buffer, pH 7.3, and centrifuged in the cold at about 10,000 r.p.m. The clear, almost colorless 9 This proportion is the optimal one, since, a l t h o u g h it ensures a good Qoi value, it is still possible to perform the (NH4)2S04 precipitation directly on the centrifuged extracts, w i t h o u t a n y dialysis to remove n-butanol. However, if small a m o u n t s of a n enzyme preparation with a Qol value of a b o u t 70 to 100 are required, especially for analytical use, one can increase the b u t a n o l concentration up to 22 to 25 ml. per 100 g. of mince a n d 260 ml. of borate buffer. In this way after incubation a n d centrifugation a n extract is obtained which can either be used as such or can be dialyzed against r u n n i n g t a p water for 12 to 16 hours, if f u r t h e r t r e a t m e n t such as freeze-drying is intended. 10 D. Keilin a n d E. F. Hartree, Biochem. J. 49, 88 (1951).

488

[74]


phosphate extract is decanted, and the protein residue is ground with 0.1 M borate buffer, p H 10, and centrifuged at high speed, at room temperature, for a b o u t 10 minutes. The resulting extract, slightly opalescent but almost colorless, is preserved, and the borate extraction is repeated three times. The borate extracts are combined. Step ~. Calcium Phosphate Gel Treatment (Acid). Fraction 3 is cooled in an ice bath, and enough 0.5 N acetic acid is added, with continuous stirring, to give a faintly acid reaction (methyl red just pink). Next, calcium phosphate gel, 0.1 vol., is added, and the p H is adjusted again. After 10 to 15 minutes, the mixture is centrifuged. T h e s u p e r n a t a n t is decanted, and the calcium phosphate precipitate with the adsorbed enzyme is washed with distilled water, care being taken to maintain a weakly acid reaction. After centrifugation and washing, the precipitate is ground in a m o r t a r with a one-fifth-saturated solution of (NH4)2S04, at a neutral to slightly alkaline pH, and centrifuged again. The (NH4)2SO4 extraction of the calcium phosphate gel is repeated two or three times; the water-clear and almost colorless supernatants are combined. Step 5. (NH4)2S04 Precipitation and Dialysis. Fraction 4 is brought to half-saturation with (NH4)~S04, and the resulting precipitate is centrifuged down and washed in the centrifuge tubes with a half-saturated solution of (NH4):S04. The final precipitate is dissolved in 0.1 M borate buffer, p H 10, corresponding to no more than one-tenth of the volume of the extract from step 4, and dialyzed in the cold against three changes of 0.01 M borate buffer, p H 10. After 36 hours, a small a m o u n t of brownish precipitate is removed by centrifugation. SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. Borate-butanol extract 2. Extract after alkaline treatment with calcium phosphate gel 3. Borate extracts after (NH4)~SO4 precipitation and dialysis 4. Extract after acid treatment with calcium phosphate gel 5. Borate extract after second (NH4)2SO4 precipitation and dialysis

Total units, Dry Specific RecovTotal thou- weight, activity, ery,• volume, Units/ml. sands mg./ml. Qo~ % ml. 5000

252

1260

6.25

40

90

5000

227

1135

4.54

50

81

1600

420

672

1.40

300

48

1600

262

419

0.37

708

30

160

2530

405

3.10

816

29

With reference to the uricase content of the original suspension (1400 thousands of units).

[74]

URICASE

489

At all stages of purification, uricase activity remains unchanged for many weeks in alkaline extracts. At the first step of the purification procedure, satisfactory stability can also be achieved by freeze-drying the preparation.

Properties Specificity. Keilin and Hartree 1 have shown the specificity of uricase for uric acid; the enzyme is not active toward substituted uric acids, such as mono-, di-, and tri-methyl or -ethyl derivatives, or the corresponding riboside (Schulern). Activators and Inhibitors. Several substances have activating properties, like dithizone, phenanthroline, a,a'-dipyridyl, thiourea, and sodium diethyldithiocarbamate (Davidson6). It appears most probable that the activation is due to the metal-binding capacity of these substances; a similar mechanism is presumably involved in the activation by ethylenediaminetetraacetate (Leonel~). Cyanide is an uricase inhibitor, in 10-4 M concentration. The cyanide inhibition is reversible, 1 and full activity can be restored if cyanide is removed by dialysis. B Most metals, such as Cu, Mn, Zn, Co, Ni, and Fe, have an inhibitory effect. Effect of pH and Temperature. Optimum pH is 9.25; 1 45 ° is the optimum temperature (Ro 13). Nature of the Enzyme. The most highly purified preparation by Davidson 6 contained 0.10 to 0.20% Fe and 0.06 to 0.09% Zn, together with traces of other metals. Holmberg's 7 preparation had a content of 0.025 % of Fe and 0.13 % of Zn. Praetorius TM has reported a Zn content of less than 0.04% in a uricase preparation, purified according to Holmberg and dialyzed against BAL. The likelihood of uricase's being a metal-containing enzyme is strongly indicated by its reversible inhibition by cyanide, but the identity of the metal remains uncertain. Mahler, Baum, and Htibscher 15 have recently reported a new procedure for the purification of uricase. Starting with pig liver mitochondria and using alkaline extraction, isoelectric precipitation and ammonium sulfate fractionation, they have obtained a preparation homogeneous on ultracentrifugation and electrophoresis. There is a gradual increase of the copper content during the purification and assuming 1 Cu a t o m / M enzyme the observed Cu content of 0.055 per cent or the purest preparation leads to a molecular weight of 122000. H W. Schuler, Z. physiol. Chem. 208, 237 (1932). 12 E. Leone, unpublished observation. 13 K. Ro, J. Biochem. (Japan) 14, 361 (1931). 14 E. Praetorius, Biochim. et Biophys. Acta 2, 590 (1948). 16 H. R. Mahler, H. Baum, and G. Hiibscher, Federation Proc. 14, 249 (1955).

490


[75]

[75] Pyrimidine Oxidase and Related Enzymes By OSAMU HAYAISHI I. Uracil-Thymine Oxidase 1,2 Uracil + 1/~ O5 -* Barbituric Acid Thymine + ~ 05-~ 5-Methylbarbituric Acid

Assay Method Principle. Since the extinction of the oxidation products is approximately three times as great as that of the substrates, the rate of increase in optical density serves as the basis for activity measurement. Reagents 0.001 M uracil or thymine solution. 2.67 × 10-3 M methylene blue solution. 0.02 M Tris buffer, pH 8.5.

Procedure. The test system (at 22 to 25°), in a quartz cell having a 1-cm. light path, contains 0.3 ml. of substrate, 0.3 ml. of the methylene blue solution, 2.3 ml. of the Tris buffer, and 0.1 ml. of enzyme. Readings are taken at 2-minute intervals (at 225 mt~ in the case of uracil and at 270 mt~ in the case of thymine) in a Model DU Beckman spectrophotometer. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount producing a density increase of 0.100 during the first 10 minutes, and the specific activity is defined as units per milligram of protein. Protein is determined by the method of Lowry et al2 With one unit or less of 'enzyme, the reaction rate is linear for about 30 minutes. Proportionality is observed between rate and the amount of enzyme in the range of 0.5 to 5 units.

Purification Procedure A strain of Mycobacterium 4 is cultured in a medium containing thymine (0.1%), K2HPO4 (0.15%), KH~PO4 (0.05%), and MgSO4'7H20 1 O. Hayaishi and A. Kornberg, J. Am. Chem. Soc. 73, 2975 (1951). 2 O. Hayaishi and A. Kornberg, J. Biol. Chem. 197, 717 (1952). 8 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4 This strain, originally isolated from rabbit feces by Dr. Schatz, Dr. Savard, and Dr. Pintner, was classified and kindly furnished to the author by Dr. T. Stadtman. It is available at the Department of Microbiology, Washington University School of Medicine, St. Louis, Missouri.

[75]

P Y R I M I D I N E OXIDASE AND R E L A T E D ENZYMES

491

(0.02%). Large-scale cultures can be conveniently made in 20-1. glass carboys, each containing 10 1. of the medium at 26 ° for 40 hours with constant shaking. The inoculum is usually made by growing the cells in 100 ml. of the same medium for 24 hours at 26 °. The cells are harvested by centrifugation in a Sharples supercentrifuge and washed once with a 0.5% NaC1-0.5% KC1 solution, The yield is approximately 0.5 to 1.0 g. (wet weight) per liter of medium. The cells can be stored at - 1 0 ° without loss of activity for a period of at least six months. Cell-free extracts prepared by grinding the cells with three times their weight of alumina (Alcoa A-301) and extracting with Tris buffer (0.02 M, pH 9.0, ten times the weight of the wet cells) are treated with ammonium sulfate (24.5 g. per 100 ml. of extract). The precipitate is removed by centrifugation, and more ammonium sulfate is added to the supernatant (10.5 g. per 100 ml. of extract). The resulting precipitate, collected by centrifugation, is dissolved in Tris buffer (0.02 M, pH 9.0) to a volume corresponding to one-twentieth of that of the original extract. This fraction contains 35.6 units/ml, and 1.2 mg. of protein per milliliter. The specific activity of crude cell-free extracts is usually about 10.

Properties Specificity. Under the conditions described above, the enzyme does not act on the following pyrimidines: barbituric acid, isobarbituric acid, 5-methylbarbituric acid, 6-methyluracil, dihydrothymine, dihydrouracil, 2-thiouracil, 2-thio-5-methyluracil, or cytosine. Effect of pH. The optimum pH of the reaction for uracil oxidation is between 8.5 and 9.0; that for thymine oxidation is between 9.0 and 9.5. Below pH 8.0 and above pH 10.0, both activities are greatly decreased. The enzyme is most stable at about pH 9.0. There is no appreciable loss of activity for at least six months on storage at pH 9.0 at - 1 0 °. Substrate A~nity. The Michaelis constants are 0.35 X 10-4 and 1.31 X 10-4 M for thymine and uracil, respectively. Identity of Uracil and Thymine Oxidase. The fact that the ratio of the rate of thymine to uracil oxidation is almost identical in cell-free extracts from either uracil- or thymine-grown cells, or in the partially purified enzyme preparations, suggests the identity of the two oxidase activities. Further indication is provided by kinetic analysis of the competitive inhibitory action of uracil and thymine. The affinity of uracil for the enzyme is the same whether it is determined with uracil as a substrate (1.31 X 10-4 M) or as a competitive inhibitor of thymine oxidation (1.10 X 10-4 M); similar results are obtained with thymine.

492


[76]

II. Barbiturase 2 Barbituric Acid -t- 2H~O--~ Urea + Malonic Acid Assay Method

Principle. The method is based on the fact that the cleavage of the pyrimidine ring is accompanied by the disappearance of the ultraviolet absorption spectrum. Reagents 0.02 M sodium barbiturate solution. 0.2 M glycylglycine buffer, pH 8.3. 2.5% solution of crystalline bovine serum albumin. 0.02 M phosphate buffer, pH 7.0.

Procedure. The test system (22 to 25 °) contains, in 1.0 ml., 0.1 ml. of barbiturate solution, 0.3 ml. of glycylglycine buffer, 0.1 ml. of albumin solution, and 0.2 mi. of enzyme. At 10-minute intervals 0.1-ml. aliquots are removed and the reaction is stopped by dilution to 3.0 ml. with phosphate buffer. Readings are made at 255 m~ in a Model DU Beckman spectrophotometer. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount producing a density decrease of 0.100 in a 10 minute interval. Specific activity is expressed as units per milligram of protein. Protein is measured by the method of Lowry et al. 3 Purification Procedure

Step 1. Preparation of Crude Extracts. Mycobacteria are cultured under essentially the same conditions as for the preparation of pyrimidine oxidase with the exception that uracil (0.1%) and glucose (0.2%) provide the sole nitrogen and carbon sources. The inclusion of glucose increases the yield of cells to about 1.5 g. (wet weight) per liter of culture medium and also increases the yield and specific activity of the enzyme three- to fourfold. Cell-free extracts prepared by grinding cells with three times the weight of dry alumina and extracting with phosphate buffer (0.02 M, pH 6.65) are lyophilized and stored at --10 °. Five hundred milligrams of lyophilized powder (obtained from 5.3 1. of culture medium) is dissolved in 20 ml. of distilled water, and insoluble material is centrifuged off and discarded. Step 2. Protamine Treatment. To the supernatant are added 20 ml. of phosphate buffer (0.02 M, pH 7.0) and 4 ml. of protamine sulfate (10 mg./ ml.). After 3 minutes, the precipitate is collected by centrifugation and extracted with 20 ml. of 0.5 M K2HPO~. The opalescent extract is diluted

[75]

PYB,IMIDINE OXIDASE AND RELATED ENZYMES

493

with 60 ml. of water. Removal of the resulting precipitate by centrifugation yields a clear, colorless solution (protamine fraction). Although this step yields little or no purification on a protein basis, it succeeds in removing essentially all the nucleic acid which is present in the cell-free extract. Step 3. Dowex Column Chromatography. Ten milliliters of the protamine fraction is adsorbed on a Dowex-1 formate column (8 cm. × 1 sq. cm.) and eluted with 0.1 M K2HPO4 at a rate of 0.3 ml./min. The eluate is tested for both urease and barbiturase activity, and the fraction between 18 and 22 ml. is observed to possess the highest specific activity of barbiturase and practically no urease activity; the urease activity appears in a later eluate. TABLE I SUMMARY OF PURIFICATION PROCEDURE

Fraction Crude extract Protamine treatment Dowex chromatography Eluate between 13.5 and 36.0 ml. Eluate between 18 and 22.5 ml.

Total activity units

Specificactivity, units/mg, protein

234 173

12.9 13.9

168 56

77.0 94.0

Properties Specificity. There is no action, as judged spectrophotometrically, on the following compounds: 5-methylbarbituric acid, orotic acid, barbital, pentobarbital, 2qthiobarbituric acid, or isobarbituric acid. Effect of pH. The enzyme exhibits a fairly sharp optimum for activity between p H 8 and 9. The Michaelis Constant. The K~ is approximately 3.37 X 10 -3 M. III. DihydroSrotic Dehydrogenase 5 0rotic Acid + D P N H + H + ~ DihydroSrotic Acid + D P N

Assay Method Principle. D P N H is generated b y the addition of glucose dehydrogenase, glucose, and D P N . The reaction is followed b y the decrease in optical density at 280 m~ in the Beckman D U spectrophotometer. Orotic acid absorbs strongly at this wavelength, whereas the product, dihydro5rotic acid, has no absorption. 5I. Lieberman and A. Kornberg, Biochim. et Biophys. Acta 12, 223 (1953).

494


[75]

Reagents Glucose dehydrogenase, approximately 2500 units/ml., prepared according to the method of Strecker and Korkes.6 0.15 M MgCI:. 1 M potassium phosphate buffer, pH 6.1. 0.01 M sodium orotate. 0.1 M cysteine, pH 7.0. 0.001 M DPN. 1 M glucose.

Procedure. The test system contains 0.1 ml. of MgC12, 0.1 ml. of phosphate buffer, 0.2 ml. of glucose, 0.04 ml. of sodium orotate, 0.2 ml. of cysteine, 0.03 ml. of DPN, 250 units of glucose dehydrogenase, and the enzyme preparation in a volume of 3.0 ml. All the components except the glucose dehydrogenase are mixed and incubated at room temperature for 5 minutes. The glucose dehydrogenase is then added, and the rate of orotate removal is followed in the Beckman DU spectrophotometer by the decrease in optical density at 280 mp. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount producing an optical density decrease of 0.100 in a 6-minute interval. In general, not more than 4 units of activity is used for the assay. Specific unit is defined as units of activity per milligram of protein, as measured by the method of Lowry et al2 Under the conditions of the assay, the rate of orotate reduction is proportional to the amount of enzyme, and, in the absence of glucose, glucose dehydrogenase, or D P N , no removal of orotate is observed. Purification Procedure

Step 1. Preparation of the Cell-Free Extract. An obligate anaerobic bacterium, Zymobacterium oroticum, 7 is grown in a medium containing 2% tryptone, 0.05% Difco yeast extract, 0.2% orotic acid, and 0.05% sodium thioglycolate. For the preparation of stock test-tube cultures, the medium is adjusted to pH 7.0 with 1 M KOH prior to autoclaving (15 minutes at 15 pounds pressure). Anaerobic conditions are maintained with a pyrogallol-Na2CO3 seal. Large cultures are grown in Erlenmeyer flasks (1 to 6 1.) without an anaerobic seal. After autoclaving for 20 to 30 minutes at 15 pounds pressure, the medium is cooled, neutralized with a sterile 50% K2CO3 solution, and sterile water is added to the neck 6H. J. Strecker and S. Korkes, J. Biol. Chem. 196, 769 (1952) ; see Vol. I [44]. Available at the Department of Microbiology, Washington University School of Medicine, St. Louis~ Missouri.

[75]

PYRIMIDINE OXIDASE AND RELATED ENZYMES

495

of the flask. The inoculum (one or two fresh stock cultures) is added promptly. Growth appears to be complete in 16 to 20 hours at 30 ° . The cells are harvested in a Sharples supercentrifuge and resuspended in 0.01 M sodium orotate (7 ml./1, of culture), potassium phosphate buffer (1 M, pH 7.0, 0.4 re.l/1, of culture), and cysteine (0.1 M, pH 7.0, 0.4 ml./1, of culture). This cell suspension is incubated in vacuo at 26 ° for 20 minutes. More active extracts appear to be obtained when this step is included in the procedure. After centrifugation, the cells are suspended in ice-cold water (about 5 ml./1, of culture), and an aliquot of the suspension (about 6 ml.) is shaken with 6 g. of glass beads s (0.10 to 0.15 ram. in diameter) in a Mickle vibrator for 15 minutes at 2 °. The mixture is centrifuged in a Servall centrifuge (at ca. 10,000 X g), and the precipitate is washed once with cold water. The volume of the combined cell-free extract is adjusted to 10 ml./1, of culture. If the purification is not init ated at once, the extract is acidified to pH 6.0 with 2 M sodium acetate buffer (pH 6.0) and stored at - 1 0 °. Step 2. Protamine Fraction. Purification of the enzyme is carried out at 0 to 2 °. One hundred milliliters of freshly prepared cell-free extract is diluted with an equal volume of water, and 15 ml. of a 1% solution of protamine sulfate (Eli Lilly) is added with stirring. After 5 minutes, the precipitate is collected by centrifugation and the supernatant solution discarded. One hundred milliliters of sodium citrate buffer (0.5 M, pH 6.0) is added to the hard and difficultly soluble precipitate. After 12 to 24 hours, the softened precipitate is dissolved to a considerable extent by homogenization with a glass pestle, 200 ml. of water is added with stirring, and the resultant stringy precipitate is discarded after centrifugation. The supernatant solution (protamine fraction) is essentially free of nucleic acid as indicated by the ratio of optical densities at 280 and 260 m~ (0.98). Step 3. Ammonium Sulfate Fraction. To the protamine fraction is now added, with stirring, 100 g. of ammonium sulfate. After 5 minutes the precipitate is removed by centrifugation, and on further addition of 25.2 g. of ammonium sulfate to the supernatant solution another precipitate is formed. This precipitate is collected by centrifugation and dissolved in 120 ml. of water (ammonium sulfate fraction). Step 4. Acid Ammonium Sulfate Fraction. Thirty milliliters of sodium formate buffer (0.5 M, pit 4.2) and then 36 g. of ammonium sulfate are added to the ammonium sulfate fraction with stirring. After 5 minutes the precipitate is removed by centrifugation, 18 g. of ammonium sulfate is added to the supernatant solution, and the precipitate that forms is 8 Obtained from Minnesota Mining and Manufacturing Company, St. Paul, Minnesota.

496


[75]

collected and dissolved in 62 ml. of sodium acetate buffer (0.01 M, pH 6.0)(acid ammonium sulfate fraction). Purification at this stage is about eightfold with an over-all yield of about 40 %. Further purification of the enzyme (three- to fivefold) could be obtained by subjecting the acid ammonium sulfate fraction to column chromatography with Dowex-1 (formate, 2% cross-linked), and eluting with phosphate buffer. The yield was too variable to warrant inclusion of this step in the routine purification procedure. TABLE II SUI~I~IARY OF PURIFICATION PROCEDURE

Fraction Cell-free extract Protamine Ammonium sulfate Acid ammonium sulfate

Total activity, units

Specificactivity, units/rag, protein

4590 3180 2420 1850

22.8 26.5 84.1 175

Properties

Substrate Specificity. No activity and no inhibition of orotate reduction is observed with uracil, cytosine, 5-methylcytosine, or thymine. Coenzyme Specificity. With TPN, the reaction rate is less than 2 % of that observed with DPN, and no inhibitory effect on the reaction with DPN is observed. Activators and Inhibitors. Although Mg ion has little effect on the initial rate of the reaction, an effect is apparent when the reaction proceeds for longer periods. Thus with 0, 2 × 10-3 M, and 5 × 10-3 M Mg ++, the decreases in optical density at 280 mu in 6 minutes are found to be 0.225, 0.240, and 0.261, respectively; at 20 minutes the density decreases are 0.355, 0.426, and 0.503, respectively. Cysteine. Freshly prepared cell-free extracts show little or no stimulation on the addition of cysteine. The reaction rate with purified enzyme preparation, however, is increased up to twofold in the presence of 0.002 to 0.007 M cysteine. Larger amounts of cysteine seem to have an inhibitory effect. Effect of pH. The optimum pH for the reaction is around 6.5. At pH 5.5 and 7.8, the initial rates of reaction were 13 and 65%, respectively, of that at pH 6.5. Further Metabolism of Dihydroiirotic Acid 9 DihydroSrotie acid is further hydrolyzed by the action of an enzyme, dihydroSrotase, to yield ureidosuccinic acid. Ureidosuccinic acid is reey9I. Lieberman and A. Kornberg~ J. Biol. Chem. in press.

[76]

ADENOSINE PHOSPHOKINASE

497

clized to 5-(acetic acid)-hydantoin; the enzyme responsible for the latter reaction is referred to as 5-(acetic acid)-hydantoinase. Ureidosuccinic acid is also converted to aspartic acid, NH3, and CO2 by an enzyme system referred to as ureidosuccinase. All these reactions are shown to be reversible, except the last-mentioned one, but only ureidosuccinase has been purified.

[76] A d e n o s i n e P h o s p h o k i n a s e Adenosine + ATP--~ 5'-AMP + ADP 2-Aminoadenosine + ATP --~ 2-Amino-5'-AMP + ADP

By ARTHUR KORNBERG Assay Method Principle. The phosphorylation of the nucleoside is initiated with small quantities of ATP which are immediately regenerated by the action of pyruvate phosphokinase (added in large excess) on phosphopyruvate. In the presence of myokinase, this leads to the accumulation of the nucleoside mono-, di-, and triphosphates. adenosine kinase Adenosine + A T P - - - ~ 5'-AMP ~- ADP pyruvate phosphokinase Phosphopyruvate + ADP ) Pyruvate ~- ATP myokinase 5'-AMP + ATP ~ * 2 ADP The amount of ADP and ATP formed is estimated spectrophotometrically as follows: hexokinase ATP ~- glucose ) Glucose-6-P + ADP myokinase 2 ADP ~ ~ ATP + 5-AMP glueose-6-phosphate dehydrogenase Glueose-6-P q- TPN ) 6-Phosphogluconate + TPNH Thus, for every mole of ATP present 2 moles of TPN are reduced, and for every mole of ADP present 1 mole of TPN is reduced. The extent of TPN reduction can be measured at 340 mg; the molecular extinction coefficient for T P N H of 6.22 X 106 sq. cm./mole is employed. 1 1B. L. Itoreeker and A. Kornberg, J. Biol. Chem. 175, 385 (1948).

498


[76]

Reagents Succinate buffer (0.33 M, pH 6.0). MgC12 (0.1 M). MnC12 (0.02 M). Glutathione (0.16 M). ATP (0.002 M). Phosphopyruvate (0.06 M). Myokinase. 2 Pyruvate phosphokinase-(acetone I). 3

Procedure. The incubation mixture contained 0.1 ml. of succinate buffer, 0.05 ml. of adenosine, 0.01 ml. of MgC12, 0.01 ml. of MnC12, 0.02 ml. of glutathione, 0.01 ml. of ATP, 0.025 ml. of phosphopyruvate, 0.02 ml. of myokinase, 0.05 ml. of pyruvate phosphokinase, adenosine phosphokinase, and water to a final volume of 0.50 ml. After 20 minutes at 21 to 23 °, the incubation mixture was placed in a boiling water bath for 3 minutes, centrifuged, and an aliquot of the supernatant fluid analyzed for ADP and ATP. 3,4 Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which leads to the accumulation of 1 pM. of kinaselabile phosphate during the test period. (ADP contains 1 mole of kinaselabile phosphate, and ATP contains 2.) Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. ~ Purification Procedure 3

Autolyzates of dried baker's yeast and of several dried beer and ale yeasts were all found to be active. Twenty-five grams of dried lager beer yeast, which yielded the most active autolyzate, was suspended in 75 ml. of 0.1 M sodium bicarbonate and incubated for 6 hours at 34 °. The mixture was centrifuged and yielded approximately 40 ml. of clear yellow autolyzate (51.2 units/ml., 1.1 units/mg, of protein). All subsequent operations were performed at 2 °, unless otherwise specified. To 40 ml. of autolyzate were added 40 ml. of water, 20 ml. of 1 N acetic acid, and then 8 ml. of nucleic acid solution (Merck, 50 mg./ml., pH 5.0). After 5 minutes, 40 ml. of 2 N acetic acid was added, and the precipitate was removed by centrifugation. The pH of the clear supernatant, which was S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148, 117 (1943); see Vol. I I [99]. 3 A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 193, 481 (1951); see Vol. I [66]. 4 A. Kornberg, J. Biol. Chem. 182, 779 (1950). 50. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 198, 265 (1951); see also Vol. I I I [73].

[76]

ADENOSINE PHOSPHOKINASE

499

approximately 4.4, was raised to 6.3 by the addition of about 12 ml. of 2 N NaOH. Salmine sulfate (Lilly, 100 mg. in 4 ml.) was added; 5 minutes later the precipitate was removed by centrifugation, yielding 120 ml. of supernatant (12.6 units/ml., 3.8 units/mg, of protein). The pH was adjusted to 5.1 with 7.0 ml. of 2 N acetic acid, the solution cooled to - 0 . 5 °, and ethanol added with mechanical stirring. The temperature was maintained just above the freezing point during the early ethanol addition and at - 5 ° thereafter. The precipitates were centrifuged at - 5 °. Forty-five milliliters of 50% ethanol and then 7.0 ml. of zinc chloride (0.5 M) were added. After 5 minutes the precipitate was removed by centrifugation. To the supernatant was added 10 ml. of 50 % ethanol, and after 5 minutes the precipitate was again removed by centrifugation. The addition of 60 ml. of ethanol to the supernatant produced a precipitate which was collected by centrifugation and dissolved in citrate buffer (0.05 M, pH 6.2) to a volume of 50 ml. (14.5 units/ml., 13.1 units/mg, of protein). Lyophilization yielded 930 mg. of a white powder which, when stored in a vacuum desiccator over CaCl~ at 2 °, was stable for eight months or more. (In the liquid state at 2 ° about one-third of the activity was lost overnight.) The dry preparation was used throughout these studies. The optical density, at 280 mtL in a light path of 1 cm., of a purified enzyme solution containing 1 mg. of protein per milliliter was 6.4. The ratio of the density at 280 m~ to that at 260 m~ was 0.53, a value corresponding to that of nucleic acid. Efforts to remove this nucleic acid, as by the use of metal salts, protamine, and ion exchange resins, were unsuccessful. Properties Adenosine phosphokinase has also been demonstrated by Caputto 6 to be present in liver and kidney. Specificity. A large number of related nucleosides were found to be inactive under conditions which resulted in the phosphorylation of adenosine and 2-aminoadenosine (Table I). (In this experiment the amount of pyruvate released from phosphopyruvate was a measure of the ability of a nucleoside to serve as a phosphate acceptor.) These compounds, when tested at equimolar levels in the presence of adenosine, showed only slight inhibitory effects (13 to 30%) on the rate of adenosine phosphorylation. Other Properties. Rates of adenosine phosphorylation by ATP, estimated by adenosine removal, were maximal at the lowest substrate concentrations which could be conveniently tested. The levels were 5 X 10-4 M for ATP and 2 × 10-4 M for adenosine. I t is noteworthy that, 6 R. Caputto, J. Biol. Chem. 189, 801 (1951).

500

[76]


TABLE I 0.8 ~M. of test compound was used. The values are expressed as micromoles of pyruvate formed, corrected for the blank. Results with the test compounds are compared with those obtained with adenosine in the same experiment. (We are indebted to Dr. J. Davoll and Dr. G. B. Brown for many of the nucleosides used in this study.) Test compound 2-Oxy-9-~-D-ribofuranosyladenine (crotonoside) 9-~-D-2-Deoxyribofuranosyladenine 9-f~-D-Ribopyranosyladenine 9-f~-D-Glucopyranosyladenine 9-a-D-Arabofuranosyladenine 9-a-L-Arabofuranosyladenine 2,6-Diamino-9-~-D-ribofuranosylpurine 2,6-Diamino-9-~-D-xylofuranosylpurine 2-Chloro-9-f~-D-ribofuranosyladenine 2,8-Dichloro-9-~-D-ribofuranosyladenine 2-Methylthio-9-/~-D-ribofuranosyladenine 2-Acetamido-9-f~-D-ribofuranosyladenine 6-Oxy-9-fl-D-ribofuranosylpurine (inosine) 2-Amino-6-oxy-9-f~-D-ribofuranosylpurine (guanosine) Uridine Cytidine D-Ribose Yeast-adenylic acid

0.02, 0.00 0.00 0.00 0.00 0.00 0.22, 0.00 0.05, 0.03, 0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.00

0.00

0.26 0.05 ~ 0.01

Adenosine 1.52, 1.41 0.91 1.18 1.18 1.34 1.34 1.41 1.34 0.68, 1.28 0.68, 1.28 0.68 1.18

0.91 1.35 1.35 1.35 0.62 1.49

This value may be attributed to an impurity of adenosine; prolonged incubation resulted in no increase of this value. when adenosine phosphokinase a n d m y o k i n a s e , t h e r e m o v a l of c o m p l e t e d , d e p e n d i n g on w h i c h M g ++ w a s r e q u i r e d a n d w a s

is c o u p l e d w i t h p y r u v a t e p h o s p h o k i n a s e a d e n o s i n e or p h o s p h o p y r u v a t e is r e a d i l y s u b s t a n c e is l i m i t i n g . p a r t i a l l y r e p l a c e d b y M n ++ ( T a b l e I I ) .

TABLE I I Mg ++ REQUIREMENT OF ADENOSINE PHOSPHOKINASE (The results are in micromoles.) Mg ++,M X 10a Mn ++, M × 108 A-Adenosine

0.33 0.29

0.54

1.7 0.62

3.3 0.70

0.33

1.7

3.3

1.7 0.33

0.46

0.55

0.50

0.62

T h e p H o p t i m u m of t h e r e a c t i o n d e t e r m i n e d e i t h e r w i t h y e a s t a u t o l y z a t e s ( p h o s p h o p y r u v a t e as d o n o r ) or w i t h p u r i f i e d a d e n o s i n e p h o s p h o k i n a s e ( A T P as d o n o r ) w a s a p p r o x i m a t e l y 6.0; a b o u t 50 % of t h e a c t i v i t y w a s o b s e r v e d a t p H 5.0 a n d 7.0. C i t r a t e b u f f e r w a s i n h i b i t o r y .

[77]

NUCLEOTIDE SYNTHESIS BY TISSUE EXTRACTS

501

[77] Nucleotide Synthesis by Tissue Extracts Adenine -4- R-5-P -]- ATP--~

AMP -I- ADP -~- P or 2AMP -[- PP

By MURRAY SAFFRAN

The formation of 5-adenylic acid from adenine, ribose-5-phosphate (R-5-P), and ATP proceeds in two steps. In the first step, R-5-P is phosphorylated by ATP. In the second step, the phosphorylated R-5-P reacts with adenine to form 5-adenylic acid. Saffran and Scarano 1 have suggested that R-5-P is phosphorylated to ribose-l,5-diphosphate; Kornberg et al. 2 have evidence for the formation of 5-phosphoribosyl pyrophosphate.

Assay Method Principle. The incorporation of C14-adenine into the acid-soluble nucleotide fraction is determined by incubating the tissue preparation with 8-Cl~-adenine, R-5-P, and ATP, precipitating the proteins with perchloric acid, isolating the acid-soluble nucleotides by barium-alcohol precipitation, washing with neutral ethanol, and determining the radioactivity in the nucleotides. Reagents

8-C~4-adenine. Dissolve about 1 mg. (about l0 Gcounts per minute) of 8-C14-adenine'HC1 (prepared according to Clark and Kalckar 3) in 1 ml. of water. Store at - 2 0 °. 0.05 M Ribose-5-phosphate. Suspend about 250 rag. of Ba ribose5-phosphate t in 4 ml. of water, and dissolve with about 25 #1. of concentrated H2SO4. Spin down the undissolved solid, and carefully remove the supernatant. Neutralize the supernatant by careful addition of 4 M KOH. Analyze the solution for ribose by the orcinol reaction, s and dilute to 0.05 M. Store the solution at - 2 0 °. 0.05 M Na ATP. 6 10% perchloric acid. 1 M. Saffran a n d E. Scarano, Nature 172, 949 (1953). 2 A. Kornberg, I. Lieberman, and E S. Simms, J. Am. Chem. Soc. 76, 2027 (1954). 3 V. M. Clark a n d H. M. Kalckar, J. Chem. Soc. 1950, 1029. 4 Available commercially or m a y be easily prepared; see Vol. I I I [28]. 5 See Vol. I I I [13]. 6 Available commercially.

502


[77]

Concentrated ammonia. 25% solution of Ba(OAc)~. Ethanol brought to neutrality with concentrated ammonia. 1 M HC1. 0.25 saturated (NH4)2S04. Tissue extract. Active extracts of pigeon liver and breast muscle are consistently obtained by the following procedure. Homogenize the tissue in the cold in 10 vol. of a medium consisting of 0.13 M KC1, 0.015 M KH2PO4, and 0.01 M MgC12, brought to pH 7.2 with 1 M K O H (about 12.5 ml./1, of medium). Spin the homogenate at 80,000 X g 7 in the cold, and dialyze the particle-free supernatant against four changes of the homogenizing medium in the cold room. It is convenient to allow the extract to dialyze overnight against the last change of medium. Divide the dialyzed extract into small volumes, and freeze. Store at - 2 0 °. The frozen extracts retain activity for a few weeks. Procedure. Thaw the tissue extract. Pipet 1 ml. of the extract into a centrifuge tube containing 10 ul. of 8-C14-adenine, 50 ul. of 0.05 M R-5-P, and 50/~l. of 0.05 M ATP. Incubate at 37 ° for 1 or 2 hours. Chill the tubes in ice for a few minutes, and add 300 ul. of 10 % HC104. After 5 minutes, spin down the proteins. Remove a 1-ml. aliquot of the supernatant liquid to another centrifuge tube, and neutralize with concentrated NH~. Add an excess (100 ~l.) of 25% Ba(OAc)2 and 4 ml. of neutralized ethanol. Stir with a thin glass rod. Let stand in the cold for 10 minutes, then spin. Decant, and discard the supernatant liquid. Wash the precipitate twice with 2 ml. of neutralized ethanol, spinning the precipitate down well after each wash. Suspend the precipitate in 400 ul. of H20. Add 100 ul. of 1 M HC1 to dissolve the precipitate. Precipitate the Ba ++ with 50 ul. of 0.25 saturated (NH4)2SO4, testing the supernatant liquid with a trace of (NH4)2SO4 after spinning to ensure that all the Ba ++ is removed. Plate 100 ul. of the supernatant liquid, and determine the radioactivity. Purification Procedure

Scarano s has described the partial purification, by alkaline (NH4)2SO4 fractionation, of the enzyme that phosphorylates R-5-P. Kornberg et al." have reported the preparation of the enzymes involved in the synthesis of nucleotides. However, these methods of concentrating the activities of either the R-5-P-phosphorylating enzyme or of the nucleotide-forming enzyme have not been described in detail. Williams and Buchanan 9 have outlined procedures for concentrating the enzymes needed for the 7 A Spinco p r e p a r a t i v e ultracentrifuge was used. s E. Scarano, Nature 172, 951 (1953). 9 W. J. Williams a n d J. M. B u c h a n a n , J. Biol. Chem. 203, 583 (1953).

[77]

NUCLEOTIDE SYNTHESIS BY TISSUE EXTRACTS

503

incorporation of hypoxanthine into IMP, in the presence of R-5-P and ATP. These enzymes may or may not be identical with those that form other nucleotides. Their preparation is described as an example of the concentration of a nucleotide-forming system. The following scheme is taken from Williams and Buchanan2 Step 1. Preparation of Crude Extract. Decapitate and bleed several pigeons. Excise the livers quickly, chill in cracked ice, and free them from gross connective tissue. Mince the liver with scissors and measure the mince by displacement of homogenizing medium in a graduated cylinder. Homogenize 1 part of the mince in 1.5 parts of a medium consisting of 0.035 M sodium phosphate buffer (pH 7.4), 0.13 M KC1, 0.04 M KHCO~, and 0.01 M MgC12. Spin the homogenate for 30 minutes at approximately 100,000 X g. Remove the supernatant carefully into other vessels. The above steps are carried out in the cold, with ice-cold medium and chilled instruments and containers. Step P. Fractionation with Ethanol. Fraction 1. Chill the particle-free extract to 0 ° in a dry ice-acetone bath. Add slowly 90% ethanol to 15% concentration by volume. During the addition of the ethanol, maintain the temperature of the extract-ethanol mixture just above the freezing point by cooling in the dry ice-acetone bath. Spin off the precipitate (fraction 1) in the cold, and keep at - 1 5 ° until lyophilized. Step 3. Fractionation with Ethanol. Fraction 2. To a second aliquot of the pigeon liver extract prepared by step 1, add ethanol slowly, as in step 2, to a concentration of 20 %. Spin down the precipitate. Decant off the supernatant carefully into another container. Discard the precipitate. Increase the ethanol concentration of the supernatant to 45% by the slow addition of 90% ethanol. Spin down the precipitate (fraction 2), and keep at - 1 5 ° until lyophilized. During the addition of ethanol in this step, maintain the temperature of the mixture just above the freezing point, as in step 2. Step 4. Lyophilization, Storage, and Use. Dissolve each fraction in small quantities of distilled ~ater. Lyophilize the solutions, maintaining the apparatus at - 1 5 °. Keep the lyophilized powders at - 1 5 ° ; they are stable for at least several days. Just before use, dissolve the dried powder in the incubating medium in a volume equal to one-half the volume of the original pigeon liver extract.

Properties Specificity. The crude extract will form nucleotide from adenine, ATP, and R-5-P or R-l-P, hut the purified enzyme will not utilize the 1-ester. 2,s Ribose-l,5-diphosphate, made by the system phosphoglucomutase

Glucose-l,6-P2 + R-1-P ¢__

) R-1,5-P2 ~- glucose-6-P

504


Glucose-6-P + TPN

Zwischenferment

[78]

) 6-Phosphogluconic acid + T P N H + H +

will replace R-5-P and ATP in the dialyzed extract.1 The base specificity of the system has not been explored. Hypoxanthine,9 adenine, 1,2 and orotic acid 2 have been reported to be incorporated into nucleotides by similar systems. Activators and Inhibitors. The unfractionated system requires Mg ++ and P O t - - - for activity. The optimal concentration of Mg ++ is about 0.01 M. In the crude extract, the optimal concentration of adenine is about 0.002 M. Fluoride has no appreciable effect on the system. In the presence of low concentrations of R-5~P, adenosine phosphates decrease the rate of incorporation of adenine. Stability to Heat. The R-5-P phosphorylating enzyme is heat sensitive and is inactivated by heating at 60 ° for 5 minutes. The adenine-incorporating activity is much more resistant to this treatment. The activity of fraction 2 in the IMP-forming system also survives heating at 60 ° for 5 minutes. 1° Other properties of the IMP-forming enzymes have not been described. ~0E. D. Korn and J. M. Buchanan, Federation Proc. 12, 233 (1953).

[78] Some Methods for the Study of de Novo Synthesis of P u r i n e Nucleotides B y DAVID A. GOLDTHWAIT a n d G. ROBERT GREENBERG

A soluble enzyme system capable of synthesizing a purine nucleotide de novo can be found in pigeon liver extracts. A tentative scheme for the biosynthesis of inosine-5'-phosphate may be outlined as follows: P--O--P--O

H

\/

Ribose-5-P

C

i -[- glutamine

A+ TP

H CIO H

I ATP, glycine o I ]

I

HCOH

I

HC I

CH20--P 5-Phosphoribosylpyrophosphate (I)

H,C--NH2 CI

O

NH

\

Ribose-5-P

Glycinamide ribotide (II)

[78]

de Novo SYNTHESIS OF PURINE NUCLEOTIDES

H--C--N

H2C--NH--CHO

t

ATP

glutamine

O

~

--~

C

" 1-C unit"

/

NH

/

\

Ribose-5'-P Formylglycinamide ribotide (III)

NH2

0

/\

+C02

--* aspartic acid

\

Ribose-5'-P 5-Amino imidazole ribotide (IV) OH C

C--N

/

C--N

CH

I

II

C

H2N

505

C~N

N

%CH / \

ATP ) "1-C unit"

H2N Ribose-5'-P 5-Amino-4-imidazoleearboxamide ribotide (V)

C--N

% CH / HC C--N %/ \ N Ribose-5'-P Inosine-5'-phosphate (VI)

Discussion of the methods of studying purine biosynthesis will be divided into six sections: (1) over-all de novo synthesis of inosinic acid (VI) by extracts of an acetone powder of pigeon liver; (2) synthesis of formylglycinamide ribotide and glycinamide ribotide (II, III) by the same preparation; (3) isolation of 5-amino-4-imidazolecarboxamide riboside from E . coli cultures; (4) conversion of 5-amino-4-imidazolecarboxamide riboside to the ribotide by a yeast enzyme; (5) activation of the l-carbon unit as a folic acid derivative; and (6) transformylation of this active l-carbon unit to glycinamide ribotide (II) and to 5-amino-4imidazole-carboxamide ribotide (V). de Novo Synthesis of Inosinic Acid P r i n c i p l e . Because of the large endogenous production of purine nucleotides in this system, it is necessary to use the incorporation of a radioactive precursor as the index of de novo synthesis. One of the simplest methods of determining purine synthesis is the incorporation of C 14 formate into a non-acid-hydrolyzable fraction. This is measured by counting a dried aliquot of the reaction filtrate as an infinitely thin layer in a glass planchet. The synthesis of methionine and serine in this preparation under these conditions is negligible. If there is incorporation of C14-formate, the hypoxanthine moiety may be isolated either by crystallization of the silver nitrate salt or by ion exchange chromatography, and the C ~4 activity of the purified material determined. The molecule may

506


[78]

be degraded by sulfuric acid hydrolysis to obtain C-2 and C-8 together, and C-6, separately.

Reagents C14-sodium formate, 0.1 M. Specific activity approximately 10,000 counts/min./micromole as an infinitely thin layer using an endwindow (2 mg./cm. ~) counter. Glutamine, 0.2 M (Nutritional Biochemical Corp., Cleveland, Ohio). Ribose-5-phosphate, 0.05 M potassium salt. Dissolve 7 millimoles of barium ribose-5-phosphate (Schwarz Laboratories, Inc., N. Y.) in 15 ml. of water and 7 ml. of 1 N HC1 solution. Pass this through a Dowex 50 column (H + form, 1.2-cm. diameter X 10 cm.), and wash the column with 50 ml. of water. Take an aliquot for pentose analysis I with 5-AMP as a standard. The solution is neutralized with KOH and diluted with water to make a 0.05 M solution. 3-Phosphoglyceric acid, potassium salt, 0.14 M. Dissolve the commercial barium salt (Schwarz Laboratories, Inc., N. Y.) in hydrochloric acid solution. Remove the barium ion either by precipitation as BaSO4 or by passage through a Dowex 50 column (H + form), and neutralize the solution with KOH. ATP, 0.04 M (Pabst Laboratories, Milwaukee, Wis.), disodium salt neutralized with NaOH. MgCl~, 0.1 M. KHCO3, 1.0 M. Glycine, 0.1 M. DL-Homocysteine, 0.1 M (General Biochemical Inc., Chagrin Falls, Ohio). Dissolve in water immediately prior to use. Boiled extract of pigeon liver. Mince 25 g. of fresh pigeon liver, place immediately in 50 ml. of boiling distilled water in a small metal Waring blendor, and homogenize for 2 minutes while the blendor is heated from the side with a small burner. Centrifuge the homogenate for 30 minutes at 5000 X g, decant the supernatant fluid, and store at - 1 3 °. Procedure. Incubation mixture: C14-Na formate 0.05 ml., glutamine 0.05 ml., ribose-5-phosphate 0.10 ml., 3-phosphoglycerate 0.20 ml., ATP 0.05 ml., MgCl~ 0.05 ml., KHCO8 0.05 ml., glycine 0.05 ml., homoeysteine 0.05 ml., boiled extract 0.2 ml., enzyme 0.5 ml. Incubation time, 30 minutes at 38 °. The reaction is stopped by the addition of 0.5 ml. of 20% trichloroacetic acid. Approximately 0.2 micromole of C14-formate is incorporated into hypoxanthine. i W. M e j b a u m , Z. physiol. Chem. 258, 117 (1939).

[78]

de Yoyo

SYNTHESIS OF PURINE NUCLEOTIDES

Analytical Methods.

DETERMINATION

OF

MEASUREMENT OF FIXATION OF C14-FORMATE.

PURINE

507

SYNTHESIS

Hydrolyze0 . 2

BY

m l . of t h e

trichloroacetic acid filtrate by adding 0.1 ml. of 3 N HC1 and heating at 100 ° for 15 minutes. Dilute to 2.0 ml. with water, and pipet a 0.5-ml. aliquot into a glass planchet (Tracerlab, Inc.). Add 5 drops of 95% ethanol, and take carefully to dryness under an infrared lamp. DETERMINATION OF PURINE SYNTHESIS BY ISOLATION OF THE SILVER

aliquot of t r i c h l o r o a c e t i c acid filtrate, add 1.0 ml. of water and 0.8 ml. of 3 N HNO3, heat at 100 ° for 45 minutes, and then add 3 ml. of carrier hypoxanthine solution (5 mg./ml, in dilute nitric acid). Neutralize with KOH to a bromothymol blue end point, and add 1 drop of 0.1 N HNO3. After addition of 1.0 ml. of 0.2 M AgNO3, allow to stand at 0 ° with occasional stirring for 20 minutes. Centrifuge. Dissolve the precipitate in 7 ml. or less of 17% HNO3 [1 vol. of COliC. HN03 (69 to 71%) and 3 vol. of water] by heating at 100 °, filter off silver chloride at 100 °, and allow the silver nitrate salt of hypoxanthine to crystallize at 0 ° for 20 minutes. Centrifuge, and wash the precipitate once with 1.0 ml. of cold 17 % HNO3. Dissolve the precipitate in 5 ml. of 17% nitric acid at 100 °, and recrystallize at 0 °. Wash the precipitate again, as before. Repeat the recrystallization and washing. After the final recrystallization wash the precipitate twice with 1.0 ml. of cold water and suspend it in 1.5 ml. of water. Decompose the silver nitrate salt by vigorous bubbling of H2S through the suspension for 15 minutes. Aerate for 15 minutes, filter off the precipitate, and dilute the filtrate to a final volume of 5 ml. Pipet 1.0 ml. of this into a glass planchet, and dry for counting. Dilute a 0.1-ml. aliquot to 10 ml. with potassium phosphate buffer (0.02 M, pH 7.0), and read in the Beckman spectrophotometer at 248 m~. Calculate the concentration of hypoxanthine using a molecular extinction coefficient of 10,500. Then calculate the specific activity of the hypoxanthine which was counted as well as the total amount of hypoxanthine synthesized per reaction vessel. NITRATE SALT OF HYFOXANTHINE. TO a 0 . 5 - m l .

DETERMINATION OF PURINE SYNTHESIS BY ISOLATION OF HYPOXANTHINE ON D O W E X 5 0 COLUMNS AND DEGRADATION BY SULFURIC ACID

HYDROLYSIS. One milliliter of trichloroacetic acid filtrate plus 1 ml. of 2 N HC1 solution is heated at 100 ° for 30 minutes to hydrolyze the purine nucleotides. Five milligrams of carrier hypoxanthine (5 mg./ml, of 1.2 N HC1) is added, and the solution is put through a Dowex 50 column (H + form, 12% cross linkage, 50 to 100 mesh, 1.5-era. diameter × 7 cm.). The hypoxanthine is eluted with 1.2 N HC1 and appears in the fractions between 75 and 225 ml. These fractions are combined and diluted to 200 ml., a 1.0-ml. aliquot is neutralized, diluted to 10 ml., and the concentration of hypoxanthine is determined from the optical density at

508


[78]

248 mu. T o the hypoxanthine isolated by the above method, hypoxanthine carrier is added to make a total of 0.2 millimole. The solution is taken to dryness in the degradation vessel (Fig. 1, C) on a steam bath. A degradation train is set up as in Fig. 1. T o the dry hypoxanthine sample in vessel C, 100 ml. of sulfuric acid (7 Vol. of conc. H2S04 plus 3 Vol. of water) is added. The vessel is heated to 150 ° and aerated by the application of v a c u u m to the outlet on F for 15 minutes. The v a c u u m is then applied to the bubbler, I, the t e m p e r a t u r e is raised to 192 to 197 °, and

i

O

E

F

G

H

I

FIG. 1. Degradation apparatus. A, U tube containing Ascarite; B, mercury valve; C, reaction vessel; D, condenser; E, Cellosolve-dry ice trap or a bead tower containing

1 N H~SO4 solution; F, U tube containing anhydrous magnesium perchlorate; G, bubbler containing 3 ml. of 2 N COs-free NaOH, and drying tube with anhydrous magnesium perchlorate; H, U tube with iodic sulfate (2); I, bubbler similar to G. For other details of purine and pyrimidine determination, see E. D. Korn, Vol. IV [26]. aeration is continued at this t e m p e r a t u r e for 30 minutes. T h e bubblers are detached, weighed, and aliquots of the alkali solutions are taken for plating as BaC03 and counted. The C02 collected in bubbler G arises from the 6-carbon (yield, 0.2 millimole); the C02 in bubbler I is derived from the iodic sulfate ~ oxidation of the CO arising from carbons 2 and 8 (yield, 0.4 millimole). Experiments with isotopically labeled adenine 3 indicate t h a t this acid hydrolysis does not result in randomization of C-4 and C-6 similar to t h a t reported to occur with uric acid. 4 Preparation of Extract. Adult pigeons (Palmetto Pigeon Plant, Sumpter, S.C.) are decapitated, their livers removed, washed, chilled in ice water, and weighed. Fifty grams of liver is homogenized in 500 ml. of M. Schutze, Ber. 77B, 484 (1944).

a W. H. Marsh, Doctoral Thesis, Western Reserve University, 1951. 4 C. E. Dalgliesh and A. Neuberger, J. Chem. Soc. 1954, 3407.

[78]


509

acetone (Merck, reagent, - 1 3 °) in a Waring blendor at full speed for 30 seconds at - 13°. The acetone solution is filtered off through a Biichner funnel, and the cake is rehomogenized with 250 ml. of acetone for 15 seconds and again collected. The homogenization with 250 ml. of acetone is repeated once. The powder is partially dried on the Bfichner funnel and then placed i n vacuo at 4 ° over concentrated }I2SO4 for 1 day. The connective tissue is screened out, and the remaining acetone powder (12.5 g.) is stored i n vacuo at - 1 3 °. One gram of acetone powder is extracted with 10 ml. of 0.05 M K2HPO4 by slow stirring at 0 ° for 30 minutes. After centrifugation at 5000 × g for 20 minutes at 0 °, the supernatant solution is used immediately in the reaction mixture, or it may be lyophilized. The protein concentration varies between 30 and 40 mg./ml. Further details of the de novo synthesis of inosinic acid may be found in the references listed, s-7

Biosynthesis of Formylglycinamide Ribotide and Glycinamide Ribotide s,9 Principle. The formyl group of the formylglycinamide ribotide is readily hydrolyzed by heating with dilute acid. Therefore the synthesis of this compound can be estimated by the incorporation of C14-formate into an easily acid-hydrolyzable form. The biosynthesis of the formylglycinamide ribotide requires a folie acid derivative. Glycinamide ribotide accumulates if the extract is treated with Dowex 1 to remove the folic acid derivative. The formation of the glycinamide ribotide is estimated by employing glycine-l-C 14 as a precursor. The amide linkage in glycinamide ribotide is not affected by heating to 100° at pH 5.4. Therefore, on treatment of an aliquot of the reaction filtrate with ninhydrin, the residual glycine-l-C TM radioactivity is converted to C1402, and the remaining radioactivity represents synthesis of the glycinamide ribotide (or formylglycinamide ribotide). Procedures are also described for the separation of the ribotides on an anion exchange column and the preparation of crude barium salts of these compounds. Reagents

Glycine-l-C TM, 0.1 M, 15,000 to 20,000 eounts/min./micromole. 5 G. R. Greenberg, J. Biol. Chem. 190, 611 (1951). 6 j. M. Buchanan, J. Cell. Comp. Physiol. 38, Suppl. 1, 143 (1951). 7 j. M. Buchanan, B. Levenberg, J. G. Flaks and J. A. Gladner, "Amino Acid Metabolism," p. 743, Johns Hopkins Press, Baltimore, 1955. s D. A. Goldthwait~ R. A. Peabody, and G. R. Greenberg, J. Am. Chem. Soc. 76, 5258 (1954). 9 S. C. Hartman, B. Levenberg, and J. M. Buchanan, J. Am. Chem. Soc. 77, 501

(1955).

510


[78]

Leucovorin, 4 mg. of the calcium salt (American Cyanamide Co.) per milliliter of water. This solution is stable in the frozen state. Tetrahydrofolic acid. Prepare according to O'Dell et al. lo as modified by Jaenicke and Greenberg. ~1 Dissolve 3 mg. of the free acid of tetrahydrofolic acid in 1 ml. of 0.05 M KHCOa and 0.2 % E D T A under petroleum ether, and keep at 0 °. It is best to make the solution fresh for each assay. Other reagents are described in the section "De Novo Synthesis of Inosinic Acid." FORMYLGLYCINAMIDE RIBOTIDE. Procedure. Incubation mixture: C14-Na formate 0.05 ml., glutamine 0.05 ml., glycine 0.05 ml., ribose-5phosphate 0.10 ml., ATP 0.02 ml., 3-phosphoglycerate 0.10 ml., MgC12 0.05 ml., leucovorin or tetrahydrofolic acid 0.05 ml., enzyme 0.2 ml. Total volume, 0.7 ml. Incubation time, 30 minutes at 38 °. One-half milliliter of 20 % trichloroacetic acid is added to stop the reaction. When tetrahydrofolic acid is employed, it may be advantageous to carry out the reaction under anaerobic conditions. Analytical Methods. To determine the total formate incorporation, pipet 0.05 ml. of the trichloroacetic acid filtrate into a glass planchet, add 5 drops of water and 5 drops of ethanol, dry, and count. The nonacid-hydrolyzable formate (counts after hydrolysis) is determined as indicated in the section "De Novo Synthesis of Inosinic Acid." Micromoles of C ~4 activity in formylglycinamide ribotide Total counts/vessel--counts after hydrolysis/vessel Counts/micromole of formate It should be emphasized that there are other compounds derived from C~4-formate which comprise approximately 5 to 10% of the total C 14 activity incorporated. Under these conditions, 0.5 to 1 micromole of the ribo tide is synthesized. GLYCINAMIDE RIBOTIDE. Procedure. Incubation mixture: C14-glycine 0.05 ml., glutamine 0.05 ml., ribose-5-phosphate 0.10 ml., ATP 0.02 ml., 3-phosphoglycerate 0.10 ml., MgC12 0.05 ml., enzyme 0.2 ml. Final volume, 0.7 ml. Incubation time, 30 minutes at 38 °. The reaction is stopped by the addition of 0.5 ml. of 20% trichloroacetic acid. Analytical Method. A 0.1-ml. aliquot of the trichloroacetic acid filtrate is pipetted carefully into the bottom of a small test tube and neutralized with 1 N N a O H using bromothymol blue. One milliliter of 1.0 M potas10 B. L. O'DeU, J. M. Vandenbelt, E. S. Bloom, and J. J. Pfiffner, J. A m . Chem. Soc. 69, 250 (1947). 11Lo Jaenicke and G. R. Greenberg, unpublished studies.

[78]


511

sium phosphate buffer, pH 5.4, 0.1 ml. of carrier glycine (0.1 M), and 1.0 ml. of ninhydrin solution (30 mg./ml.) are added. A marble is placed on the tube, which is then heated at 100 ° for 30 minutes. After cooling, 1 drop of caprylic alcohol is added, and the mixture is aerated with C02 for 15 minutes and diluted to 10 ml. A 2-ml. aliquot is pipetted into a glass planchet, dried under an infrared light, and counted. The factor to correct to an infinitely thin layer in a glass planchet has been found to be approximately 2.4, but it should be determined in each laboratory by adding in a blank run a C144abeled compound which does not react with ninhydrin. All the glycine-l-C 14 incorporated into forms not decarboxylated by ninhydrin can be accounted for by ion exchange chromatography in the fractions corresponding to glycinamide ribotide and formylglycinamide ribotide as described in the next section. ISOLATION OF GLYCINAMIDE RIBOTIDE AND FORMYLGLYCINAMIDE RIBOTIDE. The ribotides are isolated in partially purified form by chromatography on a Dowex 1 column. The elution of the compounds is followed either by radioactivity or by analysis for pentose. For large-scale preparation of the ribotides the components of the reaction mixture are increased 200-fold. The trichloroacetic acid filtrate and combined washings (5% trichloroacetic acid) of the precipitate are extracted several times with ether to remove the trichloroacetic acid and to bring the pH to at least 4. The solution is then adjusted to pH 8.2 with KOH, and 0.5 M barium acetate is added until no further precipitation of barium-insoluble material occurs (approximately 8.5 ml.). The precipitate is redissolved in a minimum amount of dilute HC1 and adjusted to pH 8 to 9 by addition of NH4OH. The precipitate is separated and washed three times with a total of 45 ml. of H~O. The combined supernatant solution and washings are diluted threefold with water and passed through a Dowex 1 column (4% cross linkage, 250 to 400 mesh, formate form, 2-cm. diameter X 15 cm.). Glycine and an unidentified compound containing C 14 from glycine are eluted at pH 8.5 with 0.02 Mammonium formate. Glycinamide ribotide is eluted with 0.05 M ammonium formate at pH 6.5 between 1800 and 2400 ml. The formylglycinamide ribotide, synthesized on a comparable scale, is also isolated by column chromatography by a similar procedure. The column is washed successively with 700 ml. of 0.05 M ammonium formate at pH 6.5 and with 600 ml. of 0.05 M ammonium formate at pH 5.2, and then the compound is eluted with 0.05 M ammonium formate at pH 5.0 in two components between the volumes 650 ml. and 2300 ml. These components are considered to be isomers, but the exact nature of the isomerization is not known. After the solutions are concentrated by lyophilization, the barium salts of both ribotides may be prepared and

512


[78]

precipitated at pH 8.2 by the addition of 4 vol. of alcohol. It must be emphasized that the barium salts of both ribotides obtained by this procedure are only partially pure. Preparation of Extract. The enzyme system employed for formylglycinamide and glycinamide ribotide synthesis is prepared from the pigeon liver acetone powder extract (see Synthesis of IMP) as follows. The extract from 10 g. of acetone powder is passed through a Dowex 1 column (bicarbonate form, 4% cross linkage, 2.9-cm. diameter × 15 cm.) over a period of 2 hours, dialyzed overnight against 0.05 M K2HP04, and lyophilized. One hundred milligrams of lyophilized powder is dissolved in 1 ml. of water. Preparation of 5-Amino-4-imidazolecarboxamide Riboside 12,13

Principle. In E. coli under sulfonamide bacteriostasis the following sequence of reactions is considered to take place: sulfa

Glycine and other inhibition --~ 5-IRMP ) Purine ribonucleotides precursors I R + P~ Amino imidazolecarboxamide riboside (IR) accumulates in the culture medium and is isolated by adsorption on charcoal, purification on ion exchange columns, and crystallization. I R has been prepared by another procedure. TM Procedure. Escherichia coli, strain B, is carried on Difco nutrient agar slants and transferred approximately monthly. The glucose-salts 15 solution is prepared by dissolving the following compounds in 500 ml. of distilled water: NH4CI 0.5 g., (NH4)2S04 0.05 g., NaC1 0.1 g., MgCI~6H~O 0.1 g., Na2HPO4.7H~O 11.4 g. and KH2P04 3.0 g. The solution is autoclaved and an equal volume of an autoclaved solution of 0.8% glucose added. For small-scale studies 10 ml. of the glucose-salts solution is pipetted into 21 × 175-ram. tubes, the additions made, the tubes plugged with cotton and autoclaved for not more than a total of 10 minutes at 15 lb. The inoculum is grown in stationary culture for 16 hours at 37 °. A reading of 150 to 170 in the Klett colorimeter with a 540-mt~ filter represents normal growth. To each liter of the culture medium containing 12Abbreviations: IR, 5-amino-4-imidazolecarboxamide riboside; 5-IRMP, 5-amino4-imidazolecarboxamide-5'-phosphoriboside; EDTA, ethylenediaminetetraacetate; FAH,, tetrahydrofolie acid. xa G. R. Greenberg, Federation Proc. IS, 745 (1954). x4E. D. Korn, F. C. Charalampous, and J. M. Buchanan, J. Am. Chem. Soe. 75, 3610 (1953). x5j. Spizizen, J. C. Kenney, and B. Hampil, J. Bacteriol. 62, 323 (1951).

[78]

de Nolo SYNTHESIS OF PURINE NUCLEOTIDES

513

30 mg. of glycine and 112 mg. of sulfadiazine is added 40 ml. of inoculum. The incubation is carried out in stationary culture at 37 ° for 11 hours with 3 1. of medium per 12-1. Florence flask. At the end of the incubation the Klett colorimeter reading should be about 50 to 80. The procedure has been carried out in 200-1. lots in a large kettle with a liquid depth of about 18 inches. The following operations are performed at room temperature. T o each liter of medium is added 5 g. of Filter-Cel (Johns Mansville). After stirring, the suspension is filtered on W h a t m a n No. 1 paper. Analysis of the clear filtrate shows 80 to 100 micromoles of nonacetylatable, diazotizable amine per liter. An aliquot of the filtrate m a y be concentrated by lyophilization and chromatographed on paper to determine that the riboside is formed (see Table I). The carboxamide compounds are detected by diazotization on the paper. TABLE I PAPER CHROMATOGRAPHY OF I R

(RI value or centimeters from starting line)

IR I Inosine D-Ribose L-Arabinose

(1)

(2)

(3)

0.42 0.56 0.34 0.53 0.45

0.24 0.56 0.17 0.28 0.20

6.7 13.4 2.4 8.2 6.8

Solvents: (1) n-Butanol:/~,¢'-dihydroxyethyl ether:water (4:1:1) and in the presence of 1 M NH~OH. (2) Solvent 1 saturated by boric acid. (3) n-Butanol saturated by water and in the presence of 1 M NH4OH. [Solvent allowed to go beyond edge of paper (40 cm.) and therefore data are recorded in centimeters.] T o the filtrate Norit A is added (1 g./1.), and the mixture is stirred for 45 minutes. All the amine is adsorbed. The Norit A is collected and dried on a Bfichner funnel. The cake is extracted by shaking for 3 hours with ten times its weight of a mixture of ethanol:concentrated ammonium h y d r o x i d e : w a t e r in the volume proportions of 5:3:2. The charcoal is again collected on a Bfichner funnel and the extraction repeated two additional times. The combined filtrates are concentrated to an oil i n vacuo with a water pump. T o dissolve the oil, 10 ml. of water is added per liter of original medium, and the solution is brought to p H 10 to 11 by addition of ammonium hydroxide. The deep amber solution is passed through a Dowex 1 (formate form, 10% cross linkage) column. For 0.5 millimole of riboside a 1.2-cm. diameter × 7-cm. column is employed.

514


[78]

The riboside is washed through the column with water, while most of the dark-colored material is retained on the column. The eluted riboside solution is a light amber color. In some experiments small quantities of the ribotide can be recovered on the column by elution with 0.2 M ammonium formate at pH 4.18. The solution is concentrated .to an oil by drying from the frozen state. The residue is dissolved in 0.01 N HC1 (100 ml. per 0.5 millimole of riboside). The acidified solution is placed on a Dowex 50 (NH4 + form) column. Since the capacity of the resin for the riboside is low, a large column is necessary. For 0.5 millimole a 2-cm. diameter X 40-cm. column is used. The column is washed with a volume of 0.01 N HC1 equal to the volume of the acid solution of riboside and then with an equal volume of distilled water. The riboside is readily eluted from the column with 0.1 N NH4OH, and the compound is measured by the optical density at 267 m~. The solution of the riboside is reduced to a small volume in vacuo and then to dryness by lyophilization. This preparation is adequate for conversion to the ribotide. The residue is dissolved in a small amount of water (less than 10 ml.) by gentle heating in a water bath and transferred to a small beaker. The riboside crystallizes at 4 ° over a period of several days. Recrystallization is brought about by dissolving the compound in a minimal quantity of water, decolorizing if necessary by heating with a very small quantity of Darco G-60, and allowing the solution to stand at 4 ° for 48 hours or more. Crystallization may be hastened by freezing the solution and then bringing it to 4 °. The crystals show slight amber color. A yield of about 30% is obtained. Analytical Methods. Non-acetylatable, diazotizable amine is determined by the method of Ravel et al. 16 The riboside may be detected on paper chromatograms by the ultraviolet method, or the diazotizable amine may be detected by spraying with a fresh solution of 8 vol. of 0.2 N HNO3 and 1 vol. of 0.1% NAN02, after 5 minutes spraying with 0.5% ammonium sulfamate and then after 3 minutes with 0.1% N-(1naphthyl)ethylenediamine dihydrochloride. Care should be taken not to get the paper too wet in the process. Data on the chromatographic behavior of I R are shown in Table I. Conversion of 5-Amlno-4-imidazolecarboxamide Riboside to the 5'Phosphoriboside ATP + I R --* ADP + 5-IRMP Principle. The reaction is catalyzed by brewer's yeast. 5-IRMP is isolated from the reaction mixture by ion exchange chromatography. (5-IRMP has been prepared enzymatically from IMP. 17) 16 j . M. Ravel, R. E. E a k i n a n d W. Shive, J. Biol. Chem. 172, 67, 1948. 17 j . G. Flaks a n d J. M. B u c h a n a n , J. Am. Chem. Soc. 76, 2275 (1954).

[78]


515

Reagents Na-phosphoglycerate and Na4 ATP. See section "De novo synthesis of Inosinic Acid." Muscle enzyme fraction, is Procedure. The following additions are made to an Erlenmeyer flask: 15 ml. of yeast autolyzate (55 mg. of lyophilized powder per milliliter of water), 105 micromoles of 5-amino-4-imidazolecarboxamide riboside, 96 micromoles of MgC12, 168 micromoles of sodium-3-phosphoglycerate, 60 micromoles of Na4ATP, 6 mg. of muscle enzyme fraction, 600 micromoles of potassium phosphate buffer, pH 7.4, and water to a volume of 21 ml. The reaction mixture is incubated for 60 minutes at 38 °, cooled and transferred to a centrifuge tube, and the flask rinsed three times with 2 to 3 ml. of water. To the combined mixture 2 ml. of 20% trichloroacetic acid is added. After centrifugation the protein precipitate is washed four times with 5 ml. each of 1% trichloroacetic acid. The combined supernatant fractions (50 to 60 ml.) are extracted in a separatory funnel three times with 150- to 200-ml. portions of ether. The ether dissolved in the aqueous phase is aerated out with N2 gas. A saturated solution of Ba(OH)2 is added to pH of 8.5 or until no further precipitation occurs. The precipitate which contains the major part of the added ATP, and also PGA and inorganic phosphate, is collected by centrifugation, redissolved in 10 ml. of 0.1 M acetic acid, adjusted to pH 8.5, and the precipitate collected by centrifugation. This procedure is repeated once more, and the precipitate is discarded. The combined supernatant fractions (about 88 ml.) containing about 81 micromoles of diazotizable amine are passed through an ion exchange column. The column is prepared from Dowex 1 (acetate form, 4% cross linkage, 200 to 400 mesh) and has the dimensions of 1.4 cm. 2 by 23 cm. The solution is added slowly to the column which is then washed with water. Unreacted riboside is found in the water eluate. Elution of the 5'-phosphoriboside is effected by 0.20 M ammonium acetate, pH 4.18, at a flow rate of 1 to 2 ml./min. The first 150 ml. of eluate contains compounds having maxima at 275 mu and 280 m~. The phosphoriboside is eluted after about 1000 ml. It is characterized in the eluates by a maximum at 267 m~ and a ratio of optical densities at 267 m~/260 m~ of 1.08-1.11 and a ratio of 1.04-1.06 for 267 m~/272 mt~. Fractions having values outside these ranges are rejected. 5-AMP is eluted directly after the carboxamide ribotide. Ammonium acetate is removed by lyophilization. The process is repeated two times by redissolving the ribotide and the remaining ammonium acetate in a small volume of water. The ammonium salt of the ribotide kept as the frozen aqueous solution has been used for most of our studies of the enzymatic is S. Rather and A. Pappast J. Biol. Chem. 179, 1183 (1949).

516


[78]

f o r m a t i o n of inosinic acid. A yield of a b o u t 50 to 5 5 % of 5 - I R M P is obtained, based on the original I R . A b o u t 2 0 % of the u n r e a c t e d I R is recovered in the w a t e r wash. 5 - I R M P has been isolated as the calcium a n d b a r i u m salts. At p H 7, 5 - I R M P shows a m a x i m u m absorption at 268 m~ and a molecular extinction coefficient of 12,800. Assay of the reaction is m a d e as described under the p r e p a r a t i v e conditions with 0.5 ml. of the y e a s t e n z y m e and with the other reagents in proportion. T h e reaction time is 30 minutes. A known volume of the T C A filtrate is c h r o m a t o g r a p h e d directly with 77 % ethanol as a solvent. T h e 5 - I R M P is determined b y diazotization after elution f r o m the paper. Some c h r o m a t o g r a p h i c d a t a of 5 - I R M P are shown in T a b l e I I . TABLE II R/

VALUES AND RELATIVE MIGRATION RATES Of COMPOUNDSa

5-IRMP 5-AMP 5-IMP 3-AMP IR

5-IRMP

AND SOME RELATED

(1)

(2)

(3)

(4)

0.23 0.24 0.21 -0.45

0.76 0.65 0.81 0.56 0.60

12.5 11.9 7.5 14.9 30.2

0.51 0.34 0.58 ---

Numbers of lcss than 1 refer to R/values, and those greater than 1 to centimeters from starting line. In the latter case the solvent was allowed to flow off the end of the paper. The chromatography is descending unless otherwise stated. The solvents are in volume proportions: (1) 77% ethanol. (2) 5% K2HPO4 layered with isoamyl alcohol (ascending). (3) n-Butanoh 50% acetic acid, 1 : 1. (4) Saturated (NH4)~S04:0.2 M Na acetate (pH 5.9):isopropanol, 79:19:2 (ascending).

Preparation of Extract. T h e e n z y m e for converting the riboside to the ribotide is obtained f r o m washed, l o w - t e m p e r a t u r e dried y e a s t (AnheuserBusch). Brewer's y e a s t is autolyzed for 31//~ hours. 19 T h e a u t o l y z a t e is dialyzed a t 4 ° against running distilled w a t e r for 24 hours and lyophilized. This almost white powder is stable indefinitely a t 4 ° in a v a c u u m desiccator. Activation of a 1 - C a r b o n Unit T h e formylation of the purine precursors m a y be divided into two steps: (1) the activation of the 1-carbon unit f r o m f o r m a t e or from serine and (2) the t r a n s f o r m y l a t i o n to glycinamide ribotide or to 5 - I R M P . T h e e n z y m e source is the same in each case. I n these experiments t e t r a h y d r o folic acid is used as the cofactor. Boiled extracts contain a cofactor(s) 19A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 193, 481 (1951).

[78]

de Noyo SYNTHESIS OF PURINE NUCLEOTIDES

517

which is more stable than tetrahydrofolie acid. General references to studies on this problem are listed. 7,13,~7,20 REACTION OF FORMATE AND TETRAHYDROFOLATE (TETRAHYDROFOLATE

FORMYLASE). Principle. Pigeon liver extract catalyzes the over-all reaction :1 HCOOH ~- folate derivative ~- ATP Mg++

Formylfolate derivative ~ ADP ~ P~ The folate compound is the easily oxidizable tetrahydrofolic acid employed in substrate quantity, and the product has the properties of N ~°-formyltetrahydrofolic acid. The reaction is measured by the fixation of C14-formate into a nonacid-volatile form when tetrahydrofolic acid and ATP are present.

Reagents Tetrahydrofolic acid. See "Biosynthesis of Formylglycinamide Ribotide and Glycinamide Ribotide." Sodium formate-C TM. 20,000 counts/micromole as counted with a 2 m g . / ~ . 2 end-window counter in a glass planchet at infinite thinness. Enzyme. The enzyme is used as obtained after dialysis or by dissolving the lyophilized powder in water (p. 519).

Procedure. To a test tube in an ice bath add the following: 0.05 ml. of enzyme, 0.05 micromole of ATP, 0.7 micromole of PGA, 1.8 micromoles of Na formate-C TM, 10 micromoles of KHCO3, 3 micromoles of MgCl2, 1.25 micromoles of FAH4 (0.2 ml.), and water to a volume of 0.5 ml. About 1 cm. of petroleum ether is layered over the reaction mixture before the addition of FAH4. Incubate for 15 minutes at 37 °. Stop the reaction by addition of 1 ml. of 10% TCA. Pipet 0.05 to 0.1 ml. into a planchet, and measure C 14 as total formate fixation as described under "Biosynthesis of Formylglycinamide Ribotide and Glyeinamide Ribotide" (Analytical 5~ethod). Appropriate controls are carried out without FAH4 and at zero time. Calculation: Micromoles FAH4.C14HO Counts fixed X filtrate volume (ml.) Counts/micromole formate-C 14 X volume of sample (ml.) Approximately 0.2 micromole of FAH4.CHO is synthesized in 15 minutes per 0.05 ml. of enzyme. ~0W. Sakami, in "Amino Acid Metabolism," p. 658, Johns Hopkins Press, Baltimore, 1955; D. A. Goldthwait, R. A. Peabody, and G. R. Greenberg, ibid., p. 765.

518


[78]

The reaction may be assayed by measurement of FAH4 CHO spectrophotometrically (see below), but this must be carried out with another protein precipitant such as 4% HC104. Liberation of orthophosphate may also be measured with appropriate controls because of apyrase activity. In this case the reduced folic compounds are removed batchwise from the HC104 filtrate on Dowex 50 (H + form) exchanger, since these compounds interfere with the phosphate analysis. Paper chromatography of the reaction filtrate (prepared by acidification with TCA anaerobically) with 1 M formic acid as a solvent (Whatman No. 1 paper) yields a compound at Rs = 0.37 which corresponds both in R / a n d , after elution with acid, in its absorption spectrum to the NS-Nl°-imidazolinium derivative of formyltetrahydrofolic acid (ACF). This compound contains more than 90% of the C 14 activity; a small quantity is found in N~°-formylfolic acid, RI 0.69, which is derived by oxidation of the product. CON'VERSION OF H-CARBON OF SERINE TO CHOFAH4.11 Principle. Serine -[- FAH4 -b T P N --* T P N H ~- NI°-CHOFAH4 ~ glycine One glycine is formed per mole of N~°-CHOFAH4. It is not known whether adenine nucleotide and phosphate are involved. NI°-CHOFAH4 is measured by conversion to ACF. Procedure. To a test tube in an ice bath add the following under a layer of petroleum ether: 0.05 ml. of enzyme, 5 micromoles of L-serine, 0.1 micromole of TPN, 1.25 micromoles of FAH4, 0.3 micromole of ATP, 10 micromoles of inorganic phosphate, 3 micromoles of MgC12, and 2.5 micromoles of MnS04.15 The reaction volume is 0.5 ml. Time 15 minutes, temperature 38 °. The reaction is stopped by addition of 0.5 ml. of 4% HC104. The protein precipitate is centrifuged down and allowed to stand under petroleum ether for 30 minutes in order to convert the reaction product to ACF. An aliquot of the filtrate (0.1 to 0.2 ml.) is transferred to a 1-ml. Beckman cuvette of 1-cm. light path, water is added to a final volume of 1 ml., and the solution is layered with petroleum ether. A reading is made at 360 m~. An aliquot is measured in the same way at zero time along with other controls. The difference in the optical density represents ACF. A change in optical density of 22 per milliliter for a 1-cm. light path is equivalent to 1 micromole of ACF. About 0.1 to 0.2 micromole of FAH4"CHO is formed in this reaction in the time given. Conversion of 5-IRMP to IMP

Principle. Transformylation from N~°-formyltetrahydrofolic acid to 5 - I R M P is measured by the disappearance of the diazotizable amine. The NS-N~°-imidazolinium salt of formyltetrahydrofolic acid is used, as it is more easily obtained than Nl°-formyltetrahydrofolic acid and under

[78]

de ~OYO SYNTHESIS OF PURINE NUCLEOTIDES

519

anaerobic conditions at neutral p H is rapidly converted to the latter. The Nl°-derivative can be synthesized directly b y reduction of N l°formylfolic acid. l°,11 The conversion of 5 - I R M P to I M P can be measured in the over-all system in the presence of C~4-formate, an ATP-regenerating system, and catalytic quantities of FAH4 by measuring C14-formate fixation and the disappearance of diazotizable amine. When serine is the 1-carbon source in the presence of catalytic quantities of cofactor the disappearance of diazotizable amine is measured. These experiments can be carried out with glycinamide ribotide as the 1-carbon acceptor. Reagents

Anhydroleucovorin (anhydrocitrovorum, ACF) (Calco Chemical Division, American Cyanamide Co.). The compound can be prepared from leucovorin by acidification and crystallization. 21 Three milligrams of ACF is dissolved in 1 ml. of 0.05 M K H C 0 3 and 0.2% E D T A under petroleum ether. I R M P - 5 , ammonium, K or Na salt, 4 ~moles/ml. Procedure. Incubation mixture: 0.1 ml. of extract, 0.08 ml. of ACF solution, 0.03 ml. of 5-IRMP. The reaction is carried out under petroleum ether at 38 ° for 20 minutes. Stop with I ml. of 10% TCA. Analyze 0.5 ml. of filtrate for non-acetylatable, diazotizable amine. Appropriate controls are made with I R M P and without ACF and vice versa and with zero time values. Some diazotizable amine color is due to the folic acid compounds. Preparation of Extract. Pigeon liver is homogenized at 0 ° with 5 vol. of 0.25 M sucrose solution containing 0.1% E D T A and centrifuged at 80,000 X g for 30 minutes. The supernatant extract is frozen overnight, allowed to stand for 2 hours at room temperature, and any precipitate is removed by centrifugation. The fraction precipitating between 0.25 and 0.60 saturation with ammonium sulfate is dialyzed against 0.01 M KHCO3 overnight at 4 °. Ammonium sulfate solution is prepared as follows: 71.5 g. of ammonium sulfate plus 98 ml. of water at room temperature, brought to p H 7.0 with NaOH, and water added to bring to a total volume of 100 ml. of solvent. T h e extract is stable to freezing and thawing.22, 2a

21D. B. Cosulich, B. Roth, J. M.: Smith, Jr., M. E. Hultquist, and R. P. Parker, J. Am. Chem. Soc. 74, 3252 (1952) ; M. May, T. J. Barrios, F. L. Barger, M. Lansford, J. M. Ravel, G. L. Sutherland, and W. Shire, ibid. 78~ 3067 (1951). 22The structure of amino imidazole ribotide (IV in Figure 1) is based on studies by S. H. Love and J. S. Gots, J. Biol. Chem. 210, 395 (1955); B. Levenberg, S. C. Hartman, and J. M. Buchanan, Federation Proc. 14~ 243 (1955). ~3Dr. Lothar Jaenicke and Mr. Richard A. Peabody kindly provided the authors with some of the procedures described here.

[79]

ACID PROSTATIC PHOSPHATASE

523

[79] Acid Prostatic Phosphatase

By G~RHARDSCHMIDT Prostatic phosphomonoesterase was discovered in 1935 by Kutscher and his associates, 1-3 who traced the frequent appearance in human urine of considerable amounts of a phosphatase with a pH optimum in the range between 5 and 6 to the admixture of semen. A systematic examination of the tissues of the male urogenital tract resulted in the detection of very high concentrations of this phosphatase in the prostate gland. In comparison with the phosphatase content of this gland and its secretion, those found in the other tissues of the male urogenital system were negligible.1 Distribution. Normal prostate glands of adults, hypertrophic glands, primary carcinoma of the prostate and their metastases contain the enzyme in concentrations of similar orders of magnitude. 4-~ The enzyme is secreted in the seminal fluid. Its concentration in normal semen is always high and often of a similar range as that in the prostate gland. 7 Before puberty, prostate glands of man and of Rhesus monkeys contain only negligible amounts of acid phosphatase, s Injections of testosterone (but not of ovarian hormones) resulted in several hundredfold increases of the concentrations of the enzyme in the prostate glands of immature Rhesus monkeys. 8 Relatively much smaller concentrations of this enzyme were found in many other human and animal tissues. Skeletal muscle and heart, however, are practically devoid of acid phosphatase. So far, the abundance of this enzyme in prostatic tissue appears to be a characteristic property of man and of monkeys. The prostate glands of dog, bull, ram, and rat were found to contain only small amounts of the enzyme2 ,s Very considerable amounts of acid phosphatase were found, however, in the preputial glands of rats 9 and in the seminal vesicles of guinea pigs. 1° The concentration of acid phosphatase in rat preputial glands did not increase on injections of testosterone, but the total yield of phosphatase 1 W. Kutscher and H. Wolberg, Z. physiol. Chem. 256, 237 (1935). 2 W. Kutscher and A. WSrner, Z. physiol. Chem. 239, 109 (1936). 3 W. Kutscher and J. Pany, Z. physiol. Chem. 255~ 169 (1938). 4 j. Fischmann, H. A. Chamberlin, R. Cubiles, and G. Schmidt, J. Urol. 59, 1194 (1948). 5 j. D. Fergusson, Lancet 251, 551 (1946). 6 A. B. Gutman and E. B. Gutman, J. Clin. Invest. 17, 473 (1938). 7 A B. Gutman and E. B. Gutman, Endocrinology 28, 115 (1941). 8 A. B. Gutman and E. B. Gutman, Proc. Soc. Exptl. Biol. Med. 41, 277 (1939). 9 A. B. Gutman and E. B. Gutman, Proc. Soc. Exptl. Biol. Med. 39, 528 (1938). ~0 H. A. Bern and R. S. Levy, Am. J. Anat. 90, 131 (1952).

524

ENZYMES I N PHOSPHATE METABOLISM

[79]

from the p r e p u t i u m was much higher after such injections, owing to the enhanced growth of glandular tissue. N o r m a l serum contains only v e r y small amounts of prostatic acid phosphatase; in serum of patients suffering from carcinoma of the prostate, elevated values of the enzyme are frequently encountered, whereas no such elevations occur in cases of benign h y p e r t r o p h y of the prostate gland. Determinations of this enzyme in serum are i m p o r t a n t for the diagnosis of prostate carcinoma, as well as for the evaluation of therapeutic measures. 11 I t is not yet clear whether an appreciable elevation can be caused b y a beginning prostate carcinoma. Specificity22

According to our present knowledge, acid prostatic phosphatase is a phosphomonoesterase in respect to the known phosphoric acid esters of living organisms, a-L-Glycerylphosphorylcholine, a-L-glycerylphosphorylethanolamine, and a-L-glycerylphosphorylserine as well as the phospholipids and the nonterminal phosphoryl groups of polynucleotides are not hydrolyzed b y the enzyme. TM Among synthetic diesters of phosphoric acid, diphenylphosphate is resistant to prostatic phosphatase, but the corresponding paranitro derivative, bis-p-nitrophenylphosphate, is hydrolyzed at a considerable rate b y the enzyme. Cohn and Volkin 14 reported t h a t another phosphatase which acts as a specific phosphomonoesterase toward the phosphoric acid esters previously examined, namely barley 3'-nucleotide phosphatase, hydrolyzes di(dinitrophenyl)phosphate. I t is conceivable t h a t nitrophenyl diesters of phosphoric acid behave exceptionally toward phosphomonoesterases because of the enhancing influence of the nitro groups on the dissociation of the hydroxyl group. Furthermore, not all phosphoric acid monoesters are substrates of prostatic phosphatase, and those which are hydrolyzed are cleaved at very different rates. 2'- and 3'-Nucleotides are hydrolyzed at faster rates than any other biological phosphoric acid monoesters. T h e hydrolysis rate of monophenylphosphate is similar to t h a t of the 2'- and 3'-nucleotides. 2'- and 3'-Adenylic acid are hydrolyzed at very similar rates; this is also the case for the 2'- and 3'-uridylic acids. Adenosine-5'-phosphate, 11A detailed discussion of the clinical application of phosphatase determinations in serum is beyond the scope of this chapter. References pertinent to this field will be found in the papers quoted in footnotes 4 and 21. 1~Some of the data on specificity of acid prostatic phosphatase are based on unpublished observations of G. Schmidt, K. Seraidarian, M. J. Bessman, and L. M. Greenbaum. is G. Schmidt, R. Cubiles, N. ZSllner, L. Hecht~ N. Strickler, K. Seraidarian, M. Seraidarian, and S. J. Thannhauser, J. Biol. Chem. 192, 715 (1951). 14W. E. Cohn and E. Volkin, J. Biol. Chem. 203, 319 (1953).

[79]


525

ribose-5-phosphate, and both glycerophosphates are hydrolyzed approximately at one-third of the rate of the 2 ~- and 3'-nucleotides. Robison ester is hydrolyzed at least five times more slowly than these nucleotides, and fructose diphosphate is completely resistant toward prostatic phosphatase. Phosphorylcholine, phosphorylethanolamine, and phosphorylserine are rapidly hydrolyzed. The rate of hydrolysis of ATP is at least thirty times slower than that of adenosine-3'-monophosphate. Tile hydrolyses of monophenylphosphate and of the 2'- and 3'-nucleotides are unimolecular reactions at least to a degree of hydrolysis of 50 %. For the other esters, the rates fall more rapidly even at early phases of hydrolysis.

Behavior of Prostatic Phosphatase toward High-Molecular Phosphorus Compounds. Owing to the growing importance of methods for end-group determinations and to the lack of a convenient chemical method for the differentiation of primary and secondary phosphoryl groups, a pure monophosphoesterase would be a highly valuable tool for the analysis of the structure of nucleic acids and phosphoproteins. The preceding paragraphs demonstrate that prostatic phosphatase has the requirements for such a tool to a limited extent--limited because an ideal end-group reagent should hydrolyze all phosphomonoester groups and none of the phosphodiester groups. Prostatic phosphatase, as stated before, is practically inactive toward the phosphomonoester groups of fructose diphosphate and hydrolyzes glucose-6-phosphate so slowly that its complete cleavage would require unreasonably long digestion times. On the other hand, it hydrolyzes nitrophenyl diesters of phosphoric acid. These observations add weight to the argument that in a substrate such as ribonucleic acid the behavior of some phosphoryl groups against phosphatase might be determined by structural conditions other than their terminal or nonterminal positions. Conclusions pertinent to the structure of the substrate must be based on the analysis of the organic fragments as well as on the amount of hydrolyzable or resistant phosphoryl groups. Whenever feasible, the interpretation of phosphatase-labile phosphoryl groups as phosphomonoester groups should be verified by titration. If the inorganic phosphate formed by prostatic phosphatase originates exclusively from phosphomonoester groups, no additional acidic groups appear in the range between pH 5 and pH 8.5. The only change of the titration curve during the titration is a slight displacement toward the alkaline side, owing to the higher pK~ of inorganic phosphoric acid in comparison to that of phosphoric acid esters. Any increase of the amount of acidic groups within the range mentioned indicates the transformation of phosphodiester groups to phosphomonoester groups or to inorganic phosphate.

526

ENZYMES IN PHOSPHATE METABOLISM

[79]

1. NUCLEIC ACIDS. Deoxyribonucleic acid prepared by any modification of E. Hammarsten's procedure is practically resistant to prostatic phosphatase. This is in agreement with the negligible amounts of terminal secondary phosphoryl groups present in highly polymerized DNA molecules. Ribonucleic acid, prepared by mild procedures in the laboratory, is slowly hydrolyzed by prostatic phosphatase until approximately 10% 13,1s,16 of its total phosphorus is liberated as inorganic phosphate. Continuation of the incubation for several days, however, results in an additional but much slower formation of inorganic phosphate which finally may amount to 40 % of the total phosphorus. This is largely due to the presence of small amounts of ribonuclease in all preparations of prostatic phosphatase available at present. Although these preparations are very useful as end-group reagents for the polynucleotide mixtures of exhaustive ribonuclease digests or for ribonuclease resistant oligonucleotides, they cannot be applied as end-group reagents for ribonucleic acids. 2. PHOSPHOPROTEINS. The action of phosphatases on the phosphoryl groups of phosphoproteins is of interest for the elucidation of the structural significance of these groups. It is obvious that phosphatase preparations used for the study of the enzymatic dephosphorylation of phosphoproteins must be carefully checked for the absence even of small amounts of proteolytic enzymes. This is particularly important in view of the fact that prolonged incubation periods are usually necessary in such experiments. Ovalbumin. Perlmann iv found that 46% of the phosphoryl groups of the ovalbumin component A1 (which has a higher electrophoretic mobility than the second component A2) are hydrolyzed by prostatic phosphatase. The ovalbumin A1 is transformed by the action of this enzyme to a protein of a lower electrophoretic mobility which resembles that of A~. Casein. Casein was fraetionated by Warner is into two components, a-casein and/~-casein. The solubility properties ~9 and the electrophoretic behavior 2° of casein suggest an even more complex composition of the mixture represented by ordinary casein preparations. According to Perlmann, 2~,~ 42% of the phosphoryl groups of a-casein are hydrolyzable by 15G. Schmidt, R. Cubiles, and S. J. Thannhauser, J. Cellular Comp. Physiol. 88, Suppl. 1, 61 (1951). 16R. Markham and J. D. Smith, Biochem. J. 52, 565 (1952). 1TG. E. Perlmann, J. Gen. Physiol. 35, 711 (1952). 18R. C. Warner, J. Am. Chem. Soc. 66, 1725 (1944). 19 K. LinderstrCm-Lang,Compt. rend. tray lab. Carlsberg 17, No. 9 (1929). 200. Mellander, Biochem. Z. 800, 240 (1939). 21G. E. Perlmann, J. Am. Chem. Soc. 74, 3191 (1952). 22G. E. Perlmann, in " A Symposium on Phosphorus Metabolism" (W. D. McElroy and B. Glass~eds.), Vol. 2, p. 167, Johns Hopk~na Press~ Baltimore, 1952.

[79]


527

prostatic phosphatase, whereas E-casein is resistant to this enzyme. Additions of f~-casein to solutions of a-casein inhibit the dephosphorylation of the latter by prostatic phosphatase. The question as to whether the phosphatase-resistant groups of phosphoproteins are diesterified requires further investigation. p H Optimum. The pH optimum of acid prostatic phosphatase is in the region between 5.3 and 5.6 for most substrates; Lundquist found, however, that the hydrolysis of calcium phosphocholine by prostate phosphatase has a pH optimum of 6.5. 23 Isoelectric Point. The isoelectric point of acid prostatic phosphatase is as pH 4.4. 3 Michaelis-Menten Constants. The Michaelis-Menten constants of acid prostatic phosphatase were determined by M. Seraidarian 24 for the hydrolysis of the following phosphoric acid esters: a-glycerophosphate, 2.96 X 10-3; ~-glycerophosphate, 2.1 X 10-3 (Ohlmeyer2~ found a Km value of 4.7 X 10-3 for the hydrolysis of f~-glycerophosphate at pH 4.5) ; yeast adenylic acid (mixture of 2'- and 3'-adenosinephosphoric acid), 2.2 X 10-3; yeast uridylic acid (mixture of 2'- and 3'-uridine phosphates), 2.3 X 10-3. Inhibitors. In its behavior to some enzyme inhibitors, prostatic phosphatase differs sharply from some alkaline phosphatase, such as intestinal phosphatase. Fluorides, which have no effect on alkaline phosphatase at low concentration, inhibit prostatic phosphatase completely at 0.01 M concentration. 2 On the other hand, cyanides, cysteine, and hydrogen sulfide, which are powerful inhibitors of alkaline phosphatase, are without appreciable influence on prostatic phosphatase. 2 Kutscher and WSrner 2 found strong and irreversible inhibitory affects of many "narcotics" such as alcohols and urethans on acid prostatic phosphatase. The use of the comparatively strong inhibitory effect of ethyl alcohol on prostatic phosphatase has been suggested as a means for the differentiation of acid prostatic phosphatase of serum from other acid phospharases (e.g., erythrocyte phosphatase)36 At present, however, the practical application of this principle for clinical tests is still in the experimental stage. In a comparative study on the acid phosphatases of the human prostate gland and of human erythrocytes Abul-Fadl and I(ing 27 reported that the former was specifically and practically completely inhibited by 25I. Lundquist, Acta Scan& Physiol. 14, 263 (1947). 24M. Seraidarian, Thesis, Science Faculty, Tufts College, 1952. 25p. Ohlmeyer, Z. physiol. Chem. 282, 1 (1945). 2sF. G. Herbert, Quart. J. Med. 39, 221 (1946). ~7M. A. M. Abul-Fadl and E. J. King, Biochem. d. 45, 51 (1949).

528


[79]

0.02 M sodium L-tartrate but not appreciably influenced by 0.5% formaldehyde. The characteristic behavior of prostatic phosphatase toward both these substances appears to be promising as a criterion regarding the prostatic origin of the acid phosphatases of serum in clinical cases. 28-30 Magnesium ions are without appreciable affect on prostatic phosphatase. 2 Prostatic phosphatase is completely inactivated by heating its solutions at neutral or weakly acid solutions at 60 ° during 5 minutes. It is unstable at room temperature, even at slightly alkaline pH ranges as well as below pH 5, but it can be kept for many months at neutral or weakly acid reaction (pH 6) in the refrigerator. The enzyme is usually inactivated by precipitation with alcohol or acetone. It has been lyophilized in active form. E n z y m e Units and Activity Determinations. Schmidt et al. 1~ defined as one unit of acid prostate phosphatase the amount of enzyme which forms 0.1 mg. of inorganic phosphorus within 15 minutes at 37 ° from a solution of " y e a s t " adenylic acid (Schwarz Laboratories; the substance is a mixture of the 2'- and 3'-adenylic acids). The incubation mixture contains 50 mg. of sodium adenylate in 10 ml. of 0.1 M sodium acetate buffer (pH 5.6) and 1 ml. of the enzyme solution. One gram of moist prostate tissue contains approximately 1500 enzyme units; for activity determination, 1 ml. of a hundredfold diluted stock solution (obtained by homogenizing the glands in 5 vol. of water) is usually a suitable amount. Phosphatase values in serum are usually expressed in King-Armstrong units per milliliter of serum. 4-9 One King-Armstrong unit is the amount of phosphatase which liberates 1 mg. of phenol from sodium phenyl phosphate at 37 ° within 1 hour; the other conditions of the hydrolysis (nature of buffer system, total volume) vary in different investigations. A procedure for phosphatase determinations in serum with sodium glycerophosphate as substrate has been described by Shinowara et al. ~1 Huggins and Talaley 3: described an assay method in which phenolphthalein phosphate was used as substrate. This substrate can be used only in comparatively low concentrations (0.3 X 10-4 M) because it inhibits the enzyme in higher concentrations. This is a serious disadvantage for kinetic studies. The use of nitrophenylphosphates as substrates for acid phosphatase in clinical determinations in serum might be adapted for clinical purposes in the future, but no reference as to their suitability can be given at present. ~8 W H. Fishman and F. Lerner, J. Biol. Chem. 200, 89 (1953). ~9 W. H Fishman, R. M. Dart, C. D. Bonner, W. F. Leadbetter, F. Lerner, and F. Homburger, J. Clin. Invest. 23, 1034 (1953). 90 E. P. Kintner, J. Lab. Clin. Med. 37, 637 (1951). 31 G. Y. Shinowara, L. N. Tones, and H. L. Reinhart, J. Biol. Chem. 142, 921 (1942). a, C. Huggins and P. Talaley, J. Biol. Chem. 1§9, 398 (1945).

[79]


529

Preparation of Purified Solutions of Prostatic Phosphatase. Prostate glands obtained by surgical enucleation (material obtained by transurethral cauterization is usually inactive) are used fresh or stored in a deepfreeze in which the full activity is preserved for years. The thawed glands are cut into small pieces with scissors and homogenized in a Waring blendor for 2 minutes. After addition of a few drops of toluene, the suspension is kept overnight in the refrigerator. The suspension is centrifuged, and the turbid supernatant is dialyzed overnight against distilled water in the cold room. To the strongly opalescent solution is added N acetic acid dropwise until a copious flocculent precipitate forms. Great caution during the acidification is essential for the success of this step during which the pH of the solution must not decrease below 5.5. This pH is considerably higher than that of the isoelectric point of prostatic phosphatase which was found to be at pH 4.4 by Kutscher and Pany2 Between pH 5.5 and 6.5, no loss of activity occurs despite the fact that a slight excess of acidity beyond the critical value of pH 5.5 results in the immediate and complete loss of the enzymatic activity. After centrifugation, a clear and usually almost colorless supernatant is obtained. This solution usually contains 300 to 400 units of the enzyme per millil i t e r - a n activity which is sufficiently high for the use of such enzyme solutions as a reagent of secondary phosphoryl groups. The solutions can be concentrated by precipitation in 0.9 saturated ammonium sulfate solution and by dissolving the precipitate in a small volume of water and subsequent removal of the salt by dialysis against distilled water in the cold. Solutions of prostatic phosphatase can be stored in the refrigerator in the presence of some toluene for many months without loss of activity. They are practically free of purine or pyrimidine deaminases, nucleotidases, phosphodiesterases, 5'-nucleotidase, or proteolytic enzymes, but they contain small amounts of heat-stable ribonuclease. So far, it has not been possible to remove the latter contamination. Further purification of the enzyme can be achieved by adsorbing contaminations on suspensions of aluminum hydroxide which is added dropwise under stirring. Prostatic phosphatase has very little affinity to this adsorbent, and it is easy to obtain in this way colorless solutions of the enzyme without appreciable loss of activity. A similar procedure for the purification of acid prostatic phosphatase was recently described by Derow and Davidson2 3 The behavior of acid prostatic phosphatase toward some reagents used for the purification of enzymes has recently been studied by London and Hudson2 4 In agreement with the observations of Schmidt, they 83 M. A. Derow a n d M. M. Davison, Science 118, 247 (1953). 34 M. London a n d P. B. Hudson, Arch. Biochem. and Biophys. 46, 141 (1953).

530


[80]

found that the enzyme (similarly to the behavior of alkaline intestinal phosphatase) is not appreciably adsorbed on aluminum hydroxide gels. It is adsorbed on kaolin or Fuller's earth at pH 4.5 when these adsorbents are applied in relatively large quantities (25 rag. per 28 mg. of protein). A considerable part of the activity can be recovered from the adsorbate by elution with citrate buffers of pH 7.0.

[80]

Intestinal Phosphomonoesterase R-O-P

+ H20 -~ P 4- ROH

By LEON A. HEPPEL Assay Method

Principle. The assay depends on measuring the formation of inorganic phosphate from sodium/~-glycerophosphate at an early stage in the reaction when the rate is linear. Reagents Sodium ~-glycerophosphate solution (0.1 M). Ethanolamine-HC1 buffer, pH 9.5 (0.1 M). Magnesium acetate (0.005 M or 0.05 M).

Procedure. The lower concentration of magnesium acetate is used in testing the crude enzyme preparation, since higher concentrations are inhibitory. Mix 1 ml. each of buffer, magnesium acetate, and sodium ~-glycerophosphate and make up to 4.8 ml. Mix this with an amount of enzyme such that hydrolysis never exceeds 5%. Incubate for 15 minutes at 38 °, and analyze for inorganic phosphate. I The reaction is stopped by the addition of 2.5 ml. of 25% (w/v) trichloroacetic acid. Any precipitate is removed by filtration through Whatman No. 42 paper, and P~ is determined on the filtrate. Definition of Unit and Specific Activity. The unit is defined as the amount of enzyme which liberates 1 ~ of inorganic phosphate per rainute at 38 °. Specific activity is expressed as units per milligram of protein N. Purification Procedure

This procedure is the method of Morton. ~ Calf intestines are collected from the slaughterhouse as soon as possible after killing, and the 1C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 2R. K. Morton, Biochem. J. §7, 595 (1954).

[80]

INTESTINAL PHOSPHOMONOESTERASE

531

mesenteric membranes are removed. The mucosa is then obtained by rinsing the lumen with water and scraping the mucosa with the edge of a plastic spatula. The mucosa is cooled to 0 ° in an ice bath. One must proceed quickly. Step 1. The mucosa is dispersed in three times its volume of distilled water (at 0°), using a Waring blendor for 2 minutes, and adjusted to pH 7.5 by slow addition of N NaOH. After hard mechanical stirring for about 30 minutes at 0 °, the dispersion is centrifuged (1800 X g, 40 minutes) in aluminum cups (1300 cc. capacity) in an International serum centrifuge (13 1.). The supernatant is poured off and filtered through a layer of well-washed cotton wool on a Bflchner funnel to remove loose fat and other aggregates. The filtrate is immediately cooled to 0 ° in a stainless steel container held in a cold bath at - 10°. The precipitate is discarded. Step 2. The filtrate at 0 to 2 ° is adjusted to pH 5 by addition of 2 M acetate buffer, pH 4.0, held for 45 minutes at 0 °, and then centrifuged (1800 × g, 40 minutes, room temperature). The white precipitate is then thoroughly dispersed in 8 1. of 0.15 M NaC1. The suspension is adjusted to pH 7.5 with 0.5 M Na~CO3, cooled to 0 °, and stirred gently to 0 to 2 ° overnight. The enzyme is then reprecipitated at pit 5 as before and similarly collected by centrifuging. At this stage the supernatant is waterclear and almost free of soluble protein. The precipitate is then washed in the cups by rubbing up into 8 1. of distilled water (at 0 °) and immediately recentrifuging. By this means the ionic strength of the material is lowered, and the last traces of soluble protein removed. The precipitate is again dispersed in distilled water at 0 ° and made up to 5 1. Step 3. About 2 1. of n-butanol is slowly added over a period of 15 minutes with vigorous mechanical stirring. The material is then heated to 38 ° for 5 minutes and immediately centrifuged in 1-1. glass cups (1800 X g, 30 minutes). The water-clear, colorless aqueous layer is removed by suction and filtered through a thick layer of Hyflo Super-Cel over Whatman No. 1 paper on a 1-1. Bfichner funnel. The filtrate is then adjusted to pH 8.5, held at 0 ° overnight, and again filtered through Hyflo Super-Cel to remove a small precipitate. Step 4. The filtrate is adjusted to pH 6.4, cooled to - 5 °, and the enzyme directly precipitated at - 5 ° by addition of ether (10% v/v) and acetone (60% v/v). After settling overnight at - 7 °, the supernatant is removed by suction, and the precipitate collected by centrifuging at - 5 °. It is often convenient to dry the precipitate at this stage and to accumulate material from several batches of mucosa before proceeding further with purification. For this purpose the precipitate is washed twice with dry acetone at - 1 5 ° followed by dry anesthetic ether at - 1 5 °, and

532

ENZYMES IN P H O S P H A T E METABOLISM

[80]

then dried, initially in a s t r e a m of nitrogen and finally in vacuo over H2SO4 and CaCl~. T h e d r y powder m a y be stored over N a O H and CaCl~ in vacuo at 0 °. I t retains activity for at least 12 months. F o r further purification it is t r e a t e d exactly as the undried precipitate. Usually, drying is omitted and the 60 % ( v / v ) acetone precipitate is dissolved in a m i n i m u m of 0.05 M Veronal-HC1 buffer at p H 6.4 and dialyzed against 0.015 M m a g n e s i u m acetate, at p H 6.4. Insoluble m a t e rial is removed b y centrifuging (20,000 X g, 1 hour, 2°). Step 5. Following addition of ether (10 % v / v ) the enzyme was precipit a t e d at - 5 ° with acetone between 35 and 48 % ( v / v ) . This fraction was dissolved in 0.05 M veronal-HC1 buffer at p H 6.4 and dialyzed against glass-distilled w a t e r (at 0 °) until free of organic solvent. Step 6. T h e solution is adjusted to p H 4.9 with 0.05 N acetic acid and heated rapidly to 48 ° for 2 minutes. A slight opalescence appears. Magnesium acetate (0.5 M) is added to give a final concentration of 0.015 M , the solution adjusted to p H 6.4, and the e n z y m e precipitated at - 5 ° with acetone between 40 and 5 0 % ( v / v ) . The slight precipitate between 40 and 5 0 % ( v / v ) acetone is dissolved in buffer and dialyzed free of organic solvent as for step 5. Step 7. T h e solution is adjusted to p H 5.5 with 0.5 N acetic acid, and successive 20-mg. lots of washed charcoal 3 are slowly added after mixing to a thick slurry in distilled water. The precipitate is removed b y centrifuging (5000 X g, 15 minutes, 2°), and the procedure is repeated until SUMMARY OF ~°URIFICATION PROCEDURE a

Step

Volume, ml.

Activity, units/ml.

Total units (X 103)

1 2 3 4 5 6 7 8

12,000 5,000 4,450 55 20 20 32 10

205 216 166 12,360 26,250 18,900 7,340 14,200

2460 1080 739 680 525 378 235 142

Total N, mg./ml, 2.12 0.82 0.03 2.05 1.96 0.94 0.11 0.17

Specific activity, units/mg. N 97 263 5,533 6,029 13,393 20,106 66,727 83,529

Yield, % 100 44 30 28 21 15 10 6

R. K. Morton, Biochem. J. in press. a Preparation of activated charcoal: 180 g. of animal charcoal is treated successively with 5 1. of 0.1 N HC1, 2 1. of 5% potassium chloride, 200 ml. of 0.1 M barium hydroxide, and finally 21. of 5% potassium chloride. After each treatment the charcoal is sucked dry over Whatman No. 42 paper on a large Biichner funnel. After the last treatment it is dried at 100° in an oven and stored in an airtight jar.

[81]

PHOSPHOMONOESTERASE OF MILK

533

about 30% of the enzymic activity is adsorbed. The solution is then centrifuged (14,000 × g, 30 minutes), adjusted to piI 8 with 0.1 N N a 0 H , and filtered on a Biichner funnel through a thin layer of magnesium carbonate over a Whatman No. 1 filter paper in order to remove colloidal material (charcoal). Step 8. The enzyme solution is adjusted to pH 6.4 with 0.1 h r acetic acid and dialyzed at 2 ° against 0.015 M magnesium acetate for 4 hours with continuous stirring. The solution is cooled to 0 °, ether added to 10% (v/v), and the enzyme precipitated at - 5 ° with acetone between 40 and 48 % (v/v). The precipitate is dissolved in glass-distilled water at 0 ° and dialyzed against glass-distilled water at 0 ° for 4 hours with frequent changes of water.

Properties Specificity. Alkaline phosphatase catalyzes the hydrolysis of various orthophosphomonoesters, phosphoamides, etc. For example, creatine phosphate, phenyl phosphate, glucose-6-phosphate, and f~-glycerophosphate are split. Pyrophosphates, such as ATP and ADP, are not split. R N A is not attacked. The enzyme also catalyzes transferase reactions. Thus, it stimulates a reaction of creatine phosphate and glucose to give glucose-6-phosphate and creatine. Effect of Inhibitors. 4 Phosphate ion inhibits the enzyme. Cyanide inhibits 50% at a concentration of 3 mM. Fluoride has no effect up to 50 raM. There is a slight enhancing effect of magnesium ions. 4 G. Schmidt and S. J. Thannhauser, J. Biol. Chem. 149, 369 (1943).

[81] P h o s p h o m o n o e s t e r a s e Typical Reaction:

of M i l k

Glycerol P ~- H~O -~ P~ -~ Glycerol

By ROBERT K. MORTON

Assay Method Principle. The rate of hydrolysis of a suitable phosphate ester is determined by estimation of liberated inorganic phosphate (P~) or of liberated alcoholic moiety (such as phenol). Kay and Graham 1 originally used sodium f~-glycerol P and later 2 phenyl P as substrates, p-Nitrophenyl P ~ has also been used. 1 H. D. Kay and W. R. Graham, J. Dairy Research 5, 63 (1933). 2 H. D. Kay and W. R. Graham, J. Dairy Research 6, 191 (1935). 3 R. Aschaffenburg and J. E. Mullen, J. Dairy Research 16, 58 (1949).

534


[81]

Reagents Sodium ~-glycerol P (0.1 M). Ethanolamine-HC1 buffer 4 (0.5 M), pH 9.95 at 20 ° (pH 9.65, 38°). 5'6 Magnesium acetate (0.2 M). TCA (30%). Enzyme. Dilute the preparation so that 0.1 ml. hydrolyzes no more than 5% of the substrate in 5 minutes at 38 °.

Procedure. Prepare a stock solution containing the following reactants: sodium ~-glycerophosphate, 0.02 M; ethanolamine-HC1 buffer, 0.05 M; magnesium acetate, 0.01 M (or 0.001 M for testing crude ext r a c t s - s e e below). The final pH should be 9.95 at 20 °. Pipet 5.0 ml. into suitable test tubes, and bring to 38 °. Add 0.1 ml. of the diluted enzyme preparation, and incubate for 5 minutes at 38 °. Stop the reaction with 2 ml. of 30% TCA. After 5 minutes, remove any precipitate by centrifuging and wash the precipitate twice with 5 % TCA. Estimate the liberated inorganic phosphate in the combined supernatant and washings by the method of Fiske and SubbaRow 7 or by any other suitable method (see Vol. I I I [147]). With partially purified enzyme preparations (after step 3 of purification procedure) no precipitate occurs on addition of TCA, and this may be omitted. The reaction is stopped by addition of the molybdate-sulfuric acid reagent of Fiske and SubbaRow, 7 and phosphate is then estimated directly. For the control tube, add TCA or molybdate-sulfuric acid to the buffered substrate prior to addition of the enzyme. Alternatively, phenyl P may be used as substrate and the liberated phenol estimated by a suitable method (see Vol. II [88]). Definition of Unit and Specific Activity. One unit of enzyme is defined as the amount which liberates 1 ~ of inorganic phosphate phosphorus per minute from ~-glycerol P under the above conditions. Because of the different activity of the enzyme with different substrates, the substrate must always be specified. Specific activity is expressed as units per milligram of protein nitrogen, determined by a micro-Kjeldahl procedure on dialyzed material (see Vol. III [145]) or as the Qp value (Engelhardt and LyubimovaS), i.e., the equivalent microliters of phosphorus liberated by 1 mg. of enzyme preparation in 1 hour at 38 °. The factor 6.25 is used to convert nitrogen values to dry weight. 4 C. A. Zittle a n d E. S. Della Monica, Arch. Biochem. 26, 112 (1950). Note t h a t all alkaline buffers change p H values markedly with temperature. 6 Other suitable amine compound~, as well as Veronal, are satisfactory as buffers. C. H. Fiske a n d Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 8 V. A. E n g e l h a r d t a n d M. N. Lyubimova, Nature 144, 668 (1939).

[81]


535

Application of Assay Method to Crude Preparations. Whole milk or b u t t e r m i l k should be dialyzed against distilled w a t e r for 12 hours to remove inorganic p h o s p h a t e which inhibits the enzyme and causes a high b l a n k value. T h e final concentration of m a g n e s i u m should not exceed 0.001 M, higher concentrations being inhibitory to crude enzyme preparations2 Purification P r o c e d u r e

T h e purification procedure described here is t h a t of Morton. 10,11 Zittle and Della Monica 12 h a v e also published a purification based on the use of butanol. I° Step 1. Preparation of Buttermilk. C r e a m of high b u t t e r f a t content (65 to 70%) is obtained b y separation of fresh whole milk at 35 ° . I t is diluted with distilled w a t e r to 45 to 5 0 % b u t t e r f a t , cooled to i0 °, and held overnight, if necessary. I t is churned to b u t t e r in a barrel churn or a reciprocating shaking machine. W h e n a fine b u t t e r grain appears, the b u t t e r m i l k is removed and the b u t t e r washed twice with minimal quantities of distilled water (at 0°). T h e b u t t e r m i l k and washings arc combined and strained through cheesecloth. Commercial b u t t e r m i l k m a y be used provided t h a t it is obtained f r o m fresh unpasteurized cream of high b u t t e r f a t content. Step 2. Treatment with Butanol. Alkaline p h o s p h a t a s e in milk and b u t termilk is not in true solution b u t associated with microsomal particles (Mortong,l~.14). These m a y be sedimented b y high-speed centrifuging (60,000 X g, 1 hour), and, where suitable e q u i p m e n t is available, this m a y be introduced into the purification procedure with considerable advantage. ~5 T h e enzyme is released from the microsomes into true solution as follows. T h e b u t t e r m i l k is heated to 32 °, and n-butanol (30 % v / v ) is added slowly with stirring. T h e emulsion is raised to 35 ° for approxim a t e l y 5 minutes and then adjusted to p H 4.95 b y cautious addition of 0.25 N acetic acid. On centrifuging, the material separates into distinct layers comprising a precipitate, a clear yellow-green aqueous layer over9 R. K. Morton, Ph.D. Thesis, University of Cambridge, 1952; Biochem. J. in press. 10R. K. Morton, Nature 166~ 1092 (1950). LIR. K. Morton, Biochem. J. 55~ 795 (1953). L2C. A. Zittle and E. S. Della Monica, Arch. Biochem. and Biophys. 35, 321 (1952). la R. K. Morton, Nature 171, 734 (1953). 14R. K. Morton, Biochern. J. 55~ 786 (1953). ~5The buttermilk is adjusted to pH 6.5 and centrifuged (60,000 × g, 1 hour). The precipitate is washed once by suspending it in 0.85 % NaC1 (at pH 6.5) and reeentrifuging. The sediment is suspended in distilled water and treated as described for buttermilk. Steps 7, 8, and 9 may then be omitted.

536

ENZ~ES

IN PHOSPHATE METABOLISM

[81]

laid b y a butanol-saturated material, and excess butanol. 16 The aqueous layer is removed b y suction and filtered through a thick layer of Hyflo Super-Cel (Johns-Manville Corp.) over W h a t m a n No. 1 filter paper on a large Bfichner funnel. T h e filtrate is brought to p H 8.5 with 0.2 N N a O H and held overnight at 5 °, when a bulky white precipitate settles. T h e sup e r n a t a n t is largely decanted, and the remainder is recovered b y centrifuging (2000 X g, 15 minutes). Step 3. Precipitation with Ether-Acetone. The clear serum is adjusted to p H 6.3 with 0.1 N acetic acid and cooled to 0 °. I t is treated in batches with addition of diethyl ether (10% v / v , - 1 5 °) and then acetone (45% v / v , - 1 5 ° ) , the t e m p e r a t u r e being maintained at - 3 to - 7 ° in a suitable low-temperature bath. T h e material is held overnight at about - 5 °, the s u p e r n a t a n t cautiously decanted, and the pink-colored precipitate collected b y centrifugation (2000 >( g, 20 minutes, - 5 ° ) . Step 4. Fractionation with Ammonium Sulfate. The precipitate is dissolved in a minimum of distilled water at 0 ° and either centrifuged (20,000 X g, 20 minutes, 0 °) or filtered overnight at 0 ° through Whatman No. 542 filter paper. T h e red-colored, water-clear solution is dialyzed against distilled water at 0 ° to remove organic solvents. Solid ammonium sulfate is added to 0.63 saturation, the solution being maintained at p H 7.1 b y cautious addition of 0.1 N N a O H . T h e red-colored precipitate is collected b y centrifugation (4000 X g, 40 minutes, room temperature) and discarded. T h e clear s u p e r n a t a n t is brought to 0.85 saturation b y addition of solid ammonium sulfate. After standing for 1 hour, the precipitate is collected b y centrifugation as before. I t is dissolved in a minimal q u a n t i t y of distilled water and dialyzed against distilled water at 0 ° to remove ammonium sulfate. Step 5. Adsorption by Charcoal. T h e solution is brought to p H 8.6 with 0.1 N NaHCO3 and diluted to about 5 mg. of protein per milliliter with distilled water. Animal charcoaP 7 is added as a slurry in distilled water. T h e material is held for about 30 minutes and then centrifuged (2000 X g, 15 minutes). The charcoal should be added cautiously in small amounts (about 2.5 g. per 100 ml. of enzyme solution), and the procedure repeated until about 10% of the enzyme is adsorbed. The p H should be maintained at about 8.6 t h r o u g h o u t this step. Peptized charcoal is finally 16The excess butanol may be recovered by distillation and is quite suitable for further preparative work. 17The charcoal is prepared as follows: About 180 g. of animal charcoal (British Drug [Houses Ltd.) is treated successively with 51. of 0.1 N HC1, 21. of 5% KC1, 200 ml. of 0.1 M BaOH, and finally 21. of 5% KC1. After each treatment the charceal is sucked dry on a Whatman No. 42 filter paper on a large Btichner funnel. After the last treatment it is dried at 100°.

[81]


537

removed by filtration of the enzyme solution through a thin layer of BaC03 overlying Hyflo Super-Cel on a Whatman No. 1 paper on a Btichner funnel. Step 6. Fractionation with Ether-Acetone. The solution is dialyzed against distilled water, adjusted to pH 6.4, and fractionated by addition of diethyl ether (10 % v/v) followed by acetone (40 to 49 % v/v) at - 5 ° (see step 3). The 40 to 49 % acetone precipitate is dissolved in the minimal amount of distilled water at 0 ° and dialyzed at 0 ° against distilled water to remove organic solvents. Any precipitate is removed by centrifuging (20,000 X g, 1 hour). Step 7. Adsorption with Charcoal. The solution is brought to pit 8.6, and step 5 is repeated, sufficient charcoal being added to adsorb about 20 % of the phosphatase activity. Step 8. Fractionation with Ammonium Sulfate. Step 4 is repeated, the fraction obtained at pH 7.2 between 0.69 and 0.82 saturation with ammoSUMMARY OF P U R I F I C A T I O N PROCEDURE a

Fraction

Specific Total Total Protein activity, Recovvolume, Units/ml. units nitrogen, units/rag, ery, ml. thousands thousands mg./ml. N %

1. Buttermilk and washings 32,500 2. Serum from butanol treatment 27,000 3. Ether-acetone precipitate 1,480 4. (NH4)~SO4 fraction, 0.63-0.85 127 5. Supernatant from charcoal adsorption 280 6. Ether-acetone fraction (40-49 % acetone) b 75 7. Supernatant from charcoal adsorption 89 8. (NH4)~SO4 fraction, 0.69-0.82 12 9. Ether-acetone fraction (42-48% acetone) b 10

80

2,600

2.80

28.6

--

65

1,755

0.20

325.0

67

1,092

1,616

1.20

910.0

62

9,230

1,172

2.25

4,102.2

45

3,340

935

0.46

7,261

36

9,800

735

1.04

9,423

28

5,730

510

0.49

11,694

20

29,000

348

2.04

14,216

13

26,800

268

1.75

15,314 ~

10

R. K. Morton, Biochem. J. 55, 795 (1953). b Limits of fractionation should be determined by small-scale trials. The figures given here should be taken as a guide only, since small changes of ionic strength, etc., may considerably alter fractionation. c Q~ 106,000. The purification compared to whole milk was 5660 times, or 535 times compared with buttermilk.

538


[81]

nium sulfate being retained. The precipitate, dissolved in the minimal amount of distilled water, is dialyzed against distilled water until free of salt. Step 9. Fractionation with Ether-Acetone. The solution is adjusted to pH 6.5 and to 0.015 M magnesium acetate, and the enzyme is precipitated by addition of diethyl ether (10% v/v) and acetone (42 to 48% v/v) at - 5 ° (see step 3). The 42 to 48 % acetone fraction is dissolved in the minimal amount of distilled water, frozen and dried in vacuo.

Properties Stability. The enzyme may be stored at 0 ° in vacuo over NaOH for about four months without loss of activity beyond an initial decline which occurs on drying the preparation. A slow, irreversible decline occurs with longer periods of storage. Dilute solutions of the enzyme may lose activity owing to irreversible denaturation if stored frozen. They may be stored more satisfactorily at 0 ° and at pH 7.2 under toluene vapor to exclude bacterial contamination. Specificity. It has been shown 9 that the purified enzyme hydrolyzes orthophosphomonoesters and related compounds such as phosphoenolpyruvate and phosphoamides (e.g., creatine phosphate). It is probable that acyl phosphates and thiophosphates are also hydrolyzed. However, phosphodiesters and pyrophosphates are not hydrolyzed by the pure enzyme. The rates at which various phosphomonoesters are hydrolyzed differs considerably (see also Effect of pH, below). For example, under optimal conditions for both substrates, the rate of hydrolysis of phenyl P is about 1.6 times that of E-glycerol P. Activators and Inhibitors. The purified enzyme is activated by several divalent metals. As indicated in the assay procedure, magnesium (10-2 M), gives maximum activation. Optimal concentrations for other metals are: zinc 10-8 M, calcium 10-~ M, and manganese, 10-3 M. Zinc and beryllium are inhibitory at 10-4 M and higher concentrations. The enzyme is inhibited 9,18 by various anions, such as phosphate, pyrophosphate, arsenate, carbonate, and borate, and by iodine. Inhibition may be both competitive and noncompetitive, and the amount of inhibition depends on both the substrate concentration and the pH of the test system. Certain amino acids, such as glycine, alanine, and cysteine, are inhibitory at high concentrations, as are other metal chelating agents. Effect of pH. The optimal ptI for hydrolysis varies with the experimental conditions, being dependent on the substrate, the concentration of the substrate, and the nature of the buffer (should this be inhibitory). 18 C. A. Zittle and E. S. Della Monica, Arch. Biochem. 26~ 135 (1950).

[82]

PHOSPHOMONOESTERASE OF BONa

539

Since different alkaline buffer systems have different temperature coefficients, it is important to make activity and pH determinations at the one temperature or to make accurate temperature corrections. When the activity is determined with either Veronal-acetate-HCl (0.05 M) or ethanolamine-HC1 (0.04 M) buffer, with 0.01 M magnesium, the following are the optimal pH values with the various substrate concentrations as shown: phenyl P (2.5 X 10-3 M), pH 10.05; ~-glycerol P (2 X 10-2 M), pH 9.65; adenosine-5-P (4 X 10-4 M), pH 9.5; creatine P (9 X 10-3 M), pH 9.55. Transphosphorylation Activity. The enzyme catalyzes the transfer of the phosphate group from suitable substrates (such as phenyl P) to certain aeceptors (such as glucose or glycerol) to form new phosphate esters 19 (see Vol. II [88]). Chemical Constitution2 The purified enzyme appears to be an unconjugated protein. It contains 16.2 % nitrogen. Tests for phosphate, carbohydrate, and nucleotide were negative2 No evidence of a dialyzable or dissociable group (except a divalent metal) essential for enzymic activity has been obtained. Relationship to Other Phosphomonoesterases. The milk enzyme differs from the alkaline phosphatase of calf intestinal mucosa (which has also been purified by a procedure similar to that described above2°). The specific activity of the purified milk enzyme is about one-fifth of that of the pure intestinal phosphatase. The optimal pH for hydrolysis of different substrates also differs for the two enzymes. The milk phosphatase is identical with the phosphomonoesterase of mammary gland and of kidney TM and either identical with, or closely related to, the phosphomonoesterases of bone and of liver. 19 R. K. Morton, Nature 172, 65 (1953). 2o R. K. Morton, Biochem. J. 57, 595 (1954). 21 S. J. Folley and H. D. Kay, Biochem. J. 29, 1837 (1935).

[82] Phosphomonoesterase of Bone By

ELLIOT VOLKIN

Assay Method

Principle. The monoesterase specifically hydrolyzes singly esterified phosphoryl groups, as in ~-glycerophosphate, monophenylphosphate, and mononucleotides, and does not attack diesterified phosphoryl groups, as in diphenylphosphate or the internucleotide phosphoryl groups of poly-

540


[82]

nucleotides. Generally speaking, a disadvantage in its use is its relatively low specific activity as compared with monoesterases from other sources (prostate, semen, potato, barley). The enzyme is activated by magnesium ion and shows optimal activity around pH 8.6 to 9.0. Protocol. One milliliter each of adenosine-2'-, -3'-, and -5'-phosphate and monosodium diphenylphosphate, each containing 100 ~, of phosphorus per milliliter; 1 ml. of 0.1 M NH4C1-NH3 buffer, pH 9.0, containing 0.005 M MgS04; 1 ml. of phosphomonoesterase preparation from beef metatarsal bone (0.10 mg. of nitrogen per milliliter); water to 5 ml. At the time intervals indicated in the table, 1 ml. was removed for inorganic phosphate assay. 1 Procedure. The protocol and table demonstrate the typical hydrolysis of the mononucleotides of adenylic acid by bone monoesterase. The absence of contaminating diesterase activity is evident by the failure of the preparation to liberate inorganic phosphate from diphenylphosphate.

Preparation Principle. No important changes have been forthcoming in the preparation of this enzyme since it was first described in 1938 by Gulland and Jackson. 2 The method consists in (a) autolysis of metatarsal bone for the extraction of total phosphatase activity 3 and (b) a preferential elution of monoesterase from diesterase activity from Norit charcoal, which adsorbs both enzymes. Increased activity results from removal of the eluting borate ion, and concentration of the enzyme by precipitation with alcohol: ether yields a more workable enzyme solution concentration. It should be noted that the method is not designed for the purification of monoesterase free of enzymes other than diesterase. Procedure. The beef foreleg bones are skinned, scraped free of tissue, sawed or crushed into small nuggets, then freed of marrow. About five times their weight of water containing a little chloroform is added, and the bones are allowed to autolyze for 7 days at room temperature. The phosphoesterases of the autolyzate are adsorbed on Norit A charcoal, by stirring 6 g. of the charcoal with 1 1. of extract at 0 to 5 ° for 1.5 hours. The charcoal is collected, washed with water, and eluted with 25 ml. of pH 8.6 Clark and Lubs borate buffer. The eluate, containing monoesterase but no diesterase, is dialyzed with cold distilled water, then precipitated at - 5 ° by the addition of 5 vol. of 2:3 methanol:ether. The precipitate is 1C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925); see also E. Volkin and W. E. Cohn, in "Methods of Biochemical Analysis," Vol. I, pp. 287-305, Interscience Publishers, New York, 1954. J. M. Gulland and E. M. Jackson, Biochem. J. 32, 590 (1938). s M. Martland and R. Robison, Biochem. J. 25, 237 (1929).

[83]

GLUCOSE-6-PHOSPHATASE FROM LIVER

541

collected, redialyzed against cold, distilled water, and a n y remaining insoluble material removed by centrifugation. The enzyme preparation m a y be stored frozen without loss of activity. DEPHOSPHORYLATIONOF THE ADENYLICACID MONONUCLEOTIDESBY BONE PHOSPHOMONOESTERASZ Percentage of hydrolysis Time, rain. Adenylic-2' 20 4O 60 240

50 8O 96 --

Adenylic-3' 42 72 95 --

Adenylic-5' Diphenylphosphate 50 78 95 --

0 0 0 0

[83] G l u c o s e - 6 - p h o s p h a t a s e f r o m Liver Glucose-6-P A- H20--* Glucose -]- P04

By MARJORIE A. SWANSON Assay Method

Principle. T h e method is based on the incubation of the specific substrate with the enzyme and determination of the liberated orthophosphate. Reagents G~6-P stock solution (0.1 M). Suspend 260 mg. of the barium salt I in 2 ml. of distilled water, and dissolve in the minimum a m o u n t of 1 N HC1. Add 72 mg. (0.5 mM) of anhydrous Na2SO4. R e m o v e the precipitated BaS04 b y centrifugation, and test the supernarant solution for complete precipitation with a v e r y small a m o u n t of Na~SO4. Bring the supernatant solution to p H 6.5 with NaOI-I, and make to 5 ml. Buffer. Dissolve 116 mg. of maleic acid in water, bring to p H 6.5 with NaOH, and make to 10 ml. Enzyme. 5 % to 10% homogenate of liver (ice-cold).

Procedure. I n t o a conical 12- or 15-ml. calibrated centrifuge tube measure 0.1 ml. of substrate solution and 0.3 ml. of buffer and bring to 37 ° in The crystalline salt G-6-P, Ba7H20 is now available commercially. For methods of preparation see Vol III [19].

542


[83]

a water bath. Add 0.1 ml. of homogenate, mix by swirling, incubate for 15 minutes, stop with 1 ml. of 10% TCA, and chill in ice. After 5 minutes, dilute to 2.5 ml., and centrifuge. Take 2-ml. aliquots of supernatant solution for determination of inorganic phosphate. No unit of activity has been defined. Under the conditions described, about 7 rag. of inorganic phosphate is liberated per gram of rat liver.

Purification Attempts to purify the enzyme have been limited by its extreme instability and its insolubility. It appears to be bound to the microsomes. These can be sedimented from isotonic sucrose at 30,000 X g, or agglutinated at pH 5.4. The precipitated microsomes may be washed at this pH to remove phosphoglucomutase. Hexose isomerase, ATPase, AMPase, and a feeble glycerophosphatase are not removed by the washing. Solubilization by means of detergents or bile salts has not been attempted. Occurrence This specific phosphatase has been found only in liver, kidney, and small intestine. ~

Properties The enzyme is apparently specific for G-6-P. After removal of the mutase by washing, G-1-P is split not at all, and fructose disappears in the same proportion that inorganic phosphate is liberated from F-6-P. Galactose phosphate and mannose phosphate have not been tested. It has an optimum pH of 6.5 and appears to require no metallic activator. Molybdate and arsenate inhibit to some extent; fluoride gives very inconsistent results. 3 Under the conditions specified, the enzyme does not appear to be completely saturated with substrate. About 10 % greater activity is obtained if the substrate concentration is doubled. A much greater decrease in activity is noted if the substrate concentration is halved. Activity is not proportional to the dilution of the enzyme; the maximum activity (for rat liver) is found with 10 to 20 mg. of tissue per milliliter of incubation mixture. It decreases with both higher and lower concentrations of enzyme. The rate of the reaction decreases with time, but not sufficiently to be a first-order reaction. Change in temperature has only a small effect, a Q10 (26 to 36 °) of 1.2 being found in the assay system described. ~ G-6-Pase is apparently located solely in the microsomes. 2'4 It there2 H. G. Hers and C. de Duve, Bull. soc chim. biol. 32, 20 (1950). 8 M. A. Swanson, J . Biol. Chem. 184, 647 (1950). M. A. Swanson, unpublished data.

[84]

FRUCTOSE-1,6-DIPHOSPItATASE FROM LIVER

543

fore becomes an indicator for the presence of microsomes, as in mitochondria prepared from liver or kidney. Homogenates prepared from liver in isotonic sucrose, KC1, NaCl, or in distilled water have the same G-6-Pase activity, but mitochondria prepared from sucrose have no G-6-Pase activity, and those prepared from salt solutions may contain 10 % to 20 % of the total tissue activity.

[84] Fructose-l,6-diphosphatase from Liver FDP + H20 --~ Pi + F-6-P

By R. W. McGILVERY

Assay Method Principle. The method is that of Pogell and McGilvery' in which the P~ liberated from FDP is determined. Reagents 0.05 M FDP. Prepare a solution of the sodium salt at pH 7.4 preferably from the purified cyclohexylammonium salt? 0.05 M boric acid-NaOH buffer, pH 9.5. 0.05 M MgS04. 0.005 M MnC12. 0.05 M cysteine-NaOH, pH 9.5. Prepare this solution daily. 0.1 M trichloroacetic acid. Enzyme. Dilute the solution to be tested to a concentration of 5 to 20 units/ml, with water (see definition below). Crude preparations should be adjusted to pH 9.5 immediately before the assay. Procedure. Place 0.i ml. of FDP, 0.4 ml. of borate buffer, 0.I ml. of MgSO4, and 0.i ml. of MnCl~ in a centrifuge tube. Warm the tube and a container with the enzyme sample in a 38 ° bath for 5 minutes. Then add 0.2 ml. of enzyme to the tube, followed immediately by 0.i ml. of cysteine previously warmed to 38 °. After 20 minutes of incubation add 1 ml. of trichloroacetic acid and determine the Pi liberated, 8 which should not exceed 1.25 micromoles from 5 micromoles of FDP. Definition of Unit and Specific Activity. One unit of the enzyme is de1B. M. Pogell and R. W. McGilvery,J. Biol. Chem. in press. See Vol. III [22]. See Vol. III [147].

544


[84]

fined as the amount which will liberate 1 micromole of P~ with linear kinetics in 1 hour under the above conditions. Specific activities are given in units per milligram of protein, estimated as 6.25 × Kjeldahl N for crude preparations and as (1.55E2s0 - 0.76E260) mg./ml, for transparent solutions. Application of Assay Method to Crude Preparations. No exact assay of crude extracts containing nonspecific phosphatases active at pH 9.5 can be made. A rough correction for contaminating activities can be made by substituting F-6-P for F D P in the assay and subtracting the value thus obtained from the FDPase assay. Preparation of the extracts at pH 4.5 (see Purification Procedure) may destroy most of the interfering enzymes, but tests should be made. In most cases, Mn ++ and cysteine should be omitted (see Properties). Purification Procedure Step 1 is essentially according to Gomori, 4 and the remaining steps are those of Pogell and McGilvery.1 Step 1. Preparation of Autolyzed Extract. Homogenize the livers from exsanguinated rabbits (6 to 8 make a convenient amount) for 2 minutes in a Waring blendor with 4 ml. of 0.005 M sodium lactate buffer, pH 3.5, per gram of tissue. Centrifuge the homogenate (specific activity of 1.3 units/rag.) for 30 minutes at 1000 X g. Transfer the supernatant, which should be near pH 4.5, to a flask in a 38 ° bath. Keep the flask in the bath for 8 hours after the temperature of the solution reaches 37 °. Chill the flask to 4 ° in ice, and adjust the solution to pH 7.0 with 1 N NaOH. Remove and discard insoluble materials by centrifugation for 60 minutes at 2500 X g. Step 2. First Ammonium Sulfate Fractionation. Bring the autolysate (specific activity of 20 to 25 units/mg.) to 2.28 M by the slow addition of solid ammonium sulfate and then to 2.75 M by the dropwise addition of 3.89 M ammonium sulfate solution. Remove and discard the precipitate by centrifugation for 20 minutes at 2500 X g. Raise the concentration of ammonium sulfate to 3.00 M by dropwise addition of 3.89 M solution. Collect the precipitated enzyme (45 to 65% from the autolysate, specific activity of 50 to 100 units/mg.) by centrifugation. Step 3. Second Ammonium Sulfate Fractionation. Dissolve the precipitate, and dilute the solution to a protein concentration of 4 to 4.5 mg./ml. with a measured volume of water. Measure the total volume, and calculate the salt concentration of the diluted solution. Remove a slight amount of insoluble material by centrifugation for 1 hour at 18,000 X g. 4 G. Gomori, J. Biol. Chem. 148, 139 (1943).

[84]

FRUCTOSE-1,6-DIPttOSPHATASE FROM LIVER

545

Adjust the supernatant to p H 7.0 with 1 N N a O H , and raise the salt concentration to 1.90 M with solid ammonium sulfate and then to 2.48 M by dropwise addition of 3.89 M solution. Centrifuge for 30 minutes at 2500 X g, and discard the precipitate. Raise the supernatant to 3.00 M ammonium sulfate b y addition of the 3.89 M solution. Collect the precipitate (25 to 35% from the autolyzate, specific activity of 100 to 120 units/mg.) b y centrifugation for 1 hour at 2500 X g. Step ~. Alumina Adsorption. Dissolve the precipitate, and dilute it to a concentration of 5 mg. of protein per milliliter with water. Bring the solution to p H 4.0 with 1 N lactic acid, and add an a m o u n t of C~ alumina 5 suspension containing an a m o u n t of alumina equal to the weight of protein. After 10 minutes of gentle stirring, collect the gel b y centrifugation. Successively wash the gel b y centrifugation with portions of 0.005 M sodium lactate, p H 4.1, and 0.1 M sodium borate, p H 8.5, equal in volume to the protein solution before addition of the alumina. Elute the enzyme b y three extractions of the gel with 0.1 M sodium borate, p H 9.25, at the centrifuge, using a volume one-third t h a t of the original solution before adsorption for each extraction. In occasional preparations, the use of more alumina with an eluting buffer of higher p H will be required. The eluate should contain ca. 2000 units per rabbit liver (10 to 15% of the activity from the autolysate) with a specific activity near 400 units/mg, of protein, representing an increase in specific activity of 300-fold over the original homogenate. SUMMARY OF PURIFICATION PROCEDUREa

Step

Fraction

1. Homogenate Autolysate 2. 2.75-3.00 M (NH4)2SO4 3. 2.48-3.00 M (NH4)~S04 4. Eluate from alumina

Totalb volume, ml.

Total units b

Specific activity, units/mg.

Recovery, %

1980 1320 --75

-122,000 56,000 40,000 16,800

1.3 20-25 50-100 100-120 400

-100 45-65 25-35 10-15

B. M. Pogell and R. W. McGilvery, J. Biol. Chem. in press. b The total values are for a preparation from 400 g. of rabbit liver. The other columns give the ranges encountered in various trials.

Properties Specificity. The enzyme has no effect on G-l-P, G-6-P, F-6-P, PGA, or L-sorbose-l-P. F-1-P and L-sorbose-l,6-di-P are hydrolyzed at rates of 6 See Vol. I [11] for preparation of alumina C~.

546

ENZYMES IN" PHOSPHATE METABOLISM

[85]

0.009 and 0.03, respectively, the rate of F D P hydrolysis, indicating a specificity toward the phosphate bond bearing a 1-relation to a 2,5-furanose ring of D-arabino configuration. Although only one of the P groups of F D P is hydrolyzed, occasional purified enzyme preparations give products having higher acid-labile P contents than does F-6-P. The cause of this anomaly is unknown. Activators and Inhibitors. Mg ++ or Mn ++ is required for activity. In crude extracts, Mg ++ is better than Mn ++, and addition of Mn ++ or cysteine in the presence of Mg ++ causes inhibition. At all stages beyond step 1, addition of Mn ++ or cysteine in the presence of Mg ++ results in stimulation and is necessary in order to demonstrate recovery of the enzyme on fractionation. The individual effects vary from one preparation to another and from step to step. The conditions given in the assay method represent a compromise. Preparations stimulated by 0.0005 M Mn ++ are slightly stimulated by Fe ++ and inhibited by Co ++, Ni ++, Cu ++, and Zn ++ at the same concentration? Fluoride at 0.01 M inhibits 60%. 4 Stability. Preparations carried through step 3 have been stored in the frozen state for 15 months with loss of less than half of the activity. After step 4, the stability is markedly lowered. The enzyme from step 2 can be incubated for 2 hours at 38 ° without substrate or activators at any pH between 4 and 9 with loss of less than half of the activity. Heating the crude extract at pH 4.6 in a 100 ° bath until its temperature rises to 70 ° causes 10 to 15% loss. At 75 ° the loss is about one-third, and at 80 ° it is greater than 90%. pH Optimum. The enzyme is most active under the assay conditions at p H 9.3 to 9.5. Serine, barbital, and borate buffers give equal values.

[85] "5" Nucleotidases 5-AMP + H~O --~ Adenosine + P By LEON A. HEPPEL and R. J. HILMOE " 5 " Nucleotidases 1 are enzymes which hydrolyze phosphate esterified at carbon 5' of the ribose or deoxyribose portions of the nucleotide molecule. Thus far, only mononucleotides have ~,een available as substrates; polynucleotides containing phosphomonoester groups at carbon 5' of the sugar have not been reported. 1j. Reis, Bull. soc. chim. biol. 16, 385 (1934).

[85]

" 5 " NUCLEOTIDASES

547

I. "5" Nucleotidase of Seminal PLasma Assay Method Reagents 1.0 M glycine-NaOH buffer, pH 8.5. 0.1 M MgC12. 5-AMP solution, kept frozen. Enzyme solution, kept frozen.

Procedure. A total volume of incubation mixture of 1.24 ml. is made to contain 0.1 ml. of buffer, 0.1 ml. of MgC12, 3 micromoles of 5-AMP, and enzyme. Incubation is for 15 minutes at 37 °, and the reaction is stopped by the addition of trichloroacetic acid in a final concentration of 5%. If necessary, the mixture is centrifuged. An aliquot is analyzed for inorganic p.2 The amount of enzyme can be varied widely because the reaction is linear until nearly all the 5-AMP is utilized. A substrate blank is usually necessary. Definition of Unit and Specific Activity. A unit of activity corresponds to the liberation of 1 micromole of P per hour, and specific activity is defined as units per milligram of protein. Protein is determined by the method of Lowry et al. 3

Purification Procedure This is from the work of Heppel and Hilmoe. ~ Bull seminal plasma is obtained, usually from an Artificial Breeder's Association. It may be stored at - 8 ° for at least six months. The following steps are at 2 °, except as noted. Step 1. Treatment with Protamine Sulfate. 110 ml. of bull seminal plasma is mixed with a solution of 550 mg. of protamine sulfate in 330 ml. of distilled water. A precipitate is removed by centrifugation and discarded. Step 2. First Ammonium Sulfate Fractionation. The supernatant is brought to pH 6.7, and the volume is adjusted to 490 ml. Ninety-five grams of ammonium sulfate is added, and the pH is adjusted to 4.2 by means of 66 ml. of 1 N acetic acid. Then 139 ml. of 0.2 M acetate buffer, pH 4.2, and 84.5 g. of ammonium sulfate are added. After 15 minutes the suspension is centrifuged for 7 minutes at 13,000 r.p.m, in a Servall type SS-1 angle centrifuge. The precipitate is discarded. To 745 ml. of super2 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 30. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4L. A. Heppel and R. J. Hilmoe, J. Biol. Chem. 188, 665 (1951).

548

ENZYMES IN P H O S P H A T E METABOLISM

[85]

n a t a n t is added 225 g. of ammonium sulfate, and the precipitate is collected by centrifugation, dissolved in 45 ml. of distilled water, and dialyzed against running 0.01 M acetate buffer, pH 6.0, for 4 hours. Step 3. Second Ammonium Sulfate Fractionation. The dialyzed material from step 2 measures 74 ml. and is turbid. I t is clarified by centrifugation and mixed with 8.2 ml. of 1 M acetate buffer, pH 6.0, and 26.4 g. of ammonium sulfate. The precipitate is removed by centrifugation and discarded. To 91.5 ml. of supernatant is added 5.6 g. of ammonium sulfate. The precipitate is collected by centrifugation, dissolved, and dialyzed as before. Step ~. Ethanol Fractionations. The dialyzed ammonium sulfate fraction (35 ml.) is brought to pH 4.6 with 4.5 ml. of 0.1 N acetic acid, and enough 1 M acetate buffer, pH 4.6, is added to bring the concentration of acetate to 0.1 M. The solution (41 ml.) is cooled to - 0 . 5 °, and successive additions of absolute ethanol are made in the following amounts (milliliters) : 3.5, 0.7, 0.5, 0.5, 0.3, 0.5, 1.0, and 2.0. The mixture is rapidly stirred during the ethanol additions and is maintained near its freezing point. After each addition there is a 5-minute wait before a brief centrifugation. The precipitates are dissolved in 0.02 M acetate buffer, pH 6.5. The best fractions are combined, and the ethanol step is repeated. No dialysis is carried out. Step 5. Heating, Followed by Alumina Gel. The ethanol fraction is adjusted to pH 6.8, warmed to 60 ° over a period of 90 seconds, and maintained at this temperature for 20 minutes. This t r e a t m e n t removes contaminating phosphatase activity. After rapid cooling small aliquots are mixed with different quantities of aluminum hydroxide gel C~ ~ (11 mg. dry weight per milliliter, aged three months at least), centrifuged, and SUMMARY OF PURIFICATION PROCEDURE a

Step Seminal plasma 1. Protamine sulfate 2. Ammonium sulfate 3. Ammonium sulfate 4. First ethanol Second ethanol 5. Heating and gel "

Volume, ml.

Total units

110 419 82 35.5 37 18.3 80

310,000 290,000 133,000 74,000 61,270 18,710 12,710

Specific activity, Yield, units/mg. % 50 111 274 476 986 1350 2500

100 93 43 24 19.8 6 4

L. A. Heppel and R. J. Hilmoe, J. Biol. Chem. 1 8 8 , 665 (1951).

5 R. Willsti~tter and H. Kraut, Ber. 56, 1117 (1923); see also Vol. I [11].

[85]

"5"

NUCLEOTIDASES

549

the supernatant solutions tested for activity and protein content. The maia lot is then treated with the amount of gel found to give the best purification and a high yield.

Properties

Specificity. Purification of bull seminal plasma with respect to the splitting of 5-AMP resulted in no change in the relative activities toward the 5' esters of inosine, uridine, nicotinamide riboside, and cytidine. Adenosine-2',5'-diphosphate is not split. A low activity against ATP and ADP remains after purification. Stability. The purified enzyme is stable for eight weeks as a frozen solution. Lyophilized samples can be stored at - 8 ° for at least six months with little loss of activity. Effect of pH. The pH optimum is 8.5. The reaction rate is proportional to the concentration of enzyme over a wide range. Substrate A~nity. The 5'-phosphates of adenosine, inosine, uridine, and cytidine are relatively tightly bound, with dissociation constants below 10-4. For nicotinamide mononucleotide the value is 1.6 X 10-3. Metal Requirements and Inhibitors. In the absence of MgC12 the rate of reaction is decreased by 70%. Neither CaC12 nor MnC12 can replace MgC12. In the presence of MgC12 the reaction is inhibited by CaC12, and this effect can be overcome by excess MgC12. Sodium fluoride inhibits the rate of splitting of 5-AMP by bull semen enzyme to the extent of 98% at 0.1 M and 73% at 0.01 M. Borate buffer (8 × 10-2 M) inhibits 85 % compared with glycine buffer of the same pH. II. "5" Nucleotidase of Snake Venom Assay Method The assay method is the same as for seminal plasma.

Purification Procedure This is taken from Hurst and Butler. 6 Ten milligrams of lyophilized venom is dissolved in 10 ml. of water and chromatographed on a column of 1 g. of shredded filter paper (Whatman No. 5) 10 cm. long and 0.8 cm. in diameter. The flow rate is i ml./min. Fifty-five milliliters of water is run through the column, followed by 80 ml. of 0.1% NaC1 and 30 ml. of 1.0% NaC1. Fractions of 10 ml. are collected, and their optical density at 260 m~ measured. The elution peak obtained with 1% NaC1 contains "5" nucleotidase with a considerable amount of phosphodiesterase removed. e R. O. Hurst and G. C. Butler, J. Biol. Chem. 193, 91 (1951).

550


[85]

Properties The unfractionated venom from many species of snakes has been tested and found to be highly specific in hydrolyzing 5'-nucleotides, but not the other isomers. 7 As is true for the bull semen enzyme, substrate affinity is high. The following enzyme-substrate dissociation constants have been obtained for Crotalus atrox venom: 5-AMP, 1,000,000 to smaller fragments without splitting off orthophosphate was found by Ingelmann and Malmgren 5-7 and occurs in some strains of Aspergillus and P e n i c i l l i u m and also in Proteus vulgaris.

Assay Methods The production of orthophosphate from tri- or hexametaphosphate can be followed b y measuring the reduction of phosphomolybdate at acid p H according to the Fiske and SubbaRow determination s or any of its modifications, e.g., L o h m a n n and Jendrassik 9 as recommended b y Kitasato, 2 use of aminonaphtholsulfonate as recommended b y Neuberg and Fischer, 1° use of ascorbic acid according to Lowry and Lopez 11 and recommended b y Ingelmann and Malmgren, 5 or Sumner's procedure 1~ as used by Krishnan and Bajaj. 13 This test is not given b y pyrophosphate, metaphosphate, or triphosphate. P present in the enzyme preparation can be removed b y previous precipitation with Mg-acetate and N H 4 0 H or cerrected for b y blank determination. Procedure (Low Molecular Weight Substrates). The substrate can be made by melting of NaH2P042 and rapid cooling of the melt, ~ or commercial tri- or hexametaphosphate preparations m a y be used if they are crystalline. The enzyme-rich cell extract is incubated in approximately IT. Kitasato, Biocliem. Z. 197, 257 (1928). 2 T. Kitasato, Biochem. Z. 201, 206 (1928). C. Neuberg and K. P. Jaeobsohn, Biochem. Z. 199, 499 (1928). 4 C. Neuberg and H. A. Fischer, Compt. rend. tray. lab. Carlsberg 22~ 366 (1938). 5 B. Ingelmann and H. Malmgren, Acta Chem. Scand. 2, 365 (1948). 6 B. Ingelmann and H. Malmgren, Acta Chem. Scand. 3, 157 (1949). 7 H. Malmgren, Acta Chem. Scand. 3, 1331 (1949). 8 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 9 K. Lohmann and L. Jendrassik, Biochem. Z. 178, 419 (1926). 19C. Neuberg and H. A. Fischer, Enzymologia 2, 241 (1937). i~ O. H. Lowry and J. A. Lopez, J. Biol. Chem. 162, 421 (1946). 12j. B. Sumner, Science 100, 413 (1944). 18p. S. Krishnan and V. Bajaj, Arch. Biochem. and Biophys. 42, 175 (1953).

578

ENZYMES

IN

PHOSPHATE

METABOLISM

[92]

0.5% solution of the substrate at the desired pH at 37 °. Samples are withdrawn at intervals, the reaction stopped with 5 to 10 % trichloroacetic or perchloric acid, filtered immediately, and the supernatant tested colorimetrically or spectrophotometrically for the amount of P present. First-order kinetics are followed if incubation does not exceed 1 hour. 14 This method is not applicable to the metaphosphate depolymerases which produce an insignificant amount of P only. The breakdown of the high molecular weight substrates is followed by measuring the decrease in viscosity. 5 Procedure (High Molecular Weight Substrates). The substrates are prepared by heating KH2PO4--~ (KPOs)n, where n = 15,000 to 20,000. The water-insoluble K salt is dissolved in buffers with excess Na + ions so that the actual substrate is (NaPO3)n. Samples of 5 ml. of 0.5% substrate solution in acetate or phosphate buffer +1.17 % NaCl (total ionic strength 0.3, of which 0.2 is due to NaC1) + 1 ml. of enzyme solution are set up in capillary Ostwald viscosimeters in a thermostat at 25 °. Viscosity measurements are taken relative to that of the buffer and spe[

\

cific viscosities (~o - 1 = vs,.) calculated. Since the viscosity of metaphosphate solutions is affected by concentration and species of salt present, an ionic factor was introduced. 5 In comparison experiments for the same substrate and substrate concentration, this factor is proportional to m~. at time = 0, and z, the relative measure of enzyme activity, is deThe value of z is found most easily as the fined as z = (~/,~.)t=0 • d(1/v,p.) dt slope of the straight line obtained by plotting the ratio of the specific vis(~.~.)~=0 cosities at time t = 0 and t = t, i.e. (~ls,.)t=t' VS. t, where t is the time from the addition of the enzyme to the beginning of the viscosity measurement plus half the outflow time. Purification Procedure Animal tissue extracts or yeast plasmolyzates containing metaphosphatase have been acetone-dried or precipitated with acetone, without further purification. A. niger mycelia were grown at 30 ° by Mann 1~ on culture media of 10 % glucose, 0.5 % NaNO3, 0.1% MgSO4-7H~O, and variable amounts of K2HP04. They were then extracted with water, dialyzed, precipitated with 3 vol. of cold acetone, redissolved in water, and purified by adsorption on Caa(PO4)2 gel A- alumina C,. The culture media separated from mycelia also show metaphosphatase activity. Dialyzed i~ O. Meyerhof, R. Shatas, and A. Kaplan, Biochem. et Biophys. Acta 12, 121 (1953). 15T. Mann, Biochem. J. 38, 339 (1944).

[92]

METAPHOSPHATASE

579

commercial takadiastase (Parke-Davis) arid clarase (Takamine) were used as the A. oryzae metaphosphatase source; A. niger and PeniciUium expansum molds grown on typical media 4,5 were simply well ground up in buffer, filtered, and the solution dialyzed. P. vulgaris 6 grown on agar plates was treated with acetone to remove lipoids, then dried and extracted by grinding with sand in water or buffer. No pure metaphosphatase has been prepared as yet. Partial purification of a metaphosphatase from rice bran, extracted with water and precipitated with ethanol, was reported by Yoshida. le On fractional precipitation with acetone this author obtained a purified enzyme in the 30 to 50 % acetone fraction.

Properties Stability. Yeast metaphosphatase loses up to 40 % of its activity after incubating for 3 hours at 33 °, but its activity is completely preserved in 50% glycerol solution, pH 7. 3,1°,u Activators and Inhibitors. Divalent metals, especially Co ++ and Mn ++ but also Fe ++ and Mg ++, activate metaphosphatase in acteone-dried liver extracts. 17 A. niger metaphosphatase activity is enhanced by Zn, Mn, Ca, Mg, and Pb and completely inhibited by Ag and Hg. 7 On the other hand, Zn ++ in A. niger growth medium prevents or reduces development of metaphosphatase activity.13 Yeast metaphosphatase is affected qualitatively the same way, but activation is less. Mg, then Mn, Co, and Zn, are potent activators. The pH optimum is displaced in the case of A. niger metaphosphatase, since activation is better at more alkaline pH if other factors remain the same. N a F inhibition of the enzyme is very slight; iodoacetie acid, taurocholic acid, and formaldehyde have no effect. NaCN almost completely inhibits the yeast enzyme, but has very little effect on A. niger activity; arsenite reduces metaphosphatase activity of yeast to 30 to 40 % without any effect on the A. niger enzyme. 7 NAN03, which strongly inhibits glucose oxidase at pH 5 in A. niger, does not affect metaphosphatase. 15 Heating at 50 ° for 10 minutes inactivates metaphosphatase without affecting pyrophosphatase. TM A purified yeast metaphosphatase is appreciably inhibited by N a F and KCN, to a lesser extent by glutathione, azide, and arsenate. Effect of pH. Optimum pH varies for metaphosphatases from different sources. Various Penicillium strains show pH optima between 4.5 and 4.8, as does P. vulgaris with a pH optimum of 4.7. 8 A. niger acts 16 A. Yoshida, J. Chem. Soc. Japan~ Pure Chem. Sect. 72, 677 (1951) [Chem. Abstr. 46, 6677 (1952)]. 17 E. Bamann and E. Heumiiller, Naturwissenschaften 9-8, 535 (1940). ~s A. Schi~ffner and F. Krumey, Z. physiol. Chem. 255, 145 (1938).

580


[93]

optimally at p H 5.7 6 against high molecular weight substrates, at p H about 415 against (NaPOa)~ or (NaPO3)~, although some strains have an optimum p H around 6 against trimetaphosphate. 13 A. oryzae is active at p H 6.6 to 7; S. pombe has a weak effect at p H 5.6, but S. cerevisiae (yeast) shows highest metaphosphatase activity at p H 7 to 7.2. 4,1° The isoelectric point of A. niger metaphosphatase has been reported as 3.1. 5 Molecular Weight Determination. Only A. niger metaphosphatase has been investigated for its physicochemical constants. The molecular weight found is approximately 33,000; sedimentation constant $20 = 3.2S; diffusion constant D20 = 8.8 × 10-7 cm.2/sec.

[93] T r i p h o s p h a t a s e B y INES MANDL and CAI~L NEUBERG

2H20 Inorganic triphosphatases splitting NasP3010 ~ 2Na2HPO4 + NaH2P04 (3 moles of orthophosphate) have been found in yeast, I A . oryzae takadiastase, and normal animal tissues (kidney, muscle)2 as well as in plants 3 and cancerous tissues. 3 Yeast appears to contain at least two different triphosphatases of this type. A . oryzae furthermore contains anH~O other type of triphosphatase which splits NasP3010 ) NaH2P04-[Na4P2OT. 4 I t is possible t h a t the triphosphatase found in myosin 5,6 is of the same type, b u t this has not been proved; however, pyrophosphate was actually isolated after cleavage b y A . oryzae takadiastase. 4 Assay Method

The production of orthophosphate can be followed b y the m e t h o d described for metaphosphatase assay (Vol. II [92]), since triphosphate, like m e t a p h o s p h a t e and pyrophosphate, does not give this reaction. Procedure. Triphosphate substrate can be prepared according to Huber's method, 7 or some commercial triphosphates m a y be used; if i C. Neuberg and H. A. Fischer, Enzymologia 2, 241 (1937). 2 C. Neuberg and H. A. Fischer, Enzymologia 2, 360 (1937). a L. Frankenthal, I. S. Roberts, and C. Neuberg, Exptl. Med. and Surg. 1~386 (1943). 4 C. Neuberg, A. Grauer, and I. Mandl, Enzymologia 14, 157 (1950). 5j. Needham, A. Kleinzeller, M. Miall, M. Dainty, D. M. Needham, and A. S. C. Lawrence, Nature 150, 46 (1942). 6 M. Dainty, A. Kleinzeller, A. S. C. Lawrence, M. Miall, J. Needham, D. M. Needham, and S.-C. Shen, J. Gen. Physiol. 27, 355 (1944). 7 H. Huber, Z. anorg, u. allgem. Chem. 230, 123 (1937).

[93]

TRIPHOSPHATASE

581

they are crystalline they are mostly monomolecular. The product must be recrystallized several times and the pH adjusted to the optimum which differs with the enzyme source. Solutions having a final substrate concentration, of 0.4 to 0.5% after addition of the enzyme are incubated at 37 °. Samples are withdrawn, diluted with 5 ml. of 7% trichloroacetic acid per milliliter, shaken, filtered, and tested for p.1

Purification Procedure Animal organs such as hog kidney or beef muscle and also tumor tissues are freed from foreign matter, chopped, and ground with sand in ten times their weight of water or dried in vacuo, then chopped and suspended in one hundred times their weight of water. 3 (Drying causes some loss of activity.) One hundred grams of fresh organs is cut up, minced for 2 hours with 300 ml. of tap water, and centrifuged after remaining in the icebox for 48 hours.2 One hundred milliliters of the aqueous extract is then precipitated with 250 ml. of acetone, filtered, dried, and taken up in 25 ml. of H20, centrifuged, and washed with another 25 ml. of H20. Both the solid (1.39 g.) and the solution (50 ml.) show triphosphatase activity, 0.2 g. of solid being more active than 7 ml. of solution. 2 Triphosphatase may also be obtained by plasmolysis of top yeast by ether or ethyl acetate2 Fresh and intact bottom yeast does not yield any triphosphatase on plasmolysis, but a solution obtained by shaking dried bottom yeast with glycerol 1:5 for 48 hours at room temperature or maceration juices prepared from this material have triphosphatase activity. 1,3,8~9 Commercial A. oryzae takadiastase (Parke-Davis) and crude potato phosphatase are dissolved in water and after filtration dialyzed.l,8,8

Properties Activators and Inhibitors. Acetone-dried liver extract triphosphatase is activated by Mg but (unlike metaphosphatase) not by Mn. Fe ++ and Co ++ activate but to a lesser extent. 1° Triphosphatase activity of myosin is doubled by Ca ++ 5.6 Of the two triphosphatases present in yeast, that obtained from dried bottom yeast is active only if Mg ions are added, but that obtained from top yeast seems to be independent of the presence of Mg2 The latter enzyme is inactivated by alkali (like pyrophosphatase and a-glycerophosphatase). Heating for one-half hour at 50 ° destroys triphosphatase activity without affecting pyrophosphatase. 9 Top yeast plasmolyzate triphosphatase is completely inactivated by

s C. Neuberg and H. A. Fischer, Enzymologia 2, 191 (1937). 9 A. Schi~ffnerand F. Krumey, Z. physiol. Chem. 255, 145 (1938). 10E. Bamann and E. Heumiiller, Naturwissenschaften 28, 535 (1940).

582


[94]

10 minutes of heating at 100°.3 Triphosphatase of frozen tumor" tissues retains its activity for months2 Effect of pH. The Mg-dependent triphosphatase from bottom yeast has an optimum pH between 7 and 8; top yeast enzyme has a more acid pH optimum2 Tumor tissue triphosphatase tested in veronal-acetate buffer acts best at pH 5.5 to 6. 3 Potato triphosphatase and A. oryzae triphosphatase are most active at neutral pH. A. oryzae enzyme is active also at pH 2.9, s but at pH 8.25 only slight cleavage is produced by takadiastase 8 or by tumor tissues (5 to 6% vs. 100% in 4 days)2 Cleavage of triphosphate to pyrophosphate and orthophosphate by A. oryzae enzyme is optimal at pH values of 6 to 6.3. 4

[94]

Myosin Adenosinetriphosphatase ATP ~- H . 0 -~ ADP ~- H,P04

By S. V. PERRY Assay Method Principle. The assay of myosin adenosinetriphosphatase (ATPase) is most simply carried out by estimating colorimetrically the inorganic phosphate liberated by the enzyme from ATP in the presence of Ca ++ under specified conditions. Reagents 0.05 M ATP (3.1 mg. of 7-minute P per milliliter) Na salt, pH 6.8. This solution may be stored at - 1 5 ° for many months without appreciable hydrolysis. 0.1 M CaCl~. 0.2 M glycine-NaOH buffer, pH 9.1, at 25 °. Enzyme. Myosin stock solution diluted with 0.5 M KC1.

Procedure. The incubation medium, containing 1 ml. of 0.2 M glycine buffer, pH 9.1, 0.2 ml. of 0.1 M CaC12, 0.3 ml. of 0.05 M ATP, and distilled water to bring the volume up to 1.8 ml., is warmed to 25 °, and 0.2 ml. of myosin solution in 0.5 M KCI is rapidly added. The contents of the tube are mixed immediately and incubated for 5 minutes at 25 °. The reaction is stopped by the rapid addition of 1 ml. of 15% trichloroacetic acid. After centrifuging or filtering, an aliquot (1 to 2 ml.) is taken for the estimation of inorganic phosphate by the method of Fiske and SubbaRow 1 or of Allen. 2 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 2 R. J. L. Alien, Biochem. J. 84, 858 (1940).

[94]

MYOSIN ADENOSINETRIPttOSPHATASE

583

Definition of Unit of Activity. The extent of ATP hydrolysis is plotted against myosin concentration, and from the gradient of the linear portion of the graph may be derived a unit of activity in terms of the volume of a hypothetical gas (in microliters) equivalent to the amount of P (in micrograms) liberated by 1 mg. of myosin in 1 hour2 For example,

QP =

(

PX~X--

+ (rag. myosin)

The concentration of protein is 6 × total N content of the myosin 8 solution determined by the micro-Kjeldahl technique. Application of Assay Method to Crude Tissue Preparations. In homogenates and crude tissue extracts satisfactory assays cannot be carried out unless it is possible to make corrections for the activity of other ATPases, apyrases, and systems which simulate ATPase activity. Nuclei have been reported to contain a Ca++-activated ATPase, and in some muscles, e.g., pigeon breast, the ATPase associated with the sarcosomes shows Ca ++ as well as Mg ++ activation. Simulated ATPase activity is shown by enzymic systems which require ATP for the synthesis of various metabolites, and muscle extracts rich in creatine will form creatine phosphate on the addition of ATP. Creatine phosphate ~ and any other acid-labile phosphate esters formed in the homogenate will be estimated as inorganic phosphate under Fiske-SubbaRow or Allen conditions. In homogenates and crude tissue extracts myosin may be present partly as actomyosin and care must be exercised in comparing the enzymic activities of this complex with that of purified myosin preparations assayed under similar ionic conditions (see Properties). For a method of differentiating between the myofibrillar (myosin) and the granular ATPases of rabbit muscle, see Perry. 5 Purification P r o c e d u r e

Myosin is most frequently prepared from rabbit skeletal muscle, and although the procedure given below is designed for use with this tissue, it can be adapted with slight modification for the extraction of myosin from skeletal and cardiac muscle of other animals. The best published preparations are Weber's ~ L-myosin and Szent-GySrgyi's7 "crystalline" myosin, but even these preparations, which have identical propers K. Bailey, Biochem. J. 86, 121 (1942). 4 C. F. Cori, J. Biol. Chem. 165, 395 (1946). 5 S. V. Perry, Biochim. et Biophys. Acta 8, 499 (1952). 6H. H Weber and H. Portzehl, Advances in Protein Chem. 7, 161 (1952). TA. Szent-GySrgyi,"Muscular Contraction." Academic Press, New York~ 1947.

584


[94]

ties, have been shown to contain small amounts (up to 10 to 15%) of impurities. 8,9 Based on the procedures of Edsall 1° and Bailey, 3 the method also incorporates features from Szent-GySrgyi's preparation and is in general similar to that of Mommaerts and Parrish. 8 Muscle is extracted with KCl-potassium phosphate buffer, pH 6.5, as recommended by Guba and Straub, 1~to give a myosin solution containing only small amounts of actomyosin, which complex can be removed by bringing the ionic strength to 0.3 as suggested by Portzehl et al. ~2 Sarcoplasmic proteins are then removed by repeated precipitation by dilution. Step 1. Preparation of Crude Extract. To obtain myosin preparations of high enzymic activity the preparation should be carried out at 0 °, and ice-cold distilled water freed from heavy metal contamination must be used throughout. An adult rabbit is killed by stunning, bled, and rapidly skinned. The dorsal and leg muscles are quickly dissected out and chilled in crushed ice. When completely cold the muscle is minced once in a meat grinder and immediately extracted with 3 vol. of cold KCl-potassium phosphate buffer, pH 6.5 (0.3 M KC1, 0.10 M KH2PO4 and 0.05 M K2HPO4). The suspension is stirred slowly for 15 minutes, and the extract is then centrifuged for 10 minutes at 600 X g. Clarification of the crude myosin extract is achieved by gentle filtration of the supernatant through a paper-pulp pad which has previously been washed with the KCl-potassium phosphate medium. At this stage 100 g. of minced muscle will give 200 to 250 ml. of extract. Step 2. Precipitation of Crude Myosin. The slow addition, with stirring, of the crude extract to 14 vol. of distilled water brings the myosin down as crystalloid particles which give a characteristic sheen to the suspension. Overnight the myosin settles well and can be removed by centrifugation. The resultant gel is transferred to a measuring cylinder, using a little of the supernatant if necessary, the volume of precipitate is noted, and the myosin is brought into solution by addition of sufficient solid KC1 to bring the ionic strength to 0.5. Step 3. Removal of Actomyosin. The pH of the solution is adjusted if necessary to approximately pH 6.6 with a small amount of solid NaHCO~ and with bromothymol blue as external indicator. Water is added to bring the ionic strength of the solution to 0.3. Any precipitate of actomyosin is then removed by centrifugation, and the clear or slightly turbid solution is reprecipitated twice as follows. s W. F. H. M. Mommaerts and R. G. Parrish, J. Biol. Chem. 188, 545 (1951). 9T. C. Tsao, Biochim. et Biophys. Acta 11, 368 (1953). 1oj. T. Edsall, J. Biol. Chem. 89, 289 (1930). ~l F. Guba and F. B. Straub, Studies Inst. Med. Chem. Univ. Szeged 3, 46 (1943). 1~H. Portzehl, G. Schramm, and H. H. Weber, Z. Naturforsch. 5b, 61 (1950).

[94]

MYOSIN ADENOSINETRIPttOSPItATASE

585

Step 4. Reprecipitation of Myosin. To induce the formation of crystalloid needles (Szent-GySrgyi's "crystalline" myosin), distilled water is added to the myosin solution slowly with stirring (glass stirrer) until the ionic strength is 0.04. Some denaturation is liable to occur at this stage, but this can be avoided b y carrying out the " c r y s t a l l i z a t i o n " within 15 minutes at 0 °. The p u r i t y of the preparation is not increased b y "crystallization," but in view of the fact t h a t it does not take place so readily with impure myosin it can be considered to some extent a control of the purity of the preparation. The precipitate is allowed to settle, centrifuged off, and the gel dissolved up b y the addition of solid KC1 to u -- 0.5. The p H is adjusted to 6.5 to 7.0, and the myosin is reprecipitated as before b y dilution to tL = 0.04. After the final precipitation the myosin gel is again dissolved b y the addition of solid KC1 to u = 0.5, and the solution is stored as concentrated as is possible at 0 ° in a flask with a trace of toluene on the stopper and containing the minimum of air space. Yield. Very approximately 100 ml. of 1% solution is obtained from 100 g. of minced muscle. Qp values at 25 ° of this preparation when fresh range from 2000 to 5000. L-Myosin constitutes about 40% of the total proteins of rabbit skeletal muscle, and consequently the ATPase activity per milligram of protein does not increase very much during the preparation. For example in one preparation the Qp values of the myosin after the first, second, and third precipitations were 3300, 4400, and 4700, respectively, measured at 25 ° . Prepared as above, myosin should liberate only one phosphate radical per molecule of A T P , but if on testing the preparation is shown to split a higher proportion of the A T P phosphate than this, adenylate kinase (myokinase) is present. This enzyme can be removed b y further reprecipitation. Reprecipitation of myosin can be carried out at least four times without the appearance of denaturation products, provided t h a t SUMMARY OF PURIFICATION PROCEDURE

(The volumes given were obtained from 100 g. of minced rabbit muscle)

Purification stage 1. Crude extract of muscle First precipitation 2. (Redissolved myosin 3. Actomyosin precipitation Second precipitation 4. Redissolved myosin Third precipitation Redissolved myosin

l

Total volume, (ml.)

pH

Ionic strength

230 3450 75 125 940 80 1000 90

6.5 6.5 6.6 6.6 6.8 6.8 6.8 7.0

0.60 0.04 0.50 0.30 0.04 0.50 0.04 0.50

586


[94]

the total time does not exceed 5 days. The preparation usually contains 5-adenylic deaminase, which is not removed by repeated reprecipitations but which does not attack A T P or ADP.

Properties Within the last decade the question of the identity of myosin and adenosinetriphosphatase has frequently been investigated and often discussed. Claims of the separation of the enzyme from myosin have been made 13,'14 but have either been withdrawn or lacked conviction. More recently, exhaustive attempts at separation employing the butyl alcohol procedure have been unsuccessful, 9 and, in view of the work with sulfhydryl reagents and on the degradation of myosin by trypsin treatment, 1~ the facts strongly suggest that ATPase activity is a specialized function of the myosin molecule. P h y s i c a l Constants. A number of determinations of the molecular weight of rabbit myosin have been made, but the most reliable appears to be that made in Weber's laboratory. 6 By the sedimentation-diffusion method the molecular weight of L-myosin was 850,000. Dubuisson 18 gives the electrophoretic mobility of his/~-myosin (considered to be identical with L-myosin) prepared from rabbit muscle as - 2 . 9 X 10-5 cm.2/v./sec, for the ascending boundary. Kinetic studies by 0uellet et al., ~7 carried out at pH 7.0 and in the presence of 0.001 M CaC12, give a Michaelis constant for myosin ATPase (probably containing some actin) of 1.4 X 10-5 M at 24.6 °. Specificity. Myosin will split I T P and U T P in addition to ATP, and no doubt when tested the other nucleoside triphosphates will be found to be hydrolyzed. Although' I T P is split at a faster rate than A T P at higher substrate concentrations, the Michaelis constant is apparently lower for ATP. 18 It is claimed th at U T P is split 3 to 6 times as fast as ATP. TM Activators. The ATPase activity of myosin is profoundly affected by the presence of ions. If the protein actin is also present, these ionic effects are modified, and consequently the enzymic behavior of myosin in some cases differs from that of actomyosin. 2° 1, B. D. Polls and O. Meyerhof, J. Biol. Chem. 163, 339 (1946). 14R. K. Morton, Nature 166, 1092 (1950). 15E. Mihalyi and A. Szent-GySrgyi, J. Biol. Chem. 201~ 211 (1953). is M. Dubuisson, Biol. Revs. Cambridge Phil. Soc. 25, 46 (1950). 17L. Ouellet, K. J. Laidler, and M. F. Morales, Arch. Biochem. and Biophys. 39, 37 (1952). is W. F. H. M. Mommaerts and K. Seraidarian, J. Gen. Physiol. $0, 401 (1947). 19H. M. Kalckar, Science 119, 478 (1954). 99W. Hasselback, Z. Naturforsch. 7b, 163 (1952).

[94]

MYOSIN ADENOSINETRIPHOSPHATASE

587

MYOSIN. In the absence of salt other than ATP at approximately neutral pH, myosin has little ATPase activity. Ca ++ activates the enzyme, the exact course of the activation depending on the other salts present. Mg ++ inhibits the activity of pure myosin either in the presence or absence of other salts, and effectively counteracts the activation obtained with Ca ++. For example, with M g : C a ratios less than 1, t h e inhibition usually exceeds 90 %.1s Potassium chloride stimulates the efizymic activity, probably in a nonspecific way, both in the absence and presence of Ca ++, and although this effect is well established in the literature there is difference of opinion regarding the qualitative aspects. This is no doubt due to the varying ionic contributions of the buffers used by the various workers. At higher concentrations of KC1 (~ = > 0.2) the activation obtained with Ca ++ falls. ACTOMYOSIN. Activated by Ca ++ and in contrast to myosin, Mg++ will activate the enzyme when other ions are absent or in low concentration. In the presence of 0.1 M KC1, Mg ++ inhibits the ATPase of actomyosin. When actomyosin is present as intact myofibrils, its Mg++-activated ATPase, however, is much less sensitive to KC1, and with fresh preparations the enzyme is strongly activated by Mg ++ in 0.1 M I£C1.21 Inhibitors. Myosin contains appreciable amounts of cysteine, and with fresh preparations free sulfhydryl groups can be readily demonstrated by the nitroprusside test. Low concentrations of heavy metals such as Cu and Hg inhibit the enzyme. Sulfhydryl reagents vary in efficiency for inhibiting myosin ATPase; 2~ e.g., alkylating agents such as iodoacetate and iodoacetamide are not very efficient and need to be used at high concentrations (0.05 to 0.01 M) to produce any effect on the enzyme. On the other hand oxidizing agents such as iodosobenzoate, hydrogen peroxide, and iodine readily bring about inhibition. A most effective inhibitor is p-chloromercuribenzoate, which, in amounts equivalent to the myosin cysteine present, brings about complete inhibition. Cysteine will reverse partial inhibition induced by p-chloromercuribenzoate. (See Barron 2~ for summary of literature.) Ethylenediaminetetraacetic acid inhibits myosin ATPase activity in 0.05 M KC1 but strangely enough activates in 0.6 M KC1. ~4 Effect of pH. When activated by Ca ++ in borate buffer, myosin ATPase shows two optima. One is pronounced at pH 9.2, and the other much smaller at pH 6.5. Similar results are obtained with glycine buffer, but at the alkaline maximum much higher activities are obtained than 21 S. V. Perry, Biochem. J. 48, 257 (1951). ~2 K. Barley and S. V. Perry~ Biochim. et Biophys. Acta 1, 506 (1947). ~s E. S. G. Barron, Advances in Enzymol. 11, 201 (1951). ~4 E. T. Friess, Arch. Biochem. Biophys. 51, 17 (1954).

588


[95]

with borate, and the reported values for the optimum v a r y from 9.0 to 9.5. In this range inactivation of the enzyme is appreciable, and values will be affected both by incubation time and temperature. In the presence of actin the activity-pH curve changes with the result t h a t two nearly equal maxima are obtained.

[95] M g - A c t i v a t e d

Muscle

ATPases

A T P + H~0 --~ A D P + P~

By W. WAYNE KIELLEY Assay Method

Reagents 0.02 M A T P solution, p H 7.0. 0.2 M histidine buffer, p H 6.8 to 7.0 (in 0.15 M KC1). 0.05 M MgC12. Enzyme. Dilute the stock enzyme solution so t h a t 0.1 ml. will hydrolyze 0.1 to 1.0 micromole of A T P under the conditions given below. 5 % perchloric acid (PCA). Reagents for determination of inorganic phosphorus (see Vol. I I I [147]).

Procedure. Tubes containing 0.1 ml. of A T P solution, 0.3 ml. of 0.2 M histidine buffer, 0.1 ml. of 0.05 M MgC12, 0.4 ml. of water, and 0.1 ml. of the diluted enzyme solution are incubated for 0 and 5 minutes at 38 °. The reaction is stopped b y adding 1.0 ml. of 5 % PCA. After centrifugation, aliquots of the supernatants are analyzed for inorganic phosphorus (see Vol. I I I [147]). Under these conditions the activity is a function only of enzyme concentration if not more than 1 micromole of the substrate is hydrolyzed. Definition of Unit and Specific Activity. Activity is expressed here in terms of micromoles of substrate hydrolyzed in 5 minutes under the conditions given above. Specific activity is expressed as micromoles of substrate hydrolyzed in 5 minutes per milligram of enzyme nitrogen (determined b y micro-Kjeldahl procedure--see Vol. I I I [145]). 1 Since the rather 1For protein nitrogen determination in fractions containing ammonium sulfate, a suitable aliquot of the enzyme solution was added to about a tenfold larger volume of 5% PCA, centrifuged, washed once with PCA, then taken up in 1.0 N NaOH and transferred to the digestion flask.

[95]

MG-ACTIVATED MUSCLE ATPASES

589

cumbersome Qp (microliters P per milligram of protein per hour) relationship has been used previously, 2 for comparative purposes these are also given in the table on page 590. Determination of Activity in Crude Muscle Homogenates. Attempts to assay unfractionated homogenates for this specific enzyme are complicated by the fact that the ATPase activity of actomyosin under some conditions is activated by Mg. Furthermore, since the isolated MgATPase may increase somewhat in activity with time, there is some uncertainty concerning the fraction of the total Mg-ATPase actually being determined. Purification Procedure The following procedure is that given by Kielley and Meyerhof 2 with only slight modification. Step 1. Preparation of Crude Extract. Chilled rabbit muscle (hind limb and back) is run through a meat chopper and then suspended in 5 vol. of cold extracting solution (0.1 M KC1, 0.04 M N a H C Q , 0.01 M Na~CO3, 0.001 M KCN) and agitated in a Waring blendor for about 1 minute. The suspension is allowed to stand for about 20 minutes and then centrifuged at 3000 X g for 15 minutes. These and all subsequent operations are carried out at approximately 5 °. The residue is resuspended in 5 vol. of the extracting solution and treated as before. The residue is then reextracted a second time. Step 2. Removal of Actomyosin. The combined extract is diluted with an equal volume of 0.001 M KCN or H20 and after standing for about one-half hour is centrifuged to remove the precipitated actomyosin. Step 3. Precipitation with Ammonium Sulfate. The enzyme is precipitated from the supernatant of step 2 by addition of ammonium sulfate-27 g. per 100 ml. of supernatant. After standing for about 1 hour, the precipitate is removed by centrifugation and washed once with 35% saturated ammonium sulfate. The precipitate is dissolved in a 1:1 dilution of the original extracting solution, and after clarifying at 10,000 X g (10 minutes) the ammonium sulfate precipitation is repeated. The precipitate is dissolved in 0.2 M KC1, 0.02 M NaHC03, 0.001 M KCN, the volume being about equal to the original weight of muscle. Step 4. Separation by Ultracentrifugation. After clarifying by centrifugation at 10,000 X g for about 15 minutes, the ATPase is sedimented by centrifugation at 70,000 X g for 1 hour. The precipitate is suspended in 0.2 M KC1, the volume being about one-fifth the previous volume. For resuspension an homogenizer is advantageous. This solution is centrifuged 2 W. W. Kielley and O. Meyerhof, J. Biol. Chem. 176, 591 (1948); 183, 391 (1950).

590

[95]


at 10,000 X g for 15 minutes, and the precipitate is discarded. A tabular summary of the preparative procedure is given below. SUMMARY OF PREPARATIVE PROCEDURE

Fraction 1. 2. 3. 4. 5.

Crude extract Actomyosin-"free" extract 0.0-0.35 (NH,):SO4 fraction Ultracentrifuged fraction Maximum activity of ultracentrifuged fraction (3 days old)

Total activity, ~M. Pi released/ Specific activity, ~M. Pi released/ 5 min./100 g. 5 min./mg N muscle 4900 2230 4600 4040 7570

2.76 2.26 28.8 93.2 174

QP 124 101 1290 4175 7800

Properties Specificity. The enzyme is specific for the removal of the terminal phosphate of ATP. Activation and Inhibition. This ATPase is activated by Mg and to some extent by Mn. It is inhibited by Ca. The enzyme is also inhibited by F - and by p-chloromercuribenzoate. Effect of pH. The pH optimum for this ATPase is about 6.8 to 7.0; at pH 7.6 and 5.6 the activity is about 50% of that at the pH optimum. These conditions hold only for the Mg and ATP concentrations given above. Physical Nature of A TPase. The behavior of the enzyme on centrifugation indicates that it is associated with submicroscopic particles, and it is probable that many other enzyme activities are also present. The particles contain a high percentage of lipid material (30 to 40% of the dry weight), most of which is phospholipid. It has been observed that the lecithinase of Clostridium welchii, which is specific for the hydrolysis of choline-containing phospholipids (giving phosphoryl choline as a product) inactivates the ATPase, the degree of inactivation being proportional to the extent of phospholipid hydrolysis. Similar Preparations. The isolation of a magnesium-activated apyrase from insect muscle has been reported by Gilmour and Calaby 3 by a procedure similar to that given here. The insect muscle enzyme differs from that of mammalian muscle in (1) pH optimum (7.8 to 8.0)--although the conditions for assay were somewhat different from those described above, a D. Gilmour and J. H. Calaby, Arch. Biochem. and Biophys. 41, 83 (1952).

[96]

POTATO APYRASE

591

(2) in specificity--removing two phosphate groups from ATP but at different rates, and (3) in behavior to centrifugation at 20,000 X g--the insect muscle enzyme does not sediment in this gravitational field.

[96] P o t a t o A p y r a s e ATP ~- 2H20 --~ 5-AYIP ~ 2H3P04 By P. S. ]~RISHNAN

Assay Method Principle. The method consists in estimating the amount of PO, liberated from ATP under standard conditions. Reagents ATP solution. The Ba salt of ATP is converted into the Na salt by precipitation of the Ba as the sulfate 1 or by the use of an ion exchanger. 2 The resulting solution is neutralized with dilute NaOH. Stored frozen, the solution is stable for several weeks. At ice-cold temperature the solution can be stored for about two weeks. For the actual assay the solution is so diluted that each milliliter contains about 500 ~, of 10-minute P04. 0.1 M succinate buffer, pH 6.5. CaC12 solution, 0.5 %, adjusted to approximate neutrality. Enzyme. The stock solution of the enzyme is suitably diluted so as to liberate about 100 ~, of PO4 in the assay. Procedure. The various components are brought to the bath temperature of 30 °. Into a 15-ml. centrifuge tube 1 ml. of the buffer solution is added, followed by 1 ml. of the enzyme solution and 0.2 ml. of CaC12 solution. After mixing, 0.3 ml. of ATP solution is run in, the contents mixed well, and the extract time noted. At the end of 30 minutes 1 ml. of chilled 20 % (w/v) TCA is added, the tube shaken, and the contents diluted to about 7 ml. with water and chilled in an ice bath. A blank is simultaneously run, which differs from the experimental tube in that the ATP solution is added at the end, after the addition of TCA. After the tubes have been chilled for about 10 minutes they are centrifuged, preferably in the cold, and the residues washed once with 3-ml. portions of 2% (w/v) TCA, the wash liquid being added to the main solution in each case. The solutions are now analyzed for PO4. The esti1p. S. Krishnan and W. L. Nelson, Arch. Biochem. 19, 65 (1948). E. C. Slater, Biochem. J. 53, 157 (1953).

592

[96]


mation is easily carried out b y direct development of color after addition of molybdate, H2S04, and reducing agent; but a more accurate value is obtained b y the isobutanol extraction method, 8 especially when the enzyme preparation is v e r y crude. The difference in P04 between the experimental and the blank runs gives the a m o u n t of P04 liberated from A T P b y apyrase. Definition of Unit and Specific Activity. One unit of the enzyme is defined as t h a t a m o u n t which in the standard test outlined above liberates 1 ~, of P04. The specific activity of the enzyme is expressed as units per milligram d r y weight or, better, per milligram of protein. Application of Assay Method to Crude Preparations. With partially purified enzyme preparations it is found t h a t the third phosphate group also is split off A T P .

Purification Procedure The following m e t h o d 4 is a modification of t h a t of Kalckar. 5 About 1 kg. of peeled p o t a t o is ground for 5 minutes in a Waring blendor with SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. Potato extract 2. (NH4)2S04 fraction, 450 g./1. extract 3. Supernatant after dialysis 4. (NH4)2S04 fraction, as in 2 and 3 5. (NH4)2SO4fraction, 30-45 g./100 ml. solution and dialysis

Total Total volume, units, ml. thousands

Specific activity, Recovery, units/mg, dry wt. %

2120

13,740

57

100

355 800

15,630 9,200

1,613

114 67

155

5,555

1,914

40

1,005

66,600

7

1 1. of neutralized M / 1 0 0 K C N , pressed through cheesecloth, and the extract centrifuged in the cold. T h e enzyme is precipitated b y the addition, with mechanical stirring, of 450 g. of powdered (NH4)~S04 for every liter of solution. T h e precipitated material is filtered b y gravity through W h a t m a n filter paper, No. 1, and allowed to drain overnight in the cold. T h e solid material is taken up in water, dialyzed in the cold against running distilled water for 24 hours, centrifuged i n the cold, and the 8 V. Bajaj and P. S. Krishnan, Arch. Biochem. and Biophys. 47, 34 (1953). 4 p. S. Krishnan, Arch. Biochem. 20, 261 (1949). 5 H. M. Kalckar, J. Biol. Chem. 153, 355 (1944).

[97]

MITOCHONDRIAL ATPASE

593

supernatant reprecipitated with (NH4)2SO4 as above. The solid material is filtered, taken up in water, dialyzed, and centrifuged clear. To every 100 mh of this solution 30 g. of finely powdered (NH4)2SO4 is added, with stirring, and the precipitated material is discarded by filtration. To the filtrate 15 g. more of the solid is added, whereupon the enzyme is precipitated. The material is filtered, dialyzed in the cold after dissolving in a small volume of water, and centrifuged clear. The various steps are outlined in the table.

Properties The purified enzyme preparation splits two phosphate groups from ATP. The preparation is, however, still contaminated with traces of other phosphatases. 6 Lee and Eiler 7 have reported an apyrase preparation from potato which catalyzes the hydrolysis of both the labile phosphate groups of ATP at temperatures above 7 ° and of only the terminal group at temperatures lower than 7 °. 6 p. S. Krishnan, Arch. Biochem. 20, 272 (1949). 7 K. Lee and J. J. Eiler, Science 114, 393 (1951).

[97] Mitochondrial A T P a s e A T P + H20 --~ A D P + P~ By W. WAYNE KIELLEY

Assay Method Reagents

0.02 M ATP solution, pH 7.0. 0.20 M histidine buffer (in 0.15 M KC1), pH 7.5. 0.05 M MgC12. Enzyme. Dilute the stock enzyme solution so that 0.1 mh will hydrolyze 0.1 to 1.0 micromole of substrate under the conditions given below. Perchloric acid (PCA), 5 %. Reagents for determining inorganic phosphorus (see Voh III [147]). Procedure. Prepare tubes containing 0.25 ml. of the ATP solution, 0.25 mh of 0.2 M histidine buffer (pH 7.5), 0.1 ml. of 0.05 M MgC12, 0.3 ml. of water, and 0.1 mh of the diluted enzyme. The tubes are incubated at 28 ° for 0 and 5 minutes; the reaction is stopped by adding

594


[97]

1.0 ml. of 5% PCA. After centrifugation, aliquots of the supernatants are analyzed for inorganic phosphorus (see Vol. III [147]). Under these conditions the reaction is a linear function of enzyme concentration when hydrolysis of the substrate does not exceed 1.0 micromole. Definition of Unit and Specific Activity. Activity is expressed in terms of micromoles of substrate hydrolyzed in 5 minutes under the conditions given above. Specific activity is expressed in micromoles of substrate hydrolyzed in 5 minutes per milligram of enzyme nitrogen (determined by micro-Kjeldahl procedure--see Vol. I I I [145]). Determination of Activity in Crude Homogenates. Mitochondria carefully prepared in isotonic sucrose (0.25 M) show very low or no ATPase activity, so that the application of this procedure to crude liver homogenates prepared as described above is of doubtful significance if maximum activity is desired. Other fractions of the cell also possess some ability to hydrolyze ATP, but whether any part of this activity is due to the same enzyme described here is not known.

Purification Procedure This procedure is taken from Kielley and Kielley. I

Step 1. Preparation of Mitochondria. Mitochondria are prepared from 6 to 7 g. of mouse liver by the isotonic sucrose fractionation procedure of Schneider: (see Vol. I [3]). Step 2. Disintegration of Mitochondria. The washed mitochondria are suspended in 20 ml. of 0.003 M K~HPO4 and treated in a chilled stainless steel micro-Waring blendor for 2 minutes (sonic vibration may also be employed). After centrifuging at 20,000 X g for 10 minutes, the supernatant is drawn off and the residue, suspended in 15 ml. of 0.003 M K2HP04, is treated as before. Step 3. Ultracentrifugal Fractionation. The combined supernatant is centrifuged at 110,000 X g for 30 minutes, the supernatant is poured off, and the pellets are suspended by homogenizing in 4.0 ml. of 0.003 M K2HP04. A tabular summary of the preparative procedure is given on page 595.

Properties

Substrate Specificity. When large amounts of enzyme are employed, ADP (or IDP) is slowly hydrolyzed. However, at enzyme concentrations comparable to those employed for activity measurement, only one phosphate is removed from ATP and ADP is not attacked. I T P is hydrolyzed at about one-third to one-half the rate for ATP. 1 W. W. Kielley and R. K. Kielley, J. Biol. Chem. 200, 213 (1953). W. C. Schneider, J. Biol. Chem. 176, 259 (1948).

[98]

INSECT ATPASE

595

Activation and Inhibition. The enzyme is activated b y Mg and is inhibited somewhat b y Ca. The enzyme is markedly inhibited b y ADP. At concentrations equal to t h a t of A T P in the incubation medium about 50 % inhibition is observed. p H Optimum. The p H optimum of the enzyme appears to be about 8.5. However, the p H - a c t i v i t y relationship is influenced b y the relative concentrations of A T P and Mg, and this optimum is observed for a molar ratio of A T P : M g of 2:1. At higher relative concentrations of Mg, the enzyme activity is increased at p H values below 8.5. Stability. The enzyme loses activity rapidly after preparation and m a y retain only about 50 % of its original activity after 24 hours of storage in the refrigerator. SUMMARY OF PREPARATIVE PROCEDURE a

Fraction Whole mitochondria in 0.003 M K_~HP04 (from 13 g. liver) Disintegrated mitochondria Supernatant after centrifugation at 20,000 X g Pellet from supernatant spun at 110,000 X g

Total activity, t~M. P released

Specificactivity, aM. P released/mg. N

415 1062

10.5 26.9

661

Not determined

556

114.0

W. W. Kielley and R. K. Kielley, J. Biol. Chem. 200, 213 (1953).

[98] Insect ATPase A T P -~ 2P + A M P B y DA~cY GILMOUR

This enzyme, which occurs in the mitochondria of insect muscle 1,2 and has been purified from whole muscle, ~ is a water-soluble, Mg-activated ATPase, which splits two phosphate groups from A T P .

Assay Method Activity is determined b y measuring orthophosphate released from 1B. Sacktor, J. Gen. Physiol. 36, 371 (1953). D. Gilmour, Australian J. Biol. Sci. 6, 586 (1953). s D. Gilmour and J. H. Calaby, Arch. Biochem. and Biophys. 41, 83 (1952).

596


[98]

ATP. P is assayed by the Fiske and SubbaRow 4 method, using a Coleman Junior spectrophotometer to measure absorption at 700 mtt. The reaction mixture consists of 0.1 ml. of 0.16 M MgC12, 0.5 ml. of sodium-ATP containing 0.3 mg. of 7-minute P per milliliter (purified by passage through a column of Amberlite IR-100 resin), and 1.0 ml. of enzyme solution containing 5 to 20 ~, of ATPase and 150 ~, of GSH in 0.05 M sodium borate-NaOH buffer, pH 8.0. The reaction mixture is made up in the cold, substrate being added last. It is incubated for 5 minutes at 42 °, and the reaction is stopped by the addition of TCA to a final concentration of 8 %. Protein is removed by filtration (Whatman No. 44 papers), and P determined on the filtrate. Preformed P is measured in a blank tube to which TCA is added immediately after the addition of substrate. Assays are usually made at three different enzyme concentrations, activity being determined from the straight-line relationship between P evolved and enzyme concentration. Protein N is determined by nesslerization after sulfuric acid digestion of a washed TCA precipitate, using the Nessler reagent of ¥anselow. ~ Activity is expressed as Qp, as defined by Bailey. e Purification Procedure

Preparation of Muscle. The best source of the enzyme is the thoracic muscle of flying insects. The larger locusts or flies are convenient material. Thoraces are separated by cutting off the abdomen and pulling off the head in such a way that the thoracic section of the gut is withdrawn with it. The ventral side of the thorax is cut away, and the flight muscles exposed by spreading out the dorsal and lateral parts. Residual fat body is removed, and the flight muscle cut out with scissors or a knife. Step 1. Preparation of Crude Extract. The dissected muscle is dropped immediately into an ice-cold solution containing 0.5 M KC1 and 0.03 M KHCO3 (pH 8.0). When sufficient muscle has accumulated, the mixture is ground with sand in a cold mortar, stirred for li/~ hours in the cold with 10 vol. of the extractant, then centrifuged for 5 minutes at 2500 × g. The supernatant, which represents the crude extract, has a Qp in the presence of Mg of about 500. Step 2. Removal of Actomyosin. Actomyosin is precipitated by dialyzing the crude extract overnight in the cold against 15 vol. of 0.005 M borate buffer at pH 8.0. It is removed by centrifugation for 10 minutes at 3000 X g, washed once with water (pH adjusted to 8.0), and centrifuged again. The two supernatants are combined, given a final centrifu4 C. H. Fiske a n d Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 5 A. P. Vanselow, Ind. Eng. Chem., Anal. Ed. 12, 516 (1940). 6 K. Bailey, Biochem. J. 36, 121 (1942).

[98]

INSECT ATPASE

597

gation at 7000 X g to remove the last traces of actomyosin, and adjusted in volume to fifteen times the original weight of muscle by addition of 0.05 M borate buffer. This fraction retains 70 % of the ATPase originally present and has a Qp of about 2000. An inorganic pyrophosphatase is also present (Qp for the hydrolysis of sodium pyrophosphate, 200). Step 3. Fractionation with Ammonium Sulfate. Solid ammonium sulfate is added to a concentration of 35%, the pH being maintained at 8.0 with NaOH. After standing for 15 minutes in the cold the precipitate is partially consolidated by centrifugation, then separated on Whatman No. 42 paper in a Biichner funnel. The paper and attached protein are extracted with 0.05 M borate buffer. Paper and undissolved protein are removed by filtering again through Whatman No. 42 paper, and the volume of the filtrate is made up to fifteen times the original weight of muscle. This step removes most of the inorganic pyrophosphatase. About 30% of the ATPase is retained, and the Qr is increased to about 3000. SUMMARY OF PURIFICATION PROCEDURE

(All figures refer to the processing of 1 g. of insect muscle)

Fraction 1. KCI extract 2. After removal of actomyosin 3. (NH4)2SO4fraction, 0-35% 4. After treatment with alumina C~

Total Total Specific volume, Units/ml., units, Protein, activity, Recovery, ml. thousands thousands mg./ml. Qp % 10

7.5

75

15.0

500

--

15

3.6

54

1.8

2000

72

15

1.5

22.5

0.5

3000

30

16

1.0

16

0.25

4000

21

Step 4. Treatment with Alumina. The last trace of inorganic pyrophosphatase is removed by treatment with alumina C~. The best concentration of alumina, which is determined by trial, is usually about 1.7 rag. of A1203 per milliliter. After standing for 15 minutes in the cold, the alumina is centrifuged off. The Q, of the supernatant varies usually between 3000 and 5000. More rarely higher values, ranging up to 7000, are encountered. About 20% of the ATPase activity originally present in the muscle is retained. Properties

Specificity. Substrates, in order of effectiveness, are ATP > ITP >> ADP > IDP. Inorganic pyrophosphate and other organic phosphate and

598


[99]

pyrophosphate esters are not split. At 42 ° ATP is hydrolyzed about ten times as fast as ADP. Activators and Inhibitors. The enzyme is optimally activated by Mg at 1 X 10-8 M. There is no activity if Mg is replaced by Na, K, or Ca. Ca inhibits in the presence of Mg. Glutathione is needed for full activity even in fresh enzyme preparations. The ATPase is inhibited by sulfhydryl reagents , p-chloromercuribenzoate producing 96% inhibition at 5 × 10-~ M, and ninhydrin 97% inhibition at 5 X 10-3 M. Iodoacetate is less effective (40% inhibition at

1 X 10-2 M). Effect of pH. The optimal pH range is 7.8 to 8.0. Effect of Temperature. The relationship between temperature and activity follows the Arrhenius equation between 0 ° and 20 °. The optimal temperature for 5-minute tests is 42 °. Activation energy for the hydrolysis of ATP is 29.6 kcal./mole; for the hydrolysis of ADP it is 16.4 kcal./mole. Activity in Relation to Substrate Concentration. The activity/log (S) curve is bell shaped, the optimal substrate concentrations being 1.6 × 10-a M for ATP and 1.4 X 10-8 M for ADP. KATe is 8.6 X 10-4 M; KADp is 3.3 X 10-3 M.

[99] Adenylate Kinase (Myokinase, A D P Phosphomutase) 2 ADP ~ ATP + AMP

By SIDNEY P. COLOWlCK Assay Method Principle. Since this reaction is accompanied by no readily detectable physical or chemical change (e.g., in light absorption, acidity, or labile phosphorus content) it is convenient to convert one of the reaction products to a derivative, the formation of which is readily detectable. For example, one may add an excess of any enzyme system which specifically removes the terminal phosphate from ATP (e.g., hexokinase or ATPase). In the presence of hexokinase and adenylate kinase, the following over-all reaction results: ADP + glucose -* G-6-P + AMP + H +

(1)

When hexokinase is present in excess, the rate of this reaction is proportional to the concentration of adenylate kinase. The resulting reaction may be followed manometrically by measuring CO2 liberation from a bicarbonate buffer, or chemically by measuring the disappearance of

[99]

ADENYLATE KINASE (MYOKINASE, ADP PHOSPttOMUTASE)

599

acid-labile phosphorus. These two procedures, which are essentially procedures for measuring hexokinase activity, are the ones originally used for the detection of myokinase. 1 These and other procedures for hexokinase assay, which should be readily applicable to the assay of adenylate kinase, are described by Crane s and will not be described in detail here. An alternative method is described below for the assay of adenylate kinase. It is based on the detection of A M P formation by the addition of the specific 5r-AMP deaminase of Schmidt. The preparation of the deaminase 3 and its use for the detection of 5-AMP formation 4 are described elsewhere in this treatise. A factor which interferes with the application of this procedure is the difference between the pH optima of the two enzymes involved. Whereas adenylate kinase is optimally active at pH 7.5, the deaminase is essentially inactive at this pH and shows a sharp optimum around pH 6. Although citrate ions shift the pH optimum of deaminase toward the neutral range, 3 they inhibit the activity of adenylate kinase because of its Mg ++ requirement. It is therefore advisable to carry out this assay in two steps, permitting the adenylate kinase reaction to proceed first, then stopping this reaction by addition of a neutral citrate buffer 3 or a strong KC1 solution, 5 and finally adding the deaminase to measure 5'-AMP. The chief advantage of this procedure is that it can be used not only for activity measurements but also for measuring other properties of the adenylate kinase system, such as the equilibrium constant or the effect of activators and inhibitors. It is obvious that certain of such measurements would be difficult or impossible with a one-stage hexokinase assay.

Reagents ADP-MgCI2-Tris mixture. Mix 1 ml. of 0.01 M Na-ADP (Vol. III [118]) with 8 ml. of 0.1 M Tris-HC1 buffer (Vol. I [16]) and 0.5 ml. of 0.1 M MgCI~. Adenylate kinase. Make appropriate dilution in cold H~0 (e.g., dilute crude muscle extract about 1:400). Citrate buffer. 0.1 M pH 6.4, prepared by addition of HC1 to trisodium citrate. Deaminase. Stock solution of fraction 3 (see Vol. II [68]) diluted to contain about 2000 units of deaminase per milliliter. 1 S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148~ 117 (1943). 2 R. K. Crane, Vol. I [33]. 3 See Vol. II [68]. 4 See Vol. I I I [121]. W. J. Bowen and T. D. Kerwin, Arch. Biochem. and Biophys. 49~ 149 (1954).

600

ENZYMES IN P H O S P H A T E

METABOLISM

[99]

Procedure. To 0.15 ml. of the ADP-MgCI.~-Tris mixture in a 3-ml. quartz cuvette, add 0.05 ml. of the diluted adenylate kinase. After 5 minutes at room temperature add 2.8 ml. of the citrate buffer to stop the reaction. Then add 0.03 ml. (60 units) of the deaminase preparation, and measure the optical density at 265 m~ exactly 15 seconds and 30 seconds after mixing. Then continue reading at 1-minute intervals until the rate of change in optical density falls to 0.001 or less per minute. Not more than 10 minutes should be required for complete deamination. To the total optical density change for the period from 15 seconds to the end of the reaction, add the change measured from 15 to 30 seconds, in order to correct approximately for the amount of reaction which occurred in the first 15 seconds after mixing. This cannot be measured directly because the deaminase itself contributes significantly to the initial absorption change at 265 m~. The total optical density change must not exceed 15 % of the actual reading at 265 mu prior to deaminase addition, since the maximum observable change with excess adenylate kinase is only 25% under the condition described here. The amount of 5'-AMP formed is, of course, proportional to the total optical density change; a reading change of 0.09 corresponds to 0.03 micromole of 5'-AS/[P under these conditions. Although the amount of AMP can also be estimated from the initial rate of the deaminase reaction 6 rather than from total optical density change, this procedure is not recommended here because of the possibility that inhibitors or activators of the deaminase might cause false estimates of AMP concentration. Definition of Unit. One unit of enzyme is defined here as that amount which causes the formation of 1 micromole of 5t-AMP per minute under the conditions of the assay. Protein may be determined nephelometrically after precipitation by trichloroacetic acid. 7 Specific activity is expressed as units per milligram of protein. Application of Assay Method to Crude Tissue Preparations. The onestage hexokinase assay is probably superior to the two-stage deaminase assay for this purpose. The presence of "apyrases," which may directly or indirectly result in dephosphorylation of ADP to form AMP, would lead to falsely high values for adenylate kinase in the deaminase assay but not in the hexokinase assay. The presence of deaminase in the crude extracts would of course invalidate the deaminase assay, but not the hexokinase assay. A potent ATPase in the crude extract could invalidate the hexokinase assay, but the excess of hexokinase added would in most cases be sufficient to "compete" successfully with the ATPase. 6 H. G. Albaum and R. Lipshitz, Arch. Biochem. 27, 102 (1950). T. Biicherp Biochim. et Biophys. Acta 1, 292 (1947).

[99]

ADENYLATEKINASE (MYOKINASE, ADP PHOSFHOMUTASE)

601

Purification Procedure The procedure outlined below is essentially t h a t originally described b y Colowick and Kalckar. ~ F u r t h e r purification by adsorption of impurities on alumina C~, followed b y fractionation with trichloroacetic acid, has been reported by Kalckar. 8 More recently, N o d a and K u b y have announced additional purification as the zinc salt2 However, the original procedure yields a preparation which is sufficiently active and free of interfering proteins for most purposes. Step 1. Preparation of Crude Extract. ~° R a b b i t skeletal muscle is cooled, ground, and extracted twice with 1 vol. of cold 0.03 N K O H 0.002 M Versene. The third extraction is with 0.5 vol. of 0.002 M Versene. Step 2. Acidification and Heating. The combined extracts are acidified with 0.05 volume of 2.0 N hydrochloric acid and heated as rapidly as possible to a temperature of 90 °. After 3 minutes at this temperature, the solution is cooled rapidly and neutralized to p H 6.0 to 6.5 with 2 N sodium hydroxide. A very large precipitate is formed which is removed b y filtration. The resulting filtrate shows about sixfold purification and serves as a convenient source of adenylate kinase for m a n y purposes. I t m a y be stored as a solution in the refrigerator for several weeks without appreciable loss in activity. Information on stability at - 1 5 ° is not available. SUMMARY OF PURIFICATIONPROCEDUREa

Fraction

Total protein~ mg.

Total units, ~M./min.

Specific activity, ~M./min./mg.

1. Crude extract (100 ml.) 2. Filtrate after heating in acid (ca. 100 ml.)

2370 320

2250 1500

0.82 4.68

The specific activity recorded here for the crude extract is about one-tenth of that originally observed. ~,c It should be noted, however, that the adenylate kinase may not be saturated with substrate under the conditions of the present assay, in which the ADP concentration is 8 X 10-~ M, as compared with 1.3 X 10-2 M in the original assay, c b S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148, 117 (1943). c H. M. Kalckar, J. Biol. Chem. 148, 127 (1943). 8 H. M. Kalckar, J. Biol. Chem. 148, 127 (1943). 9L. Noda and S. A. Kuby, Federation Proc. 14, 261 (1955). 10The use of alkaline Versene for extraction is primarily for the purpose of isolating 3-phosphoglyceraldehyde dehydrogenase (see Vol. I [60]). The preparation of adenylate kinase was a by-product in this particular case. In the original studies, distilled water was used for extraction.

602


[99]

Step 3. Concentration by Salting Out. F o r convenience in storage, or for removal of nucleotides and other small molecules f r o m fraction 2, the a d e n y l a t e kinase m a y be salted out b y adding a m m o n i u m sulfate to 0.8 saturation. T h e resulting precipitate m a y be filtered off and stored in the cold as a paste or dissolved in the minimal volume of water. N o purification results f r o m this step.

Properties Stability. 1 T h e m o s t r e m a r k a b l e p r o p e r t y of the a d e n y l a t e kinase of muscle is its resistance t o w a r d acid and heat. I n 0.1 N hydrochloric acid at 100 ° , its half-life is almost 30 minutes. Distribution. E a r l y studies 1 on distribution were in error b e c a u s e it was not realized t h a t the stability of the enzyme varies with the source. When various tissues were assayed after boiling with 0.1 N HC1, it a p p e a r e d t h a t muscle was the m a j o r source, none of the e n z y m e being found elsewhere, except for small a m o u n t s in brain and heart. Hence the n a m e m y o k i n a s e was adopted. L a t e r it was shown t h a t when other tissues such as liver 11 and yeast 12 are assayed w i t h o u t boiling in acid, the e n z y m e is readily detected. I t was therefore suggested 13 t h a t the n a m e adenylate kinase would be more appropriate. A s y s t e m a t i c reinvestigation of distribution and stability of the e n z y m e would be desirable. I t is not y e t clear whether the adenylate kinase a c t i v i t y found in h e a r t , ' brain, I and spleen 14 after boiling in HC1 represents all the e n z y m e present in those tissues or just t h a t r e m n a n t which survives the acid t r e a t m e n t . Intracellular Distribution and Function. T h e adenylate kinase is present in the m i t o c h o n d r i a of liver ~5 and muscle tissue. ~6 T h e e n z y m e is p r e s u m a b l y necessary whenever A M P is to serve as a p h o s p h a t e acceptor or A D P as a p h o s p h a t e donor. Evidence for a specific role of the e n z y m e in the relaxation of contracted muscle has been reviewed b y Bailey. ~7 Specificity. Until recently, little had been known concerning the specificity of this e n z y m e except t h a t I D P would not serve as subs t r a t e in place of ADP.lS T h e recent studies of L i e b e r m a n et al., 19 S t r o m -

1: A. V. I/:otel'nikova, Chem. Abstr. 43, 6263 (1949). 12R. E. Trucco, R. Caputto, L. F. Leloir, and N. Mittleman, Arch. Biochem. 18, 139 (1948). 1~S. P. Colowick, in "The Enzymes" (Sumner and Myrb$ick, eds.), Vol. II, Part A, p. 148, Academic Press, New York, 1951. 14E. M. Uyeki, Federation Proc. 14, 295 (1955). 15p. Siekevitz and V. R. Potter, J. Biol. Chem. 200, 187 (1953). 16A. Kityakara and J. W. Harman, J. Exptl. Med. 97, 553 (1953). ~7K. Bailey in "The Proteins" (Neurath and Bailey, eds.), Vol. II, Part B, pp. 1053-5, Academic Press, New York, 1954. is A. Kleinzeller, Biochem. J. 36, 729 (1942). 19I. Lieberman, A. Kornberg, and E. S. Simms, J. Am. Chem. Soc. 76, 3608 (1954)

[99]

ADENYLATEKINASE (MYOKINASE, ADP PHOSPHOMUTASE)

603

inger et al. 2° and others (see in footnote 3 Strominger et al. 2°) have revealed the existence of enzyme systems in yeast and animal tissues which might be termed "nucleoside monophosphate kinases." According to Heppel and Strominger (personal communication) there may be two enzymes involved in liver, one of which is specific for A T P as phosphate donor: A T P + X M P --~ ADP + X D P and the other of which is specific for AMP as acceptor: X T P + AMP --* ADP + X D P In both cases, X may be adenosine, guanosine, uridine, or cytidine. However, according to Lieberman et al., 19 the muscle enzyme with which we are concerned here works only with adenine nucleotides. Activators and Inhibitors. The adenylate kinases of muscle 5,8 and liver 15 are Mg++-activated. Activating effects of Ca ++ have also been described. 5,15 Fluoride 15,~-23 citrate, and Calgon ~ are inhibitors by virtue of their metal-binding action. This is the basis of the finding 2~that fluoride prevents AMP, but not ADP, from functioning as a phosphate acceptor in respiring mitochondria. The adenylate kinase of muscle can be inactivated by warming with H:02 and reactivated by glutathione (GSH) or cysteine. 1 If the enzyme is not subjected to oxidants, glutathione and cysteine are without effect on the activity and need not be added to the assay system. The muscle enzyme is inactivated by commercial pepsin. p H O p t i m u m . Kalckar 8 reported that the muscle enzyme was maximally active at pH 7.5. Bowen and Kerwin 5 also report a value of about 7.5 for assays in the absence of 5/[g++ but find an optimum at pH 6 when Mg ++ is present. Since the latter figures were obtained by the deaminase " r a t e assay" instead of the "extent assay," the value found by Kalckar would appear to be the more reliable figure for the pH optimum of the enzyme. Equilibrium Constant. Kalckar s showed that the adenylate kinase reaction could be demonstrated in either direction. Difficulty was experienced by both Kalckar 8 and Bowen and Kerwin ~ in reaching the same equilibrium position from the two directions. Starting with ADP, all investigators ~,s,24 agree that somewhat more than half is utilized at 2oj. L. Strominger, L. A. Heppel, and E. S. Maxwell, Arch. Biochem. and Biophys. 52, 488 (1954). 2I S. S. Barkulis and A. L. Lehninger, J. Biol. Chem. 190, 339 (1951). 22A. V. Kotel'nikova, Chem. Abstr. 45, 198 (1951). 28E. C. Slater, Biochem. J. 53, 521 (1953). 24L. V. Eggleston and R. Hems, Biochem. J. 52, 156 (1952).

604


[99]

equilibrium. Eggleston and H e m s ~4 report a constant of 0.444 for this reaction a t p H 7.4 and 25 ° with 0.01 M MgC12. According to Bowen and Kerwin, 5 the a p p a r e n t equilibrium constant increases significantly with M g ++ concentration, because of the f o r m a t i o n of a M g - A T P complex with a higher binding constant t h a n t h a t for the M g - A D P complex. The Adenylic Acid Effect. When the s u b s t r a t e for the reaction is A D P , there is a v e r y rapid falling off in the rate of the reaction with time, because of the a c c u m u l a t i o n of 5-AMP, which appears to be strongly inhibitory to the muscle enzyme. 1.8 A T P also appears to be s o m e w h a t inhibitory with the liver enzyme. 15 Slater 23 points out t h a t the 5 - A M P inhibition is readily observed even when a large excess of hexokinase and glucose is present, which m u s t certainly prevent appreciable back reaction of A M P with A T P in a homogeneous system. I t appears t h a t the A M P inhibition is therefore best interpreted as being due to a v e r y high affinity of A M P for the protein, relative to t h a t of A D P . 25 However, it m u s t be k e p t in mind t h a t A M P would not be expected to be an inhibitor of the e n z y m e when the latter is catalyzing the reaction of AS~[P and A T P . I t therefore appears t h a t Slater's ~3,26 use of A M P as an " i n h i b i t o r " of this e n z y m e in a particulate s y s t e m generating A T P oxidatively f r o m A D P could conceivably lead to erroneous results. The generated A T P m i g h t react with A M P within the particles preferentially to reaction with hexokinase and glucose present externally. This could account for the low values for a p p a r e n t A T P synthesis actually observed b y Slater in such a system. ~5Slater23 presents data indicating that ADP has a high affinity for the enzyme, but the conclusion does not appear to he warranted, since the AMP:ADP ratio was maintained constant as the ADP concentration was varied. From the consideration mentioned in footnote a in the accompanyfng table, as well as from the strong inhibitory effect of AMP, it would seem that ADP may actually have a rather low affinity for the enzyme. 26 E. C. Slater, Nature 166, 982 (1950).

[I00]

ATP~CREATINE TRANSPHOSPHORYLASE

605

[lC0] ATP-Creatine Transphosphorylase ATP -b Cr ~ ADP -b C r o P

By LAFAYETTE NODA, STEPHEN KUBY, and HENRY LARDY Assay Method

Principle. The formation of Cr--~P from ATP and Cr is followed by determination of the acid-molybdate labile P of C r o P . Reagents 60% HCIO4. 5 % (NH4)6MoTO24"4H~O. Reducing agent. 0.2 g. of recrystallized 1-amino-2-naphthol-4-sulfonic acid, 12 g. of NaHSO3, and 2.4 g. of Na2SO~ in distilled water to make 100 ml. 0.08 M creatine (soluble at room temperature but not at 3°). 0.10 M MgS04. 0.005 M ATP (Na+), pH 7. 0.40 M glycine (Na+), pH 9.0. A stock reaction mixture of creatine, MgS04, and glycine is prepared by mixing 3.0, 0.6, and 2.4 vol. of their respective solutions. This mixture may be stored at 3 °. For each experiment 2 vol. of the ATP solution is added to 6 vol. of stock mixture to form the reaction mixture.

Procedure. To each incubation tube, 8 ml. of the reaction mixture (containing ATP) is added, and, after equilibrating in a 30 ° bath, 2 ml. of the enzyme is added at its required dilution (1:5000 to 1 : 125,000, depending on the fraction and protein concentration; dilution made with 0.001 M glycine, pH 9, just before use). A 2.0-ml. zero-time .aliquot (removed within 20 seconds after addition of the enzyme) is pipetted directly into Evelyn tubes containing 1.0 ml. of molybdate solution, 0.8 ml. of perchloric acid, and distilled water to bring the total volume to 9.6 ml. Succeeding aliquots were similarly removed at desired times (usually 5, 10, and 15 minutes). No deproteinization is necessury since the amount of protein in the reaction mixture (0.2 ~, to 4 ~//ml.) is too low to interfere. After standing for exactly 30 minutes at room temperature (average about 23°), 0.4 ml. of reducing agent is added. After exactly 10 minutes for color development, the inorganic phosphate is determined with the Evelyn colorimeter (660-m# filter) or the Beckman B spectrophotometer

606


[100]

(660 m~) equipped to handle Evelyn tubes. Suitable blanks and standards are also set up with each run. The zero-time aliquot is used to correct for any inorganic phosphate present in the ATP sample and the small amount of phosphate liberated from ATP by the acid-molybdate. C a l c u l a t i o n o f A c t i v i t y U n i t s . Throughout the concentration ranges of ATP, Cr, and Mg ++ that have been investigated, the enzymatic catalysis does not follow good zero-order or first-order kinetics. The enzymatic activity may be determined from the extrapolated initial velocity under conditions approaching maximal velocity (0.004 M ATP, 0.004 M MgSO4, 0.024 M creatine, 0.096 M glycine-NaOH, pH 9.0 buffer; 30°), since under these conditions the reaction is first order with respect to the enzyme. However, for routine fractionation work, determinations of the initial velocities proved to be somewhat tedious, and the more empirical but reliable method described here was devised for following the enzymatic activity. Under the conditions described below, the reaction follows an apparent second-order kinetics with respect to ATP. The empirical rate equadx

tion may be simply expressed as ~/ = kE[ATP]2[Cr]°; setting kE = k'; dx "'" dt -

x

k'[ATP]2' which integrates to k ' t - a ( a - x ) ' where a is the ini-

tial concentration of ATP, x the amount disappearing in time t (equal to the amount of Cr~-~P formed), and E the enzyme concentration. Using the concentration scale in micromoles per milliliter, the time scale in minx x utes, and the setting a = 1.0 ~M./ml., k't = 1- - -. x A plot of 1 - x vs. t should be linear with a slope proportional to k' (i.e., proportional to the enzyme concentration); this holds up to 40 to 50 % of equilibrium which is attained under these conditions when 80 % of the terminal ~ P of ATP has been transferred to Cr. One unit of enzyme is defined as that amount of enzyme per milliliter of reaction mixture which will catalyze the transphosphorylytic reaction between 0.001 M ATP and 0.024 M creatine in the presence of 0.006 M MgSO4, at pH 9.0 (glycine buffer) and 30 °, and yield an apparent second-order velocity constant, k ' (defined above), equal to 1.0 ml. (micromoles) -1 (minute) -1. The determination of k' must of course be restricted to the apparent second-order portion of the reaction (usually where k' is in the range 0.015 to 0.025). For routine purposes, three measurements are usually made within the apparent second-order range; the k' values are calculated individually, then averaged and the units of the enzyme preparation determined per milliliter. When familiarity is gained with the activity and protein concentration of each fraction, dilutions can be made so that a 2.0-ml. zero-time and 10-minute aliquot are sufficient.

[100]

ATP~CREATINE TRANSPHOSPHORYLASE

607

T h e a p p a r e n t l y anomalous enzyme kinetics result from a severe product inhibition of this highly reversible system, and an inhibition of the reaction (ca. 20%) at the ratio of M g + + / A T P = 6.0; the rate of change of the reaction curve with time tends to slope off rapidly, giving rise to the empirical rate expression described above.


Step 1. Preparation of Crude Extract. T h e b a c k and leg muscles of a rabbit are excised i m m e d i a t e l y after decapitation and thrust into ice. The muscles are passed through a chilled m e a t grinder and homogenized in a Waring blendor (3 minutes per batch) using 2 1. of cold 0.01 M KC1 per kilogram of ground muscle. The thick h o m o g e n a t e is gently stirred for 15 minutes in the cold room before draining and squeezing through cheesecloth. Step 2. Purification with Ethanol. Solid NH4C1 is added to fraction 1 to a concentration of 0.10 M, and the p H is brought to 9.0 with 5 M NH4OH. After stirring for 1/~ hour in an ice b a t h to allow complete precipitation of inorganic salts (primarily MgNH4P04), 1.5 vol. of cold ethanol is added. After stirring for 2.5 hours at 20 °, the denatured protein and salts are removed b y centrifugation for 1/~ hour at 1000 X g and the clear, pale yellow solution is retained. Step 3. Precipitation and Fractional Extraction. T o fraction 2 2.0 M MgSO4 (pH 8.5) is added, with stirring, to a final concentration of 0.03 M, and cold 9 5 % ethanol (1.5 times the volume of 2 M MgS04 used) is added. After stirring for 1/~ hour at 20 °, the precipitate is collected b y centrifugation. T h e precipitate is twice thoroughly resuspended and extracted at 0 ° with 0.07 M MgAc2, p H 9.0, in volumes equal to 6 % and 4 % of the volume of fraction 1. E a c h time the insoluble portion is separated at 2000 X g for 20 minutes. T h e exact volumes of 0.07 M MgAc2 used for extraction and of the combined extracts are noted for the purpose of calculating the a m o u n t of alcohol in the extract. ~ l The calculation, though purely empirical, has been found to be reliable and reproducible and is illustrated by the following example: The volume of combined 0.07 M MgAc~ extracts, 200 ml., minus the volume of 0.07 M MgAc2 used, 160 ml., is assumed to be 60 per cent (v/v) of 95% alcohol, and thus represents 40 X 0.60 = 24 ml. of 95% alcohol. As an approximation there is 200 - 24 = 176 ml. of aqueous 0.36 solution. The volume of 95% alcohol required is thus 176 X ~ - 24 -= 75 ml. of 95% alcohol to bring the extract to 36% (v/v) of 95% alcohol. The alcohol required to raise the concentration from 36 to 50%, neglecting the 36% alcohol precipitate, is also calculated on the basis of the original extract volume; i.e., the alcohol required for 50% concentration minus the alcohol for 36% is: 176 - (75 -{- 24) = 77 ml. of 95% alcohol. In the recrystallization step a similar calculation is made to determine the amount of alcohol and NH40H introduced from the first crystalline sediment.

608


[100]

Step 4. Alcohol Fractionation. To fraction 3 (0 ° and p H 8), cold ethanol is slowly added with stirring to 36% (concentrations of alcohol are expressed in terms of volume per cent of 95% ethanol--assuming t h a t the volumes are additive). After ~ hour at 0 ° the precipitate is removed b y centrifugation at 2000 X g for 20 minutes. E t h a n o l is added to the clear supernatant solution to a final concentration of 50%, and after 1/~ hour at 0 ° the precipitate is collected. The precipitate is dissolved in 30 to 50 ml. of 0.05 M ammonium citrate, p H 9, and dialyzed against 1 1. of 0.05 M ammonium citrate for 5 to 6 hours with stirring and then against two changes of 14 1. of 1.7 X 10-3 M N H 4 O H at about 3 °. A trace of precipitate formed in the dialysis bag is centrifuged off to obtain a clear, colorless solution. The preparation at this stage is usually 85 to 95% pure (45 to 50 units/rag.), and the yields are good (70 to 85%); if lyophilization is desired, one should substitute 0.01 M glycine-NaOH buffer, p H 9.0, for the weakly ammoniacal solution used for the above dialysis. The lyophilized powder appears to be quite stable if kept in the refrigerator. However, lyophilization should not be a t t e m p t e d if crystals are desired. Crystallization and Recrystallization. T h e solution is diluted with 1.7 × 10 -3 M N H 4 O H to a protein concentration of 20 to 30 mg./ml, and while stirring efficiently at 0 ° is slowly brought to 56 % ethanol. The final concentration of N H 4 O H is brought to 3.0 X 10 -3 M b y the addition of 5 M NH4OH. The solution in a small E r l e n m e y e r flask is covered with Parafilm and is allowed to stand at - 1 0 ° with occasional opening and swirling of the flask. ~ Apparently crystallization is induced as the result of a slow loss Of NH~. However, if the N H , O H concentration drops too low, much amorphous precipitate will form which m a y be redissolved b y addition of a trace a m o u n t of 5 M NH4OH. Sometimes a small a m o u n t of amorphous denatured protein forms and must be centrifuged off before crystallization occurs. W i t h o u t seeding, crystallization m a y take two weeks. The protein crystallizes in the form of masses of large elongated needles. Seed crystals m a y be kept in the ammoniacal ethanol (tightly stoppered) for about three m o n t h s at - 1 0 °, after which t h e y begin to deteriorate. T h e crystals are collected at - 1 0 ° b y centrifugation for 1 hour at 2000 X g or preferably 1/~ hour at 15,000 X g and washed with 60% Crystallization is facilitated by the cautious addition of small aliquots, 0.5 -- 2 ml., of gaseous CO~ to the air space in the crystallizing flask (conveniently by needle and syringe from a Dewar of dry ice). Crystallization is allowed to proceed until the crystalline mass first tends to settle, leaving a clear solution above it. This usually required about 2 to 3 days after the first crystals appeared.

[100]

ATP-CREATINE TRANSPHOSPHORYLASE

609

E t O H containing 0.003 M N H 4 O H at - 10 ° and recentrifuged as before2 T h e crystals are then dissolved in 0.003 M N H 4 O H (20 to 30 rag. of protein per milliliter), a n y insoluble material is separated b y centrifugation, and the alcohol and N H 4 O H added to 5 6 % and 3 X 10 -3 M, respectively, taking into account the 6 0 % alcohol retained in the precipitate and the a m o u n t of N H 4 O H used for dissolution. 1 Recrystallization at - 10 ° is hastened b y seeding from the first crop. After complete crystallization (usually a b o u t 2 d a y s after seeding) the p r o d u c t is collected and washed, dissolved in 0.01 M glycine, p H 9.0, and dialyzed against the same buffer at ca. 3 ° . The protein is stable in concentrated solution (3 to 5 %) for several weeks in the refrigerator. If desired, it m a y be lyophilized or placed in the cold room at - 1 0 ° ; a 5 % protein solution will not readily freeze at this t e m p e r a t u r e . A typical purification is summarized in the table. SUMMARY OF PURIFICATION PROCEDURE

(Initially, 1.0 kg. of rabbit skeletal muscle)

Fraction

Total protein, mg.

1. 0.01 M KC1 ext. 40,000 2. 20°, 60% EtOH supernatant 9,050 3. 0.07 M MgAc2 ext. of MgSO~ ppt. 3,250 4. 0.07 M MgAc~, 36-50% EtOH 2,970 5. Crystals 2,480 6. Recrystallized 2,150

Total units

Units/ mg.

Purification

Yield, %

(189,000) a 179,000

(4.73) ~ 19.8

(1.0) 4.18

(100.0) 94.7

44.0 46.5 52.0 52.3

9.31 9.84 11.0 11.1

75.7 73.1 68.2 59.5

143,000 138,000 129,000 112,500

a Small correction was made for trace amount of ATPase activity by assaying in absence of creatine and for trace amount of myokinase activity by substituting ADP for ATP. Other fractions are free of these contaminants. Properties

Purity. T h e crystalline enzyme prepared b y the above procedure is homogeneous as determined b y electrophoresis in several different buffers at p H values of 5.5 to 8.9, b y sedimentation and b y solubility. Specificity. T h e enzyme is specific for the c o m p o n e n t s shown in the The volume of alcohol used for washing is about one-third to one-half the aqueous volume before addition of the alcohol. If crystallization is incomplete the mother liquor and washing are retained and allowed to stand at - 10° until further crystallization takes place (facilitated sometimes by the further cautious addition of gaseous CO~).

610


[101]

initial equation. Neither ADP nor I T P can serve as donors of ~-~P; arginine or creatinine will not serve as acceptors of ~-~P. Activators. The enzyme has an absolute requirement for divalent cations such as Mg ++ or Mn ++. Ca ++ is about half as effective as Mg++. Ba ++ appears to be inactive; Zn ++ and Cu ++ inhibit. In the forward direction the optimum concentration of Mg ++ is equal to the concentration of ATP. In the reverse direction near maximum velocity is achieved by concentrations of Mg ++ equal to that of ADP, but the optimum concentrations of ~Ig ++ are somewhat higher. pH Optimum. The reaction velocity is greatest at pH 9 for the forward reaction and at pH 6 to 7 for the reverse direction. Michaelis constants measured at these respective optimum pH values and at 38 ° are as follows: ATP, 6 X 10-4; Cr, 1.9 X 10-2; ADP, 1 X 10-3; Cr~-~P, 5 X 10-3; all in moles X liter-L Turnover. Under optimum conditions, 1 mole of enzyme (80,000 g.) catalyzes the phosphorylation of 25,000 moles of Cr (forward direction) or 150,000 moles of ADP (reverse direction) per minute at 38 °.

[101] C o u p l i n g of P h o s p h o r y l a t i o n

with Oxidation

By F. EDMUND HUNTER, JR. The direct measurement of oxidative phosphorylation by determining the disappearance of inorganic phosphate or the formation of an organic phosphate compound is a very useful and widely applied procedure. However, if significant amounts of the organic phosphate formed are split by phosphatases or further metabolized with the release of inorganic phosphate, indirect assays must be used. Direct Assay

Principle. The enzyme system and substrate are incubated in a medium containing inorganic orthophosphate and a phosphate acceptor system. Oxygen consumption and the removal of inorganic phosphate are measured. Equipment. A Warburg apparatus or other suitable respirometer for measuring oxygen uptake. 1 W. W. Umbreit, in " M a n o m e t r i c Techniques and Tissue Metabolism" (Umbreit, Burris, and Stauffer, eds.), Chapter 1, Burgess Publishing Co., Minneapolis, 1949.

[101]

COUPLING OF PHOSPHORYLATION WITH OXIDATION

611

Reagents

Oxygen. Although oxygen may be used in the respirometer, air is usually satisfactory. 1 Medium. 2 For each component the final concentration in a 3.0-ml. reaction mixture will be indicated. In addition, a suggested volume and concentration of stock solution will be included in parentheses. Mixed medium can be prepared as convenient. If stock solutions are made essentially isotonic, any component may be omitted and the volume made up with isotonic KC1 or sucrose. 1. Potassium phosphate buffer, pH 7.4, 0.01 to 0.013 M (0.4 ml. of 0.1 M). Tris or glycylglycine buffer may be used in studies with lower phosphate levels. 2. Cytochrome c, 1.0 to 1.5 X 10-~ M (0.05 ml. of 1%). Routinely used by most workers, added cytochrome c is essential in many systems. 3. MgC12, 0.005 to 0.0075 M (0.2 ml. of 0.1 M). Must be added after the phosphate has been well diluted with other additions to avoid precipitation of magnesium phosphate compounds, especially if NaF is being used. 4. ATP-AMP. If an additional phosphate acceptor is used, only catalytic amounts of ATP, 0.001 to 0.002 M, pH 7.4 (0.05 ml. of 0.1 M), are needed. If AMP is to be the final phosphate acceptor, 0.005 to 0.01 M (0.25 ml. of 0.1 M) must be used. 3 5. Hexokinase. Yeast hexokinase, 4 approximately 20% pure, together with glucose is commonly used as a final phosphate acceptor system (0.05 ml. of 5 to 10 mg. of protein per milliliter, 20% pure). This provides a large excess of transferring activity. 6. Glucose, 0.02 M (0.2 ml. of 0.3 M). Sometimes creatine kinase 5 and creatine have been used in place of hexokinase and glucose. The lability of phosphocreatine and unfavorable position of the equilibrium with ATP are disadvantages. 7. NaF, 0.01 to 0.02 M (0.2 ml. of 0.15 M). This inhibitor is commonly used to reduce ATPase and phosphatase activity. It is essential with broken cell preparations but is less critical with intact, washed mitochondria. 2 8. Potassium malonate, pH 7.4.0.01 to 0.02 M (0.3 ml. of 0.1 M). 2j. H. Copenhaver, Jr., and H. A. Lardy, J. Biol. Chem. 195, 225 (1952). 3W. W. Kielley and R. K. Kielley, J. Biol. Chem. 191, 485 (1951). 4See Vol. I [32]. See Vol. II [100].

612


[101]

This inhibitor is added only when it is desired to prevent oxidation beyond the succinate step. 9. Isotonic KC1 (0.15 M) or sucrose (0.25 M). Used to adjust final volume. Sucrose preferable for long incubations. 10. DPN or TPN (0.0002 to 0.0005 M). Essential in some systems. See later discussion. Substrate. 0.005 to 0.01 M, pH 7.4 (0.3 ml. of 0.1 M). Controls without substrate must be run with each experiment. Enzyme system. The amount should yield an oxygen consumption of 3 to 10 microatoms per 30 minutes. Homogenate representing 50 to 300 mg. or mitochondria representing 300 to 500 mg. of original tissue have been used (1.0 ml. of a suspension of mitochondria from 10 g. of tissue in 20 ml. of isotonic sucrose). Incubation Procedure. PREPARATION OF FLASKS. The flasks are kept in cracked ice while being prepared. All components except the hexokinase and glucose are placed in the main compartment of the vessel, with the enzyme preparation being added at the last moment. The hexokinase and glucose are placed in a side arm and tipped in after temperature equilibration. In this way the rather labile phosphorylating systems are never without the protective effects of substrate, ATP, and some oxidative activity. Moreover, the periods of net phosphate uptake and measured oxygen consumption correspond exactly. If the vessels have a second side arm, 0.3 to 0.5 ml. of 30 to 50 % perchloric acid or TCA should be placed there. TEMPERATURE. A temperature of 30 ° is commonly used. If the preparation deteriorates rapidly, temperatures as low as 15° may have advantages. EQUILIBRATION PERIOD. Short equilibration periods (5 minutes) are desirable, but must be demonstrated adequate by experiment. Control flasks should be deproteinized at the end of the equilibration period to give the true zero-time phosphate value. INCUBATION PERIOD. The incubation period is 15 to 30 minutes. STOPPING TEE REACTION. If possible, perchloric acid or TCA should be tipped in to stop the reaction. Otherwise immediate chilling and prompt deproteinization must be carried out. Mix thoroughly, and filter or centrifuge. Measurement of Phosphate Esterified. Inorganic phosphate is determined by any suitable procedure. 6 The ratio between inorganic phosphate removed and atoms of oxygen consumed is the P:O ratio. Under 6O. H. Lowry and J. A. Lopez, J. Biol. Chem. 162, 421 (1946); see also Vol. III [114].

[101]


613

certain circumstances, inorganic pyrophosphate may accumulate, and i n o r g a n i c p h o s p h a t e r e m o v e d is n o t e q u i v a l e n t t o o r g a n i c e s t e r f o r m e d . 7 When changes in phosphate are small, greater accuracy can be achieved TABLE I NUMBER OF PHOSPHORYLATIONS PER ATOM OF OXYGEN CONSUMED WITH DIFFERENT SUBSTRATES

Reaction studied

Number of oxidation steps undergone by substrate

~-Hydroxybutyrate -* acetoacetate D P N H --* D P N ~-Ketoglutarate -* succinate Succinate -~ fumaratef Pyruvate -~ acetate Pyruvate --* acetoacetate Citrate --* succinate Glutamate -~ succinate Malate -* (?) Oxalacetate --* (?) Pyruvate --* 3C02 W 2H~O Caprylate -* (?) Proline -* (?)

1 1 1 1 1 1 2 2 --5 ---

P: Oa Probable in Observed intact cell References 2.5 1.5 3.6 1.7 -2.6 2.6 2.6 2.4 2.1 2.5 1.6 2.1

3.0 3.0 4.0 2.0 4.0 3.0 3.5 3.5 --3.0 ---

b, c c b, d, e b, d, e b b b g g e, g h g

When more than one-step oxidation of the substrate occurs, the P: O ratio will be the average for the two or more steps. b j. H. Copenhaver, Jr., and H. A. Lardy, J. Biol. Chem. 195, 225 (1952). c A. L. Lehninger, J. Biol. Chem. 190, 345 (1951). d H. A. Krebs, A. Ruffo, M. Johnson, L. V. Eggleston, and R. Hems, Biochem. J. 54, 107 (1953). F. E. Hunter, Jr., in "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 1, p. 297, The Johns Hopkins Press, Baltimore, 1951. / With succinate as substrate, the reaction seems largely confined to a single step in short experiments. o R. Cross, J. V. Taggart, G. A. Coco, and D. E. Green, J. Biol. Chem. 177, 655 (1949). h H. A. Lardy, in "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 1, p. 387, The Johns Hopkins Press, Baltimore, 1951. by determining the G-6-P formed, s Occasionally ATP or creatine phosphate formation has been measured. The theoretical and experimental P : O r a t i o s o b t a i n e d w i t h v a r i o u s s u b s t r a t e s a r e s u m m a r i z e d in T a b l e I. 7 R. J. Cross, J. V. Taggart, G. A. Covo, and D. E. Green, J. Biol. Chem. 177, 655 (1949). 8 E. C. Slater, Biochem. J. 53, 521 (1953).

614


[101]

Indirect A s s a y When direct assay cannot be applied, indirect methods have been used to estimate the phosphorylating activity. These assays are of several types. 1. Ability of an oxidation to maintain an ATP-organic phosphate pool at a constant level2 2. Rate of incorporation of p32 into organic phosphates, particularly ATP. 1° If critical conditions are met, quantitative calculations can be made from indirect assays. 1° Application of A s s a y to Crude E n z y m e Preparations

Qualitative detection of oxidative phosphorylation is easily made by noting the removal of some inorganic phosphate or the incorporation of p32 into ATP, but quantitative assay requires careful consideration of possible side reactions and losses which may occur. These are greater and less easy to control or evaluate with crude enzyme systems. Comparisons between tissues are difficult, for the losses due to phosphatases and other factors may be quite different. E n z y m e Preparations 11 Slices. Experiments are confined to indirect assays because of the limited permeability of cells to phosphate, ATP, and acceptor systems. Homogenates. In spite of phosphatase activity, considerable net uptake of inorganic phosphate may be obtained, so limited direct assays may be possible. Mitochondrial Suspensions. Preparations from liver, heart, and kidney have proved exceedingly useful for study of efficiency (P:O ratios) and some studies on mechanisms. Most of the citric acid cycle oxidations and accompanying phosphorylations of a cell occur in the mitochondria. 12 Isolated mitochondria show fairly high efficiency in conversion of substrate energy to ATP energy. ~,13 Even greater efficiency within the cell seems unlikely, but not impossible. The rate of oxidation in mitochondria is determined by pH and by the availability of phosphate acceptors. In addition, recent work suggests that other cellular components may influence the rate even in the presence of excess acceptor systems. ~4 9 V. R. Potter, G. G. Lyle, and W. C. Schneider, J. Biol. Chem. 190, 293 (1951). 10 H. A. Krebs, A. Ruffo, M. Johnson, L. V. Eggleston, and R. Hems, Biochem. J. 54,

107 (1953). xl See Vol. I [1, 2, 3]. 12W. C. Schneider, J. Histoehem. and Cytochem. 1, 212 (1953). 18F. E. Hunter, in "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 1, p. 297, The Johns Hopkins Press, Baltimore, 1951. x4R. B. Johnson and W. W. Aekermann, J. Biol. Chem. 200, 263 (1953).

[101]


615

A g r e a t a d v a n t a g e of m i t o c h o n d r i a is t h e v e r y low A T P a s e a c t i v i t y . Modified Mitochondria. W a s h e d i n s o l u b l e r e s i d u e s / KC1 a g g r e g a t e d m i t o c h o u d r i a , a n d h y p o t o n i c a l l y t r e a t e d m i t o c h o n d r i a 1~ h a v e b e e n u s e d advantageously at times. However, such preparations show greater ATPase activity, cytochrome c and coenzyme requirements, and may s h o w less p h o s p h o r y l a t i o n . P r o p e r t i e s of t h e P h o s p h o r y l a t i n g S y s t e m s

Requirements. M g ++ is e s s e n t i a l for p h o s p h a t e i n c o r p o r a t i o n i n t o A T P a n d for f u r t h e r t r a n s f e r s . A D P ( A M P ) is e s s e n t i a l e i t h e r as a n a c c e p t o r s y s t e m or as a t r a n s f e r m e c h a n i s m t o a s e c o n d a r y a c c e p t o r s y s t e m . W h e t h e r c y t o c h r o m e c, D P N , T P N , etc., n e e d be a d d e d d e p e n d s on t h e e n z y m e p r e p a r a t i o n a n d s u b s t r a t e used. TABLE II EXAMPLES OF AGENTS ~TtnCH UNCOUPLE AEROBIC PHOSPHORYLATION

Approximate molar concentration required for

Uncoupling agent Gramicidin Dinitrocresol Usnic acid Dinitrophenol Mannitol hexanitrate Sodium azide Thiopental Aureomycin 2,4-Dichlorophenoxyacetic acid Quinacrine Arsenite Nitrite

50% uncoupling 4 2 6 1 3 2 4 5 5 2

X 10-7 X 10-6 X 10-6 X 10-5 X 10-~ X 10-4 X 10-4 X 10-4 X 10-4 X 10-8 -1 X 10-2

Essentially complete uncoupling 4 1 2 1 2 2 1

X 10-6 X 10-s X 10-5 X 10-4 X 10-4 X 10-a X 10-3 --5 X 10-3 1 X 10-~ --

The exact percentage uncoupling with a given conccntration of agent will vary with substrate and type of enzyme preparation. Phosphorylations occurring below the DPN level in the electron transport chain are not uncoupled by many of these agents.

Lability. T h e c o u p l i n g of p h o s p h o r y l a t i o n w i t h t h e s e o x i d a t i o n s is e a s i l y i n a c t i v a t e d . I n c u b a t i o n of t h e e n z y m e s y s t e m w i t h o u t s u b s t r a t e , e x p o s u r e t o low t o n i c i t y , t r e a t m e n t w i t h s u r f a c e a c t i v e a g e n t s , a n d m a n y o t h e r c o n d i t i o n s c a u s e a d e c r e a s e or c o m p l e t e loss of p h o s p h o r y l a t i o n . Uncoupling Phenomenon. A n u m b e r of c h e m i c a l a g e n t s , for e x a m p l e 2 , 4 - d i n i t r o p h e n o l , h a v e a v e r y s e l e c t i v e effect in e l i m i n a t i n g or u n c o u ~5A. L. Lehninger, J. Biol. Chem. 190, 345 (1951).

616


[101]

pling phosphorylation with no inhibition of oxidation. The mechanism is not understood. A summary of a number of the uncoupling agents is given in Table II. Comments Qualitative detection of phosphorylation can be made without measuring oxygen consumption. Quantitatively the disappearance of substrate or formation of product may be substituted for and at times preferred to the measurement of oxygen consumption. The addition of few coenzymes is necessary with mitochondrial preparations, but in assaying altered mitochondria and crude tissue preparations the addition of cytochrome c, DPN, TPN, etc., is usually essential. When oxidative phosphorylation occurs, it does not prove that the phosphorylation is coupled with the first-step oxidation of the substrate. The oxidation may proceed through several steps of the cycle or even go to completion. Malonate is fairly effective for blocking at the succinate step. Direct measurement of products will determine how far oxidation is proceeding. The single-step oxidation of malate, isocitrate, and ~-hydroxybutyrate is exactly equivalent to the oxidation of D P N H or T P N H . However, with the oxidation of a-ketoglutarate to succinate and pyruvate to acetate, there is an additional phosphorylation below the D P N - T P N level.

[102]

PANTOTHENATE-SYNTHESIZING ENZYME

619

[102] P a n t o t h e n a t e - S y n t h e s i z i n g E n z y m e By G. DAVID NOVELLI

Assay Method Principle. The enzyme catalyzes the following reaction :1 Pantoate ~- ~-alanine -~ ATP --~ Pantothenate ~ AMP ~ PP In the presence of an excess of ATP the reaction proceeds to completion. The course of the reaction may be followed by measuring the formation of any of the products, but, since crude extracts are contaminated by ATPase, myokinase, and inorganic pyrophosphatase, the most accurate measure of the reaction is the microbiological assay of pantothenate. This assay procedure may be applied to resting cell suspensions, crude homogenates, or extracts. The method of isolation and the characterization of this enzyme was worked out by Dr. W. K. Maas, 2 and we are indebted to him for making available much unpublished information. Procedure. To a series of tubes containing, per milliliter, 10 uM. of ATP, 100 uM. of KC1, 20 ~M. of ~-alanine, 20 ~M. of K pantoate, 10 ~M. of MgS04, and 100 uM. of Tris buffer, pH 8.5, is added varying amounts of enzyme solution. The tubes are incubated at 25 ° for 30 minutes, and the reaction is stopped by heating in a boiling water bath for 3 minutes. The pantothenate is determined microbiologically with a pantothenate auxotroph of Escherichia coli as described by Maas and Davis2 Definition of Unit and Specific Activity. A unit of enzyme activity is defined as that amount resulting in the synthesis of 1 ~M. of pantothenate under the above conditions. Specific activity is expressed as units per milligram of protein. Protein is measured by the turbidimetric method of Bficher. 4

Purification Procedure Step 1. Preparation of Acetone-Dried Cells. Escherichia coli w, ACTCC 9637, is grown with aeration at 37 ° for 24 hours in a minimal medium enriched with yeast extract and hydrolyzed casein2 The cells are harvested by centrifugation with a Sharples centrifuge and washed twice with distilled water. An acetone powder of the cells is prepared by stirring the cells into 20 vol. of ice-cold acetone. The powder is collected on a Bfichner funnel, washed with 10 vol. of acetone, and then with 10 vol. of ether. w. K. Maas and G. D. Novelli, Arch. Biochem. and Biophys. 43, 236 (1953). 2W. K. Maas, J. Biol. Chem. 198, 23 (1952). 8W. K. Maas and B. D. Davis, J. Bacteriol. 60, 733 (1950). 4T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).

620

COENZYME AND VITAMIN METABOLISM

[102]

The powder is finally dried in a vaccum desiccator over P205 and paraffin chips. Step ~. Preparation of Dialyzed Extract. The acetone powder is extracted by suspending it in ten times its weight of 0.01 M phosphate buffer, pH 7.1. The suspension is allowed to stand with occasional stirring for 1 hour, after which the debris is removed by centrifugation. Equally active extracts have been obtained at room temperature and in the cold. The extract is dialyzed against 20 vol. of distilled water; the fluid is changed twice. Step 3. Precipitation with 0.6 Saturated Ammonium Sulfate. The extract from step 2 is brought to 0.6 saturation with solid ammonium sulfate. The precipitate is collected by centrifugation at 15,000 r.p.m, in the Servall angle head in the cold room. The supernatant is discarded. The precipitate is resuspended in one-fifth its original volume of 0.01 M phosphate buffer, pH 7.1. Step 4. Protamine Treatment. The solution from step 3 is treated with 2% protamine sulfate using 0.6 mg. of protamine sulfate per milligram of nucleic acid in the extract. (The nucleic acid is arbitrarily estimated by determining the turbidity produced by an aliquot of the extract when treated with 0.5 ml. of 2% protamine sulfate in 5.0 ml. of 0.01 M phosphate buffer at pH 6.0 and comparing with a standard curve prepared with yeast nucleic acid.) The precipitate is centrifuged off and discarded. Step 5. Fractionation with Ammonium Sulfate. The solution from step 4 is fractionated with solid ammonium sulfate, and the fraction precipitating between 0.25 and 0.5 saturation is collected on the centrifuge. The precipitate is dissolved in 0.01 M phosphate buffer, pH 7.1, using 0.3 vol. of the solution from step 4. Step 6. Second Protamine Treatment. The solution from step 5 is treated again with 2 % protamine sulfate, now using 2 mg. of protamine sulfate per milligram of nucleic acid. The precipitate is centrifuged off and discarded. SUMMARY OF PURIFICATION PROCEDURE Step

Specific activity


Recovery, %

2 3 4 5 6 7 8

1.32 1.37

3870 3260 1810 1750 1500 83O 700

86 55 54 46 25 22

2.4 5.1 8.5

[102]

PANTOTHENATE-SYNTHESIZING ENZYME

621

Step 7. Fractionation with Ammonium Sulfate. The solution from step 6 is again fractionated with ammonium sulfate. The fraction precipitating between 0.3 and 0.46 saturation is collected and dissolved in a minimum volume of 0.01 M phosphate buffer, pH 7.1. Step 8. Treatment with Calcium Phosphate Gel. The solution is now treated with ~g vol. of calcium phosphate gel (26 mg. of solids per milliliter) at pH 6.0 in the cold. The precipitate is centrifuged off and discarded. The enzyme in the supernatant is then purified six-fold. The summary of the purification is given in the table.

Properties Stability. The enzyme is very stable. Storage in distilled water at 6 ° for a week or longer resulted in no detectable loss in activity. The acetone powder may be extracted at room temperature, and the enzyme may be subjected to prolonged dialysis against distilled water without loss of activity. pH Optimum. The enzyme is active over the pH range 7 to 9 with an optimum at pH 8.5. At pH 7.0 to 7.5 phosphate and Tris buffers yield identical activities. At pH 8 to 9 Tris and ammonium hydroxide yield identical activities. Substrate A~inities. The Michaelis constant as determined by the graphical method of Lineweaver and Burk for ~-alanine was 0.46, 0.57, 0.74, and 0.38, and for pantoate it was 1.52, 1.93, and 3.54. Although variable, these data indicate that the affinity for f~-alanine is several times greater than for pantoate. The enzyme is specific for pantoate, pantoyl lactone being inactive with the isolated enzyme, although the lactone has about one-third the activity of pantoate in resting ceils. ATP is the energy source and undergoes a depyrophosphorylation during the reaction. For optimal activity a high concentration approximately that of the substrates is required. Above optimal concentrations, ATP becomes inhibitory. A 50 % inhibition is seen when ATP is in sixfold excess over the substrates. Activators. The enzyme requires both monovalent and divalent cations. The monovalent cation requirement is satisfied by either K + or NH4 +. Ammonium ion is slightly better than potassium. Sodium ions are distinctly inhibitory even in the presence of K + or NH4 +. The requirement for these cations is fairly high, being of the order of 100 to 200 mM. The chlorides are as active as the sulfates. The divalent cation requirement is satisfied by either Mn ++ or Mg ++. Mg ++ is considerably superior to Mn ++. The optimum concentration is 10 mM., above which they become slightly inhibitory. Ca ++ and Zn ++ are not stimulatory and at 10-mM. concentration are slightly inhibitory.

622

COENZYME AND VITAMIN METABOLmM

[103]

Rate. The equilibrium of this reaction lies far to the side of synthesis. Within the limits of measurement the reaction proceeds to the complete conversion of the substrates. In crude extracts the rate is approximately 0.5 uM./mg./hr, at 25 °. Between 15 ° and 45 ° the rate increases three times for each 10 ° rise in temperature. During the 30-minute testing period the rate is constant. NOTE. The purified enzyme is relatively free from ATPase, myokinase, and inorganic pyrophosphatase. With the purified enzymes, rate studies are conveniently measured by running the reaction in Warburg vessels at pH 8.0 in a bicarbonate buffer and following the liberation of COs. COs is produced in a bicarbonate buffer because the over-all reaction results in a net production of acid. Also with the purified enzyme, the reaction may be followed by measuring the formation of A M P with 5-adenylic deaminase (see Vol. II [68]) or by following the production of inorganic pyrophosphate.

[103] T h i a m i n a s e

By AKIJI FUJITA I. Thiaminase from the Viscera of Clam (Meretrix meretrix) Pm.CHs'Th + q- B H ~ Pm'CHs'B q- Th q- H + (Thiamine)

(Base)

Assay Method Principle. The following method (Fujita et al. 1) is based on the fact that the enzymatic activity is interrupted by metaphosphoric acid and the remaining thiamine is determined fluorometrically by the thiochrome method after adsorption on Permutit (HennessyS). As the crude enzyme preparation contains the necessary base, its addition is usually not necessary.

Reagents 0.1 M citric acid-NaOH buffer, pH 5.5. 10% metaphosphoric acid. Enzyme. The viscera of clams are thoroughly ground with a small amount of water and sand, and the mixture is adjusted to pH 4.5 with 1 N HC1 and diluted tenfold. The mixture is kept at 30 ° for 1 A. Fujita, Y. Nose, S. Kozuka, T. Tashiro, K. Ueda, and S. Sakamoto, J. Biol. Chem. 196, 289 (1952). 2 D. J. Hennessy, Ind. Eng. Chem. Anal. Ed. 18, 216 (1941).

[103]

THIAMINASE

623

15 minutes with occasional stirring, and then centrifuged. The supernatant is used for the experiments after suitable dilution.

Procedure. The experimental samples contain enzyme solution (1.0 ml.), 0.02 M citrate buffer of pH 5.5 (2.0 ml.), thiamine (1 ~,), and water in a final volume of 10 ml. For full activity of the enzyme, aniline in a final concentration of 10-3 M is added; in all cases where specific activity was determined aniline was present. Suitable controls are carried out by omitting thiamine and with samples with heat-inactivated enzyme (20 minutes at 100°). After incubation at 50 ° for 1 hour, the samples are deproteinized with 5 ml. of 10% metaphosphoric acid, whereby the enzymatic activity is almost completely interrupted. The supernatant is adjusted to pH 5 with 1.0 N NaOH, and thiamine is determined, after its adsorption on Permutit, by a modification of the thiochrome method. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which decomposes 1 ~ of thiamine in 1 hour under the above conditions, whereby the enzyme concentration is adjusted so that the decomposed thiamine attains more than 50%. Specific activity is expressed as units per milligram of protein. Protein is determined spectrophotometrically by measurement of optical density E (in a cell with a 1-cm. light path) at 280 m~. The protein concentration (mg./ml.) corresponds to 0.625 X E. Purification Procedure

For purification the adsorption by alumina C~ is found to be the most suitable. The adsorption by tricalcium phosphate and acetone precipitation are found also to be useful. Fractionation with ammonium sulfate (most suitable at 0.8 saturation), magnesium sulfate, or sodium sulfate (most suitable at 0.5 saturation) and precipitation with ethanol or methanol were found to be scarcely promising, the specific activity and the yield being low. The removal of nucleic acid by protamine sulfate was found not to be especially helpful. Step 1. Preparation of Crude Extract. Clam viscera is extracted with water as described under Reagents, Enzyme. Step 2. Precipitation with Acetone. To 40 ml. of the ice-cold supernatant of the centrifuged extract (1:10) is added 60 ml. of acetone which has been cooled by solid CO2. The acetone in the precipitate is removed by evacuation. The precipitate is dissolved in 40 ml. of 0.07 M ice-cold phosphate buffer, pH 6.5, and centrifuged. Step 3. Adsorption by Tricalcium Phosphate. Forty milliliters of icecold 15 mg./ml, tricalcium phosphate solution is added to the ice-cold

624

[103]


supernatant of step 2; the solution is adjusted to pH 6 and stirred for 15 minutes. After centrifugation in the cold, the supernatant fluid is discarded; the precipitate is washed with water and eluted with 40 ml. of 0.2 M Na~HPO~. Step ~. Adsorption by Alumina C~. To the supernatant fluid of step 3 is added 40 ml. of 6.0 mg./ml, alumina C~. The solution is adjusted to pH 7 and stirred for 15 minutes in the cold. It is centrifuged in the cold, the supernatant fluid is discarded, and the precipitate is washed with water. It is eluted with 40 ml. of 0.2 M Na:HP04 solution. This solution, when stored in the refrigerator in frozen state, can be kept for several days without detectable loss of activity. TABLE I SUMMARY OF PURIFICATION PROCEDURE FOR CLAM ENZYME

Fraction 1. Raw extract 2. Acetone precipitation 3. Eluate from Ca-phosphate 4. Eluate from Alumina C~

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 40 40

54 52

2160 2080

5.1 0.91

10.6 57.1

100 96

40

36.5

1460

0.44

82.8

68

40

28.8

1150

0.11

262

53

The specific activity of this final solution can usually be attained with adsorption twice by alumina C, alone, without any other treatment. The purification procedure is summarized in Table I.

Properties Specificity. The shellfish enzyme is specific for thiamine, thiamine pyrophosphate, and thiamine derivatives with the 4-amino group of the pyrimidine moiety intact. The 2-position of the pyrimidine moiety or the 5-position of the thiazole moiety can be substituted (lVIurata3). It has no action on thiamine derivatives with a substituted 4-amino group of the pyrimidine moiety, thiothiamine, namely 3-[2'-methyl-4 taminopyrimidyl- (5')]-methyl-4-methyl-5-f~-hydroxyethylthiazole-2-thione (Murata3), thiamine disulfide of Zima,4 allithiamine, namely 2-(2'-methyl4'-aminopyrimidyl-5')-methylformamino-5-hydroxy-A 2-pentenyl- (3)-allyl 8 K. Murata, Bull. Cherr~ Soc. Japan 23, 37 (1950). 4 O. Zima and R. R. Williams, Bet. deut. chem. Ges. 78, 941 (1940).

[103]

THIAMINASE

625

disulfide of Fujiwara et al. 5 and Matsukawa et al. 6 and thiamine propyl disulfide of Matsukawa et al. (SakamotoT). Activators and Inhibitors. The activity of clam and crucian (Carassius carassius) enzyme is markedly activated by a series of aromatic amines, the amino group of which is directly attached to the benzene ring, as aniline and its derivatives as well as heterocyclic amines, such as pyridine, and quinoline, and further some sulfhydryl compounds in a final concentration of 10-3 M (Fujita ct al.1). The activation of crucian enzyme is qualitatively about the same as clam enzyme but quantitatively far less intensive. Sealock et al. s found that m-aminobenzyl-(3)-4-methylthiazolium salt increases the activity of carp enzyme. 2-Methyl-4-aminopyrimidine derivatives inhibit clam and crueian enzyme (Fujita et al.1). Sealock et al. s found that o-aminobenzyl-(3)-4-methylthiazolium salt is the most potent inhibitor to the carp enzyme, and the analogous 2-methyl compound is almost equally efficient, but the 2-4-dimethylthiazolium compound is distinctly less active. In experiments with crude clam and crucian enzyme, FeS04, Fe2(SO4)3, CuS04, and MnS04, in a final concentration of 10-3 M, showed more or less inhibitory action (Fujita et al.1). Effect of pH. The clam enzyme exhibits a sharp optimum for activity at pH 5.0 in citrate buffer (Fu]ita et al.~), the crude enzyme of P a p h i a philippinarum at pH 5 (Fujita et al2), that of Corbicula sandai at pH 5.5 (Kaminishi~°), and the crude crucian enzyme at 6.0 in phosphate buffer (Fujita et al.ll). Sealock et al. ~2 reported the optimum pH of the carp enzyme to be 9.1; Fabriani et al. 1~ reported 4.4. Reddy et al. 14 showed two optima of 3.6 and 6.5 for muscle enzyme. Tenmatay ~5 observed two optima of 3.6 and 9.0 for the quahog clam (Venus mercuriana). Effect of Temperature. The crude enzyme of P a p h i a philippinarum shows an optimum temperature of 60 ° (Fujita et al2), that of crucian enzyme is 43 to 45 ° (Fujita et al.~l). Sealock et al. ~2 found the optimum temperature for the carp enzyme to be 60 °. 5 M. Fujiwara and H. Watanabe, Proc. Japan Acad. 28, 156 (1952). 6 T. Matsukawa and S. Yurugi, Proc. Japan Acad. 28, 146 (1952). 7 S. Sakamoto, Vitamins (Japan) 7, 360 (1954). 8 R. R. Sealock and A. H. Livermore, J. Biol. Chem. 177, 153 (1949). 9 A. Fujita and I. Numata, Seikagaku 18, 63 (1944). l0 K. Kaminishi, Seikagaku 22, 45 (1950). 11 A. Fujita, S. Kozuka, K. Yamazaki, K. Kaminishi, and E. Hasegawa, Seikagaku 22, 205 (1950). 12R. R. Sealock, A. H. Livermore, and C. H. Evans, J. Am. Chem. Soc. 65, 935 (1943). 13 G. Fabriani, A. Fratoni, and M. A. Spadoni, Quaderni nutriz. 10, 98 (1947). ~4 K. K. Reddy, K. V. Giri, and R. Das, Enzymologia 12, 238 (1945). ~ A. L. Tenmatay, Thesis, Fordham University 1950; cited by J. D. Barnhurst and D. J. Hennessy, J. Am. Chem. Soc. 74, 353 (1952).

626


[103]

Heat Inactivation. T h e crude e n z y m e of Paphia philippinarum is ina c t i v a t e d b y heating at 90 ° for 15 minutes (Fujita et al2), t h a t of Corbicula sandai at 100 ° for 10 minutes (Kaminishil0), and crucian e n z y m e at 90 ° for 10 minutes (Fujita et al.11). Necessity of Oxygen. Oxygen is not necessary for the action. Velocity Constants. Sealock et al. 12 tested the carp e n z y m e and ass u m e d the reaction to be monomolecular; the reaction constants k =

co

log c

were found to be practically constant within 120 minutes, n a m e l y 2.3 X 10 -8. According to the view of F u j i t a et al. 18the reaction does not proceed m o n o molecularly b u t bimolecularly b y the base exchange reaction.

II. Thiaminase from the Culture Media of Bacillus thiaminolyticus Matsukawa et Misawa Assay Method Principle. T h e principle is the same as for clam enzyme, described above.

Reagents E n z y m e . A 3-day aerobic culture of B. thiaminolyticus on ordinary b r o t h y f r o m which thiamine has been largely removed b y kieselguhr, is centrifuged, and the s u p e r n a t a n t fluid is used as the enz y m e source. Other reagents are the same as for clam enzyme.

Procedure. T h e procedure is also the s a m e as for clam enzyme, except t h a t the e n z y m e solution is incubated at 30 ° for 1 hour. Purification Procedure F o r purification the adsorption b y alumina C~ is found to be the m o s t promising. The adsorption b y tricalcium p h o s p h a t e and acetone precipitation are also found to be useful. F r a c t i o n a t i o n with a m m o n i u m sulfate ( o p t i m u m : 0.5 saturation) showed relatively lower specific a c t i v i t y and is A. Fujita, Y. Nose, K. Ueda, and E. Hasegawa, J. Biol. Chem. 196, 297 (1952). ~7The culture media used for the study on thiaminase is the ordinary broth culture. It is adjusted to pH 4.5 and 3 g. of kieselguhr is added per 100 ml. and shaken vigorously, whereby the thiamine is largely removed. After centrifugation the supernatant is adjusted to pH 6.5 and sterilized. The bacilli are cultivated in this media at 37 ° for 3 days. At this time the enzymic activity becomes most potent; thereafter it decreases gradually [H. Saiki, T. Kishida, T. Tashiro, and M. Yamadori, Kitasato Arch. Exptl. Med. 28, 121 (1951)].

[103]

THIAMINASE

627

yield. Fractionation with sodium sulfate (optimum: 0.7 saturation) or magnesium sulfate and precipitation with ethanol or methanol were found to be scarcely applicable because of very low specific activity and yield. Step 1. Preparation of Crude Enzyme. Step 1 is the same as described under Reagents, Enzyme, for clam enzyme. Step 2. Adsorption by Alumina C~. To 40 ml. of the ice-cold crude enzyme is added 40 ml. of ice-cold 0.07 M phosphate buffer, pH 6, the pH being adjusted to 6. Forty milliliters of ice-cold alumina C~ (6.0 mg./ml.) is mixed in the cold for 15 minutes and centrifuged. The precipitate is washed with water and centrifuged. The precipitate is eluted with 40 ml. of 0.2 M Na2HPO4 in the cold and centrifuged. Step 3. Second Adsorption by Alumina C~. The eluate from the alumina is adjusted to pH 6, and 40 ml. of ice-cold alumina is mixed and stirred in the cold for 15 minutes. After centrifugation the supernatant fluid is discarded, and the precipitate is washed with water and centrifuged. The precipitate is eluted with 40 ml. of 0.2 M Na~HPO4. After centrifugation the supernatant fluid is used for the experiment. See Table II for a summary of the purification procedure. TABLE II SUMMARY OF PURIFICATION PROCEDURE FOR BACTERIAL ENZYME

Fraction 1. Raw extract 2. First eluate from alumina 3. Second eluate from alumina

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 40

106

4240 9.05

11.7

100

40

74.8

2990 0.23

325

71

40

53.5

2140 0.066

806

51

Properties Specificity. The specificity is the same as for shellfish thiaminase (Murata, TM SakamotoT). The substrate specificities of the two enzymes are given in Table III. Activators and Inhibitors. The crude enzyme is also activated by aromatic amines, heterocyclic amines, and sulfhydryl compounds as shellfish enzyme, but the degree of activation by aniline derivatives is relatively low. That by heterocyclic amines and sulfhydryl compounds, however, is very remarkable. It is also activated by many pyrimidine is K. Murata and A. Ueba, J. Biochern. (Japan) 88~ 309 (1951).

628


[103]

d e r i v a t i v e s a n d sulfa d r u g s such as h o m o s u l f a n i l a m i d e , c o n t r a r y t o fish a n d shellfish e n z y m e ( F u j i t a et al.1). Effect of pH. T h e c r u d e e n z y m e e x h i b i t s a s h a r p o p t i m u m for a c t i v i t y , f a l l i n g t o o n e - h a l f of t h e o p t i m u m a t p H 4.5 a n d 6.5 (Tashiro19). Effect of Temperature. T h e c r u d e e n z y m e s h o w s a n o p t i m u m t e m p e r a t u r e a t 30 t o 37 ° , t h e a c t i v i t y f a l l i n g r e m a r k a b l y a t 20 ° a n d 50 ° (Tashiro19). Heat Inactivation. T h e c r u d e e n z y m e is c o m p l e t e l y i n a c t i v a t e d b y h e a t i n g a t 100 ° for 20 m i n u t e s . Necessity of Oxygen. O x y g e n is n o t n e c e s s a r y for its a c t i o n . TABLE I I I ~UBSTRATE SPECIFICITY OF THIAMINASE a

CH3

I

N(~=C--R1

C~C--R~

I ~ ®1

R2--C(~ C - - C H 2 - - - - N IIv It N(~)--CH

®/

+/ ~

/ CH--S Decomposition by

R1

R2

R~

OH NH--CH3 NH2 NH2 NH2 NIt2 NH~

CHs CH3 CH2OH Et CHs CH3 CH~

CH2CH:OH CHRCH~OH CH~CH~OH CH~CH~OH CH2CH20--Ac C00Et CH~CH~C1

Shellfish BMM b -+ + + + +

+ + + + +

Taken from K. Murata and A. Ueba, J. Biochem. (Japan) 38, 309 (1951). b BMM = Bacillus thiaminolyticus Matsukawa and Misawa. Murata [Bull. Chem. Soc. Japan 23, 37 (1950)] examined the substrate specificity of the enzyme and found that the amino group in the 4-position of pyrimidine is necessary for the enzymic action. If the amino group is substituted by another group, without any further change in the pyrimidine moiety, the decomposition does not take place at all. But, if the amino group of the pyrimidine moiety is intact, the methyl group in the 2-position of the pyrimidine and the side chain in the 5-position of the thiazole moiety can be exchanged without loss of activity. For the detection of the reaction the thioehrome method or diazo method was used. ~9 T. Tashiro, Seikagaku 23, 239 (1952).

[104]

FOLIC AClD CO~JUGASE

629

[1041 Folic Acid Conjugase

By HARRY P. BROQUIST Assay Method Principle. After t r e a t m e n t with folic acid conjugase, the sample is assayed for folic acid content b y microbiological procedures employing Streptococcus faecalis or LactobaciUus casei as described elsewhere. 1-~ Treatment of Sample with Folic Acid Conjugase. Folic acid in natural materials occurs commonly in the form of polyglutamates which contain two or more additional glutamic acid molecules joined in ~,-peptide linkages to the glutamate radicle of the parent molecule. These polyglutamates are termed " c o n j u g a t e s . " The folic acid conjugases m a y be described as a group of enzymes which act on conjugates of folic acid to release substances having "folic acid a c t i v i t y " for S. faecalis or L. casei. These enzymes are distributed quite widely in nature and m a y be divided into two groups: (1) the type present in chicken pancreas, having a p H optimum of 7.0 to 8.0, and (2) the type widely distributed in animal tissues, especially liver and kidney, with an optimum p H around 4.5. Recent evidence indicates that the folic acid activity of natural materials is due in part to citrovorum factor (CF). When yeast extract, a potent source of folic acid heptaglutamate, is treated with folic acid conjugase, the apparent CF content markedly increases, 4,5 from which it has been inferred t h a t CF, like PGA, exists in conjugated form. However, recent work of Silverman and Keresztesy s indicates that !0-formylfolic acid appears as an early product of the action of liver enzymes on bound folic acid, after which 10-formylfolic acid m a y be converted b y other liver enzymes to CF. Although the precise mechanism is not known whereby folic acid conjugates are converted to P G A or CF, it seems advisable to follow the present practice of liberating folic acid activity from its conjugates with crude tissues high in folic acid conjugase. Treatment of Sample with Chicken Pancreas. Homogenize 1 g. of sample with 100 ml. of 0.05 M phosphate buffer, p H 7.2. Autoclave for 1E. E. Snell, in "Vitamin Methods, Microbiological Methods in Vitamin Research," p. 327, Academic Press, New York, 1950. "Methods of Vitamin Assay," p. 231, Interscience Publishers, New York, 1951. 3E. C. Barton-Wright, "The Microbiological Assay of the Vitamin B-Complex and Amino Acids," p. 73, Pitman Publishing Corp., New York, 1952. 4 C. H. Hill and M. L. Scott, J. Biol. Chem. 196, 189 (1952). 50. P. Wieland, B. L. Hutchings, and J. H. Williams, Arch. Biochem. and Biophys. 40, 205 (1952). e M. Silverman and J. C. Keresztesy, Federation Proc. 12~ 268 (1953).

630


[104]

15 minutes at 15 pounds, cool to 37 °, and incubate with 20 mg. of desiccated chicken pancreas 7 under toluene for 24 hours at 37 °. After incubation autoclave the sample for 5 minutes, and then clarify it b y centrifugation or filtration. I t is then ready for microbiological assay. A blank to correct for the folic acid content of the chicken pancreas preparation should be included b y carrying out the above procedure in the absence of sample. Desiccated chicken pancreas can be obtained from Difco Laboratories, Detroit, Michigan, or it can be obtained b y preparing an acetone-dried powder of fresh chicken pancreas.

Alternative Procedures The conjugate-splitting enzyme from hog kidney m a y also be used. The procedure is similar to the m e t h o d just described; desiccated hog kidney 3 or a water extract of fresh hog kidney 1,2 is used. The p H optim u m is 4.5, b u t if a prolonged period of incubation is employed, destruction of C F m a y occur, since C F is extremely labile to mild acid. TakadiaFOLIC ACID (FA) AND CITROVORUM FACTOR (CF) CONTENT OF NATURAL MATERIALS BEFORE AND AFTER TREATMENT WITH FOLIC ACID CONJUGASE PREPARATIONS

Microbiological activity

Sample studied

Enzyme source

Before After enzyme enzyme treatment treatment References FA con(dry tent, ~ ~/g. weight)

Aqueous extract of plasmolyzed yeast Fleisehmann Type 3 yeast extract Difco yeast extract Difco yeast extract Liver Fraction L (Wilson) Liver Fraction L (Wilson)

Hog kidney Chicken pancreas

2.5

50

3.0 53.9 CF content, ~ (dry -y/g. weight) Hog kidney 0.7 59.7 Chicken pancreas 0.7 39.0 Hog kidney 6.1 15.0 Chicken pancreas 6.1 8.1

b c e e e e

FA content determined by microbiological assay with LactobaciUus casei. b O. D. Bird, B. Bressler, R. A. Brown, C. J. Campbell, and A. D. Emmet t, J. Biol. Chem. 159, 631 (1945). P. R. Burkholder, I. McVeigh, and K. Wilson, Arch. Biochem. 7, 287 (1945). CF content determined by microbiological assay with Leuconostoc citrovorum. *V. M. Doctor and J. R. Couch, J. Biol. Chem. 200, 223 (1953). P. R. Burkholder, I. McVeigh, and K. Wilson, Arch. Biochem. 7, 287 (1945).

[105]

d-BIOTIN OXIDASE

631

stase has been suggested for the release of folic acid from its conjugates, 8 but it has been found by some to give irregular results owing to hydrolysis of the folic acid conjugate in the takadiastase by conjugases present in the samples to be assayed. Several experiments giving typical data of the increase in folic acid or citrovorum factor activity from natural materials after treatment with two sources of folic acid conjugase are illustrated in the accompanying table. s V. H. Cheldelin, M. A. Eppright, E. E. Snell, and B. M. Guirard, Univ. Texas Publ. No. 4237, 32 (1942).

[105] d - B i o t i n O x i d a s e B y J. H.

QUASTEL

An enzyme capable of oxidizing d-biotin (Baxter and Quastel ~) with liberation of C02 is present in guinea pig kidney and liver. Rat liver and kidney are only about one-tenth as active as the guinea pig tissues. Slices of guinea pig brain and pigeon liver show no activity. So far, oxidation of d-biotin has been studied only in tissue slices, usually slices of guinea pig kidney cortex. No success has yet attended efforts to prepare a kidney homogenate that retains biotin oxidase activity; it is possible, however, that a mitochondrial preparation will be active.

Assay Method The substrate for the investigation made so far I has been d-biotin carboxyl-C 14. This substance is prepared ~by refluxing sodium cyanide-C 1. with d-3,4-(2'-ketoimidazolido)-2-(co-bromobutyl)thiophane, followed by hydrolysis of the resulting nitrile without prior isolation. The specific activity ranges from 10,000 to 200,000 c.p.m, per milligram in different preparations. The procedure in studying biotin oxidase activity is as follows. In the main compartments of Warburg manometric vessels are placed tissue slices, radiobiotin solution, Ringer-phosphate solution, and any other additions to a total volume of 3.0 or 3.2 ml. The center wells contain 0.2 ml. of 20 % NaOH solution, and the side arms 0.2 ml. of 8 N H,SO,. 1 R. M. Baxter and J. H. Quastel, J. Biol. Chem. 201, 751 (1953). 2 S. B. Baker, D. E. Douglas, and A. E. Seath, Nuclear Sci. Abstr. 5, 802, abstr. 5158

(1951).

632


[105]

The gas phase is air. The vessels are attached to the manometers and incubated at 37 ° usually for 3 hours. At the end of the incubation period, the acid is tipped into the main compartment. This stops further enzymatic activity and serves to drive off any CO2 trapped in the solution. After 20 minutes or more, the units are taken from the bath, and the alkali is removed from the center well. The carbonate present is precipitated as barium carbonate by addition of barium chloride after addition of sufficient sodium carbonate to give a precipitate of about 50 mg. After standing for several hours, usually overnight, the precipitate is filtered off in a weighed sintered glass crucible and assayed for radioactivity.

Properties The destruction of biotin, measured by the release of radioactive carbon dioxide, is accompanied by loss of growth-promoting activity for yeast. 1 The destruction that occurs aerobically is reduced by 90% on replacing air with nitrogen in the gas phase, and 80 to 90 % by the presence of sodium azide at a concentration of 0.01 M. The Michaelis constant (Kin) of the d-biotin-biotin oxidase system is approximately 6 × 10-~ M. d-Biotin oxidation by guinea pig kidney cortex is inhibited by sodium malonate and stimulated by sodium fumarate. It is also inhibited by the presence of a variety of fatty acids, the inhibitory effect increasing with length of the carbon chain. Of the four-carbon acids, n-butyrate, isobutyrate, and crotonate, n-butyrate is the most potent inhibitor, the inhibition being noncompetitive. The results are consistent with the view that biotin oxidation is accomplished by breakdown of the fatty acid component, the carboxyl group being removed as part of a 2-carbon fragment similar to that derived from fatty acids, and that this is subsequently oxidized to carbon dioxide and water via the citric acid cycle. d-Biotin oxidation is inhibited competitively by norbiotin, whose affinity for the enzyme involved is about one-tenth of that of d-biotin. Bis-homobiotin is an inhibitor of the enzyme, its affinity being about five times as great as that of d-biotin. Other biotin analogs that are inhibitors of d-biotin oxidation are desthiobiotin and/-biotin.'

[106]

PANTETHEINE KINASE

633

[106] Pantetheine Kinase B y G. DAVID N0VELLI

Assay Method The enzymatic phosphorylation of pantetheine to form 4'-phosphopantetheine is measured by converting the product to CoA by a second incubation with a protamine-treated extract of acetone-dried pigeon liver. 1 Such an extract is no longer able to carry out the phosphorylation of pantetheine, but it is still effective in synthesizing CoA from phosphopantetheine. This extract also contains the enzymes for the acetylation of sulfanilamide and thus the conversion of phosphopantetheine to CoA, and the quantitative measure of the latter may be carried out simultaneously. Reagents

Assay enzyme. A crude extract of pigeon liver acetone powder (prepared as described in Vol. I [101]) is treated with an equal volume of acid-washed Dowex-1 to remove CoA. The supernatant is treated two times with 1/.~0vol. of 2% protamine sulfate. After removal of the protamine precipitate, the supernatant is free of pantetheine kinase, although it can still effect the conversion of phosphopantetheine to CoA as well as the CoA-dependent acetylation of sulfanilamide. This enzyme is called PRS, protaminetreated supernatant. Pantetheine (stock solution containing 0.1 mM./ml, which must be in the reduced form). ATP (0.05 M) pH 7.0. MgC12 (0.1 M). Phosphate buffer (1 M), pH 7.2. Reagents for CoA assay s (see Vol. I [101] and Vol. III [132]). Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which results in the synthesis of one unit of phosphopantetheine, expressed in CoA units, in 30 minutes under the conditions described below. Specific activity is expressed as units per milligram of protein. Protein is determined by the turbidimetric method of Biicher2 Procedure. To a series of tubes are added 0.05 ml. of stock pantetheine (0.005 ~M), 0.10 ml. of ATP solution (5 ~M), 0.05 of MgC12 (5 ~M),

L. Levintow and G. D. Novelli, J. Biol. Chem. 207, 761 (1954). 2N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948). 3T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).

634


[106]

0.1 ml. of phosphate buffer, und enzyme containing between 0.5 and 2.0 units in a final volume of 0.5 ml. The incubation is carried out at 37 ° for 30 minutes, after which the tubes are immersed in a boiling water bath for 3 minutes to inactivate the enzymes. The tubes are cooled in an ice bath, and to each tube is added 0.1 ml. of cysteine HC1 ( M / l ) , 0.5 ml. of CoA assay mix, 2 0.1 ml. of M/1 Tris buffer, pH 8.0, and 0.15 ml. of assay enzyme PRS to a final volume of 1.5 ml. The tubes are further incubated at 37 ° for 90 minutes. During this period any phosphopantetheine formed in the first incubation is converted to CoA. Since the CoA assay mix contains ATP, Ac, and sulfanilamide, the sulfanilamide will be acetylated in proportion to the amount of CoA present. The reaction is stopped by adding 3.5 ml. of 5% TCA. The tubes are centrifuged, and residual sulfanilamide is determined on a 1.0-ml. aliquot by the method of Bratton and Marshall. 4 This value is compared with control tubes containing 0, 1, and 2 units of CoA. From these values the amount of CoA formed in the test samples can be calculated. This reflects the amount of phosphopantetheine formed by pantetheine kinase in the first incubation. A control tube with the assay enzyme PRS and pantetheine should be included to show that PRS is free from pantetheine kinase. Purification Procedure

Step 1. Preparation of Crude Extract. Acetone-dried pigeon liver powder is extracted with 10 parts of ice-cold 0.05 M KHC03 by stirring for 30 minutes at 5 °. The insoluble residue is removed by centrifuging in a Spinco preparative centrifuge at about 50,000 X g. The pH of the clear, red supernatant solution is usually 7.2 and contains about 41 mg. of protein per milliliter. If the supernatant is to be used to make PRS it is treated with an equal volume of acid-washed Dowex-1; otherwise fractionation is carried out without Dowex treatment. This supernatant should not be aged, since pantetheinekinase is largely destroyed by aging at this stage. Step ~. Protamine Treatment. To the supernatant from step 1 is added, dropwise, with stirring, ~ 0 vol. of 2% protamine sulfate. The precipitate is removed by a Servall centrifuge in the cold room at 5 °. The precipitate is discarded, and the supernatant is treated with ~ 0 vol. of protamine sulfate. The precipitate contains pantetheine kinase and is collected by centrifugation. The supernatant is saved to make PRS. Step 3. Elution from Protamine Sulfate. The gummy precipitate from the second protamine treatment is suspended in 0.05 M phosphate buffer, pH 7.0. Fifty milliliters of buffer is used for each 200 ml. of crude 4A. C. Bratton and E. K. Marshall, Jr., J. Biol. Chem. 128, 537 (1939).

[106]

PANTETHEINE KINASE

635

extract used in step 2. A Potter-Elvehjem homogenizer, employed manually as a mortar and pestle, is useful for dispersing the precipitate. The suspension is stirred for several minutes, centrifuged in the Servall centrifuge at 1~,000 r.p.m., and the precipitate discarded. Step 4. Adsorption and Elution from Calcium Phosphate Gel. The supernatant from step 3 is treated with 75 ml. of calcium phosphate gel (dry weight 29 mg./ml.) which adsorbs almost all the activity. Elution is achieved by a single treatment with 25 ml. of cold 0.2 M K2HPO4. The eluate is dialyzed for 12 hours against 0.04 M KC1 at 5 ° without loss of activity. Attempts to purify the enzyme by further employing ammonium sulfate or cold ethanol fraction invariably result in large losses of activity. A summary of the purification is presented in the table. SUMMARY OF PURIFICATION PROCEDURE

Fraction Original extract Extract of second protamine treatment Eluate from calcium phosphate gel

Protein, Specific T i m e s Recovery, mg./ml, activity purified % 42.8 13.7 10.7

2.8 28.5 41

10 15

80 50

Properties Specificity. The purified enzyme seems to be specific for pantetheine. It has no action on pantothenate or on pantethine unless the S--S group is reduced to --SH. Stability. The enzyme is largely inactivated after standing for 4 hours at 25 °. It is completely inactivated by heating to 50 ° for 10 minutes. The partly purified preparation very slowly loses activity when stored in the frozen state at - 1 0 °, appreciable loss being noted after two months. Effect of Ions. The reaction between pantetheine and ATP does not proceed in the absence of divalent cations. Mn ++ is about one-fourth more effective than Mg ++. Ca ++ is about half as effective as Mn ++, and Co++, Cu ++, and Fe ++ are inactive. The reaction also exhibits a requirement for phosphate ions. If phosphate is excluded, the reaction proceeds at only one-fourth to one-half the maximal rate. Effect of pH. With Mn ++ as activating ion the peak of maximal activity is near pH 6.5, with Mg ++ the peak is nearer pH 7.2. Substrate A.~nity. Maximal rates are observed with pantetheine concentrations of 2 X 10-4 M, and the Michaelis-Menton constant with re-

636

CO:ENZYM:E AND VITAMIN METABOLISM

[107]

spect to pantetheine is about 10-5. An ATP concentration of 6 )< 10-~ M is required for maximal activity, and the reaction rate diminishes again if the ATP concentration exceeds 3 X 10-~ M.

[107] T h i a m i n o k i n a s e Thiamine ~ ATP --~ T P P ~ A M P

By H. G. K. W:ESTENBRINK The phosphorylation of thiamine by ATP under the influence of protein precipitated by ammonium sulfate from Lebedew juice was discovered by Weil-Malherbe. 1

Assay Method

Principle. The method to be described was developed by SteynParv~. ~ The enzyme is incubated with thiamine and ATP in phosphate buffer at pH 7.0 in the presence of Mg ions. The reaction is stopped by boiling at pH 3, and the T P P formed is determined by the manometric method (decarboxylation of pyruvate under the influence of alkalinewashed dried brewer's yeast and TPP). Reagents 0.02 M ATP solution (potassium-salt). 0.1 M MgSOt solution. 0.1 M phosphate buffer, pH 7.0. 0.1 M phosphate buffer, pH 6.2. O.O5 N HCh Brewer's yeast dried at room temperature. 2-Methyl-4-amino-5-ethoxymethylpyrimidine ("pyrimidyl"), mg./ml. 10% KOH. 2.5% sodium pyruvate in 0.1 M phosphate buffer, pH 6.2. 0.2 M Na~HPO~ solution, pH about 10. 0.1 M MnCl~ solution. Standard solutions of T P P (0.2, 0.1, and 0.05 ~//ml.).

15

Procedure. The reaction mixture has a volume of 5 ml. and contains: thiamine-HC1, 5 mg.; 0.02 M K-ATP, 0.5 ml.; 0.1 M MgSO4, 0.5 ml.; enzyme solution; 0.1 M phosphate buffer, pH 7.0, to volume. 1 H. Weil-Malherbe, Biochem. J. 33, 1997 (1939). E. P. Steyn-Parv~, Biochim. et Biophys. Acta 8, 310 (1952).

[107]

THIAMINOKINASE

637

Incubate at 27 ° for an appropriate length of time (usually 60 minutes). Transfer 1 ml. of the reaction mixture into 5 ml. of boiling 0.05 N HC1 at the beginning and the end of the period of incubation, and continue the boiling for 1 minute to stop the reaction. After cooling adjust to pH 6.2 with 10% KOH and make up to 10 ml. with 0.1 M phosphate buffer, pH 6.2. Spin down the protein precipitate, and take an aliquot for determination of TPP. Comments. 1. The method described can also be applied to crude tissue preparations, homogenates, and cell suspensions. 2. The high amount of thiamine (5 mg.) is required only when crude enzyme preparations from baker's yeast are used, as they contain much phosphatase, which would decompose the TPP formed if not inhibited by a relatively high concentration of thiamine2 3. In Weil-Malherbe's experiments 1 the thiaminokinase is not separated from apocarboxylase. He therefore measures the C02 production from added pyruvate, during the formation of TPP by the thiaminokinase. It is obvious that this procedure cannot give exact measurement of thiaminokinase activity. The same applies to the procedure of NguyenVan-Thoai and Chevillard, 4 who omit to stop the thiaminokinase action before determining TPP. Determination of T P P ~ (range 0.05 to 0.2 ~/ml.). Two-tenths milliliter of a 2.5% solution of sodium pyruvate in 0.1 M phosphate buffer, pH 6.2, is placed in the side arm of a conical Warburg vessel (capacity 10 to 15 ml.). One milliliter of the solution containing a suitable amount of TPP and 0.5 ml. of a suspension of alkaline-washed yeast (both pH 6.2) are measured into the main compartment. After 10 minutes' equilibration in a water bath of 27 °, the pyruvate is tipped in. Readings of the COs evolved are taken 10, 20, and 30 minutes later. To obtain a standard curve for calculating results, each run must include besides the test solutions a blank and three solutions of known TPP content, viz., 0.05, 0.1, and 0.2 ~,/ml. Preparation of A l k a l i n e - W a s h e d Yeast. 3 Brewer's yeast is dried in a thin layer at room temperature. One gram of the dried yeast is suspended in 20 ml. of distilled water. An equal volume of 0.2 M Na2HP04 solution, which has previously been brought to a pH of about 10 with concentrated NaOH, is added. The final pH should then be about 8.5 and can be adjusted if necessary. The temperature should be 16 to 20 °. After 5 minutes of stirring the yeast is separated by centrifugation for 1 minute at 3000 r.p.m. The supernatant is discarded, and the yeast is rapidly washed three times on the centrifuge with 40 ml. of distilled water. The H. G. K. Westenbrink and E. P. Steyn-Parv6, Intern. Z. Vitaminforsch. 21, 461 (1950). Nguyen-Van-Thoaiand L. CheviUard, Bull. soc. chim. biol. $1, 204 (1949).

638


[107]

whole procedure must be carried out as rapidly as possible to minimize denaturation of the apocarboxylase and should not require more than 15 minutes in all. Then 1 ml. of 0.1 M MnC12 and 0.25 ml. of a " p y r i m i d y l " solution, containing 15 mg./ml., are added to the washed yeast, which is suspended to a volume of 6 ml. with 0.1 M phosphate buffer, pH 6.2. This amount of yeast suspension suffices for ten Warburg flasks. Comments. 1. Leuthardt and Nielsen 5 use apocarboxylase prepared according to Weil-Malherbe, 1 as they had difficulty in adequately removing T P P from brewer's yeast by alkaline washing. The former preparation is much more laborious, especially when purification is pursued so far that thiaminokinase is also removed. Therefore the use of alkalinewashed dried brewer's yeast is to be preferred. According to extensive experience in the author's laboratory this washing never fails, provided that the exact pH at which the yeast should be treated is determined in preliminary experiments. If the pH is too low, not all TPP is removed; if it is too high, the apocarboxylase is damaged. For a certain yeast the suitable pH may be somewhat higher or lower than 8.5. It may be necessary to redetermine this pH after the dried yeast has been stored for some time, as it sometimes tends to shift as the yeast ages. 2. Usually 0.2 rag. of thiamine is added to each gram of alkalinewashed yeast instead of 3.75 mg. of " p y r i m i d y l " in order to inhibit the yeast phosphatase. In this case the adding of thiamine should be avoided as the alkaline-washed yeast is not absolutely free of thiaminokinase. Purification Procedure The best material to prepare the enzyme from is fresh yeast. Preparations have also been made from rat liver 5 and dog liver, 4 but they will not be described here as the most potent samples obtained are much less active than those derived from yeast. Step 1. Preparation of Crude Extract. A. FROM BREWER'S YEAST.4 Two hundred grams of freshly pressed brewer's yeast is triturated with 20 to 30 g. of NaC1. After plasmolysis, 1 1. of 0.2 M phosphate solution, pH 10.1, is added. After 10 minutes the mixture, which has a pH of 9.4 to 9.6, is centrifuged. The supernatant is discarded, and the yeast residue is suspended in 200 ml. of distilled water. The suspension is kept at 37 ° for 2 to 3 hours and then centrifuged again. The supernatant contains the enzyme and can be further purified by fractionation.e 5 F. L e u t h a r d t and H. Nielsen, Helv. Chim. Acta 35, 1196 (1952). Crude and dialyzed extracts of brewer's yeast form a b o u t 8 -y of T P P per milligram of protein N in 60 minutes. Crude extracts of baker's yeast form 5 to 7 -y of T P P under the same conditions. Dialyzed baker's yeast extracts form from 22 to 30 -y.

[107]

TIIIAMINOKINASE

639

B. FROM BAKER'S YEAST.2 Fresh, pressed baker's yeast, as delivered by the factory, is washed three times with 10 vol. of distilled water. Portions of 10 g. of the washed yeast, contained in metal centrifuge tubes, are immersed in a mixture of acetone and solid CO2 (temperature approximately - 7 0 °) for 15 minutes and then thawed by dipping the tubes in cold water. This process of alternate freezing and thawing is repeated twice. Solid KC1 is then added to the liquefied yeast until a molarity of 0.5 is obtained, and the mixture is shaken in a water bath of 37 ° for 3 hours. After standing overnight in a refrigerator, the yeast macerate is centrifuged, and a slightly opalescent light brown fluid, with a good thiaminokinase activity, is obtained. Forty grams of fresh yeast yields about 25 ml. of extract. Comments. 1. The thiaminokinase activity of both crude extracts described above does not differ much. The extract from brewer's yeast contains more carboxylase, i.e., more preformed TPP. Lebedew juice is much less active. 2. If large amounts of baker's yeast are frozen at a time, the freezing time must be lengthened considerably, and the extracts are not so active as when small portions are frozen. Step 2. Fractionation with A m m o n i u m Sulfate. 2 During fractional pre~ cipitation of the yeast extract with saturated ammonium sulfate solution the bulk of the thiaminokinase comes down when the amount of ammonium sulfate solution in the mixture is raised from 45 to 60 vol. %. The increase of the activity per milligram of nitrogen as compared with the original extract is about tenfold. After the precipitate is dissolved in distilled water the enzyme is reprecipitated by fractionation with saturated ammonium sulfate solution, but it separates when the amount of ammonium sulfate in the mixture is raised from 55 to 65 vol. %. This precipitate is completely free of phosphatase. However, the activity per milligram of nitrogen is not higher than that of the first precipitate. Comments. 1. In the case of baker's yeast extracts the increase of activity per milligram of protein N as a consequence of ammonium sulfate precipitation depends in part on removal of unknown inhibitors (inorganic and organic), for the activity is increased about fivefold simply by dialysis of the crude extract against 0.02 M KC1. 2. According to Nguyen-Van-Thoai and Chevillard 4 the enzyme precipitates at somewhat lower ammonium sulfate concentrations, but from their communication it is not clear whether they have allowed for the lower solubility of ammonium sulfate in yeast extracts as compared to water and for the volume constriction occurring when solid ammonium sulfate is dissolved in water or aqueous solutions.

640


[108]

3. Salting-out analysis according to Derrien shows 2 that the ammonium sulfate precipitates of yeast extracts contain several protein components, incompletely separated in the salting-out diagrams, so that it will not be possible to purify the enzyme further by ammonium sulfate fractionation without incurring enormous losses.

Properties Activators and Inhibitors. 2 The baker's yeast enzyme is activated by Mg, Mn, and orthophosphate. Maximal activation with 5~g is attained at 2.10 -2 M. At a lower concentration level (2.10 -3 M) Mn is slightly superior to Mg as activator, but with increasing concentration the stimulation by Mn decreases to zero, whereas the influence of Mg increases steadily. For orthophosphate the optimal concentration is about 2.10 -3 M; strong inhibition is observed in M phosphate. The crude extracts from baker's yeast contain inhibitors, both inorganic and organic, which can be removed by dialysis. Effect of pH. The enzyme has a broad range of optimal action between pH 6 and 7.5. 3 Stability. The enzyme is much more stable in the crude or dialyzed extracts than after ammonium sulfate fractionation. 2

[108] F l a v o k i n a s e Riboflavin + ATP --* F M N + ADP

By EDNA B. KEARNEY

Assay Method Principle. The F M N formed during the enzymatic reaction is measured in a protein-free filtrate by an adaptation of the method of Burch et al. 1 The method 2 is based on the different distributions of F M N and riboflavin between benzyl alcohol and water. After extraction of the protein-free filtrate with benzyl alcohol, the flavin content of the water phase may be estimated spectrophotometrically at 450 m~ or fluorometrically, and the F M N content is calculated from a formula derived from the distribution coefficients of the flavins between benzyl alcohol and the aqueous solution (see Vol. I I I [141]). Reagents Riboflavin stock solution. 37.6 mg. of riboflavin is dissolved in 500 ml. of H20 (2 × 10-4 M); any undissolved material is ill1H. B. Burch, O. A. Bessey, and O. I-I. Lowry, J. Biol. Chem. 175, 457 (1948). 2 E. B. Kearney and S. Englard, J. Biol. Chem. 198, 821 (1951).

[108]

FLAVOKINASE

641

tered off, and the actual concentration is determined by light absorption at 450 m~, using the molar extinction coefficient, = 12.2 X 106 sq. cm. mole-~. The solution should be stored in the cold in a dark bottle, and the concentration should be redetermined occasionally if it is used over a period of months. Acid solutions of riboflavin are more stable but the solubility is no greater. More concentrated solutions may be made in alkali, but these must be used immediately, since the flavin deteriorates rapidly in alkaline solutions. 0.75 M Tris-HC1 buffer, pH 8.2 at 30 °. ATP, 3 MgSO4 at any convenient concentration. 17.5 % trichloroacetic acid. 2.4 M K2HP04. Benzyl alcohol. Merck reagent grade; need not be redistilled. The reagent is saturated with H20 at room temperature. Chloroform. Reagent is saturated with H20 at room temperature. Enzymatic Reaction. The enzyme is assayed in low-actinic test tubes in a 5.0-ml. reaction mixture containing 7.5 × 10-2 M Tris buffer, 1 X 10-~ M riboflavin, 1.0 × 10-3 M ATP, 1.0 X 10-3 M MgSO4, and 50 to 150 units of enzyme. (See definition below.) The mixture is incubated for 2 hours at 30 °, and the reaction is stopped by the addition of 2.0 ml. of trichloroacetic acid. Analysis. The solutions should be protected from light as much as possible throughout the following procedure. The reaction mixture obtained above is boiled for 10 minutes to hydrolyze any FAD which may have been formed, and the solution is cooled and filtered. A 5.0-ml. aliquot of the filtrate is neutralized with 1.25 ml. of 2.4 M K2HPOt, and the light absorption is measured at 450 m~. Five milliliters of the neutralized filtrate is then bubbled for 30 seconds in 20 × 150-mm. test tubes with 12.5 ml. of benzyl alcohol. After brief centrifugation, the benzyl alcohol layer is quantitatively removed, and the residual benzyl alcohol is removed from the aqueous phase by bubbling with 5.0 ml. of chloroform. The solution is again centrifuged, and the aqueous layer is pippetted into a cuvette for measurement of light absorption at 450 m~. Calculation. The flavin content of the initial 5.0-ml. reaction mixture is calculated from the light absorption of the unextracted sample as millimicromoles of total flavin per 5.0 ml. ; this is value A in the equation below. Value B is identically calculated from the light absorption after extraction. 8For critical experiments ATP should be freed from ADP and AMP. Methods for the purification of ATP are given in Vol. III [1] 7].

642


[108]

Millimicromoles of F M N per 5.0 ml. of reaction mixture = 1.25B - 0.125A 4 This value may be corrected for the slight loss of flavin in the deproteinization step by multiplying by the factor, millimicromoles riboflavin added/value A. This correction factor is very low except with crude enzymes, where the protein precipitate is bulky. Definition of Unit and Specific Activity. One unit is that amount of enzyme which catalyzes the synthesis of 1 millimicromole of FMN in 2 hours at 30 ° per 5.0 ml. of reaction mixture under the conditions of assay given above; specific activity is defined as units per milligram of protein. Protein concentration is determined by light absorption at 280 m~ at pH 7.0, and this value is related to the dry weight at various stages of purification. Applicability of Assay Method to Crude Preparations. The assay conditions described above are recommended only for the most highly purified enzyme preparations (steps 4 to 5 below). For less purified stages (steps 2 to 3), the final concentration of MgSO4 should be 3 × 10-4 M, since 1 X 10-3 M MgSO4 causes a slight inhibition. The presence of phosphatases and other hydrolytic enzymes in crude preparations (e.g., yeast autolyzate) complicates the assay; the effect of interfering enzymes is largely eliminated by the inclusion of 1 M K F in the reaction mixture.

Purification Procedure Preparations of flavokinase from brewer's yeast s and intestinal mucosa 5 have been described. The detailed procedure for the isolation of the enzyme from intestinal mucosa has not yet been reported, and the preparation has not yet been satisfactorily freed of phosphatase activity.5 The starting material is a thoroughly washed, low-temperature-dried beer yeast, Lot D-422, obtained from Anheuser-Busch, Inc. 4 The distribution coefficients of riboflavin and F M N between benzyl alcohol and the neutralized trichloroacetic acid filtrate are 3.6 and 0.044, respectively, corresponding to 10% and 90% remaining in the aqueous phase after extraction with benzyl alcohol. ~ Then, A = Riboflavin -{- F M N or B = 0.1 riboflavin + 0.9 F M N

Riboflavin -~ A -- F M N

Substituting for riboflavin in equation 2, B = 0.1A - 0.1 F M N + 0.9 F M N and FMN

B - 0.1A 0.8

S. Englard, Federation Proc. 11, 208 (1952).

1.25B -- 0.125A

(1) (2)

[10~]

FLAVOKINASE

643

Step 1. Autolysis. In essence, the conditions of autolysis determine the effectiveness of the entire purification procedure and should be adhered to as rigidly as possible. These conditions are so chosen as to give the most satisfactory compromise between yield and purity, with relatively little contamination by interfering enzymes. Five hundred grams of yeast is allowed to autolyze in 1 1. of water at 35 to 36 ° with effccient stirring. At the end of 2 hours at this temperature, 500 ml. of water is added, and the mixture is cooled rapidly to 30 °. Stirring is continued for 15 minutes, at which time the yeast is cooled to about 10° and centrifuged for 30 to 45 minutes at 4500 to 5000 r.p.m. Step 2. Ammonium Sulfate Precipitation. The supernatant fluid obtained above is cooled to 0 °, and the pH adjusted, if necessary, to pH 5.9 to 6.0. Solid ammonium sulfate is added to give 0.40 saturation (calculated as 71.5 g. per 100 ml. = 1.0 saturation), and the mixture is stirred for 45 minutes. The precipitate is collected by centrifugation for 45 to 60 minutes at 5000 r.p.m., redissolved in about 30 to 40 ml. of water, and dialyzed in the cold against running distilled water for 16 to 18 hours. The heavy precipitate formed during dialysis is centrifuged down at 10,000 r.p.m, for 30 minutes and discarded. Step 3. Precipitation at pH 5.0 and Adsorption of Impurities with Alumina. The dialyzed enzyme is diluted with water to give 8 to 10 rag. of protein per milliliter (E2s0 = 13.2 for 1% protein) and brought to pH 5.0 at 0 ° by the addition of 0.2 to 0.3 ml. of 1 N acetic acid. The precipitate formed is not centrifuged off. Aluminum hydroxide gel C~ ~ is added in the ratio of 60 to 80 mg. of gel per gram of protein. After 15 minutes of stirring, the mixture is centrifuged for 30 minutes at 10,000 r.p.m., and the gel is discarded. These two steps should remove about 45 to 50% of the protein present in the dialyzed solution. Step ~. Fractionation with Ammonium Sulfate at pH 6.0 and 7.2. The enzyme solution obtained in the previous step is adjusted to pH 6.0 at 0 ° with 0.4 to 0.5 ml. of 0.5 M K2HP04 and brought to 0.42 saturation with solid ammonium sulfate. The suspension is stirred for 30 minutes at 0 ° and then centrifuged for 15 minutes at 10,000 r.p.m. The precipitate is dissolved in 0.015 M phosphate buffer, pH 7.2, in half the initial volume (end of step 3), and the ammonium sulfate concentration is calculated, taking into consideration the volume of the precipitate, which is 0.42 saturated with respect to ammonium sulfate. The solution is centrifuged if not clear, and the ammonium sulfate concentration of the supernatant solution is raised again to 0.42 saturation with solid ammonium sulfate. The suspension is stirred for 15 minutes and then centrifuged for e For the preparation of alumina C~, see Vol. I [11].

644


[108]

10 minutes at 10,000 r.p.m. T h e precipitate is dissolved in a small volume of water and dialyzed for 2 to 3 hours against running distilled water at 0 °. The dialyzed enzyme is centrifuged free of any precipitate which forms and is lyophilized to dryness. Step 5. Differential Denaturation. Much of the protein present in the lyophilized powder will not go into solution at 0 ° in water or various buffers, and a continuous denaturation and precipitation of protein is apparent thereafter. The following procedure, however, results in a solution which remains water-clear for several hours at 0 ° and also effects considerable purification. The lyophilized powder is gently resuspended in water and 0.2 M succinate buffer, p H 6.0, is added to give a final molarity of 0.05 M and 5 to 6 mg. of enzyme per milliliter. The suspension is kept at 0 ° for 45 minutes, and the insoluble proteins are removed by 10 minutes of centrifugation at 18,000 r.p.m, at 0 °. Solutions of flavokinase at this stage of p u r i t y deteriorate rapidly and, therefore, the enzyme should not be dissolved until just before the assay. The most highly purified fraction still gives a linear reaction rate in the assay described for 1 hour but not for 2 hours. SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. Centrifuged autolyzate 2. (NH4)~SO4ppt., 0.40, pH 6 3. pH 5.0 precipitation and A1 C~ adsorption 4. 0.42 (NH4)~SO~precipitation at pH 6.0 and 7.2 5. Differential denaturation

Total Total Specific volume, units, Protein, activity, ral. Units/ml. thousands mg./ml, units/mg. 770 82

52 450

40 37

90 15.4

0.57 30

128

260

37

4.9

55

15 296

1378 642

20 19

12.4 2.2

112 290

Properties Specificity. The purified enzyme acts on riboflavin, dichloroflavin [6,7dichloro-9-(D-l'-ribityl)isoalloxazine], and arabitylflavin [6,7-dimethyl-9(D-l'-arabityl)isoalloxazine], but not on isoriboflavin [5,6-dimethyl-9(D-l'-ribityl)isoalloxazine], 6-carbon alcohol derivatives of isoalloxazine such as galactoflavin, sorbitylflavin, and dulcitylflavin, alloxazine, or ring compounds of other than the isoalloxazine configuration. 7 None of these inhibits the phosphorylation of riboflavin. 7 E. B. Kearney, J. Biol. Chem. 194, 747 (1952).

[108]

FLAVOKINASE

645

Besides ATP, A D P can also act as a phosphate donor, but the maximal velocity in the presence of the latter is only half t h a t given by ATP. Although purified fractions still contain myokinase, 8 there is as yet no valid reason to believe t h a t A D P acts by virtue of prior conversion of A T P b y myokinase, since Km values for A T P and A D P are identical and since the V .... with A D P is much lower than with ATP. Kinetics. The enzyme follows a zero-order reaction under the assay conditions given above. At 30 °, the optimal p H range is 7.8 to 8.5. At p H 7.8, the apparent temperature optimum is 38 °. The enzyme is saturated b y 1 X 10-4 M riboflavin and half-saturated b y 1.0 X 10 -5 M. The Km for A T P as determined from the Lineweaver-Burk type of plot is 1.7 X 10-5 M, and t h a t for A D P is 1.6 × 10-5 M. Activators and Inhibitors. Mg ++, Zn ++, Co ++, and Mn ++ all activate the enzyme, but only Mg ++ exhibits fairly constant behavior. Relatively impure preparations are activated optimally b y 3 X 10-4 M 1Vig++, and the Km is 7.5 X 10-5 M. With the most highly purified preparations, the observed Km for Mg++ is 2.1 X 10-4 M; no inhibition is evident at high concentrations; and 9 X 10 -4 M Mg ++ is necessary to saturate the enzyme. The maximal activity in the presence of Mn ++ is low, although higher concentrations of this cation are noninhibitory. The extent of activation b y Co ++ is variable, although often much greater than b y Mg++; at high concentrations Co ++ is inhibitory. Activation b y Zn ++ is manifest only at certain stages of purification (end of steps 4 or 5 but not step 2); however, at these stages the activity in the presence of Zn ++ is far greater than in the presence of Mg ++. (Specific activity with 9 X 10 -4 M Zn++ = 352, with 1 X 10-3 M Mg ++ = 241.) The Km for Zn ++ is 9.0 X 10-5 M. Ca ++ does not produce activation but is inhibitory in the presence of other metals. 5-AMP is a competitive inhibitor of flavokinase (KI = 2.5 X 10 -5 M). Lumiflavin, when present in excess over riboflavin, inhibits phosphorylation of the latter. s S. Englard, J. Am. Chem. Soc. 75, 6048 (1953).

646


[109]

[109] Pyridoxal Kinase from Brewer's Yeast Pyridoxal + ATP --* Pyridoxal-5-phosphate + ADP

By I. C. GUNSALUS and W. E. RAZZELL

Assay Method Principle. Hurwitz 1 measures pyridoxal phosphate, formed by kinase action, by the activation of a crude tyrosine apodecarboxylase preparation from Streptococcus faecalis. ~ The assay is proportional to pyridoxal phosphate over the range of 1 to 10 muM. (1 to 10 units). After phosphorylation of pyridoxal by the specific kinase, ATP is removed by treatment with a potato apyrase 3 to prevent further phosphorylation by the kinase contained as an impurity in the decarboxylase preparation. Reagents 0.3 M phosphate buffer, pH 6.85. 0.05 M Na~ATP. 0.05 M pyridoxal hydrochloride, neutralized to pH 6.85. 0.01 M MgSO4. 0.1 M succinate buffer, pH 6.5. 0.045 M calcium chloride. 1 M sodium acetate, pH 5.5. 0.03 M tyrosine suspension, pH 5.5.

Enzymes Tyrosine apodecarboxylase (1500 #l. of COs per hour per milliliter; i.e., ~ ca. 10 mg. of dried cells). See text. Potato apyrase, 400 units/ml. Prepared according to Krishnan. ~

Procedure. Pyridoxal kinase activity is measured by adding 10 to 100 units in 1.2 ml. or less to a 13 × 100-mm. Pyrex test tube containing 0.2 ml. each of the phosphate buffer, sodium ATP, pyridoxal, and magnesium sulfate solutions, and enzyme plus water to 2 ml. Incubate for 2 hours at 30 °, and immerse in a boiling water bath for 3 minutes to stop the reaction; then dilute to 5 ml. and filter. ATP is removed with apyrase by adding 0.8 ml. or less of the diluted reaction mixture, containing about 1.6 ~M. of ATP, to a 13 × 100-mm. test tube containing the following: 0.5 ml. of succinate buffer, 0.2 ml. of calcium chloride, 0.5 ml. (200 units) of apyrase, and enzyme plus water 1 j . Hurwitz, J. Biol. Chem. 205, 935 (1953). 2 W. D. Bellamy a n d I. C. Gunsalus, J. Bacteriol. 50, 95 (1945). s p. S. Krishnan, Vol. I I [96].

[109]

PYRIDOXAL KINASE FROM BREWER'S YEAST

647

to 2 ml. Incubate for 30 minutes at 30 °, and stop the reaction by chilling it in an ice bath. Then dilute to 5 ml. with water. Pyridoxal Phosphate Assay. Transfer an aliquot containing 1 to 10 units of pyridoxal phosphate in not more than 1.8 ml. to the main compartment of a Warburg flask containing 0.2 ml. of acetate buffer (see reagents) plus 0.5 ml. (750 units) of tyrosine apodecarboxylase enzyme and water to 2.5 ml. Equilibrate for 10 minutes at 30 °, tip from the side arm 0.5 ml. of the tyrosine suspension, and measure the CO~ released during the first 10 minutes. Definition of Units. The pyridoxal kinase unit is 1 m~M. of pyridoxal phosphate formed in 2 hours at 30 ° in the protocol indicated. (Hurwitz's unit was defined at 33.5°; the unit is redefined at 30 ° to coincide with all other measurements in his publication.) The apyrase unit is the quantity of enzyme required to liberate 1 ~, of inorganic phosphate in 30 minutes at 30 ° in the presence of 300 ~ or more of ATP phosphorous. The tyrosine decarboxylase unit is the quantity of enzyme liberating 1 ~l. of C02 per 60 minutes at 30 ° in the protocol specified above. 4 The specific activity, in all cases, is the units per milligram of protein.

Preparation of Assay Enzymes Potato apyrase is prepared as described by Krishnan.3 Tyrosine apodecarboxylase can be prepared by growing Streptococcus faecalis, strain R, for 15 (___3) hours at 37 ° in the medium of Bellamy and Gunsalus. ~ The cell crop is harvested by centrifugation, washed once in a small volume of distilled water, resuspended in distilled water, and pipetted with stirring into 10 vol. of - 2 0 ° acetone. After 15 minutes the cells are collected on a Bfichner funnel, washed once with acetone, and once with cold ( - 2 0 °) ether. Tyrosine apodecarboxylase is prepared by suspending 20 mg./ml, of acetone powder in M/50 phosphate buffer, pH 5.5, followed by incubation for 20 hours at 37°. 5 The debris is removed by centrifugation, and the extract containing the tyrosine apodecarboxylase is lyophilized and stored in a desiccator at 0 °. The enzyme is stable indefinitely under these circumstances. Alternatively, the tyrosine apodecarboxylase can be extracted by the methods outlined in Vol. I [7]. For most purposes, acetone- or vacuum-dried cells are suitable for pyridoxal phosphate assay. 6,7 A water suspension of cells is lyophilized, 4 Tyrosine decarboxylase assay can be r u n at 37 °, with greater activity per u n i t of cells or extract. 5 5 H. M. R. Epps, Biochem. J . 38, 242 (1944). 6 W. W. U m b r e i t a n d I. C. Gunsalus, J. Biol. Chem. 179~ 279 (1949). 7 I. C. Gunsalus a n d W. W. Umbreit, J. Biol. Chem. 170, 415 (1947).

648


[109]

preferably in a petri dish containing not over 10 ml. of cell suspension, in a 250-mm. desiccator containing 2 to 3 pounds of Drierite by evacuation with a good vacuum pump. The Qco, (N) of such cells is approximately 2000, i.e., 200 units per milligram in the presence of excess pyridoxal phosphate, and approximately 5 units in the absence of this coenzyme. The cells contain an excess of pyridoxal kinase and are thus not suitable for pyridoxal phosphate assay unless devoid of either ATP or free pyridoxal.

Purification Procedure Hurwitz ~ used low-temperature-dried brewer's yeast (Anheuser Busch strain BSC) as a source of the kinase. Suspend 500 g. of dried yeast in 1 1. of water and stir for 2 hours at 35 °. Add 300 ml. of water, and cool to 30 ° with an additional 15-minute stirring. Then cool to 10° in an ice bath, and remove the cell debris by centrifugation. (This is essentially the autolysis procedure of Kearney and Englard. 8) All enzyme fractionations are carried out at 0 °. Precipitate the kinase by addition of 28 g. (0.4 saturation) of ammonium sulfate per 100 ml. of extract with stirring at 0 ° for 40 minutes. Remove the precipitated enzyme by centrifugation. Discard the supernatant, suspend the precipitated enzyme in 60 ml. of water, and dialyze against distilled water for 15 hours. Remove the precipitate by centrifugation, and discard. Adjust the clear supernatant to pH 5 with normal acetic acid, and stir for 30 minutes. Remove the precipitate by centrifugation, and add 1/~0 vol. of 2 M acetate buffer, pH 5, 0 ° (final concentration 0.067 M). Add ethanol cooled to - 10° slowly over a period of 15 minutes to a final concentration of 12% by volume. Recover the precipitate by centrifugation, and dry over Drierite at 0 ° in a vacuum desiccator. Owing to the removal of phosphatases, the recovery of kinase as compared to the autolyzed extract is 230% with a purification of 440-fold. The enzyme is stable at this stage and can be used for most experiments. The enzyme can be further purified by another ammonium sulfate fractionation and a heat step with a recovery of 40 to 50% (yield equal to activity measurable in autolyzate) at 1000-fold purification.

Properties The purified enzyme obtained by alcohol fractionation is stable at 30 ° in the presence of pyridoxal but is rapidly inactivated in the absence of the substrate. The kinase is specific for the substituted pyridine molecule; i.e., pyridoxine and pyridoxamine are phosphorylated at a rate approximately equal to that for pyridoxal. Other kinases, for glucose and ribos E. B. Kearney a n d S. Englard, J. Biol. Chem. 193, 821 (1951).

[110]

DEPHOSPHO-COA KINASE FROM PIGEON LIVER

649

flavine, for example, are also present in the alcohol-precipitated protein fraction, but their ratio compared to the pyridoxine phosphorylating enzyme varies with stage of purification, and they are thus considered to be impurities. The enzyme is also specific for ATP; I T P and inorganic phosphate neither phosphorylate nor inhibit. Adenine, adenosine, AMP, and ADP are inhibitors of the kinase. Fluoride, 0.1 M, is also inhibitory to the kinase. There is a rathei sharp pH optimum at 6.9, half-maximal activity being exhibited at pH 6.5 and 7.5.

[110] D e p h o s p h o - C o A K i n a s e f r o m P i g e o n L i v e r Mg+ ATP ~ D P C o A - ) CoA ~- ADP Enzyme By T. P. WANG Assay Method

Principle. Dephosphorylated coenzyme A (DPCoA), in contrast to CoA, is inactive in the routine phosphotransacetylase assay.l.2 The activity of DPCoA kinase is thus followed by the arsenolysis of acetyl phosphate by a phosphotransacetylase method in which the CoA formed from the phosphorylation of DPCoA is assayed. Reagents DPCoA. Prepared by dephosphorylation of CoA (see Vol. III [136]), 20 units. ATP (0.04 M). DPCoA kinase, 0.4 saturation, (NH4) 2SO~ fraction. Cysteine hydrochloride (0.1 M). MgCl2 (0.05 M). Tris buffer (0.5 M), pH 8.2. Phosphotransacetylase and reagents for arsenolysis of acetyl phosphate. Procedure. Twenty units of DPCoA, 2 micromoles of ATP, 5 micromoles of MgC12, 3 micromoles of cysteine HC1, 40 micromoles of Tris, and 0.05 rag. of enzyme are incubated at 37 ° for 30 minutes. The reaction mixture is then treated in a boiling water bath to stop the reaction and E. R. Stadtman, J. Biol. Chem. 196, 527 (1952); see Vol. I [98]. 2 If the level of phosphotransacetylase is increased 100 fold there is some activity with the DPCoA.

650

[I10]

CO:ENZYME AND VITAMIN METABOLISM

to denature any acetyl phosphatase that might be present in the enzyme preparation. It is necessary to observe this step, since the hydroxamic acid method for acetyl phosphate could not tell whether the disappearance of acetyl phosphate is obtained through the action of phosphotransacetylase or is the result of hydrolysis by acetyl phosphatase. The heated mixture is then assayed for its CoA content by the phosphotransacetylase method of Stadtman. 1 Definition of Units and Specific Activity. A unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of 1 unit of CoA per 30 minutes under the above conditions. Specific activity is then the amount of CoA (in units) formed per milligram of protein per 30 minutes. The above procedure can be used for crude tissue preparations as well. Purification Procedure s

Steps 1 and 2. The source of the DPCoA kinase is the same pigeon liver acetone powder used in the preparation of D P N kinase. ~ Steps 1 and 2, extraction with K H C 0 3 and precipitation with protamine, are also the same as described for D P N kinase. The only difference is that the D P N kinase stays with the protamine precipitate whereas the DPCoA kinase activity remains in the supernatant (see Vol. II [111]). SUMMARY OF PURIFICATION PROCEDURE

Fraction KHCO3 extract Protamine supernatant Acetone fraction (NH4) 2S04 fraction, 0.4 saturation (NH4)2SO4 fraction, 0.6 "

Volume, ml.

Protein, mg./ml,

51 138 60 4 9

30.9 6.3 5.8 9.9 21.7

Specific Yield, activity units 34.1 34.3 69.7 153 74.3

53,900 29,600 24,200 6,050 14,500

Step 3. Acetone Precipitation. To the protamine supernatant, cooled to almost 0 ° in a salt-ice bath, is added slowly with stirring a stream of 150 ml. of cold acetone ( - 1 5 ° ) . The temperature of the solution is kept below - 3 ° after the addition of the first few milliliters of acetone. The precipitate formed is centrifuged at 18,000 r.p.m, for 10 minutes and dissolved in 50 ml. of Tris buffer, 1 M, pH 8.2. Part of the precipitate does not go into solution. The acetone fraction is then dialyzed against 6 of cold solution of 0.02 M KHCOa and 0.2 To KC1 for 6 hours. Some precipitate formed during the dialysis plus the undissolved material originally present is removed by centrifugation. a T. P. Wang and N . O . Kaplan, J . Biol. Chem. 206, 311 (1954) ; see also Vol. I I [111].

[110]

DEPHOSPHO-COA KINASE FROM PIGEON LIVER

651

Step ~. (NH4)2SO~ Fractionation. The acetone fraction is then fractionated with solid (NH4)2S04. Two protein fractions, precipitating at between 20 and 40% and 40 and 60% saturation with respect to (NH~)~SO~, are collected. The precipitates are dissolved in a small volume of 0.2 % KC1. The total yield is about 38% (both (NH4)~SO~ fractions combined), and purification is 4.5-fold (40% (NH4)2SO~ fraction). The procedures used in the purification of DPCoA kinase are given in the table.

Properties Specificity. Because of the lack of assay method, compounds structurally related to DPCoA such as deamino-DPCoA have not been tested with the DPCoA kinase. But among the phosphorylating agents tried, only ATP is active. Neither I T P nor ADP can be used to replace ATP. Metal Requirement. In addition to DPCoA, ATP, and enzyme, Mg ion is required for the phosphorylation of DPCoA to CoA. However, addition of fluoride ion (0.02 M) with or without inorganic phosphate (0.02 M) does not inhibit the reaction. Inhibitors. In contrast to D P N kinase, the DPCoA kinase is not inhibited to any significant extent by adenosine, 2'-, 3'-, and 5'-adenylic acids, ADP, ADPR, D P N H , DPN, and deamino-DPN. The enzyme is stable at - 15 ° for at least two months without losing activity. Repeated freezing and thawing also do not significantly alter the activity of the enzyme. The activity of DPCoA kinase can also be followed by means of the firefly bioluminescence system, since CoA but not DPCoA is capable of giving a secondary stimulation on the light production. ~ When DPCoA, cysteine, and the kinase are added to the firefly bioluminescence system consisting of luciferase, luciferin, ATP, Mg ion, and oxygen, the secondary light production is proportional to the activity of the kinase within certain limits. From experiments using this system, the Km for DPCoA has been estimated to be 3 X 10-~ M. The partially purified preparation DPCoA kinase requires cysteine for maximal activity. 4 W. D McElroy and J. Coulombre, in preparation.

652


[111]

[iii] D P N Kinase from Pigeon Liver ATP q- DPN

Mg ++

Enzyme

) TPN -f" ADP

By T. P. WANG Assay Method

Principle. The TPN-specific isocitric dehydrogenase from pig heart 1 which does not react with DPN is used for the assay of TPN. The amount of TPN formed under a specified condition serves, in turn, as a measurement of the DPN kinase activity. Reagents A T e (0.04 i ) . DPN (90 % purity). Dissolve 25 mg. in 1 ml. of water. DPN kinase. MgC12 (0.05 M). Tris buffer (0.05 M), pH 7.5. Isocitric dehydrogenase (0.5 to 0.6 saturation, (NH4)2SO~ fraction of pig heart extract). Isocitrate (0.05 M). Prepared by hydrolyzing isocitric lactone with acid and then neutralized with NaOH.

Procedure. One-tenth milliliter each of DPN, ATP, DPN kinase, MgCl~, and Tris buffer are incubated together at 37 ° for 60 minutes. One-half milliliter of water is then added to the mixture, and the diluted mixture is heated in boiling water for 2 minutes. After the coagulated protein has been removed by centrifugation, an aliquot of the clear supernatant is pipetted into a Beckman cuvette which contains enough Tris buffer, pH 7.5, to make the final concentration of the buffer around 0.05 M and the final volume 2.9 ml. Then 0.05 ml. of isocitrate is added with 0.1 ml. of the isocitric dehydrogenase, and readings are taken at 30-second intervals thereafter. In general, the reaction should be completed in about 2 minutes. The increase in reading at 340 mu indicates the formation of T P N H from TPN which is formed from the phosphorylation of DPN by ATP in the presence of DPN kinase. Definition of Unit and Specific Activity. Enzyme activity under the above conditions is expressed in micromoles of TPN formed per hour. The specific activity, defined as the unit of enzyme activity per hour per milligram of protein, 2 is used as the index of purity. 1A. Graffiinand S. Ochoa, Biochim. et Biophys. Acta 4, 205 (1950); see Vol. I [116]. 20. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

[111]

DPN KINASE FROM PIGEON LIVER

653

The same procedure can be used for crude tissue preparation, provided that a suitable amount of nicotinamide is present to inhibit the activity of DPNase. 3 Purification Procedure 4

Acetone powder of pigeon liver prepared according to Kaplan and LipmamP is used as the starting material for purification of DPN kinase. Step 1. Preparation of Crude Extract. Six grams of pigeon liver acetone powder is extracted with 70 ml. of ice-cold 0.02 M K H C Q . The extraction is facilitated by using a Tenbroeck homogenizer kept in an ice bath. (All subsequent operations are made at 0 to 5°.) The homogenate is then centrifuged at 18,000 r.p.m, for 15 minutes to remove the insoluble residue. The supernatant should be dark red in color. Step 2. Protamine Precipitation. A 0.2 % protamine sulfate solution in 0.04 M Tris buffer, pH 7.5, is added slowly with stirring to the above crude extract. The precipitate forms immediately during this addition. As soon as no further precipitation occurs, the addition of protamine solution is discontinued. In general, about 2 vol. of the protamine solution is required for 1 vol. of the crude extract. The protamine precipitare, collected by centrifugation at 4000 r.p.m, for 10 minutes, is extracted twice with 20 ml. each of an 0.2 M acetate buffer, pH 5.0. The two acetate extracts are combined, and the residue is discarded. Step 3. Adsorption on Alumina C~. 6 An equal volume of water is add e d to the acetate extract. To the diluted extract is gradually added 18.5 ml. of alumina C~ (dry weight 16.3 mg./ml.). After 10 minutes of stirring, the mixture is centrifuged at 4000 r.p.m, for 10 minutes. The supernatant is discarded. The alumina residue is then eluted three times with 18.5 ml. each time of 0.2 M phosphate buffer, pH 7.5, and discarded. Step 4. (NH4)2SO~ Precipitation. A solution of (NH~)2SO4, saturated at room temperature with a pH of 7.1, is added to the phosphate eluate to give 0.5 saturation. The precipitate, collected by centrifugation, is dissolved in 4.5 ml. of 0.02 M phosphate buffer, pH 7.5. The over-all yield of the enzyme from the acetate extract is 64%, and the purification from the crude extract is 46.5-fold. The low specific activity in the crude extract is probably due to the presence of inhibitors or competitive enzymes such as ATPase, DPNase, nucleotide pyrophosphatase, etc., in the crude extract. The steps in the purification of the DPN kinase are summarized in the table. P. Handler and J. R. Klein, J. Biol. Chem. 143, 49 (1942). 4 T. P. Wang and N. O. Kaplan, J. Biol. Chem. 206, 311 (1954). 5 N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948). 6 Alumina C~, see Vol. I [11].

654


[111]

SUMMXRY OF PURIFICXTION PROCEDURE

Fraction KHC08 extract Acetate extract Acetate eluate (NH4)2S04 fraction

Volume, ml.

Protein, rag./ml,

Specific activity

Yield, units

51 37 55 6.5

30.9 5.2 1.8 10.7

0. 0178 0. 469 0. 768 0. 828

28.2 a 90 75.6 57.7

a See text for explanation. This procedure has been used b y several workers besides the a u t h o r with the same or b e t t e r yield a n d / o r higher purification. Properties

Specificity. The D P N kinase from pigeon liver is specific for D P N with no activity on d e a m i n o - D P N , D P N H , and D P C o A . However, the enzyme will catalyze the phosphorylation of the 3-acetylpyridine analog of DPN.7 T h e requirement of A T P is also specific; neither A D P nor I T P can be used to replace A T P . The K~ for D P N is 6 × 10-4 M. Metal Requirements. Besides D P N and A T P , metal ions such as M g ++ or Mn++ are necessary for D P N kinase activity. T h e optimal concentration of M n ++ is about 1.8 X 10-3 M, and t h a t of Mg ++ a b o u t 0.9 X 10-2 M. At the optimal concentration of M n ++, the a m o u n t of T P N formed is about 25 to 30 % higher than t h a t formed at the optimal concentration of Mg ++. However, at higher concentration, M n ++ shows some inhibitory effect, whereas Mg ++ does not even at 1.8 X 10-2 M. Inhibitors. A group of compounds related to D P N have been found to have an inhibitory effect on D P N kinase. With the exception of dea m i n o - D P N , the more the compound resembles D P N , the greater inhibition it exhibits. For example, adenosine, 2-adenylic acid, and 3-adenylic acid are less inhibitory t h a n 5-adenylic acid, which, in turn, is not so inhibitory as A D P or A D P R . And among all the compounds tested, D P N H is the most p o t e n t inhibitor. E v e n at a concentration of 1.5 X 10-3 M, D P N H gives an inhibition of over 84% when the concentration of D P N is 2.5 X 10-8 M. The inhibition of D P N H on D P N kinase can be overcome b y the increase of D P N concentration. In other words, the inhibition of D P N H appears to be competitive. When D P N H is treated with a snake venom nucleotide pyrophosphatase, its inhibitory effect becomes greatly reduced to the level of t h a t of 5-adenylic acid. The reduced N M N seems to have no effect on D P N kinase at all. I t is interesting to note t h a t d e a m i n o - D P N has no inhibition on D P N kinase. Since d e a m i n o - D P N is a hypoxanthine derivative instead of an

[112]

NUCLEOTIDE PYROPHOSPHATASE

655

adenine compound, the amino group on the adenine ring m a y be responsible for the inhibitory effect of the adenine compounds. In this connection, it m a y be worth mentioning t h a t I T P , also a hypoxanthine derivatire, cannot be used as a phosphate donor. The presence of an amino group in the purine ring in both the phosphate donor and the phosphate acceptor seems essential for the activity of D P N kinase from pigeon liver. Other Properties. D P N kinase can be dialyzed against 0.02 M N a H C O s at 4 ° for at least 16 hours without losing any activity. I t is also stable toward freezing and thawing. Storage at - 1 5 ° for four months resulted in little loss of activity. The enzyme has been used successfully in preparing large quantities of T P N of high purity.7 7 T. P. Wang, N. O. Kaplan, and F. E. Stolzenbach, J. Biol. Chem. 211, 465 (1954).

[112] Nucleotide Pyrophosphatase 1 DPN TPN FAD ATP +

+ + + 2

H20 H20 H20 H20

--+ N M N + 5'-AMP --+ N M N + Adenosine-2',5'-diphosphate --+ Riboflavin Phosphate + 5'-AMP --* 5 ' - A M P + 2 P

By ARTHUR KORNBERQ Assay Method Principle. The method depends on the spectrophotometric determination of the removal of D P N . 2 In crude preparations the possible contributions to D P N removal b y other DPNases can be checked b y demonstrating the persistence of the pyridinium linkage 3 or the appearance of 5 ' - A M P ? Reagents

D P N (0.02 M), p H ca. 6. KH~PO4-K:HP04 (0.5 M), p H 7.0. Procedure. A mixture containing 0.1 ml. of D P N , 0.2 ml. of phosphate buffer, 1 to 5 enzyme units, and water to a final volume of 1.0 ml. was

1A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 182, 763 (1950). 2 A. Kornberg, J. Biol. Chem. 182, 779 (1950). 3j. W. Huff and W. A. Perlzweig, J. Biol. Chem. 167, 157 (1947). H. M. Kalckar, J. Biol. Chem. 167, 445 (1947).

656

COENZYME A N D

VITAMIN METABOLISM

[112]

incubated for 20 minutes at 38 °. An aliquot of 0.10 ml. was pipetted directly into a cuvette containing (in 2.8 ml.) all the components needed for the reduction of D P N except the enzyme (i.e., alcohol dehydrogenase), and an initial reading was taken. After the addition of enzyme, reduction of D P N was complete in about 5 minutes. The concentration of D P N before and after incubation with nucleotide pyrophosphatase was based on the use of an extinction coefficient 5 of 6.22 × 106 cm.2/mole. Definition of Unit and Specific Activity. One unit of enzyme is defined as t h a t a m o u n t which causes the splitting of 1 micromole of substrate (i.e., D P N ) per hour. Specific activity is expressed as units per milligram of protein. Protein was determined by a b i u r e t procedure 6 for crude preparations and by ultraviolet light absorption 7 for purified preparations.


Step 1. Extraction. Two hundred grams of peeled Maine potatoes was extracted with 400 ml. of 0.40 saturated ammonium sulfate for 90 seconds in a Waring blendor. The extract was filtered on fluted papers at 2 °. F r o m 5 kg. of potatoes, 10 1. of filtrate was obtained. The use of Celite as a filter aid resulted in some loss of enzyme activity. The yield obtained b y extraction of potatoes with 2 vol. of water (aqueous extract, see the table) approximated t h a t of the a m m o n i u m sulfate fraction. Step 2. Ammonium Sulfate Fraotionation. T o 10 1. of filtrate was added 2 kg. of solid ammonium sulfate. Filtration was at 2 ° through a 50-cm. fluted paper with an arrangement for automatic refilling to permit collection of the precipitate on a single paper and completion of the filtration overnight. T h e brownish black precipitate was scraped from the paper and dissolved with water to a volume of 550 ml. The dark solution was dialyzed against running tap water (at 8 to 18 °) for 90 minutes in cellophane sacs. The volume after dialysis was 660 ml., and the p H 5.5 to 5.6. F o u r such batches of dialyzed ammonium sulfate fractions were combined (ammonium sulfate, see the table) and fractionated with ethanol. Step 3. Ethanol Fractionation. The dialyzed a m m o n i u m sulfate fraction was brought to p H 4.4 with acetic acid (61 ml. of 1 M). The solution was cooled to - 0 . 5 ° , and 95% ethanol was added with mechanical stirring. The t e m p e r a t u r e was maintained just above the freezing point during the early ethanol additions and at - 5 ° thereafter. The precipitates 5 B. L. Horecker a n d A. Kornberg, J. Biol. Chem. 176, 385 (1948). 6 T. E. Weichselbaum, Am. J. Clin. Path., Tech. Sect. 10, 40 (1946). 7 O. W a r b u r g a n d W. Christian, Biochem. Z. 310, 384 (1941-1942); see Vol. I I I [73].

[112]


657

were centrifuged off at 0 ° and dissolved in water. Fraction 2 was refractionated as indicated (see the table). These two fractionations have been carried out four times with little variation from the results of the first trial. A t t e m p t s to standardize a third ethanol fractionation were unsuccessful. Fractions with high specific activity were obtained in good yield, but minor variations in temperature, time, and speed of ethanol addition influenced the a m o u n t of SUMMARY OF PURIFICATION PROCEDURE

Ethanol Volume Specific (95 %) of Total activity, added, fraction, activity, Yield, units/ ml. ml. units % mg. protein

Step Aqueous extract Ammonium sulfate Ethanol fraction I-1 Ethanol fraction 1-2 Ethanol fraction II-2a Ethanol fraction II-2b Ethanol fraction II-2c Ethanol fraction III-2c-1 Ethanol fraction III-2c-2 Ethanol fraction III-2c-3 Ethanol fraction III-2c-4• Ethanol fraction III-2c-5b Calcium phosphate, first adsorption Fractions 2c-3, 4, 5 Eluate 1 Eluate 2 Eluate 3 Calcium phosphate, second adsorption, eluate Fraction 2c-4 was the 2c-3 after standing at b Fraction 2c-5 was the 2c-4 after standing at

397 438 41 27 30 44 11 5.5

2750 1000 680 178 160 162 36 36 30 25 21

350,000 317,000 58,500 230,000 15,850 61,000 125,000 13,300 34,000 30,700 21,400 25,700

91 18 72 7 27 54 11 27 24 17 20

2.9 5.4 1.8 15.5 2.0 15.6 84.7 18.8 76.5 202 278 216

254 100 100 100

77,500 52,000 7,700 910

67 10 1

220 1625 1065 530

75

38,300

74

2200

precipitate which appeared in the supernatant of fraction - 1 0 ° for 3 hours. precipitate which appeared in the supernatant of fraction - 1 0 ° for 18 hours.

ethanol required. Accordingly, this fractionation was carried out by collecting several ethanol fractions and combining the best (see the table). Step 4. Calcium Phosphate Adsorption. The combined ethanol fractions (Nos. 2c-3, 4, and 5) (pH 4.4) were diluted with water to give a protein concentration of 1.5 mg./ml. Calcium phosphate gel 8 (202 ml., aged 2 months, dry weight 7.9 mg./ml.) was added, and the mixture was stirred mechanically for 5 minutes at room temperature. The precipitate 8 D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124~ 397 (1938).

658


[112]

was collected by centrifugation and washed four times with 100 ml. of 0.1 M potassium phosphate buffer, pH 7.4. The enzyme was eluted with three portions of 100 ml. of 0.20 saturated ammonium sulfate adjusted with ammonia water to pH 7.5. To concentrate eluates 1 and 2 to a small volume, 36 g. of solid ammonium sulfate was added to each. The precipitates, collected in a highspeed centrifuge, were dissolved in water to yield a protein concentration of 2 mg./ml. The yield in this step was 92%, and the specific activity was unaltered. A second calcium phosphate adsorption increased the specific activity to 2200 units/rag, with a yield of 80 %. Fifteen milliliters of the ammonium sulfate concea,trate of eluate 1 above were diluted to 300 ml., adsorbed with 15 ml. of calcium phosphate gel, washed three times with 150 ml. of 0.05 M potassium phosphate buffer, pH 7.0, and eluted with 75 ml. of 0.20 saturated ammonium sulfate, pH 7.5. The entire purification procedure resulted in a 750-fold purification with an over-all yield of 11%. The yield may be improved by combining and reprocessing some fractions of lower purity. The term "purified enzyme" in this report refers to a calcium phosphate eluate (or ammonium sulfate concentrate) with a specific activity of 1625 units or more per milligram and "crude enzyme" to the aqueous extract (see the table). Variations in Activity in Potatoes. The age and variety of potato influenced the nucleotide pyrophosphatase activity. Eleven varieties of Maine potatoes, which were harvested at the same time and stored under identical conditions prior to the initial assay, differed widely (3.3 to 21.9 units/ml.). Aging at 3 ° resulted in a variable increase in activity. Since the protein concentration was relatively uninfluenced by the variety of potato or its age, the specific activity reflected the enzyme activity. The low activity was not due to the presence of an inhibitor, since an extract of a potato of high activity (55 units/ml.) tested in the presence of an equal volume of an extract of a potato of low activity (3.3 units/ml.) was not inhibited. Stability. The enzyme is remarkably stable at 0 to 5 °. Preparations of varying purity (including the most purified) have shown no detectable loss of activity over a period of six years. However, some dilute solutions (50 ~, of protein per milliliter) lost 50 % of their activity in 5 days. There was no inactivation on incubation for 20 minutes at 38 ° at pH 3.2 in 0.1 M citrate buffer, or at pH 9.3 in 0.1 M glycine buffer. There was complete inactivation on incubation for 15 minutes at 38 ° at pH 12.5, or for 10 minutes at 38 ° at pH 1.4. The enzyme was not inactivated by freezing or by a 9-hour dialysis against running distilled water.

[112]


659

Properties Effect of pH. As measured with DPN as substrate, there is a broad optimum between pH 6.5 and 8.5, and a decrease of activity of about 50% at pH 4:0 and 9.0. Enzyme inactivation did not contribute to this result, and a twofold increase in DPN concentration did not change the result. Specificity. Certain evidence suggests that the same enzyme splits DPNH, TPN, FAD, and ATP. This evidence is the relative constancy of ratios of these activities during the course of purification and kinetic data. There are weaker indications that thiamine pyrophosphate is a substrate. Park 9 has demonstrated that UDPG is very likely a substrate of nucleotide pyrophosphatase. The conclusion of Novelli et al. 1° that the enzyme from potatoes which splits CoA at the pyrophosphate bond is a different enzyme is based only on the fact that the pH optimum for this activity is much lower than that reported for DPN. Other Phosphatase Activities. The purified preparations still contained detectable amounts of phosphatase activity toward several phosphate esters including inorganic pyrophosphate, 5'-AMP, NMN, glucose-6-phosphate, and glycerophosphate. However, these activities were very low, splitting of inorganic pyrophosphate being only 3 % as active and the other activities being less than 1% of the rate for DPN splitting. There was no indication during the course of purification that any of these activities became stabilized with reference to DPN splitting, as was the case with adenyl pyrophosphate and thiamine pyrophosphate splitting. Michaelis constants determined for DPN, TPN, and ATP were as follows: 1.5 X 10-4 M, 3.0 X 10-3 M, and 2.0 X 10-3 M, respectively. Activators and Inhibitors. DPN splitting was not stimulated by Mg ++ or Ca ++ and was inhibited about 50% by NaF (0.1 M) only in the presence of phosphate (0.05 M). Somewhat different results were obtained with other nucleotides as substrates.

g J. T. Park, J. Biol. Chem. 194, 885 (1952). 10G. D. Novelli, F. J. Schmetz, Jr., and N. O. Kaplan, J. Biol. Chem. 206, 533 (1954).

660


[113]

[113] Animal Tissue D P N a s e (Pyridine Transglycosidase 1) N + R P P R A ~ H 2 0 -* N ~- R P P R A + H + N + R P P R A -]- X -~ X + R P P R A ~- N

By NATHAN O. KAPLAN

Assay Method 2 Principle. T w o m e t h o d s are used for following the cleavage of D P N . One is based on the cyanide reaction of D P N 2 This involves determining the D P N level b y cyanide before and after incubation with the enzyme. Cyanide reacts only with the q u a t e r n a r y nitrogen form of D P N and will not react with adenosine diphosphate ribose or free nicotinamide. A second m e t h o d involves following the splitting of D P N b y assaying with y e a s t alcohol dehydrogenase. T h e cyanide procedure is of value in t h a t it will signify the cleavage of the nicotinamide ribose link. B y the use of the alcohol dehydrogenase assay, it is not possible to ascertain whether splitting occurs at the nicotinamide glycosidic bond or at the p y r o p h o s p h a t e grouping. 3 Reagents Crystalline y e a s t alcohol dehydrogenase (Vol. I [79]).

M/1 KCN. 0.1 M phosphate, p H 7.2. 0.005 M D P N .

Determination of Enzymatic Activity. 2 T h e reaction consists of 0.3 ml. of the 0.1 M phosphate, 0.5 micromole of D P N , and e n z y m e (approxim a t e l y 1 unit) in a total volume of 0.6 ml. After incubation at 37 ° for 8 minutes, 3 ml. of the m o l a r cyanide is added, and the mixtures are read at 325 m~ (which is the absorption m a x i m u m of the D P N cyanide complex). F o r determination of the action with alcohol dehydrogenase, 3 ml. of a mixture containing 0.5 M ethanol and 0.02 M nicotinamide 4 in 0.1 M 1The name pyridine transglycosidase has been given to the enzyme, since the enzyme has been found not only to induce hydrolysis of DPN at the nicotinamide ribose linkage but also to promote a transfer of adenosine diphosphate ribose from one pyridine grouping to another. In the equation N -- nicotinamide, R ~- ribose, P = phosphate, A = adenine, and X -- a pyridine compound structurally related to nieotinamide. 2L. J. Zatman, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 200, 197 (1953). a S. P. Colowick, N. O. Kap]an, and M. M. Ciotti, J. Biol. Chem. 191, 473 (1951). 4 The nicotinamide is added to prevent further cleavage of DPN at the end of the incubation period.

[113]

ANIMAL TISSUE DPNAS~

661

glycine NaOH buffer, pH 9.5, were added. Readings are taken at 340 mg before and after the addition of crystalline yeast alcohol dehydrogenase, a Definition of Unit. A unit of enzyme is that amount which will cleave 1 micromole of DPN per hour under the conditions described. Application to Crude Extracts. The enzyme is usually found in the insoluble fractions of tissue homogenates; the assay technique is applicable to whole homogenates. Purification Procedure As yet no success has been achieved in solubilizing the enzyme, and as a result no extensive purification of the enzyme has been made. However, some purification has been obtained by differential centrifugation. The preparation of the DPNase from beef spleen and pig brain will be described. These two enzymes, although carrying out an identical cleavage of DPN, differ with respect to their sensitivity to isonicotinic acid hydrazide and their activity in forming DPN analogs. Preparation of Spleen Enzyme. 6 Two hundred grams of fresh beef spleen was homogenized in a Waring blendor with ice and water for 4 minutes and made to 750 ml. with ice water and 15 ml. of M NaHCO3. The connective tissue was removed by centrifugation at 2000 r.p.m, for 10 minutes; the resulting red-brown opaque supernatant fluid can be stored in the deep-freeze for several weeks without more than 10 to 20 % loss in activity. A 100-ml. portion of this crude material was centrifuged at 18,000 r.p.m. 7 for 15 minutes, and the residue resuspended in 60 to 70 ml. of 0.02 M N a H C Q and respun at 3000 r.p.m, for 15 minutes. The supernatant fluid was then centrifuged at 18,000 r.p.m, for 15 minutes, and the residue, which is devoid of red pigment, resuspended in 80 ml. of 0.02 M N a H C Q . s The suspension was finally centrifuged at 18,000 r.p.m. for 1 minute and the opaque supernatant separated from the loose precipitate. The supernatant contains material of the highest specific activity, usually about 35 units/mg, of protein, and represents an approximately sevenfold purification from the crude homogenate. Protein was determined by the method of Lowry et al2 6 The splitting can also be followed manometrically, since acid is produced when D P N is cleaved a t the nicotinamide ribose link. 2 6 F r o m the procedure of Z a t m a n et al. 2 7 Carried out on an I n t e r n a t i o n a l centrifuge. 8 W h e n the enzyme is used in chromatographic experiments, distilled water is used instead of NaHCO3 for the final suspension of residue in order to avoid gas production on acidification of the column. 9 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, a n d R. J. Randall, J. Biol. Chem. 195~ 265 (1951); see also Vol. I I I [73].

662


[113]

Further high-speed centrifugation always sedimented more of the enzyme, and attempts to render the enzyme soluble by heat, repeated freezing and thawing, salt, deoxycholate, and digitonin were unsuccessful. The highest specific obtained to date has been less than 1% of that of the DPNase purified from Neurospora (see Vol. II [114]). Preparation of Pig Brain Enzyme. TM Whole pig brain (150 g.) was blended with 500 ml. of ice water in a Waring blendor. After passing through cheesecloth, the homogenate was centrifuged for 20 minutes at 20,000 X g and the precipitate made up to 300 ml. with ice water. This was then centrifuged for 40 minutes at 20,000 × g, after which the precipitate was suspended in 150 ml. of ice water and treated for 15 minutes in the 10-kc. sonic oscillator (Raytheon). The suspension was finally centrifuged at 20,000 X g for 20 minutes, and the relatively small amount of precipitate discarded. The supernatant fluid is colloidal and contains nearly all the DPNase activity. This fluid contains about 20 units of enzyme per milliliter and can be stored at - 1 5 ° for at least a month without loss in activity.

Properties pH Optimum. The optimum for the beef spleen system is 7.2; the pig enzyme has roughly the same optimum. Specificity. Both the beef spleen and pig brain enzymes attack only the oxidized form of DPN, and not reduced DPN. Oxidized TPN is attacked by the beef spleen DPNase at 50% the rate of DPN; the pig brain enzyme also will cleave TPN. Deamino-DPN is split by the spleen enzyme at 23 % the rate of DPN. Nicotinamide riboside and mononucleotide are not hydrolyzed by either the spleen or brain enzymes. The pig brain enzyme will liberate the pyridine component from a number of DPN analogs. These include the 3-acetylpyridine, isonicotinic acid hydrazide (INH), marsilid, 1~ isonicotinamide, and ethyl nicotinate analogs of DPN. The beef spleen enzyme does not act on the isonicotinic acid hydrazide, marsilid, and isonicotinamide analogs but will split the 3-acetylpyridine analog. Inhibitors. Nicotinamide has been found to be a potent inhibitor of animal tissue DPNase. The spleen enzyme is inhibited 50 % by a nicotinamide concentration of 1.5 × 10-3 M. The inhibition has been shown to be of a noncompetitive nature. By the use of C14-1abeled nicotinamide, it has been possible to show that this inhibition involves an exchange reaction between free nicotinamide and the bound nicotinamide of DPN. TM 10 L. J. Zatman, N. O. Kaplan, S. P. Colowick, a n d M. M. Ciotti, J. Biol. Chem. 209~ 467 (1954). 11 Marsilid is the isopropyl derivative of I N H . 12 For a description of D P N prepared with labeled nicotinamide, see Vol. IV [34].

[113]

ANIMAL TISSUE DPNASE

663

I N H is a p o t e n t inhibitor of the beef spleen system. On the other hand, this same compound does not inhibit the cleavage of D P N b y the pig brain catalyst. T h e effect of I N H on D P N a s e s from various species was found to fall into two distinct categories, those which were strongly inhibited b y the compound and those which are not inhibited. The t e r m " I N H - s e n s i t i v e " enzyme has been used to refer to the beef spleen enzyme, which is inhibited, whereas the pig brain enzyme has been referred to as an " i n s e n s i t i v e " enzyme, because it is not inhibited b y the I N H.I3 A s u m m a r y of the effect of I N H on a n u m b e r of different D P N a s e s is given in the table. INH

SENSITIVITY OF VARIOUS ANIMAL TISSUE D P N A s E S ~

" I N H sensitive . . . . INH insensitive" Beef spleen Beef brain Lamb spleen Lamb brain Goat spleen Goat brain

Rat spleen Rat brain Mouse spleen Mouse brain Mouse lymphosarcoma Rabbit spleen

" I N H sensitive" " I N H insensitive" Pigeon brain Duck brain

Rabbit brain Horse spleen Horse brain Human spleen Human prostate Frog spleen

From L. J. Zatman, N. 0. Kaplan, S. P. Colowick, and M. M. Ciotti, J. Biol. Chem. 209, 453 (1954). I t has been found t h a t the " I N H - i n s e n s i t i v e " systems will f o r m the I N H analog of D P N v e r y readily. Although the " s e n s i t i v e " enzymes are strongly inhibited b y I N H , the analog is still formed b u r n t a b o u t 1 % the rate of the " i n s e n s i t i v e " systems. I°,I~ T o date the following D P N analogs have been synthesized with the pig brain enzyme as catalyst: I N H , I°,15 marsilid, 1° isonicotinamidel 1° 3-acetylpyridine, I6 and ethyl nicotinate. I7 Alivisatos and Woolley h a v e reported the synthesis of the 4-amino 5-carboxamido imidazole analogue of D P N f r o m D P N and the imidazole; this reaction is catalyzed b y the beef spleen system, is 1~L. J. Zatman, N. O. Kaplan, S. P. Colowiek, and M. M. Ciotti, J. Biol. Chem. 209, 453 (1954); Bull. Johns Hopkins Hosp. 91, 211 (1952). 14D. S. Goldman, J. Am. Chem. Soc. 76, 2841 (1954). 15L. J. Zatman, N. O. Kaplan, S. P. Colowiek, and M. M. Ciotti, J. Am. Chem. Soy. 75, 3293 (1954). 16N. O. Kaplan and M. M. Ciotti, J. Am. Chem. Soe. 76, 1713 (1954). 17N. O. Kaplan and M. M. Ciotti, in preparation. is S. G. A. Alivisatos and D. W. Woolley, J. Am. Chem. Soc. 77, 1065 (1955).

664


[114]

[114] N e u r o s p o r a D P N a s e + N R P P R A ~- H 2 0 --* N + R P P R A q- H +

B y NATHAN O. KAPLAN Assay Methods

T h e procedures used are identical with those described for the animal e n z y m e (Vol. I I [113]). T h e only difference is t h a t the incubation time is for 71/~ minutes at 37 °. Unit of Activity. One unit of a c t i v i t y is t h a t a m o u n t of e n z y m e which causes cleavage of 0.01 micromole of D P N u n d e r the conditions of t h e assay. Protein was determined b y the m e t h o d of L o w r y et al.1 Purification P r o c e d u r e T h e e n z y m e has been purified from m a t s grown in a zinc-deficient medium. ~ B y the inclusion of only a small a m o u n t of nitrate instead of the usual a m m o n i u m nitrate of the normal Fries medium, it has been possible to obtain m a t s of s o m e w h a t higher specific a c t i v i t y 2 The m e d i u m used for the growth of the mold is as shown in the table. M a k e up in triple-distilled water. Medium

G./1.

Medium

Sucrose KH2PO4 MgSO4.7H20 NaC1 CaC12 Biotin Na tetraborate

20 1 0.5 0.1 0.1 5 X 10-6 8.8 X 10-s

(NH4)sMo40~4 FeC13.6H20 CuCl: MnCI~.4H20 Na tartarate Na nitrate

G./1. 6.4 9.6 2.7 7.2

X 10-5 X 10-4 × 10-4 X 10-5 5.0 1.0

Step 1. Preparation of Crude Extracts. 2 T h e mycelia m a t s are collected in a Biichner funnel, and washed with triple-distilled water. F r o m 10 1. of m e d i u m 40 to 50 g. of m a t s (wet weight) is obtained. The m a t s are frozen for at least 1 to 3 hours, then homogenized in a Ten Brock glass homogenizer in three times their weight of 0.1 M p h o s p h a t e buffer at p H 7.5 and centrifuged in the Servall at 13,000 r.p.m, for 10 minutes 10. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. I I I [73]. 2 A. Nason, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 188, 397 (1951). A. Nason, N. O. Kaplan, and H. A. Oldewurtel, J. Biol. Chem. 201, 435 (1953).

[114]

NEUROSPORA DPNASE

665

at + 4 °. The supernatant, which is cell-free and turbid, is used as the enzyme source. N e a r l y all the enzyme is found in the extract. Step 2. Acidification. 4 The crude extract is adjusted to p H 5 with HC1 at 0 °. The resulting precipitate is discarded; the acid filtrate contains 80% of the initial activity, with a twofold increase in purity (see the summary). SUMMARY OF PURIFICATION PROCEDURE a

Recovery, % Total units Units/rag. protein units/mg, protein Crude extracts 1,600,000 pH 5 filtrate 1,260,000 pH 5, 60% acetone precipitate 609,000 pH 2.7, 60% acetone precipitate 480,000

2,650 4,980 25,800 85,600

-79 39 30

a 250 ml. of crude extract used in this experiment.

Step 3. Acetone Fractionation. Acetone is added to the acid filtrate of step 2 at 0 ° to a concentration of 35%. This precipitate contains little activity. The supernatant is then brought to 60% acetone, and the precipitate is dissolved in 0.1 M K~HPO4. This precipitate has an activity about five times t h a t found in the previous step. Step 4. pH 2.7 Acetone Precipitate. 5 The dissolved 60% precipitate from step 3 is brought to p H 2.7 at 0 ° b y adding normal HC1 dropwise. Acetone is then added to 60%. The resulting precipitate, spun at 0 °, is triturated with 5 ml. of 0.1 M phosphate buffer, p H 7.5, and the denatured protein is removed. The soluble protein represents a 30% yield with about 30-fold purification from the initial extract. I t is of interest to note t h a t to obtain the same specific activity from normal Neurospora mats 2 a 1500-fold purification would be required. Properties

Stability. The enzyme can be kept in the deep-freeze without loss of activity. I t slowly becomes inactivated at 4°; this is particularly true with very dilute solutions of the DPNase. Dialysis against a variety of buffers resulted in no loss of activity. Heating at 80 ° at p H 5 for 2 minutes completely destroys the activity of the enzyme. However, little loss occurs after heating at 55 ° for 2 minutes in the p H range 3 to 5. N. 0. Kaplan, S. P. Colowick, and A. Nason, J. Biol. Chem. 191, 473 (1951). 6 In the routine preparation of the enzyme, the final step has been omitted. This preparation has been used extensively for the determination of DPN (see Vol. III [128]).

666


[114]

Trichloroacetic acid only partially precipitates the enzyme; the precipitated activity can be recovered b y dissolving the precipitate in 0.1 M K2HPO4. Some of the enzyme is present in the trichloroacetic acid filtrate. The enzyme appears to be stable in such filtrates for a period of at least two weeks at 4 ° . p H Optimum. The enzyme operates over a v e r y broad p H range (3 to 9). The nature of the buffer does not influence the Neurospora DPNase, and citrate, phosphate, and acetate can be used interchangeably. Effect of Metal. Zinc, manganese, ferric, calcium, and magnesium ions in concentrations of 0.05 M have no effect on the activity of the enzyme. Fluoride, cysteine, Versene, and cyanide at 0.01 M do not inhibit the enzyme. Effect of D P N Concentration. The Km for D P N has been found to be approximately 5 X 10 -4 mole/1. Inhibition by Nicotinamide. Unlike the animal tissue DPNase, the Neurospora enzyme is quite insensitive to nicotinamide. Only at v e r y high concentrations (0.1 M) is the enzyme inhibited. The inhibition of nicotinamide is competitive in contrast to the noncompetitive inhibition observed with the beef spleen system. 6 Formation of D P N Analogs. The enzyme operates by a different mechanism from t h a t of the animal tissue DPNase, and it does not form D P N analogs. Specificity. The enzyme attacks both D P N and T P N . 4D e a m i n o - D P N 7 is split at a much slower rate t h a n is D P N . The various analogs of D P N (i.e., isonicotinie acid hydrazide, 3-acetylpyridine) are not cleaved b y the enzyme. A synthetic nucleotide, dinicotinamide ribose 5'-pyrophosphate, is split at about one-third the rate of D P N . s I t is interesting to note t h a t only one nicotinamide is removed from the compound b y the Neurospora enzyme. Reduced D P N or T P N , nicotinamide mononucleotide, and nicotinamide riboside are not hydrolyzed b y the enzyme. 9 Effect of Deficiencies on the Concentration of Enzyme. Neurospora mats grown on a zinc-deficient medium show an approximate 10- to 20-fold increase in the level of DPNase. Other metal deficiencies have little or no effect on the D P N a s e concentration. 2 A nitrogen deficiency also produces a v e r y marked elevation in enzyme. 3 Neurospora m u t a n t s grown on minimal levels of the specific nutrient also exhibit high DPNase.1 6L. J. Zatman, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 200, 197 (1953). N. O. Kaplan, S. P. Colowick, and M. M. Ciotti, J. Biol. Chem. 194, 579 (1952). s L. Shuster, N. O. Kaplan, and F. E. Stolzenbach, J. Biol. Chem. in press. The alpha isomer of I)PN is also not split by the Neurospora enzyme [N. O. Kaplan, M. M. Ciotti, F. E. Stolzenbach, and N. R. Bachur, J. Am. Chem. Soc. 77, 815 (1955)].

[115]

DEPHOSPHO-COA PYROPHOSPHORYLASE

667

[115] Dephospho-CoA Pyrophosphorylase B y G. DAVID !X~OVELLI

Assay Method Principle. This enzyme catalyzes the reversible condensation between ATP and phosphopantetheine to yield dephospho-CoA and inorganic pyrophosphate. 1 The enzyme has not yet been separated from dephosphoCoA kinase (see Vol. II [110]); therefore, starting with ATP and phosphopantetheine the product of the over-all reaction is CoA. CoA is measured by means of the phosphotransacetylase 2 assay (see Vol. I [98]). Alternatively, the reaction may be measured by the determination of inorganic pyrophosphate. Crude preparations are usually contaminated with ATPase and inorganic pyrophosphatase, however, making such measurements less reliable. In more purified preparations inorganic pyrophosphatase is eliminated, but dephospho-CoA kinase is still present. The latter enzyme displaces the equilibrium in favor of CoA synthesis, and thus the inorganic pyrophosphate level is a direct measure of the condensation reaction. Reagents

4'-Phosphopantetheine, 0.02 M. ATP, 0.1 M. MgCI~, 0.1 M. Cysteine HC1, 0.1 M. Tris buffer (M/l), pH 7.7. Reagents for phosphotransacetylase assay for CoA 2 (see Vol. I [98] and Vol. III [132]). Dephospho-CoA pyrophosphorylase. Procedure. The enzyme activity is measured under the following conditions: 0.2 uM. of phosphopantetheine, 5 to 10 ~M. of ATP, 5.0 ~M. of Mg ++, 10.0 tiM. of cysteine, 50 tLM. of Tris buffer, pH 7.7, and enzyme sufficient to make 10 to 20 units of CoA when CoA is being measured or 1 to 2 uM. of CoA when pyrophosphate is being measured. Incubation is carried out at 37 ° for 60 minutes in a final volume of 1.0 ml. The reaction is stopped by boiling for 3 minutes in a final volume of 1.0 ml. The reaction is stopped by boiling for 3 minutes, and CoA is measured with the

1M. Hoagland and G. D. Novelli, J. Biol. Chem. 207, 767 (1954). E. R. Stadtman, G. D. Novelli, and F. Lipmann, J. Biol. Chem. 1911365 (1951).

668


[115]

phosphotransacetylase assay, or pyrophosphate by the time-color development method of Flynn et al. 3 Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which effects the synthesis of 1 unit of CoA or the liberation of 0.0025 ~M. of inorganic pyrophosphate in 1 hour at 37 ° under the conditions specified below. Specific activity is expressed as units of enzyme per milligram of protein. Protein is determined by the turbidimetric method of Biicher. 4 Purification Procedure

Step 1. Preparation of the Crude Extract. Fresh hog liver obtained from the slaughterhouse and kept in ice during transport to the laboratory is put at once through a chilled meat grinder and then blended in a Waring blendor for 45 seconds with an equal volume of cold 0.15 M KC1. This homogenate is filtered through cheesecloth and centrifuged in the preparative Spinco at 40,000 X g for 11/~ hours at 5 °. This process yields a red translucent supernatant from which most microsomes are removed. Higher speeds would be more satisfactory, but this is sacrificed in order to process larger volumes. The removal of particulate protein by this centrifugation procedure effects a fourfold purification. In addition the removal of nuclei eliminates a potent inhibitor. Step 2. Protamine Treatment. The supernatant from step 1 is diluted with 2 vol. of water and treated with 0.06 vol. of 2% protamine sulfate. The resulting precipitate is removed by centrifugation and discarded. To process large volumes the refrigerated Sharples operating at 50,000 r.p.m. is employed. This step removes some of the pantetheine kinase and a large amount of nucleic acid, the remaining microsomes, and inert protein. Step 3. Ammonium Sulfate Fractionation. The supernatant from step 2 is brought to pH 8.0 with Tris, and solid ammonium sulfate is added to 0.25 saturation. The precipitate is centrifuged off at 5 ° and discarded. The supernatant is raised to 0.38 saturation by the addition of solid ammonium sulfate. The resulting precipitate, which contains the bulk of the activity, is collected by centrifugation, and the supernatant is discarded. For large volumes, the precipitate may be conveniently collected in a single Sharples bowl. Step 4. Treatment with Calcium Phosphate Gel. The precipitate from step 3 is dissolved in water to give 50 rag. of protein per milliliter and is then treated with 2.5 vol. of calcium phosphate gel (dry weight, 36 mg./ml.). The gel with its adsorbed protein is quickly spun off and discarded, and the pH of the supernatant is restored to above pH 7 by the addition of 0.05 vol. of M Tris, pH 8.0. Finally the protein is cona R. M. Flynn, M. E. Jones, and F. Lipmann, J. Biol. Chem. 211, 791 (1954). 4 T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).

[115]

DEPHOSPHO-COA PYROPHOSPHORYL£SE

669

centrated by bringing the solution to 0.7 saturation with ammonium sulfate. The precipitate is collected and dissolved in a minimum volume of water. The purification procedure is summarized in the table. SUMMARY OF PURIFICATION PROCEDURE

Fraction Hog liver homogenate Hog liver supernatant Protamine supernatant Ammonium sulfate fractionation Calcium phosphate gel treatment Over-all

Specific activity

Purification, -fold

1.5 3.0 10.0 50 200 200

2 6.6 33.0 132 132

Recovery,% 600 50 80 40

Properties

Stability. The enzyme is quite stable when kept in the frozen state at - 1 0 °, no appreciable diminution of activity being noted in three months. pH Optimum. Dephospho-CoA pyrophosphorylase is active over the pH range from 6.5 to 8.5 with an optimum near pH 7.5. Mg Requirement. Mg ++ is required for activity of the enzyme. The optimum concentration appears to be around 2 × 10-3 M. Higher levels of Mg ++ appear to activate a latent inorganic pyrophosphatase. Rate. The rate of the over-all reaction, i.e., phosphopantetheine -~ dephospho-CoA--* CoA appears to be linear up to about 80% conversion of phosphopantetheine. It is necessary that the substrate be in the reduced form. The enzyme is totally inactive with the oxidized substrate. Cysteine or H~S have been found suitable in keeping the substrate reduced. Reaction Catalyzed. Dephospho-CoA pyrophosphorylase catalyzes the reversible reaction: Phosphopantetheine ~ ATP ~--Dephospho-CoA + P-P

(1)

Because the enzyme preparations are always contaminated with dephospho-CoA kinase, it has not been possible to measure the equilibrium of reaction 1. However, the presence of this contaminating enzyme makes possible the following dismutation. Dephospho-CoA ~ P-P ~--ATP + P-pantetheine Dephospho-CoA -b ATP--* CoA + ADP • Dephospho-CoA ~- P-P--~ CoA nu ADP ~ P-pantetheine

(sum)

(2) (3) (4)

This stoichiometry has been observed, indicating the reversibility of reaction 2.

670


[116]

[116] D P N P y r o p h o s p h o r y l a s e N M N -~- A T P . ~ D P N + P P B y ARTHUR KORNBERG

Assay Method Principle. The method is based on the initial rate of formation of D P N starting with a large excess of A T P and N M N . D P N is measured spectrophotometrically after its total reduction by the alcohol dehydrogenase system. 1 The extinction coefficient of 6.22 X 106 cm.2/mole at 340 m/~ ~ was used. Reagents ATP (0.02 M). N M N (0.05 i ) . Glycylglycine buffer (0.25 M), pH 7.4. MgC12 (0.15 M). Nicotinamide (2 M). Procedure. The incubation mixture contained 0.1 ml. of ATP, 0.05 ml. of N M N , 0.2 ml. of glycylglycine buffer, 0.1 ml. of MgC12, enzyme (1 unit or less), and water to make a final volume of 1.0 ml. To avoid interference by D P N nucleosidase, 0.1 ml. of nicotinamide was added in assays of crude liver fractions. After incubation at 38 ° for 20 minutes, 1.0 ml. of 10% trichloroacetic acid was added. The supernatant solution was neutralized with 2 N N a O H with the aid of an internal indicator (bromothymol blue or phenol red), and 1.0 ml. was pipetted into each of two absorption cells for D P N analysis. With the purified yeast and liver enzymes, the use of trichloroacetic acid was unnecessary and analyses were performed directly on aliquots of the incubation mixtures. Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the amount causing the synthesis of 1 ~M. of D P N per hour, and specific activity as units per milligram of protein. Proportionality to enzyme concentration was observed in this test with crude as well as with purified preparations when 1 unit or less was present in the test system. Protein concentration was determined by a nephelometric method 3 with the Beckman spectrophotometer at 340 mt~. i A. Kornberg, J. Biol. Chem. 182, 779 (1950). 2 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175~385 (1948). 8T. Biicher, Biochim. et Biophys. Aeta 1~ 292 (1947).

[116]

DPN PYROPHOSPHORYLASE

671

Purification Procedure T h e enzyme has been purified from both liver and yeast, but most extensively from the latter (see the table). Only the preparation of the liver enzyme is described here because the starting material is more uniform and more readily obtained and the most purified fraction, although only 5 % as pure (on a protein basis) as the purified yeast enzyme, is more stable and is adequate for equilibrium studies. Purification of Liver Enzyme. Homogenates of rat liver and brain carried out D P N synthesis from N M N and A T P in the presence of nicotinamide. A convenient source of the enzyme was hog liver, from which active, stable acetone powders were prepared. Fresh hog liver (100 g.) was homogenized in acetone ( - 1 0 °, 500 ml.) in a Waring blendor. The residue collected on a Biicher funnel were resuspended in cold acetone, filtered off, and dried at room temperature. Ten grams of powder was extracted with 100 ml. of 0.1 M Na2HPO~ for 10 minutes at room temperature. Subsequent operations were at 3 ° unless otherwise indicated. The residue was separated b y centrifugation and discarded. To the extract (see the table) was added 16 g. of ammonium sulfate; the precipitate was removed b y centrifugation and discarded. Eight grams of ammonium sulfate was added to the supernatant, and the precipitate collected b y centrifugation was dissolved in water to a volume of 40 ml. This fraction was reprecipitated b y adding 8 g. of ammonium sulfate, centrifuging, and dissolving the resulting precipitate in water (ammonium sulfate, see the table). This fraction was diluted with water (23 °) to 94 ml. and adsorbed on 9.4 ml. (75 mg.) of calcium phosphate gel 4 during a 5-minute period. The gel was washed three times with 36 ml. of cold 0.02 M phosphate buffer (pH 7.0) and eluted with 37 ml. of cold SUMMARY OF PURIFICATION PROCEDURES

Step Yeast Autolyzate Final step Liver Extract Ammonium sulfate Adsorption, ammonium sulfate

Volume of fraction, ml.


Specific Over-all activity, yield, units/ % mg. protein

210 5

716 313

44

O. 24 453

80 13 4

202 163 57

81 28

0.11 0.71 12.4

D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124, 397 (1938).

672


[116]

0.5 M K2HPO4. To the clear eluate was added 6.3 g. of ammonium sulfate; the precipitate was collected by centrifugation and dissolved in water (adsorption, ammonium sulfate). This fraction was 100 times as active as the acetone powder extract on the basis of protein content and represented a yield of 28 %. The stability of this preparation was greater than that of the purified yeast enzyme; no significant loss of activity was detected during storage at 3 ° for one to two weeks.

Properties Specificity. N M N H and D P N H replace the corresponding oxidized nucleotides as formulated in the following equation: N M N H + ATP ~ D P N H + PP Nicotinamide nucleoside, ADP, TPN, FAD, orthophosphate, and metaphosphate are not substrates for this enzyme. Activators and Inhibitors. Mg ++ is required. The Michaelis constant is 5 × 10-4 M and 2 X 10-4 M for the yeast and liver enzymes, respectively. Stimulation by Mn ++ is relatively small. 2,4-Dinitrophenol (1 × 10-4 M) and fluoride (0.05 M) do not inhibit this enzyme. Substrate A~nities and Maximal Rates. Each substrate was tested in the presence of an optimal concentration of the corresponding reactant. The dissociation constants of the liver enzyme-substrate complexes calculated from the values were as follows, in moles per liter: 1.5 X 10-4 for N M N ; 4.6 X 10-4 for ATP; 0.83 × 10-4 for DPN; and 1.9 X 10-4 for PP. With use of the optimal substrate concentrations, values for the maximal reaction velocities were obtained. The ratio of V.... for DPN synthesis to V.... for DPN breakdown was 0.48 for the purified liver enzyme and 0.42 for the purified yeast enzyme. The ratio was 0.39 for the same yeast enzyme when tested after six weeks of storage, at which time it had lost 86 % of its activity.

[117]

FAD PYROPHOSPHORYLASE

673

[117] F A D P y r o p h o s p h o r y l a s e FM N -[- A T P ~ FAD -{- P P

By ARTHUR KORNBERG Assay Method Principle. The method I is based on the initial rate of formation of FAD, starting with A T P and FMN. The FAD is determined by the method of Warburg and Christian, 2 which involves the measurement of 02 consumption as a function of FAD concentration in the oxidation of DL-alanine by D-amino acid oxidase. ATP served not only as a substrate but also as a competitive inhibitor of FAD hydrolysis by the nucleotide pyrophosphatase present in the various enzyme fractions. Reagents

MgCI2 (0.15 M). ATP (0.02 M). F MN (2 X 10-4 M). KH:PO,-K2HP04 (0.25 M), pH 7.5. Procedure. The incubation mixture contained 0.05 ml. of MgCl~, 0.1 ml. of ATP, 0.1 ml. of FMN, 0.1 ml. of phosphate buffer, 3 to 10 units of enzyme, and water to a final volume of 1.0 ml. After incubation for 6 to 15 minutes at 37 °, the mixture was immersed in boiling water for 3 minutes, cooled, and centrifuged. An aliquot of the supernatant fluid was assayed for FAD by the D-amino acid oxidase test. Definition of Unit and Specific Activity. One unit of enzyme is defined as th at amount causing the synthesis of 1 millimicromole of FAD per hour and specific activity as units per milligram of protein. Protein concentration was determined by a nephelometric method ~ with the Beckman spectrophotometer at 340 m~.

Purification Procedure One hundred grams of dried beer yeast 4 was autolyzed with 300 ml. of 0.1 M sodium bicarbonate (saturated with a mixture of 95% N2 and 5% COs) for 24 hours at 23 °. All subsequent operations, including storage of solutions, were carried out at 3 °, unless otherwise specified. The 1A. W. Schrecker and A. Kornberg, J. Biol. Chem. 182, 795 (1950). 20. Warburg and W. Christian, Biochem. Z. 298, 150 (1938). 3T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947). A. Kornberg, J. Biol. Chem. 182, 779 (1950).

674

[117]


mixture was centrifuged, and the supernatant (autolyzate, see the table) diluted with water to 324 ml. T h e precipitate obtained b y adding 108 g. of ammonium sulfate was centrifuged, dissolved in 60 ml. of water, and dialyzed against running, demineralized water for 1 hour. T h e dialyzed solution (fraction I) was diluted to 75 ml. with w a t e r and mixed with 75 ml. of 0.1 M sodium acetate buffer (pH 5.0). After 5 minutes, 12 ml. of 95% ethanol was added dropwise with mechanical stirring at 0 ° to - 1 °, and the precipitate collected b y centrifugation was discarded. T o the supernatant was added another 23 ml. of 95% ethanol at - 2 °. T h e precipitate was centrifuged and dissolved in 45 ml. of water and sufficient 0.1 N N a O H to give a nearly neutral solution which was then adjusted to p H 5.85 by cautious addition of 0.02 N acetic acid (fraction II). T o the solution was added 13.8 ml. of aluminum hydroxide gel C~ 5 (dry weight 15.5 g./1.). The suspension was centrifuged after 10 minutes, and the adsorbate washed with 13 ml. of 0.02 M sodium acetate buffer (pH 6.0) and eluted with three 14-ml. portions of 0.02 M phosphate buffer (pH 7.7). The combined eluates (fraction I I I ) were diluted to 48 ml. with water, and 1.0 ml. of 1.0 N acetic acid was added with mechanical stirring at 0 °, followed b y 6.9 ml. of 95% ethanol at - 1 ° to - 2 °. T h e precipitate was centrifuged and dissolved with 20 ml. of water and sufficient 0.1 N N a O H to give a nearly neutral solution which was then adjusted to p H 6.0 with 0.02 N acetic acid (fraction IV). I t was then treated with 21.4 ml. of calcium phosphate gel 8 (dry weight 8.2 g./1.) and centrifuged after 10 minutes. The adsorbate was washed with 17 ml. of 0.02 M sodium acetate buffer (pH 6.0) and eluted with four 4.3-ml. portions of 0.01 M phosphate buffer (pH 7.7). The combined eluates SUMMARY OF PURIFICATION PROCEDURE

Fraction Autolyzate, from 100 g. dried yeast I. Ammonium sulfate II. Ethanol III. Aluminum hydroxide gel eluate IV. Ethanol V. Calcium phosphate gel eluate

Volume, ml.


162 66 48 41 26 17

4340 7070 4060 3480 3340 1970~

Specific activity, Yield, units/ % mg. protein 163 57 86 96 59

0.91 7.4 20.2 30.2 50.7 83.0

a In repeated preparations, the total activity of fraction V varied between 1600 and 2800 units and the specific activity between 51 and 86 units/mg. 5 R. Willst~itter and H. Kraut, Ber. 66, 1117 (1923). 6 D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124, 397 (1938).

[118]

UDPG PYROPHOSPHORYLASE FROM YEAST

675

(fraction V) were clear and colorless. The protein content varied between 1.3 and 1.9 mg./ml. This fraction represents an over-all yield of 45% and a 91-fold purification as compared to the autolyzate. The enzyme (fraction V), at 3 °, lost 20 to 30% of its activity in 4 days and 58% in 11 days. Since the most purified enzyme fractions contained high concentration of nucleotide pyrophosphatase and inorganic pyrophosphatase, specific inhibitors were employed to avoid their interference in balance studies of FAD synthesis and pyrophosphorolysis.

Properties pH Effect. At pH 6.0 the enzyme is 10%, and at pH 8.4 64%, as active as at pH 7.5. Specificity. ADP does not replace ATP, and metaphosphate and inorganic orthophosphate do not replace PP. With riboflavin in place of F M N and with increased amounts of enzyme, a small amount of FAD is synthesized, indicating the presence of a riboflavin kinase 7 in the preparation. Activators and Inhibitors. Mg ++ is required. The optimal concentration is around 1.5 X 10-s M. Higher concentrations are slightly inhibitory. Dissociation Constants. The values for FMN, ATP, and FAD are, respectively, 1.4 X 10-6 M, 1.2 X 10-5 M, and < g and resuspending them in 0.15 M phosphate buffer, pH 7.0, with the aid of a hand-driven groundglass homogenizer. The washing should be repeated once. The heart muscle particle suspension should contain an active succinic oxidase system. This activity can be tested by (1) measuring the 02 uptake manometrically in the presence of succinate; 18 (2) measuring the half-time for reduction of the cytochromes after addition of substrate;17 (3) measuring the rate of oxidation of succinate by observing the formation of fumarate (increase in optical density at 250 m~).17 Purified Cytochrome Oxidase Preparation. A preparation derived from the heart muscle particles can be made 19 which contains cytochromes a plus a~, has very high cytochrome c oxidase activity, and has the advantage that it is optically clear and is free of cytochrome c. Thus it is also useful in testing for the activity of cytochrome c. First Stage Preparation. Heart muscle extract is prepared as described in steps 1 through 4 above, except that the phosphate buffer should be 0.1 M instead of 0.02 M and the final volume adjusted to 300 ml. Sodium cholate is added to the extract to make a final concentration of 0.8%. The extract is then digested with "1-300" trypsin preparation of the Nutritional Biochemicals Co. (0.2 mg./ml, of digest) for 1 to 2 hours at 4 °. At the end of this time there should be a considerable clarification observed in the cloudy extract when it is compared with a sample of undigested cholate extract. Solid ammonium sulfate is then added to make the digest 0.4 saturated, and the mixture is allowed to stand in the cold for several hours. The precipitate which forms is removed by centrifugation, and the supernatant fluid is decanted off and made up to 0.5 saturation with ammonium sulfate. The precipitate is collected by centrifugation (7000 >< g for 20 minutes is sufficient) and dissolved in 0.1 M Na2HPO4, using a volume of about 10 ml. for each 100 ml. of cholate extract digested. The whole procedure is carried out at 4 ° . Second Stage Preparation. The first stage preparation contains in addition to cytochromes a and as some pigment with an absorption peak in the reduced form at 560 m,. This latter pigment can be removed by diluting the first stage preparation with about 4 vol. of cold 0.1 M Na2HPO4 and centrifuging at 20,000 >< g for 1 hour. The sticky red precipitate is then dissolved in a mixture of 13 % ammonium sulfate plus 0.5 % sodium cholate. If the final solution is not absolutely clear, it is cen17 B. Chance, J. Biol. Chem. 197, 557 (1952). is D. Keilin a n d E. F. Hartree, Biochem. J. 44, 205 (1949). 1~ L. S m i t h a n d E. Stotz, J. Biol. Chem. 209, 819 (1954).

740

RESPIRATORY ENZYMES

[131]

trifuged hard for 15 to 20 minutes. Most of the cholate in the preparation can be removed by dialyzing it against cold 13 % ammonium sulfate. If there is insufficient digestion of the particulate mixture, the resulting preparation will become cloudy on standing. The preparation has very strong cytochrome c oxidase activity and can be stored in the frozen state for several months without measurable loss of oxidase activity. If it is diluted with a mixture of 13 % ammonium sulfate plus 0.5% sodium cholate or with 13% ammonium sulfate, the preparation remains optically clear. When diluted with water or buffer, cloudiness will develop with a loss of activity, unless the preparation is diluted as much as 100 times. When diluted 100 times with cold distilled water, a water-clear solution is obtained which will retain its cytochrome c oxidase activity unchanged for at least 24 hours, if kept in an ice bath.

[131] C y t o c h r o m e b ( M a m m a l s )

By

E L M E R STOTZ

Cytochrome b of mammalian tissue is a hemoprotein which in the reduced (ferrous) form has absorption bands centered at 564 mp (a), 530 mp (B), and 430 mp (7). l-a In heart muscle extracts which contain both an active succinic dehydrogenase and cytochrome oxidase, the addition of succinate causes reduction of cytochrome b and aeration causes its oxidation. ~ This hemoprotein does not appear to combine with carbon monoxide or cyanide. 4,5 Ball 6 estimated the oxidation-reduction potential of cytochrome b in heart muscle extracts to be - 0 . 0 4 (Eo' at pH 7.0). The proximity of this potential to that of the succinate-fumarate system, coupled with the belief that the cytochromes react in sequence in the succinoxidase system, has led to the assumption that succinate is the direct reductant of cytochrome b in the succinoxidase system; indeed, from studies with narcotics and other agents Keilin and H a r t r e d ,s have stated their belief that cytochrome b functions as a link between succinate and cytochrome c. Nevertheless, Chance, 9 from studies 1 D. 2 D. 3 D. 4 D. s D. 6 E. 7 D. 8 D. 9 B.

Keilin, Proc. Roy. Soc. (London) B98, 312 (1925). Keilin, Proc. Roy. Soc. (London) B100, 129 (1926). Keilin a n d E. F. Hartree, Proc. Roy. Soc. (London) B127, 167 (1930). Keilin, Proc. Roy. Soc. (London) B104, 206 (1929). Keilin a n d E. F. Hartree, Nature 141, 870 (1938). G. Ball, Biochem. Z. 295, 262 (1938). Keilin a n d E. F. Hartree, Proc. Roy. Soc. (London) B129, 277 (1940). Keilin a n d E. F. Hartree, Biochem. J. 44, 205 (1949). Chance, Nature 169, 215 (1952).

[131]

CYTOCHROME b

(MAMMALS)

741

on the rate of reduction of the cytochromes by succinate, concluded that cytochrome b does not form a link between succinate and cytochrome c. It is clear that elucidation of the interactions of cytochrome b awaits separation of the components of the succinoxidase system. In this direction, Clark et al.10 have described a preparation of cytochrome b which was free of cytochrome c but rich in succinic dehydrogenase; addition of succinate caused reduction of the cytochrome b and the latter catalyzed the reduction of cytochrome c by a succinic dehydrogenase preparation which could not otherwise reduce cytochrome e by addition of succinate. 11 This preparation, in conjunction with cytochrome c and the purified cytochrome oxidase of Smith and Stotz, 12 reconstituted a system which caused the rapid oxidation of succinate.10 Other reports on the purification of cytochrome b include those of Eichel et al. 13 and Hfibscher and Kiese. 14 The former method involves an extraction of heart muscle particles with 2 % deoxycholate, and the latter an ammonium sulfate fractionation of a cholate extract of heart muscle. Neither report provides an assay for cytochrome b, and, although the report of Kiese contains a spectrum which indicates good separation of cytochrome b from the other cytochromes, the method of preparation is not sufficiently detailed to permit ready duplication. Since the preparation of Clark et al. 1° resulted from purification of the factor linking succinate with cytochrome c, and this activity paralleled the intensity of the cytochrome b absorption spectrum, the assay and preparation employed by these investigators have been chosen for more detailed description.

Assay Method The method is based on the spectrophotometric determination of the rate of reduction of cytochrome c in the presence of succinate and an excess of succinic dehydrogenase. With the availability of a succinic dehydrogenase preparation which does not link directly with cytochrome'c, l~ it has been shown that the rate of cytochrome c reduction is proportional to a factor other than succinic dehydrogenase. 16 The assay method ~0H. W. Clark, H. A. Neufeld, C. Widmer~ and E. Stotz, J. Biol. Chem. 210, 851 (1954). 11C. Widmer, H. W. Clark, H. A. Neufeld, and E. Stotz, J. Biol. Chem. 210, 861 (1954). 12L. Smith and E. Stotz, J. Biol. Chem. 209, 819 (1954). 1~B. Eichel, W. W. Wainio, P. Person, and S. J. Cooperstein, J. Biol. Chem. 183, 89 (1950). 14G. Hiibscher and M. Kiese, Naturwissenschaften 22, 524 (1952). 16H. A. Neufeld, C. R. Scott, and E. Stotz, J. Biol. Chem. 210, 869 (1954). 16By definition such a factor can be called SC factor (succinate-cytochrome c), a term previously used by F. B. Straub [Z. physiol. Chem. 272, 219 (1942)].

742

RESPIRATORY ENZYMES

[131]

is not unlike that of Cooperstein et al. 17 for succinic dehydrogenase, which, at least in heart muscle extracts, appears to be critical for the factor linking succinic dehydrogenase with cytochrome c rather than for suceinic dehydrogenase itself. P r o c e d u r e . To a 3-ml. Beckman cuvette (10-mm. light path) are added the following: 1.0 ml. of 0.1 M phosphate buffer, pH 7.4; 0.2 ml. of 0.3 M sodium succinate; 0.2 ml. of neutralized 0.02 M potassium cyanide; 1.2 ml. of distilled water; 0.1 ml. of succinie dehydrogenase; TM and 0.1 ml. of the solution to be tested for its ability to link succinic dehydrogenase and cytochrome c (SC activity). To initiate the reaction 0.2 ml. of 2 X 10-4 M cytochrome c solution is added. Readings are taken at 550 mu every 30 seconds against a " b l a n k " solution containing all the above constituents except the cytochrome c and the factor being assayed. After an initial period of about 1 minute, the plot of optical density against time is essentially a straight line, and the slope of this line is proportional to the amount of SC factor employed. A unit of SC factor activity is defined as that amount which produces a change of 1.0 in optical density per minute at 28 °. Purification Procedure

A 400-g. portion of pig heart muscle, previously trimmed of fat, is ground twice and washed by constant stirring with 2 ~ 1. of distilled water for about 15 minutes. The muscle residue is allowed to settle, and, after the supernatant has been poured off, the residue is strained and squeezed through a layer of shroud cloth. This procedure is repeated twice more with water and once with 0.01 M (phosphate) buffer, pH 7.4. The pressed, washed muscle is divided into two equal portions, and each portion is homogenized for 5 minutes in the Waring blendor with 500 ml. of 0.1 M (phosphate) buffer, pH 7.4. The homogenates are centrifuged at 1800 X g for 25 minutes in the cold. The resulting supernatants are saved, and the two residues are each re-extracted two more times by homogenizing for 3 minutes in the blendor with 300-ml. portions of the phosphate buffer, followed by centrifugation as before. All the supernarants are then combined to give about 1900 ml. of phosphate extract with a purity of about 1.0 SC factor units/mg. This extract is stable for several days at 4 ° . 17S. J. Cooperstein, A. Lazarow, and N. Kuffess, J. Biol. Chem. 186, 129 (1950). 18In tests with heart muscle extracts or with any of the fractions described under Purification Procedure, it has been determined that succinic dehydrogenase is already present in excess and further addition of this enzyme is unnecessary. For the assay to be valid, however, it is necessary to prove that this is true, in which case it is necessary to employ a succinic dehydrogenase preparation which itself is unable to link with cytochrome c.

[131]

CYTOCHROME b

(MAMMALS)

743

All subsequent operations are carried out at 4 ° . Step 1. The p H of the phosphate extract is adjusted to 5.4 while the mixture is stirred mechanically in an ice bath and cold 1.0 M acetic acid is slowly added. The resulting mixture is centrifuged for 45 minutes at 1800 X g, and the supernatant fluid is discarded. The precipitate is resuspended in 500 ml. of 0.1 M phosphate buffer, p H 7.4, b y homogenizing for 30 seconds in the Waring blendor. The protein concentration of this solution is determined b y a sulfosalicylic acid turbidity method, 19 and the protein concentration is adjusted to 12 mg./ml. Step 2. To this mixture is added 1.25 ml. of 40% sodium cholate for every 100 ml. of solution, to yield a mixture containing 0.5% sodium cholate. T o every 100 ml. of resulting solution, 54 ml. of 0.1 M phosphate buffer, p H 7.4, saturated with ammonium sulfate, is added. The mixture is centrifuged for 30 minutes at 1800 × g. The supernatant fluid is saved and made up to 0.5 saturation with ammonium sulfate b y adding 30 ml. of the buffered ammonium sulfate solution to each 100 ml. After centrifugation as above, the precipitate is suspended in 0.1 M disodium phosphate to make a volume about one-eighth that of the extract used for ammonium sulfate fractionation. This solution (0.35 to 0.50 fraction) is allowed to stand overnight at 6 ° . Step 3. The precipitate present is discarded after centrifugation for 1 hour at 18,000 X g. The ammonium sulfate concentration of the supern a t a n t fluid is measured b y nesslerization after removal of the protein by tungstic acid 2° and then adjusted to 8.0% ammonium sulfate b y dilution with 0.1 M disodium phosphate. Step .~. To each 100 ml. of this solution is added 43 ml. of buffered ammonium sulfate solution, and the mixture is centrifuged for 15 minutes at 12,000 X g. The supernatant fluid is saved, and 16.7 ml. of buffered ammonium sulfate is added to each 100 ml. After centrifugation as above, the precipitate is resuspended in 0.1 M disodium phosphate to make a volume equal to one-eighth t h a t of the solution used for step 4. The solution obtained is a clear red solution which in SC factor activity is purified thirtyfold and concentrated sixtyfold over the heart 19An appropriate amount of protein solution (0.05 to 0.2 ml.) is diluted to 5.0 ml. with distilled water, 10 ml. of 3% sulfosalicyclie acid is added, and the turbidity read in a Klett colorimeter using the No. 44 blue filter. The protein content is compared with turbidities produced by known amounts of crystalline egg albumin under the same conditions. 2o Two-tenths milliliter of sample is diluted to 8.0 ml. with distilled water, followed by addition of 1.0 ml. of 10% sodium tungstate and 1.0 ml. of 1 N H2SO4. After mixing and centrifuging, an aliquot of the supernatant fluid is diluted to 45 ml. with distilled water, and 5.0 ml. of Nessler's solution is added. Colorimetric comparisons arc made against standard ammonium sulfate solutions.

744

RESPmATOnV ENZYMES

[132]

muscle extract used as the starting material. On spectrophotometric examination the solution may show a slight absorption peak at 553 m~ which is absent if the solution is treated with ferricyanide. Addition of dithionite produces a strong absorption band at 562 mt~ and a shoulder centered at 553 m~. Succinate causes only a partial reduction of the material absorbing at 562 mtL and complete reduction of the material absorbing at 553 mp. Addition of ascorbic acid selectively produces the 553-mu band with little change at 562 mtL. The preparation is rich in succinic dehydrogenase but apparently devoid of cytochrome c. Addition of succinate and cytochrome c to the preparation causes immediate reduction of the cytochrome c. It is concluded that cytochrome b is the predominant hemoprotein present in the preparation, but that at least one other hemoprotein, tentatively designated cytochrome 553, is also present. The role of the latter component in the linking of succinic dehydrogenase with cytochrome c is not yet clear. The positions of the absorption bands of the reduced components are as follows: Cytochromeb Cytochrome 553

562 m~ 553 mt~

530 mt~ 522 mtL

430 m~ 417 m~

[132] C y t o c h r o m e b G r o u p ( B a c t e r i a ) B y A. M.

PAPPENHEIMER,JR.

The heme-containing pigments which have been called cytochrome b represent a group of proteins which not only differ from one another in physicochemical properties but probably differ as well in their catalytic function in the chain of respiratory enzymes. Thus, although all members of the b group show a and ~ absorption bands in the visible spectrum and a strong ~, or Sorer band in the violet region, the exact position of these bands varies from one bacterial species or strain to another. In the table, the positions of the a, 2, and ~, bands for the cytochrome b components of certain representative bacterial species and for yeast are given. I t can be seen that they may be classified into groups according to the position of their a bands. 1. Cytochrome b with absorption maximum 562 to 565 mt~. 2. Cytochrome bl at 560 mu. 3. Cytochrome b2 of yeast at 556 mtL. 4. A component absorbing at 554 mt~ which may or may not belong to this group and which is characteristic of Acetobacter. 1 1L. Smith, Bacteriol Revs. 18, 106 (1954).

[132]

CYTOCttROME b GROUP (BACTERIA)

745

Finally, the recently discovered cytochrome e ~ with an a band at 552 to 553 m~ at liquid air temperature is present in Bacillus lichenformis as well as in yeast and various animal and insect tissues. Recent studies suggest t h a t cytochrome e should be considered with the b group2 DIFFERENCE SPECTRA OF CYTOCHROME b IN CERTAIN BACTERIAa

Position of maxima for reduced pigment, m~ Strain

a

-y

Type

Bacillus subtilis Staphylococcus albus Sarcina lutea

564 564 565 562

525 523 523 523

430 422 427 430

Aerobacter aerogenes Escherichia coli Azotobacter chroococcum Corynebacterium diphtheriae c

560 560 560 560

530 533 530 524

430 432 428 429

bl

Acetobacter pasteurianum Acetobacter suboxydans

554 554

523 525

428 422

?

Delft yeast a

557

528

424

b~

Baker's yeast

This table has been constructed from the data of Smith? Except for the bands of C. diphtheriae and of yeast, all values are for difference spectra, i.e., difference in extinction between oxidized and reduced pigments. bL. Smith, Bacteriol. Revs. 18, 106 (1954). A. M. Pappenheimer, Jr., and E. D. Hendee, J. Biol. Chem. 171, 701 (1947). d C. A. Appleby and R. K. Morton, Nature 173, 749 (1954). I n addition to the similarity in their absorption spectrum, the various members of the b group have certain other characteristics in common. Most of the cytochrome b's appear to be autoxidizable at an appreciable rate, although far more slowly than is cytochrome oxidase. T h e y do not combine with CO, H C N , or HN3, although all the group probably contain protoheme as their prosthetic moiety. Their oxidation potential lies below t h a t of cytochrome c, and in the few cases studied their position in the respiratory chain lies below t h a t of cytochrome c. Little is known of the function of the bacterial cytochrome b group. Cytochromes b and bl are both concerned in the oxidation of succinate. 4,5 2 D. Keilin and E. F. Hartree, Nature 164, 254 (1949). a A. M. Pappenheimer, Jr., and C. M. Williams, J. Biol. Chem. 209, 915 (1954). 4D. Keilin and C. H. Harpley, Biochem J. 35, 688 (t941). s A. M. Pappenheimer, Jr., and E. D. Hendee, J. Biol. Chem. 180, 597 (1949).

746

RESPIRATORY ENZYMES

[132]

Cytochrome b~ of yeast appears to be closely associated with lactic dehydrogenase activity (see Vol. I [68] for preparation and properties of yeast lactic dehydrogenase). The cytochrome bl of E. coli is not only involved in succinate oxidation 4 but may also be an integral part of the nitrate reductase system. 6 None of the cytochrome b group has been isolated in pure form as a soluble pigment with the possible exception of cytochrome b2 of yeast. 7,7~ All other members appear to be associated with large mitrochondrial and microsomal fragments. Preparation and Partial Purification of Cytochrome bl from Corynebacterium diphtheriae 5 Bacterial Cultures. The Toronto strain of Corynebacterium diphtheriae is grown in 5- to 10-1. lots in Povitsky bottles as a pellicle on the surface of Mueller and Miller's casein hydrolyzate medium s containing 1.5 % maltose. Just before inoculation 25 mg. of FeSO4"7H20 per liter is added as a sterile solution. The pH of the medium should be below 7.5 at the time of harvesting. After 6 to 7 days' growth at 34 °, the bacteria are collected by centrifugation and are washed three times with saline in the centrifuge. Extraction of Cytochrome bl from the Bacterial Cells. Washed cells from 5 1. of culture are suspended in 150 to 200 ml. of saline, and the thick suspension (6 to 8 mg. bacterial N per milliliter) is disrupted for 30 minutes in a 9000-cps sonic oscillator (Raytheon Corp., Waltham, Massachusetts) in 25-ml. charges. If a sonic oscillator is not available, the cells may be broken up by grinding. 9 Partial Purification of Cytochrome bl. The purification procedure by differential centrifugation is entirely empirical, and no rigid procedure can be outlined. The Qo, (succinate, KCN) and Qo~ (succinate, MB, KCN) were 146 and 4070 ~l./mg. N/hr., respectively, for a crude diphtherial extract containing 7.6 mg. N/ml. The purification of this particular lot was accomplished as follows: 180 ml. of extract was centrifuged for 30 minutes at 10,000 r.p.m, in a refrigerated International centrifuge to remove bacterial debris. The turbid supernatant was then centrifuged at 12,000 r.p.m. (ca. 18,000 X g) in a Servall centrifuge, Model SS-2, for 30 minutes. Then 6R. Sato and F. Egami, Bull. Chem. Soc. Japan 22, 137 (1949). 7 S. J. Bach, M. Dixon, and L. C. Zerfas, Biochem. J. 40, 229 (1946). 7~C. A. Appleby and R. K. Morton [Nature 173, 749 (1954)] have isolated the lactic dehydrogenase of yeast as a crystalline hemoprotein containing flavin. s j. H. Mueller and P. Miller, J. Immunol. 40, 21 (1941). 9 G. KalnitSky, M. F. Utter, and C. H. Werkman, J. Bacteriol. 49, 595 (1945).

[132]

CYTOCHROME b GROUt (BACTERIA)

747

25 ml. of fatty upper layer of low activity was removed and discarded. Next 130 ml. of a clear red layer containing more than 60% of the total succinic oxidase activity was removed from a small amount of sediment and centrifuged in the Servall at 16,000 r.p.m. (ca. 30,000 )< g) for 3 hours. Once again three layers were obtained: an inactive yellow upper layer, a clear reddish-brown layer with considerable activity, and a highly active dark wine-red transparent sediment at the bottom of the tube. The sediment was suspended in 0.02 M phosphate buffer, pH 7, and homogenized for 20 minutes in the sonic oscillator. Thirty-five milliliters of dark-red solution was obtained which was clarified by centrifugation in the Servall for 30 minutes at 12,000 r.p.m. This solution contained about 30% of the total original succinic oxidase activity. Its Qo2 (succinate, KCN) was 440 and Qo2 (succinate, MB, KCN) was 14,000 ;~l. O2/mg. N/hr. This represents a purification of 3- to 3.5-fold, on the basis of both hemin content and specific activity.

Properties The above preparation contained 3.9 mg. N/ml. and 1.09 ~, hemin per milligram of nitrogen. Solutions of the purified pigment are dark red in color, clear by transmitted light, but show a pronounced Tyndall effect. After reduction with dithionite or with succinate, the color changes to a more pinkish hue and intense absorption bands centered at 560 and 529 m~ become readily visible with a hand spectroscope. The Soret band of the oxidized pigment shows a maximum absorption at 415 m# which shifts to 429 m# on reduction. When examined in the ultracentrifuge, cytochrome bl is polydisperse, and no distinct sedimenting components can be identified. On standing, even in the cold, aggregation occurs, and within 1 or 2 days solutions become turbid without, however, appreciable loss in enzymatic activity. Thus, although cytochrome bl cannot be regarded as a truly soluble protein such as cytochrome c, freshly purified preparations, in contrast to similarly prepared material from mammalian tissues, yield clear solutions which can readily be studied in an ordinary Beckman spectrophotometer. The preparations can be dried from the frozen state or can be precipitated at pH 4.8 in the cold without loss of activity. However, reconstitution of dried or precipitated b~ in buffer always yields turbid solutions. When purified preparations are heated to 60 ° at pH 7.6, aggregation occurs and the solution rapidly becomes turbid. Actual coagulation does not occur until 75 °, at which temperature 75% of the succinic oxidase activity is lost in 30 minutes. Assay of Enzymatic Activity. Diphtherial cytochrome b~ preparations

748

RESPIRATORY ENZYME$

[132]

catalyze the oxidation of succinate to fumarate. Unlike the bl of E. coli, 4 the reaction is not affected by M/400 KCN. Since available evidence suggests that for the diphtherial system under these conditions autoxidation of bl is the rate-controlling step, manometric determination of succinie oxidase activity in the presence of cyanide can be used as a method of assay. The total volume in each Warburg vessel is 2.2 ml., including 0.2 ml. of 20% NaOH in the center well and 0.2 ml. of M / 5 sodium succinate in the side arm. Phosphate buffer, pH 7.8, to give a final concentration of M/IO, 0.1 ml. of M/20 KCN, and a suitable quantity of enzyme are placed in the vessel itself. Control vessels without substrate should always be included but usually show no oxygen uptake. Qo2 values may be calculated from the oxygen consumed during the first 30 minutes and expressed as microliters per milligram of nitrogen per hour. The enzyme shows a sharp pH optimum at 7.8. Other Methods of Assay. Since succinic oxidase activity as measured above has always proved proportional to succinic dehydrogenase activity, any method for measuring the latter (see Vol. I [121]) would appear suitable for assay of diphtherial cytochrome bl. Succinic dehydrogenase may be determined manometrically in the presence of methylene blue and cyanide or by the anaerobic ferricyanide method of Quastel and Wheatly. '° Both methods are more sensitive than the one described above, because the autoxidation of methylene blue is more rapid than that of cytochrome bl and because oxidation of reduced bl by either methylene blue or ferrieyanide is more rapid than its autoxidation by molecular oxygen. N~

Qco,(Suce., ferrieyanide) = 4Qo,(suec., MB, KCN) = 120Qo,(SUCC., KCN) Suecinate oxidation may also be determined spectrophotometrically by following the rate of reduction of the nonautoxidizable dye 2,6-dichlorophenolindophenol (Price and Thimann). H 10j. H. Quastel and A. H. M. Wheatley, Biochem. J. 32, 936 (1938). 11C. A. Price and K. V. Thimann, Arch. Biochem. and Biophys. 33, 170 (1951).

[133]

CYTOCHROME C (MAMMALS)

[133] Cytochrome

c

749

(Mammals)

By KARL-GusTAV PAUL Fe +++ (Cytochrome Oxidase) -t- Fe ++ (Cytochrome c) ~-Fe ++ (Cytochrome Oxidase) % Fe +++ (Cytochrome c) Fe +++ (Cytoehrome c) + R - ~ Fe ++ (Cytochrome c) + R

Assay Methods Spectrophotometric Method. The concentration of cytochrome c in a solution of unknown strength is determined, after suitable dilution with 0.05 to 0.15 M phosphate buffer of pH 6 to 8 and reduction with Na2S204, from the difference in light absorption between the a band (e550~ = 27.7 mM -1 X cm.-1) 1 and the minimum (e536,n~= 7.7 mM -1 X cm.-1) 1 between the a and ~ bands of the hemochromogen spectrum of reduced cytochrome c. Since the ~ band is very sharp, the readings have to be taken at every 0.5 m~ around 550 m~. The highest value is used. The above molar absorption coefficients have been found for several cytochrome e preparations obtained according to the method of Keilin and Hartree, 2 with Beckman DU spectrophotometers and with Na2S204 as reducing agent. Cytochrome c is, under certain conditions, e.g., extreme pH values and aging in solution, converted to a catalytically inactive form which is oxidizable by 02 even at neutral pH. k partial destruction of cytochrome c to the autoxidizable pigment cannot easily be detected by visual spectroscopy but can be determined according to Tsou. 8 This test should be made on every preparation prior to its use. The solution to be examined is suitably diluted with phosphate buffer of pH 7.3, final buffer concentration 0.05 to 0.15 M, and the optical density at 550 mu is determined after reduction with Na2S~O~. A current of CO is passed through the solution for 10 minutes, and the density at 550 mu is redetermined. The per cent of autoxidizable cytochrome c is given by 162(Drod.- D,ed+co)/D~d. where D~d. and D~od+coare the first and second readings. ~ 1 K. G. Paul, Acta Chem. Scand. 5, 389 (1951). 2 D. Keilin and E. F. Hartree, Biochem. J. 39, 289 (1945). C. L. Tsou, Biochem. J. 49, 362 (1951). 4 Tsou's method presupposes t~hat the molar absorptions of active and inactive cytochrome c are the same at 550 m~ after reduction. The latter gives, however, probably about 5 % higher absorption [D. L. Drabkin, J. Biol. Chem. 146, 605 (1942); E. Boeri, H. Baltscheffsky, R. K. Bonnichsen, and K. G. Paul, Acta Chem. Scand. 7, 831 (1953)].

750

RESPIRATORY ENZYMES

[133]

Manometric Method. The catalytic activities of different specimens of cytochrome c can be compared in the system O2/cytochrome oxidase/cytochrome c/succinate-succinic dehydrogenase. Succinic oxidase ( -- cytochrome oxidase ~- cytochrome c -F succinic dehydrogenase) preparations from kidney cortex are deficient in cytochrome c, which thus becomes the determining link. (For the preparation of succinic oxidase, ~,8 see Vol. I [121].) Reagents 0.4 M Na-succinate in water. 0.12 M phosphate buffer, pH 7.3. Kidney cortex preparation.

Procedure. Kidney cortex preparation (0.2 m l . ) + cytochrome c (2>k4ao, k4 c a n be o b t a i n e d , since in this case

dx

d--£ ~-" k4aoe

(14a)

and 1

A~5

k4 ~ -2- X - aoe At

(14b)

T h e e x p e r i m e n t a l p r o c e d u r e is t h e s a m e for b o t h m o d i f i c a t i o n s of t h e t e s t . Reagents 20 m M 10 m M 10 m M 40 m M --~10 -7

g u a i a c o l (0.22 ml. of g u a i a c o l in 100 ml. of w a t e r ) . p h o s p h a t e buffer, p H 7.0. h y d r o g e n p e r o x i d e s o l u t i o n (for kl d e t e r m i n a t i o n ) . h y d r o g e n p e r o x i d e s o l u t i o n (for k4 d e t e r m i n a t i o n ) . M p e r o x i d a s e solution.

Procedure. T h e v a l u e s b, c, d, a n d f d e p e n d on t h e c o n s t a n t t o be measured and are read from Table II. TABLE II AMOUNTS OF REAGENTS ADDED TO THE CUVETTE AND THE FINAL CONCENTRATION OF THESE REAGENTS FOR THE TWO MODIFICATIONS OF THE GUAIACOL ASSAY

Constant to be determined

Buffer Guaiacol [Guaiucol] H~02 stock [H202] Df ~ klxo k4a0

Units

Symbol used in text

kl

k4

ml. ml. M mM M cm. -1 ---

b c a0 d x0 f ---

1.0 2.0 1.3 X 10-2 10 3.3 X 10-5 0.2 300 4300

2.9 0.05 3.3 X 10-4 40 1.3 X 10-4 0.8 1170 110

Approximate final reading of optical density at 470 m~.

772

R~SPIRATORV ~NZVMES

[136]

A spectrophotometer or direct-reading colorimeter suitable for a wavelength region of 470 m~ m a y be used. The assay is carried out at room temperature. One of a pair of cuvettes of 1-cm. p a t h is filled with water, the other with b ml. of phosphate buffer, c ml. of guaiacol, and about 10-9 M peroxidase (see below). The initial optical density is read at 470 m~, and the optical density scale is offset to a value 0.050 greater t h a n this reading. The shutter is closed, and 10 td. of d m M hydrogen peroxide is added as a drop at the end of a stirring rod (the final concentration is x0). The solution is rapidly stirred, and a stop watch is started. The shutter is opened, and the time required (At) for the galvanometer to reach the null point is measured; it should be between 15 and 30 seconds. The end point of the reaction should correspond to an optical density of f. Determination of the Enzyme Concentration. The peroxidase concentration can be determined spectrophotometrically on the basis of the molecular extinction coefficients given on pp. 799, 812, 817. Alternatively, the dry weight of the enzyme used m a y be divided b y the molecular weight of the peroxidase (pp. 798, 808, 817) to give an effective concentration for an impure enzyme solution. Calculation of the Results. The hydrogen peroxide utilized in the formation of a tetraguaiacol solution t h a t gives an optical density of 0.05 4 × 0.050 at 470 m# is 26.6 m M -- 7.5 ~M, since 4 moles of H202 is required to form 1 mole of tetraguaiacol and since the latter has an extinction coefficient of e = 26.6 cm. -1 m M -1 at 470 m~. 9 This 7.5 ~M of H202 corresponds to Ax of equations 13b and 14b, and At (units sec.) to the measured time interval. Both x0 and e (units M. X 1.-~) can be measured spectrophotometrically (see above). F r o m equations 13b and 14b the rate constants k~ and k4, respectively, can thus be calculated: 1 Ax 7.5 X 10-8 1 .22 kl × . . . . (15) xoe At 3.3 X 10-6 X e At e At Experiments with pure horseradish peroxidase gave under the conditions of the test kl = 0.89 × 107 at 20 °, and 1.03 X 107 at 30 ° . T h e value of kl should be 0.9 × 107 at 25°. 1~ k4 =

Ax 7.5 X 1 0 - ~ 1 1 X -aoe At 3.3 X 10-~ X e At

__

10-2 e At

2.2 X

(16)

Actual experimental values were k4 = 2.2 X 10 s at 20 °, and 3.1 × 10 ~ at 30°; k4 should be 3.3 × 105 at 25 °.17 16B. Chance, Arch. Biochem. 22, 224 (1949). 1~B. Chance, Arch. Biochem. 24, 410 (1949).

[136]

ASSAY OF CATALASES AND PEROXIDASES

773

The value of h~ for lactoperoxidase, verdoperoxidase, and yeast peroxidase (cytochrome c peroxidase) is of the same order of magnitude as that for the horseradish enzyme, but experimental tests have not been made to determine whether this value can actually be obtained in the guaiacol test with any but the horseradish enzyme. Accuracy and Limitations of the Method. The results should be accurate to a few per cent. The main sources of error are caused by the use of this method with crude cell extracts. In some cases there are substances present that interfere with the formation of tetraguaiacol.17~ B. The Pyrogallol Test

Principle. The traditional test for peroxidase activity is the formation of purpurogallin from pyrogallol (Willstiitter and StolllS). This method was devised before the mechanism of peroxidase action was fully understood. The original experimental conditions seem not to be suitable for measuring k4 of equation 12, since klxo--~h~ao, and it was demonstrafed 17 that the enzymatic reaction proper was terminated (the enzyme-substrate compounds had disappeared) before all the end product (purpurogallin) had been formed. This shows that intermediate products must be involved and that the color formation is not really a direct measurement of enzyme kinetics. The assay will nevertheless be described for those who wish to correlate their data with those of the older literature. Reagents. All solutions should be made up in glass-distilled water, and rigidly cleaned vessels should be used. Pyrogallol (two times resublimed), 1.25 g. in 500 ml. (20 mM). Hydrogen peroxide, 12.5 rag. in 500 ml. (0.74 raM). Phosphate buffer, pH 7.0, 10 mM. Peroxidase, about 1 ~, in 500 ml. (5 X 10-11 M). Procedure. In one of the modified procedures the reagents are made up in a 500-ml. volume, and the reaction is allowed to proceed for 5 minutes after which time it is stopped by adding 5 ml. of 5 N H2SO4. The purpurogallin is extracted three times with ether, alcohol is added, and the solution is made up to a known volume. The concentration of purpurogallin is determined spectrophotometrically at 430 m~, where the extinction coefficient e = 2.47 cm. -I mM-L 19 A large number of varia17~I t m u s t be ascertained, therefore, t h a t no secondary reactions w i t h guaiacol or tetraguaiacol occur. Failure to take such reactions into account can give rise to misleading conclusions. Thus, P. George [J. Biol. Chem. 201, 413 (1953)l observed t h a t HOC1 reacts with guaiacol or its oxidation products directly. 18 R. Willsti~tter and A. Stoll, Ann. 416, 21 (1917). 19 A. C. Maehly, unpublished experiments.

774

RESPIRATORY ENZYMES

[136]

tions of this procedure h a v e been used, b u t as long as the concentration of the reactants is unchanged, the same result should be obtained. Calculation of Results. The activity of peroxidase is traditionally expressed b y the purpurogallin n u m b e r (PZ). PZ is the n u m b e r of milligrams of purpurogallin formed b y 1 mg. of e n z y m e under the conditions of the s t a n d a r d test. T h e milligrams of e n z y m e present are determined directly b y a d r y weight determination or spectrophotometrically.~° The value of PZ for pure peroxidase as determined b y the test is 1020 according to Theorell and Maehly, 23 and 1220 according to Keilin and Hartree. 24 T h e value of PZ reached with equation 12 and the k n o w n values of kl and k4 is 1500 (Chance17), which is s o m e w h a t in excess of t h a t actually realized under the assay conditions. TABLE III MISCELLANEOUS PEROXIDASE TESTS

Peroxidase Horseradish Yeast MyeloLacto-

Donor employed

Investigator

Cf. page

Mesidine Cytochrome c Uric acid Dihydroxyphenylalanine

Paul and Avi-Dor~ Altschul et al.b Agner ~ Polls and Shmukler ~

794 813

a K. G. Paul and Y. Avi-Dor, Acla Chem. Scand. 8, 649 (1954). b A. M. Altschul, R. Abrams, and T. R. Hogness, J. Biol. Chem. 136, 777 (1940). c K. Agner, personal communication. d B. D. Polls and H. W. Shmukler, J. Biol. Chem. 201, 475 (1953) L i m i t a t i o n s of the Method. Considerable care is necessary to obtain reproducible results in the purpurogallin test (see Reagents). I n addition, the test gives v e r y low values for lacto- and verdoperoxidase, owing to the fact t h a t these enzymes are not s a t u r a t e d with peroxide u n d e r the conditions of the test and are to some extent i n a c t i v a t e d b y the high H202 concentration. T h u s their respective PZ values of 71 (Theorell and /kkeson 25) and 41 (Agner 26) cannot be converted into a specific rate constant. Direct m e a s u r e m e n t s of k4 from the kinetics of an enzyme-subs t r a t e c o m p o u n d of lactoperoxidase in the presence of pyrogallol gives a

20The molecular weight of horseradish peroxidase is 40,200 according to Theorell and Ehrenberg, 21 and 39,800 according to Cecil and Ogston. 22 21 A. Ehrenberg, personal communication; cf. A. C. Maehly, Vol. II [143], p. 808. ~2R. Cecil and A. G. Ogston, Biochem. J. 49, 105 (1951). 23 H. Theorell and A. C. Maehly, Aeta Chem. Scan& 4, 422 (1950). 24 D. Keilin and E. F. Hartree, Biochem. J. 49~ 88 (1951). ~s H. Theorell and/~. ~keson, Arkiv Kemi, Mineral. Geol. 17B, No. 7 (1943) ~e K. Agner, Acta Physiol. Seand. 2, Suppl. 8 (1941).

[137]

LIVER CATALASE

775

value of 7 × 106, considerably greater t h a n the value for the horseradish enzyme. ~7

C. Other Peroxidase Assays Those working on the purification of peroxidases h a v e usually developed their own particular assay s y s t e m t h a t should p r o b a b l y be followed b y those who wish to duplicate the preparations. Table I I I lists some of these methods, which, however, do not clearly define a single reaction velocity constant. The guaiacol test is satisfactory for the enzymes listed in T a b l e I I I , although it has not y e t been proved t h a t accurate values of kl and k4 can be obtained in all cases. ~7B. Chance, J. Am. Chem. Soc. 72, 1577 (1950).

[137] L i v e r C a t a l a s e B y JAMES B. SUMNER and ALEXANDER L. DOUNCE

I. Introduction N e x t to urease, beef liver catalase is possibly the easiest enzyme to obtain in crystalline condition. The reasons for this are the unusual stability of this enzyme, its insolubility in w a t e r at its isoelectric point, and its relatively high concentration in beef liver. Since the p r e p a r a t i o n of crystalline catalase from beef liver b y S u m n e r and Dounce 1 crystalline catalases h a v e been obtained from a n u m b e r of other sources. These are: l a m b liver, 2 horse liver, 3 beef erythrocytes, 4 horse kidney and h u m a n liver, 5 guinea pig liver, 8 Micrococcus lysodeikticus, 7 and pig liver. 8 Recently a m e t h o d has been reported but not described for obtaining crystalline catalase from rat liver2 Methods for preparing catalase from the livers of animals other t h a n the ox are generally rather involved and for t h a t reason will not be described here. 1j. B. Sumner and A. L. Dounce, J. Biol. Chem. 121, 417 (1937). 2 A. L. Dounce, J. Biol. Chem. 143, 497 (1942). 3 A. L. Dounce and O. D. Frampton, Science 89, 300 (1939). 4 M. Laskowski and J. B. Sumner, Science 94, 615 (1941). 5 R. K. Bonnichsen, Acta Chem. Scand. 1, 114 (1947); Arch. Biochem. 12, 83 (1947). s R. K. Bonnichsen, Acta Chem. Scan& 2, 561 (1948). 7 D. Herbert and A. J. Pinsent, Nature 160, 125 (1947). 8 N. K. Sarkar and J. B. Sumner, Enzymologia 14, 280 (1951). g R. E. Greenfield and V. E. Price, Proe. Am. Assoc. Cancer Research 1, 21 (1953).

776

RESPIRATORY ENZYMES

[137]

II. M e t h o d of Sumner and Dounce I

Put beef liver through a household meat grinder four times, and mix 3O0-g. portions with 400-ml. portions of 35 % dioxane. 9a After stirring for 4 or 5 minutes, place on fluted filters (32 cm., Schleicher and Schuell, No. 595) and cover with watch glasses. Allow to filter into 500-ml. graduates overnight at room temperature. The next day add to every 100 ml. of filtrate 20 ml. of dioxane with stirring, and set the material in the ice chest. After 12 or more hours, filter and refilter in the ice chest until the solution is practically clear. Then precipitate the catalase by adding 10.2 ml. of dioxane to every 100 ml. of filtrate. Allow the material to stand in the ice chest overnight, and then filter off the precipitated catalase, refiltering if necessary. The residue on the filter paper must stand in the ice chest until all the liquid has drained through the filter paper. Then open up the filter paper and place it upon several dry filter papers. Serape the precipitate off with a spatula, and place it in a beaker. Depending on the yield of precipitate, stir with 5 to 15 ml. of water for each 300 g. of liver, and add a few drops of pancreatic amylase in order to digest the glycogen. Then filter the solution at room temperature, and extract the residue a second time. Crystals of catalase can be obtained from the combined filtrates in one of two ways: (1) Chill the combined filtrates, and add saturated ammonium sulfate cautiously until a haziness appears. The catalase crystallizes as fine needles almost immediately. Keep the material in the ice chest, and add ammonium sulfate until a good crop of crystals is obtained. However, it is not advisable to add a great excess of ammonium sulfate, since the catalase crystals, which are present as needles, are difficult to centrifuge down from solutions of high specific gravity. 2. Adjust the pH of the combined filtrates described above to approximately 5.7, the isoelectric point of catalase, and then dialyze for a day or two against a number of changes of distilled water. Catalase crystallizes out as very thin plates and occasionally as prisms. Observe the suspension microscopically, and stop the dialysis when appreciable numbers of spheroids appear. Catalase crystals may be stored suspended in ammonium sulfate solution for considerable time without appreciable loss in activity. The crystals can also be stored in a suspension in distilled water, but in this case they are apt to undergo gradual deterioration, owing to bacterial action, unless they have been crystallized from a sterile solution. 9~Dr. Roger Young of this laboratory has purified dioxane from such impurities as hydrochloric acid and ferric chloride by shaking each liter of dioxane with 20 ml. of saturated sodium hydroxide, separating the two liquids in a separatory funnel, and then filtering the dioxane layer through filter paper.

[137]

LIVER CATALASE

777

III. Method of Sarkar and Sumner s

Grind fresh beef liver in a meat grinder, putting the material through the grinder four times. To every kilogram of ground material add 1500 ml. of distilled water and mix well. Mix 1 vol. of 95 % alcohol with 1 vol. of c.p. chloroform, and add 480 vol. of this mixture to every 1000 vol. of the liver suspension. Shake violently for 30 seconds, and filter through 32-cm. Balston No. 12 folded filter papers at room temperature. When most of the liquid has filtered through, for every kilogram of liver pour about 500 ml. of distilled water on the residue to wash more catalase through. Cautiously bring the filtrate to pH 5.7 by adding glacial acetic acid, centrifuge, and remove protein impurity. Add 250 ml. of tricalcium phosphate suspension which is at about pH 5.7 (30 to 35 mg./ml.) to every liter of filtrate, and mix for 10 to 15 minutes. At once centrifuge down the adsorption complex, and discard the supernatant. Elute the catalase from the adsorption complex with 200 ml. of 0.1 M phosphate buffer of pH 8.0 (for about 2 kg. of liver used at the start), followed by centrifuging. Repeat this elution once more, using 200 ml. of the pH 8.0 phosphate buffer. To the combined eluates add solid ammonium sulfate, using 30 g. for every 100 ml., mix, and allow to stand in the ice chest overnight. Next day centrifuge the catalase down at high speed in a refrigerated centrifuge and discard the supernatant. Mix the sediment with just enough water to make a mushy suspension, transfer to a dialyzing parchment, and dialyze in the ice chest against several changes of distilled water. If a whitish or buff colored precipitate forms, it should be centrifuged down and discarded. The catalase will crystallize if it is from beef liver. This method will give satisfactory results starting with as little as 100 g. of beef liver. IV. Employment of the Tswett Column

Catalase can be purified by being adsorbed on tricalcium phosphate in a Tswett column at pH 5.7 and later by eluting with phosphate buffer of pI~ 8.0. However, since most preparations of tricalcium phosphate are rather impermeable to water, it is probably better to adsorb on Celite. V. Other Methods Kitagawa and Shirakawa obtained crystalline beef liver catalase by means of a method involving precipitations with acetone and ammonium sulfate. 1° 1oM. Kitagawa and M. Shirakawa, J. Agr. Chem. Soc. (Japan) 67, 794 (1941).

778

RESPIRATORY ENZYMES

[137]

Dounce ~ has prepared crystalline beef liver catalase, using dilute acetone. Mosimann, 11 Bonnichsen, 5 and Tauber and Petit 1~ have still other methods. The procedure of Herbert and Pinsent ~ for the crystallization of bacterial catalase has been mentioned already. Recently Brown 13 has claimed that he has succeeded in separating beef liver catalase into two components through a new procedure. VI. RecrystaUization of Catalase The original method described by Sumner and Dounce 1 advised dissolving the catalase crystals in water plus the least possible amount of 9.6% phosphate buffer of pH 7.4. However, if any considerable amount of ammonium sulfate is present, the catalase crystals will not dissolve. One can add a little solid sodium chloride and warm the preparation to 40 ° in order to dissolve the catalase. However, it is better to recrystallize catalase as follows: Centrifuge down the catalase crystals, and discard the supernatant liquid. Stir up the crystals with very little water, and add a few milliliters of 6 to 9 % phosphate buffer of pH 7.4 to 7.8. Place this suspension of catalase crystals in a dialyzing parchment, and dialyze in the ice chest against distilled water. As soon as most of the ammonium sulfate present has dialyzed away the crystals will dissolve, provided that enough water is present. The solution can then be centrifuged to remove any whitish protein impurity that may be present. Further dialysis will result in the precipitation of a part of the catalase as prisms, provided that the solution is sufficiently concentrated. In order to precipitate the catalase as plates it will be necessary to dialyze against changes of distilled water for several days. If the catalase solution is brought to pH 5.7 by adding acid potassium phosphate, dialysis will cause the catalase to precipitate as needles. If catalase has been crystallized originally by dialysis, recrystallization is extremely simple. ~4 The crystals are centrifuged down and dissolved in the minimal amount of sodium chloride solution at a final concentration of 10% NaC1. A volume of sodium chloride solution of about ten to twenty times the volume of the centrifuged crystalline precipitate usually suffices. Buffer is unnecessary, but reasonable care should be taken to keep the sodium chloride concentration correct. After the catalase is completely dissolved, as can be observed by the disappearance of the silkiness of the solution on swirling, amorphous conII W. Mosimann, Arch. Biochem. and Biophys. 3~ 487 (1951). 12H. Tauber and E. L. Petit, J. Biol. Chem. 195, 703 (1952). 13G. L. Brown, Biochem. J. 51, 569 (1952). 1~j. B. Sumner and A. L. Dounce~ J. Biol. Chem. 127~439 (1939).

[137]

LIVER CATALASE

779

taminating protein is centrifuged down with high speed, and the catalase is recrystallized b y dialysis for 24 hours against several changes of distilled water. The crystals appear as plates. If prisms are desired, the p H must be increased to 7.0 to 7.5 by the addition of M / 2 Na2HPO4 before dialysis. Catalase crystallizes as needles from ammonium sulfate; as extremely thin plates from distilled water on dialysis at or near the isoelectric point (pH 5.7); and as prisms when dialyzed from distilled water above the isoelectric point. All three crystalline forms are intraconvertable. The yield of crystals obtained b y dialysis is best when plates are obtained by dialyzing at the isoelectric point. Both prisms and plates are soluble in sodium chloride at a final concentration of 10%.

VII. Estimation of Catalase Activity In 1927 von Euler and Josephson ~5 described a method for the determination of catalase activity which in all probability is the best yet devised for solutions of pure or partially purified catalase. The method is as follows: Chill 50 ml. of approximately 0.01 N hydrogen peroxide 16 to 0 ° in a b a t h of chopped ice. The peroxide should be 0.0067 M in phosphate buffer of p H 6.8. Add 1 ml. of properly diluted catalase, mix rapidly, at once remove a 5-ml. sample, and immediately blow it into a small flask containing 5 ml. of 2 N sulfuric acid. Remove other samples in a similar manner at 3, 6, 9, and 12 minutes. Titrate the peroxide in the flasks with 0.005 N permanganate. Calculate the monomolecular velocity constants, using the formula: 1 A K =~logl0A -x where K = monomolecular velocity constant, t = time in minutes, A = milliliters of permanganate used at zero time, and A - x = milliliters of permanganate used at 3, 6, 9, and 12 minutes. Construct a graph using minutes plotted against K values. The K value for zero time is found b y extrapolation. This value represents the catalase units at the dilution employed in the analysis. T o obtain the p u r i t y or Katalasefahigkeit of the catalase the equation used is: K Kat.f . = g. catalase per ml. 15H. yon Euler and K. Josephson, Ann. 452, 158 (1927). le We find it best to prepare this by diluting 30% hydrogen peroxide which has been distilled in ~acuo. The redistilled peroxide must be kept in the ice chest.

780

R E S P I R A T O R Y ENZYMES

[137]

For crystalline catalases the K a t . f . value m a y range from 30,000 to 60,000, depending on the source of the catalase. In 1904 Jolles 17 described a m e t h o d for the estimation of catalase activity in which the unused hydrogen peroxide was determined b y adding potassium iodide and a drop of ammonium molybdate, followed by titration with thiosulfate in the presence of starch. This method is applicable to homogenates, where the permanganate m e t h o d is poor because of reduction of the permanganate b y material in the homogenate. T h e method, as modified b y Sumner and Dounce, TMis: Add 1 ml. of properly diluted catalase solution (e.g., h u m a n blood diluted 1 to 1000) to 50 ml. of ice-cold 0.01 N hydrogen peroxide in M / 1 5 0 phosphate buffer, p H 6.8. At once withdraw a 5.0-ml. aliquot, and blow this into 5 ml. of 10% sulfuric acid. Withdraw other samples after 3, 6, 9, and 12 minutes. Add to each sample 1 ml. of 5 % potassium iodide and 0.5 ml. of saturated molybdic acid solution. Mix well, and allow to stand for about 3 minutes. Add a few drops of soluble starch solution and titrate to the end point, using 0.005 N thiosulfate. The plotting of titration values against minutes of time and the calculation of K values and K a t . f . values are exactly as described for the procedure of yon Euler and Josephson. If it is desired to express the a m o u n t of catalase present, a catalase unit can be taken as the a m o u n t of catalase required to give a calculated K0 value of 1. TM This m e t h o d has been employed b y Dounce and Shanewise 2° for the estimation of catalase in rat liver and appears to be highly reliable. A similar m e t h o d has been described b y Balls and Hale. 21 (See also Schwartz et al. ~2) Bonnichsen, Chance, and Theorell 2a have modified the m e t h o d of yon Euler and Josephson b y employing much greater quantities of catalase and much shorter periods of digestion. Sarkar and Sumner s have claimed t h a t this modification is no improvement. Feinstein 24 has published a method for catalase activity wherein perborate is employed in place of hydrogen peroxide. Walker 25has determined catalase activity b y means of the polarograph. i~ A. Jolles, Mi~nch. Med. Wochschr. 51, 2083 (1904); quoted from It. yon Euler, Chemie der Enzyme, II Teil, 3 Abschnitt, p. 73. J. F. Bergman, Mfinchen, 1934. 18j. B. Sumner and A. L. Dounce, unpublished. 19E. Tria, J. Biol. Chem. 129, 377 (1939). ~0A. L. Dounce and A. B. Shanewise, Cancer Research 10, 103 (1950). 2~A. K. Balls and W. S. Hale, J. Assoc. O~c. Agr. Chemists 15, 483 (1932). 2~E. J. Katz, L. Holt, and S. Schwartz, Natl. Nuclear Energy Ser. Div. IV, 23, 283 (1951). ~3R. K. Bonniehsen, B. Chance, and H. Theorell, Acta Chem. Scand. 1, 685 (1947). 24R. N. Feinstein, J. Biol. Chem. 180, 1197 (1949). 2~B. S. Walker, Proc. Federation Am. Sac. Exptl. Biol. 1, 140 (1942).

[138]

BLOOD CATALASE

781

Perlman and Lipmann 2e have described a manometric method for catalase activity. However, Theorel127 states t h a t a t t e m p t s to estimate catalase monometrically have been made repeatedly, but without success. This statement is probably true if one is concerned solely with an investigation of the kinetics of catalase action, but from the standpoint of determining catalase concentrations it does not apply. Dounce 2s has devised a manometric method for catalase activity and a m a n o m e t e r to be employed in this method. Beers and Sizer 29 have described the determination of catalase by means of a manometric method. ~6G. E. Perlmann and F. Lipmann, Arch. Biochem. 7, 159 (1945). 27H. Theorcll, in "The Enzymes" (J. B. Sumner and K. Myrbgck, eds.), Vol. 2, Part 1, p. 397, Academic Press, New York, 1951. 2s A. L. Dounce, Natl. Nuclear Energy Ser. Div. VI, 1, 270 (1949). 29R. F. Beers, Jr., and I. W. Sizer, Science 117, 710 (1953).

[138] B l o o d C a t a l a s e B y ROGER BONNICHSEN

Assay Method Principle. M a n y methods have been devised for the determination of catalase. 1 The m e t h o d described below, developed b y Bonnichsen, Chance, and Theorell, 1 is based on the determination of the a m o u n t of H202 split b y the enzyme in 15 and 30 seconds at room temperature and extrapolating to zero time. The H202 is measured b y titration with permanganate. The rapid titration technique minimizes the effect of catalase destruction t h a t takes place during the reaction. The method is rapid and sufficiently accurate for most purposes. (For other methods, see Vol. II [136].) Reagents

0.01 M phosphate buffer, p H 7. H~O2 solution (0.7 ml. of Perhydrol to 50 ml. of water). 2 % sulfuric acid. 0.01 M permanganate solution. Enzyme. The catalase is diluted with phosphate buffer to about 300 ~g per ml. Procedure. Fifty ml. of buffer is pipetted into a 100-ml. Erlenmeyer flask, and 2 ml. of H202 solution is added. Two ml. is withdrawn and

1R. K. Bonnichsen, B. Chance, and H. Theorell, Acta Chem. Scan& 1, 685 (1947).

782

RESPIRATORY E N Z Y M E S

[138]

blown into a 25-ml. E r l e n m e y e r flask containing a few drops of 2 % sulfuric acid. The enzyme is conveniently added to the buffer solution on a small watch glass, and the time is noted. Fifteen and 30 seconds later another 2 ml. is withdrawn and blown into 2 % sulfuric acid. The a m o u n t of H~O2 in the three samples is determined with permanganate. Definition of Units and Specific Activity. T h e first order reaction constant can be used as a measure of the catalase activity. T h e a m o u n t of catalase in the solution can be calculated from the density of the Soret band at 405 m~. There is some u n c e r t a i n t y in the literature a b o u t the value of the react[0n constant for pure catalase. Bonnichsen, Chance, and Theorell 1 give a value of 3.5 × 107 1. × mole -I X sec. -1. Deutsch 2 gives a value as high as 6.6, which agrees with the value found b y Agner2 There is also a disagreement about the millimolar extinction at 405 m~. T h e values v a r y from 380 to 420 mM. -1 cm. -1 for pure catalase. ~,4 The millimolar extinction at 280 m~ is more constant, 280 mM. -1 cm. -1.1 Application of Assay Method to Crude Tissue Preparations. The abovementioned m e t h o d can be used. The first 2-ml. sample is in this case taken a few seconds after the addition of the enzyme, and this sample is used as zero value. For preparative purpose it is more convenient to use Kat.f. described b y von Euler and Josephson 5 to express the activity and the p u r i t y of the enzyme.

Kat.f . =

K g. of enzyme in the 50-ml. vessel

X0 1 where K = log10 - ~ t ' X0 = initial H202, and X = H~02 at t minutes. Purification P r o c e d u r e The following m e t h o d can be used to prepare catalase from horse and pig blood. Preparation from beef blood has been described b y Laskowski and Sumner, e from h u m a n blood b y H e r b e r t and Pinsent. 7 If the m e t h o d is applied to beef blood, precaution must be taken when the hemoglobin is denatured with alcohol-chloroform as this catalase is more sensitive t h a n t h a t from other animals. If h u m a n blood is used the adsorption procedure used b y H e r b e r t and Pinsent should be carried out after step 2. H. F. Deutsch, Acta Chem. Scand. 6, 1516 (1952). 3 K. Agner, Arkiv Kemi, Minerol. Geol. B17, No. 9 (1943). 4 H. Theorell and A. Ehrenberg, Arch. Biochem. and Biophys. 41, 443 (1952). 5 H. von Euler and K. Josephson, Ann. 452, 158 (1927). 6 M. Laskowski and J. B. Sumner, Science 94, 615 (1941). D. Herbert and J. Pinsent, Biochem. J . 45, 203 (1948).

[138]

BLOOD CATALASE

783

Step 1. The blood corpuscles are washed twice with 0.9% NaCl to remove the plasma. They are then laked by addition of twice the volume of distilled water. Step 2. Tsuchihashi's 8 method is used to remove the hemoglobin. To each liter of the solution is added 500 ml. of an alcohol-chloroform mixture (1 part of chloroform to 3 parts of ethanol), while the solution is stirred vigorously for about 30 minutes. The denatured hemoglobin is removed by centrifugation or filtration. The alcohol-chloroform is then evaporated in vacuo. It is convenient to concentrate the solution at this step. The volume should be about 50 ml. for each liter of blood used as starting material. The evaporated solution is dialyzed overnight against water. The spectrum of the catalase is now clearly visible in a hand spectroscope. The spectrum can from now on be used to follow the catalase concentration. The purity is usually about 10 %. Step 3. To the dialyzed solution is added 2 M acetate buffer, pH 4, until the pH of the whole solution is about 4.0. The solution is allowed to stand for several hours. A brownish precipitate that forms is centrifuged and discarded. To the solution is added phosphate buffer, pH 6, until the solution is 0.1 M with regard to phosphate. To each 100 ml. of solution is added 60 ml. of acetone at room temperature. The precipitate is centrifuged and discarded. Further addition of 50 ml. of acetone precipitates the enzyme which is dissolved in 0.01 M phosphate, pH 7. The purity is now about 30 %. Step 4. It is possible at this purity by repeated ammonium sulfate fractionations to get crystalline enzyme, as described by Deutsch. 2 The first precipitate on addition of the salt should be discarded. Several fractions of partly crystalline material will be obtained by the first fractionation. Each of these fractions can be recrystallized to get pure enzyme. Another way of getting crystalline materials is to dialyze the catalase for several days against large volumes of distilled water. The catalase will then precipitate in a semi-crystalline form together with some impurities. The precipitate is dissolved in weak ammonia, pH about 9 in the solution. The catalase will rapidly dissolve, leaving some colored impurities undissolved. This procedure is repeated, and this time the catalase will crystallize from water. If this procedure does not yield crystalline catalase, ammonium sulfate should be tried. To the dialyzed solution solid ammonium sulfate is added to about 38"% saturation. From there on small drops of a saturated ammonium sulfate solution are added until a faint turbidity appears. The solution is centrifuged, and the inactive precipitate discarded. Very small amounts of the salt are again added to the solution with stirring, and the catalase will begin to crystallize. After 8 M. Tsuchihashi, Biochem. Z. 140, 63 (1923).

784

RESPIRATORY ENZYMES

[139]

a few hours most of the catalase has crystallized and can be centrifuged. The procedure requires sgme practice in protein crystallization and has to be varied a little with each new batch. The catalase can also be crystallized from alcohol solution at 4 °. The catalase solution is made 0.02 M with respect to phosphate buffer, pH 6, and cooled to 0 °. Ninety per cent ethanol is added dropwise with stirring. When the ethanol is 15% (v/v), the solution is left standing in the cold. In a few hours the catalase crystallizes. Occasionally the ethanol concentration has to be increased to 20%. To get pure catalase the procedure is repeated two or three times. The yield is about 400 mg. of catalase from each liter of blood corpuscles.

Properties The catalase has a red color at neutral pH. The molecular weight is 225,000. I t contains 1.1% protohemin, which equals 4 heroins per molecule and 0.09 % iron. The nitrogen content is 16.8 %. The catalase spectrum in a 0.001 M phosphate buffer shows bands in the visible at 623, 583, 535, and 505 m~. The Soret band is situated at 405 m~. As mentioned above, the millimolar extinction of the Sorer band varies in different preparations from 380 to 420 mM. -1 cm. -1. This is probably due to damage to the bonds between the hemin and the protein in part of the catalase. The millimolar extinction of the protein band at 280 mp seems more constant, 280 mM. -I cm. -1. Deutsch and Ehrenberg 9 have measured the paramagnetic susceptibility of horse blood catalase prepared by Deutsch. 2 T hey found the susceptibility per iron atom to be constant and equal to 13,390 c.g.s.e.m.u, between pH 4.8 and 10.4. g H. F. Deutsch and A. Ehrenberg, Acta Chem. Scan& 6, 1552 (1952).

[139]

Catalase

from

Bacteria

(Micrococcus lysodeikticus)

2H202~2H20+02

By

DENIS HERBERT

Catalase has been isolated in a pure state from only one species of bacterium, Micrococcus lysodeikticus (Herbert and Pinsenti). It is not known whether the catalases of other bacteria are similar, but the protein constituents of the enzymes would be expected to differ. 1D. Herbert and J. Pinsent, Biochem. J. 43, 193 (1948).

[139]

CATALASEFROM BACTERIA (Micrococcus lysodeikticus)

785

Assay M e t h o d

Principle. Catalase is allowed to act on hydrogen peroxide under defined conditions, and the peroxide remaining after known time intervals is determined by iodometric titration. The original isolators 1 of the enzyme used the assay method of yon Euler and Josephson. 2 Bonnichsen et al. 3 have since pointed out the disadvantages of this method (enzyme destruction by H202, causing falling values of k), and the following procedure, based on their ideas, has been found to be an improvement. Procedure. Pipet 5.0 ml. of H202 (0.01 M) in phosphate buffer (0.01 M, pH 6.8) into each of five test tubes (6 × 1 inch), and stand them in a bath at 25 °. To one tube add 1.0 ml. of enzyme rapidly (blowout pipet), simultaneously starting a stop watch. Stop the reaction after ca. 15 seconds by adding ca. 2 ml. of N H2SO4 rapidly from a small cylinder. Repeat the procedure with three of the remaining tubes of H~O2 using reaction times of ca. 30, 45, and 60 seconds; with the fifth tube the initial H:O2 concentration is obtained by adding the H2SO4 before the enzyme. The H202 remaining in each tube is determined by adding 0.5 ml. of 10% KI, one drop of 1% ammonium molybdate, and, after the tubes have stood for 3 minutes, titrating the liberated iodine with 0.01 N sodium thiosulfate (starch-iodide indicator), the whole contents of each tube being titrated in situ. Calculation of Results. For each of the four time intervals calculate the observed first-order velocity constant as

(where So is the H202 concentration at zero time, and S is the concentration remaining after t seconds), and take the mean of the four values. They should agree to within 5%, and there should be no tendency for the values to fall with increasing reaction time. The value of ]Cobs.is a measure of the concentration of catalase in the reaction mixture. If w is the dry weight (mg.) of enzyme preparation used in each test, then its concentration in the reaction mixture is c = w / 6 (g./1.). The specific catalase activity of the preparation is k = kobs. (1. X g.-1 X sec. -1) C

The specific catalase activity of pure, crystalline M . lysodeikticus cata2 H. yon Euler and K. Josephson, Ann. 452, 158 (1927). s R. K. Bonnichsen, B. Chance, and H. Theorell, Acta Chem. Scand. 1, 685 (1947); see also Vol. II [136, 138].

786

RESPIRATORY ENZYMES

[139]

lase (k0) is 230 1. × g.-1 X sec. -1 at 25°. 4 Hence the purity of the enzyme preparation is k/230, and the concentration of pure catalase in the reaction mixture is $ = kob~./230 (mg./ml.). Remarks. The amount of enzyme taken for assay should be enough to give a time for disappearance of half the initial H~02 of 10 to 100 seconds. Very dilute solutions of catalase are somewhat unstable and should be diluted immediately before testing. Permanganate titration of the H20~ may be used with purified enzyme, but the iodometric titration is essential (to avoid blank reactions with protein) when assaying crude preparations or whole bacteria. Purification Procedure

Principle. An essential preliminary to purification is the extraction of enzyme from the bacterial cells, which is effected by lysing them with lysozyme. Catalase is stable to ethanol-chloroform, which may be used to remove nucleoprotein and other extraneous material; a subsequent step involves partitioning of the enzyme in the two-phase three-component system ethanol-(NH4)2SO4-H20. Other procedures are conventional. Step 1. Preparation and Lysis of the Bacterial Suspension. Micrococcus lysodeikticus (strain N.C.T.C. No. 2665) is grown on agar in large enameled trays 5 for 40 hours at 35 °, and the growth is suspended in 0.5% NaC15 to a density of 4% (on a bacterial dry weight basis). Crystalline lysozyme6 is added (1 mg./g, bacteria), and lysis is allowed to proceed for 2 hours at 30 °. Step 2. Ethanol-Chloroform Treatment. After lysis the cell suspension is transformed to a translucent jelly, owing to the liberation of intracellular nucleoprotein. It is stirred vigorously, 0.1 vol. of M acetate buffer (pH 5.6) added, and cooled to 0 °. Ethanol, precooled to - 1 0 °, is added slowly to a concentration of 33 % v/v. The gelatinous precipitate is centrifuged off, washed with an equal volume of 0.1 M acetate, pH 5.6, containing 33 % ethanol, and the washings added to the first supernatant. The combined supernatants are treated with 0.2 vol. of CHC13, shaken on a fast mechanical shaker for 15 minutes, and centrifuged. Two liquid layers separate with a thick layer of denatured protein at the interface. The top layer is siphoned off. Step 3. First Ethanol-(NH4)~S04 Partition. The top layer from step 2 is treated with ~ 0 vol. of M sodium acetate, and solid (NH4):SO4 is added (30 g. to each 100 ml.). On standing in large separating funnels 4B. Chance and D. Herbert, Biochem. J. 46, 402 (1950). 5See ref. 1 for details of growth media and optimum conditionsfor lysis with lysozyme. 6 G. Alderton and H. L. Fevold, J. Biol. Chem. 164, 1 (1946).

[139]

CATALASEFROM BACTERLk (Micrococcus lysodeikticus)

787

two liquid phases separate, the lower containing most of the (NH4)2SO4 and the upper most of the ethanol, some (NH4)2SO4, and water. The smaller top layer, which is pale brown and contains all the catalase, is separated. Step ~. Second Ethanol-(NH4)~SO~ Partition. The top layer from step 3 is treated with an equal volume of CHC13, shaken for 15 minutes, and centrifuged; the top layer is removed. (Some protein is removed by this treatment, but its main purpose is to reduce the ethanol concentration of the aqueous layer.) Solid (NH4)2S04 (23 g. to each 100 ml.) is added to the top layer, and two layers again separate. The top layer, which is much the smaller, is dark brown and contains all the catalase i it is removed in a separating funnel and dialyzed against several changes of 0.05 M acetate (pH 5.6). Step 5. First (NH~)2S04 Fractionation. The dialyzed solution from step 4 is roughly fractionated with (NH4)2SO4 by adding successive portions of the salt and centrifuged off the resulting precipitates. No attempt is made to standardize the procedure exactly, the aim being to divide the material into a series of roughly equal fractions; the precipitates are dissolved in 0.05 M acetate, pH 5.6, and assayed for catalase and total protein. In a typical experiment, three fractions precipitated betweea the levels 25 and 35% (w/v) (NH4)~S04 contained together 80% of the initial catalase and only 38 % of the total protein; these were combined for the next step. Step 6. Second (NH4) 2S04 Fractionation. The combined fractions from the last step, dissolved to a protein concentration of ca. 3 %, are refractionated by the dropwise additiou of 50% (w/v) (NH4)2S04 solution adjusted to pit 5.6 with NH4OH. Again the procedure is empirical, a succession of small fractions being taken at gradually increasing salt concentrations, the precipitates being separately assayed and the purest reserved for the next step. The color of the precipitates is a useful guide to fractiouation, those containing inert protein being visibly paler than the dark-brown precipitates containing the purest enzyme. Step 7. Crystallization. The purest fraction from the last step is treated with just enough 50 % (NH4)2SO4 to precipitate all the catalase. The precipitate is centrifuged down and redissolved by adding distilled water drop by drop, very slowly and with good stirring, until all but a trace has dissolved; this is centrifuged off. The supernatant is then saturated with amorphous catalase, nearly all of which crystallizes on standing for 24 hours at room temperature, leaving an almost colorless superna° tant. Recrystallization can be effected by repeating the procedure but is usually unnecessary, as the crystals first obtained are virtually pure enzyme.

788

[139]

RESPIRATORY ENZYMES SUMMARY OF PURIFICATION PROCEDURE

Stage 1. Lysed bacteria 2. Ethanol-chloroform treatment 3. First ethanol-(NH4)~S04 4. Second ethanol-(NH~)2S04 partition 5. First (NH4)2SO4fractionation 6. Second (NH4)~SO~ fractionation 7. Crystals

Total Total volume, protein, ml. g. 5085

203

Total catalase, g.

k I.g. sec.

Yield, %

1.94

22.2

100

7610 2760

29.5 8.9

1.60 1.46

12.5 37.8

83 75

600 61

5.4 2.1

1.14 0.90

49.5 99

59 47

63 --

0.74 0.37

0.49 0.37

151 230~

25 19

Expressed in the units of yon Euler and Josephson, ~ this specific activity of the pure enzyme corresponds to a Katalasefdhigkeit (Kat.f.) of 98,000. Properties

General. T h e pure e n z y m e crystallizes f r o m (NH4)2S04 as regular octahedra which are isotropic when viewed between crossed polaroids; it m a y also be crystallized b y exhaustive dialysis against distilled water, when similar crystals are formed. Strong solutions of the e n z y m e h a v e a brown-red color resembling t h a t of methemoglobin and are v e r y stable at alkaline or neutral p H values b u t readily d e n a t u r e d below p H 5. Prosthetic Group. Bacterial catalase solutions h a v e a characteristic absorption s p e c t r u m in the visible region, with three bands centered at 506, 545, and 631 m~ (in p h o s p h a t e buffer at p H 6.8). These are due to a h e m a t i n prosthetic group which m a y be split off the colorless protein c o m p o n e n t b y t r e a t m e n t with acetone-HC1 and has been identified as p r o t o h e m a t i n I X (identical with t h a t of hemoglobin). This catalase contains no biliverdin. T h e h e m a t i n content of the crystalline e n z y m e is 1.09%. T h e e n z y m e gives a typical hemochromogen s p e c t r u m on t r e a t m e n t with N a O H and Na2S204 and forms characteristic compounds with cyanide and azide.1 Physicochemical Properties. T h e crystalline e n z y m e is homogeneous in the ultracentrifuge and has a sedimentation constant of 11 X 10 -13. T h e h e m a t i n content of 1.09% corresponds to a molecular weight of 58,000 X n, where n is the n u m b e r of h e m a t i n groups per molecule. F r o m the sedimentation constant it can be deduced t h a t n = 4 and the molecular weight is 232,000.

[14~

PLANT CATALASE

[140]

789

P l a n t Catalase

By ARTHUR W. GALSTON Assay Method All assays for activity of fractions were made b y the Bonnichsen et al.1 modification of the von Euler and Josephson ~ technique. The relative p u r i t y of each fraction was expressed in terms of its Kat.f. 2 D r y weights of enzyme used, needed for calculation of Kat.f,, were obtained b y dialyzing 1.0 ml. of the preparation against 2 1. of distilled water (1 °) for 24 hours, then drying overnight at 105 ° on a previously tared watch glass and weighing. Purification Procedure 3 Freshly harvested spinach leaves are washed in tap water and stored in a cold room at W2 to 4 °. The leaves are transferred to large stainless steel vats, covered with commercial acetone previously chilled to - 1 5 °, and permitted to sit for at least 2 hours at this temperature. The cold, partially dehydrated leaves are now very brittle and are easily reduced to small pieces with a turmix (large Waring-type blendor). The blending is complete in 30 to 60 seconds, and the slurry is now gravity-filtered in the cold, the filtrate being discarded. The residue is washed several more times with portions of cold acetone, and the resulting gray-green material is permitted to dry overnight in a ventilated hood. The acetone powder is extracted repeatedly with ice-cold 0.1 M Na2HPO~, yielding a filtrate of Kat.f. approximately 50 and an inactive residue, which is discarded. The filtrate is half-saturated with solid (NH4)2SO~ and permitted to stand overnight in the cold. The precipitate, which contains all the activity, is redissolved in a small volume of 0.1 M Na~HPO4. This preparation, after clarification b y filtration or centrifugation, has a Kat.f. of about 180. Three successive fractionations with 20%, 7%, and 20% saturated (NH4)2SO4 are carried out, the precipitates being saved in each instance. The product has a Kat.f. of about 920, shows a faint absorption band at 630 m~ when viewed with a hand spectroscope, and gives absorption peaks at 275 m~, 330 m~, 405 m~, and 625 m~ in a Beckman spectrophotometer. The catalase solution is adjusted to p H 6.5 and made 0.1 M with respect to N a acetate b y addition of the solid salt. Cautious fractionation 1R. K. Bonnichsen, B. Chance, and H. Theorell, Acta Chem. Scand. 1, 685 (1947) ; see Vol. II [136, 138]. 2 H. yon Euler and K. Josephson, Ann. 452, 158 (1927). 3 A. W. Galston, R. K. Bonnichsen, and D. I. Arnon, Acta Chem. Scand. 5, 781 (1951).

790

RESPIRATORY ENZYMES

[140]

with s a t u r a t e d (NH4)2804, also adjusted to p H 6.5, yields several precipitates, v a r y i n g in Kat.f. f r o m 1200 to 10,000. T h e m o s t active preparations are dialyzed for 2 days in the cold against p H 7.14 p h o s p h a t e buffer of ionic s t r e n g t h 0.1 and t h e n subjected to p r e p a r a t i v e electrophoresis (18 milliamperes, 330 volts, 4 hours), yielding five fractions, three on the anodic side, one in the b o t t o m cell, and one f r o m the cathodic side. T h e Kat.f. and s p e c t r o p h o t o m e t r i c d a t a for these fractions, as well as Kat.f.'s of all other fractions in the p r e p a r a t o r y scheme, are shown in the table.

Kag.f.

VALUES AND SPECTROPHOTOMETRIC CHARACTERISTICS OF VARIOUS FRACTIONS OF THE SPINACH LEAF CATALASE PREPARATION

Fraction 1. Na2HP04 extract of acetone powder 2. Ppt. of fraction 1 insol, in 50% satd. (NH02SO4 redissolved in 0.1 M Na2HPO~ 3. Ppt. of fraction 2 insol, in 20% satd. (NH~)2S04 redissolved in 0.1 M Na2HPO4, sol. in 7% sat& (NH4)2S04 4. Ppt. of fraction 3 insol, in 20% satd. (NH4):SO~ redissolved in 0.1 M Na2HP04 5. Ppts. obtained from adjusting fraction 4 to pH 6.5, making 0.1 M with respect to Na acetate, then cautiously fractionating with solid (NH4)2SO4 6. Electrophoretic fractions of redissolved fraction 5 Cathodic Bottom cell Anodie No. 2 Anodic No. 1 Anodic (top cell)

Kat.f.

Ratio E2a0 ~

E4o5 m.w

50

--

180

--

425

--

920

--

10,100

--

23,600 9,020 12,100 7,320 2,620

1.54 2.07 2.07 2.37 2.54

T h e cathodic fraction of Kat.f. 23,600 yields needle-like crystals after concentration, dialysis against 0.1 M Na2HP04, and addition of solid (NI-I4) 2SO4 to a b o u t 12 % saturation. These needles, when redissolved in 0.1 M Na2HPO~, give a good catalase absorption s p e c t r u m (Fig. 1).

Properties T h e e n z y m e contains 0.049 % Fe, a p p r o x i m a t e l y half the value for a pure 4 - h e m a t i n catalase. T h e prosthetic group is p r o t o h e m a t i n , as shown b y p r e p a r a t i o n of a typical pyridine h e m o c h r o m o g e n on the acetoneHC1 s u p e r n a t a n t of an aliquot of the enzyme. T h e e n z y m e is inhibited b y K C N and NAN3, 50% inhibition being produced b y 5 X 10 -e M K C N and 2 × 10 -~ M NAN3. N a diethyldithioc a r b a m a t e is without effect on activity.

[141]

PEROXIDASE (LIVER)

791

The enzyme is completely inactivated by 10 minutes of incubation at 60 ° but is indefinitely stable at 1° between pH 5.3 and 8.9. The activity is optimal between pH 5.3 and 8.0, falling off quickly at more acid values and slowly at more alkaline values. 1"8!

f

1.6 1.4

4O5

1.2

~'

~ 1.0 ~ 0.8 0.6

0.4

~

510 545 ~t ~

620

0.2 I

I

I

220

300

400

I

~00

600

|!

700

Wavelength, m/s Fro. 1. The absorption spectrum of eleetrophore~ically prepared spinach leaf catalase. Kat.f. = 23,600. Concentration of the enzyme approximately 0.5 mg. per 1 ml.

[141] P e r o x i d a s e (Liver) By

MARGARET J . H U N T E R

Assay Methods Heme. Measurements at 405 m~ were performed on a Beckman spectrophotometer to establish the presence of heme-containing compounds and, in the fractionation procedures, 405/280 ratios were used as indices of purification. Catalase. The method described below was developed by Bonnichsen et al. 1 Slight modifications in technique were adopted. REAGENTS

0.01 M hydrogen peroxide. 0.01 N potassium permanganate. 2 % sulfuric acid. 1 R. K. Bonnichsen, B. Chance, a n d H. Theorell, Acta Chem. Scand. 1, 688 (1947).

792

RESPIRATORY ENZYMES

[141]

Enzyme diluted with 0.01 M phosphate buffer, pH 7, to approximately 0.02 %. PROCEDURE. The procedure was as in Bonnichsen's paper with the exceptions that the enzyme + peroxide was stirred with a magnetic stirrer and that the aliquots were removed from the mixture as quickly as possible, the time of reaction being taken as the time of half-emptying the pipet. All aliquots (usually four) were removed within 60 seconds of the addition of the catalase to the peroxide solution. D E F I N I T I O N OF UNIT AND SPECIFIC ACTIVITY X

2.3 l o g k = over-al] reaction constant = x0 ct where x0 = hydrogen peroxide concentration at t = 0, x = hydrogen peroxide concentration at t (sec.), and c = concentration of enzyme (moles). Duplicates gave agreement within 5 %. Peroxidase. As it proved impossible to remove the catalase from the peroxidase-active fraction and still maintain peroxidase activity, no accurate assay for peroxidase was possible. The presence of peroxidase activity was routinely demonstrated by the addition of 0.03 ml. of 0.02 M guaiacol to 3 ml. of solution to which 0.03 ml. of 0.02 M methyl hydrogen peroxide had previously been added. A red color developed. Crystalline beef liver catalase gave no reaction with guaiacol. Further proof of the existence of a heme-containing compound other then catalase was demonstrated by calculating c, the concentration of catalase, from Ely, measurements for catalase at 405 m~, and from hydrogen peroxide activity measurements. The latter figure was always much lower than the former (one-third to one-half). This was also shown by measurements at 425 m~ before and after the addition of 0.03 ml. of 0.02 N KCN when Ae425, the change in molar extinction at 425 m~, was always much less than for the crystalline catalase. 2 Purification Procedure

Step 1. Preparation of Crude Extract. Beef livers were perfused, frozen, and comminuted. The resultant frozen powders were fractionated in ethanol-water mixtures as described by Cohn et al.3 The a u t h o r wishes to express her t h a n k s to Dr. B r i t t o n Chance for his help on the spectrophotometric analyses. 3 E. J. Cohn, D. M. Surgenor, a n d M. J. H u n t e r , " E n z y m e s a n d E n z y m e Systems," p. 129, H a r v a r d University Press, Cambridge, 1951.

[141]

PEROXIDASE (LIVER)

793

All catalase and peroxidase activity was concentrated in fraction C~. This fraction contained 16 g. of dry protein per kilogram of fresh liver. The proteins of this fraction were insoluble in 9% ethanol, pH 5.8 acetate-phosphate buffer, F/2 0.02, at - 3 °, but were soluble in 0% ethanol, pH 5.8 acetate-phosphate buffer, F/2 0.15, at ~ 1 °. Step 2. A 10% solution of fraction C3 was dialyzed against pH 7.4 phosphate buffer, I'/2 0.01, at -t-1°, for 12 hours. The solution was then dialyzed against pH 6.2 phosphate buffer, F/2 0.05, at -[-1°, for 12 hours. A heavy, non-heme-containing precipitate was obtained. This was centrifuged and discarded. The brown supernatant (approximately 3 % protein) had a 405/280 ratio of 0.3 to 0.35. The supernatant was dialyzed against 10% ethanol, pH 7.1, F/2 0.1, at --3 °, and any precipitate so obtained was discarded. The alcohol concentration in the supernatant was raised to 15%, and the suspension was kept at - 5 ° for 12 hours. The suspension was then centrifuged at - 5 °, the precipitate washed with 15% ethanol, pH 7.1 phosphate buffer, F/2 0.1, at --5 °, and recentrifuged. The washed precipitate was dissolved in pH 7.5 phosphate buffer, F/2 0.1, at -l-1°, to give a 5% protein solution. The 405/280 ratio of this solution was 0.54. Spectrophotometric analyses of this fraction with cyanide and hydrogen peroxide activity measurements soon showed that another heme-containing protein was present as well as catalase. Cytochrome c was found absent by Warburg analyses, and the absence of reduced or oxidized hemoglobin, methemoglobin, and hematin was confirmed by various solubility and spectophotometric analyses. It was found, however, that this fraction had the ability to oxidize guaiacol after the addition of methyl hydrogen peroxide, and that sodium hydrosulfide caused the Soret band maximum to move from 405 m~ to 410 m~. The spectrum of catalase does not change in the presence of sodium hydrosulfite. Step 3. If the above fraction was adjusted to pH 4.2 with 0.1 N acetic acid at -[-1° and after 12 hours dialyzed against pH 7.1 phosphate buffer, I'/2 0.05, at ~-1 °, a brown heme-containing precipitate was formed. The supernatant was found to be 95 % pure catalase (405/280 = 0.92) which readily could be crystallized at its isoelectric point. The residue, which redissolved at pH 4.5, had a 405/280 ratio of 0.35 and showed about 75 % of one component by electrophoretic analysis at this pH. From its change in solubility characteristics it had obviously been denatured, and it gave no color reaction with methyl hydrogen peroxide and guaiacol, and did not react with hydrogen peroxide. After crystalline catalase had been subjected to pH 4.2 at 0 °, there was no observable change in its solubility behavior or activity to hydrogen peroxide. Many attempts were made to separate the catalase and guaiacol-oxi-

794

RESPIRATORY ENZYMES

[149. ]

dizing protein, without losing the activity of the latter. I n every case, however, when crystalline catalase was obtained, the solution lost its power to oxidize guaiacol and a heme-protein was precipitated at neutral p H ' s . This heme-protein m a y be similar to the catalase or the heme-prorein reported b y Brown. 4 4 G. L. Brown, Biochem. J. 51, 569 (1952).

[142] Myeloperoxidase 1 B y A. C. ~/[AEHLY Distribution. Myeloperoxidase (abbreviated M y P O ) has been isolated b y Agner 2 f r o m e m p y e m i c fluid, f r o m leucocytes of a p a t i e n t with empyemic leukemia, and f r o m cbloroleukemic infiltrates. M y P O seems to occur in all myelonic leucocytes, especially in the eosinophylic granules, b u t not in l y m p h o c y t e s . An historical review is found in Agner's paper. ~

Assay Methods T h e a c t i v i t y determination of peroxidases is discussed in Vol. I I [136]. Agner 4 r e c o m m e n d s a special assay for M y P O based on the oxidation of uric acid b y the peroxidase and H202. 4~ Principle. T h e disappearance of the ultraviolet absorption b a n d of uric acid at 290 m~ is measured spectrophotometrically during the course of the action of M y P O on this donor. Solutions 0.01 M uric acid (stable for several days). 0.07 M p h o s p h a t e buffer, p H 7.3. 15 m M h y d r o g e n peroxide (stable for weeks at 0°). Procedure. T o 2.0 ml. of buffer and 0.15 ml. of uric acid solution is added M y P O solution containing a b o u t 60 to 120 ~/of the enzyme. T h e 1 Originally called verdoperoxidase (VPO) by Agner. 2 The name referred to the color of the enzyme due to its prosthetic group, a green hemin. Since lactoperoxidase (Vol. I I [144]) also contains a green hemin, the newer name myeloperoxidase (MyPO) suggested by Theorell [Arkiv Kemi, Mineral. Geol. 17B~ No. 7 (1943)] is more appropriate, especially since the other known peroxidases carry names derived from their sources. MPO could be mistaken for milk peroxidase. 2 K. Agner, Acta Physiol. Scan& 2, Suppl. 8 (1941), M.D. thesis. a K. Agner, Advances in Enzymol. 3, 137 (1943). 4 K. Agner, in press (personal communication). 4~About the oxidation of uric acid by peroxidase cf. K. G. Paul and Y. Avi-Dor, Acta Chem. Scand. 8, 637 (1954).

[142]

MYELOPEROXIDASE

795

volume is brought to 3.0 ml. b y the addition of more buffer, and the optical density at 290 m~ is measured. 5 At to, 20 ~l. of H202 solution (micropipet) is stirred into the cuvette, and a stop watch is started. The optical density at 290 m~ is read at regular intervals for a period of 10 minutes, and the initial rate of oxidation obtained from the resulting plot is used for the calculation of the enzyme activity. Calculation of the Enzyme Concentration. AgneP found that, under the conditions of the test, 0.5 X 10-6 mole of uric acid is oxidized per minute b y 10 3" of M y P O per milliliter of assay solution. This figure, together with the concentrations of H202 and of uric acid used in the test, allows us to calculate the rate constant, k4 (see Vol. I I [136]), for the reaction of the M y P O - H 2 0 , complex with uric acid as a donor. An equation given b y Chance e is used for the computation.

dx k4=d[Xp

1 ....

Xa

(1)

where k4 = the second-order reaction velocity constant.

dx

d-t = the rate of disappearance of the substrate (H20~).

a = the concentration of the donor (uric acid). p .... = the steady-state concentration of the enzyme substrate complex. Since p .... has not been measured for this case it will be replaced b y p

....

=

f X e

(2)

where e = enzyme concentration. f = factor relating p .... and e (degree of " s a t u r a t i o n " of the enzyme with substrate in the steady state). Since 10 3' of M y P O (molecular weight assumed to be equal to the equivalent weight, see p. 798) oxidizes 0.5 X 10-~ mole of uric acid per minute and 4 moles of H202 is required to oxidize 1 mole of uric acid, 4 we get

dx = 0.19 mole sec. -1 for e -- 1.0 molar dt -

-

Using equations 1 and 2 and solving for f X k4, we obtain dx 1 f X ~:4 = ~- X X-----e ~

(3)

5 The increment of optical density due to the subsequent addition of H~O~ is only about 0.001 unit and needs not to be taken into account. B. Chance, Arch. Biochem. 24, 410 (1949).

796

RESPIRATORY ENZYMES

[142]

and inserting

dx - - = 0.19 mole sec. -1 dt e = 1.0 molar a = 0.5 X 10-3 molar we get, at p H 7.3 and ca. 25 °, f X k4 = 380 M -1 sec. -1 Using this value and solving equation 3 for e we arrive at

dx e = 5.3 X d--/

(4)

Under the conditions of the standard assay m e t h o d and using the extinction coefficient for uric acid [e = 12.2 _+ .5 cm. -1 mM-1] 6awe can express the enzyme concentration in terms of the decrease of optical density at 290 m~ and obtain dD e = 1.72 )< 10-~ X d~(5) where e = concentration of M y P O in the cuvette. dD d--/ = decrease of optical density (log~0 Io/I) per second. Preparation

T h e main problem in obtaining M y P O is the isolation of leucocytes in sufficient quantities. Agner 2 used h u m a n e m p y e m a as the starting material. I t contains M y P O in v e r y high concentrations (about 0.5 g./1.) but is difficult to obtain. In our laboratories ox blood was u s e d / w h i c h is easy to collect in large amounts b u t contains only very small concentrations of M y P O (about 5 mg./1, of blood). T h e isolation of the leucocytes is achieved b y repeated gentle centrifugation and removal of erythrocytes and of serum b y suction. A short outline of the procedure follows. TM The top layer of cells (about 50% leucocytes) is diluted with 4 parts of 0.01 M phosphate (pH 7.2) and homogenized in a Waring blendor for 20 seconds. The solution is incubated with trypsin for 3 hours at room t e m p e r a t u r e and an equal volume of ethanol is added in the cold. T h e enzyme is extracted from the precipitate with 0.1 M phosphate (pH 7.2) and fractionated with a m m o n i u m sulfate. T h e fraction of 40 to 60 % saturation contains most of the activity e~ E. S. Canellakis and P. P. Cohen, J. Biol. Chem. 213, 397 (1954). 7 Alternatively, packed human blood cells obtained from Sharp and Dohme (Philadelphia, Pa.) were used. 7~ W. J. Steele, in cooperation with L. Smith and A. C. Maehly, unpublished experiments.

[142]

MYELOPEROXIDASE

797

and is collected by centrifugation. Extraction with 5 % ammonium sulfate yields MyPO of about 50% purity. It can be further purified (to > 8 0 % purity) by electrophoresis, but this procedure involves sizable losses. In the following paragraphs the isolation and purification of MyPO from empyema according to Agner 2,3 is described. Crude MyPO from Empyema. The mixture of 1 1. of empyema, 2 1. of water, and 1 1. of ether is shaken with 1.6 kg. of ammonium sulfate, centrifuged, and after removal of the etherical and aqueous solutions by suction the semisolid middle layer is dissolved in water. About 3.6 1. of viscous opaque solution is obtained. Purification Procedure

Step 1. Purification by Barium Acetate. About 470 ml. of saturated barium acetate solution is added to the crude extract described above. 8 The heavy precipitate is removed by centrifugation, and the supernarant, A, is used for step 2. Step 2. Alcohol Fractionation. An equal volume of 95% EtOH 9 is stirred into solution A, and the precipitate is removed by centrifugation at high speed (Sharpies centrifuge). Next 0.27 vol. of EtOH is added (final concentration of EtOH about 65 vol. %), and the precipitate is centrifuged down and dissolved in about 170 ml. of water, solution B. The supernatant is discarded. Step 3. Ammonium Sulfate Fractionation. Solution B is incubated with an equal volume of saturated ammonium sulfate solution and centrifuged. The precipitate is discarded, and the supernatant is again treated with the same amount of ammonium sulfate solution as above. The green precipitate is dissolved in 60 ml. of water and gives a clear brownish green solution, C. Step 4. Second Barium Acetate Purification. Solution C is treated with saturated barium acetate solution as described in step 1. The precipitate is removed by centrifugation and discarded. Saturated ammonium sulfate solution is added to the supernatant to a final saturation of 60%, and the precipitate is collected by centrifugation and dissolved in water. The solution is dialyzed vs. distilled water, and if a precipitate forms in the dialysis sack the contents of the sack are centrifuged. The supernatant is saved, and the precipitate is dissolved in 1% NaC1 and again treated with barium acetate and ammonium sulfate. Step 5. Electrophoresis. Electrophoresis is carried out in a phosphate buffer of pH 6.8 and u = 0.1. The fraction moving toward the cathode 8 The proper amount must be determined by pilot experiments for each preparation. A large excess yields opalescent solutions after eentrifugation. 9 EtOH stands for ethanol.

798

RESPIRATORY ENZYMES

[142]

with a mobility of 2.0 × 10 -5 cm. ~ volt - I sec. -1 is collected. T h e p u r i t y is determined spectrophotometrically b y measuring RZ (Reinhcitszahl) or be at 637 or 475 m~ (see p. 801). A b o u t 160 rag. of purified M y P O is obtained. TABLE I SUMMARY OF PURIFICATION PROCEDURE

Fe, Volume gequiv, Crude extract Mter step 1 Mter step 2 After step 3 After step 5

3.6 1. -170 ml. 60 ml. --

8.5 7.4 5.2 4.3 2.9

MyPO, Total yield, mg. % 470 410 287 240 160

100 87 61 51 35

Physical Properties These d a t a were obtained b y Agner2 1. Molecular Weight. F r o m the iron content, an equivalent weight of 56,700 is obtained. Direct determinations of the molecular weight are not available. 2. Isoelectric Point. At p H 6.8, ionic strength 0.1, and 0 °, the electrophoretic mobility was found to be 2.0 X 10 -5 cm. ~ volt -J sec. -1 toward the anode. T h e isoelectric point lies at p H ~ 10. 3. Solubility. I n distilled water, M y P O is easily soluble. I n a m m o n i u m sulfate, it is soluble at < 50 % saturation, insoluble at > 65 % saturation. 4. pH Stability (for 10 minutes of t r e a t m e n t ) . At p H 13.8, 52 % of the a c t i v i t y was left ( N a O H ) ; at p H 12.8, 100% ( N a O H ) ; at p H 1.2, 0 % (HC1); with 0.66 N acetic acid, 100%. 5. Thermal Stability (after 10 minutes of t r e a t m e n t ) . At 60 °, 100% of the a c t i v i t y was left; at 70 °, 95 %; at 80 °, 94 %; at 90 °, 0 %. Chemical Analysis of Agner's MyPO. ~,3 Only the content of iron, copper, and nitrogen were determined quantitatively, with the following results: Element

Content, %

Number of atoms per gram equivalent of iron

Fe Cu N

0.0985 0.0010 17.15

1 5 i ? 475 65.2 637 16.7 Red. -b HN~ -i 0.3 460 ? 615 ? HF -i 10 Same spectrum as ox. M y P O CO and OH' Do not react as judged by lack of inhibition a n d the absence of spectral changes -

-

-

-

a K. Agner, Acta Physiol. Scand. 2, Suppl. 8 Ox. stands for oxidized MyPO, red. for its form; the other agents react with the form a stands for active, i for inactive. '~ The concentration of reagent t h a t leads to ~ is the wavelength in m~.

(1941). reduced form. H F reacts with either indicated. 50% inhibition.

I e = log~0 7/0 cm. --1 m M -1. The values are calculated from Agner's paper, where ,

the d a t a are plotted in terms of the absorption coefficient, /~ = I n ~

cm. ~

mole-1. g Weak band. h B. Chance, Advances in Enzymol. 12, 153 (1951). 10 H. Theorell, Arkiv Kemi, Mineral. Geol. 16A, No. 8 (1942). n B. D. Polls a n d H. W. Shmukler, J. Biol. Chem. 201, 475 (1953); see also Vol. I I [144].

800

[142]

R E S P I R A T O R Y ENZYMES

TABLE III DONORS USED IN DETOXICATION EXPERIMENTS DESCRIBED IN THE TEXT a Common name

Alternative name

D e t o x i c a t i n g effect

Aromatic amines Aniline o-Toluidine m-Toluidine p-Toluidinc Mesidine o-Anisidine o-Chloroaniline p-Chloroaniline o-Nitraailine m-Nitraniline p-Nitraniline A n t h r a n i l i c acid o-Phenylenediamine p-Phenylenediamine Benzidine o-Tolidine

-2-Methylaniline 3-Methylaniline 4-Methylaniline 2,4,6-Trimethylaniline 2-Methoxyaniline 2-Chloroaniline 4-Chloroaniline 1-Amino-2-nitrobenzene 1-Amino-3-nitrobenzene 1-Amino-4-nitrobenzene 2-Aminobenzoic acid 1,2-Diaminobenzene 1,4-Diaminobenzene 4, 4 ' - D i a m i n o b i p h e n y l 4,4'-Diamino-3,3'-dimethylbiphenyl

+ + + -+ + + --+ --

Other amines and amine derivatives Phenethylamine Tyramine Acetanilide Phenylurea N-Methylaniline N,N-Dimethylaniline

1-Amino-2-phenylethane p-(~-Aminoethyl)phenol N-Phenylacetamide ----

-+ -

Hydroxybenzene 1,2-Benzenediol 1,3-Benzenediol 1,4-Benzenediol /~-(p-Hydroxyphenyl)alanine /3- ( 3 , 4 - D i h y d r o x y p h e n y l ) a l a n i n e

+ + + ---

1,4-Cyclohexadienedione Benzopyrrol -2,3-Indolenedione a - A m i n o - 3 - i n d o l e p r o p i o n i c acid 4 - H y d r o x y - 2 - p y r r o l i d i n e c a r b o x y l i c acid a - A m i n o - 5 - i m i d a z o l e p r o p i o n i c acid 4-Imidazole~thylamine

+

Phenols Phenol Pyrocatechol Resorcinol Hydroquinone Tyrosine Dopa

Other donors Quinone Indole Indoleacetic acid Isatin Tryptophan Hydroxyproline Histidine Histamine

K. Agner, J. Exptl. Med. 92, 337 (1950).

---

[143]

PLhNT PEROXIDASE

801

Measurement of Enzyme Purity by Spectrophotometry. Two spectrophotometric tests are available for checking the degree of purification of MyPO obtained. One of them is based on the ratio of the heights of the bands in the Soret region and in the protein region. The ratio D43o/D27~ can be called RZ, as in the case of horseradish peroxidase (Vol. II [143]). Agner 2 found the RZ to be 0.79. The other test measures the increase of optical density on reduction of the fully oxidized peroxidase. This measurement is performed either at 637 m~, where ~red. - -

~ox. =

1.08

cm. -I

X

mM

-1

or at 475 m~, where ered. -- Cox. = 49.5 cm. -1 X mM -~ for the purified enzyme. Neither kinetic, magnetic, nor titration data on myeloperoxidase are at present available. Physiological Role. Although the physiological function of M y P 0 is by no means clearly established, it was shown that one function of the enzyme could very well be the detoxication of bacterial toxins. Experiments in this field were performed by Kojima and others, 12 but the most intensive research is again due to Agner. 13 He placed a cellophane bag containing solutions of MyPO and diphtheria toxin into a solution of 0.05 mM H202 and about 0.05 mM donor. Dialysis was allowed to proceed for 24 hours at pH 7.1 (phosphate buffer). The incubated toxin solutions were assayed by injection into guinea pigs. Agner found that certain donor substances are oxidized to products which destroy the toxic properties of diphtheria toxin (the immunological properties of the toxin are initially unaffected). Table III lists the substances which were tested for their antitoxic action under the experimental conditions described above. 12S. Kojima, J. Biochem. (Japan) 14, 95 (1931-32). is K. Agner, J. Exptl. Med. 92~ 337 (1950).

[143] P l a n t P e r o x i d a s e 1

By A. C. MAEHLY Distribution. Peroxidase has been found in most plant cells that were investigated and seems to be a normal component of such cells. Partial purification of the enzyme has been achieved from several sources, nora1For over-all reaction equation see p. 764.

802

RESPIRIkTORY ENZYMES

[143]

bly f r o m figs, 2 b u t the e n z y m e has not been isolated from plants in a pure state except in the case of horseradish peroxidase (henceforth design a t e d b y H R P ) . This e n z y m e has been p r e p a r e d b y Keilin and coworkers 8,4 and has been crystallized b y Theorell. 5 I t s enzymatic, physical, and chemical properties h a v e been studied extensively, b u t its physiological role is still largely unknown. Reaction M e c h a n i s m . T h e chemical reactions underlying the function of peroxidase are not known in detail, b u t m a n y properties of the intermediates and the kinetics of e n z y m e action are well understood, in cont r a s t to m o s t other enzymes. This is due to the work of Keilin and coworkers, 3,4 of George, 8-8 and especially of Chance, who published a great n u m b e r of papers and reviews on this subject2 -~2 According to Chance the reactions are in principle the following: F o r m a t i o n of green, active e n z y m e s u b s t r a t e c o m p o u n d I: kl H R P + H~O2 ~- C o m p l e x I (e) (x) k2

(1)

Transition of complex I to red, active complex I I :

k7 Complex I ~- A H --~ Complex I I -~- A (a) (p)

(2)

R e c o v e r y of e n z y m e b y reduction of complex I I : k~ Complex I I ~- A H --~ H R P ~ A ~ 2 H 2 0

(3)

T o a certain degree, spontaneous decomposition of complex I I and the J. B. Sumner and M. J. Howell, Enzymologia 1, 133 (1936). D. Keilin and T. Mann, Proc. Roy. Soc. (London) B122, 119 (1937). D. Keilin and E. F. Hartree, Biochem. J. 49, 88 (1951). s H. Theorell, Enzymologia 10, 250 (1942). 6 p. George, Advances in Catalysis 4, 367 (1953). 7 p. George, Biochem. J. 54, 267 (1953). s p. George, J. Biol. Chem. 9-01, 413 (1953). 9 B. Chance, Advances in Enzymol. 12, 153 (1951). i0 B. Chance, in "The Enzymes" (J. B. Sumner and K. Myrb~ck, eds.), Vol. 2, Part 1, p. 428, Academic Press, New York, 1951. 11B. Chance, in "Modern Trends in Biochemistry and Physiology" (E. S. G. Barron, ed.), p. 25, Academic Press, New York, 1952. 1~B. Chance, in "Investigation of Rates and Mechanisms of Reactions" (S. L. Friess and A. Weissberger, eds.), Vol. 8 of "Technique of Organic Chemistry," p. 627, Interscience, New York, 1953.

[143]

PLANT PEROXIDASE

803

formation of complex I I I from complex II and an excess of H20~ can occur: k3 Complex I I --* H R P Jr P Complex II ~ H~02--* Complex III, inactive

(4) (5)

The meaning of A and AH is explained in footnote 13; e is enzyme concentration; x is substrate concentration; p is concentration of the rate limiting enzyme substrate compound II; and a stands for the concentration of free donor. P means products without specifying their nature. Assay Methods. The general assay methods for peroxidases are described in Vol. l I [136]. A further detailed paper on activity determinations was published by Maehly and Chance. 14 For this reason, only a physical measurement of enzyme purity will be included in this chapter. The RZ Unit. In the later stages of purification and generally when purified H R P solutions are used a spectrophotometric measure of the purity is very convenient. The ratio of the optical density at 403 mg (due to the hemin group) to that at 275 mg (due to the protein) is determined. This ratio has been called RZ (for Reinheitszahl) and was found to be 3.04 for pure crystalline HRP. la I t is recommended that the purity of H R P be characterized in terms of RZ. The ratio Sorer band to protein band is a very convenient way to express the purity of hemoproteins in general. Preparation of Crystalline HRP. Theorell and co-workersT M as well as Keilin and co-workers a,4 have worked out preparative procedures for HRP. Theorell's group crystallized the enzyme several times, whereas Keilin's group, although not succeeding in this, claims to have obtained an even purer preparation as iudged by the molecular weight. 17 The following procedure has been successfully used several times in this laboratory. Steps 1 to 3 follow essentially Keilin's method; the others follow Theorell's procedure quite closely. The whole preparation, except for electrophoresis and alcohol fractionation, is carried out at room temperature. Step 1. Crude Extract from Horseradish Roots (Calculated for 100 kg.). The roots are either collected in the spring when they begin to sprout, la AH = donor, in the reduced form; A = donor, oxidized, cf. also p. 808. 14A. C. Maehly and B. Chance, in "Biochemical Analysis" (D. Glick, ed.), Vol. 1, p. 357, Interscience, New York, London, 1954. ~5H. Theorell and A. C. Maehly, Acta Chem. Scand. 4, 422 (1950). 16H. Theorell, Arkiv Kemi, Mineral. Geol. 16A, No. 2 (1942). 17Btt see footnote 41.

804

RESPIRATORY ENZYMES

[143]

or sprouting is achieved b y allowing the roots to lie in running w a t e r for several days. TM T h e roots, including the peroxidase-rich skin, are then minced mechanically. 19 This and the following operations should be carried out in the open air, and rubber gloves should be used ~° since irritating allylisothiocyanate is liberated. T h e mince is pressed out with a hydraulic press, and 30 1. of w a t e r is added to the d r y material. After standing and occasional stirring for several hours, the mass is pressed out again, and this operation is repeated once more. A b o u t 100 1. of extract is obtained. Step 2. First Ammonium Sulfate Fractionation. 2z T o 100 1. of extract 68 kg. of a m m o n i u m sulfate is added gradually and with vigorous mechanical stirring, which is continued for 1 hour after the last addition of salt. T h e solution is then left standing undisturbed overnight close to an aspirator. A solid layer collects at the top of the solution, and m o s t of the liquid is then r e m o v e d b y suction and discarded. T h e remaining suspension is washed into centrifuge cups with small a m o u n t s of saturated a m m o n i u m sulfate solution and gently centrifuged. T h e colorless solution is sucked off, and the remaining semiliquid paste (about 6 1.) is dialyzed vs. t a p w a t e r using h e a v y cellophane tubing. 22 After 1 day, the tubing is changed and dialysis continued until the solution is salt-free (2 to 3 days). ~3 T h e contents of the tubing are centrifuged to give a b o u t 10 1. of solution. N e x t 312 g. of a m m o n i u m sulfate per liter is added, and the solution is left standing overnight. T h e rather loose precipitate, a, is filtered off t h r o u g h large fluted filters. T o each liter of the filtrate 250 g. of a m m o nium sulfate is added. T h e precipitate, b, containing m o s t of the activity, sticks to the walls of the container when the s u p e r n a t a n t is poured off. Precipitates a and b are each suspended in a m i n i m u m a m o u n t of water, dialyzed salt-free and centrifuged, yielding solutions A and B. 18Daily a sample of about 50 g. is minced, pressed out, and the activity of the juice is measured. When the activity reaches its maximum, the whole batch of roots is used for the preparation. For Pennsylvania roots 3 days' sprouting gave the best results. 19 The firms delivering the roots usually have facilities to carry out the mincing operation. ~0If a good hood and gas mask are available, the operations may be carried out indoors. 21In this step ammonium sulfate "purum" can be used after pulverization. ~ Sometimes a filtration on Bfichner filters is necessary at this point to keep the volume of the suspension small. Kieselguhr (Celite) was found to absorb a fair amount of HRP and should be avoided. ~a Crude HRP preparations attack cellophane tubing. It is therefore safer not to use running water but to replace the dialysis water every few hours and to use mechanical stirring.

[143]

PLANT PEROXIDASE

805

Step 3. Purification with Calcium Phosphate Gel. Solution A (about 1 1.), containing a b o u t 15% of the total activity, needs further purification. T h e solution is treated with 100 g. of calcium p h o s p h a t e gel 24 per liter and, after 5 minutes standing, centrifuged. T h e s u p e r n a t a n t is t r e a t e d again in the same way, and the precipitates are discarded. T h e n 242 g. of a m m o n i u m sulfate per liter is added, and after several hours the precipitate is collected b y centrifugation and discarded. T h e s u p e r n a t a n t is treated with 205 g. of a m m o n i u m sulfate per liter, the precipitate centrifuged off, suspended in a little water, and dialyzed salt-free. T h e resulting solution is added to solution B of step 2 and the combined solutions (~-~4 1.) are the starting material for step 4. Step 4. Alcohol Fractionation. 25 T o each liter of solution from step 3, 4 1. of 95% E t O H 26 is added slowly and with mechanical stirring. T h e s u p e r n a t a n t is carefully decanted and discarded. T h e precipitate, which sticks to the walls of the container, is dissolved in 3 1. of w a t e r with the help of a rubber policeman, and 2 1. of 9 5 % E t O H is added as above. 27 Centrifugation yields solution C and a deposit which is stirred up with 500 ml. of 50% E t O H , centrifuged, and the s u p e r n a t a n t added to solution C ( ~ 5 1.). This solution is e v a p o r a t e d in vacuo and below 40 ° (cooling b y ice water or refrigerated brine) to a volume of 250 ml. F o u r volumes of 9 5 % E t O H is again added in the cold, and after centrifugation the deposit is dissolved in 150 ml. of water, yielding solution D. Step 5. Electrophoresis. Buffer solution is prepared b y adding N N a O H to 20 1. of 0.03 M Na2HPO4 until the p H is 10.3. Solution D is dialyzed against this buffer, and p r e p a r a t i v e electrophoresis is carried out in a large Tiselius apparatus, if necessary in batches. T h e H R P migrates slowly toward the anode; brown-colored impurities wander in the same direction b u t at m u c h higher rates. 28 A b o u t every 24 hours the impurities are removed b y sucking out the protein solution f r o m the top c o m p a r t ~ The gel is prepared as follows:~ To a 10-l. glass jar containing 1 I. of tap water (if the city water is of high quality) and 250 ml. of 0.6 M CaCl~ are added, with stirring, 250 ml. of 0.4 M Na3PO4, followed by N acetic acid until the pH is 7.3. The jar is then filled with water, the precipitate washed six times by decantation, centrifuged down, and suspended in water to give 400 ml. of a thick cream. ~5Step 4 should be performed at 0°, or ice-cooled solutions and vessels should be used. 26EtOH stands for ethanol. In Europe, the same alcohol is labeled 96 %. 27Sometimes it is advantageous to use more dilute solutions of the enzyme at this point, but the ratio 3 1. of solution to 2 1. of EtOH must be maintained. Pilot runs are recommended for this stage of the preparation. 28 Since all compounds migrate toward the anode, it is wisest to fill the protein solution into the bottom cell and into all cathodic cells, whereas all anodic cells are filled with buffer.

806

RESPIRATORY ENZYMES

[1~]

m e n t on the anode side and replacing it b y buffer. 29 F r o m time to time the protein solution is carefully moved back into its original position. When no more impurities can be removed (after 3 to 10 days), the electrophoresis is stopped. T h e contents of each cell c o m p a r t m e n t from each electrophoresis experiment are collected separately, and their RZ values are measured. Fractions with similar RZ values are combined, and those with RZ ~ 0.20 are discarded or added at step 2 of the next preparation. Step 6. Second Ammonium Sulfate Fractionation. ~° The progress of purification can now be followed spectrophotometrically b y determining the RZ. Activity tests need only be performed as an occasional check. The electrophoresis fraction with the lowest RZ is treated with 31 g. of ammonium sulfate per 100 ml. (50 % saturation), left standing for several hours, and centrifuged. The precipitate is set aside if its RZ > 0.20, and 13.5 g. of ammonium sulfate is added to every 100 ml. of the supern a t a n t (70 % saturation). After a few hours of standing, the suspension is centrifuged. The s u p e r n a t a n t and all loose particles are poured off completely and set aside if RZ > 0.20 after addition of water. T h e precipitate (as free from s u p e r n a t a n t as possible) is dissolved in the electrophoresis fraction which is next in purity. T h e procedure is repeated until the purest fraction is reached. The last precipitate is dissolved in a small a m o u n t of water and centrifuged to give solution E. All fractions below 50% and above 70% saturation having RZ > 0.20 are combined, brought to about 90% saturation with solid ammonium sulfate, and centrifuged. T h e precipitate is dissolved in little water and dialyzed. The resulting solution is refractionated, and the fraction from 50 to 70% saturation is added to solution E, which then is dialyzed salt-free. Step 7. Solubility Curve and Crystallization. Centrifuge tubes (15 ml.) are filled with aliquots of solution E, and saturated ammonium sulfate solution is added to each tube so t h a t the final degree of saturation ranges in steps from 50 to 70%. The tubes are well stoppered and left standing overnight. ~1 The next morning they are centrifuged, and the concentration of H R P (at 403 or 500 m~) and of total protein (at 275 m~) in the s u p e r n a t a n t determined spectrophotometrically and plotted as a function of per cent ammonium sulfate concentration. F r o m this graph, the values of ammonium sulfate saturation for " 1 0 % H R P precipita2g Sometimes a colored compound is seen migrating toward the cathode. This compound was called paraperoxidase by Theorel116and is regarded as an artifact of the preparation method. a0 From here on, analytical-grade ammonium sulfate should be used. 31If crystallization occurs in some of the tubes, the solubility values obtained for those samples cannot be relied on. The crystals should be saved for inoculation at a later stage.

[143]

PLANT PEROXIDASE

807

tion" and "90% HRP precipitation" are determined2 2 All samples are p o o l e d , d i a l y z e d salt-free, a n d a d d e d t o t h e r e s t of t h e s o l u t i o n . F o r c r y s t a l l i z a t i o n t h e c o n c e n t r a t i o n of H R P is a d j u s t e d t o a b o u t 0 . 5 % (--~0.1 r a M ) , a n d solid a m m o n i u m s u l f a t e is a d d e d t o give t h e l o w e r of t h e t w o s a t u r a t i o n v a l u e s d e t e r m i n e d b y t h e s o l u b i l i t y curve. A f t e r s t a n d i n g o v e r n i g h t t h e s o l u t i o n is c e n t r i f u g e d , t h e p r e c i p i t a t e , F, s e t aside, a n d t h e s u p e r n a t a n t t r e a t e d d r o p w i s e w i t h s a t u r a t e d a m m o n i u m s u l f a t e s o l u t i o n u n t i l c r y s t a l l i z a t i o n begins.3~ A f t e r t h e a p p e a r a n c e of t h e first c r y s t a l s t h e a d d i t i o n of a m m o n i u m s u l f a t e s o l u t i o n is cont i n u e d v e r y s l o w l y ( d u r i n g s e v e r a l h o u r s ) u n t i l t h e u p p e r v a l u e of t h e s a t u r a t i o n d e t e r m i n e d b y t h e c u r v e is r e a c h e d . A f t e r 24 h o u r s t h e c r y s tals are spun down and the mother liquor combined with precipitate F. T h e r e s u l t i n g s o l u t i o n s h o u l d b e p u r e e n o u g h for m o s t p u r p o s e s b u t can be p u r i f i e d f u r t h e r b y a s e c o n d or t h i r d f r a c t i o n a t i o n . TABLE I SUMMARY OF PURIFICATION PROCEDURE

(for 100 kg. of roots)

1. 2. 3. 4. 5. 6.

Fraction

Total volume, 1.

Concentration m g./ml,

Total amountj g.

Recovery, %

RZ a

Raw juice First (NH4)2S04 precipitate Before EtOH fractionation After EtOH fractionation Before crystallization Crystals

100 10 4 0.15 0.82 --

0.27 2.3 2.6 48 5.0 --

27.0 b 23.5 b 10.5 7.2 4.1 2.0

--100 68 39 19

---0.32 1.4 3.0 c

" The meaning of RZ is explained on p. 803. b Some oxidase activity is probably present. c Sometimes RZ values of only 2.8 were obtained. The value 3.0 is reached only after several recrystallizations. Specificity. T h e s u b s t r a t e s p e c i f i c i t y ( e q u a t i o n 1) of H R P is h i g h : o n l y H20~, M e O O H , 84 a n d E t O O H 35 c o m b i n e ecith H R P t o g i v e a c t i v e e n z y m e s u b s t r a t e c o m p l e x e s . 36,a7 33 These values for Swedish roots were ~ 5 3 % and 60 % saturation,~S and for Pennsylvania roots ~ 5 8 % and 62 % saturation (A. C. Maehly, unpublished data). 33 A silky shine is observed when a crystallizing suspension is swirled in strong light. Inoculation with crystalline HRP is helpful at this stage. 34 MeOOH stands for methyl hydrogen peroxide. 35 EtOOIt stands for ethyl hydrogen peroxide. 38 p . George~-S found that certain oxidizing agents form compounds with HRP which are apparently identical with those formed by the action of hydroperoxides. The mechanism of these oxidations is currently being investigated in several laboratories. This problem is, e.g., the subject of recent papers by R. R. Fergusson and B. Chance, Science, and J. Am. Chem. Soc, in press. ~ P. George suggested to replace the denotation "complex" by "compound."

808

RESPIRATORY ENZYMES

[143]

T h e s p e c i f i c i t y of t h e s e e n z y m e s u b s t r a t e c o m p l e x e s for h y d r o g e n d o n o r s ( e q u a t i o n s 2 a n d 3) is q u i t e low. S e v e r a l score s u b s t a n c e s a r e e a s i l y o x i d i z e d (see also T a b l e I V ) , m a i n l y p h e n o l s , a m i n o p h e n o l s , dia m i n e s , i n d o p h e n o l s , a s c o r b i c acid, l e u c o d y e s , a n d c e r t a i n a m i n o acids. 3s E x t e n s i v e lists of t h e s e c o m p o u n d s w e r e p u b l i s h e d b y K a s t l e a n d P o r c h 39 a n d b y Joslyn.40 P h y s i c a l P r o p e r t i e s . 1. Molecular Weight. T h e m o l e c u l a r w e i g h t is 40,200 a c c o r d i n g t o T h e o r e l l a n d E h r e n b e r g , 41 39,800 a c c o r d i n g t o Cecil a n d O g s t o n . 42 2. Isoelectric Point. T h e i s o e l e c t r i c p o i n t is 7.2. 43 3. Solubility. F i v e g r a m s is s o l u b l e in 100 ml. of w a t e r if t r a c e s of s a l t s a r e p r e s e n t . I n a m m o n i u m s u l f a t e t h e e n z y m e is s o l u b l e u p t o 58 % s a t u r a t i o n b u t i n s o l u b l e a b o v e 62 % s a t u r a t i o n . 4. p H Stability. ~4,45 T h e p H s t a b i l i t y is p H 5.5 t o 12 in t h e p r e s e n c e of fluoride, p H 4.5 t o 12 in t h e p r e s e n c e of o t h e r h a l o g e n ions, N3' a n d C N ' , a n d p H 3.5 t o 12 in t h e a b s e n c e of all ions l i s t e d a b o v e . 38 I. W. Sizer, Advances in Enzymol. 14, 129 (1953). 39 j. H. Kastle and M. B. Porch, J. Biol. Chem. 4, 301 (1908): ~0 M. A. Joslyn, Advances in Enzymol. 9, 613 (1949). 41 The value of 44,100 reported by Theorell [Arkiv Kemi, Mineral. Geol. 15B, No. 24 and 16A, No. 2 (1942)] was based on a sedimentation constant of s~0 -- 3.85 X 10 I3, determined in an oil turbine centrifuge. R. Cecil and A. G. Ogston [Biochem. J. 48, 592 (1948)] as well as S. Schulman [Arch. Biochem. and Biophys. 44, 230 (1953)] and several others have recently shown that the values for s20 obtained with ~his type of centrifuge are about 7-11% higher than those found with a Spinco ultracentrifuge. Most of this deviation was found to be due to the fact that the sample cell reaches a several degrees higher temperature than the environment during the run. A sample of crystalline and electrophoretically pure HRP prepared by K. G. Paul was recently used to determine the sedimentation constant. The determination was carried out by A. Ehrenberg at Professor Theorell's institute using a Spinco instrument. A single run at a protein concentration of 0.5% gave a value of s~0 = 3.59 X 1013 (personal communication from A. Ehrenberg). The deviation from the oil turbine value cited above is 7 % and thus in the range usually encountered on comparing data on the two types of instruments. The same value would be arrived at if it is assumed that the temperature of the oil turbine centrifuge cell was 2.8 ° higher than that assumed by Theorell in his earlier calculations. The new value for s2°0leads to a molecular weight for HRP of 40,200, when leaving all other magnitudes unchanged. Cecil and Ogston 42 found s~0 = 3.48 X 10 ia and arrived at a molecular weight of 39,800. 42 R. Cecil and A. G. Ogston, Biochem. J. 49, 105 (1951). 43 H. Theorell and A..~keson, Arkiv Kemi, Mineral. Geol. 17B, No. 7 (1943). 44 A. C. Maehly, in "Enzymes and Enzyme Systems" (J. T. Edsall, ed.), p. 81, Harvard University Press, Cambridge, Mass., 1951. 45 A. C. Maehly, Biochim. et Biophys. Acta 8, 1 (1952).

[143]

PLANT PEROXIDASE

809

5. Thermal Stability. If w a r m e d for 15 m i n u t e s t h e e n z y m e is s t a b l e u p to 63°. 15 A t r o o m t e m p e r a t u r e H R P is s t a b l e for weeks. 46 P a r t i a l rec o v e r y of a c t i v i t y a f t e r cooling of h e a t e d s o l u t i o n s h a s b e e n r e p o r t e d (e.g., b y B a c h a n d Wilensky47). 6. Titration. Acid base t i t r a t i o n curves were p u b l i s h e d b y Theorell. 471 M i c r o t i t r a t i o n t e c h n i q u e s h a v e b e e n a p p l i e d to H R P b y M a e h l y (in preparation). 7. Oxidation Reduction Potential. T h e redox p o t e n t i a l of H R P was r e c e n t l y d e t e r m i n e d b y H a r b u r y 47b a n d f o u n d t o be r e m a r k a b l y low, v a r y i n g f r o m E0' = - 0 . 2 0 7 0 a t p H 6.08 to E0' = - 0 . 2 7 8 7 a t p H 7.71. C h e m i c a l A n a l y s i s . Q u a n t i t a t i v e d a t a are a v a i l a b l e o n t h e c o n t e n t i n t h e following g r o u p s or e l e m e n t s : 43,4s ( T a b l e I I ) . TABLE II COMPOSITION OF HRP Element or group C H

O (by difference) N S Fe Protohemin Carbohydrates Arginine Histidine Lysine

Content, % 47.0 7.25

32.0 13.2 0.43 0.127 1.36 ~,b 1.30" 18.4 ~ 6.91 0.71 4.06

Groups per mole ---

-416 6 1 1 1 -18 2 12

Recently K. G. Paul, H. Thcorell, and /~..~keson [Acta Chem. Scan& 7, 1284 (1953)] have redetermined the extinction coefficient of pyridine ferroprotoporphyrin (new value e557 = 34.7 mM -1 X cm.-1). The figures for the hemin content listed in this paper are recalculated on the basis of the new value. Keilin and Hartree b have found a hemin content of 1.61% for their HRP preparation, but Paul et al. found 1.36% hemin for the same preparation when using ~7 = 34.7 mM -1 X cm.-1 in the pyridine hemochromogen test. b D. Keilin and E. F. Hartree, Biochem. J. 49, 88 (1951). c Determined by weighing the humin formed on acid hydrolysis. 48 Growth of bacteria or fungi in aqueous ttRP solutions is very rare even at room temperature. 4~ A. Bach and B. Wilensky, Biochem. Z. 226, 482 (1930). 471H. Theorell, Arkiv Kemi, Mineral. Geol. 16A, No. 14 (1943). 4 7 b H. A. Harbury, Dissertation, Johns Hopkins University, Baltimore, 1953. 48 H. Theorell, Arkiv Kemi, Mineral. Geol. 16A, No. 8 (1942).

810

RESPIRATORY ENZYMES

[143]

TABLE III R A T E AND EQUILIBRIUM CONSTANTS OF ENZYME SUBSTRATE COMPLEXES OF

HRP~ (pH 4.7; 25 to 30 °)

Substrate H202 MeOOH" EtOOH/

k~, b M -~ scc. -~ 0 . 9 X l0 T 1.5 X 106 3.6 X 106

k~ -~ k3, b sec. -~

kT, b,~ sec. -1

K, d M

0.10 0.15 --

3.5 3.9 3.8

10 -s 10 -7 --

B. Chance, Arch. Biochem. 2 2 , 2 2 4 (1949). b See equations 1 to 4 on p. 802. No added donor; varies from preparation to preparation due to endogenous donor. k2 + k3 d Apparent equilibrium constant for complex II; K = k~ ; see equations 1 and 4. M e 0 0 H stands for m e t h y l hydrogen peroxide. J E t 0 0 H stands for ethyl hydrogen peroxide. T A B L E IV VALUES OF k4 FOR THE REACTION OF H R P H20~ COMPLEX I I WITH VARIOUS DONORS a

(at 25 to 30 °) Donor type Phenols

Amines

Enediols

Others

Name p-Hydroxydiphenyl Hydroquinone IIydroquinone m o n o m e t h y l ether Catechol Catechol m o n o m e t h y l ether (guaiacol) Resoreinol Pyrogallol o-Phenylenediamine m-Phenylenediamine Aniline p-Aminobenzoic acid Reductone c Ascorbic acid Dihydroxymaleic acid Uric acid Leucomalachite green DPNH Nitrite

k4, M -1 sec. -1 8 3 2 2 2.5 3 3 5 1 7 1 1 2 2 2 3 3

X )< )< X X X X )< X X X X X X X X X 17

107 106 10 e 106 106 l05 105 107 106 104 103 106 104 104d 104 l0 s 10 ~

pH 7.0 7.0 7.0 7.0 6.7 b 7.0 6.7 b 7.0 7.0 7.0 7.0 4.2 4.7 4.0 7.0 4.7 7.0 7.0

B. Chance, in " T h e E n z y m e s " (J. B. Sumner and K. Myrbi~ck, eds.), Vol. 2, P a r t 1, p. 428, Academic Press, New York, 1951. b B. Chance, Arch. Biochem. 24, 410 (1949). c H. yon Euler and C. Martius, Svensk Kern. Tidskr. 45, 73 (1933). d Measured at 4 ° b y B. Chance, J . Biol. Chem. 197, 577 (1952).

[143]

PLANT PEROXIDASE

811

The following amino acids have also been shown to be present in H R P : 49 a l a n i n e , g l y c i n e , l e u c i n e , m e t h i o n i n e , p h e n y l a l a n i n e , p r o l i n e , s e r i n e , t h r e o n i n e , t y r o s i n e , v a l i n e , a s p a r t i c , g l u t a m i c , a n d c y s t e i c a c i d s . 5° Spectroscopic, Magnetic, and Kinetic Data on HRP and Its Derivatives. The spectroscopic and kinetic data on the enzyme substrate comp o u n d s a n d d e r i v a t i v e s of H R P a r e s u m m a r i z e d i n T a b l e s I I I t o V I . T h e m a g n e t i c s u s c e p t i b i l i t y of m a n y of t h e H R P c o m p o u n d s h a v e b e e n TABLE V THE SPECTRA OF THE ENZYME SUBSTRATE COMPLEXES OF H R P (wavelengths in m~) Substrate H~02 H~O~ H20~ H202 H20~ H20~ H20~ MeOOH~ MeOOH MeOOH EtOOH z

Compound I II II II III IIIf III I II IV I-IV

pH

X

e~

Ref.

k

~-

7.0 410 48 b --5.4 419 91 b . . . 7.0 418 95 b 527 8.5 9.0 418 93 b . . . 5.4 418 102 b . . . 7.0 416 106 b 546 9.9 9.0 416 116 b . . . 7.0 410 48 h i --4.7 419 91 h j 530 ? 7.0 410 ? j 557 k ? T h e spectra of the E t O O H c o m p l e x e s are of the M e O O H c o m p l e x e s . b

k

e~

Ref.

657 c ? d . 558 8 . 5 ~ b . . 583 8.6 d . 660 ? i 560 ? i 675 ? i the same as those

a T h e new value for the extinction coefficient of pyridine hemochromogen at 557 m~ found b y Paul et al. (see Table II, footnote a) was used for calculating the extinction coefficients in this a n d the following table. Theorell's* former value or H R P at 403 m~ becomes thus e~03 = 89.5 cm. -1 X m M -~. Keilin a n d Hartree ~ found e4o3 = 91.0. * H. Theorell, Enzymologia 10, 250 (1942). b B. Chance, Arch. Biochem. and Biophys. 41, 404 (1952). c Measured at a t e m p e r a t u r e of 4 °. d D. Keilin and E. F. Hartree, Biochem. J. 49, 88 (1951). ' Keilin a n d Hartree d found a lower E for this b a n d . f Complex I I I is best obtained from complex I I direetly, r a t h e r t h a n from the free enzyme. g M e O O H stands for m e t h y l hydrogen peroxide. h Recalculated according to the d a t a of B. C h a n c e ) B. Chance, Arch. Biochem. 9.1, 416 (1949). i B. Chance, in " T h e E n z y m e s " (J. B. Sumner a n d K. Myrbfiek, eds.), Vol. 2, P a r t 1, p. 428, Academic Press, New York, 1951. Weak b a n d . E t O O H stands for ethyl hydrogen peroxide.

49 A. C. Maehly a n d S. Pal6us, Acta Chem. Scan& 4, 508 (1950). 69 Oxidation product from cysteine a n d cystine.

812

RESP~R~ORr E ~ Z ~ E S

%

O~

dtl

I~

[143]

r~. v

%

% L-~

v

~fr

%

i>. •~

%

dll

0

Z 0 r~ 0

II

II

It

il

o

•F~~r2

,'-, X

XX

X

~

~g

~ Z ~

r~

M4

-~ ~ ~ ~4~m

~

©

o

t.

~ i ~ •

0

~. ~

°

[144]

LACTOPEROXIDASE

813

remeasured recently by Theorell and Ehrenberg, 51 using improved techniques of high sensitivity. A summary of the presently available data can be found in a paper by Chance and Fergusson. 5~ 61H. Theorell and A. Ehrenberg, Arch. Biochem. and Biophys. 41, 442 (1952). 52 B. Chance and R. R. Fergusson, in "The Mechanism of Enzyme Action" (W. D. McElroy and B. Glass, eds.), p. 389, The Johns Hopkins Press, Baltimore, 1954.

[144] L a c t o p e r o x i d a s e

By B. DAVID POLIS and H. W. SHMUKLER Assay Method The peroxidase concentrations of turbid milk fractions can be determined with a modification 1 of the purpurogallin test of Sumner and Gjessing. ~ With homogeneous solutions of the enzyme, a more convenient method results from the oxidation of dihydroxyphenylalanine (dopa) by peroxidase 1 and H~.O~ to a red derivative. Reactions are carried out in 1-cm. square cuvettes holding 3 ml. of solution. The reaction mixture is composed of 0.08 M phosphate buffer, pH 7.0, 16.67 × 10-4 M dopa, 2 X 10-4 M H202, and a suitable dilution of the enzyme. When the dopa is added as the last reagent, a zero-order reaction is obtained that can be determined by taking absorbency readings every 15 seconds at 475 m~ for 2 minutes. By adjusting the concentration of the lactoperoxidase to catalyze the utilization of 10% of the hydrogen peroxide per minute, the velocity constant obtained for the zero-order reaction may be expressed directly in terms of the turnover number of moles of hydrogen peroxide per milligram or mole of lactoperoxidase per minute. Protein concentrations may be determined by the optical density at 280 m~ or by the biuret reaction. A good approximation to the specific activity of the lactoperoxidase preparation is given by the ratio of the optical densities at wavelengths 412 and 280 m~.

Isolation Procedure The commercial skim milk used for the isolation of lactoperoxidase was processed most conveniently in 50-1. batches. A. The casein of 50 1. of milk is coagulated with rennet at room temperature and separated from the whey by straining through muslin bags. B. Salt Fractionation. (1) The whey protein is precipitated by the adB. D. Polis and H. Shmukler, J. Biol. Chem. 201, 475 (1953). J. B. Sumner and E. C. Gjessing, Arch. Biochem. 2, 291 (1943).

814

RESPIRATORY ENZYMES

[144]

dition of solid ammonium sulfate to a final concentration of 2.8 M, p H 6.0, and filtered overnight at 3 °. (2) T h e precipitate is redissolved in water to a protein concentration of 3%. Sodium t e t r a b o r a t e is added to 0.1 M, ammonium sulfate to 1.5 M, and the precipitate is filtered off. 3 (3) T h e salt concentration of the 1.5 M filtrate is increased to 1.9 M. The precipitate is filtered at 3 ° . (4) All the peroxidase in the 1.9 M filtrate is then precipitated at 2.5 M a m m o n i u m sulfate. (5) T h e precipitate of step 4 is redissolved in 0.1 M borax solution to a 2 % protein concentration, and ammonium sulfate is added to 1.9 M. The 1.9 M salt filtrate is then brought to 2.3 M. After standing for 1 hour at room temperature, a brown-green, sticky cake forms t h a t floats on the surface of the salt solution. This is filtered through glass wool, and the excess ammonium sulfate solution is removed from the precipitate b y kneading it into a ball. C. Chromatography. The ionic composition of the lactoperoxidase obtained b y salt fractionation is best controlled for c h r o m a t o g r a p h y b y adjusting the p H to neutrality with 0.5 M KH~P04 and then dialyzing against 20 col. of water for 2 hours with continuous stirring of the peroxidase within the membrane. In this way the ammonium sulfate concentration of the crude lactoperoxidase is reduced to about 0.02 M with only 10% loss in enzyme activity. The peroxidase is then diluted to a final concentration of 2 % protein in 0.1 M K2HP04, p H 9. C h r o m a t o g r a p h y is accomplished first on columns of calcium phosphate to about half-maximal purity. Final purification is attained with silica-Celite columns. 1. CALCIUM PHOSPHATE CHROMATOGRAPHY. Glass columns, 50 × 4 cm., are packed with 30 to 40 g. of calcium phosphate 4 contained between layers of glass wool on stainless steel screens. T h e columns must be packed in a m a n n e r t h a t prevents channeling and y e t permits swelling of the calcium phosphate particles without forming an impermeable block. This is accomplished b y tamping successive layers of a b o u t 10 g. of the adsorbent at a time to an approximate height of 4 cm. and then wetting the columns with 50 ml. of water. Flow rates of 1 to 3 ml./min. 3 See A. A. Green and W. L. Hughes, Vol. I [10], for calculation of (NH4)~SO4saturation. 4Stoichiometric concentrations of calcium chloride and disodium acid phosphate are combined in solutions made alkaline to phenolphthalein with ammonium hydroxide. The gel is washed free of chloride by decantation over a period of '~l~veek,~filtered, dried overnight at 70°, then ground in a mill to a powder fine enough to pass through a 90-mesh sieve.

[144]

LACTOPEROXIDASE

815

can then be maintained through these columns at pressures of 50 to 80 cm. of mercury. In the first chromatographic purification step, a 2 % protein solution in 0.1 M K2HPO4, pH 9, is filtered through successive 30-g. columns of calcium phosphate under such conditions that the peroxidase containing filtrate from one column is refiltered through a second column until the efituent contains no peroxidase. About three-quarters of the total protein passes through the columns and appears in the final filtrate. The columns are then washed with 0.1 M sodium tetraborate solution until the borate filtrate is protein-free. Subsequent elution of the columns with 0.5 M phosphate yields an effluent with a 25-fold increase in peroxidase purity. For further purification, the 0.5 M effluent is diluted to 0.1 M salt and 0.2 % protein concentration and again filtered through calcium phosphate columns. In this step, half the number of columns of the previous step is used. After the columns have been washed with borate, the peroxidase is again eluted with 0.5 M K:HPO4 to yield a fraction with hail maximal purity. 2. SILICA-CELITE CHROMATOGRAPHY. Final purification is accomplished by chromatography on silica-Celite columns. The combined eluares from the calcium phosphate columns are diluted to 0.1 M phosphate and passed through a column composed of a mixture of 2 parts of silicic acid to 1 part of Celite 5 at pressures of 50 to 80 cm. of mercury. With a ratio of 20 mg. of protein to 1 g. of adsorbent, approximately threequarters the length of a 30-g. column is saturated with enzyme. Both the lactoperoxidase and a red protein are adsorbed from 0.1 M solutions of K2HPO4. In contrast to the behavior on calcium phosphate, the red protein is held more firmly than the lactoperoxidase on silica-Celite, and as a result the lactoperoxidase is preferentially displaced with 0.5 M phosphate. The red protein remaining is eluted from the column with 1 M K2HP04, and the column may be regenerated by displacing the excess phosphate with water. Spectrophotometric analysis of the effluent aliquots permits the combination of the effluent from the column into fractions of comparable purity. Chromatography of the subfraction combinations concentrates the lactoperoxidase through steps of graded purity into the 0.5 M eluate with a ratio of optical densities D412/D28o equal to 0.9. D. Crystallization. The purified lactoperoxidase is obtained from the silica-Celite columns as a reddish-black solution in 0.5 M K:HPO4. This is precipitated completely by the addition of 4 M K2HPO4 at room tem5 Merck's reagent-grade silicic acid and Johns-Manville Celite analytical filter aid gave reproducible results without any special preparation.

816

RESPIRATORY ENZYMES

[144]

perature to a final concentration of 2.5 M and is filtered off with suction with the aid of 0.5 g. of Celite (analytical filter aid grade) per 10 ml. of solution. T h e enzyme then is eluted with 1 M K2HPO4, and sufficient 4 M K~HP04 is added to form a slight turbidity (2.2 M.). Approxim a t e l y 0.2 g. of Celite per 10 ml. of solution is added, and the precipitate is filtered off. The filtrate is then stored in a beaker covered with filter paper at 5 °. Crystals form after a few weeks. The crystalline enzyme is stable in the concentrated phosphate solution for over a year. SUMMARY OF ISOLATION PROCEDURE FROM

100 L. OF SKIM

MILK a

Specific activity

Fraction

Protein, Pyrogallol g. testb

Whey 715 2.3 M (NH4)2SO4 ppt. 207 0.5 M K~HPO4 eluate from Ca3(P04)~ First eluate 4.4 Second eluate 1.6 0.5 M K~HP04 eluate from sflica-Celite Fourth eluate 0.5 Crystallization from 2.2 M K~HP04 Second crop 0.25

D~12 Lactoperoxidase

D2s~-o yield, mg.

0. 049 1.09

2870 1850

58

0.19 0.42

974 764

122

0.9

500

122

0.9

250

27

B. D. Polis and H. Shmukler, J. Biol. Chem. 201,475 (1953). b Specific activity by the pyrogallol test is defined arbitrarily as the milligrams of purpurogallin formed in 20 seconds by 1 mg. of protein.

Properties In the presence of hydrogen peroxide, lactoperoxidase catalyses the oxidation of m a n y phenols and aromatic amines. In this respect, its behavior is similar to horseradish peroxidase, 6 with the exception t h a t pure lactoperoxidase has no apparent action on tyrosine. T h e enzyme binds phosphate strongly, causing a shift in the isoelectric point and also producing a threefold activation of dopa peroxidation. T h e reaction with dopa is especially interesting, since a competitive inhibition of the dopa oxidation is produced b y increasing concentrations of H20:. Lactoperoxidase exists in two forms t h a t have been designated A and B, differing primarily in electrophoretic mobility, spectrophotometric constants, and the rate of reaction with dopa. Lactoperoxidase B, obtained primarily from spring milk, is similar to the enzyme prepared 6j. B. Sumner and G. F. Somers, "Chemistry and Methods of Enzymes," 3rd ed., Academic Press, New York, 1952,

[145]

PLANT TYROSINASE (POLYPIIENOL OXIDASE)

817

by Theorell. 7.8 Crystalline lactoperoxidase A is a heme-protein containing 0.069% iron and 15.56% nitrogen. Its molecular weight is 82,000. The enzyme is isoelectric in 0.1-~ Veronal buffer at pH 8.0, in 0.1-~ phosphate buffer at pH 6.8. It is isoionic at pH 9.6. For pure preparations, the optical densities at 280 and 412 m~ are 1.541 and 1.390 at a concentration of 1.0 g./1. With dopa as a substrate, the turnover number of lactoperoxidase A (D412/D2so = 0.89) was 1500 moles of H~O~ per minute per mole of enzyme. The turnover number of lactoperoxidase B, (D412/D~so = 0.77) was 1013. 7 H. Theorell and ~. ,~keson, Arkiv Kemi, Mineral. Geol. 17B, No. 7, p. 1 (1943). H. Theorell and K. G. Paul, Arkiv Kemi, Mineral. Geol. 18A, No. 12, p. 10 (1944).

[145] P l a n t Tyrosinase (Polyphenol Oxidase)

By CHARLES 1%. DAWSON and RICHARD J. MAGEE Cresolase and Catecholase Activities The darkening of mushrooms, potatoes, apples, and many other plants and plant products on injury to the tissue is the result of the enzymatic oxidation of certain mo/aohydric and o-dihydric phenols. The enzyme responsible for these oxidations is called tyrosinase, for the phenolic amino acid, tyrosine, was the first experimental substrate. The purified enzyme is commonly prepared from the edible mushroom, PsaUiota campestris, and p-cresol and catechol have been most frequently employed as experimental substrates. Consequently, the two activities of the mushroom enzyme have come to be known as the "cresolase" and "catecholase" activities. The ratio of catecholase to cresolase activities (cat/cre ratio), as measured by the assay methods described below, is about 5:1 in crude extracts of the mushroom. In the course of purification this ratio is usually increased greatly in favor of the catecholase activity. Such "high catecholase" preparations of tyrosinase often contain little or no cresolase activity and appear to be very similar to the "polyphenoloxidase" and "catechol oxidase" described in the literature.l.~ It is also possible to prepare from the same starting material a purified enzyme possessing a cat/cre ratio comparable to or even lower than that of the original crude extract; such preparations have been termed "high cresolase" preparations of tyrosinase. No preparation has yet been obtained which possesses only cresolase activity. D. Keilin and T. Mann, Proc. Roy. Soc. (London) B125, 187 (1938). z F. Kubowitz, Biochem. Z. 292, 221 (1937).

818

RESPmXWORY ENZYMES

[145]

Assay of Cresolase Activity Principle. The method for measuring cresolase activity depends on the fact that, within a certain range of enzyme concentration, the rate of oxygen consumption during the oxidation of p-cresol is proportional to the amount of enzyme present. It must be noted that the oxidation of monophenols by tyrosinase does not set in immediately on mixing of the enzyme and substrate but is characterized by an induction period, the length of which may vary with the source and purity of the enzyme and with the presence of oxidizing or reducing agents2 An advantage of the assay method 4 described here is the fact that usually the induction phase of the reaction is over by the time the first reading is taken. Reagents p-Cresol solution (0.037 M). Dissolve 100 mg. of redistilled p-cresol in 25 ml. of water. Such solutions are sufficiently stable at room temperature to be used over a period of several weeks. 0.2 M Na2HP04--0.1 M citric acid buffer, pH 7.0. Gelatin solution. Add 750 mg. of gelatin to 150 ml. of water, and warm with stirring until the gelatin has dissolved. Add one crystal of thymol as preservative. Store at 5 °. This solution should be freshly made every week. Enzyme. Dilute the cold stock solution of enzyme with ice-cold water to obtain a dilution containing between 1 and 2.5 units of cresolase activity per milliliter. (See definition below.) Procedure. Manometers of the Warburg type are used with respirometer flasks of about 40-ml. volume. Prior to diluting the enzyme, prepare the reaction mixture by adding to each of the flasks 4.0 ml. of buffer, 1.0 ml. of p-cresol solution, 1.0 ml. of gelatin solution, and 3.0 ml. of water. Then dilute the enzyme, and add immediately 1.00-ml. aliquots of the enzyme dilution to the reaction mixtures. Attach the flasks at once to the manometers with the manometer stopcocks open, place in a thermostat at 25 __ 0.01 °, and shake at 120 oscillations per minute. After 10 minutes close the stopcocks and take readings at 5-minute intervals for 20 or 30 minutes, the extent of the period of linear rate. Activity measurements should be run in duplicate, along with a control containing the reaction mixture without enzyme. Definition of Unit and Specific Activity. One unit of cresolase activity is defined as that amount of enzyme which causes an oxygen uptake of 3 C. A. Bordner and J. M. Nelson, J. Am. Chem. Soc. 61, 1507 (1939). 4 M. F. Mallette and C. R. Dawson, J. Am. Chem. Soc. 69j 466 (1947).

PLANT TYROSINASE (POLYPHENOL OXIDASE)

[145]

819

10 ~l. per minute during the linear portion of the reaction. 5 Specific activity is expressed as units per milligram of dry weight. Dry weights are determined by the method of Mallette et al. e A useful spectrophotometric method is also available. ~

Assay of Catecholase Activity Principle. Although several useful colorimetric methods for the determination of eatecholase activity have been described, 1,8 the chronometric method,9 described below, is used in the writer's laboratory because it is less complicated by the possible side reactions of o-benzoquinone and because it permits the accurate evaluation of the rate of enzymatic oxidation of catechol during the period 15 to 150 seconds after the start of the reaction. Because the initial reaction course can be determined with greater precision, the chronometric method is superior to earlier manometric procedures. 5 The method involves several measurements of the time required for a given quantity of enzyme to produce from catechol certain amounts of o-benzoquinone. The amount of o-benzoquinone involved is that which is equivalent to a known amount of added ascorbic acid. As long as the ascorbic acid is present, no o-benzoquinone exists in the system (it is reduced as rapidly as formed). The end point, which is the first appearance of o-benzoquinone as indicated by a starch-iodide indicator, corresponds to the depletion of the ascorbie acid. If the measurement is run a number of times using different known amounts of ascorbic acid with a constant amount of enzyme and catechol, a series of end points is obtained which measures the production of o-benzoquinone as a function of time for that amount of enzyme. Reagents

H20 should be doubly distilled from an all-Pyrex apparatus to reduce copper content to less than 0.05 ~,/ml. Ascorbie acid solution (0.0057 M). Dissolve 100 rag. of ascorbic acid in 100 ml. of water containing 100 mg. of metaphosphoric acid (HPOa). This solution should be freshly made on the day of using. Catechol solution (0.182 M). Dissolve 1.00 g. of catechol in 50 ml. 5 M. Graubard and J. M. Nelson, J. Biol. Chem. 111, 757 (1935). 6 M. F. Mallette, S. Lewis, S. R. Ames, J. M. Nelson, and C. R. Dawson, Arch. Biochem. 16, 283 (1948). 7I. Z. Eiger and C. R. Dawson, Arch. Biochem. 21, 181 (1949). 8 j. D. Ponting and M. A. Joslyn, Arch. Biochem. 19, 47 (1948). W. H. Miller, M. F. Mallette, L. J. Roth, and C. R. Dawson, J. Am. Chem. Soc. 66, 514 (1944).

820

RESPIRATORY ENZYMES

[145]

of water. This solution should be freshly made on the day of using. 0.4 M Na2HP04--0.2 M citric acid buffer, pH 5.1. Starch-iodide indicator solution. To a mixture of 25 ml. of 10 % K I solution and 25 ml. of 2 N H~S04, add 0.5 g. of pyrogallol and 5 ml. of a 1% starch solution. This indicator solution can be used for several determinations. Enzyme. Dilute the cold stock solution of enzyme with ice-cold water so that 1 ml. of the dilution contains an amount of enzyme that will produce the end point in 40 to 60 seconds when 50 mg. of catechol and 3 mg. of ascorbic acid are used as described under Procedure. This dilution, which contains an enzyme concentration of about 30 units/ml., is kept in a small ice bath until the activity measurement is completed. Procedure. A 300-ml. round-bottom three-neck flask is clamped in a 25 ° thermostat. In one neck of the flask is placed a rubber stopper holding a glass capillary tube bent as a siphon so that through it the reaction mixture may be sampled dropwise (about 2 drops per second) into the starch-iodide indicator solution (see Fig. 1). Another neck of the flask

A,r

/Capillary siphon

s ' rer

thermostat

~ ~-- ~ ~ s~Starch'i°dide /t / ~=-*~ solution J ~/~Lamp

Fro. 1. A p p a r a t u s used in t h e chronometric m e t h o d for t h e m e a s u r e m e n t of catecholase activity.

contains a rubber stopper holding a glass tube through which air is bubbled to stir the contents of the flask at a medium rate. The center neck of the flask is open and is used for introducing the reagents which (except for the enzyme) are held until needed in separate flasks immersed in the same thermostat. The starch-iodide indicator solution is placed in a crystallizing dish illuminated from beneath through a white opalescent glass and is located close to the thermostat. This indicator solution should be mechanically stirred with a glass stirrer at a slow, constant rate.

[145]

PLANT TYROSINASE (POLYPHENOL OXIDASE)

821

The first step is the preparation of the proper enzyme dilution and the determination of the optimum concentration of catechol to be used. In the flask are placed 10 ml. of buffer and enough water so that the total volume will be 100 ml. after all the reagents have been added. The enzyme is diluted, and 1.0 ml. of the dilution is added to the reaction mixture through a small funnel, followed by 3 ml. of ascorbic acid solution and a known volume of rinse water. The siphon tube is then adjusted in position and the air bubbler inserted, the center neck of the flask being left open. The reaction is initiated by rapidly introducing 2.5 ml. of catechol solution from a small Erlenmeyer flask, and simultaneously the stopwatch is started. As soon as possible the siphon is put into operation by momentarily stoppering the center neck. The end point is indicated by the first fleeting appearance of a blue color at the point where the reaction mixture drops into the starch-iodide indicator solution. If the reaction time does not fall close to the desired 40 to 60 seconds, the enzyme should be rediluted. Once the proper dilution is obtained, the end point determination is repeated three or four times using the same amounts of enzyme and ascorbic acid but varying the catechol concentration until the quantity giving the shortest reaction time has been determined. Purified high catecholase preparations generally have an optimum catechol concentration of about 20 mg. per 100 ml., whereas crude preparations and high cresolase preparations generally show maximum activity at about 100 mg. of catechol. The second step is the actual rate measurement and the calculation of the enzyme activity. A reaction rate curve is obtained using the optimum amounts of enzyme and catechol and varying amounts of ascorbic acid, usually from 1 to 4 mg., so that the end points lie in the range of 20 to 100 seconds. The reciprocal of the milligrams of ascorbic acid used is then plotted against the reciprocal of the reaction time in seconds. The slope of this straight line is divided by 2.62 X 10-8 to give the "units of catecholase activity" present in the reaction. A utilization of 2.62 X 10-3 mole per second of ascorbic acid corresponds to the production of o-benzoquinone at the rate of 1.49 )< 10-s mole per second. Definition of Unit and Specific Activity. Prior to the development of the chronometric method, a unit of catecholase activity was defined as that amount of enzyme which causes an oxygen uptake of 10 ~1. per minute. TM This rate of oxygen uptake is equivalent to an o-benzoquinone production in the chronometric method of 1.49 X 10-8 mole per second. Specific activities are expressed as units per milligram of dry weight (see assay of cresolase activity). io D. C. Gregg and J. M. Nelson, J. Am. Chem. Soc. 62~ 2500 (1940).

822

RESPIRATORY ENZYMES

[145:

Purification P r o c e d u r e T h e procedure described below has been developed and used in the writer's l a b o r a t o r y during m a n y years of investigation on this e n z y m e I n this procedure the enzyme usually undergoes a b o u t a 15-fold increase in purity, and the final product, useful for most purposes, is a b o u t 30 ~( pure as judged b y the criteria of Mallette and Dawson. n As shown by the d a t a in Table I, the procedure through step 7 yields a high catechol. ase enzyme (cat/cre ratio 30 to 40). Suggestions for the preparation oJ high cresolase tyrosinase are given after step 7. TABLE I SUMMARY OF PURIFICATION PROCEDURE a

Fraction

Total Specific Units/ml., units, activity, Total thousands thousands Dry units/mg. volume, weight, Cat/ere ml. Cat Cre Cat Cre mg./ml. Cat Cre ratio

1. H20 extract 19,000 0.33 0.03 2. After acetone fractionation 5,200 1.07 0.13 3. (NH4),.SO, fraction 2,000 2.96 0.23 4. Dialyzed enzyme, after alumina treatment 2,400 1.26 0.07 5. After lead subacetate treatment 2,400 0.53 0.015 6. Eluate from alumina 210 8.00 - 7. Second lead subacetate fraction 55 7.60 0.22

6260 570 2.23

148 14.3

10

5570 675 3.85 5920 460 5.97

304 34.3 497 37.7

9 13

3020 168 3.68

343 19.0

18

1270 7.6 1680 - -

1.00 5.67

525 14.5 1410 - -

36 --

418 12.3

7.02

1080b 32

34

a Typical data taken from I. Z. Eiger and C. R. Dawson, Arch. Biochem. 21~ 181 (1949). b The decrease in specific activity from step 6 to 7 reported here is exceptional There is often a twofold improvement in specific activity at this stage. Procedures which yield essentially pure high catecholase and higI cresolase tyrosinase preparations have also been developed, b u t thest methods are considerably more laborious. T h e y have been described ii detail elsewhere. 6 Step 1. Preparation of Crude Extract. T h i r t y pounds (ten baskets) o: the c o m m o n mushroom, Psalliota campestris, is passed through a m e a grinder into 50 1. of acetone which has previously been chilled with dr~ ~ M. F. Mallette and C. R. Dawson, Arch. Biochem. 23, 29 (1949).

[145]

VLANT 'rYROSINASE (POLYPHENOL OXIDASE)

823

ice. T h e suspended pulp is collected b y suction filtration on filter p a p e r and pressed dry in a hydraulic press. T h e pressed pulp, which weighs 3 to 4 pounds, is frozen b y being placed in contact with d r y ice for at least 4 hours. I t m a y be stored in this frozen condition for several months, if desired. T h e frozen pulp is then broken up, suspended in a b o u t 20 1. of water, and allowed to stand overnight in the refrigerator a t a b o u t 5 ° in order to extract the enzyme. The suspension is filtered through cheesecloth, and the pulp wrapped in chain cloth and squeezed d r y under hydraulic pressure. Step ~. Fractionation with Acetone. An a m o u n t of acetone 1.5 times the volume of the filtrate from step 1 is added, and the resulting precipit a t e is filtered on Celite. 1~ The precipitate and Celite pad are suspended and stirred in 6 1. of cold water (5 °) to redissolve the protein. T h e solution is then refiltered through another Celite pad. Step 3. Fractionation with Ammonium Sulfate. Sufficient solid (NH4)~SO4 is dissolved in the cold solution from step 2 to m a k e the solution 0.35 M (0.6 saturation) in (NH~)2SO4. After stirring for a few minutes, the mixture is filtered on Celite, and the precipitated protein on the pad is washed with more cold 0.35 M (NH4)~S04. The precipitated protein is then redissolved b y stirring in a b o u t 2.4 1. of w a t e r as in step 2. Step 4. Partial Removal of Color with Alumina Gel. T o the solution from step 3 is added one-tenth its volume of a suspension of alumina gel. 18 After thorough mixing, the alumina and the adsorbed material, consisting mainly of dark-colored pigments, are filtered on Celite and discarded. T h e filtrate is dialyzed overnight against cold running w a t e r (15 to 20 hours). ~2Filtrations employing filter aids such as Celite (Johns-Manville No. 535) or infusorial earth (Fisher Scientific Co., white calcined powder) are carried out with suction in a Bfichner funnel using a quarter-inch pad of the filter aid on the filter paper. 13Alumina gel reagent (based on the method of Willst{itter14). Dissolve 170 g. of A12(SO4)3.18H20 in 2 1. of warm H20 (60°) in a 12-1. round-bottom flask, and to the solution add 2 1. of warm H20 containing 50 g. of (NH4)2S04. While swirling the flask, add slowly about 150 ml. of concentrated NH4OtI to produce a heavy precipitate of AI(OH)~. Fill the flask with hot tap water, stir, and, after allowing the precipitate to settle (30 to 40 minutes), add a little more concentrated NH4OH. When no further precipitate forms on the addition of NH4OH, siphon off the clear solution from above the precipitate, and refill the flask with warm H:O. Mix thoroughly and allow to settle. Usually about twelve washings with hot tap water, about four a day, are required to bring the pH of the washings to below 8.0. Then wash the AI(OH)3 with distilled H20 until the washings are at pH 7.0. Finally, make up the suspension to a volume of 2 1. with distilled H20. (Dry weight 0.144 g. per 10 ml. of the final suspension.) 14 R. Willstiitter, "Untersuchungen fiber Enzyme," p. 575. Springer, Berlin, 1928.

824

RESPIRATORY ENZYMES

[145]

Step 5. Further Removal of Color with Lead Subacetate Reagent. Lead subacetate reagent 15 is added dropwise to the dialyzate from step 4 until the solution is definitely cloudy, le Filtration on Celite produces a solution of lighter brown color. T h e precipitate is discarded. Step 6. Adsorption of Tyrosinase to Alumina. T h e filtrate from step 5 is treated with 0.2 its volume of alumina gel reagent. The alumina with the adsorbed crude enzyme is filtered on Celite in as small a funnel as feasible, and the filtrate is discarded. T h e tyrosinase is eluted at room t e m p e r a t u r e b y stirring the filter pad and gel in about 200 ml. of 0.2 M Na2HPO4 at room t e m p e r a t u r e for 30 minutes. T h e eluate is filtered through Celite and dialyzed against running tap water. Step 7. Fractionation with Lead Subacetate. A volume of cold acetone 0.1 times the total volume of the dialyzate is added, followed b y just enough lead subacetate reagent, added dropwise, to produce cloudiness.iS T h e resulting mixture is filtered and the filtrate similarly fractionated two to three more times with lead subacetate. N o acetone is added for these further fractionations, b u t each time just enough of the lead reagent is added to produce cloudiness. Each precipitate is dissolved with stirring in 30 to 60 ml. of 0.2 M NasHPO4 and the resulting solution refiltered on Celite. The filtrates are assayed for catecholase and cresolase activities. Fractions having the maximum activity and the desired c a t / cre ratio are usually combined. Such preparations are dialyzed against doubly distilled (copper-free) water at refrigerator temperature for 3 to 4 days before a copper assay is conducted or the specific activity determined. Preparation of High Cresolase Tyrosinase. The crude extract from step 1 is purified through step 4 as already described. If the resulting solution possesses a c a t / c r e ratio of about 15 or less, it is then subjected to the stepwise (NH4)2SO4 fractionation described below. If, however, the ratio is higher t h a n 15, it is usually helpful to fractionate four or five times at this point with lead acetate and acetone, as described in step 7. The resulting precipitates are dissolved in 0.2 M Na2HPO4, and the activity ratio of each fraction is determined. In this way it is possible to collect and combine the fractions having the lowest c a t / c r e ratios. 1~Lead subacetate reagent. Grind 420 g. of Pb(C~H30~)r3H~O and 140 g. of PbO together in a mortar, and mix with 1400 ml. of H20 in a glass-stoppered bottle. Shake thoroughly, allow to stand for a week at room temperature, and filter. Dilute one volume of the filtrate with 10 vol. of water. 16It is usually advisable to estimate the amount of lead subacetate reagent required by first adding it dropwise to a small aliquot of the dialyzate. Both pigment and enzyme are precipitated by this reagent. Therefore care must be taken not to precipitate too much of the enzyme. If not enough of the reagent is used, a colloidal dispersion results which passes through the filter.

PLANT TYROSINASE (POLYPHENOLOXIDASE)

[145]

825

The combined fractions are then dialyzed against cold water overnight and subjected to the stepwise (NH4)2S04 fractionation described in the next paragraph. The (NH4)2S04 fractionation is performed as follows: 22.5 g. of (NH4)2SO4 is dissolved in each 100 ml. of the dialyzed enzyme solution. Four additional fractions are then precipitated b y each time adding to the filtrate an a m o u n t of (NH4)2S04 equivalent to 7.6 g. for each 100 ml. of the original dialyzed enzyme solution. Each precipitate is collected separately b y filtration on Celite and redissolved in cold water. The cat/cre ratio decreases with increasing (NH4)2S04 concentration.

Properties Constitution of the Enzyme. I t appears likely t h a t mushroom tyrosinase as it exists in nature has a copper content of less than 0.1% and a molecular weight in excess of 200,000. However, highly purified enzymes (judged b y electrophoretic and ultracentrifuge criteria) possess the properties summarized in Table II. TABLE II PROPERTIES OF EXTENSIVELY PURIFIED HIGH CATECHOLASE AND HIGH CRESOLASE TYROSI NASEa

Units/mg. Units/7 Cu Type

Cu,b %

Cat

Cre

Cu in dry Molecular active weight Homoge-weight of frac- Cat/cre neity, active tion, Cat Cre ratio % fraction %

High catecholase 0.206 2130 48 4400 95 48 90-100% 100,000 0.25 High cresolase 0.028 856 536 237 149 1.6 75 -0.036 M. F. Mallette and C. R. Dawson, Arch. Biochem. 23, 29 (1949). b Copper analyses performed by the methods of 0. Warburg and H. A. :Krebs, Biochem. Z. 190, 143 (1927), and S. Ames and C. R. Dawson, Ind. Eng. Chem., Anal. Ed. 17, 249 (1945). The Priming Reaction in Monophenolase Action. Addition of small amounts of catechol to the monophenol-tyrosinase system removes the characteristic induction period. Oxidizing agents, such as potassium ferricyanide or laccase, greatly lengthen the induction period, whereas reducing agents such as ascorbic acid markedly shorten it. A theory of monophenolase action consistent with these facts has been proposed2 ,17 Specificity. Tyrosinase is the only enzyme known to catalyze the direct aerobic oxidation of monophenols. M a n y monophenols have been iv j M. Nelson and C. R. Dawson, Advances in Enzymol. 4, 99 (1944).

826

RESPIRATORY ENZYMES

[145]

investigated and found to be oxidized by this enzyme. Among those most commonly studied are tyrosine, phenol, p-cresol, 3,4-dimethylphenol, and 4-t-butylphenol. The corresponding o-dihydric phenols are also commonly used as experimental substrates. In addition, the enzymatic oxidation of adrenaline, pyrogallol, and numerous other substituted catechols 18 has been investigated. The tyrosinase oxidation of certain high molecular weight substrates, such as proteins 19 and tea tannins, has been reported. Inhibitors. Substances known to complex with copper, such as potassium cyanide, diethyldithiocarbamate, hydrogen sulfide, carbon monoxide, potassium ethyl xanthate, sodium azide, salicylaldoxime, p-aminobenzoic acid, sulfathiazole, thiouracils, thioureas, cysteine, glutathione, and BAL inhibit the enzyme. Certain of these agents have been found to inhibit the monophenolase and o-dihydric phenolase activities to about the same extent. 2° Reagents, such as iodoacetamide, p-chloromercuribenzoate, trivalent arsenic ion, and cupric ion, which react with free sulfhydryl groups do not inhibit polyphenoloxidase. 4-Nitrocatechol and 4-nitrophenol are competitive inhibitors. Stability. As a general rule, the cresolase activity of the enzyme is less stable than the catecholase activity. Conditions which cause protein denaturation (heating to 60 °, vigorous shaking) result in a serious loss of enzyme activities of both kinds, but an increase in cat/cre ratio. Purified tyrosinase solutions containing in excess of 1 mg. of enzyme per milliliter show little loss in activity over a period of several months if they are buffered at pH 7, inoculated with a few drops of toluene as antiseptic, and stored in the refrigerator. Highly diluted enzyme solutions, however, may exhibit a significant loss in activity within 15 to 20 minutes even at 5 °. Reaction Inactivation. One of the most characteristic features of the enzymatic oxidation of catechol by the purified enzyme is the marked inactivation of the enzyme that occurs during the reaction. 21,~2 Reaction inactivation of the enzyme during the oxidation of monophenols is much less pronounced. p H Optima. At pH below 5, tyrosinase rapidly loses activity. For the enzymatic oxidation of catechol the most satisfactory range of pH is 5.5 to 7. There is no marked optimum in this range. The oxidation of p-cresol shows an optimum in the region of pH 6 to 7, dependent to some extent on the state of purity of the enzyme. is M. L. Cushing, J. Am. Chem. Soc. 70, 1184 (1948). 19I. W. Sizer, J. Biol. Chem. 163, 145 (1946). ~0D. C. Gregg and J. M. Nelson, J. Am. Chem. Soc. 62, 2500 (1940). 9~I. Asimovand C. R. Dawson, J. Am. Chem. Soc. 72, 820 (1950). ~ L. L. Ingraham, J. Corse, and B. Makower, J. Am. Chem. Sac. 74, 2623 (1952).

[146]

MAMMALIAN TYROSINASE

827

Substrate Optima. The optimum concentrations of catechol and cresol are dependent on the cat/cre ratio of the tyrosinase preparation under examination, and on the structures of the phenol or catechol being used as substrate.

[146] M a m m a l i a n Tyrosinase

By AARON BUNSEN LERNER

Tyrosinase //%~, /

//

OH

\

//

OH

CH2CHCOOH I NH2

Tyrosine

NH

\\

\\

,~0

~0

C,H2~HCOOH NH2

Dopa

Melanin

Three methods are given for the assay of mammalian tyrosinase. Each has its merits, and the choice of assay depends in part on the type of material available for analysis.

1. Manometric Assay Method 1,2

Principle. The rate of oxygen utilization of dopa-tyrosinase mixtures in the conversion of dopa to melanin is measured with a Warburg type of respirometer in the usual manner. Dopa and not tyrosine is usually chosen as the substrate, because dopa is rapidly oxidized as soon as it comes in contact with oxygen and tyrosinase. With tyrosine, an induction period precedes the onset of the maximal rate of oxidation; however, if there is no objection to waiting for the induction period to be completed, tyrosine can be used as the substrate. Reagents Dihydroxyphenyl-L-alanine (dopa), 1.0 mg./ml. Add 5 mg. of dopa to 5.0 ml. of 0.1 M sodium phosphate buffer at pH 6.8. Heat the A. B. Lerner, T. B. Fitzpatrick, E. Calkins, and W. H. Summerson, J. Biol. Chem. 178, 185 (1949). A. B. Lerner and T. B. Fitzpatrick, Physiol. Rev. 30, 91 (1950).

828

RESPIRATORY ENZYMES

[146]

tube in boiling water, shaking occasionally, until the dopa dissolves--usually 1 to 5 minutes. The buffer should be made with water deionized by means of ion exchange resins or with doubledistilled water. The solution should be prepared freshly each day. 0.1 M sodium phosphate buffer, pH 6.8, is made with ion-free water. Enzyme. Tyrosinase obtained usually from melanomas or pigmented eye tissue diluted with the phosphate buffer so that 2 to 5 units of enzyme will be present in a 3-ml. mixture with dopa.

Procedure. The substrate, 1.0 mg. of dopa in 1 ml. of phosphate buffer, is added from the side arms of 15-ml. Warburg vessels to 2 ml. of the tyrosinase solution after 10 minutes of equilibration of the solutions at 38 °. Oxygen uptake is recorded every 10 minutes and plotted against time. From the slope of the curve, the rate of reaction can be determined. Definition of Unit and Specific Activity. One activity unit is the amount of enzyme required to catalyze the absorption of 1 gl. of oxygen per minute by 1 mg. of substrate when oxidation is proceeding at a maximal rate. 2. H i s t o c h e m i c a l A s s a y M e t h o d 3,4

Principle. Incubation of tyrosine or dopa with properly prepared fresh tissue containing melanocytes results in the formation and deposition of melanin granules in the cytoplasm of the melanocyte at the site of tyrosinase action in the cell. The amount of melanin granule formation visualized under the microscope gives an approximation of the quantity of enzyme present. The formation of melanin granules from dopa as the substrate is usually but not always specific, because dopa can be oxidized to melanin not only in the presence of tyrosinase but also in the presence of active oxidizing systems such as the cytochrome one. With tyrosine as substrate the reaction is specific, because only tyrosinase will catalyze the oxidation of tyrosine to dopa. The dopa in turn is oxidized and polymerized to melanin. Reagents L-Tyrosine or L-dopa (1.0 mg./ml.) in 0.1 M sodium phosphate buffer, pH 6.8. As described previously in the manometric assay method, tyrosine or dopa is dissolved in phosphate buffer made with ion free water. 0.1 M sodium phosphate buffer, pH 6.8, made with ion-free water. s T. B. Fitzpatrick, S. W. Becker, A. B. Lerner, and H. Montgomery, Science 112, 223 (1950).~ 4 A. B. Lerner,'and-T."B. Fitzpatrick, "Pigment Cell Growth," p. 319, Academic Press, New York, 1953.

[146]

MAMMALIAN TYROSINASE

829

Enzyme. Fresh tissue containing tyrosinase, such as melanomas, skin, or ciliary bodies, is cut into small pieces and placed in 5 to 30 ml. of buffer. Procedure. Fresh tissue is cut into slices 1 to 3 ram. thick and placed in a 5 % solution of formalin for 30 minutes at 5 °. The fixed tissue slices are washed once in 0.1 M phosphate buffer and placed in 5 to 30 ml. of freshly prepared tyrosine or dopa solution and allowed to remain for 12 to 15 hours at 5 °. The tissue slices are then reimmersed in fresh tyrosine or dopa solution and left in an incubator at 37 ° for 24 hours. As a control, one or two slices are treated just as described except that phosphate buffer without added tyrosine or dopa is used. Gross examination of the tissue slices at the end of the final incubation may reveal a darkening of the specimens incubated in tyrosine or dopa solutions but no color change in the controls. To prepare the slices for histologic examination, further fixation is done by immersing them in a 10% solution of formalin for 3 hours. Then they are dehydrated, cleared in toluene, embedded in paraffin, sectioned at 15 ~, and counterstained. Comparison of the amount of melanin granule formation in the cytoplasm of the melanocytes in the tissues incubated with tyrosine or dopa versus the controls give an approximation of the tyrosinase present. In heavily pigmented tissue, this histochemical method is of no value in estimating the presence of tyrosinase because usually it is not possible to differentiate between preformed melanin and that resulting from the incubation. For pigmented tissues, the following radioactive tyrosine method is used. 3. Radioactive Tyrosine Assay Method 4.5 Principle. Carbon-14-1abeled tyrosine on incubation with fresh tissue containing melanocytes is converted to radioactive melanin. By determining the quantity of melanin formed with a Geiger counter, an estimation is gained of the quantity of tyrosinase present. Reagents

C14-Labeled DL- or L-tyrosine (1.0 mg./1 ml.) in 0.1 M sodium phosphate buffer, pH 6.8, is prepared as described previously. Although DL-tyrosine can be used, it should be kept in mind that only the L-tyrosine will be converted to melanin. L-Tyrosine is readily obtained from the DL mixture, e 0.1 M sodium phosphate buffer, pH 6.8. Enzyme. Small pieces of tissue (5 to 50 mg.) are placed in a cold 5T. B. Fitzpatrick and A. Kukita, to be published. 6A. B. Lerner, J. Biol. Chem. 181, 281 (1949).

830

RESPIRATORY ENZYMES

[146]

mortar at 5 ° and by direct downward pressure are mashed with a cold pestle. The crushed moist tissue is placed on a slide exposed to air. It becomes dry in 3 to 5 minutes. The slide is immersed in 70% alcohol for 3 minutes, then removed from the solution and allowed to dry in air. Procedure. Each slide is incubated with 100 ~, of radioactive DL-tyrosine for 24 hours in the presence of 1000 units of penicillin. It is washed with running cold tap water for 5 hours, dried, and measured for radioactivity with a Geiger counter. Control slides show no radioactivity because only tyrosinase converts tyrosine to the insoluble product, melanin. It seems that because of either a lack of cofactors or inactivation during drying other enzymic processes which metabolize tyrosine are not functioning. As yet, no unit of activity has been proposed for results obtained by this method. The results are expressed in terms of number of counts per microgram of tissue on a dry-weight basis. Choice of Method and Properties of Tyrosinase. The choice of method of analysis depends on the concentration of enzyme available, the type of tissue to be analyzed, and the technical facilities at hand. When large quanti Lies of tyrosinase are available, as would be the case with a malignant melanoma or ciliary bodies, the manometric method is simple and rapid. The histochemical method is good for melanomas and skin when an approximation of tyrosinase activity within the cell is desired. The radioactive procedure is by far the most sensitive. Although localization of activity cannot be seen as in the histochemical procedure, it is excellent when working with small quantities of tissue and is essential for studying pigmented tissue. Mammalian tyrosinase is a relatively stable enzyme which requires copper for activity. It is located in the cytoplasm of melanocytes. In the conversion of tyrosine to melanin approximately five atoms of oxygen are used. Tyrosinase from different sources varies in activity. 7 The enzyme prepared from plants is obtained in colloidal solution. However, tyrosinase from mammalian tissue is retained on cytoplasmic particles. Plant tyrosinase is less specific in its action than mammalian tyrosinase. Some plant tyrosinases catalyze the oxidation of many phenol derivatives and o-dihydroxyphenyl compounds at a greater rate than the oxidation of tyrosine and dopa. With mammalian tyrosinase, tyrosine and dopa are oxidized at a much greater rate than any other substance structurally related to these amino acids. D-Tyrosine is not oxidized in the presence of mammalian tyrosinase. Unlike plant tyrosinase, mammalian tyrosin-

A. B. Lerner, Advances in Enzymol. 14~ 73 (1953).

[147]

ASCORBIC ACID OXIDASE

831

ase is not inactivated during the enzymic oxidation of a suitable substrate. Tyrosinase from grasshopper eggs occurs as a protyrosinase and must first be activated before it can exert any catalytic action on tyrosine or related derivatives. Usual activating agents are distilled water, sodium chloride, detergents, or changes in pH or temperature. Tyrosinase in human skin must also be activated before it can readily catalyze the oxidation of tyrosine to melanin. Irradiation of skin with ultraviolet light before excision can activate the enzyme. However, tyrosinase obtained from malignant melanomas appears to exist in an active state and requires no special treatment in order to function at maximal rate.

[147] Ascorbic Acid O x i d a s e Ascorbic Acid + ~/~O2~ Dehydroascorbic Acid -4- H~O B y CHARLES R. DAWSON and RICHARD J. ]VIAGEE

Assay Method Principle. The method 1,~ most generally used depends on the fact that, within a certain range of enzyme concentration, the rate of oxygen consumption during the oxidation of L-ascorbic acid is proportional to the amount of enzyme present. Reagents

H~O used in the reaction mixture and in the preparation of all reagents should be doubly distilled to reduce the copper content to less than 0.05 3,/ml. L-Ascorbic acid solution (0.028 M). Dissolve 250 mg. of L-ascorbic acid in 50 ml. of water containing 50 mg. of metaphosphoric acid. This solution should be freshly made every day. 0.2 M Na2HPO4--0.1 M citric acid buffer, pH 5.7. Gelatin solution. Add 750 rag. of gelatin to 150 ml. of water, and warm with stirring until the gelatin has dissolved. Add about 2 rag. of thymol as preservative, and store at 5 °. This solution should be freshly made every week. Enzyme. Dilute the stock solution of enzyme so as to obtain a solution containing between 1 and 2.5 units of enzyme per milliliter. (See definition below.) Usually this is best accomplished 1p. L. Lovett-Janison and J. M. Nelson, J. Am. Chem. Soc. 62, 1409 (1940). W. H. Powers, S. Lewis, and C. R. Dawson, J. Gen. Physiol. 27, 167 (1944).

832

RESPIRATORY ENZYMES

[147]

with a series of dilutions made with ice-cold water. The final dilution is made so as to contain 2 ml. of gelatin solution per 10 ml. of final volume. If a precipitate forms when the first subdilution is made, this solution is discarded, and a fresh dilution is made with ice-cold 0.1 M acetate buffer, pH 5.6.

Procedure. Manometers of the Warburg type are used with respirometer flasks of about 40-ml. volume. Prior to diluting the enzyme, prepare the reaction mixtures by adding to the main compartment of each flask 4.0 ml. of the phosphate-citrate buffer, 1.0 ml. of gelatin solution, 1.0 ml. of L-ascorbie acid solution, and 3.0 ml. of water. Then dilute the enzyme as described above, and add immediately 1.00-ml. aliquots to the side arms of the flasks. Place the flasks in a thermostat at 25 +_ 0.01 °, and equilibrate for 15 minutes with slow shaking. After initiating the reaction by tipping the enzyme from the side arms into the reaction mixtures, take readings every 2 minutes for at least 10 minutes while the flasks are shaking at about 120 oscillations per minute. Take the average of the three most constant consecutive values for the oxygen consumed in 2 minutes, and calculate the rate of oxygen uptake per minute. Activity measurements should be run in duplicate, along with a control containing the reaction mixture without enzyme. Definition of Unit and Specific Activity. One unit of ascorbic acid oxidase activity is defined as that amount of enzyme which causes an initial rate of oxygen uptake of 10 ~l. per minute. Specific activity is expressed as units per milligram of dry weight. Dry weights are determined by the technique of Mallette et al.3 with one modification. When samples of highly purified enzyme (purified at least up to step 5, Purification Procedure) are being measured, these are first dialyzed against cold 0.1 M acetate buffer, pH 5.7. A correction is therefore necessary for the dry weight of the buffer. Purification Procedure

Lovett-Janison and Nelson 1 attempted the preparation of highly purified ascorbic acid oxidase from eleven different plants and found the yellow summer squash, Cucurbita pepo condensa, to be the most satisfactory source. Procedures developed since that time have resulted in the preparation of solutions of enzyme which are homogeneous electrophoretically and in the ultracentrifuge. 2,4 The procedure described below is less time consuming and produces s M. F. Mallette, S. Lewis, S. R. Ames, J. M. Nelson, and C. R. Dawson, Arch. Biochem. 16, 283 (1948). 4 F. J. Dunn and C. R. Dawson, J. Biol. Chem. 189, 485 (1951).

[147]


833

in much better yield an enzyme that is about 80 % pure as compared with the properties of homogeneous ascorbic acid oxidase. It has been recently developed in the writers' laboratory with the help of Mr. Stanley Lewis. Step 1. Preparation of Crude Extract. Four hundred pounds (10 bushels) of yellow squash are peeled. The rinds are minced to a fine pulp in a meat grinder, and the juice is squeezed through cheesecloth. The pulp is then wrapped in canvas and subjected to hydraulic pressure to remove all the juice. Step 2. Fractionation with Ammonium Sulfate. Enough solid Na2B40:10H20 is added to the juice to bring the pH to about 7.6. The crude juice is then treated with 1 M Ba(C2H~02)2 (10 ml./1, of juice) and made 1.6 M with respect to (NH4)2SO4 (0.3 saturation) by adding the solid salt at room temperature. The precipitate is allowed to settle overnight in the refrigerator so that the supernatant fluid is removable almost entirely by siphon, requiring centrifugation of only the settled material. The precipitate is discarded. The supernatant is treated with an amount of (NH4)2S04 equal to that previously added, and the resulting precipitate, after filtration, is either immediately treated according to step 3b or filtered on Celite 5 and stored at - 1 5 ° until needed (step 3a). Step 3. Refractionation with Ammonium Sulfate. (a) The frozen precipitate from step 2 is slurried in 10 1. of cold water and allowed to stand for 2 hours. The solution is then filtered, and the enzyme is reprecipitated from the filtrate by adding 4200 g. of (NH~)2S04. (b) The precipitate is filtered and redissolved by slurrying the pad and precipitate in 2.5 1. of cold water. After filtration, the resulting solution is dialyzed against several changes of distilled water in the refrigerator until a yellow precipitate forms (about 24 hours). This precipitate is discarded. Step 4. Adsorption of Enzyme to Alumina. To the solution is added one-fifth its volume of alumina gel reagent. 8 The alumina with its adsorbed protein is filtered immediately. The protein is eluted by placing the alumina and filter pad in a small amount of 0.2 M Na~HPO4 and stirring to a thick paste. The mixture is then diluted to about 800 ml. with 0.2 M Na~HP04, stirred for 30 minutes, and refiltered. The filtrate is dialyzed against several changes of water in the refrigerator. Step 5. Fractionation with Acetone. The dialyzed enzyme solution is treated with one-fifth its volume of cold acetone (5 °) and filtered immediately. The filtrate is treated with a second quantity of acetone equal to that used initially. The precipitate is removed by immediate filtration on a fresh filter pad. The process is repeated until about seven precipitates have been collected. A green to blue precipitate is obtained in the 5 All filtrations are c a r r i e d o u t u s i n g Celite as d e s c r i b e d in f o o t n o t e 12 in Vol. I I [145]. GSee f o o t n o t e 13 in Vol. I I [145].

834

RESPIRATORY ENZYMES

[147]

region of the fourth, fifth, or sixth acetone t r e a t m e n t s . T h e precipitates are redissolved s e p a r a t e l y in 150 to 200 ml. of 0.2 M N a ~ H P 0 4 and assayed for e n z y m e activity. T h e green or blue fractions i n v a r i a b l y contain the m o s t a c t i v i t y and are dialyzed against doubly distilled (copper-free) water. Usually during this dialysis, especially with more concentrated solutions, the e n z y m e precipitates as a blue a m o r p h o u s protein. This blue protein is redissolved in a small a m o u n t of 0.1 M acetate buffer and dialyzed against several changes of copper-free 0.1 M acetate buffer in preparation for the determination of copper content and specific activity. An overall yield of a b o u t 18 % (see the table) is obtained b y combining fractions of similar specific activity. SUMMARY OF PURIFICATION PROCEDURE

Fraction

Total volume, Units/ml., ml. thousands

1. Crude extract 50,0O0 0.05 2. (NH4)~SO4fraction . . . 3. Repreeipitation with (NH4) 2SO~ 3,000 0.26 4. Eluate from alumina 800 1.88 5. Acetone fractions 5 and 6 combined after dialysis 15.0 30.00

Total Specific units, activity, Recovery, thousands units/mg. %

2,500 .

1.2

--

. 780 545

110 290

31 22

450

1600

18

Properties

Constitution of the Enzyme. Ascorbic acid oxidase f r o m squash is a blue protein having a molecular weight of 150,000, a copper content of 0.25%, and properties of a globulin. 4 M a x i m a in its absorption spectra a p p e a r at 288 and 605 m~. H o m o g e n e o u s ascorbic acid oxidase has been shown to possess a specific a c t i v i t y of 2000 u n i t s / m g , and 750 u n i t s / 7 of copper. ~ Specificity. Purified ascorbic acid oxidase shows m a r k e d specificity for L-ascorbic acid. D-Ascorbic acid and a n u m b e r of other ene-diols similar in structure to ascorbic acid are also oxidized although m u c h more slowly. Monophenols are not oxidized alone or during the oxidation of ascorbic acid. Polyhydric phenols such as catechol and h y d r o q u i n o n e are not oxidized. Several studies concerning the specificity of ascorbic acid oxidase f r o m various sources h a v e a p p e a r e d in the literature. 8 7 The activity of the enzyme is more than one thousand times that of an equivalent amount of ionic copper. 8 For references, see C. R. Dawson and W. B. Tarpley in "The Enzymes" (J. B. Sumner and K. Myrbiick, eds.), Vol. II, Part 1, p. 492, Academic Press, New York, 1951.

[147]


835

Activators and Inhibitors. Ascorbic acid oxidase is inhibited by cyanide, sodium sulfide, diethyldithiocarbamate, 8-hydroxyquinoline, and potassium ethyl xanthate. 8 It is not inhibited by ethylenediaminetetraacetate. ~ Recently, it has been found that the enzyme is inhibited by certain metallic ions including cupric ions. As a result, low concentrations of certain metal-complexing agents appear to activate the enzyme. 10.11 In higher concentrations, these agents inhibit the enzyme. Stability. The stability of solutions of ascorbic acid oxidase appears to depend on the source and degree of purity of the enzyme and the protein concentration of the solution. Experience indicates that in general the stability of the enzyme increases with increase in purity, i.e., increase in specific activity. Enzymes of maximum specific activity appear to be stable for long periods of time in concentrated solutions at refrigerator temperatures. Enzymes of specific activity in the region of 1400 to 1700 units/mg, usually undergo a slow denaturation even at refrigerator temperatures. Losses of 5 to 10 % of the activity per week are not uncommon. Highly diluted solutions of the purified enzyme, such as are required for activity measurements, undergo rapid loss of activity on standing. This effect is minimized by making the final dilution with a dilute solution of gelatin. ~ Reaction Inactivation. When the enzyme functions in the aerobic oxidation of ascorbic acid, it undergoes pronounced inactivation. 12,~3 The inactivation does not appear to be due to a rupture of the copper-toprotein bond. 14 Effects of p H and of Substrate Concentration. Solutions of ascorbic acid oxidase rapidly and irreversibly lose their activity at pH values below 4. The pH optimum for enzyme activity in citrate-phosphate buffer is about 5.6, regardless of the source and purity of the enzyme. The effect of substrate concentration appears to depend on the purity of the enzyme under investigation.2

9 Recently observed in these laboratories. l0 C. L. Gemmfll,J. Biol. Chem. 192, 749 (1951). H E. Frieden, Federation Proc. 11, 215 (1952). 1~H. G. Steinman and C. R. Dawson, J. Am. Chem. Soc. 64, 1212 (1942). 13W. H. Powers and C. R. Dawson, J. Gen. Physiol. 27, 182 (1944). 14C. R. Dawson,in "Copper Metabolism" (W. D. MeElroyand B. Glass, eds.), p. 18, The Johns Hopkins Press, Baltimore, 1950.

836

RESPIRATORY ENZYMES

[148]

[148] C a r b o n i c A n h y d r a s e ( P l a n t a n d A n i m a l ) By E. RoY WAYGOOD

CO2 ~- HsO ~- H2CO3~ H+ ~- HCO3When COs is dissolved in water it is slowly hydrated to carbonic acid which spontaneously ionizes according to the above equation. Hydration occurs in the pH range 6.5 to I0.0, whereas the reverse dehydration takes place in the pH range 5.5 to 7.5.1 This reversible reaction is catalyzed by many oxy-acid buffers, 2 including phosphate, cacodylate, Veronal, chromate, borate, selenite, and also by the enzyme carbonic anhydrase which catalyzes both phases of the reaction equally.', Assay Methods

Principle. There are two main methods for the determination of carbonic anhydrase activity. 1. Manometric Method. Activity may be determined by measuring the increased rate of COs output when carbonic acid, supplied in the form of a bicarbonate solution, is dehydrated by shaking with a buffer (usually phosphate, pH 6.6 to 6.8) and enzyme. The hydration of carbon dioxide is measured by the increased rate of COs uptake when gaseous COs is shaken with a buffer (usually Veronal, pH 8.0) and enzyme. 2. Colorimetric Method. When a solution of COs is mixed with an alkaline buffer, the pH drops rapidly, owing to the reaction COs-}OH---+ HC03- (above pH 8.0) accompanied and followed by the hydration of COs (below pH 10.0). The decrease in time taken for the buffer containing enzyme to drop to a specified pH is used as a measure of catalytic rate. The lower pH value is determined by a sharp change in the color of an appropriate indicator when the buffering capacity of the solution changes markedly. Procedure. (1) Manometric Procedure. Clark and Perrin 4 have suggested that Meldrum and Roughton's boat-manometric method 5 should be retained provisionally as the standard procedure for measuring enzyme activity. The procedure with elegant refinements which afford a more sensitive means of studying the kinetics of the enzyme under a 1F. J. W. Roughton and V. H. Booth, Biochem. J. 40, 309, 319 (1946); F. J. W. Roughton, Harvey Lectures, Series 39, p. 96 (1943-4). F. J. W. Roughton and V. H. Booth, Biochem. J. 32, 2049 (1938). 3 M. Kiese and A. B. Hastings, J. Biol. Chem. 132, 281 (1940). 4 A. M. Clark and D. D. Perrin, Biochem. J. 48, 495 (1945). 5 N. U. Meldrum and F. J. W. Roughton, J. Physiol. (London) 80, 113 (1933).

[148]

CARBONIC ANHYDRASE (PLANT AND ANIMAL)

837

wider range of conditions 1,2 has been described in considerable detail elsewhere. ~-7 However, for purely routine studies on the distribution or purification of the enzyme and the effects of inhibitors and activators, the use of the W a r b u r g a p p a r a t u s is recommended, since it comprises one of the standard items of equipment available in m a n y laboratories and suffers from no more serious disadvantages than does the b o a t apparatus. Nevertheless, for the accurate interpretation of d a t a derived from a n y manometric procedure measuring carbonic anhydrase activity, the operator m u s t be fully aware of the limitations imposed on the method b y the reaction system. These have been discussed in detail b y R o u g h t o n et al. 1,2,8 and b y Clark and Perrin. 4 Although a n u m b e r of investigators 9-1~ have described reliable methods using the W a r b u r g technique, the one described below, developed by K r e b s and Roughton, 14 is recommended because of its simplicity and the use of a low concentration of phosphate which becomes increasingly inhibitory toward the enzyme at high concentrations. KREBS-t~OUGHTONWARBURGTECHNIQUE. For experiments at 0 °, 2 ml. of 0.1 M N a 2 H P 0 4 and 0.1 M KH2PO4 in the proportion 3.2 are placed in the main c o m p a r t m e n t of a W a r b u r g flask with 0.2 ml. of water or enzyme. One milliliter of 0.1 M N a H C 0 3 is placed in the side arm. At zero time, after equilibration, the two solutions are mixed, shaken at 120 to 180 oscillations per minute, and pressure changes recorded at 30-second intervals over a period of 5 minutes or more. T h e pressure change due to the control (nonenzymically catalyzed reaction) as well as the reaction catalyzed b y small amounts of the enzyme is a linear function of time until a b o u t one-third (ca. 80 mm.) of the final pressure change has been attained. F o r experiments at 15 ° and above, 1 ml. of phosphate buffer and 1 ml. of 0.05 M N a H C 0 3 are used. DEFINITION OF UNIT AND SPECIFIC ACTIVITY. Pressure changes are converted to microliters of C02 b y the use of flask constants (kco,) in the conventional manner. ~ The increased C02 evolved in 30 seconds (i.e., 6 H. Van Goor, Enzyrnologia 13, 73 (1948). 7 F. J. W. Roughton and A. M. Clark, in "The Enzymes" (J. B. Sumner and K. Myrbtick, ed.), Vol. 1, Part 2, p. 1250, Academic Press, New York, 1951. s F. J. W. Roughton, J. Biol. Chem. 141, 129 (1941). 9 M. Leiner and G. Leiner, Biochem. Z. 311, 119 (1942). 10 W. C. Stadie, B. C. Riggs, and N. Haugaard, J. Biol. Chem. 161, 175 (1945). n M. D. Altschule and H. D. Lewis, J. Biol. Chem. 180, 657 (1949). 12E. R. Waygood and K. A. Clendenning, Can. J. Research C28, 673 (1950) ; Science 113, 177 (1951). 13R. U. Byerrum and E. H. Lucas, Plant Physiol. 27, 111 (1952). 14H. A. Krebs and F. J. W. Roughton, Biochem. J. 43, 550 (1948). 15W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques and Tissue Metabolism," Burgess Publishing Co., Minneapolis, 1949.

838 ~

RESPIRATORY

ENZYMES

[148]

over-all rate - nonenzymic rate) in the region of 60 to 80 mm. of pressure can be used to calculate Qco, on a nitrogen or other appropriate basis. In the case of labile highly purified enzymes (see later), or when the effect of activators or inhibitors is being studied, it m a y be more accurate to express the enzymic activity as the difference between the true initial unimolecular velocity constants (k, - k,) of the enzymically and the nonenzymically catalyzed reactions, respectively, according to the methods of Mitchell et al. '8 and Clark and Perrin. 4,~7 F r o m the equation a of the first-order reaction kt = 2.303 log a - x' log (a - x) is plotted against t and the initial slope times 2.303 gives the value of the velocity constant. PRECAUTIONS. Phosphate has a strong catalytic effect on the dehydration of carbonic acid, ~ and the Q10 = 2.9 for this reaction differs markedly from the Q~0 = 1.4 for the enzymically catalyzed reaction (over-all r a t e - non-enzymic rate). ~8 Accordingly, in order to work within the range of strict proportionality between activity and enzyme concentration, the apparatus is limited to the use of enzyme concentrations which increase the nonenzymic activity up to sixfold at 0 o and twofold at 38 ° . If measurements are made at higher concentrations of enzyme, the reaction is limited b y the diffusion of COs and should be corrected b y applying the theoretical concepts and experimental treatments of Roughton. 8 According to Roughton el al., ~,7,8 in most cases, when diffusion is limiting the apparent rate m a y be corrected b y applyR,~Ro

ing the formula R - R~ - R~ where R0 is the apparent rate and Rm is the maximum observable rate in the presence of a large a m o u n t of enzyme. R~ is a function of liquid volume and the dimensions of the flask. As yet it has only been determined for the boat apparatus. When highly purified enzymes from blood are used, a proportion m a y be inactivated b y impurities or adsorption. Such enzyme preparations give a sigmoid relationship between activity and enzyme concentration and furthermore are subject to progressive inactivation during shaking. These errors m a y be overcome b y observing strict precautions in cleanliness of glassware, acid washing, use of doubly distilled water, or b y stabilizing the enzyme with 0.05 % peptone.l.4.~9 Or, on the other hand, since these errors result in deviations from the first-order reaction, during the latter part of the reaction time, the initial slope of the plot log (a -- x) le C. A. Mitchell, U. C. Pozzani, and R. W. Fessenden, J. Biol. Chem. 160, 283 (1945). 17A. M. Clark, Nature 168, 562 (1949). ~8F. J. W. Roughton, J. Physiol. (London) 107, 12P (1948). 19D. A. Scott and J. R. Mendive, J. Biol. Chem. la9, 661 (1941); 140, 445 (1941).

[148]

CARBONm ANHYDRASE (PLANT AND ANIMAL)

839

vs. t will give a true value for the velocity constant (k~) independent of the inactivation of the enzyme. 4,~7 Clark 4,~7 has made use of this method to distinguish between stabilizations and activations. By calculating the percentage decrease (D) in the value of k, during the first 100 seconds of reaction, the sensitivity of the preparation may be determined. In a similar way, the degree of stabilization (S) is calculated as 100(1 - d/d ~) where d and d ~are the values of D in the presence and the absence of stabilizing or activating agents, respectively. 2. Co!orimeoric Method. Colorimetrie techniques are usefu) for rapid tests but have a restricted range regarding the conditions under which enzyme activity can be investigated. Owing to the relatively large inhibitions of the enzyme by CO~= in the original method of Brinkman 2° and Philpot and Philpot, 2~ a more reliable technique using Veronal buffer has been developed by Roughton and Booth s and is being used successfully in other laboratories ~,s3 (C. A. Mawson, personal communication). CO2-VERONAL INDICATORMETHOD. Veronal buffer (3 ml. of 0.022 M Veronal in 0.022 M Na salt, pH 7.95), three drops of bromothymol blue, and 2.3 ml. of distilled water (or 0.3 ml. of enzyme and 2.0 ml. of water) are mixed in a 15-ml. stoppered weighing bottle and placed in ice water for 15 minutes. Five milliliters of ice-cold water saturated with COs (0.071 M) is added anaerobically from a long nozzled all-glass syringe. The time is observed for the pH to drop to 6.3, determined with the aid of a bromothymol standard at this pH. The solutions are mixed in less than 1 second without bubbling or loss of COs. The control time averages 90 seconds, compared to about 64 seconds in the presence of 14.3 p.p.m, of a crude chloroform preparation (Step 2, p. 841). DEFINITION OF UNIT. The enzyme unit E.U. -

to - -

t

t

, where t and

to

are time of reaction in the presence and the absence of catalyst, respectively. Since the velocity constant of the uncatalyzed reaction (k~) calculated from the experimental data was found to be 0.0022 mole/1./sec., which agrees well with the accepted value of 0.0021 at 0 °, the validity of the method is established. Accordingly, Roughton and Booth ~ calculated the rate of enzymic hydration of COs in moles per liter per second as follows, allowing a period of 1 second for mixing. R

--

Ro

Ro

to - -

t-

t

1

2~ R. Brinkman, J. Physiol. (London) 80, 171 (1933). 21 F. J. Philpot and J. St. L. Phflpot, Biochem. J. 80, 2191 (1936). 22 K. M. Wilbur and W. G. Anderson, J. Biol. Chem. 176, 147 (1948). .,3 E. R. Trethewie and A. J. Day, Australian J. Exptl. Biol. Med. Sc/. 27, 429 (1949).

840

RESPIRATORY ENZYMES

[148]

where R and R0 are the rates in the presence and the absence of catalyst, respectively. Ro = k~ [average COs] Then Rate = R - R 0 -

((tt 0- - t 1) ) ]c, [average COs]

since k~ = 0.00225 mole/k/see, and [average COs] = 0.0307 mole under the experimental conditions. Therefore, Rate of enzymic hydration of COs

_ (to -- t)

(t - 1) 6.91 X 10-5 mole/1./sec.

The figures for the all-liquid method are approximately 70 % of those expected from manometric data. The discrepancy of 30% may be put down to the inhibitory action of the indicator and experimental error. Wilbur and Anderson 22 have used an automatic syringe to introduce the COs, and the pH changes are measured electrometrically. RAPID-FLOW COLORIMETmCM~.THOD. All methods of assay heretofore discussed are subject to a mixing error and are restricted to the use of low concentration of enzyme owing to diffusion limitations. Accordingly, the development of the Hartridge-Roughton rapid-flow technique, which minimizes these errors and photoelectrically records the color change of a pH indicator, opens up a wider range of conditions under which the enzyme can be studied. Clark and Perrin 4 have used the rapid-mix, quickstop method of Chance. 24 A saturated solution of COs is rapidly mixed at room temperature with Veronal buffer, pH 8.6 and pK 8.0, in a modified Millikan 25 microapparatus. The flow through a capillary tube is stopped in milliseconds, and in the presence of phenol red the pH of the solution is recorded continuously as a function of time. Much higher temperatures and enzyme concentrations may be used in this technique. U s e of C a r b o n i c A n h y d r a s e as a B i o c h e m i c a l T o o l

In decarboxylation or other reactions where gaseous COs is produced, a question of some significance arises as to whether CO2 or HCOa- is the primary product. In systems rapidly producing COs into the gaseous phase, carbonic anhydrase will slow down the rate owing to the accelerated hydration of the COs. On the other hand, it is inferred that if HCO3is the primary product the rate would be initially accelerated. Krebs and Roughton ~4have shown that COs is the primary product of yeast carbox2~B. Chance, J. Franklin Inst. 229, 455 (1940). 25G. A. Millikan, Proc. Roy. Soc. (London) A155, 277 (1936).

[148]


841

ylase and the urease reaction. Hansl and Waygood 26 have confirmed their findings and in addition have shown that C02 is the primary product of the plant pyruvic, glutamic, oxalacetic, and a-ketoglutaric decarboxylation systems. More recently, Conway and O'Malley ~7 have concluded from their theoretical and experimental treatments that HC03- may be produced concomitantly with CO2 in the latter two reactions. Source and Purification of the Enzyme Animal. Mammalian red blood cells are the richest source. Among other tissues, the pancreas, gastric mucosa, and kidney contain comparable amounts. The enzyme is absent from plasma and other body fluids with certain exceptions. 5,6 Many methods of purification have been described in the literature (see Van Goor 6) culminating in a crystalline preparation of ammonium carbonic anhydrase by Scott and Fisher. 2s In general, however, investigators have used one or all of three stages of purification, either lysed red cells or the crude chloroform preparation of Meldrum and Roughton 1,5 or highly purified enzymes prepared by the particular method of the investigator. Since many laboratories have successfully used the highly purified preparations of Keilin and Mann 29 and because of its relative simplicity the details of their procedure and those of the crude preparations are described. Step 1. Lysed Red Cells. The red blood cells of defibrinated ox blood are centrifuged from the serum and washed three times with an equal volume of 0.9 % NaC1. The washed cells are hemolyzed with an equal or half-volume of distilled water. Step 2. Crude Chloroform Preparation.1 To 10 ml. of lysed red cells are quickly added 8 ml. of 40 % ethanol and 4 ml. of chloroform. The mixture is stirred in a centrifuge tube for 3 minutes to a thin sludge and allowed to stand for 20 minutes. After centrifugation for 10 minutes at 3500 r.p.m., a three-phase system is formed consisting of a supernatant layer of enzyme solution, a central layer of denatured protein, and a bottom layer of chloroform. Step 3. Method I I of Keilin and Mann. 29 Dialysis of Alcohol-Chloroform Extract. The filtered enzyme solution is dialyzed for 24 hours against running water. Step 4. Total Precipitation with Ammonium Sulfate. The fluid is saturated with (NH4)2S04, and the precipitate after filtering on a Btichner 36 N. 37 E. 3s D. 39 D.

Hansl and E. R. Waygood, Can. J. Botany 30, 306 (1952). J. Conway and E. O'Malley, Biochem. J. 54, 154 (1953). A. Scott and A. M. Fisher, J. Biol. Chem. 144, 371 (1942) ; Nature 153, 711 (1944). Keilin and T. Mann, Biochem. J. 34, 1163 (1940).

842

RESPIRATORY ENZYMES

[148]

funnel is dissolved in a small a m o u n t of water, dialyzed against running tap water and centrifuged. Step 5. Fractional Precipitation with Ammonium Sulfate. T h e supern a t a n t fluid is saturated 4 5 % with respect to (NH4)2S04 and filtered. The filtrate is completely saturated with (NH4)2SO4 and, after filtering, the precipitate is dissolved in water and dialyzed. Step 6. Purification with Alumina C~ Gel. T h e solution is treated with three successive 5-ml. ( = 100 mg.) portions of alumina C~ gel at p H 6.8 and centrifuged, the cakes being discarded each time. Step 7. Fractional Precipitation with Ammonium Sulfate. The above solution is made 50 % saturated with (NH4)2SO4 and, after filtering, completely saturated. The precipitate is dissolved in water and dialyzed against distilled water until free from salt. SUMMARY OF PURIFICATION PROCEDURE a

Step

Total volume, ml.

Absolute E.U.

1 2-3 4 5 6-7

2000 2250 110 85 50

4,000,0002 1,488,000 1,100,000 765,000 500,000

M1./E.U.

Specific activity, E.U./mg.

Zn, %

Recovery, %

0.0005 0.0015 0.0001 0.0001 0.0001

14.3 384 588 830-1000 2220

0.C024 --0.15-0.17 0.33

-37 27 19.5 12

Method II [D. Keilin and T. Mann, Biochem. J. 34, 1163 (1940)]. b Calculated from Step 1, Method I [D. Keflin and T. Mann, Biochem. J. 34, 1163 (1940)]. Plant. Bradfield 8° and Waygood and Clendenning TM found the enzyme to be ~ocalized only in the cytoplasm of leaf tissue. The enzyme is absent from root tissue2 ° Sirois and Waygood (unpublished) recommend spinach (Spinacea oleracea) as a source of the enzyme, since it has the highest specific activity of all plants tested, b u t m a n y other sources are available, n o t a b l y leaves of New Zealand spinach (Tetragonia expansa), nast u r t i u m (Tropaeolum majus), lamb's quarters (Chenopodium album), and beet (Beta vulgaris). 12.13,30.31 Sibly and Wood 3~ have purified the plant enzyme b y the m e t h o d of Keilin and Mann, b u t the percentage recovery was low. We have found t h a t it is essential to protect the plant enzyme with cysteine at all stages during purification, and the following procedure is recommended. Midribs are removed from spinach leaves, and 200 g. is ground in a m o r t a r with 50 ml. of cysteine (final concentration 0.005 M). The brei is 30j. R. G. Bradfield, Nature 159, 467 (1947). sz p. M. Sibly and J. G. Wood, Australian J. Sci. Research B4, 500 (1951).

[148]


843

pressed through nylon, and the crude juice is centrifuged for 20 minutes at 15,000 X g. The supernatant fluid (200 ml., A = 15 E.U./mg.) is shaken with 75 g. of Ca3(P04)2 and centrifuged. The cake is eluted with 0.2 M phosphate, pH 6.6, in 0.005 M cysteine. After an initial precipitation with 30 % alcohol, the enzyme is totally precipitated at a concentration of 75% ethanol. The white powder is completely soluble in water. The solution, when dialyzed against 0.005 M cysteine and lyophilized, yields a preparation containing 300 E.U./mg. and 0.05 % Zn with no other metals. The enzyme unit is that defined by Waygood and Clendenning. 12

Properties Specificity. Carbonic anhydrase is specific for the reaction CO2 + H20 ,~- HsCO3. It is doubtful if it catalyzes the reaction COs + O H - --* HC03- which predominates above pH 11.0 and is significant between pH 8 and 10.1,~,3,7 Since the latter occurs as a primary reaction succeeded by, or accompanying, the enzyme-catalyzed reaction, it may introduce errors in some colorimetric methods. There are numerous processes which are limited by the reaction COs + H20 ~---H~CO~, and their rates may be increased by the addition of enzyme, e.g., the deposition and solution of CaCO3, and the dissociation of carbamate into C02. 5,7 Stability. Crude chloroform preparations of the animal enzyme are stable for long periods in the dry state but are progressively inactivated in solution. Purer preparations are less stable. Plant carbonic anhydrase is stable in the dried form but deteriorates rapidly in solution. It is also much less stable during dialysis than the animal enzyme. Cysteine (0.005 to 0.01 M) protects the plant enzyme in solution and during dialysis. 30 Nature of the Enzymes. Carbonic anhydrase is a Zn-protein compound. ~9,32 Keilin and Mann's preparation ~9 and others contained 0.033% Zn, whereas Scott and Fisher's crystalline preparation 28 contained 0.02% Zn and had a molecular weight of 30,00023 The latter value corresponds to 1 atom of Zn per molecule. Our partially purified preparations of the plant enzyme contain 0.05 % Zn with no other metals. Sibly and Wood81 calculated ~hat their preparations of the plant enzyme contained 0.056 % Zn. The failure of dialysis to remove zinc from either animal 29 or plant carbonic anhydrase (Sirois and Waygood, unpublished) and the fact that Tupper et al. 34 have shown that Zn 6~ ions in the medium do not exchange a2 D. Keflin and T. Mann, Nature 144, 442 (1939). 33 M. L. Peterman and N~. ¥. ttakala, J. Biol. Chem, 14§, 701 (1942). 34 R. Tupper, R. W. E. Watts, and A. Wormalls, Bioehem. J. 50, 49 (1952).

844

RESPIRATORY ENZYMES

[148]

with the zinc moiety of the enzyme from blood indicates that the metal is firmly entrenched in the protein molecule. The evidence that we have been able to provide in favor of the view that plant carbonic anhydrase is a Zn-protein is as follows. The Zn content of the purified enzyme preparation increases proportionately with increasing specific activity during prolonged dialysis against cysteine. No other metals are present, but the possibility that Zn is present in a nonspecific protein is not excluded. Inhibition by cyanide and azide (see later) is also indicative of the metalloprotein nature of the enzyme in plants. The kinetics of the enzyme from blood have been studied in detail by Roughtou and Booth 1 using the refined boat method. Byerrum and Lucas 1~ give some kinetic data for the enzyme from plants. Since it is doubtful if all the necessary precautions were observed in their experiments, the results await confirmation. Effect of Enzyme Concentration. Provided that diffusion is not a limiting factor, there is a linear relationship between activity and concentration of crude preparations of the enzyme. When diffusion is limiting, the apparent rate may be corrected by Roughton's formula (see above). In the case of highly purified preparations a dilution effect may occur, giving a sigmoid relationship between activity and enzyme concentration. As pointed out earlier, this may be overcome by stabilizing the enzyme. Effect of Substrate Concentration. The value of the Michaelis constant (Kin) for the crude chloroform preparation is 0.009 M C02 (+0.001 M) at 0°. 1 The value is independent of pH, although Kiese ~5 has reported values at 1° of 0.0012 M at pH 7.4, rising to 0.0022 M at pH 9.3 for a highly purified enzyme. It is not certain whether Kiese took all the necessary precautions. Calculations from Leiner's data for a stabilized highly purified enzyme gives K~ = 0.0075 M + 20%. T M Turnover Number. The rapidity of the reaction is shown by calculations from the data of Roughton et al. 1,~ which gives a maximum turnover number of the order of 9.6 X 107 at 0 ° and pH 7.3 for the enzyme from blood. The highest turnover number previously recorded is 5 X 106 for catalase. The value of 1.8 × 108 for the velocity constant of combination of enzyme and substrate is also indicative of the rapid reaction and is of the same order as that for catalase. Effect of pH. The activity of the enzyme from blood is at a minimum at pH 6.5 and gradually increases to fivefold the activity at pH 10.0. There is evidence that the activity is increased below pH 6.0.1 The isoelectric point is at pH 5.3. 3~ 35M. Kiese, Biochem. Z. 307, 400 (1941). 36M. Leiner, Biochem. Z. 315, 31 (1943).

[148]


845

Effect of Temperature. Recently corrected values give Q10 = 2.9 for the nonenzymic rate and Q10 - 1.4 for the enzymic rate (over-all rate nonenzymic rate). TMA plot of the log activity against l I T shows a linear relationship. Inhibitors. Heavy metal poisons--e.g., cyanide, azide, and sulfide-inhibit the activity of carbonic anhydrase from blood to the extent of 50% at concentrations ranging from 10`4 to 10-e M. 7 Whereas the activity of the plant enzyme is strongly inhibited by azide (70 to 90 % at 10-3 M), 12,3° cyanide, according to Bradfield 3° and Sibly and Wood, 31 only inhibits at much higher concentrations (65 to 75 % at 10-3 M; no inhibition at 10-3 M). However, Waygood and Clendenning ~2 have reported 50 to 75% inhibition of the activity of a crude and dialyzed enzyme preparation from Tradescantia fluminensis by 10-3 M HCN. These values have been confirmed by Sirois and Waygood (unpublished) using stabilized semipurified preparations from spinach leaves. The activity of a crude spinach preparation is inhibited 50% by 2.4 X 10-3 M HCN, whereas the activity of the purified enzyme is inhibited 50% by 1.25 X 10-3 M HCN. Nevertheless, the relative insensitivity of the plant enzyme to cyanide, compared with the enzyme from blood, constitutes one of the more important differences between the two enzymes. The markedly inhibitory effect of low concentrations of sulfanilamide and related substances containing the --S02NH2 group has been extensively investigated. 37-4° N-Substituted sulfonamides are ineffective.89 Thiophene-2-sulfonamide and p-sulfonamidobenzoic acid are eight to twelve times as effective as sulfanilamide which inhibits the activity of the enzyme from animals 50% at 10-7 M. 3s,39 A new and more specific inhibitor and less toxic drug, Diamox 6063 (2-acetylamino-l,3,4-thiadiazole-5-sulfonamide) has recently been reported. 4° In contrast, Bradfield a° and Sibly and Wood ~ found that sulfanilamide at a concentration of 10-5 M caused little or no inhibition of the activity of the plant enzyme from two different sources, thus indicating another important difference between the animal and the plant enzyme. The inhibitory effect of anions on the activity of the enzyme from blood in decreasing order I - > NO3- > Br- > C1- > acetate > SO4--. The effect of C1- is especially interesting, since it appears to cause inactivation by forming an inactive complex with the enzyme. The divalent ion COs-- also causes inhibition of enzyme activity, especially at high pH values.

37T. Mann and D. Keilin, Nature 146, 164 (1940). as H. W. Davenport, J. Biol. Chem. 168, 567 (1945). 39H. A. Krebs, Biochem. J. 48, 525 (1948). 40T. H. Maren, Trans. N. Y. Acad. Sci. 16, 53 (1952).

846

RESPIRATORY ENZYMES

[148]

Activators, Stabilizers and Protectors. Claims of activating substances reported in the literature are now suspect, owing to the questionability of the methods used. Clark and Perrin 4 have shown convincingly that apparent activation of the enzyme by certain substances, e.g., boiled horse plasma and glutathione, is due to the restoration of the activity of purified enzymes, lost by adsorption or the effect of impurities. No activation occurs when the enzymes are stabilized by 0.05 % peptone; thus these substances are acting as stabilizers. Indeed the stabilizing power of plasma obscured a weak inhibitory effect.4 Scott and Mendive 19 report a number of stabilizers of which horse serum (1:40) and 0.05% peptone are the most effective. We have used 0.025% gelatin to stabilize the plant enzyme. One of the most important differences between plant and animal carbonic anhydrase is the dependency of the former on free - - S H groups. Thus Bradfield 3° discovered that cysteine efficiently protected the plant carbonic anhydrase while standing or during dialysis. Furthermore, the enzyme was completely inhibited by 5 X 10-4 M p-mercuriochlorobenzoate, which has little effect on animal carbonic anhydrase. Sibly and Wood 31 have confirmed these findings and also demonstrated complete inhibition of activity by 5 X 10-3 M arsenite, another mercaptide-forming compound. The inhibition caused by both these substances could be reversed by cysteine o r glutathione. Iodoacetate does not cause inhibition, but the presence of sulfhydryl groups on plant carbonic anhydrase has been confirmed polarographically. 3~ The important papers of the Japanese workers, Kondo et al., 4~ on the kinetics of spinach carbonic anhydrase were not available to the author at the time of writing. They have reported the isolation of a metal-free electrophoretically homogeneous protein, stabilized by 0.1 M NaC1 having carbonic anhydrase activity. 41 K. Kondo, H. Chiba, and H. Kawai, Bull. Research Inst. Food Sci. Kyoto Univ. 8, 17-27 (1952); 13, 1-59 (1954). (English abstracts.)

[149]

DEHYDROASCORBIC REDUCTASE

847

[149] Dehydroascorbic Reductase D H A ~- 2 GSH--~ GS'SG ~ AA By M. A. JOSLYN

Assay Method Principle. The method used by Crook, I Crook and Morgan, ~ and Bukin 3 was based on measurement of ascorbic acid produced b y enzymecatalyzed reduction of dehydroascorbic acid (DHA) in the absence of oxygen. The method described below was developed b y Yamaguchi 4 and used for the determination of the distribution of dehydroascorbic acid reductase in the various tissues of the developing pea plant (Pisum sativum) and for assay of enzyme preparations from pea tissue. ~ I t is based on the determination of ascorbic acid produced b y reduction of dehydro-L-ascorbic acid in the presence of reduced glutathione in an evacuated Thunberg tube. The reduced ascorbic acid was determined by the m e t h o d of Loeffler and Ponting 6 from measurement of the concentration of unreduced 2,6-dichlorophenolindophenol in an E v e l y n photoelectric colorimeter with a 515-m~ filter. The reduction of dehydroascorbic acid is carried out at pH 6.3, 25 °, with 1.14 X 10-8 M dehydroascorbic acid, 2.28 X 10 -8 M glutathione, and 10 mg. of dry enzyme preparation in a total volume of 10 ml. of reaction mixture (sufficient to produce about 1 mg. of reduced ascorbic acid per 10 ml. of reaction mixture). Reagents

Dehydroascorbic acid. Crystalline dehydro-L-ascorbic acid was prepared b y the method of K e n y o n and Munro 7 from iodine-oxidized L-ascorbic acid in methanol. The hydriodic acid formed was removed b y precipitation with PbCOa. Excess lead was precipitated with H2S, the PbS removed. The residual methanol was removed by evaporation and the dehydroascorbic acid recrystallized from absolute ethanol at 0 °. Two milligrams of the dehydroascorbic acid were dissolved in 1 ml. of 0.1% acetic acid. E. M. Crook, Biochem. J. 35, 226 (1941). E. M. Crook and E. J. ~Iorgan, Biochem. J. 38, 10 (1944). 8V. N. Bukin, Biokhimiya St 60 (1943). 4 M. Yamaguchi, Ph.D. Thesis, University of California, Berkeley, 1950. 5 M. Yamaguchi and M. A. Joslyn, Plant Physiol. 26, 757 (1951); Arch. Biochem. and Biophys. 38, 451 (1952). s H. J. Loeffier and J. D. Ponting, Ind. Eng. Chem. Anal. Ed. 14~ 846 (1942). J. Kenyon and N. Munro, J. Chem. Soc. 158 (1948).

848

RESPIRA.TORY ENZYMES

[149]

Glutathione solution. Seven milligrams of pure reduced glutathione dissolved in 1 ml. of copper and iron-free redistilled water. 0.1 M phosphate buffer, pH 6.3 (SCrensen). 2,6-Dichlorophenolindophenol solution. Thirteen milligrams of pure reagent freshly dissolved in 1 1. of redistilled water, standardized so that 1 ml. of 1% metaphosphorie acid and 9 ml. of dye will give a 15-second reading of about 30 on the Evelyn photoelectric colorimeter with a 520"mt~ filter. 6 % metaphosphoric acid. Sixty grams of metaphosphoric acid dissolved in 900 ml. of water and diluted to 1 1. Store at 0 °, and dilute to 1% before use. 1% oxalic acid. Oxygen-free nitrogen. Procedure. Pipet 1 ml. of enzyme solution and 7 ml. of phosphate buffer into the main part of the Tam and Wilson 8 modified Thunberg tube, or 8 ml. of tissue extract at pH 6.3 (equivalent to 1 g. of tissue). Pipet 1 ml. of dehydroascorbic acid solution into the side arm. Pipet 1 ml. of glutathione solution into a glass tube, 2 mm. diameter X 25 mm., small enough to slide down the side arm; place some lanolin on the rim of this tube so that the solutions do not mix when the tube is inserted into the side arm or splash out during evacuation. Evacuate the Thunberg tube by oil pump, controlling foaming during evacuation. After the first evacuation relieve with oxygen-free nitrogen and evacuate again. Repeat the evacuation twice. Close the side arm, place the tube in constant temperature bath, and allow 15 minutes to reach bath temperature. After the tube and contents are brought to temperature, start the reaction by mixing the contents of the tube. Stop the reaction after 10 minutes at 25 ° or 5 minutes at 40 ° by quickly adding 40 ml. of 1% metaphosphoric acid (40 ml. of 1% oxalic acid may be used also, but this is not so satisfactory for crude preparations of enzyme or for tissue extract). Determine the amount of ascorbic acid present by pipetting 1 ml. of the resulting solution into an Evelyn colorimeter tube, adding 9 ml. of indophenol, and measuring the galvanometer deflection 15 seconds after mixing. Correct for nonenzymatic reduction of DHA by GSH at the same time by running a control Thunberg tube without added enzyme. The quantity of DHA reduced is proportional to time for the first 10 minutes at 25 °. The nonenzymatic reduction is appreciable at pH 6.9 [optimal for reduction by the enzyme in pea tissue (Joslyn and Yamaguchi~); Bukin 3 reported somewhat lower optimal pH for cabbage juice (6.7 to 6.8); and Crook and Morgan ~ also found this to be optimal for

8 R. K. Tam and P. W. Wilson, J. Bacteriol. 41, 529 (1941).

[149]

D E H Y D R O A S C O R BREDUCTASE IC

849

cauliflower preparations], but it is negligible at pH 6.3. For plant tissue juices and enzyme preparations of low activity, pH 6.3 is preferable to pH 6.8 for higher accuracy. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which causes the reduction of 1 mg. of dehydroascorbie acid in 10 minutes at 25 °. Specific activity is expressed as units per milligram of protein or per gram of dry weight of tissue. Enzyme activity is determined at pH 6.3 instead of the optimal pH of 6.9 used for purified enzyme to reduce correction for nonenzymatic reduction and at 25 ° instead of the optimal temperature of 40 ° because of rapid inactivation at 40 ° or above. The type and concentration of buffer markedly influence enzyme activity. "In pressed pea juice, activity was higher in orthophosphate buffer than in metaphosphate, citrate, or acetate buffer of the same concentration (0.1 M) at pH 6.3. This was also true for the purified enzyme. With the SOrensen orthophosphate buffer, increasing the concentration of phosphate from 10-4 M to 1 M resulted in increase in activity up to sharp optimum at 0.4 M. Metallic ions at low concentration (10-5 to 10-3 M) had no effect on activity but at 10-3 M and above produced appreciable inhibition (particularly true of Cu++). Purification Procedure The reductase is not widely distributed in plant tissue and is most active in the Cruciferae and Leguminosae. In the developing pea plant the enzyme is most active in the immature pea seed and in leaflets and stlpules, but activity in the petioles and tendrils and in stems and roots is low. As the pea seeds mature, the enzyme activity decreases; the activity in dry pea seeds is less than one-fourth that in the immature peas on~both the dry weight and protein basis. In the pods the activity decreases by about 50% on dry weight basis but remains constant on the protein basis. Plant tissue iuice is prepared by freezing the tissue with solid C02, macerating, and pressing at about 0 ° in a laboratory hydraulic press. To 100 ml. of the pressed juice at 0 °, add very slowly and with constant stirring 100 ml. of saturated (NH~)2S04, prepared by the method of Wildman and Bonner, 9 "adjusted to pH 6.8, and cooled to 0% Separate by filtration the inactive sediment formed on standing for 5 minutes, and discard. To 192 ml. of the resulting filtrate, add 288 ml. of cold saturated (NH4) 2SO4 to bring the solution to 80 % saturation. Separate the precipitate by centrifugation, redissolve in a small amount of 0.1 M phosphate buffer, and dialyze against phosphate buffer in a cellophane bag at 0 ° 9 S. G. Wfldman and J. Bonner, Arch. Biochem. 14, 381 (1947).

850

RESPIRATORY ENZYMES

[149]

for 8 hours. After this preliminary dialysis reprecipitate as above and dialyze again. Prolonged dialysis, particularly against water, decreases activity of enzyme. The final dialyzed preparation is preserved by lyophilization. The lyophilized preparation had an activity on a protein N basis over ten times that of the pressed juice. Tissue preparations are easily made by freezing the tissue, macerating in mortar, and expressing in a laboratory hydraulic press at 5000 p.s.i. This juice has reductase activity of that obtained from tissue ground at 0 ° in a Potter-Elvehjem 1° homogenizer. After centrifugation at 0 ° and filtration with diatomaceous filter aid, the pressed juice may be preserved by freezing and storage at - 1 0 to - 2 0 ° for several weeks with little loss in activity.

Properties The partially purified enzyme is more specific for the GSH component than the ascorbic acid component. Bukin a reported that thiolactic and thioglycolic acids could not replace GSH for the reductase in white cabbage. Crook 1 reported reduction of DHA by cysteine and thiolactic acid by the reductase in cauliflower. Yamaguchi and Joslyn ~ found that with cysteine and thioglycolate as hydrogen donors the nonenzymatic reduction was too rapid to permit accurate measurement of enzymatic reduction. Dehydro-D-araboascorbic acid and reductic acid (1,2,3-triketocyclopentane) are reduced but not so rapidly as DHA. The loss in activity of the reductase during dialysis, particularly against distilled water, was not due to loss of metallic catalysts (Mn ++ or Mg ++) or heat-stable coenzyme. The addition of Mn ++, Mg ++, or boiled juice did not restore activity. Addition of dihydrocozymase (coenzyme I) was without effect, contrary to results obtained by Bukin. 3 Optimal conditions for reduction of DHA for reductase in pea seeds were pH 6.9, 40 °, and D H A / G S H 2:1 (molar ratio). At constant DHA concentration, the reduction increased with increase in GSH at first rapidly up to 7 mg. per 10 ml., then more slowly. At constant GSH, the reduction of DHA increased rapidly up to 2 mg. per 10 ml. and then gradually decreased. At a D H A / G S H ratio of 2:1 (2:7 on weight basis), the quantity of ascorbic acid formed increased linearly with time at pH 6.3 and 25 ° for the first 10 minutes and then decreased. The reaction apparently was of zero order at first but became more complex later. 10 V. R. P o t t e r a n d C. A. E1vehjem, J. Biol. Chem. 114, 495 (1936).

[150]

CYPRIDINA AND FIREFLY LUCIFERASE

851

[150] Cypridina and Firefly Luciferase Cypridina (C. hilgendorfii) - LH2 + O2 ~ Light Firefly (Photinus pyralis) - LH2 + O2 + Mg++-+ ATP --~ Light B y WILLIAM D. MCELROY

Assay Method Luciferase is the enzyme which catalyzes the oxidation of luciferin (LH2) in the presence of molecular oxygen to yield visible radiation and unknown products. 1 Luciferin and luciferase differ, depending on the organism used. It is necessary, therefore, to specify the source of both substances. Since the intensity of the light emitted depends on both enzyme and substrate concentration, the quantitative determination of these substances can be made by measuring the light intensity by a suitable photocell arrangement. The Farrand photofluorometer is satisfactory. Qualitative estimations can be made with the naked eye. With the purified enzymes described below, light can be obtained when these are diluted l: 1000, which is still visible to the dark-adapted eye. Procedure for Cypridina. Dilute the stock enzyme 1:100 with 0.06 M KH2PQ'Na2HP04 buffer, pH 7.0, containing 0.01 M NaC1. Immediately prior to testing the luciferase preparation, 0.10 ml. of stock luciferin is placed in 10 ml. of phosphate-NaCl buffer in the reaction vessel of the light-measuring apparatus. Samples of the diluted enzyme are then added with sufficient buffer so that the final volume of the reaction mixture is 20 ml. Light emission is recorded until the reaction is complete. The firstorder velocity constants, k, are obtained from a semilog plot. Procedure for Firefly. The reaction mixture consists of 0.1 ml. of luciferase, 1 ml. of luciferin, 1 ml. of 0.01 M MgS04, and 1 ml. of 0.004 M ATP made up to a volume of 10 ml. with 0.05 M glycine buffer, pH 8.0. The reaction is initiated by adding ATP and recording the initial maximum light intensity, the latter being proportional to the enzyme concentration. Definition of Unit. One unit of enzyme is-defined as that amount which gives 1 unit of light in arbitrary values under the above stated conditions. Specific activity is expressed either as light units or as rate per milligram of protein. Protein is determined by the method of Lowry et al. 2 1 E. N. Harvey, "Bioluminescence." Academic Press, New York, 1952. -~O. H. Lowry, N. 0. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

852

RESPIRATORY ENZYMES

[150]

Cypridina Luciferase Purification Procedure

The following procedures are based on the original report of McElroy and Chase. 3 A crude active luciferase can be obtained from air-dried Cypridina by grinding the organisms first in a mortar and then in a Ten Brock tissue grinder. Starting with 20 g. of dried material, the first extraction is made with 100 ml. of ice-cold distilled water. After centrifugation at 3000 r.p.m, in an International refrigerated centrifuge for 15 minutes, the residue is re-extracted with an additional 50 ml. of water. The supernatants are combined and cooled to 10 °. The pH is rapidly adjusted to 4.5 with 1 N HC1, and the resulting precipitate is removed by centrifugation in the cold. After adjustment of the pH of the supernatant with NaOH to 7.5, cold acetone is added to 35 % saturation by volume. The temperature is kept between 0 and - 2 ° during this procedure. After 15 minutes at - 2 ° the precipitate is removed by centrifugation and discarded. Additional cold acetone is added to the supernatant to raise the saturation by volume to 55%. The mixture is kept at - 2 ° for 30 minutes. The resulting precipitate is removed by centrifugation and dissolved in 60 ml. of water (fraction 1). The pH is adjusted to 7.5, and solid (NH4)2S04 is added to 40% saturation. The pH is maintained at 7.5 with NaOH. After cooling at 0 ° for 15 minutes, the precipitate is removed by centrifugation and discarded. Solid (NH4)2SO4 is added to raise the per cent saturation to 65, while the pH is maintained at 7.5. After 15 minutes at 0 ° the precipitate is removed and dissolved in 15 ml. of water (fraction 2). Further purification is achieved by adsorbing onto and eluting from calcium phosphate gel. Five milliliters of calcium phosphate gel (1.6% by weight) is added to 15 ml. of fraction 2, and the pH is adjusted to 7.0. The mixture is stirred for 10 minutes at 5 °, after which the gel is removed by centrifugation in the cold. The pH of the supernatant is maintained at 7.0, and 60 ml. of calcium phosphate gel is added. After standing for 15 minutes, the gel is removed by centrifugation. All the luciferase is removed from the supernatant by the latter procedure. The enzyme is removed from the gel by washing three or four times with 25-ml. portions of 10% (NH4)2SO4 (pH 7.3). The combined eluates are again treated with (NH4)2S04, as described above, and the resulting precipitate, obtained with 40 to 65% (NH4)2SO4 saturation, is dissolved in 10 ml. of water (fraction 3). The activities of the various fractions are presented in Table I. 8 W. D. McElroy and A. M. Chase, J. Cellular Comp. Physiol. 38, 401 (1951).

[150]


853

Preparation of the Substrate--Luciferin. A crude Cypridina luciferin prepared b y making a fresh hot-water extract can be used in assaying for the enzyme. Since Cypridina luciferin is autoxidizable, it is necessary to make up a fresh solution each time. A stable preparation can be prepared, and this has been described in detail b y Anderson. 4 TABLE I PURIFICATION OF

Cypridina LUCIFERASE a Rate constant

Fraction

Protein, mg./ml, k/ml. k X dilution

Crude extract 14.0 Fraction 1 2.4 (0.35-0.65 saturated acetone) Fraction 2 1.1 (0.40-0.65 saturated (NH4)2S04) Fraction 3 0.08 (calcium phosphateoelution)

Specific activity, rate/mg. protein

Recovery, %

1.56 0.81

156 162

11 68

100 46

4.46

446

405

32

1.00

100

1250

4.0

The preparation of the various fractions and the method of enzyme assay are described in the text. The k values were obtained from a plot of log (a - x) vs. time. In all cases, except for fraction 1, the enzyme preparations were diluted 100 times for assay. Fraction 1 was diluted 200 times.

Properties Specificity. Cypridina luciferase is known to catalyze only the luminescent oxidation of Cypridina luciferin. I t does not catalyze the oxidation of luciferins from other sources. Activators. N o requirements for the usual inorganic ions have been observed for the purified enzyme preparations. M a x i m u m activity of the enzyme depends on the presence of a high salt concentration. M a x i m u m activity is obtained with 0.01 M NaC1 and 0.06 M phosphate. pH and Temperature Effects. The enzyme has a rather sharp p H optim u m at 7.0 and a temperature optimum at approximately 25 °. A t 50 ° the enzyme loses approximately 50% of the original activity in 2 hours. Chase 5 has studied the temperature inactivation in some detail. The purified enzyme is very stable when kept in the frozen state, very little loss of activity occurring in five months under these conditions. I t has also been kept at refrigerator temperature for at least four weeks without appreciable loss of activity. 4 R. S. Anderson, J. Gen. Physiol. 19, 301 (1935). 6 A. M. Chase, J. Gen. Physiol. 33, 535 (1950).

854

RESPIRATORY ENZYMES

[150]

Firefly Lucifeiase Purification Procedure The following procedure is based on the original report of McElroy and Coulombre. 8 Five grams of the dried lanterns of Photinus pyralis is ground with sand and extracted three times with a total volume of 100 ml. of H20. The pH of the extract is adjusted to 8 with NaOH, and the solution is placed in the deep-freeze. After freezing and thawing, the inactive precipitate is removed by centrifugation. Twenty-five milliliters of a calcium phosphate gel (16.7 mg./ml.) is centrifuged, and the supernatant discarded. The extract is then thoroughly mixed with the gel, and the pH adjusted to 8. After 15 minutes the mixture is centrifuged and the gel discarded. The supernatant (preparation 2) is considerably more active than the crude extract. Ninety milliliters of the calcium phosphate gel is centrifuged and subsequently mixed with 90 ml. of preparation 2. The pH is maintained at 8. After 15 minutes the mixture is centrifuged and the supernatant discarded. In this latter step most of the luciferase is adsorbed onto the gel while the majority of the luciferin remains in the supernatant. To remove the residual luciferin as well as inactive protein, the gel is washed twice with cold alkaline water and then with a 2 % solution of (NH4)2SO4 at pH 8. Elution of the enzyme is obtained by washing the gel twice with a 7% solution of (NH4)2SO~ at pH 8 (preparation 3). The final volume of combined eluates of preparation 3 is 95 ml. Preparation 3 is then fractionated with (NH4)2SO4 in successive steps of 10% saturation up to 50% saturation and then in units of 2 to 3% saturation up to 65 %. The pH during this procedure is maintained at 8.0. The major part of the active enzyme is recovered between 57 and 65% (NH4)2S04 saturation. The latter precipitate is dissolved in 25 ml. of water (preparation 4), and the enzyme is readsorbed onto calcium phosphate gel as described above. The supernatant is discarded. The enzyme is eluted with 7% (NH4)2SO4 at pH 8 (preparation 5) and precipitated by adding solid (NH4)2SO4 to 70% saturation (pH 8). The precipitate is dissolved in 5 ml. of H20, and the pH is adjusted to 8 (preparation 6). A further treatment of preparation 6 with the low concentration of calcium phosphate gel removes some inert protein (preparation 7). The activity of the various fractions is summarized in Table II. In this procedure the enzyme was purified approximately seventy times on a protein basis with a total recovery of 15 %. In addition, the preparation is completely free of luciferin, and under these conditions no light is emitted on the addition of ATP. Preparation of the Substrate--Luciferin. 6 Most of the firefly luciferin 6 W. D. McElroy and J. Coulombre, J. Cellular Comp. Physiol. 39, 475 (1952).

[150]


855

remains in the supernatant after the calcium phosphate gel treatment. The supernatant is adjusted to p H 3.5 and extracted two times with an equal volume of redistilled ethyl acetate. All the active luciferin passes into the ethyl acetate. The ethyl acetate is removed b y vacuum distillation, and the active luciferin is dissolved in a small volume of water. This crude preparation can be used for enzyme assay. F u r t h e r purification is TABLE II PURIFICATION OF FIREFLY LUCIFERASE

Preparation 1. 2. 3. 4. 5. 6. 7.

Crude extract Supernatant, first Ca3(PO,)~ gel Eluate, second Ca.~(PO4)2gel 57-65 % (NH4)2SO, precipitate Eluate, third Ca3(PO4)~gel 70 % (NH4)2S04 precipitate Supernatant, fourth Ca3(PO4)2 gel

Light units/ml., Protein, Specificactivity, volts mg./ml, volts/mg, protein 57 166 80 105 92 210 182

10.1 6.3 0.82 0.60 0.33 0. 627 0. 465

5.7 26 98 175 279

335 391

achieved by adsorbing the luciferin on an acid (2 N HC1)-treated Dowex 50 column (mesh size less than 80). The column is washed thoroughly with 2 N HC1 and finally water. The tuciferin is slowly developed on the column b y a weak solution of N H 4 0 H (1.5 %). The luciferin migrates down the column in a sharp band and is finally eluted. The luciferin can be readily followed on the column b y its brilliant yellow-green fluorescent band. The eluates containing the active luciferin are again extracted with ethyl acetate, and the latter is concentrated by vacuum distillation. The luciferin is finally concentrated in water. At neutral p H luciferin has two characteristic adsorption maxima, one major peak at 330 m~ and a secondary peak at 263 m~. The exciting wavelength for fluorescence corresponds to the adsorption peak at 330 m~. The concentrated luciferin is slightly yellow in alkaline solution b u t changes to a colorless solution in weak acid. In the former case the fluorescence on ultraviolet activation is an intense yellow-green, whereas in the latter case the fluorescence changes to a pale red. The luciferin can be maintained for several weeks without an appreciable loss of activity, either frozen in the aqueous solution or in the dried state. In aqueous solution at p H 3.5 and 100 ° complete inactivation occurs in 15 minutes and approximately 50 % loss of activity in 5 minutes. At p H 10 less than 5 % inactivation occurs in 20 minutes at 80 °. The inactive luciferin can be removed from the active b y extraction with ethyl acetate at p H 3.5. Under these conditions only the latter is removed from the aqueous phase.

856

RESPIRATORY ENZYMES

[151]

Properties Specificity. The only known function of firefly luciferase is that of catalyzing the luminescent oxidation of firefly luciferin. Oxidation of the latter depends, however, on the presence of ATP and Mg ++ ions. Activators and Inhibitors. Luciferin, Mg, and ATP are the only known requirements. Mn can replace Mg, but cobalt and iron are considerably less active. Calcium is inhibitory. No known phosphorylated compound will replace ATP. Among those which have been tested are ADP, UTP, ITP, CP, and AcP. 7 The enzyme is not affected by arsenate, cyanide, and azide, but it is strongly inhibited by pyrophosphate, various amines, copper, and p-chloromercuribenzoate, s Inhibition by the latter can be reversed by glutathione. Benzimidazole, benztriazole, and substituted derivatives inhibit the reaction by competing with luciferin (unpublished). pH and Temperature Effects. The pH optimum is at 7.8, and maximum light intensity is observed at 26 ° . The purified enzyme is stable for months in the frozen state but rapidly loses activity on repeated thawing and freezing. The enzyme is rapidly denatured by bubbling air through the solution or by exposing it to various surfaces such as shredded cellophane or glass beads. Activity is rapidly lost on dialysis and cannot be restored by the dialyzate or a crude boiled extract. Note added in proof: The firefly enzyme has recently been obtained in crystalline form (A. A. Green and W. D. McElroy, to be published). 7 W. D. McElroy, J. Biol. Chem. 191, 547 (1951). 8 W. D. McElroy, J. W. Hastings, J. Coulombre, and V. Sonnenfeld, Arch. Biochem. and Biophys. 46, 399 (1953).

[151]

BACTERIAL LUCIFERASE

857

[151] B a c t e r i a l L u c i f e r a s e O D P N H ~- F M N + RC

-{- Os -~ Light

\ H

or

O FMNH2 ~- RC

-[- O, --~ Light

\ H

By ARDA A. GREEN and WILLIA~ D. MCELROY Assay Method

Bacterial luciferase is a flavoprotein which catalyzes the oxidation of reduced D P N by numerous oxidants such as FMN, ferricyanide, quinones, and various dyes. In the presence of F M N and a long-chain fatty aldehyde the oxidation of D P N H is accompanied by light emission. ~Pyridine nucleotides are not essential for light emission, since chemically reduced F M N and aldehyde will support luminescence. The intensity of the light emitted depends on the concentration of these various components. Quantitative measurements can be made by a suitable photocell arrangement such as the Farrand photofluorometer. Qualitative estimations can be made with the naked eye. Procedure. The reaction mixture consists of 0.5 ml. of 0.1 M phosphate buffer, pH 6.8, 1.0 ml. of saturated dodecyl aldehyde, 0.2 ml. of riboflavin phosphate (2 X 10-4 M), 0.05 ml. of 1% bovine albumin, 0.2 ml. of D P N H (7.0 × 10-4 M for maximum luminescence), 0.05 or 0.1 ml. of diluted enzyme, and water to a total of 2.5 ml. The reaction is initiated with D P N H , and the light intensity recorded for various time periods. Definition of Unit. One unit of enzyme is defined as that amount which gives 1 unit of light in arbitrary values under the above stated conditions. Specific activity is expressed as light units per milligram of protein. Protein is determined by the method of Lowry et al3 Growth of the Organism. The salt-water bacterium Achromobacter fischeri was grown under forced aeration or by shaking in the following 1 W. D. McElroy and B. L. Strehler, Bacteriol Revs. 18, 177 (1954). 20. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193~ 265 (1951).

858

RESPIRATORY ENZYMES

[151]

medium: NaC1, 30 g.; Na2HP04, 5.3 g.; KH~P04, 2.1 g.; (NH4)~HPO4, 0.5 g.; MgSO4, 0.1 g.; glycerol, 3 ml.; peptone, 1 g.; and water, 1 1. The pH was adjusted to 7.1 to 7.3 with NaOH. At the peak of luminescence (15 to 20 hours) the cells were harvested by high-speed centrifugation. With good aeration approximately 4 g. (wet weight) of cells per liter is obtained. Purification Procedure

The following procedures are based in part on the original report of McElroy et al2 A crude bacterial extract is obtained by lysing the cells in distilled water (1 g. wet weight per 15 ml. of water). After thoroughly mixing in the cold, the debris and unlysed cells are removed by centrifugation at high speed in a Servall centrifuge. An active isoelectric precipitate is formed by adjusting the pH to 4.0 to 4.3 with HC1. The precipitate is separated by centrifugation in the cold, then suspended in water (one-tenth the volume used for lysing) and dissolved by the addition of N/IO NaOH to pH 6.8 to 7.0. This materially increases the volume and brings the final protein concentration to between 15 and 20 mg./ml. The centrifuged supernatant of the lysed cells contains about one-half of the total protein in the cells, and 85 to 90% of this protein is found in the isoelectric precipitate. Thus, the isoelectric precipitation is a concentration rather than a purification procedure, except for the removal of certain inhibitors, especially riboflavin. These inhibitors make it impossible to properly assay the initial extract. Ammonium sulfate precipitation has proved to be the only practical method of purification. To the solution of the isoelectric precipitate, fraction A, is added 0.1 vol. of Na4P~07 (0.2 M adjusted to pH 7 with HC1). Solid ammonium sulfate is then added with stirring, and the final pH is adjusted to 6.8. All pH values are read on the Beckman glass electrode pH meter without correction for salt concentration. Successive precipitates are separated by centrifugation in the high-speed Servall centrifuge, dissolved in 0.02 M pyrophosphate, and the pH readjusted to 6.8. The initial fractionation procedure yields four fractions. Fraction B. The precipitate obtained at 1.35 M ammonium sulfate (0.33 saturation) is voluminous and contains about 70% of the total protein. It is washed once with 1/~ vol. of 1.35 M ammonium sulfate and the precipitate discarded, since it contains a negligible amount of activity. Fraction C. The combined supernatants from the previous step are brought to 2.05 M ammonium sulfate (0.50 saturation). The precipitate contains about 5 % of the protein and 5 % of the activity. 3 W. D. McElroy, J. W. Hastings, V. Sonnenfeld, and J. Coulombre, J. Bacteriol. 67, 402 (1954).

[151]

BACTERIAL LUCIFERASE

859

Fraction D. The supernatant from fraction C is brought to 2.67 M ammonium sulfate (0.65 saturation). This fraction contains 15 to 20% of the total protein and 90 % of the activity. In fact, it may assay substantially higher total activity than fraction A, owing to assay in a more concentrated solution and to the removal of inhibitors. Fraction E. A fraction at 2.87 M ammonium sulfate contains so little protein and so little activity that it is not worth taking. Fraction D thus contains essentially all the luciferase with a specific activity which may vary from 1200 to 3000 light units per milligram of protein but is about five times the specific activity of fraction A. The variation is due largely to the particular strain of bacterium used. Besides protein impurity this preparation contains large amounts of nucleic acid. Attempts to remove this by protamine sulfate or manganese ion were unsuccessful. The fractionation was followed by optical density measurements at 280 and 260 mu. It was found that the nucleic acid tended to precipitate in the higher ammonium sulfate fractions. Thus, after repeated fractionation the worst fractions are almost pure nucleic acid and the best have a "280 to 260 ratio" of about 1.2. Fraction D is repeatedly refractionated in ammonium sulfate. The point of firstprecipitation depends on the concentration of the proteins and the character of the protein impurities but usually occurs around 2 M ammonium sulfate. Small increments of the solid salt are added so that the protein is divided into convenient amounts. All fractions are analyzed for protein, enzymatic activity, and ultraviolet absorption. Similar fractions are combined and refractionated. The table presents the results from a single run including the isoelectric precipitate, A, the first active ammonium sulFRACTIONATION OF LUCIFERASE BY AMMONIUM SULFATE

Fraction A D Dl D~ D3 D4 D5 86

Ml.

Total L.U. ~

186 26 2.5 5 9 8 3

986,000 1,639,000 54,000 210,000 864,000 384,000 3,000

Total protein, E280 b E ..... mg. L . U . / m g . protein ~ E . . . . /1000 L. U. mtL 2880 670 58 101 230 182 28

341 2420 925 2080 3760 2100 107 5200

0.705 0.712 0.89 0.78 0.79 0.74 0.57 1.24

9.8 1.41 1.7 1.04 0.62 1.75 11.8 0.29

260 260 265 265 263 260 260 275

L. U. = light units as defined in text. b E = optical density determined in a Beckman D U spectrophotometer in a 1-cm. cell.

860

RESPIRATORY ENZYMES

[151]

fate precipitate, D, and a series, D1 to Ds, derived from D. If the per cent protein and activity of the subfractions of D are calculated with the values for D taken as 100 %, 90 % of the protein and 93 % of the activity are recovered. Further fractionation may be accompanied by loss of activity. We have tried stabilizing the enzymes by the addition of cysteine, FMN, or the aldehyde without marked success. In fact, cysteine and the aldehyde, in the presence of the enzyme, form an inhibitor. In the last row of the table are given the characteristics of one of the best fractions we have obtained. About 70 % of this protein is in a symmetrical peak on electrophoresis, and activity seems to be correlated with this peak. The sedimentation constant is consistent with a molecular weight of about 100,000. Properties

Stability. Bacterial luciferase slowly loses activity even in the frozen state. It is rapidly inactivated at temperatures above 40 °. The enzyme can be dialyzed in the cold without great loss of activity with 0.02 M pyrophosphate buffer and 2 X 10-5 M FMN. Various attempts to demonstrate a metal requirement by dialysis against metal-free buffers have been unsuccessful. Effect of Inhibitors. The enzyme is particularly sensitive to various SH reagents, p-Chloromercuribenzoic acid at 4 X 10-e M inhibits light emission approximately 50%. This inhibition can be reversed by glutathione. Riboflavin is a potent inhibitor of luminescence (2 X 10 -e M gives approximately 50 % inhibition). It appears to compete with FMN. Cyanide and Versene likewise inhibit, as does copper, iron, and other heavy metals. Various quinones and ferricyanide inhibit light emission by removing reduced DPN. As indicated below, this inhibition is due to the rapid reduction of these compounds by D P N H in the presence of bacterial luciferase. Reduction of Dyes and FMN. In the absence of aldehyde bacterial luciferase catalyzes the reduction of methylene blue, various quinones, ferricyanide, and F M N by D P N H without light emission. In the presence of aldehyde there is a competition between light emission and the reduction of the various compounds." F M N does not appear to be required for the reduction of quinones and ferricyanide. 4 Requirements for Light Emission. Bacterial luciferase catalyzes a lightemitting reaction in the presence of oxygen, reduced FMN, and a longchain aldehyde. There is an absolute requirement for all these compo4 W. D. McElroy and A. A. Green, Arch. Biochem. and Biophys. §6t 240 (1955).

[152]

ASSAY AND PROPERTIES OF HYDROGENASES

861

nents, and all are utilized during the reaction. Cormier and Strehler 5 have shown that a number of long-chain aldehydes will function in the reaction. Dodecyl or tetradecyl aldehyde are excellent substrates. With F M N various reducing agents will support light emission. Reduced safranin T, indigotrisulfonate, and rosindulin GG are all effective. Strehler et al. e reported that reduced riboflavin would support luminescence in crude extracts. Our studies with the purified enzyme indicate, however, that F M N is an absolute requirement. Reduced D P N and T P N are both effective reducing agents and appear to be the natural substrates for light emission. Chemically reduced F M N is the most effective substrate for light emission and the evidence shows quite clearly that pyridine nucleotides are not required for luminescence. Kinetics. The kinetics of light emission starting with reduced F M N indicate that two F1VINH~ molecules combine with luciferase. It seems likely that a complex interaction between oxygen, two reduced F M N molecules, and aldehyde is necessary for luminescence. The concentration of reduced F M N which gives approximately one-half maximum light intensity is 2.5 X 10-8 M. The relationship between light intensity and enzyme concentration is proportional when reduced F M N is used but is nonlinear when D P N H and F M N is used. This latter effect appears to be due to the autoxidation of reduced F M N which is formed from D P N H and FMN. pH and Temperature Optimum. Light emission has been observed over a pH range of 5.5 to 8.5, with a peak at 6.8. At the latter pH the temperature optimum is approximately 27 °, which is remarkably similar to that observed in the intact bacterium. 5 M. J. Cormier and B. L. Strehler, J. Am. Chem. Soc. 75, 4864 (1953). 6 B. L. Strehler, E. N. Harvey, J. J. Cheng, and M. C. Cormier, Proc. Natl. Acad. Sci.

U.S. 40, 10 (1954).

[152] Assay and Properties of Hydrogenases By ANTHONY SAN PIETRO

Hydrogenase activity may be defined as the enzymatic activation of molecular hydrogen. The main methods which have been employed to assay hydrogenase activity, in cell-free preparations, are as follows: (a) The reduction of some substrate by molecular hydrogen. Various acceptors which have been used in this assay system include methylene

862

RESPIRATORY ENZYMES

[152]

blue 1 and other dyes, ~ ferricyanide, I,~ nitrate, 4,5 fumarate, 5 oxygen; 6 etc. (see also Vol. I I [129]). (b) The exchange reaction between molecular hydrogen and h e a v y water or between deuterium gas and normal water. 8,6-9 (c) The ortho-para hydrogen conversion reaction. 8 (d) Evolution of molecular hydrogen from a variety of substrates.m0.li The ability to catalyze any one of the above reactions has been used to demonstrate hydrogenase activity. However, catalysis of the exchange reaction has been generally assumed to be a fundamental p r o p e r t y of hydrogenase and is perhaps the most ideal assay system. 3,6,7,12 As noted b y Fisher et al. 6 this assay circumvents any difficulties connected with the activation of the reducible substance and directly measures the primary reaction catalyzed by hydrogenase. Unfortunately, the exchange reaction has not found widespread application probably owing to the lack of the necessary facilities. Most investigators have used the reduction of various acceptors b y molecular hydrogen as an assay for hydrogenase. As pointed out b y H y n d m a n et al. ~ interpretation of results from these studies is handicapped b y the necessity for intermediate electron carriers or additional enzymes for activation of the acceptors. The present chapter is concerned primarily with a description of those assay systems which make use of molecular hydrogen as substrate (methods (a), (b), and (c) above). For purposes of comparison, it will be assumed t h a t the source of the enzyme is Proteus vulgaris for each of the assays described.

Assay Methods 1. Exchange Reaction. s,8 P r i n c i p l e . The exchange reaction between molecular hydrogen and h e a v y water (H2 ~ H D O ~ H D W H20) results in the appearance of deuterium in the gas phase. The rate of the i H. Gest, J. Bacteriol. 63, 111 (1952). 2 W. K. Joklik, Australian J. Exptl. Biol. and Med. Sci. 28, 321 (1950). 8L. A. Hyndman, R. H. Burris, and P. W. Wilson, J. Bacteriol. 65, 522 (1953). 4 A. I. Krasna and D. Rittenberg, J. Bacteriol. 68, 53 (1954). H. Gest, in "Phosphorous Metabolism" (W. D. MeElroy and B. Glass, eds.), Vol. II, p. 522, The Johns Hopkins Press, Baltimore, Md., 1952. H. F. Fisher, A. I. Krasna, and D. Rittenberg, J. Biol. Chem. 209, 569 (1954). 7 W. Curtis and E. J. Ordal, J. Bacteriol. 68, 351 (1954). 8 A. I. Krasna and D. Rittenberg, J. Am. Chem. Soc. 76, 3015 (1954). 9 A. I. Krasna and D. Rittenberg, Proc. Natl. Acad. Sci. (U.S.) 40~ 225 (1954). 10It. I). Peck and H. Gest, Bacteriol. Proc. p. 117 (1955). n H. Gest, Bacteriol. Revs. 18~ 43 (1954). 1~L. Farkas and E. Fischer, J. Biol. Chem. 167, 787 (1947).

[152]


863

reaction is followed b y removing samples of gas at various time intervals and analyzing for deuterium with a mass spectrometer. 13

Reagents 0.15 M phosphate buffer, p H 6.7. 99.5 per cent D20.14 Na2S204. Hydrogen gas rendered O~-free by passage over hot copper. Enzyme. Use an a m o u n t of enzyme to give a first order rate constant, k, of 1 X 10-3 per minute or a change in the deuterium concentration of the gas phase of 0.2 atom per cent excess per hour. 1~ For calculation of k see Definition of Activity below.

Procedure. The reaction is carried out in 30-ml. flasks fitted with a stopcock for evacuation, as illustrated in Fig. 2 of the paper by H o b e r m a n and Rittenberg. 16 Each flask contains enzyme (e.g. 0.5 ml. containing 1 mg. of total N), 0.5 ml. of 99.5% D20 and 0.15 M phosphate buffer, pH 6.7, to give a final volume of 5 ml. After cooling in ice for 10 minutes, approximately 1 mg. of solid Na2S204 is added 17 and the flasks are immediately evacuated b y a mechanical pump for an additional 10 minutes. The flasks are filled to approximately 40 cm. pressure with hydrogen, reevacuated, and refilled with hydrogen to between 30 and 60 cm. pressure. The flasks are shaken at room temperature on a r o t a r y shaker at 100 r.p.m, and aliquots of gas removed at intervals for analysis. Definition of Activity. The activity is defined in one of two ways; either as the value of dC/dt, in which C is the deuterium concentration in atom per cent excess of the gas phase and t is in minutes or as the value of the first order rate constant, k. This latter value is calculated from the slope of the curve of log (Coo - C) against time where C and C~¢ are the atom per cent excess deuterium in the gas phase at time t and at equilibrium, 18 See Vol. IV [21]. 14 H e a v y water m a y be obtained on allocation from the Atomic Energy Commission a n d purchased from the Stuart Oxygen Company, San Francisco, California. ~ The exchange reaction a n d the ortho-para hydrogen conversion reaction involve (1) solution of hydrogen in the medium a n d (2) activ~,tion of the dissolved hydrogen by the enzyme. At low enzyme concentration, the rate is proportional to the enzyme concentration a n d the rate limiting step is the activation of the dissolved hydrogen by the enzyme. At high enzyme concentration, the reverse is true, t h a t is, the rate is i n d e p e n d e n t of the enzyme concentration a n d the rate limiting step is the solution of hydrogen in the medium. Under the conditions described herein, the rate of the reaction is proportional to the enzyme concentration. ~6 H. H o b e r m a n a n d D. Rittenberg, J. Biol. Chem. 147, 211 (1943). 17 The a m o u n t of hydrosulfite necessary to activate a particular cell-free system seems to be dependent solely on the a m o u n t of oxygen dissolved in the water. 6

864

RESPIRATORY ENZYMES

[152]

respectively, and t is in minutes. The equilibrium value C~ found after 24 hours is 3.30 atom per cent excess deuterium and is identical to t h a t resulting from catalysis b y active platinum. Specific activity m a y be defined as the activity per rag. of total N. 2. Conversion Reaction. 8 Principle. The conversion reaction is defined as the reaction which converts parahydrogen to normal hydrogen. is The rate of the conversion reaction is determined b y measuring the concentration of parahydrogen in the gas phase at various time intervals according to the thermal conductivity m e t h o d of Bonhoeffer and Harteck. 19,2° This m e t h o d is based on the different thermal conductivities of the hydrogen modifications and involves the measurement of the resistance of a thermal conductivity cell containing the gas sample to be analyzed.

Reagents 0.15 M phosphate buffer, p H 6.7. Na2S20~. 50 to 60 per cent parahydrogen m a y be prepared b y adsorbing purified hydrogen on a degassed coconut charcoal catalyst at 60 to 70 ° K. TM The time necessary to establish spin equilibrium is dependent on the activity of the charcoal used; at the end of this time, the gas is desorbed. Enzyme. Use an a m o u n t of enzyme to give a first order rate constant, k', of 2 X 10 -3 per minute. ~5For calculation of k' see p. 865.

Procedure. The reaction is carried out in the same flasks as for the exchange reaction. ~s The procedure is identical to t h a t used for the exchange reaction with the exceptions t h a t no h e a v y water is added and the gas phase is parahydrogen. T h e flasks are shaken as previously described. Samples of gas are removed at various time intervals and analyzed for the concentration of parahydrogen in the following manner. The thermal conductivity cell is immersed in liquid nitrogen (77 ° K.) and its filament heated b y a direct current. The sample to be analyzed is introduced into the conductivity cell and the pressure adjusted to 30 ram. The resistance of the cell is measured b y making it one leg of a Wheatstone bridge. The bridge is balanced b y a slide wire resistance. is The term normal hydrogen denotes the equilibrium mixture at room temperature of ortho- and parahydrogen (25 per cent para and 75 per cent ortho). The term parahydrogen is used for hydrogen containing more than 25 per cent parahydrogen. A discussion of the various nuclear spin isomers of hydrogen can be found in the book by Farkas. 19 ~gA. Farkas, "Orthohydrogen, Parahydrogen and Heavy Hydrogen," Cambridge University Press, New York, 1935. s0 K. F. Bonhoeffer and P. Harteek, Z. physik. Chem. B4, 113 (1929).

[152]


865

The reading of the slide wire, R, is proportional to the concentration of parahydrogen. Definition of Activity. The activity is defined as the value of the first order rate constant, k'. The value of this constant is calculated from the slope of the curve of log AR versus time, in minutes, where AR is the difference between the value of R when the conductivity cell is filled with the gas sample and when filled with normal hydrogen. Specific activity may be defined as the activity per rag. of total N. 3. Reduction of Methylene Blue. Principle. The method is based on the measurement of hydrogen uptake manometrically in the presence of methylene blue as the hydrogen acceptor.

Reagents 0.15 M phosphate buffer, pH 6.7. 0.05 M methylene blue solution. Fieser's solution. 21 Dissolve 20 g. of KOH in 100 ml. of water and add 2 g. of sodium anthraquinone-f~-sulfonate and 15 g. of Na2S204 to the warm solution with stirring until dissolved. The blood red solution is ready for use when it has cooled to room temperature. Hydrogen gas which has been purified by passage over hot copper to remove oxygen. Enzyme. Use an amount of enzyme to give a hydrogen uptake of about 500 ~l. per hour.

Procedure. Each Warburg cup contains enzyme (e.g. 0.25 ml. containing 0.5 rag. of total N) and 0.15 M phosphate buffer, pH 6.7, in the main compartment, 0.2 ml. of methylene blue in the side arm, and 0.2 ml. of Fieser's solution in the center well with filter paper, total liquid volume 3.0 ml. The vessels are flushed with hydrogen for 5 minutes, closed and equilibrated for 1 hour. After equilibration, the reaction is started by tipping in the methylene blue from the side arm. Definition of Specific Activity. The specific activity, QH,(N), is generally defined as the rate of hydrogen uptake, in td. per hour, per rag. of total N. Preparation and Properties ~2,2~

1. Proteus vulgaris. 4,6 Growth Medium. Proteus vulgaris is grown at 37 ° for twelve to twenty hours in 15 liters of broth of the following 21 A. I. Vogel, " A Textbook of Practical Organic Chemistry," Longmans, Green and Co., New York, 1948. 22 See Vol. II [129] for description of hydrogenase of Clostridium kluyveri. 28 The oxidation and evolution of molecular hydrogen by microorganisms has recently been reviewed by Gest. 11

866

RESPIRATORr ENZYMES

[152]

composition: Na2HPO4, 7.93 g. ; KH~PO4, 1.47 g. ; glucose, 5.0 g.; nutrient broth (Difco), 10.0 g.; NaC1, 5.0 g.; casamino acids (Difco), 5.0 g.; FeSO4.7H20, 0.005 g.; and H20, 1 liter. The bacteria are harvested by centrifugation in a Sharples supercentrifuge and washed twice with water. The cell paste may be stored for several weeks in the cold without appreciable deterioration. Preparation of Cell-free Extract. 6 g. of the bacterial paste is diluted to 25 ml. with water and treated for 20 minutes in a Raytheon 9 kc. oscillator. The cell debris is removed by centrifugation at 13--20,000 )< g. The extract is generally stored in the cold under hydrogen. The activity appears to reside in the particulate elements of the cell and may be sedimented by centrifugation at higher speeds (Krasna and Rittenberg, personal communication). Nitrogen values, as determined by the Kjeldahl procedure, are in the range of 3 to 4 mg. of total N per ml. Properties. The properties described below are based on experiments with intact cells (prior to 1954) and with cell-free extracts (1954 et seq.). It has been suggested 16 that the enzyme is an iron-porphyrin-protein complex which is active only in the reduced (ferrous) form. The evidence presented in support of this conclusion is: (a) The active enzyme is inhibited by carbon monoxide and the inhibition is partially reversed by light. With equal volumes of hydrogen and carbon monoxide, 80% inhibition occurs in the dark and 55% in the presence of light; (b) The exchange activity is depressed when the cells are preincubated in the presence of oxygen. Complete inactivation occurs when the bacterial suspension is shaken with oxygen for 24 hours. This inactivation by oxygen is reversible and the enzyme can be reactivated by incubation under hydrogen or by the addition of glucose, pyruvate, formate, succinate, 24 fumarate or sodium hydrosulfite; (c) The addition of cyanide anaerobically to the actively exchanging system in 10-2 M concentration is without effect. When cyanide is added to the system under aerobic conditions in 10-3 M concentration, the enzyme is completely and irreversibly inactivated. Oxygen inhibits hydrogenase by two different mechanisms; namely, oxidation and oxygenation. 6 The oxygenated enzyme is inactive and can be reactivated by any process which removes oxygen; for example, addition of hydrosulfite, 17 or glucose oxidase and glucose, or physically by degassing. 6 Hydrosulfite is to be preferred since it cannot only deoxygenate the enzyme, but also reduce it. Neither thioglycolate nor hypophosphite at a concentration of 10-2 M causes any appreciable activation of the oxygenated enzyme. 8 ~In the experiments of Farkas and Fischer,1~ the addition of succinate had no restoring effect on the partially inactivated system.

[152]

ASSAY AND P R O P E R T I E S OF HYDROGENASES

867

Heavy suspensions of P. vulgaris fail to reduce methylene blue under hydrogen when the system is made 2% with urethane; whereas the exchange reaction is not inhibited under these conditions.18 When added to the exchanging system (which is operating under anaerobic conditions), the following compounds are without effect on the rate of the exchange reaction: 1% fluoride, 2% urethane, l0 -3 M iodoacetate, 10-2 M K2S2Os and 0.08 M malonate.16 Aerobic incubation with Ag+ results in complete inhibition of the exchange reaction at a concentration of 10-2 M, 85% inhibition at 10-3 M and is without effect at 10-4 M.IG The pH optimum for both the exchange reaction 16 and the reduction of nitrate by molecular hydrogen 4 is approximately 7. For the reduction of nitrate by molecular hydrogen, the system is saturated at a concentration of 0.02 % nitrate. The reduction of one mole of nitrate is accompanied by the uptake of close to one mole of hydrogen. Nitrite is not reduced further by this system in the presence or absence of benzyl viologen. Both nitrate and nitrite inhibit the deuterium exchange reaction. 4 In each case, the inhibition is 27% with an inhibitor concentration of 10-~ 31. The rate of hydrogen oxidation with fumarate is increased by the addition of fumarate to the growth medium. 1~ However, the level of the exchange activity is unaffected by the presence or absence of fumarate during growth. The rate of the exchange reaction is retarded by the addition of fumarate. The effect is related to the fumarate reduction activity of the cells; the higher the hydrogenation activity toward fumarate, the lower is the rate of exchange in the presence of fumarate. The enzyme is completely inhibited by high concentrations of nitric oxide. 9 At nitric oxide concentrations of 2 × 10-3, 8 × 10-3 and 1 × 10-1 vol. per cent in the gas phase, the per cent inhibition is 87, 93 and 97, respectively. With 1 vol. per cent or more, the inhibition is not reversible even by the addition of sodium hydrosulfite. With inhibitor concentrations of less than 1 vol. per cent, the inhibition could be partly reversed by removal of the nitric oxide-hydrogen mixture and its replacement with hydrogen. Nitrous oxide is not an inhibitor of hydrogenase. 2. Escherichia coli. The preparation described is essentially that reported by Gest. Growth Medium. E. coli, strain B, is grown in deep stationary culture for 8 to 24 hours at 37 ° in a medium of the following composition: Difco peptone, 5 g. ; glucose, 10 g. ; Difco beef extract, 3 g. ; NaC1, 5 g. ; distilled water, 1 liter; pH adjusted to 7.5. Preparation. 12 g. (wet weight) of 8-hour-old cells are washed with

868

RESPIRATORY ENZYMES

[152]

25 ml. of water and ground in a mortar by hand with 30 g. of alumina (Alcoa A-303). After extracting the paste for 20 minutes with 60 ml. of water and removing the alumina, etc., by centrifugation, the brown colored extract is treated with ~ 0 vol. of 1 M MnC12 which precipitates a considerable amount of nucleoprotein. The precipitate is removed and the hydrogenase precipitated by 50% saturation with (NH,)2SO4. This precipitate is dissolved in water and the residual insoluble particles removed by centrifugation. The highest specific activity reported, at this stage of purification, is 30,000 ~l. hydrogen per hour per mg. protein N at 37 ° with methylene blue as the acceptor. The enzyme is generally stored at 5 ° under hydrogen. Properties. The activity of the cell-free enzyme is markedly depressed by oxygen and this inactivation is partially reversed by prolonged incubation with H2.1,~,25 The ability of various reducing agents to reactivate the enzyme varies with the " n a t u r e " of the enzyme preparation. Ferrous sulfate, thioglycolic acid, GSH, ammonium polysulfide and sodium hydrosulfide, individually, at a concentration of 10-3 M, rapidly activates the ammonium sulfate-precipitated enzyme to the same degree as prolonged exposure to H2. 2 With the pre-aerated crude extract, incubation under hydrogen for only 5 minutes in the presence of Na2S204 causes a comparable stimulation; however, reducing agents other than Na2S204 are without effect under these conditions. 2 Cyanide at a concentration of 10-2 M inhibits aerated preparations about 9 % whereas it causes a 47 % inhibition after two hours anaerobic incubation. 28 Carbon monoxide inhibits the enzyme and the inhibition is not reversible by light. 36 A somewhat constant inhibition of about 70 % is obtained when the carbon monoxide/hydrogen ratio is varied from 9:1 to 1 : 1. 38 The above-mentioned results with cyanide and carbon monoxide are different from those found for Proteus vulgaris. In contrast to the particulate preparations from anaerobically grown cells, the soluble enzyme reduces methylene blue (and a few oxidationreduction indicators of the type of methylene blue) but shows no activity with nitrate, fumarate, ferricyanide, or oxygen. 2,5 Cell-free preparations which show high activities with methylene blue do not catalyze the reduction of cytochrome c or pyridine nucleotides ~,2 (cf. Vol. II [129]). The enzyme is not inhibited by 1,10-phenanthroline, a,a'-dipyridyl, or diethyldithiocarbamate in concentrations up to 0.01 M. The hydrogenase of E . coli shows almost maximal activity over a broad pH range (5.5-8.5). 3 ~5K. J. C. Back, J. Lascelles, and J. L. Still, Australian J. Sci. Research 9, 25 (1946). ~ W. K. Joklik, Australian J. Exptl. Biol. and Med. ,.~ci. 28, 332 (1950).

[152]


869

3. Desulfovibrio desul]uricans. This organism, which exhibits unusually high hydrogenase activity, has been used by Sadana and Jagannathan ~7as starting material for the preparation of the most active hydrogenase fraction described to date. Preparation. 27 "Hydrogenase was extracted from acetone-dried bacteria (specific activity 5 X 104 td. per hour per mg. N) with 0.2 M phosphate buffer, pH 6.4, and centrifuged at 18,000 × g for 2 hours. The supernatant was heated at 60 ° for 10 minutes and centrifuged to remove the denatured proteins. The supernatant, which contains hydrogenase, was then adjusted to pH 4.5 and the small precipitate formed was removed by centrifugation." Hydrogenase was then precipitated by adjustment of the pH to 4.0 and redissolved in 0.1 M Tris buffer at pH 7.0. The solution was adjusted to pH 5.0 with acetic acid, heated at 50 ° for 2 minutes, and centrifuged to give a water clear pale pink solution.28 The recovery was about 90% and the enzyme had a specific activity of 2.5 X 108 ~l. per hour per mg. N at 34 ° with methylene blue as the acceptor. Properties. Like other hydrogenase preparations, this enzyme is sensitive to oxygen and activated by sulfhydryl compounds. The purified enzyme could be stored in the frozen state without loss of activity. It could be precipitated by (NH4)2S04 between 70 and 100% saturation at pH 5.5 and was not sedimented by centrifugation for one hour at 80,000 X g. The activity with riboflavin-5'-phosphate as hydrogen acceptor was less than 5 % of that obtained with methylene blue. 4. Clostridium pasteurianum. 29 Preparation. Cell-free extracts are prepared by subjecting the organism to sonic vibrations. The purification of the enzyme involves the following procedures: separation of the particulate fraction by centrifugation, removal of impurities with protamine sulfate, followed by zinc hydroxide gel and finally fractional precipitation with (NH4)2S04. Properties. The enzyme is not sedimented by centrifugation at 144,000 X g for 30 minutes and has a specific activity of 7.5 X 105 ~I. per hour per mg. protein N with methylene blue as the acceptor. Treatment of the enzyme with ammonium sulfate, followed by dialysis, reduces the activity with either methylene blue or cytochrome c to a very low level. The activity with methylene blue can be restored by the addition of FAD; it is necessary to add both FAD and Mo to restore cytochrome c activity. The presence of phosphate is required only for the metal catalyzed reaction. 27 j . C. Sadana a n d V. J a g a n n a t h a n , Biochim. et Biophys. Acta 14, 287 (1954). 28 This information was kindly furnished b y Dr. Sadana. 22 A. L. Shug, P. W. Wilson, D. E. Green, a n d It. R. Mahler, J. Am. Chem. Soc. 76, 3355 (1954).

870

RESPIRATORY ENZYMES

[152]

T h e difference spectrum of the purified enzyme reveals maxima at 390 m~ and 450 m~ and resembles t h a t of riboflavin. F A D in boiled extracts is demonstrable b y spectroscopic and enzymatic tests. Participation of the flavin in the action of the enzyme is indicated b y the negligible difference in spectrum between the "oxidized e n z y m e " and the " r e d u c e d e n z y m e " in an evacuated cuvette. This observation is interpreted to mean t h a t the interaction of hydrogen with flavin is reversible; at low pressures of hydrogen reduced flavin is oxidized to H2 gas and oxidized flavin. 5. Other Sources. Hydrogenase has been reported in cell-free preparation from a variety of other bacteria; A zotobaeter vinelandii,3 A zolobaeler agile, 3° Micrococeus aerogenes, 7 Rhodospirillum rubrum, l Hydrogenomonas ruhlandii, 3~ Micrococcus lactilyticus, 32 and Hydrogenomonas facilis. ~3 Addendum: Sadana and J a g a n n a t h a n (Biochim. et Biophys. Acta, in press) have been able to demonstrate an activation of the hydrogenase of Desulfovibrio desulfuricans by ferrous chloride, with an enzyme purified by a modified procedure, which includes acetone precipitation and adsorption on calcium phosphate gel. T h e y have found in addition t h a t the enzyme is cyanide-sensitive. 2s

a0 M. Green and P. W. Wilson, J. Bacteriol. 65, 511 (1953). a~L. Packer and W. Vishniac, Biochim. et Biophys. Acta 17~ 153 (1955). 3 : H. Whiteley and E. J. Ordal, Bacteriol. Proc. p. 117 (1955). 3~D. E. Atkinson and B. A. McFadden, J. Biol. Chem. 210, 885 (1954).

List of Abbreviations (Selected from Volumes I and II) A

Ac, acetate ACF, anhydrocitrovorum factor Ac-SCoA, acetyl coenzyme A ADP, adenosine diphosphate ADPR, adenosine diphosphate ribose AMe, S-adenosylmethionine 2'-AMP, 2'-adenylic acid (a adenylic acid) 3'-AMP, 3'-adenylic acid (b adenylic acid) 5'-AMP-5'-adenylic acid (muscle adenylic acid) AR, adenosine ATP, adenosine triphosphate ATPase, adenosine triphosphatase B

BAL, British anti-lewisite BCG, bacillus of Calmette and Guerin (strain of M. tuberculosis) C CDR, cytosine deoxyriboside CF, citrovorum factor CHOFAH4, Nl°-formyltetrahydrofolic acid CoA, coenzyme A CoASH, coenzyme A, reduced

Cr, creatine CR, cytidine CTP, cytidine triphosphate D

DAP, dihydroxyacetone phosphate DFP, diisopropyl fluorophosphate DNA, deoxyribonucleic acid DNase, deoxyribonuclease DNP, 2,4-dinitrophenol DP-enzymes, diisopropyl phosphate enzymes DPN, diphosphopyridine nucleotide DPNase, DPN nucleosidase DPNH, diphosphopyridine nucleotide, reduced DPCoA, dephospho-coenzyme A E

EDTA, ethylenediaminetetraacetate (Versene) F

FAD, flavin adenine dinucleotide FAH4, tetrahydrofolic acid FAH4CHO, Nl°-formyltetrahydrofolic acid FDP, fructose-l,6-diphosphate FMN, flavin mononucleotide 871

872

LIST OF ABBREVIATIONS

F-l-P, fructose-l-phosphate F-6-P, fructose-6-phosphate G

GA, glyceraldehyde Gal-I-P, galactose-l-phosphate GAP, glyceraldehyde-3-phosphate GPC, glycerophosphorylcholine GPE, glycerophosphorylethanolamine G-l-P, glucose-l-phosphate G-6-P, glucose-6-phosphate GR, guanosine GSH, glutathione GSSG, glutathione, oxidized H

HxR, inosine

I, 5 (4) amino-4(5)-imidazolecarboxamide IAA, iodoacetate IDP, inosine diphosphate 5'-IMP, 5'-inosinic acid INH, isonicotinic acid hydrazide IR, 5 (4)-amino-4(5)-imidazolccarboxamide riboside IRMP, 5 (4)-amino-4 (5)-imidazolecarboxamide ribotide ITP, inosine triphosphate K

KG, a-ketoglutarate L LTPP, lipothiamide pyrophosphate

M

MB, methylene blue ]~[-6-P, mannose-6-phosphate N

NS/IeN, N 1-methylnicotinamide NMN, nicotinamide mononucleotide NR, nicotinamide riboside NTZ, neotetrazolium O OAA, oxalacetate P PEP, phosphoenolpyruvic acid 3-PGA, 3-phosphoglyceric acid PGA-P, 1,3-diphosphoglyceric acid PNPA, p-nitrophenol acetate P, orthophosphate, inorganic PP, pyrophosphate, inorganic PuR, purine riboside PyR, pyrimidine riboside R

RNA, ribonucleic acid RNase, ribonuclease R-l-P, ribose-l-phosphate R-5-P, ribose-5-phosphate T TCA, trichloroacetic acid THAM, tris(hydroxymethyl) aminomethane TPNH, triphosphopyridine nucleotide, reduced

LIST OF ABBREVIATIONS

TPP, thiamine pyrophosphate TPN, triphosphopyridine nucleotide Tris, tris(hydroxymethyl) aminomethane TTZ, 2,3,5-triphenyltetrazolium

UDPGal, uridinediphosphogalacrose UDR, uracil deoxyriboside UR, uridine UTP, uridine triphosphate X

U

UDPG, uridinediphosphoglucose

873

XR, xanthosine

Author Index T h e numbers in parentheses are footnote numbers and are inserted to enable the reader to locate a cross reference w h e n the author's n a m e does not appear at the point of reference in the text.

A Aas, K., 140, 153 Abbott, L. D., 328 Abderhalden, E., 97 Abraham, E. P., 121, 123, 710, 711(12) Abrams, R., 774 Abul-Fadl, M. A. M., 527 Ackermann, W. W., 614 Adams, E., 84, 87, 92, 97, 101, 102, 104(15, 19) Adelberg, E. A., 250, 253(16) Adler, E., 221, 224(2), 707, 710, 711(4, 12) Aggeler, P. M., 149 Agner, K., 765, 768, 774, 782, 794, 795, 796, 797, 798, 799, 800, 801 Aihara, D., 232 ~keson, ,~., 752, 754(11), 774, 808, 809, 811,817 Alais, C., 77 Albaum, H. G., 600, 735 Alberty, R. A., 25, 688, 707, 708(6), 710(6), 711(6) Albrecht, G. S., 79, 80(9) Alderton, G., 786 Alexander, B., 347 Alexander, H. E., 433 Alicino, J. F., 120 Alivisatos, S. G. A., 663 Alkjaersig, N., 144, 145 Allen, F. W., 433 Allen, M. B., 421, 422(1) Allen, P., 755, 757 Allen, R. J. L., 582 Allfrey, V., 437, 445(2), 447(2) Allgdn, L. G., 442 Almy, L. H., 315 Altschul, A. M., 774 Altschule, M. D., 837 Ambros, O., 64 Ambrose, J. A., 9 Ames, S. R., 819, 822(6), 825, 832 Anderson, A. E., 25 Anderson, D. G., 720, 721(9) Anderson, L., 415, 729 Anderson, R. S., 853

Anderson, W. G., 839, 840 Andrews, E. B., 162 Anfinsen, C. B., 224, 225(6), 429, 435(13) Anslow, W. P., Jr., 364, 367(14) Anson, M. L., 3, 34, 55, 77, 79, 80(1), 81 Appleby, C. A., 745, 746 Archibald, R. IV[., 351,357, 358, 359, 365, 368, 375 Armstrong, A. R., 328 Armstrong, M. D., 311, 312(3) Arnon, D. I., 789 Aschaffenburg, R., 533 Asenjo, C. F., 56/57, 63 Asimov, I., 826 Astrup, J., 158, 162, 165, 166 Atkinson, D. E., 870 Audrain, L., 37, 53 Auerbach, G., 97 Avi-Dor, Y., 774, 794 Awapara, J., 178 Ayengar, P., 215, 217(13) Azarkh, R. M., 318 B

Bach, A., 809 Bach, S. J., 746 Bachur, N. R., 666 Back, K. J. C., 868 Bagdy, D. D., 160 Bagot, E. A., 371 Bailey, K., 160, 583, 584, 587, 596, 602 Bajaj, V., 577, 579(13), 580(13), 592 Baker, S. B., 631 Balcazar, M. R., 56/57, 62, 64 Ball, E. G., 449, 482, 483, 485, 740, 754 Balls, A. K., 13, 14, 23, 24, 35, 37, 56/57, 58, 59, 60, 61, 62, 63, 780 Baltscheffsky, H., 749 Bamann, E., 579, 581 Bancroft, F. W., 140 Bard, R., 417 Bardawill, C. J., 357, 360(6), 365(6), 688, 7O8 Bardos, T. J., 519 Barger, F. L., 519 Barker, H. A., 730, 731 875

876

AUTHOR INDEX

Barkulis, S. S., 603 Barnes, B. A., 450, 452(16) Barnes, F. W., Jr., 178 Barnett, R. C., 721 Barnhurst, J. D., 625 Barron, E. S. G., 434, 587 Barsky, J., 396 Bartlett, G. R., 203 Barton-Wright, E. C., 629, 630(3) Bartz, Q. M., 7 Baudet, P., 76 Bauer, E., 727 Baum, H., 489 Baxter, R. M., 631, 632(1) Beadle, G. W., 168, 235, 320 Bean, R. C., 37, 48(8), 51, 53 Becker, S. W., 828 Beers, R. F., Jr., 764, 781 Behrens, O. K., 93, 12 3 Beinert, H., 485 Bellamy, W. D., 646, 647 Bender, A. E., 200, 202(3), 211 Bendich, A., 148, 149 Bentley, R., 556 Bergeim, O., 316 Berger, J., 89, 93(6), 108 Bergeret, B., 333 Bergmann, M., 21, 66, 84, 89(5), 93(5), 100, 101(14), 102, 114, 399 Bergold, V. G., 35 Bern, H. A., 523 Bernheim, F., 202 Bernheimer, A. W., 447 Bernstein, P., 22 Berridge, N. J., 69, 70(2), 74, 75, 76(11), 77(11) Bertoye, A., 166 Bessey, O. A., 640 Bessman, M. J., 524 Bettelheim, F. R., 8, 160 Bier, M., 32, 35, 37, 5.1, 53 Binkley, F., 311, 312(3), 318, 320, 322(22) Bird, O. D., 630 Birnbaum, S. M., 107, 108(21), 109(21), 112(2), 114, 115, 117(1), 118(1, 10), 119(9), 382, 399 Blanehard, M., 209, 210(9), 211(9) Blaschko, H., 177, 191, 196, 197, 390, 393 Blau, K., 21 Bloch, K., 342, 343(2, 3, 4), 344

Block, R. J., 457 Bloom, E. S., 510, 519(10) Bodansky, O., 447, 735 Boeri, E., 749, 753, 754 Boeseken, J., 290 BSttger, I., 442 Bolomey, R. A., 433 Bonhoeffer, K. F., 864 Bonner, C. D., 528 Bonner, D. M., 253 Bonner, J., 849 Bonnichsen, R. K., 749, 765, 768, 770(9), 772(9), 775, 778, 780, 781, 782, 785, 789, 791 Booth, V. H., 836, 837(1, 2), 838(1, 2), 839, 841(1), 843(1, 2), 844(1), 845(1) Bordner, C. A., 818, 825(3) Borek, E., 267, 268(1), 269(1), 338, 341(7) Borsook, H., 260, 347, 348, 415 Bovarnick, M. R., 203, 698 Bowen, W. J., 599, 603, 604 Bradfield, J. R. G., 842, 843(30), 845, 846 Brahinsky, R. A., 36, 40, 50, 51 Brand, E., 311, 435 Brandenberger, H., 178 Bratton, A. C., 406, 407, 634 Braun-Men~ndez, E., 124, 136 Braunshtein, A. E., 318, 348 Brenner, M., 21 Bressler, B., 630 Brinkhaus, K. M., 140 Brinkman, R., 839 Brodie, A. F., 695, 697, 698 BrSmel, H., 200, 201, 204(2), 212 Brown, C. S., 755 Brown, D. M., 9, 17(17), 24, 59, 83, 89, 91(8), 93(8), 433, 568 Brown, G. L., 778, 794 Brown, H., 486 Brown, K. D., 9, 18(14), 25, 436, 447 Brown, R. A., 630 Brown, R. K., 450, 452(16) Brown, W. T., 203 Buchanan, J. M., 448, 453(9), 502, 503, 504, 509, 512, 514, 517(7, 17), 519 Biicher, T., 290, 291(7), 335, 404, 483, 600, 602(7), 619, 633, 668, 670, 673, 704 Bueding, E., 406

AUTHOR INDEX Bukin, V. N., 847, 848, 850 Bull, H. B., 35 Bullock, I. H., 160 Bumpus, F. M., 136 Burch, H. B., 640 Burk, D., 698 Burkholder, P. R., 630 Burris, R. H., 837, 862, 870(3) Burton, H. S., 123 Burton, K., 203, 204(17), 211 Butler, G. C., 549, 561, 562, 563 Buzzell, A., 35 Byerrum, R. U., 837, 842(13), 844(13) C Cahill, G. F., ~311 Calaby, J. H., 590, 595 Calkins, E., 827 Cammarata, P. S., 172, 178, 179(4), 180(4), 182(9), 289 Campbell, C. J., 630 Campbell, E. W., 149 Canellakis, E. S., 796 Cannan, R. K., 51, 167 Cantoni, G. L., 254, 255(3), 256(2), 257, 259, 260(4), 262 Caputto, R., 499, 602 Carlisle, C. H., 435 Carpenter, D. C., 56/57, 61 Carpenter, F. H., 433 Carroll, R. T., 160, 161(50) Carter, C. E., 111, 382, 433, 434, 442, 461 Carter, J. R., 145 Castafieda, M., 56/57, 62, 64 Cavalieri, L. F., 442 Cecil, R., 774, 808 Ceriotti, G., 434 Chain, E., 121 Chamberlin, H. A., 523, 528(4) Chance, B., 732, 733, 734, 735(8), 739, 740, 750, 764, 765, 767, 768, 769, 770(9), 772, 774, 775, 780, 781, 782, 785, 786, 789, 791, 795, 799, 802, 803, 807, 810, 811, 812, 813, 840 Chantrenne, H., 347, 348(6), 349(6) Charalampous, F. C., 448, 453(9), 512 Chargaff, E., 148, 149, 320, 322(23), 433, 441, 445, 446(35), 447 Chase, A. M., 852, 853

877

Chatagner, F., 333 Cheldelin, V. H., 631 Chen, S. Y., 302 Cheng, J. J., 861 Cherbuliez, E., 76 Chevillard, L., 637, 638(4), 639 Chiba, H., 846 Chinard, F. P., 376 Christian, W., 203, 213, 247, 255, 257, 260(5), 290, 302, 305, 308, 407, 416, 656, 673, 676, 698, 699, 700, 712, 713, 715(1), 722 Christman, J. F., 320, 322(26) Chung, C. W., 422 Ciminera, J. L., 166 Ciocalt~u, V., 3, 55, 329, 559 Ciotti, M. M., 308, 310(27), 475, 476(1), 477(1), 660, 662, 663, 666, 681, 683, 684(7, see 8), 685(7), 686(1, 2), 687(1) Clark, A. M., 836, 837, 838, 839, 843(7), 844(7), 846 Clark, D. G. C., 166 Clark, H. W., 741 Clark, V. M., 501 Clark, W. G., 198, 199(9) Clendenning, K. A., 837, 842, 843, 845 Cliffton, E. E., 166 Cohen, G. H., 218 Cohen, P. P., 170, 172, 176(1), 177(1), 178, 179(4), 180(4), 182(9), 289, 341, 350, 351(2), 354, 355, 796 Cohen, S. S., 149 Cohen-Bazire, G., 233, 237 Cohn, E. J., 450, 452(16), 792 Cohn, W. E., 433, 524, 540 Collier, H. B., 37, 38(13), 54(13) Collinson, E., 434 Colowick, S. P., 233, 237(3), 308, 310(27), 311, 411, 471, 475, 476(1), 477(1), 498, 599, 601, 602, 603(1), 604(1), 660, 661(2), 662, 663, 664, 665, 666, 681, 682, 683, 684(7, see 8), 685(7), 686(1, 2, 5), 687(1), 726, 728 Conn, E. E., 719, 720, 721, 722 Connors, W. M., 76, 77(15), 298, 299 Consden, R., 172 Conway, E. J., 22, 472, 841 Cook, R. P., 387, 388(5) Cooke, R., 472

878

AUTHOR INDEX

Coon, R. W., 151 Cooper, E. J., 443 Cooperstein, S. J., 741, 742 Copenhaver, J. H., Jr., 611, 613, 614(2) Cori, C. F., 583 Cormier, 1V[.J., 861 Corran, H. S., 707, 709(2), 710(2), 711 Corse, J., 826 Cosulich, D. B., 519 Couch, J. R., 630 Coulombre, J., 651, 854, 856; 858 Covo, G. A., 613, 615(7) Crandall, D. I., 292, 293, 294, 295(15), 299(12) Crane, R. K., 599 Crewther, W. G., 32 Crook, E. M., 847, 848 Cross, R., 613, 615(7) Cubiles, R., 433, 436, 523, 524, 526, 528(4, 13) Cunningham, L. W., Jr., 20, 35 Curtis, W., 862, 870(7) Cushing, M. L., 826 Cutolo, E., 675, 676(1) Czaky, T. Z., 417 D

Dabrowska, W., 443 Dainton, F. S., 434 Dainty, M., 580, 581(5, 6) Dalgliesh, C. E., 249, 508 Damodaran, M., 388 Darling, S., 158, 162 Dart, R. M., 528 Das, N. B., 221, 224(2) Das, R., 625 Davenport, H. E., 753 Davenport, H. W., 845 David, M. M., 357, 360(6), 365(6), 688, 7O8 Davidson, J. N., 486, 489 Davie, E. W., 19, 26, 27(12) Davis, A. R., 56/57, 62 Davis, B. D., 300, 301, 303, 305, 307(3), 309(5, 14), 311(25a), 380, 619, 686 Davis, N. C., 84, 87(4), 92, 97, 98, 99(10), 100(10), 101, 103, 104(17), 105(16, 17, 19) Davis, W. B., 63

Davis, W. W., 136 Davison, M. M., 529 Dawson, C. R., 818, 819, 822, 825, 826, 831, 832, 834, 835 Day, A. J., 839 De Baun, R. M., 76, 77(15) de Duve, C., 542 Dekker, C. A., 433 del Campillo, A., 303 del Capella de Fern~indez, M., 56/57, 63 Della Monica, E. S., 534, 535, 538, 557 Delory, G. E., 562 Delwiche, C. C., 267 Delwiche, E. A., 315, 316(4), 317(4) De Maria, G., 80, 81(11) Dent, C. E., 192 de Robertis, E., 157 Derouaux, G., 450, 452(16) Derow, M. A., 529 Desnuelle, P., 8, 9(7), 14(7), 16(7), 23, 26, 36(13), 315, 318(3) Desreux, V., 4 Detolle, P., 166 Deutsch, H. F., 37, 49, 51, 53, 765, 782, 783, 784 Devlin, T. M., 770 Dewan, J. G., 221, 707, 710(1), 711 Dickman, S., 434 Dietrich, L. S., 195 Dillon, R. T., 115 Dische, Z., 438 Dixon, M., 18, 482, 484(1), 746 Doctor, V. M., 630 Dogson, K. S., 324, 325, 326, 327, 328, 332 Doherty, D. G., 21 Dole, V. P., 446 Dolin, M. I., 213 Doty, D. M., 559 Doughty, C. C., 377, 378(16) Douglas, D. E., 631 Dounce, A. L., 379, 483, 765, 775, 776, 778, 780, 781 Drabkin, D. L., 749 Dreskin, O. H., 150 Dreyfuss, J.-C., 161 Driscoll, P. E., 84, 87(2) Dubnoff, J. W., 347, 348, 357 Dubos, R. J., 427, 432, 434(4) Dubuisson, M., 586 Ducay, E. D., 37, 48(8), 51, 53

AUTHOR INDEX Duckert, F., 155 Duke, A. J., 51, 53 Dunn, F. J., 832, 834(4) Dunn, M. S., 33 Durand, M. C., 432, 433(22) Durrum, E. L., 457 Duthie, E. S., 49, 122 du Vigneaud, V., 93 Dyckerhoff, H., 97 l] Eakin, R. E., 514 Edelhoch, H., 688, 707 Edlbacher, S., 202, 203 Edman, P., 136 Edsall, J. T., 164, 584 Edson, N. L., 170 Egami, F., 417, 746, 759 Egan, R., 25 Eggleston, L. V., 603, 604, 613, 614 Ehrenberg, A., 754, 774, 782, 784, 808, 813 Eichel, B., 741 Eiger, I. Z., 819, 822 Eiler, J. J., 593 Eirich, F., 35, 53 Eiscnberg, M. A., 19 Elam, D. W., 7 Elkins, E., 80 Ellfolk, N., 386, 387, 388, 390(10) Elliott, W. H., 264, 337, 338(2), 339(2) Ellis, D., 84, 85, 86, 87(3) Ellis, W. J., 56/57, 64 Ells, V. R., 435 Elowe, D., 691, 710 Elsden, S. R., 759 Elvchjcm, C. A., 704, 850 Emmett, A. D., 630 Engel, F. L., 370 Engel, M. G., 370 Engelhardt, V. A., 534 Englard, S., 640, 642, 645, 648, 675 Eppright, M. A., 631 Epps, H. M. R., 187, 188, 189, 647 Epstein, E., 157 Errera, M., 382 Evans, C. H., 625, 626(12) Evans, H. J., 408, 411, 414, 415(2) Evans, W. C., 273

879 F

Fabre, C., 8, 23, 26, 36(13) Fabriani, G., 625 Fager, J., 442 Fahcy, J. L., 153, 157 Fankuchen, I., 24, 435 Farkas, L., 862, 864, 866, 867(12) Farr, A. L., 234, 320, 401, 409, 412, 417, 428, 438, 454, 469, 472, 476, 490, 492(3), 494(3), 498, 547, 552, 566, 652, 661, 664, 666(1), 727, 730, 851, 857 Fasciolo, J. C., 136 Fearon, W. R., 486 Feigl, F., 406, 407 Feinstein, R. N., 780 Feldman, L. I., 171, 172, 173(11), 175(11), 176(11), 177(11), 178 Fellig, J., 434 Ferguson, J. H., 154, 159, 164, 166 Fergusson, J. D., 523, 528(5) Fergusson, R. R., 807, 813 Fermi, C., 64 Ferry, J. D., 160 Fessenden, R. W., 838 Fevold, H. L., 786 Fiale, S., 162 Filitti-Wurmser, S., 754 Fischer, E., 862, 866, 867(12) Fischer, F. G., 442 Fischer, H. A., 577, 579(4, 10), 580(4), 581, 582(8) Fischmann, J., 523, 528(4) Fisher, A. M., 841, 843 Fisher, H. F., 862, 863(6), 865(6), 866(6) Fisher, J., 186, 187(1), 188(1), 189(1) Fishman, W. H., 528 Fiske, C. H., 530, 534, 540, 547, 551, 571, 577, 582, 596 FitzGerald, P. L., 433 Fitzpatrick, T. B., 827, 828, 829 Flaks, J. G., 509, 514, 517(7, 17) Florey, H. W., 121 Florey, 5/[. E., 121 Flynn, J. E., 151 Flynn, R. M., 668 Fodar, P. J., 114 Folin, O., 3, 55, 260, 329, 559 Folley, S. J., 539

880

AUTHOR INDEX

Fones, W. S., 117, 118 Fontaine, T. D., 56/57, 63 Ford, J. H., 120 Forro, F., Jr., 35 Fouts, J. R., 396 Fowden, L., 178 Fowler, D. I., 192 Fraenkel-Conrat, H. L., 37, 48, 51, 53 Frampton, O. D., 765, 775 Frankenthal, L., 580, 581(3), 582(3) Fraser, P. E., 170, 382 Fratoni, A., 625 Frederieq, E., 37, 49, 51, 53 Freedman, R. I., 443 Fried, M., 21, 65 Friedemann, T. E., 170, 316, 730 Frieden, E., 835 Friedkin, M., 448, 449, 456 Friess, E. T., 587 Frisch-Niggemeyer, W., 442 Fromageot, C., 315, 318(3), 333 Fruton, J. S., 9, 21, 64, 65, 66, 68, 84, 85, 86, 87(2, 3), 100, 114 Fu, S.-C. J., 117, 118(10), 399 Fuchs, H., 253 Fujita, A., 622, 625, 626, 628(1), 7!9, 732 Fujiwara, M., 625 G Gailey, F. B., 755 Gale, E. F., 170, 171, 173(3, 4, 13), 174(3, 4), 176(3, 4), 177(13), 182, 187, 189, 241, 320, 322(24) Galston, A. W., 789 Garkovi, P. G., 94 Garner, R. L., 427 Garrison, L., 123 GavarrSn, F. F., 56/57, 62, 64 Gay, H., 441 Gehrig, R. F., 375, 376, 378(14) Gemmill, C. L., 835 George, P., 770, 773, 802, 807 Gerber, C., 64 Gerischer, W., 700, 713 Gest, H., 862, 865, 867, 868(1, 5), 870(1) Gibbs, R. J., 51, 53 Gilbert, J. B., 114 Gilbert, L. M., 432, 441, 442 Gillespie, J. M., 450, 452(16)

Gilmour, D., 590, 595 Gilvarg, C., 301 Giri, K. V., 625 Gjessing, E. C., 813 Gladner, J. A., 8, 9(9), 12, 19, 20, 21, 509, 517(7) Glendening, M. B., 149 Goddard, D. R., 719 Goebel, W. F., 434 Goldblatt, H., 124, 125(1), 126(4), 133(2), 134(5, 8), 135 Goldman, D. S., 663 Goldthwait, D. A., 509 Gollan, F., 134 Gomez, C. G., 171, 173(16), 175(16) Gomori, G., 292, 544, 546(4) Gordon, A. H., 172, 711 Gordon, M., 233, 470 Gorini, L., 32, 37, 53 Gornall, A. G., 357, 360(6), 365(6), 688, 7O8 Gots, J. S., 519, 695, 697 Graffiin, A., 652 Graham, W. R., 533 Grassmann, W., 64, 83, 88, 93, 97, 100, 105, 107, 115, 384 Graubard, M., 819 Grauer, A., 580, 582(4) Greco, A. E., 400, 436, 447, 568 Green, A. A., 136, 814, 856, 860 Green, D. E., 170, 172, 174(5), 175(5), 209, 210(9), 211(9), 225, 226(1), 485, 559, 613, 615(7), 707, 709(2), 710(1, 2), 711, 869 Green, M., 870 Green, N. M., 20, 32, 33, 35, 39, 40, 50, 51, 53, 78 Green, R. H., 559 Greenbaum, L. M., 524 Greenberg, D. M., 56/57, 62, 63(11), 289 Greenberg, G. R., 509, 510, 512, 517(11, 13), 518(11), 519(11) Greenfield, R. E., 775 Greenstein, D. S., 767 Greenstein, J. P., 107, 108(21), 109(2!), 110, 111, 112(2), 114(1, 2), 115, 117(1), 118(1), 119(9), 298, 299, 300(20), 318, 319, 380, 382, 383(2), 384(2), 399, 434, 442 Gregg, D. C., 821, 826

AUTHOR INDEX Gr~goire, J., 442 Griese, A., 700, 713 Grimm, P. W., 755 Grisolia, S., 350, 351(2), 355 Grob, D., 49 Grossowicz, N., 267, 268(1), 269(1), 338, 339, 341(7) Grunert, R. R., 302 Guba, F., 584 Gtinther, G., 221, 224(2), 707, 710, 711(4, 13) Guest, M. M., 158 Guevara-Rojas, A., 134 Guirard, B. M., 631 Gulland, J. M., 540, 561 Gunsalus, C. F., 275, 276, 277, 278(8) Gunsalus, I. C., 170, 171, 173(11), 175(11), 176(la, 11, 17), 177(la, 11), 178, 182, 183(18), 212, 215(1), 216, 236, 237(11), 238, 239(2), 241(2), 275, 276, 277, 278(8), 303, 319, 320, 322(19), 324, 646, 647 Gurd, F. R. N., 450, 452(16) Gutcho, S., 694 Gutfreund, H., 24, 35 Gutfreund, K., 160 Gutman, A. B., 328, 523, 528(6-9) Gutman, E. B., 328, 523, 528(6-9) Gutmann, H. R., 64 H

Haas, E., 124, 125, 126(4), 133(2), 134(5, 8), 135, 694, 697, 699, 703, 704, 711, 713, 715, 718(11, 12) Haehn, H., 388 Hakala, N. V., 843, 844(33) Hale, W. S., 63, 780 Halkerston, I. D. K., 330 Halliday, D., 317 Halvorson, H. O., 213, 215(6) Hamilton, P., 115 Hampil, B., 512, 518(15) Hand, D. B., 379 Handler, P., 202, 653 Hankinson, C. L., 76 Hansl, N., 841 Hanson, H. T., 83, 94, 95, 96 Happold, F. C., 238, 242 Harbury, H. A., 809

881

Harman, J. W., 602 Harpley, C. H., 733, 745, 746(4), 748(4) Harrer, C. J., 697, 699, 703(2) Harteck, P., 864 Hartley, B. S., 24 Hartman, S. C., 509, 519 Hartree, E. F., 202, 203(6), 235, 303, 405, 455, 482, 485, 487, 489, 657, 671, 674, 705, 727, 732, 733, 735, 737, 738, 739, 740, 745, 749, 750, 751(2), 753, 754, 769, 774, 802, 809, 811, 812 Harvey, E. N., 851, 861 Hasegawa, E., 625, 626 Hashimoto, K., 281, 282(11) Haskins, F. A., 167, 470 Hasselback, W., 586 Hastings, A. B., 836, 843(3) Hastings, J. W., 856, 858 Hatch, B., 442 Haugaard, N., 837 Haugcn, G. E., 170, 316, 730 Hawk, P. B., 316, 762 Hayaishi, O., 229, 245, 249, 250, 251, 252, 281, 490, 492(2), 688, 707 Hayano, M., 354 Hayflick, L., 166 Hearn, W. R., 65, 68 Heatley, N. G., 121 Hecht, L., 433, 524, 526(13), 528(13) Heise, R., 195 Hellerman, L., 203, 698 Hellstrom, H., 707, 710(3), 711(3) Helmer, O. iV[., 134, 136 Hems, R., 603, 604, 613, 614 Hendee, E. D., 745, 746(5) Hendley, D. D., 722 Heneage, P., 197 Hennessy, D. J., 622, 625 Henry, R. J., 120, 123(7), 124(7) Henry, S. S., 178 Henstell, H. H., 443 Heppel, L. A., 404, 405, 449, 453, 462, 463(18), 482, 484(4), 547, 548, 566, 568, 569, 575, 576, 603 Herbert, D., 764, 765, 768, 775, 778, 782~ 784, 785(1), 786, 788(1) Herbert, F. G., 527 Herbst, R. M., 290 Hern~ndez, A., 62 Herriott, R. M., 4, 5(3), 7, 13

882

AUTHOR INDEX

Hers, H. G., 542 Hess, W. C., 311 Hesselvik, L., 51 Heumiiller, E., 579, 581 Heuser, G. F., 698 Heyde, W., 83, 88, 93, 97, 100, 105, 107, 115 Hill, C. H., 629 Hill, F. W., 698 Hilmoe, R. J., 404, 405, 453, 462, 463(18), 547, 548, 566, 568, 569, 575, 576 Hilton, S., 72, 74 Himwich, W. A., 336, 337(5) Hinman, R. L., 241 Hird, F. J. R., 289 Hirs, C. H. W., 9, 12, 435, 436(54) Hirschmann, D. J., 53 Hoagland, M., 667 Hoberman, H., 863, 864(16), 866(16), 867(16) Hoff-JCrgensen, E., 464 Hoffmann-Ostenhof, O., 442 Hogden, C. G., 66, 213, 721, 722 Hogeboom, G. H., 168, 486 Hogness, T. R., 694, 697, 699, 703(2), 704, 711, 715, 718(12), 754, 774 Holden, M., 136 Holmberg, C. G., 486, 489 Holmes, B., 434 Holt, L., 780 Holter, H., 77 Holtz, P., 195 Homburger, F., 528 Hoogerheide, J. C., 217, 218(4) Hoover, S. R., 58 Horecker, B. L., 449, 482, 484(4), 497, 656, 670, 676, 694, 695, 699, 704, 711, 715, 718(12), 735, 754, 755, 760 Horn, F., 376 Horowitz, N. H., 211 Hotchkiss, R. D., 458 Houghton, J. A., 66 Housewright, R. D., 120, 123(7), 124(7), 171, 173(16), 175(16), 176 Howard, A., 441 Howell, M. J., 802 Huber, H., 580 Hudson, P. B., 529 Hiibscher, G., 489, 741

Huff, J. W., 257, 454, 655 Huggins, C., 528 Hughes, D. E., 219, 382 Hughes, W. L., 814 Hultquist, M. E., 519 Hunter, F. E., 613, 614 Hunter, M. J., 792 Hurst, R. 0., 549, 561, 562, 563 Hurwitz, J., 646 Hussey, C. V., 157 Hutchings, B. L., 629 Hyndman, L. A., 862, 870(3)

Ichihara, T., 388 Ingelmann, B., 577, 578(5), 579(5, 6), 58O(5, 6) Ingraham, J. L., 284 Ingraham, L. L., 826 Ingram, V. M., 25, 68 Irving, G. W., Jr., 56/57, 63 Itahashi, M., 759 Izumiya, N., 118, 399

Jackson, E. M., 540, 561 Jacobs, G., 436, 447 Jacobsen, C. F., 8, 15, 16 Jacobsohn, K. P., 388, 577, 579(3) Jaenicke, L., 510, 517(11), 518(11), 519(11) Jaffa, W. G., 56/57, 63, 64 Jagannathan, V., 869 Jakoby, W. B., 253 Jang, R., 13 Jansen, E. F., 11, 12, 13, 14, 23j 24, 35, 56/57, 61 Jeffreys, C., 35 Jelinek, V. C., 120 Jendrassik, L., 254, 577 Jennings, M. A., 121 Johnson, C. A., 134 Johnson, M., 613, 614 Johnson, M. J., 89, 93(6), 97, 108, 755 Johnson, R. B., 614 Johnson, S. A., 149 Johnston, R. B., 21, 64, 342, 343(3) Joklik, W. K., 862, 868

AUTHOR INDEX Jolles, A., 780 Jolliffe, N., 406 Jones, M. E., 65, 68, 668 Jones, W., 427 Josephson, K., 768, 779, 782, 785, 788, 789 Joslyn, M. A., 808, 819, 847, 848, 850 Jul6n, C., 163 Jungner, G., 442 Jungner, I., 442 K

Kahnt, F. W., 450, 452(16) Kalckar, H. M., 448, 449, 456, 458, 469, 470, 473, 474, 480, 481(2), 482, 485, 486, 498, 501, 586, 592, 599, 601, 602(1), 603, 604(1, 8), 655, 675, 676(1) Kallio, R. E., 318 Kalnitsky, G., 459, 746 Kamehora, T., 281, 282(11) Kamen, M. D., 759 Kamin, H., 203 Kaminishi, K., 625, 626(10, 11) Kapeller-Adler, R., 394 Kaplan, A, 578 Kaplan, N. O., 233, 237(3), 308, 310(27), 311, 411, 475, 476, 477, 552, 553(4), 554, 633, 634(2), 650, 653, 655, 659, 660, 661(2), 662, 663, 664, 665, 666, 681, 682, 683, 684(7, see 8), 685(7), 686(1, 2, 5), 687(1), 726, 728, 758, 759(1), 760(1) Kassell, B., 311 Kastle, J. H., 808 Katz, E. J., 780 Katz, S., 160 Katz, Y. J., 124, 125(1), 134, 135 Kaufman, S., 8, 15, 21, 22(40), 23(40), 24, 34, 36(29), 90 Kawai, H., 846 Kay, H. D., 533, 539 Kazal, L. A., 36, 40, 50, 51 Kazenko, A., 9, 17(12), 18(13) Kearney, E. B., 171, 172(15), 203, 205, 207(2), 208(2), 209, 333, 640, 642 (2), 644, 648, 675 Keckwick, R. A., 715 Keil, H. L., 73

883

Keilin, D., 102, 202, 203(6), 235, 303, 405, 455, 482, 485, 487, 489, 657, 671,674, 693, 698, 705, 727, 732, 733, 735, 737, 738, 739, 740, 745, 746(4), 748(4), 749, 750, 751(2), 753, 754, 769, 774, 802, 809, 811, 812, 817, 819(1), 841, 843, 845 Keith, C. K., 9, 18(13) Kenney, J. C., 512, 518(15) Kenyon, J., 847 Keresztesy, J. C., 629 Kerwin, T. D., 599, 603, 604 Kielley, R. K., 350, 594, 595, 611 Kielley, W. W., 589, 594, 595, 611 Kies, M. W., 56/57, 62 Kiese, M., 741, 836, 843(3), 844 Kilby, E. F., 24 Kimmel, J. R., 59 King, E. J., 328, 428, 429(10), 527, 559, 562 Kingsley, R. B., 115, 117(1), 118(1), 119(9) Kintner, E. P., 528 Kirberger, E., 290, 291(7) Kirchheimer, W. F., 396 Kishida, T., 626 Kitagawa, IVI., 777 Kitasato, T., 577 Kityakara, A., 602 Kjeldgaard, N. 0., 449, 485 Klausmeyer, R., 417 Kleczkowski, A., 435 Klein, J. R., 202, 203, 653 Klein, P. D., 157 Kleiner, I. S., 73 Kleinzeller, A., 580, 581(5, 6), 602 Klenow~ H., 448, 449, 458, 482, 485 Kline, D. L., 164 Knivett, V. A., 376, 377(11), 378(11) Knox, W. E., 242, 244, 246(3), 247, 249 (5), 250(13), 252, 253(13), 287 Kocholaty, W., 217, 218(4) Kodama, T., 732 Koerner, J. F., 561, 565 Kohlstaedt, K. G., 134 Ko]ima, S., 801 Koller, F., 155 Kondo, K., 846 Korkes, S., 303, 494, 729 Korn, E. D., 448, 453(9), 504, 508, 512

884

AUTHOR INDEX

Kornberg, A., 448, 453, 454, 473, 490, 492(2), 493, 496, 497, 498, 501, 502 (2), 503(2), 504(2), 516, 550, 551, 602, 655, 656, 670, 673, 676, 677, 682, 720, 754 Korzenovsky, M., 376, 377(13), 378(13) Kotake, Y., 249 Kotel'nikova, A. V., 602, 603 Kozloff, L. M., 436 Kozuka, S., 622, 625, 626(11), 628(1) Krasna, A. I., 862, 863(6), 864(8), 865 (4, 6), 866(6), 867(4, 9) Kraut, H., 455, 484, 548, 567, 576, 674, 702, 706 Krebs, H. A., 170, 178, 182(14), 200, 202 (3), 203(8), 204(8), 211(1), 613, 614, 825, 837, 840, 845 Kreshaw, B. B., 37, 48(7), 53 Krishnan, P. S., 577, 579(13), 580(13), 591, 592, 593, 646, 647 Krueger, R., 347 Krumey, F., 579, 581, 582(9) Kubacki, V., 9, 25 Kubowitz, F., 817 Kuby, S. A., 601 Kukita, A., 829 Kun, E., 695 Kunitz, M., 4, 5(3), 8, 9(1), 11(4), 12(1, 4), 14(3), 16, 17, 19, 24, 25, 26, 27(4), 28(4, 8), 29, 30(4), 31, 32(8), 33, 34, 35, 36, 37, 38, 40(4), 44, 46, 49, 50, 51, 53, 427, 429, 431, 433(2, 5), 434 (2), 435(2), 437, 438, 439, 441, 442, 443(3), 462, 571, 574, 576 Kurfess, N., 742 Kurnick, N. B., 437 Kutscher, W., 523, 527, 528(2), 529 L Lacombe, G., 375, 376(3) La Du, B. N., Jr., 289 Lagrain, A., 434 Laidler, K. J., 586 Laine, T., 387 Lajtha, A., 269, 270(7), 271(7) Laki, K., 159 Lamanna, C., 433 Lamfrom, H., 124, 125, 126(4), 133(2), 134(5, 8)

Lampen, J. O., 448, 458, 461, 478, 479(2), 480(2) Lamy, F., 146 Lanchantin, G. F., 162 Landwehr, G., 347 Lansford, M., 519 Lardy, H. A., 611, 613, 614(2) Larrieu, YI. J., 147 Lascelles, J., 868 Laskowski, M., 9, 17(12, 17), 18(13, 14), 19(20), 20(20), 24, 25, 37, 38, 46(6), 47, 48, 49(12, 14), 50, 51, 53, 54(14), 436, 437, 442(4), 443, 447, 775, 782 Laskowski, M., Jr., 37, 46(6), 47, 48, 49 (14), 50, 51, 53, 54(14) Laufer, S., 62, 63, 64 Laurell, C.-B., 140 Lawrence, A. S. C., 580, 581(5, 6) Lawrence, J. M., 316, 317(7) Lazarow, A., 742 Lea, D., 434 Leadbetter, W. F., 528 Leanza, W. J., 192 Lederle, E., 764 Ledou×, L., 434 Lee, K., 593 Lee, M., 117, 118 Legge, J. W., 169 Lehmann-Echternacht, H., 442 Lehninger, A. L., 301, 302(7), 556, 603, 613, 615 Leidy, G., 433 Lein, J., 238 Leiner, G., 837 Leiner, M., 837, 844 Leitch, R. H., 72 Leloir, L. F., 124, 136, 170, 172(5), 174(5), 175(5), 602 LelVIay-Knox, iV[., 287 Lemberg, R., 169 Lenhoff, H. M., 758, 759(1, 2), 760(1, 2) Lennox, F. G., 56/57, 64 Leone, E., 486, 489 Leopold, H., 388 LePage, G. A., 676 Lerner, A. B., 827, 828, 829, 830 Lerner, F., 528 Leuthardt, F., 115, 178, 182(12), 347, 348, 638

AUTHOR INDEX

Leuthardt, F. M., 114, 319, 380, 382, 383 (2), 384(2) Levenberg, B., 509, 517(7), 519 Lever, W. F., 450, 452(16) Levintow, L., 115, 117(1), 118(1), 341, 633 Levy, G. B., 120, 122 Levy, R. S., 523 Lewis, C. J., 53 Lewis, H. A., 124, 125(1), 134, 135 Lewis, H. D., 837 Lewis, J. H., 154, 164, 166 Lewis, J. I. M., 332 Lewis, S., 819, 822(6), 831, 832, 835(2) Li, S.-O., 77 Lichstein, H. C., 170, 176(1, la), 177(1, la), 320, 322(25, 26) Lichtenstein, N., 341 Lieberman, I., 493, 496, 501, 502(2), 503 (2), 504(2), 602 Linderstr0m-Lang, K., 89, 115, 526 Lindsay, A., 203, 698 Lineweaver, H., 37, 53, 56/57, 59, 60, 698 Lipmann, F., 65, 268, 338, 555, 556, 633, 634(2), 653, 667, 668, 781 Lipschitz, R., 600 Little, H. N., 400, 402 Little, J. A., 561, 562 Liu, C. H., 450, 452(16) Livermore, A. H., 625, 626(12) Loaeza, F., 62 Loeffier, H. J., 847 Loeliger, A., 155 Loftfield, R., 752 Logan, M. A., 375, 376(4), 378(4) Lohmann, K., 254, 577 Lominski, I., 376 London, M., 529 Longsworth, L. G., 51, 757 Loomis, E. C., 143 Loomis, W. D., 264, 266(2), 267 Lopez, J. A., 577, 579(11), 612 Lorand, L., 145 Lorber, L., 757 Lorenz, L., 49 Loring, H. S., 433, 458 Louhivuori, A., 389 Love, S. H., 519 Lovelace, F. E., 56/57, 61 Lovett-Janison, P. L., 831, 832

885

Lowry, O. H., 234, 320, 401, 409, 412, 417, 428, 438, 454, 469, 472, 476, 490, 492, 494, 498, 547, 552, 566, 577, 579(11), 612, 640, 652, 661, 664, 666(1), 727, 730, 851, 857 Lucas, E. H., 837, 842(13), 844(13) Liidtke, K., 195 Lundquist, I., 527 Lustig, H., 356 Lyle, G. G., 614 Lyubimova, M. N., 534 M

Maas, W. K., 619 McBride, T. J., 160, 161(50) McCann, S. F., 38 MeCarty, M., 433, 438, 441(8), 442(8), 443(8), 444(8), 446, 447 McClaughry, R. I., 147, 157 McCubbin, J. W., 136 McDonald, M. R., 26, 29, 429, 432, 434, 435, 444 McElroy, W. D., 476, 477, 651, 852, 854, 856, 857, 858, 860 McFadden, B. A., 870 MacFadyen, D. A., 115, 428, 565 MeGilvery, R. W., 350, 543, 544, 545, 546(1) McGinty, D. A., 157 McIlwain, H., 459, 479 McInnes, D. A., 51 McLaren, A. D., 53 McMeekin, T. L., 74 MeNutt, W. S., 468 McVeigh, I., 630 Maehly, A. C., 765, 768, 773, 774, 796, 803, 807, 808, 809(15), 811, 812 Magasanik, B., 172, 176(21), 433 Mahler, H. R., 489, 688, 689(2), 691, 692, 707, 708(6), 710, 711(6), 869 Makower, B., 826 Mallette, M. F., 433, 818, 819, 822, 825, 832 Malmgren, H., 577, 578(5), 579(5, 6, 7), 580(5, 6) Mamelak, R., 217, 218(3), 219, 220 Mandl, I., 580, 582(4) Mann, T., 102, 578, 579(15), 580(15), 802, 812, 817, 819(1), 841, 843, 845

886

AUTHOR INDEX

Manson, E. E. D., 123 Manson, L. A., 448 Mapson, L. W., 719 Maren, T. H., 845 Margoliash, E., 752, 754(13) Maritz, A., 207, 208(5) Markham, R., 433, 434, 466, 526, 568 Mars, P. H., 47, 50, 51, 53 Marsh, W. H., 508 Marshak, A., 442 Marshall, E. K., Jr., 406, 407, 634 Martin, A. J. P., 172, 435 Martin, J. B., 559 Martius, C., 810 Martland, M., 540 Massart, L., 434 Mathies, J. C., 560 Matsukawa, T., 625 Matsuoka, H., 281, 282(11) Mattenheimer, H., 77 Matter, M., 155 Maver, M. E., 400, 436, 447, 568 Mawson, C. A., 839 Maxwell, E. S., 603 May, M., 519 May, S. C., Jr., 13 Mayr, O., 384 Mehl, J. W., 221 Mehler, A. H., 228, 242, 244, 246(3), 247, 249(5) Meister, A., 110, 114, 170, 171(2), 172 (12), 173(12, 14), 174(14), 176(2, 12), 177(12), 178, 182, 188, 189(10), 289, 298, 299, 341, 380, 381(3, see 6), 382 (3, see 6), 385(14) Mejbaum, W., 506 Mela, P., 269, 270(7), 271(7) Meldrum, N. U., 836, 837(5), 841, 843(5) Mellander, O., 526 Mendive, J. R., 838, 846 Metzler, D. E., 177, 320, 322 Meyerhof, O., 578, 586, 589 Miall, M., 580, 581(5, 6) Michaelis, L., 226 Michelson, C., 264, 266(2) Mihalyi, E., 586 Miller, A., 269 Miller, I. L., 250, 253(16) Miller, K. D., 162 Miller, L., 66

Miller, P., 746 Miller, W. H., 819 Miller, Z. B., 436 Millikan, G. A, 840 Milstein, S. W., 427, 428(6) Milstone, J. H., 140, 156 Mims, V., 190, 191(1), 192(1) Mingioli, E. S., 303, 305, 309(14), 380 Mirsky, A. E., 437, 445(2), 447(2) Mishuck, E., 35, 53 Mitchell, C. A., 838 Mitchell, H. K., 167, 233, 238, 470, 476, 477 Mitchell, M. B., 167 Mitsuhashi, S:, 305, 307, 311(25a), 686 Mitsui, H., 417 Mittelman, D., 450, 452(16) Mittelman, N., 602 Miura, Y., 434 Miyaji, T., 442 Mocquot, C., 77 Mommaerts, W. F. H. M., 584, 586, 587 (18) Monod, J., 233, 237 Montgomery, H., 828 Moore, D. H., 148 Moore, S., 79, 435, 436(54) Morales, M. F., 586 Morell, D. B., 485 Morgan, E. J., 847, 848 Mori, T., 759 Morrison, P. R., 160 Morrison, R. B., 376 Morton, R. K., 112, 530, 532, 535, 537, 538(9), 539, 557, 558, 559(1, 2), 560, 586, 745, 746 Mosimann, W., 778 Mouton, R. F., 450, 452(16) Mtiller, A. F., 178, 182(12) Mueller, G. C., 676 Miiller, H. R., 21 Mueller, J. H., 746 Mfillertz, S., 164 Mullen, J. E., 533 Munch-Petersen, A., 675, 676(1), 677 Mufioz, J. M., 124, 136 Munro, N., 847 Muntz, J. A., 434 Murata, K., 624, 627, 628 Murphy, R. C., 162

AUTHOR INDEX

Murray, C. W., 37, 53 Mycek, M. J., 21, 64 N

Najjar, V. A., 186, 187(1), 188(1), 189(1), 421, 422(1), 676 Nakamura, Y., 434 Narrod, S. A., 215, 217(12) Nason, A., 233, 237(3), 408, 411,414, 415 (2, 7), 664, 665, 666(2, 3, 4), 725, 728 Needham, D. M., 580, 581(5, 6) Needham, J., 580, 581(5, 6) Negelein, E., 200, 201, 204(2), 212, 700, 713 Neidle, A., 268 Neilands, J. B., 752, 755, 750 Nelson, J. M., 818, 819, 821,822(6), 825, 826, 831, 832 Nelson, N., 457 Nelson, W. L., 591 Nemchinskaya, V. L., 442 Neuberg, C., 356, 577, 579(3, 4, 10), 580, 581, 582(3, 4, 8) Neuberger, A., 249, 508 Neufeld, E. F., 308, 310(27), 311, 411, 681, 682, 683, 684(7, see 8), 685(7), 686(2, 5) Neufeld, H. A., 741 Neuman, R. E., 98, 100(12) Neurath, H., 8, 9(9), 11, 12, 15, 19, 20, 21, 22(40), 23(40), 26, 27(12), 32, 33, 34, 35, 36(29), 77, 78, 80(2), 81(11) Newton, B. L., 166 Nguyen-Van-Thoai, 637, 638(4), 639 Nicholas, D. J. D., 414, 415(7) Nielsen, H., 347, 348, 638 Nienburg, H., 97 Nikiforuk, G., 471 Nisrnan, B., 218, 220 Nisonoff, A., 178 Nitschmann, H., 77 Nocito, V., 170, 172(5), 174(5), 175(5) 209, 210(9), 211, 225, 226(1) Noda, L., 601 Nokayama, T., 249 Nord, F. F., 32, 35, 37, 51, 53 Norris, E. R., 7, 480, 482(1) Norris, L. C., 698

887

Northrop, J. H., 4, 5(3), 7, 8, 9(1), 12(1), 16, 24, 25, 26, 27(4), 28(4), 29, 30(4), 35, 36, 37, 38, 46, 53, 58, 429, 439 Nose, Y., 622, 626, 628(1) Novelli, G. D., 619, 633, 659, 667 Numata, I., 625, 626(9), 719 Nutting, M. D. F., 13 O

Oehoa, S., 303, 652, 681 O'Dell, B. L., 510, 519(10) Oginsky, E. L., 375, 376, 378(14) Ogston, A. G., 774, 808 Ohlmeyer, P., 527 O'Kane, D., 696 Oleott, H. S., 37, 48(8), 51, 53 Oldewurtel, It. A., 664, 666(3) Olitzky, P. K., 434 Olivard, J., 177 Olsen, N. S., 203 Olson, J. A., 224, 225(6) O'Malley, E., 841 Ordal, E. J., 862, 870 Osato, R. L., 66 Oser, B. L., 762 Otey, M. C., 382, 448 Oullet, L., 586 Overend, W. G., 432, 441, 442(17) Owades, P., 269 Owren, P. A., 140, 145, 146, 151,153, 154, 160

Packer, L., 870 Page, E. W., 195 Page, I. H., 136 Palade, G. E., 168 Pal~us, S., 752, 811 Palmer, K. J., 24 Pany, J., 523, 527(3), 529 Pappas, A., 344, 351, 356, 358(1), 515 Pappenheimer, A. M., Jr., 745, 746(5) Park, J. T., 659 Parker, R. P., 519 Parks, R. E., Jr., 22, 23(45) Parrish, R. G., 584 Paseyro, P., 157 Patwardhan, V. N., 178

888

AUTHOR INDEX

Paul, K. G., 736, 749, 752, 753, 754, 774, 794, 808, 809, 811, 817 Paulsen, M. M. P., 150 Peabody, R. A., 509, 519 Peanasky, R. J., 37, 49(12), 50 Peck, It. D., 862 Pedersen, K. O., 715 Peeters, G., 434 Peiree, J. D., 134 Pele, S. R., 441 Perlmann, G. E., 526, 781 Perlzweig, W. A., 454, 655 Permin, P. IV[., 166 Perrin, D. D., 836, 837(4), 838(4), 839, 846 Perry, S. V., 583, 587 Person, P., 741 Peterjohn, H. R., 461 Peterman, M. L., 843, 844(33) Petit, E. L., 778 Petrack, B., 356, 359, 360(10), 364, 365 (2), 367(14) Pfiffner, J. J., 510, 519(10) Pfister, K., 192 Pfister, R. W., 21 Phillips, P. H., 302 Philpot, F. J., 839 Philpot, J. St. L., 839 Pillard, E., 35 Pinsent, J., 765, 775, 778, 782, 784, 785 (1), 786, 788(1) Plantl, A. A., 136 Plass, R., 707, 711(4) Plaut, G. W. E., 22, 23(45), 415, 729 Plesner, P., 448 Ploeser, J. M., 458 Pogell, B. M., 543, 544, 545, 546(1) Poilroux, M., 8, 9(7), 14(7), 16(7) Polglase, W. J., 92 Polls, B. D., 586, 774, 799, 813, 816 Pollock, M. R., 120, 122(8), 123 Ponting, J. D., 819, 847 Porch, 1V[.B., 808 Porter, I. A., 376 Porter, R. R., 435 Portzehl, H., 583, 584, 586(6) Potter, V. R., 602, 603(15), 604(15), 614, 693, 704, 754, 758 Poup~, F., 726 Powers, W. H., 831, 832(2), 835

Pozzani, U. C., 838 Praetorius, E., 489 Price, C. A., 748 Price, V. E., 110, 114, 448, 775 Pricer, W. E., Jr., 473, 498, 516, 550, 551, 655 Pucher, G. W., 267 Pullman, M. E., 726, 728 Putnam, F. W., 77, 80(2)

Q Quastel, J. H., 217, 218(3), 219, 220, 387, 388(4), 631, 632(1), 748 Quick, A. J., 140, 149, 157 R

Racker, E., 358, 407~ 722 Rall, T. W., 301, 302(7) Randall, R. J., 234, 320, 401, 409, 412, 417, 428, 438, 454, 469, 472, 476, 490, 492(3), 494(3), 498, 547, 552, 566, 652, 661, 664, 666(1), 727, 730, 851, 857 Rao, K. R., 109, 114(1), 117, 119(9) Ratner, S., 209, 210(9), 211(9), 225, 226 (1), 344, 351, 356, 358(1), 359, 360 (10), 364, 365(2), 367(14), 515 Raub, A., 194 Ravdin, R. G., 292, 293, 299(12) Ravel, J. M., 514, 519 Ravin, H. A., 22 Raynaud, M., 218 Reddy, K. K., 625 Reid, J., 311, 312(3) Reif, A. E., 693 Reinhart, H. L., 528 Reis, J., 546 Reissig, J. L., 319, 320, 321(18), 322 Reissig, M., 157 Richardson, E., 124, 125(1), 134, 135 Rieche, A., 764 Riggs, B. C., 837 Rittenberg, D., 21, 862, 863, 864(8, 16)~ 865(4, 6), 866(6, 16), 867(4, 9, 16) Ro, K., 489 Roberts, D., 448 Roberts, E., 215, 217(13) Roberts, I. S., 580, 581(3), 582(3)

AUTHOR INDEX Robinson, D., 328 Robinson, D. S., 107, 108, 109, 112, 114

(2) Robinson, H. W., 66, 213, 721, 722 Robison, R., 540 Roche, J., 375, 376(3) Rodkey, F. L., 754 Roholt, 0. A., Jr., 371 Roll, P. M., 433 Rosebrough, N. J., 234, 320, 401, 409, 412, 417, 428, 438, 454, 469, 472, 476, 490, 492(3), 494(3), 498, 547, 552, 566, 652, 661, 664, 666(1), 727, 730, 851, 857 Rosenthal, N., 150 Rosenthal, R. L., 150 Ross, H. E., 341 Roth, B., 519 Roth, J. S., 427, 428(6), 434, 436 Roth, L. J., 819 Rothen, A., 435 Rothschild, Lord, 769 Roughton, F. J. W., 767, 836, 837, 838, 839, 840, 841, 843(1, 2, 5, 7), 844(1, 7), 845(I, 18) Roush, A., 480, 482(1) Rovery, M., 8, 9(7), 14(7), 16(7), 23, 26, 36 Rowen, J. W., 448, 453, 454 Rowsell, E. V., 172, 289 Roy, A. B., 326, 328, 330, 331, 332(6, 16) Rudman, D., 171, 172, 173(12), 176(12), 177(12), 380, 381(3, see 6), 382(3, see 6) Rtifenacht, K., 21 Ruffo, A., 613, 614 Russell, J. A., 375, 376(2) S

Sable, H. Z., 478, 479(2), 480(2) Sacktor, B., 595 Sadana, J. C., 869 Saenz, A. C., 434 Saffran, M., 501, 504(1) Saiki, H., 626 Sailer, E., 21 Saito, H., 388 Sakami, W., 517 Sakamoto, S., 622, 625, 627(7), 628(1)

889

Salamon, I. I., 305 Salazar, W., 62 Samuels, P. J., 342 Sanders, A. G., 121 Sarkar, N. K., 688, 689(2), 692, 707, 708 (6), 710(6), 711(6), 775, 777, 780 Sato, R., 746, 759 Saunders, J. P., 336, 337(5) Scarano, E., 262, 501,502, 503(8), 504(1) Schachter, D., 348, 349(9), 350(9) Sch~ffner, A., 579, 581, 582(9) Schaffer, N. K., 13 Schales, O., 136, 182, 188, 190, 191(1, 3), 192(1, 3), 193(2), 195, 196, 198, 199(6) Schales, S. S., 136, 182, 190, 191(1, 3), 192(1, 3), 193(2), 195, 198 Schepartz, B., 295 Schlamowitz, M., 427 Schieich, H., 114 Schmetz, F. J., Jr., 659 Sehmid, J., 147 Schmid, K., 450, 452(16) Schmidt, G., 433, 436, 469, 470, 471, 474, 523, 524, 526, 528(4, 13), 533 Schmidt, G. C., 375, 376(4), 378(4) Schmitz, A., 49 Schneider, C. L., 149 Schneider, W. C., 168, 350, 486, 594, 614, 695 Sch5berl, A., 311, 312(3) Seholefield, P. G., 203 Schott, H. F., 198, 199(9) Schou, M., 269, 339 Schramm, G., 584 Schrecker, A. W., 673 Schubert, M. P., 226 Sehuler, W., 489 Schulman, S., 808 Schutze, M., 508 Schwander, H., 77 Schwartz, S., 780 Schwarzenbach, G. M., 193 Schwert, G. W., 8, 11, 15, 19, 21, 22(40), 23(40), 24, 34, 36, 78, 80(5, see 11), 81(11) Scott, C. R., 741 Scott, D. A., 838, 841, 843, 846 Scott, E. W., 403 Scott, M. L., 629, 698

890

AUTHOR I N D E X

Scouloudi, H., 435 Scudi, J. V., 120 Seale, B., 178 Sealock, R. R., 625, 626(12) Seath, A. E., 631 Seegers, W. H., 141, 143, 144, 145, 147, 151, 153, 156, 157, 158, 162 Seller, A., 160 Sekine, T., 376 Seligman, A. M., 22, 347 Sera, Y., 229, 232 Seraidarian, K., 433, 524, 526(13), 528 (13), 586, 587(18) Seraidarian, M., 433, 524, 526(13), 527, 528(13) Sevag, M. G., 317, 435 Shanewise, A. B., 780 Shapiro, B., 555, 556(2) Shapiro, H. S., 441 Shapot, V. S., 442 Shatas, R., 578 Shelton, E., 695 Shemin, D., 290 Shen, S.-C., 580, 581(6) Sheppard, E., 53 Shinn, M. B., 403 Shinowara, G. Y., 528 Shipley, R. E., 134 Shirakawa, IV[., 765, 777 Shive, W., 514, 519 Shmukler, H. W., 774, 799, 813, 816 Shug, A. L., 869 Shugar, D., 435 Shulman, S., 160 Shupe, R. E., 9, 18(14) Shuster, L., 475, 477, 552, 553(4), 554, 666 Sibly, P. M., 842, 843(31), 845, 846 Sidwell, A. E., 754 Siekevitz, P., 602, 603(15), 604(15) Silverman, M., 629 Simms, E. S., 501, 502(2), 503(2), 504(2), 602 Simms, H., 431 Singer, T. P., 171, 172(15), 203, 205, 207(2), 208(2), 209, 333, 434 Sinsheimer, R. L., 561, 565 Sistrom, W. R., 282 Sizer, I. W., 764, 781, 808, 826 Slade, H. D., 376, 377(12), 378(12, 16) Slamp, W. C., 377, 378(16)

Slatcr, E. C., 560, 591, 603, 604, 613, 693, 737, 750 Sleeper, B. P., 274 Sloane-Stanley, G. H., 197 Slonim, N. B., 89, 92(9) Smathers, W. M., 149 Smith, C. L., 443 Smith, E. E. B., 675, 676(1) Smith, E. L., 9, 17(17), 21, 24, 59, 78, 83, 84, 87(4), 89, 91(8), 92(9), 93(5, 7, 8, 13), 94, 95, 96, 97, 98, 99(10), 100(10, 12), 101(14), 102, 103, 104 (15, 17), 105(16, 17), 107(20), 108, 100(22, 23, 24), 118, 134, 399 Smith, H. P., 140, 157 Smith, J. D., 433, 466~ 526 Smith, J. M., Jr., 519 Smith, J. N., 328 Smith, K. M., 434 Smith, L., 732, 733, 735, 736(12), 739, 741, 744, 745, 761,796 Smith, V. A., 84, 87(2) Smolens, J., 435 Smyrniotis, P. Z., 676 Smythe, C. V., 315, 316, 317(7) Snell, C., 411 Snell, E. E., 177, 320, 322, 468, 629, 630(1), 631 Snell, F. D., 411 Snell, N. S., 53 Snellman, O., 163 Snoke, J. E., 21, 22(40), 23(40), 34, 36(29), 342, 343(4), 344 Snyder, H. R., 241 Soares, M., 388 Sober, H. A., 171, 173(14), 174(14), 178, 182, 188, 189(10), 382 Soda, T., 330 SSrbo, B. H., 334, 335(1), 336, 337 Somers, G. F., 816 Somogyi, M., 457 Sonnenfeld, V., 856, 858 Soulier, J. P., 147 Sourkes, T., 197 Spackman, D. H., 89: 91(8), 92, 93(8, 13), 104(19) Spadoni, M. A., 625 Speck, J. F., 337, 338(1), 339 Spencer, B., 324, 325, 326, 327(2), 328, 332 Spicer, D. S., 36, 40, 50, 51

AUTHOR INDEX Spizizen, J., 512, 518(15) Spray, R. S., 233 Sprinson, D. B., 21, 320, 322(23) Sprissler, G. P., 7 Sreenivasan, A., 289 Sri Ram, J., 37 Stadie, W. C., 203, 837 Stadtman, E. R., 649, 650, 667, 730, 731 Stafford, H. A., 720, 721 Stage, A., 166 Stanier, R. Y., 245, 249, 250, 251, 252, 273, 274, 275, 276, 277, 278(8), 282, 284 Stanley-Brown, M., 140 Stanly, A. R., 233 Stannard, J. N., 735, 755 Stauffer, J. F., 837 Steele, W. J., 796 Stefaniak, J. J., 755 Stefanini, M., 149 Stein, W. H., 79, 435, 436(54) Steiner, R. F., 35, 53 Steinman, H. G., 835 Stephens, W. D., 726 Stephenson, M., 320, 322(24) Sterndorff, I., 166 Stewart, B. T., 213, 215(6) Stewart, E., 694 Steyn-Parvd, E. P., 636, 637, 639(2), 640(2) Stickland, L. H., 217, 218(1), 220 Still, J. L., 868 Stitch, S. R., 330 Stoll, A., 773 Stolzenbach, F. E., 475, 655, 666 Stotz, E., 298, 299, 300(20), 739, 741, 754, 761 Stout, B. K., 73 Straub, F. B., 584, 707, 708, 709(2), 710, 711(2), 741 Strecker, It. J., 224, 494 Strehler, B. L., 857, 861 Strickler, N., 433, 524, 526(13), 528(13) Strominger, J. L., 603 Struyvenberg, A., 242 Stumpf, P. K., 264, 266(2), 267 Sturtevant, J. M., 53 SubbaRow, Y., 530, 534, 540, 5471 551, 571, 577, 582, 596 Subramanian, S. S., 388

891

Suda, M., 281, 282, 292, 295 Sujishi, K., 295 Sullivan, M. X., 311 Sullivan, R. A., 76, 77(15) Sumiki, Y., 388 Summerson, W. H., 13, 762, 827 Sumner, J. B., 378, 379, 483, 577, 696, 765, 775, 776, 777, 778, 780, 782, 802 813, 816 Surgenor, D. M., 450, 452(16), 792 Sutherland, G. L., 519 Swanson, M. A., 542 Swenson, T. L., 37 Sylvdn, B., 163 Sz£ra, S., 160 Szent-GySrgyi, A., 484, 583, 586 T Tabor, H., 228, 229 Taggart, J. V., 348, 349(9), 350(9), 613, 615(7) Taha, S. M., 730 Takeda, Y., 292, 295 Takeuchi, M., 229, 232 Talaley, P., 528 Tallan, H. H., 68 Tam, R. K., 848 Tamm, C., 441 Tanaka, T., 295 Taniguchi, S., 417 Taquini, A. C., 136 Tarpley, W. B., 834, 835(8) Tashiro, T., 622, 626, 628 Tatum, E. L., 168, 235, 320 Tauber, H., 21, 37, 48, 53, 55, 56/57, 62, 63, 64, 73, 778 Taylor, E. S., 187, 189 Tenmatay, A. L., 625 Teply, L. J., 688, 707 Tepperman, J., 735 Terminiello, L., 37, 51, 53 Terrell, A. J., 725 Thannhauser, S. J., 433, 436, 474, 524, 526, 528(13), 533 Thayer, P. S., 211 TheoreU, H., 482, 714, 715(8), 751, 752, 753, 754, 758, 765, 768, 770(9), 772(9), 774, 780, 781, 782, 785, 789, 791,794, 799, 802, 803, 807(16), 808, 809, 811, 812, 813, 817

892

AUTHOR INDEX

Thimann, K. V., 748 Thomas, J., 325, 327(2) Thomas, L., 21 Thomas, R., 432, 433(22) Thompson, C. B., 241 Thompson, R. H. S., 427 Thompson, R. R., 56/57, 62 Thorne, C. B., 171, 173(16), 175(16), 176 Thornley, B. D., 72, 74 Tice, S. V., 170, 171(2), 173(14), 174(14), 176(2), 178, 182, 188, 189(10), 382 Tietze, F., 28, 35 Tinoco, I., Jr., 160 Tiselius, A., 470 Tissieres, A., 167 Toeantins, L. M., 160, 161(50) Todd, A. R., 433 Tones, L. N., 528 Tonhazy, N. E., 170, 174(7), 178 Torriani, A.-M., 122 Tosi, L., 753 Toyoda, J., 417 Trano, Y., 197 Trautmann, M. L., 443 Trethewie, E. R., 839 Tria, E., 780 Troll, W., 167 Trucco, R. E., 602 Tsao, T. C., 584, 586(9) Tsou, C. L., 749, 752(3) Tsuchida, M., 250, 274 Tsuchihashi, M., 783 Tsuda, N., 388 Tulpule, P. G., 178 Tupper, R., 843 Tuttle, L. C., 65, 268, 338, 556 Twigg, G. H., 120 Tytell, A. A., 375, 376(4), 378(4)

Uroma, E., 450, 452(16) Utter, F. M., 459, 746 Uyeki, E. M., 602

Vallee, B. L., 78 van Creveld, S., 150 Vandenbelt, J. M., 143, 510, 519(10) Vandendriessche, L., 432 van der Burg, B., 73 van der Scheer, A. F., 73 Van Goor, H., 837, 841 Vanhoucke, A., 434 van Orden, L. S., 396 Vanselow, A. P., 596 Van Slyke, D. D., 115, 358, 365, 368 Varin, R., 77 Vaughan, J., 66 Vely, V., 698 Vennesland, B., 719, 720, 721 Vercauteren, R., 442 Vernon, L. P., 688, 689(2), 692, 707, 708(6), 710(6), 711(6), 759 Verwey, W. F., 166 Vickery, H. B., 267 Vignos, P. J., Jr., 262 Vinet, G., 218, 220 Virtanen, A. I., 387, 388, 389 Vishniac, W., 870 Vlitos, A. J., 330 Vogel, A. I., 865 Volkin, E., 433, 524, 540 yon Euler, H., 221, 224(2), 707, 710, 711(3, 4, 13), 768, 779, 780, 782, 785, 788, 789, 810 von Lebedev, A., 713 von Schoenebeck, O., 97

U

W

Uber, F. M., 435 Ueba, A., 627, 628 Ueda, K., 622, 626, 628(1) Umbarger, H. E., 172, 176(21) Umbreit, W. W., 170, 174(7), 176(la), 177(la, 11), 178, 182, 183(18), 216, 236, 237(11), 238, 239(2), 241(2), 320, 322(25), 324, 610, 611(1), 647, 837

Waelsch, H., 267, 268(1), 269(1), 270(7), 271(7), 338, 339, 341(7) Wagner, A., 311, 312(3) Wagner, W. H., 273 Wainfan, E., 267, 268(1), 269(1), 338, 341 (7) Wainio, W. W., 741 Wakerlin, G. E., 134 Waley, S. G., 21

V

AUTHOR INDEX Walker, B. S., 780 Walker, J. B., 367, 376 Walser, A., 202 Walti, A., 56/57, 61 Wang, T. P., 458, 475, 477, 478, 479(2), 480(2), 554, 650, 653, 655 Warburg, O., 203, 213, 247, 255, 257, 260(5), 290, 302, 305, 308, 407, 416, 656, 673, 676, 698, 699, 700, 712, 713, 715(1), 722, 825 Ware, A. G., 141, 143, 151, 153, 156, 158, 162 Warner, E. D., 140, 145 Warner, R. C., 526 Wasserman, A. E., 166 Watanabe, H., 625 Watts, R. W. E., 843 Waugh, D. F., 146 Waygood, E. R., 837, 841, 842, 843, 845 Webb, M., 432, 441, 442(17), 443, 444, 447(31) Weber, H. H., 583, 584, 586(6) Weichselbaum, T. E., 255, 257, 260(6), 656 Weil-Malherbe, H., 559, 636, 637, 638 Weisel, P., 757 Weiss, U., 301, 305, 309(5) Werkheiser, W. C., 457 Werkman, C. H., 376, 377(13), 378(13), 459, 746 Werle, E., 194 Werner, A. E., 369 Wertheimer, E., 555, 556(2) Westenbrink, H. G. K., 637 Wheatley, A. H. M., 748 Whitby, L. G., 203 White, N. G., 170, 174(7), 178 White, S. G., 149 Whiteley, H., 870 Whitfeld, P. R., 568 Widmer, C., 741 Wieland, O. P., 629 Wiggans, D. S., 68 Wilbur, K. M., 839, 840 Wilcox, P. E., 19 Wildman, S. G., 849 Wilensky, B., 809 Williams, C. M., 745 Williams, J. H., 629

893

Williams, J. N., Jr., 289 Williams, R. R., 624 Williams, R. T., 328 Williams, V. R., 322 Williams, W. J., 502, 503, 504(9) Williamson, D. H., 382 Willstiitter, R., 64, 455, 484, 548, 567, 576, 674, 702, 706, 773, 823, 833(13, see 6), 834(4) Wilson, K., 630 Wilson, P. W., 759, 848, 862, 869, 870 Wilson, T. G. G., 759 Winitz, M., 68 Winniek, T., 56/57, 62, 63(11) Winzler, R. J., 457 Wise, W. S., 120 Wiss, O., 202, 203, 249, 250, 253 WSrner, A., 523, 527, 528(2) Wolberg, H., 523 Wood, J. G., 842, 843(31), 845, 846 Wood, W. A., 171, 176(17), 212, 215(1), 217(12), 236, 237(11), 238, 239(2), 241(2), 319, 320, 322(19), 324 Woods, D. D., 217, 218(2), 219 Woodward, C., 74, 75 Woodward, G. E., 343, 428, 719 Woolf, B., 387, 388(4, 5) Woolley, D. W., 663 Work, E., 39, 40, 50, 51, 53, 202 Wormalls, A., 843 Wosilait, W. D., 725, 728 Wright, R. D., 37, 48(7), 53 Wroblewski, F., 447 Wu, F. C., 9, 19(20), 20(20), 37 Wurmser, R., 754 Y

Yamada, T., 417 Yamadori, M., 626 Yamaguchi, M., 847, 848, 850 Yamazaki, K., 625, 626(11) Yanari, S., 342, 343(4), 344 Yaniv, H., 301 Yanofsky, C., 236, 238, 319, 320, 321(18), 322(19), 323(32) Yefimochkina, E. F., 348 Yoshida, A., 579 Young, R., 776

894

AUTHOR INDEX

Yudkin, W. H., 114 Yurugi, S., 625 Z Zahler, P., 77 Zamenhof, S., 433, 441, 445, 446(35), 447 Zapp, J. A., 203 Zatman, L. J., 660, 661(2), 662, 663, 666, 681, 686(1), 687(1)

Zeller, E. A., 207, 208(5), 390, 393, 394, 395(9), 396, 550 Zerfas, L. C., 746 Zima, O., 624 Zittle, C. A., 382, 384(7), 434, 441, 534, 535, 538, 557 ZSllner, N., 433, 434, 524, 526(13), 528(13) Zweig, G., 457

Subject Index Note on use of indexes for Volumes I and I I . Under the name of a given enzyme, inclusive pages are listed for the chapter in which it is described. Under this heading, the assay method, purification procedure and properties are not ordinarily listed, since these regularly appear within a few pages of each other and in the order mentioned. Only when unusual topics are covered, or when the organization of the material is more complex than usual, do sub-entries other than source appear under the name of the enzyme. Instead, all items of interest within each chapter have been listed under their own names. Thus, the names of individual sources, substrates, eoenzymes, activators and inhibitors of the enzymes are listed as major entries. The index may therefore be used to ascertain (1) the various enzymes which arc obtainable from a given bacterial, plant or animal source, (2) the various enzymes which are activated (or inhibited) by a given metal ion, eoenzyme, or other agent, and (3) the various fates of a given substrate. Other useful lists include one for crystalline enzymes and one for adaptive enzymes. Enzymes marked by asterisk (*) are covered in detail in Volume I.

Acetylation, enzymes for, in liver extracts~ 633 Acacia, Acetyldehydroalanine, fibrinoplastic action of, 159-160 as substrate for dehydropeptidase II, presence of calcium in, 156 109 use in two-stage prothrombin assay, 141 Acetyl-L-glutamate, Accelerin, see Serum Ac-globulin role in citrulline synthesis, 355 Acetaldehyde, p-Acetylphenylsulfate, formation by nitroethane oxidase, 400 as substrate for arylsulfatases, 327 Acetal phosphatides, Acetyl-L-phenylalanine ethyl ester, presence in thromboplastin, 150 as substrate for trypsin and ehymoAcetate, trypsin, 23 activation of 5'-AMP deaminase by, Acetyl phosphatase, 472 action at phosphorus-oxygen bond, formation by reduction of glycine, 217 556 inhibition of erythrocyte carbonic from animal tissues and bacteria, 555anhydrase by, 845 556 5-(Acetic acid)-hydantoinase, interference in acetyl-phosphate assay, cyclization of ureidosuecinic acid by, 650 496-497 Acetyl phosphate, Acetoacetate, CoA assay by arsenolysis of, 649 conversion of tyrosine to, 287-300 hydrolysis of, 555-556 formation by fumarylacetoacetate 3-Acetylpyridine analog of D P N , 654, hydrolase, 298-300 662, 663 Acetobacter sp., phosphorylation of by D P N kinase, eytochrome al as terminal respiratory 654 enzyme of, 732, 733, 734 3-Acetylpyruvate, eytoehrome component absorbing at action of hydrolase on, 299 554 mu in, 744-745 N-Acetyl-L-tyrosine ethyl ester, Acetonitrile, as substrate for trypsin and chymoinhibition of aspartase by, 388 trypsins, 23 Aeetyl amino acids, Ae-globulin, see Plasma Ac-globulin and action of acylases on, 116, 118, 119 Serum Ac-globulin 895 A

896

SVBJEC~ INDEX

8-C ~4-Adenine, Achromobacter fischeri, incorporation into nueleotide fraction, growth of, 857-858 501-502 luciferase from, 857-861 mechanism of hydrolytic nueleosidase Acid-soluble deoxypentose compounds, action studied with, 464 release by DNase, 437 Adenine deoxyriboside, "Acid-soluble phosphorus," R! values for, 466 measurement in RNase assay, 427 in transdeoxyribosidase reaction, 464, Acridine, inhibition of RNase by, 434 468 Acriflavine, bioassay of, 464 inhibition of DPNH cytochrome c reAdenosine (AR), ductase by, 698 inhibition of DPN kinase by, 654 Actin, of inosine cleavage by, 463 effect of on pH curves for myosin of pyridoxal kinase by, 649 ATPase, 588 nucleosidase action on, 459, 463 modification of ion effects on myosin Adenosine-3'-benzylphosphate, ATPase by, 586-587 3'-adenylic acid formation from, by Actomyosin, intestinal diesterase, 570 ATPase activity of, 589 Adenosine compounds, activation by Mg, 589 assay of with deaminase, 474-475 ion effects on ATPase activity of, 587 direct deamination of, 475 removal from Mg-activated ATPases, resistance of 2'-derivatives of to 589, 596 deaminase, 477 from myosin, 584 Adenosine deaminase, N-Acyl amino acids, see also under anonspecific, Amino acids, from takadiastase, 475-478 as substrates for carboxypeptidase, 78 affinities of various substrates for, Acyl phosphates, 478 as probable substrates for alkaline specific, phosphatase, 538 from calf intestinal nmcosa, 473-475 Acylpyruvase, assay of 3'-nucleotidase with, 551 probable identity with fumarylacetoseparation of from phosphatase, acetate hydrolase, 298 473, 474 Adaptive enzyme(s), see also Aromatic Adenosinediphosphate (ADP), rings, cleavage of, activation energy for hydrolysis of, 598 hydroxylamine reductase from Neuroactivation of 3,-glutamyltransferases spora crassa as, 411-417 by, 266, 272 kynureninase, bacterial, as, 253 deaminase action (non-specific) on, nitrate reductase from Neurospora as, 477, 478 411, 415 inhibition of D-amino acid oxidase by, nitrogen gas forming systems of Ps. 203 stutzeri and B. subtilis as, 423 of DPN kinase by, 654 tryptophan peroxidase of liver as, 244 of glutamine synthetase by, 339, 342 246, 253 of GSH formation by, 343 of Pseudomonas as, 245 of liver mitochondrial ATPase by, Adenine, 595 as acceptor of deoxyriboside group, 468 of pyridoxal kinase by, 649 inhibition of pyridoxal kinase by, 649 insect muscle ATPase action on, 597 R / v a l u e for, 466 liver mitochondrial ATPase action on, spectrophotometric assay of, 458 594 synthesis of nucleotides from, 501-504

SUBJECT INDEX myokinase action on, 598 " 5 " nucleotidase action on, 549 in oxidative phosphorylation assay, 615 as phosphate acceptor in citrullinase reaction, 377, 378 phosphorylation of by phosphocreatine, 605 in phosphorylation of riboflavin, 645 spectrophotometric assay via glucose6-phosphate dehydrogenase, 497 Adenosine-2',5'-diphosphate, cleavage of by potato adenosine-5phosphatase, 550 resistance of to "5" nueleotidases of seminal plasma and snake venom, 549, 550 TPN conversion to, by nucleotide pyrophosphatase, 655 Adenosine diphosphate phosphomutase, see Adenylate kinase Adenosine diphosphatc ribose (ADPR), action of nonspecific deaminase on, 477, 478 formation and transfer of by animal tissue DPNase, 660 formation of by Neurospora DPNase, 664 inhibition of DPN kinase by, 654 Adenosine monophosphate, see 2'-,3'- and 5'-Adenylic acids Adenosine-5-phosphatase, see also 5'Nucleotidase, from potato, 550 Adenosine phosphates, inhibition of nucleotide synthesis by, 504 Adenosine phosphokinase (adenosine kinase), in baker's yeast, 498 from brewer's yeast, 497-500 specificity of, 499, 500 in liver and kidney, 499 Adenosine triphosphatase (ATPase), in E. coli, 619 effect on arginine synthesis, 358, 359 on assay of adenylate kinase, 598 on glutamine synthetase reaction, 338, 339 on GSH synthesis, 343

897

in liver, 542, 593-595, 615, 653, 667 microsomes, 542 mitochondria, 593-595, 615 activity of intact versus modified, 594, 595, 615 from muscle, 588-591 possible identity of with myosin, 586 Adenosine triphosphatase, Mg-activated, from muscle (insect), 590-591, 595-598 comparison with mammalian enzyme, 590-591 occurrence in muscle mitochondria, 595 from muscle (rabbit), 588-591 simulation of by actomyosin, 589 Adenosine triphosphate (ATP), activation energy for hydrolysis of, 598 activation of ~-glutamyltransferase (brain) by, 272 complex of with Mg, 604 as component of firefly luciferase system, 651, 851, 854, 856 specificity for, 856 formation of by adenylate kinase, 598 by citrullinase, 376-377 with muscle enzyme fraction and phosphoglycerate, 515 by oxidative phosphorylation, 613 in formylation of glycinamide ribotide, 505, 510 of 5-IRMP, 519 of tetrahydrofolic acid, 517 hydrolysis of by apyrase, 591 by deaminase (non-specific), 477, 478 by myosin, 586 by 5-nucleotidase, 549 by nucleotide pyrophosphatase, 655, 659 by prostatic phosphatase, 525 incorporation of p~2 into as measure of oxidative phosphorylation, 614 inhibition of adenylate kinase by, 604 of arginine synthesis by, 356 of citrulline synthesis by, 355 of FAD hydrolysis by, 673 of pantothenate-synthesizingenzyme by, 621 phosphorylation of adenosine by, 497 of dephosphooCoA by, 649, 651 of DPN by, 652, 654

898

SUBJECT INDEX

of nucleoside monophosphates by, 6O3 of pantetheine by, 633, 635 of pyridoxal by, 646, 649 of riboflavin by, 640, 645 of thiamine by, 636 speetrophotometric estimation via glucose-6-phosphate dehydrogenase, 497 in synthesis of active methionine, 254 of amino-imidazolecarboxamideribotide, 514-515 of arginine, 356-357, 364 of citrulline, 350 of dephospho-CoA, 667-669 of DPN from NMN, 670 of DPNH from NMNH, 672 of FAD from FMN, 673 of glutamine, 337 of glutathione, 342 of glycinamide ribotide, 504, 509-512 of hippuric acid, 346, 348, 349 of IMP, 505, 518-519 of nucleotide from adenine and R-5-P, 501-504 of pantothenate, 619 of 5-phosphoribosyl pyrophosphate, 5O4 Adenosine triphosphate-creatine transphosphorylase (creatine kinase), from rabbit muscle, 605-610 crystallization of, 608-609 kinetics of, anomalous nature of, 606-607 S-Adenosylmethionine (AMe), methylation of guanidinoacetic acid by, 260 of nicotinamide by, 257 as product of methionine-activating enzyme, 254 Adenylate kinase (myokinase, ADP phosphomutase), distribution of, 602 in E. coli, 619 in flavokinase preparations, 645 nomenclature, basis for, 602 from rabbit muscle, 598-604 as byproduct of 3-phosphoglyceraldehyde dehydrogenase preparation, 601

removal of from myosin, 585 role in muscle relaxation, 602 Adenylic acid (s), bone phosphatase action on 2'-, 3r-, and 5'-, 540, 541 potato phosphatase action on 2'- and 3'-, 550 suppression of at high pH, 550 prostatic phosphatase action on 2'-, 3'and 5'-, 524-525 kinetics of, 525 2'-Adenylic acid (2'-AMP) (yeast adenylic acid, isomer A), activation of Pseudomonas transhydrogenase by, 683, 685-686 dependence on nature of substrate, 685-686 cleavage by non-specific phosphatases, 552 inhibition of DPN kinase by, 654 3'-Adenylic acid (3'-AMP) (yeast adenylic acid, isomer B), action of nonspecific deaminase on, 477, 478 cleavage by non-specific phosphatases, 552 inhibition of DPN kinase by, 654 paper chromatography of, 516 as substrate for 3'-nucleotidase, 551553 5'-Adenylic acid (5'-AMP, adenosine5'-phosphate), deaminase action on, non-specific, 477, 478 specific, 469 in glutathione reductase assay, 720 inhibition of adenylate kinase by, 604 of D-amino acid oxidase by, 203 of DPN kinase by, 654 of flavokinase by, 645 of pyridoxal kinase by, 649 in isocitric dehydrogenase system (DPN-linked) of yeast, 682 molecular extinction of, 469 in oxidative phosphorylation assay, 611, 615 paper chromatography of, 516 phosphatase (AMPase) for in microsomes, 542

SUBJECT I N D E X

as phosphate acceptor in citrullinase reaction, 377, 379 in nucleoside monophosphate kinase reaction, 603 in respiring mitochondria, 603 fluoride effect on, 603 as product of adenylate kinase action, 598 of apyrase action, in insect, 590-591, 595-598 in potato, 591 of nucleotide pyrophosphatase action, 655 of pantothenate synthesizing system, 619, 622 of thiaminokinase action, 636 as substrate for " 5 " nucleotidases: 547-549, 550, 561 synthesis of from adenine and R-5-P, 501-504 from adenosine, 497 5'-Adenylic acid deaminase (Schmidt's deaminase), in assay of adenylate kinase, 599 of AMP derivatives, 473 of pantothenate-synthesizing enzyme, 622 from rabbit muscle, 469-473 as byproduct of preparation of other muscle enzymes, 470 presence of in purified myosin, 586 Adenyl pyrophosphate, see Adenosine triphosphate Adenylyl uridylic acid, as substrate for spleen phosphodiesterase, 568 Adrenaline, see Ephinephrine Aerobacter aerogenes, cytochrome bl in, 745 5-dehydroquinase in, 305-307 5-dehydroshikimic reductase in, 304 7-glutamyltransferase (GTF) in, 269 nucleotide transhydrogenase in, 308 quinic dehydrogcnase from, 307-311 transaminase in, 173-174 tryptophan synthetase formation in, 237 Aerobic phosphorylation, see Phosphorylation, oxidative

899

A gkistrodon piscivorus (water moccasin), 5'-nucleotidase in venom of, 561 separation of from phosphodiestero ase, 563, 564 phosphodiesterase activity of venom of, 565 Agmatine, action of diamine oxidase on, 396 L-Alaninamide, hydrolysis by amidase, 399 by leucine aminopeptidase, 92 Alanine, D-amino acid oxidase and, 171, 176, 199, 200, 613 formation by kynureninase, 249 inhibition of alkaline phosphatase by, 538 in peptide B from fibrinogen, 160 transaminase for valine and, 176 transamination with pyridoxamine phosphate in C. welchii, 173 ~-Alanine, enzymatic conversion to pantothenate, 619 formation by aspartic acid decarboxylase, 188 Alanine racemase, in Bacillus spores, 215 from S. faecalis, 212-215 L-Alanyl dipeptides, action of acylase I on, 118 ~-Alanyl dipeptides, resistance of to glycylglycine dipeptidase, 109 L-Alanylglycylglycine, as substrate for aminotripeptidase, 87 ~-Alanyl-L-histidine (carnosine), activity of carnosinase on isomers of, 93, 94, 96 carbobenzoxy derivative of, resistance to carnosinase, 96 presence in muscle, 93 Albumin, serum, as protective agent in enzyme assays, 346, 483, 492, 724 Alcohol(s), inhibition of prostatic phosphatase by, 527 *Alcohol dehydrogenase, yeast, DPN assay with, 660, 670

900

SUBJECT INDEX

DPNH generation with, 694, 707 nitroaryl reductase assay with, 407408, 411 Aldehyde(s), formation of by amine oxidase~ 390 by diamine oxidase, 394 long chain aliphatic, as component of bacterial luciferase system, 857, 860, 861 combination of with cysteine to form inhibitor, 860 as substrate for xanthine oxidase, 484 *Aldehyde oxidase, reduction of dinitrobenzene by, 409 Alkaptonurics, homogentisate oxidase and, 294 Alkylsulfatases, 324, 330 Allantoin, formation of by uricase, 485 D-Allocystathionine, as substrate for cleavage enzyme~ 312 L-Alloisoleucinamide, hydrolysis of by leucine aminopeptidase, 92 Alloxazine nucleotide, see also Flavin nucleotides, reversible dissociation from old yellow enzyme, 712 Allylisothiocyanate, from horseradish, 801-813 Alum, in clarification of rennet, 73 Alumina gel, preparation of, 823 Amberlite IRC-50, chromatography of Ustilago eytochrome c with, 756-757 Amberlite XR-64, purification of cytochrome c with, 752 Amidase, action on amino acid amides, 397 chymotrypsins as, 22-23 trypsin as, 34-36 Amides, resistance to action of iminodipeptidase, 100 as substrates for leucine aminopeptidase, 91 Amine oxidase, see also Diamine oxidase and Monoamine oxidase, from steer plasma, 390-393 Amines, activation of thiaminase by, 625, 627

determination of non-acetylatable, diazotizable, 574 inhibition of diamine oxidase by, 396 of firefly luciferase by, 856 of ~-glutamyltransferase (bacterial) by, 269 oxidation of by horseradish peroxidase complex II, 810 by lactoperoxidase, 816 by myelopcroxidase, 800 role in detoxication of diphtheria toxins, 800 a-Amino acid(s), activation of D-amino acid oxidase by, 202 N-aeyl derivatives of, hydrolysis by aminoaeylases, 113, 115, 117-118 effect of variation of acyl group, 118 amides and esters of as substrates for trypsin, 32, 34-36 inhibition of ~-glutamyltransferase (bacterial) by, 269 of ~-glutamyltransferase (brain) by, 272 of Neurespora kynurenir,ase by, 253 liberation of by amidase, 397 oxidation by peroxidase complex II, 8O8 presence of in horseradish peroxidase, 811 as product of aminotripeptidase action, 83 reduction of, 217-220 resolution of by means of aminoacylases, 119 substances competing with, in D-amino acid oxidase reaction, 203 N-substituted, as substrates for Damino acid oxidase, 202 transamination among aliphatic, 171 among aromatic, 171 among D-forms of, 171, 175-176 Amino acid(s), aromatic, amides and esters of as substrates for chymotrypsins, 21 inhibition of carboxypeptidase by D-form of, 79 Amino acid acylase I (dehydropeptidase II, soluble acylase I), from hog kidney, 109-114, 115-119

SUBJECT INDEX

action as acylase, 115-119 heat stability of, 117 specificity of, 117-118 action as dehydropeptidase, 109-114 preparation of, 116-117 Amino acid acylase II (aspartic acid acylase), from hog kidney, 115-119 preparation of, 117, 119 specificity for acyl aspartic acids, 119 Amino acid acylase III, evidence for, 118 Amino acid amidase, from hog kidney, 397-400 Amino acid amides, participation in transaminase reactions, 170 role of manganous ion in destruction of, 398 Amino acid decarboxylases, from animals, 195-199 3,4-dihydroxyphenylalanine (dopa) decarboxylase, 195-199 other amino acid decarboxylases, 199 from bacteria, 185-189, see also under names of individual amino acids listed below, arginine, 187 aspartic acid, 188 glutamic acid, 186-187 histidine, 187 lysine, 188-189 ornithine, 189 tyrosine, 188 inhibition of by carbonyl reagents, 241 from plants, 190-194 glutamic acid decarboxylase, 190194 other amino acid decarboxylases, 194 D-Amino acid oxidase, assay of alanine racemase with, 212 assay and identification of FAD with, 200, 673, 698 occurrence with glycine oxidase, 227 requirement for FAD, 227 from pig kidney, 171, 212 in measurement of transaminations involving D-alanine, 176 from sheep kidney, 199-204

901

L-Amino acid oxidase(s), detection of, 179 in Neurospora crassa, 211 in rabbit liver, 213 interference in racemase assay, 213 from rat kidney, 209-211 in rat liver, 209 from snake venom, 205-209 in tissue slices, apparent presence of, 211 Amino acid racemases, 212-217, see also under names of individual enzymes, alanine racemase, 212-215 glutamic acid racemase, 215-217 Amino acid reductases, from Cl. sporogenes, 217-220 evidence of separate enzymes for, 220 other sources of, 218 Aminoacrylic. acid, as probable intermediate in tryptophanase reaction, 238 Aminoacylase(s), see also Amino acid acylase I and Amino acid acylase II, resolution of racemic a-amino acids by, 119 2-Aminoadenosine (2,6-diamino-9-~-Dribofuranosylpurine), phosphorylation of, 497 ~-Aminoadipic acid, as substrate for glutamine synthetase, 341 p-Aminobenzoic acid, estimation of, 350 inhibition of tyrosinase by, 826 oxidation of by peroxidase complex II, 810 as product of aromatic biosynthesis, 300 m-Aminobenzyl-(3)-4-methylthiazolium salt, activation of thiaminase by, 625 o-Aminobenzyl-(3)-4-methylthiazolium salt, and related compounds as inhibitors of thiaminase, 625 DL-~-Amino-n-butyramide, hydrolysis by leucine aminopeptidase, 92

902

SUBJECT INDEX

a-Aminobutyric acid, transaminase for valine and, 176 -r-Aminobutyric acid, formation by glutamie acid deearboxylase, 182, 186 L-a-Aminobutyryl-L-histidine, activity of earnosinase on, 96 L-Aminocaproie acid, as substrate for ~-amino acid oxidase, 211 p-Aminodimethylaniline, reaction with sulfide to form methylene blue, 315 Aminodinitrotoluene, reduction of trinltrotoluene to, 406 p-Aminohippurie acid, enzymatic synthesis of, 350 estimation of, 350 3-Amino-4-hydroxydichloroarsine, inhibition of aspartase by, 388 m-Amino-p-hydroxyphenylarsenoxide, inhibition of amino acid reductases by, 220 5 (4)-Amino-4(5)-imidazoleearboxamide, as acceptor of deoxyriboside group, 468 conversion to DPN analog by spleen DPNase, 663 paper chromatography of, 513 R/values for, 466 5 (4)-Amino-4(5)-imidazoleearboxamide riboside, enzymatic synthesis of, 448, 453 conversion to ribotide by yeast enzyme, 514-516 isolation of from E. coli cultures, 512514 paper chromatography of, 513, 516 5 (4)-Amino-4-(5)-imidazolecarboxamide ribotide, characterization by spectrum, 515 isolation of by ion exchange chromatography, 514-516 molecular extinction coefficient of, 516 paper chromatography of, 516 as precursor of inosine-5'-phosphate (5'-IMP), 505, 518-519 of riboside in sulfa-treated E. coli, 512 preparation from inosinie acid (IMP), 514

recovery from sulfa-treated E. coli, 514 5-Amino imidazole ribotide, basis for structure of, 519 conversion to 5-amino-4-imidazolecarboxamide ribotide, 505 2-Amino-2-methyl-l,3-prepanediol, glutamic dehydrogenase and, 224 2-Amino-4-nitrophenol, inhibition of quinone reductase by, 729 p-Amino-ornithuric acid, enzymatic synthesis of, 350 Aminopeptidases, 83-93 aminotripeptidase (tripeptidase), 8387 leucine aminopeptidase, 88-93 Aminophenols, oxidation by peroxidase complex II, 808 a-Aminosulfonic acids, aliphatic, inhibition of L-amino acid oxidase by, 208 Aminotripeptidase(s) (tripeptidase), 8387 from calf thymus, 84, 85-86 from horse erythrocytes, 84-85 leueine aminopeptidase and metalactivated dipeptidases as contaminants in, 87 presence in prolinase preparations, 97 solubility in ammonium sulfate solutions, 84 in swine intestinal mueosa, 84 table of specificity of thymus and erythroeyte enzymes, 87 Amino-unsaturated acid, as probable product of desulfhydrases, 315 ~-Aminovaleric acid, formation by reduction of ornithine or proline, 217 L-a-Aminovaleric acid, as substrate for L-amino acid oxidase, 211 Ammonia, citrulline synthesized from COs, ornithine and, 350 formation from adenosine, 473 from adenosine compounds, 475 by 5'-adenylic acid deaminase, 469 by amino oxidase, 390

SUBJECT INDEX

by amino acid amidase, 397-400 by arginine desimidase, 374 by asparaginase, 383 by aspartase, 386 by eathepsin C, 64 by citrullinase, 374 by cystathionine cleavage, 311-314 by bacterial enzyme, 314 by liver enzyme, 311 by eysteine desulfhydrase, 315-318 by cytosine nucleoside deaminase, 478 by dehydropeptidases, 110, 111 by diamine oxidase, 394 by exocystine desulfhydrase, 319 by glutaminase, 380 by glyeine oxidase, 225 by guanase, 480 by histidase, 228 by hydroxylamine reduetasc, 416 by leueine aminopeptidasc, 88 by reduction of glycine, 217 by L serine (L-threonine) dehydrase, 319 322 by tryptophanase, 238 in glutamie dehydrogenase reaction, 220 in glutamine synthetase reaction, 337 in 5-glutamyltransferase reaction, 263, 267, 269 inhibition of v-glutamyltransferase (brain) by, 272 measurement of, 204, 316, 397 for assay of L-amino acid oxidase, 204 Ammonium chloride, inhibition of nitroethane oxidase by, 402 Ammonium ion, activation of pantothenate-synthesizing enzyme by, 621 of tryptophanase by, 242 effect of on RNase, 433 inhibition of mammalian L-amino acid oxidase by, 211 Ammonium polysulfide, see Sulfide Ammonium sulfate, effect of on protein solubility in aqueous acetone, 132 equation for changes in saturation of, 571

903

inhibition of arginine-synthesizing system by, 359 of hydrogenase by, 732 measurement of concentration of by conductivity, 391 nomogram for concentration of, 18 Amylamine, amine oxidase action on, 393 *Amylase, in preparation of crystalline liver eatalase, 776 Anana sativa, see Pineapple Ai~giotonase, 135, 136 Angiotonin (hypertensin), bioassay of, 136 formation by renin, 124, 135 preparation of, 135 136 ultraviolet absorption of, 136 Anhydroleueovorin (anhydrocitrovorum factor) (ACF), chemical intereonversion of N ~°-formyltetrahydrofolic acid, and leucovorin, 518, 519 in formylation of IRMP to IMP, 519 spectrophotometric measurement of, 518 Aniline, as base in thiaminase reaction, 623 detoxication of diphtheria toxin by myeloperoxidase in presence of, 800 oxidation by peroxidase complex II, 810 Aniline citrate, decarboxylation of oxalacetate with, 170-171, 174-175 preparation of solution of, 174 Anions, inhibition of L-amino acid oxidase by bi- and trivalent, 208 of DPNH cytochrome c reduetase by, 692 of erythrocyte carbonic anhydrase by, 845 monovalent, stabilization of purine nueleosidase by, 460 o-Anisidine (2-methoxyaniline), detoxication of diphtheria toxin by myeloperoxidase and, 800

904

SUBJECT INDEX

Anthranilic acid, absorption maximum for, 250 formation by kynureninase, 249 inhibition of kynurenine formamidase by, 246, 249 as precursor of catechol, 273 tryptophan peroxidase and, 244 Antifibrinolysin,from ox lung, 165 Antifoam agent, 756 Antihemophilic factor (AHF, PTC), activation by thrombin, 157 assay of, 147-148 concentration in oxalated horse plasma, 147 as heat labile factor in thromboplastin, 139, 147-148 purification from plasma, 149 stability of, 150 Antimycin, inhibition of DPNH cytochrome c reductase activity of mitochondria by, 693 Antipenicillinase serum, effect on penicillinase activity, 123 Antirenin, neutralization of pressor effect of renin by, 134 production in human subjects, 129, 134 Antisera, inhibition of pancreatic and streptococcal DNases by respective, 447 Antithrombin, 158, 162, 163 in defibrinated plasma, 162 heparin cofactor and, 163 Antithromboplastin(s), 160-162 lipid antithromboplastin from brain, 161 protein antithromboplastin from muscle, 161-162 Apocarboxylase, preparation for thiamine pyrophosphate assay, 637, 638 Apurinic acid, disintegration by Mg ++, 441 resistance to DNase action, 441 Apyrase(s), see also Adenosine triphosphatase, from insect muscle, 590-591, 595-598

interference with adenylate l~inase assay by, 600 from potato, 591-593, 646 removal of ATP by, 646 temperature effect on nature of products, 593 L-Arabinose, paper chromatography of, 513 Arabitylflavin, phosphorylation by flavokinase, 644 Arachain, protease from peanut, 57, 63 Arachis hypogen, see Peanut Arginase, in assay of arginine-synthesizing system, 356-357, 359, 360, 364-365 from beef liver, 357, 358 effect of heat on, 370 from horse liver, 368-374 physical-chemical tests of purity of, 373-374 seasonal variation in content of, 371 L-Arginine, action of arginase on, 368 activation of DNase by, 442 content in horseradish peroxidase, 809 enzymatic synthesis of, 356-367 condensing enzyme system for, 359-364 liver enzyme, 360-362 yeast enzyme, 360, 362-364 over-all reaction, 356-359 from kidney, liver and yeast, 357, 358-359 splitting enzyme for, 364-367 preparation from beef liver or pig kidney, 365-367 hydrolysis of to eitrulline and NHs by desimidase, 3741 inhibition of splitting enzyme for arginine synthesis by, 367 pK of a-amino group, 368 reduction of, 217 stabilization of arginase by, 373 as substrate for L-amino acid oxidase, 2O8 Arginine decarboxylase, from E. coli, 187 resolution of, 189

SUBJECT INDEX in measurement of transaminase reactions, 171 Arginine desimidase, distribution of, 376 from yeast, 375-376 Arginine dihydrolase system~ 374-378 arginine desimidase component of, 374, 375-376 citrullinase component of, 374, 376378 L-Argininosuccinic acid, as intermediate in synthesis of arginine, 359, 364-365, 367 Aromatic biosynthesis, bacterial enzymes for, 300-309 5-dehydroquinase, 305-307 5-dehydroshikimic reductase, 301304 quinic dehydrogenase, 307-309 scheme for, 300 Aromatic rings, cleavage of, in aerobic bacteria, 273-287 enzymes in scheme for, benzaldehyde dehydrogenases (TPN- and DPN-linked), 280-281 benzoylformic carboxylase, 273, 278-280 lactonizing and lactone-splitting enzymes, 282-284 L(~)-mandelic acid dehydrogenase, 273, 274, 277-278 mandelic acid racemase, 273, 274, 276-277 protocatechuic acid oxidase, 284287 pyrocatechase, 273, 274, 281-282 reaction sequences for, 273 Arsenate, activation of -y-glutamyltransferase by, 264, 267, 272 inhibition of alkaline phosphatase by, 538 of DNase by, 442 of D P N H cytochrome e reductase by, 692 of glucose-6-phosphatase by, 542 of metaphosphatase by, 579 of purine nucleosidase by, 460

905

replacement of phosphate, Mg ++ and ADP requirements of citrullinase by, 377, 378 stabilization of pyrimidine nucleosidase by, 459 Arsenicals, inhibition of aspartase by, 388 Arsenious oxide, inhibition of eysteine desulfhydrase by, 317 Arsenite, see also Arsenious oxide, effect of on metaphosphatases, 579 inhibition of amino acid reductases by, 220 of arginine desimidase by, 376 of D P N H cytochrome c reductase by, 693 of hydrogenase by, 732 of plant carbonic anhydrase by, 846 reversal by cysteine or glutathione, 846 as uncoupling agent, 615 Arsenolysis, CoA assay by, 649 Arsenoxides, organic, inhibition of amino acid reductases by, 220 Arylsulfatases, from marine mollusks, 332 from ox liver, 330-332 preparation of arylsulfatase A, 330-332 of arylsulfatase B, 332 from takadiastase, 328

Ascaris, digestion of by plant proteinases, 55 trypsin inhibitor from, 37, 54 Asclepain, protease from mllkweed~ 56, 61-62 amorphous form of, from leaves, 62 activation by sulfite, 62 crystallization of, from latex, 62

Asclepias speciosa, mexicana, syriaca, see Milkweed Ascorbate, activation of homogentisate oxidase by, 292 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292, 295 of tyrosine-oxidizing system by, 287, 288, 289

906

SU-B J E C T I N D E X

in catecholase assay, 819 cytochrome c reduction by, 754 determination of, 847-848 effect on absorption bands of cytochrome b, 744 formation by dehydroascorbic reductase, 847-850 oxidation by peroxidase complex II, 808, 810 L-Ascorbate, as substrate for ascorbic acid oxidase, 831-835 comparison of with D-form and other ene-diols, 834 Ascorbic acid oxidase, from yellow squash, 831-835 absorption spectrum of, 834 reaction inactivation as characteristic of, 835 L-Asparaginase, from guinea pig serum, 383-384 in yeast, 384 Asparagine, comparison of rate of cleavage of D- and IMsomers by asparaginase, 384 ~-keto aeid-mediated deamidation of, 382 transamination with aliphatic keto acids, 171 Asparagine transaminase, distinction from asparaginase, 382 Aspartase, 386-390 determination of L-aspartic acid with, 389, 390 in protein hydrolysates, 390 from propionic acid bacteria, 386-387, 388 from Ps. fluorescens, 387 Aspartie acid, action of D-serine (D-threonine) dehydrase on, 324 N-acyl derivatives of, as substrates for acylase II, 119 arginine synthesis from, 356-357, 364 as component of transaminase system, 180 conversion to fumaric acid and ammonia, 386 formation by asparaginase, 383

inhibition of glutamic dehydrogenase by, 224 measurement by chloramine-T method, 170-171 quantitative determination by aspartase, 389, 390 in protein hydrolysates, 390 as C-terminal residue, effect of on iminopeptidase, 100 transaminase reactions involving, 170, 172, 174, 175, 176, 179-182 measurement of formation of oxalacetate in, 174, 175, 179-182 Aspartic acid acylase, see Amino acid acylase II Aspartic acid decarboxylase, from C1. welehii, 182, 188 measurement of transaminase reactions with, 171 L-Aspartic diamidc, hydrolysis by leucine aminopeptidase, 92 ~-L-Aspartyl-L-histidine, activity of carnosinase on, 96 AspergiUus sp., metaphosphatase in, 577, 578, 579

Aspergillus niger, glucose oxidase in, inhibition by nitrate, 579 growth of, 578

Aspergillus oryzae, commercial takadiastase and clarase from, 579 triphosphatase in takadiastase from, 580, 582 Atabrine, see Quinacrine Aureomycin, as uncoupling agent, 615 Auric salts, inactivation of renin by, 134 8-Azaguanine, deamination by guanase, 482 enzymatic synthesis of riboside and deoxyriboside of, 448 Azide, compound of catalase with, 788 of ferricytochrome c with, 755 of horseradish peroxidase with, 812 and cytochrome b, lack of reaction between, 745

SUBJECT INDEX determination of cytochrome c peroxidase with, 764 effect on activity and spectrum of myeloperoxidase, 799 inhibition of D-amino acid oxidasc by, 203 effect of pH on, 203 of aspartase by, 388 of d-biotin oxidation by, 632 of carbonic anhydrase by, 844, 845 of catalase by, 764 of cytochrome c peroxidase by, 763 pH dependence of, 763 of homogentisate oxidase by, 295 of hydroxylamine reductase by, 419 of liver glutathione reductase by, 725 of metaphosphatase by, 579 of nitrate reductase by, 415 of nitroaryl reductase by, 410 of nitroethane oxidase by, 402 of spinach catalase by, 790 of tryptophan peroxidase by, 246 of tyrosinase by, 826 as uncoupling agent, 615 Azotobacter spp. transhydrogenase in, 686 Azotobacter agile, hydrogenase in, 870 Azotobacter chroScoccum, cytochrome bl in, 745 Azotobacter vinelandii, cytochrome c in, 759 cytochrome c peroxidase in, 764 glutamic-oxalacetic transaminase in, 184 hydrogenase in, 870 B

Bacillus cereus, penicillinase from, 120-124 growth medium for, 122 Bacillus lichenformis, cytochrome e in, 745 Bacillus subtilis, action of lysozyme on, 421 cytochrome a3 in, 734 cytochrome b in, 745 enzymes for nitrogen gas formation from, 420-423 growth of thermophilic strain of, 421

907

protease from, 80 transaminase in, 173, 176 Bacillus thiaminolyticus, growth of, 626 thiaminase from culture medium of, 626-628 Bacteria, see also under names of individual species, catalase in, 765 cytochromes a, as and a3 in, 732 cytochrome b group in, 744-748 dcsulfhydrases in, 318 desulfinases in, 333 press for extraction of, 219 L-serine (L-threonine) dehydrase in, 320 transaminases in, 170-177 distribution of, 172, 173-4, 176 Bacterium cadaveris, lysine decarboxylase from, 188-189 Barbital, see Veronal Barbiturase, from Mycobacterium, 492-493 Barbituric acid, hydrolysis to urea and malonic acid, 492 Barium carbonate, use in purification of prothrombin, 144-145 Barium ion, inhibition of DPNH cytochrome c reductase by, 692 Barley, 3'-nucleotide phosphatase in, 524 phosphatase in, 540 Beet (Beta vulgaris), carbonic anhydrase in leaf of, 842 Benadryl, inhibition of amine oxidase by, 393 Bentonite, in purification of soybean trypsin inhibitor, 41 Benzaldehyde, formation by benzoylformie carboxylase, 273, 277, 278-280 by amine oxidase, 390 molar extinction coefficient for, 390 oxidation of, 273, 277 pyridine nucleotide requirement for, 277

908

SUBJECT INDEX

Benzaldehyde dehydrogenases (TPN- and DPN-linked), from Ps. fluorescens, 273, 280-281 Benzidine (4, 4'-diaminobiphenyl), detoxication of diphtheria toxin by myeloperoxidase and, 800 sulfate determination with, 324-326 Benzimidazole (s), acceleration of RNase action by, 434 competition with firefly luciferin, 856 Benzoic acid, S-benzoyl-CoA formation from, 346 conversion to hippuric acid, 346 and derivatives, inhibition of D-amino acid oxidase by, 203 formation by benzaldehyde dehydrogenase, 273, 280, 281 inhibition of venom L-amino acid oxidase by, 208 o-Benzoquinone, detection of in chronometric assay of tyrosinase, 819 p-Benzoquinone (p-quinone, quinone), photochemical reduction of, 726 reaction with glycyglycine, 729 reduction by cysteine and GSH, 728 by DPNH, 725, 728 Benzoquinone acetic acid, inhibition of homogentisate oxidase by, 294 reduction by DPNH, 728 L-(~-Benzoyl)-alanine, as substrate for liver kynureninase, 253 a-Benzoylargininamide, as substrate for trypsin, 36 Benzoyl-L-arginine ethyl ester, as substrate for trypsin, 36 a-Benzoyl-L-arginine methyl ester, cross reactivity with trypsin and chymotrypsins, 21 as substrate for trypsin, 36 S-Benzoyl-CoA, condensation with glycine, 346, 349 speetrophotometric measurement of, 349-350 as intermediate in hippuric acid synthesis, 346 Benzoylformic acid, conversion to benzaldehyde, 273, 277, 278-280

formation by mandelic acid dei~ydrogenase, 273, 277 Benzoylformic carboxylase, from Ps. fluorescens, 273, 278-280 a-Benzoyl-L-lysinamide, as substrate for trypsin, 36 Benztriazole, competition with firefly luciferin, 856 Benzyl alcohol, distribution of riboflavin and F M N between water and, 642 Benzylamine, molar extinction coefficient for, 390 as substrate for amine oxidase, 390, 393 a-Benzyl-i-argininamide, as substrate for assay of papain, 59 2-Benzylglyoxaline, inhibition of penicillinase by, 123 Benzylpenicillin, as substrate for penicillinase, 120-121 Benzylviologen, reduced, oxidation by amino acids, 217, 218 Beryllium ion, inhibition of alkaline phosphatase by, 538 Beta vulgaris, see Beet Bicarbonate, in carbonic anhydrase assay, 837 distinction from CO2 as product of enzymatic decarboxylations, 840841 in over-all system for I M P synthesis, 506 Bioluminescence, enzyme systems for, 851-861 Biotin, L-amino acid oxidase and, 211 Biotin analogs, inhibition of d-biotin oxidation by /-biotin and, 632 d-Biotin carboxyl-C 14, oxidation by liver slices, 631, 632 preparation of, 631 d-Biotin oxidase, in kidney and liver slices, 631-632 Bis-p-nitrophenylphosphate, hydrolysis by prostatic phosphatase, 524

SUBJECT INDEX Bisulfite, inhibition of hydroxylamine reductase by, 419 Blood clotting, 139-166, see also under names of individual components, antifibrinolysin, 165 antithrombin, 162 antithromboplastins, 160-162 cofactor of heparin, 163 convertin (SPCA, Factor VII, cothromboplastin), 155-156 fibrinogen, 158-160 fibrinolytic activators from tissues, 166 hypothesis of, 139-140 plasma Ac-globulin (proaccelerin, Factor V), 151-153 pro-convertin (SPCA precursor), 153154 pro-fibrinolysln (plasminogen), 163165 prothrombin, 140-146 serum Ac-globulin (accelerin), 153 thrombin, 156-158 thromboplastin (thrombokinase), 146151 Blood plasma, see Plasma Boiled juice (kochsaft), from pig heart, as source of flavin, 407, 416 Bone, alkaline phosphatase from, 539-541 phosphodiesterase in, 540 Borate, catalysis of CO~ hydration by, 836 complex of with a-hydroxy acids, 290 with carbohydrates, 291 inhibition of alkaline phosphatase by, 538 of ATPase (myosin) by, 588 of DNase by, 442 of "5" nucleotidase of seminal plasma by, 549 stabilization of enol form of p-hydroxyphenyl pyruvate by, 290 Borohydride, sodium, reduction of flavin component of cytochrome c reductase by, 691 Bottom yeast, see Yeast, brewer's Brain, adenylate kinase (myokinase) in, 602

909

DPNase (pyridine transglycosidase) from, 662 I)PN pyrophosphorylase in, 671 glutamic acid decarboxylase in, 199 glutamic-oxalacetic transaminase activity of, 184 L-glutaminase in, 382 glutamine synthetase from, 339, 341342 ~-glutamyltransferase (Mn-dependent) from, 269-271 lipid antithromboplastin from, 161 pyridine nucleotide transhydrogenase in, 681, 687 thromboplastin from, 139, 149 Brewer's yeast, see Yeast, brewer's British anti-lewisite (BAL) (2,3-dimercaptopropanol), activation of aspartase by, 388 of citrullinase by, 378 of guanidinoacetate methylpherase by, 263 effect on metal content of uricase, 489 inhibition of DPNH cytochrome c reductase activity of mitochondria by, 693 of tyrosinase by, 826 Bromelia pinguin, see Maya plant Bromelin, protease from pineapple, 56, 62-63 as meat tenderizer, 55 Bromoacetyl amino acids, action of acylase I on, 118 n-Butanol, extraction of aspartase from bacteria with, 387 extraction of kidney with borate and, 486, 487 Butylamine, amine oxidase action on, 393 4-t-Butylphenol, oxidation by tyrosinase, 826 n-Butyrate, inhibition of d-biotin oxidation by, 632 Butyryl phosphate, hydrolysis of, 556 C Cabbage, dehydroascorbic acid reductase in, 848, 85O

910

SUBJECT INDEX

Caeodylate, catalysis of CO2 hydration by, 836 Cadaverine (1,5-diaminopentane), as substrate for diamine oxidase, 396 Cadmium ion, activation of iminodipeptidase by, 100 inhibition of aminotripeptidase by, 87 of aspartase by, 388 of prolidase by, 105 Calcium carbonate, carbonic anhydrasc effect on deposition and solution of, 843 Calcium chloride, in assay of potato apyrase, 591, 646 of prothrombin, 140 of rennin by milk-clotting, 69 Calcium ions, activation of actomyosin ATPase by, 587 of adenylate kinase by, 603 of alkaline phosphatase by, 538 of ATP-creatine transphosphorylase by, 610 of metaphosphatase by, 579 of myosin ATPase by, 587 of pantetheine kinase by, 635 of proconvertin by, 154 of prothrombin by, 145 of thromboplastin by, 150 of triphosphatases by, 581 effect on isoelectrie point of trypsin, 35 on stability of trypsin, 32 on trypsinogen transformation, 26 inhibition of condensing system for arginine synthesis by, 364 of D P N H eytochrome c reductase by, 692 of firefly luciferase by, 856 of flavokinase by, 645 of glutamine synthetase by, 342 of glycyl-L-leucine dipeptidase by, 107 of inorganic pyrophosphatase by, 575 of insect muscle ATPase by, 598 of liver mitochondrial ATPase by, 595 of Mg-Activated ATPase by, 590 of " 5 " nucleotidase of seminal plasma by, 549

antagonism by magnesium ion, 549 of pantothenate-synthesizing enzyme by, 621 of penicillinase by, 123 of RNase by, 433 of thrombin activity by, 160 role in blood clotting mechanism, 152, 154, 158, 160 Calcium phosphate, chromatography of lactoperoxidase on, 814 of liver catalase on, 777 preparation for chromatography, 814 preparation of gel of, 214, 572, 805 Calgon, inhibition of adenylate kinase by, 603 Canavanine, formation of canavaninosucclnic acid from, 367 Cancerous tissue, dehydropeptidases in hepatic, 114 triphosphatase in, 580, 581, 582 Cantaloupe fruit, glutathione reductase in, 721 Caper spurge (Euphorbia lathyris, cerifera), protease (euphorbain) from latex of, 57, 64 Capryl alcohol, inhibition of apparent L-amino acid oxidase by, 211 Caprylate, P : 0 ratio during oxidation of, 613 Carassius carassius (crucian), thiaminase in, 625 Carbamate, carbonic anhydrase effect on dissociation of, 843 Carbamyl-n-glutam ate, role in citrulline synthesis, 350-355 Carbitol acetate (diethylene glycol monoethyl ether acetate), fractionation of arginase with, 373 Carbobenzoxyglycyl-L-amino acids, as substrates for carboxy peptidase, 78, 79 Carbobenzoxy-L-phenylalanine, as substrate for a-chymotrypsincatalyzed oxygen exchange reaction, 21

SUBJECT INDEX Carbobenzoxy-L-tyrosine ethyl ester, as substrate for trypsin and chymotrypsin, 23 Carbohydrates, content of in horseradish peroxidase, 809 presence of in thromboplastin, 150 Carbon dioxide, bicarbonate formation from hydroxyl ion and, 843 catalysis of reversible hydration of, 836-841, 843-846 by carbonic anhydrase, 836-841, 843-846 by oxy-acid buffers, 836, 838 citrulline synthesized from ornithine, ammonia and, 350 distinction from HC03- as product of enzymatic decarboxylations, 840, 841 formation by citrullinase, 374 by uricase, 485 velocity constant for uncatalyzed hydration of, 839, 840 Carbonate ion, inhibition of alkaline phosphatase by, 538 of carbonic anhydrase by, 839, 845 Carbonic acid, see Carbon dioxide Carbonic anhydrase, 836-846 assay methods for, 836-840 colorimetrie procedures for, 836, 339-840 CO2-Veronal indicator method, 839-840 rapid-flow method, 840 Krebs-Roughton manometric procedure, 836-839 limitation by COs diffusion rate, 838, 844 distinction between enzymatic COs and HC03- production by use of, 840-841 from erythrocytes, 838, 841-842, 843-846 crystalline preparation of, 841 inactivation during shaking, 838 prevention by peptone, 838 Keilin and Mann method for purification of, 841-842

911

zinc content of, 842, 843 sources of, 841, 842 from spinach, 836-840, 842-846 cyanide and sulfonamide insensitivity of, 845 essential sulfhydryl groups in, 846 evidence for zinc-protein nature of, 844 isolation as metal-free homogeneous protein, 846 zinc content of, 843 in Tradescantia fluminensis, 845 Carbon monoxide, complex with ferrocytochrome c, 754 compound with horseradish peroxidase, 812 detection of inactive form of cytochrome c by, 749 effects on spectra of cytochrome a group, 733, 734 inhibition of hydrogenase by, 866, 868 light-reversal of in P. vulgaris, 866 distinction from E. coli enzyme, 868 of tryptophan peroxidase by, 246 reversal by light, 246 of tyrosinase by, 826 non-reactivity with cytochrome b, 745 Carbonyl reagents, see also under names of individual reagents, inhibition of L-amino acid oxidase by, 2O8 of bacterial transaminases, by, 177 of diamine oxidase by, 396 of pyridoxal phosphate-requiring enzymes by, 241, 253 *Carboxylase (a-keto acid), in squash, 192, 193 inhibition by phenol, 192 in yeast, C02 versus HCO3- as product of, 840 Carboxyl groups, liberation by peptidases, estimation by Grassmann and Heyde procedure, 83, 88, 93-94, 97, 100, 105-106, 107-108 Carboxylic acids, inhibition of carboxypeptidase by aromatic and heteroeyclic, 79

912

SUBJECT INDEX

,-y-Carboxymethyl-Aa-butenolide, absorption spectrum of, 282 as lactone intermediate in/~-ketoadipic acid formation, 273, 282, 284 ~-Carboxymuconic acid, conversion of to B-ketoadipic acid, 273, 285 formation by protocatechuic acid oxidase, 273, 284-287 molar extinction coefficients for, 285 ~-Carboxymueonic acid decarboxylase, separation from protocatechuic acid oxidase, 286 Carboxypeptidase, from beef pancreas, 77-83, 118 acylases I and I I I compared with, t18 crystallization of, 82-83 effect on chymotrypsins, 8 failure to activate procarboxypeptidase, 80 formation from zymogen and pancreatic exudate, 81 physical properties of, 78 presence of zinc in, 78 resistance to DFP, 78 stability of, 78 substrates for, 78 strt~ctural analogs of as inhibitQrs, 79 Carica papaya, see Papaya Carnosinase, sources of, 94 from swine kidney, 93-96 Carnosine, see/~-Alanyl-L-histidine Carp, thiaminase in, 625 Carrots, glutamic acid decarboxylase in, 194 Casein, action of prostatic phosphatase on aform of, 526-527 inhibition by f~-casein, 527 proteolysis by rennin, 77 as substrate for trypsin and chymotrypsins, 19, 33-34 Catalase(s), assay of, direct spectrophotometric method for intact cells, 769

iodometric titration method, 785, 789 manometric method, 768-769, 781 perborate as oxidizing agent for, 780 permanganate titration method for, 768, 779, 781-782, 791-792 polarographic method, 780 ultraviolet spectrophotometric method for, 764-768 calculations for specific activity of, 767, 785 from liver, 775-781, 791-794 crystallization of, 776-779, 793 isoelectric point of, 776 separation into two components, 778 from M. lysodeikticus, 784-788 absorption bands for, 788 molecular weight of, 788 in Ps. fluorescens, 764 association with cytochrome c peroxidase, 764 from red blood cells, 781-784 absorption bands for, 784 crystallization of, 783-784 extinction coefficients for, 782, 784 molecular weight of, 784 paramagnetic susceptibility of, 784 sensitivity to alcohol-chloroform treatment, 782 from spinach leaves, 789-791 absorption bands of, 789 crystallization of, 790 role in assay of alanine racemase, 212, 213 of D-amino acid oxidase, 199 of L-amino acid oxidase, 199 of diamine oxidase, 394 of glycine oxidase, 225 of tryptophan peroxidase, 244, 246 of xanthine oxidase, 483 separation from D-amino acid oxidase, 2O0 from L-amino acid oxidase, 209 from various sources, 209, 765, 775 crystallization of, 775 molecular weights, extinction coefficients and specific activities of, 765 in Zwisehenferment preparations, 712, 718 inhibition by cyanide, 712

SUBJECT INDEX

Catechol, oxidation b y peroxidase complex II, 810 by pyrocatechase, 281-282 by tyrosinase, 819 Catechol monomethyl ether, see also Guaiacol, oxidation by peroxidase complex II, 810 Catecholase (catechol oxidase, polyphenol oxidase), see also Tyrosinase, tyrosinase as, 817, 819-821 assay of, 819-821 Catechols, substituted, oxidation by tyrosinase, 826 Cathepsin C, from beef spteen, 64-68 action on esters, 68 hydrolytic action of, 64 transamidation by, 65, 68 insoluble polymeric peptides as products of~ 68 pH dependence of, 68 Cations, divalent, inhibition of aminotripeptidase by, 87 of D P N H cytochrome c reductase by, 692 pantothenate synthesis requiremen~ for monovalent and, 621 Cauliflower, dehydroascorbic reductase from, 849, 85O Cclite, chromatography of liver catalase on, 777 Cellulose column, separation of venom phosphodiesterase from monoesterase by, 562-563 Cephalosporin P, inactivation by crude penicillinase, 123 Cetyltrimethylammonium bromide, inhibition of aspartic acid decarboxylase by, 188 reversal by pyruvate, 188 Charcoal, preparation of activated, 532 Chelate compound(s), formation by cobalt in glycylglycine dipeptidase action, 109 by manganese ion in prolidase reaction, 105

913

Chenopodium album, see Lamb's quarters Chloralosane, effect on renin action, 134 Chloramine-T, measurement of aspartate with, 170171

ChloreIla pyrenoidosa, arginine desimidase in, 376 Chloride, activation of 5'-AMP deaminase by, 472 effect on I~amino acid oxidase by, 208 inhibition of D P N H cytochrome c reductase by, 692 of erythrocyte carbonic anhydrase by, 845 stabilization of metal-free carbonic anhydrase by, 846 Chloroacetyl-amino acids, action of acylases on, 117, 118, 119 of dehydropeptidases I and II on, 113 of liver peptidase on, 114 Chloroaeetyldehydroalanine, preparation of, 110 as substrate for dehydropeptidase II, 109, 110 for liver dehydropeptidase, 114 Chloroacetyldehydroamino acids, action of dehydropeptidases I and I I on, 113 Chloroacetyldehydroleucine, as intermediate in synthesis of glycyldehydroleucine, 110 Chloroacetyl-L-glutamate, role in citrulline synthesis, 355 Chloroacetyl-L-phenylalanine, as substrate for carboxypeptidase, 78 Chloroaniline, detoxication of diphtheria toxin by myeloperoxidase and o and p forms of, 800 3-Chloro-4, 4'-diaminodiphenyl sulfone, effect on prothrombin activation, 145 Chloroform, activation of profibrinolysin by, 165 use in denaturation of hemoglobin, 102-103 p-Chloromercuribenzoate, activation and inactivation of RNase by, 434

914

SUBJECT INDEX

inhibition of aspartase by, 388 of bacterial luciferase by, 860 of D P N H cytochrome c reductase by, 693, 698 of firefly luciferase by, 856 reversal by glutathione, 856 of glutamic dehydrogenase by, 224 of glutamine synthetase by, 342 of homogentisate oxidase by, 295 of hydroxylaminc rcductase by, 419 irreversibility of, 419 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 of insect muscle ATPase by, 598 of Mg-activated ATPase by, 590 of myosin ATPase by, 587 of nitrate reductase by, 415 of plant carbonic anhydrase by, 846 reversal by cysteine or glutathione, 846 of prolidase by, 105 of tryptophan synthetase by, 237 Chloroplasts, Hill reaction with glutathione reductase system and, 722 dl-Chloropropionyl-amino acids, action of acylase I on, 118 Cholate, in purification of cytoehrome oxidase, 739 solubilization of eytochrome b with, 743 Cholesterol, presence of in heparin eofaetor, 163 in thromboplastin, 150 Chondrosulfatases, 324 Chromate, catalysis of C02 hydration by, 836 Chromatography, column, purification of barbiturase by, 493 of eatalase on calcium phosphate or celite by, 777 of eytoehrome e (bacterial) on kaolin by, 762 of dihydroSrotic dehydrogenase by, 496 of lactoperoxidase on calcium phosphate by, 814 of lactoperoxidase on silica celite by, 815

Chromatography, paper, of purine and pyrimidine bases and nucleosides, 457 for qualitative measurements of transaminations, 171, 172, 173, 175 separation of deoxyribosides by, 465, 466 Chymotrypsin (s), 8-26 action of on amides and esters, 21 cross-reactivity with trypsin on a synthetic substrate, 21 determination of activity of, 19-26 colorimetric test for amidase activity, 22-23 manometric assay of esterase activity, 23-26 milk clotting test, 20 potentiometric determination of esterase activity of, 23 substrates for, 23 spectrophotometric method of Kunitz, 19-20 modification of, 20 effect of carboxypeptidase on, 8 isoelectric points of, 25 molecular weights of, 24 optical factors for, 19 standard activity curves for, 20 terminal groups of, 8 a-Chymotrypsin, see also Diisopropyl phosphate a-chymotrypsin, action of on carbobenzoxy-L-phenylalanine (oxygen exchange), 21 as contaminant of a-chymotrypsinogen, 11-12 crystallization of, 12-13 formation of from a-chymotrypsinogen, 8, 12 hydrolysis of C - - C bond by, 21 transformation into fl- and -y-chymotrypsins, 8-14 transpeptidation and, 21 ~-Chymotrypsin, crystallization from pancreas, 14-15 7-Chymotrypsin, crystallization of from pancreas, 14 ~-Chymotrypsin, see also Diisopropyl phosphate ~-chymotryFrin, formation of from a-chymotrypsinogen, 8, 15-16

SUBJECT I N D E X

7r-Chymotrypsin, formation of from a-chymotrypsinogen, 8 Chymotrypsin B, comparison with a-chymotrypsin, 9 crystallization from pancreas, 9, 18 formation from chymotrypsinogen B, 9, 18 a-Chymotrypsinogen, chromatographic purification of, 12 conversion to ~-chymotrypsin, 8, 12 to ~r- and ~-chymotrypsins, 8, 15, 16 crystallization of, 8, 10, l l mechanism of activation by trypsin and by chymotrypsin, 8-9 Chymotrypsinogen B, activation to chymotrypsin B, 9, 18 crystallization of, 17, 18 deoxyribonuclease and, 10, 17 Chymotrypsinogen(s), 8-26 as byproduct of DNase preparation, 439 isoelectric points of, 25 molecular weights of, 24 Ciliary bodies, tyrosinase from, 829, 830 Citrate, activation of 5'-AMP deaminase by, 469, 472 of cystathionine cleavage enzyme by, 313 of prostatic phosphatase by, 557 of prothrombin by, 145 inhibition by triaminodiphenyl sulfones, 145 stimulation by 3-chloro-4,4'diaminodiphenyl sulfone, 145 effect on pH optimum of 5'-AMP deaminase, 473, 599 inhibition of acetyl phosphatase by, 556 of adenosine kinase by, 500 of adenylate kinase by, 599, 603 of aspartase by, 388, 390 of carnosinase by Mn ++ plus, 96 of DNase by, 442, 447 of D P N H eytochrome e reductase by, 692 competition with cytochrome c, 692

915

of iminodipeptidase by, 100 of leucine aminopeptidase by, 93 of phosphatases by, 313 of plasma Ae-globulin by, 152 of prolidase by, 105 P : O ratio during oxidation of, 613 Citrovorum factor (CF), see also Leucovorin, effect of folic acid conjugase on content of, in natural materials, 629 formation from 10-formylfolic acid by liver enzymes, 629 lability to acid, 630 Citrullinase (citrulline ureidase), in bacteria, 374, 376-378 ATP generation by, 376-377 distribution of, 378 Citrulline, action of citrullinase on, 374 arginine synthesis from, 356-357, 364 formation by arginine desimidase, 374 role of heavy metals in colorimetric determination of, 351,359 as substrate for L-amino acid oxidase, 2O8 Citr ulline-synthesizing system, from rat liver, 350-355 enzyme E1 for synthesis of "Intermediate," 350, 355 enzyme E~ for conversion of "Intermediate" and ornithine to citrulline, 350, 355 preparation of "Intermediate," 352353 Clam, see Meretrix meretrix, Venus mercuriana Clarase (Takamine), metaphosphatase in, 579 CIostridia, amino acid reductases in, 218 Clostridium kluyveri, cytochrome c peroxidase in, 764 hydrogenase from, 729-732 Clostridium pasteurianum, hydrogenase from, 869-870 Clostridium perfringens, arginine desimidase in, 375, 376 citrullinase in, 378 Clostridium septicum, ornithine decarboxylase from, 189

916

SUBJECT I N D E X

Clostridium welchii, aspartic acid decarboxylase from, 182, 188 glutamic-oxalacetic transaminase activity of, 84 L-glutaminase in, 382 growth of, 187, 188 histidine decarboxylase from, 187 inactivation of ATPase by lecithinase from, 590 transaminase in, 173, 176, 182 for pyridoxamine phosphatealanine system, 173 Clot-dissolving system, mode of action of, 140 Clotting, see Blood clotting and Milk clotting Cobaltous ion, activation of acylase I by, 119 dependence on nature of substrate, 119 activation of arginine desimidase by, 376 of citrullinase by, 378 of 3,-glutamyltransferase (brain) by, 272 of glycylglycine dipeptidase by, 107, 109 mechanism of, 109 of inorganic pyrophosphatase by, 575 of metaphosphatase by, 579 of triphosphatase by, 581 effect of on firefly luciferase system, 856 on flavokinase, 645 inhibition of aspartase by, 388 of 5-dehydroquinase by, 307 of fructose diphosphatase by, 546 of RNase by, 434 of D-serine (D-threonine) dehydrase by, 324 of splitting enzyme for arginine synthesis by, 367 of tryptophan synthetase by, 237 of uricase by, 489 Coenzymes, see also under names of individual coenzymes, metabolism of vitamins and, 619-677 requirement by modified mltochondria, 615

Coenzyme A, cleavage by potato enzyme, 659 effect on firefly bioluminescence system, 651 formation in liver extracts, from dephospho CoA, 649-651 from phosphopantetheine and ATP, 633, 667, 669 measurement of, 633, 649-650, 667-668 reagents for, 633 3'-nucleotidase action on, 553 removal of with Dowex-1, 349, 633 role in hippuric acid synthesis, 346, 348, 349 Colostrum, trypsin inhibitor from, 37, 46-48, 50, 51, 52 Convertin [serum prothrombin conversion accelerator (SPCA), Factor VII, cothromboplastin], complexes of with thromboplustin and plasma Ac-globulin, 151 purification of from plasma, 155-156 from serum, 156 role in clotting mechanism, 139-140, 150, 155, 156 Copper, see also Cupric ion, catalysis of leuco MB oxidation by, 716 complexing agents for, effect on ascorbic acid oxidase, 835 on tyrosinase, 826 content in ascorbic acid oxidase, 834 comparison with activity of ionic copper, 834 content in tyrosinase from mushroom, 825 in uricase, 489 inhibition of bacterial luciferase by, 860 of firefly luciferase by, 856 of glycine oxidase by, 227 of nitroethane oxidase by, 402 of D-serine (D-threonine) dehydrase by, 324 of tryptophan synthetase by, 237 of uricase by, 489 requirement for mammalian tyrosinase activity, 830 Corbicula sandal, thiaminase in, 625

SUBJECT INDEX Cortisone, effect on tryptophan peroxidase activity of liver, 246 Corynebacterium diphtheriae, cytochrome bl in, 745 growth of, 746 Cothromboplastin, see Convertin Coupled phosphorylation, see Phosphorylation, oxidative Cream, xanthine oxidase from, 483 Creatine, enzymatic phosphorylation of, 605 in oxidative phosphorylation assay, 611 synthesis by guanidinoacetate methylpherase, 260 Creatine kinase, see Adenosine triphosphate-creatine transphosphorylase Creatine phosphate, see Phosphocreatine p-Cresol, as substrate for tyrosinase, 818, 826 Cresolase, see also Tyrosinase, Monophenolase, tyrosinase as, 817, 818-819 assay of, 818-819 Crotalus atrox venom, "5" nucleotidase action of, 550 Crotonate, inhibition of d-biotin oxidation by, 632 Crucian, see Carassius carassius Cruciferae, dehydroascorbic reductase in, 849 Crystallization, of asclepain (protease) from latex of milkweed, 62 of ATP-creatine transphosphorylase, 608-609 of carbonic anhydrase from erythrocytes, 841 of carboxypeptidase from pancreas, 82-83 of catalase from beef liver, 776-779, 793 from M. lysodeikticus, 787 from red blood cells, 783-784 from spinach leaves, 790 of chymopapain (protease) from latex of papaya, 61 of a-chymotrypsin from pancreas, 1213

917

of ~o and ~-chymotrypsins from pancreas, 14-15 of ehymotrypsin B from pancreas, 9, 18 of chymotrypsinogen B from pancreas, 17-18 of cystathionine cleavage enzyme from pig liver, 312 of DNase from beef pancreas, 440--441 of DP-a-chymotrypsin, 14 of DP-~-chymotrypsin, 8, 16 of ficin (protease) from latex of fig, 61 of firefly luciferase, 856 of L-glutamic dehydrogenase from liver, 223-224 of glutathione reductase from yeast, 723 of inorganic pyrophosphatase from yeast, 573-574 of lactic dehydrogenase (cytochrome b2) from yeast, 746 of mexieain (protease) from latex of cuaguayote, 62 of old yellow enzyme, 714 of papain (protease) from latex of papaya, 59, 60 of pepsin from commercial pepsin, 5 of pepsinogen from swine mucosa, 6 of peroxidase from horseradish, 806-807 from milk, 815-816 of rennin from commercial rennet, 75-76 of rhodanese, 334-337 of ribonuclease from pancreas, 430-432 of trypsin from pancreas, 26, 29-31 of trypsin complex with trypsin inhibitor from colostrum, 47-48 from lima bean, 48-49 from pancreas, 38-39 from soybean, 44-46 of trypsin inhibitor from pancreas, 36, 38-40 from soybean, 42-44 of trypsinogen from beef pancreas, 26, 27-29 of urease, 378-379 Crystal violet, inhibition of glutamine synthetase by, 342

918

SUBJECT INDEX

Cuaguayote (Pileus mexicanus), crystalline protease (mexicain) from latex of, 56, 62 Cucumber fruit, glutathione reductase in, 721 Cucurbita pepo condensa, see Squash, yellow Cupric ion, see also Copper, activation of -y-glutamyltransferase (bacterial) by, 269 of p-hydroxyphenlpyruvate enolketo tautomerase by, 291 inhibition of arginine desimidase by, 376 of ascorbie acid oxidase by, 835 of ATP-creatine transphosphorylase by, 610 of dehydroaseorbie reductase by, 849 of 5-dehydroquinase by, 307 of DNase by, 442 of DPNH eytochrome e reductase by, 692 of fructose diphosphatase by, 546 of myosin ATPase by, 587 of nitrate reductase by, 415 of organic nitrate reductase by, 405 of quinone reductase by, 728 of renin by, 134 of RNase by, 434 of thiaminase by, 625 of tryptophan peroxidase by, 246 Cyanide, activation of guanidinoacetate methylpherase by, 263 of papain by, 59 in assay of cytochrome bl, 746-748 of cytochrome c reductase, 689 of diaphorase, 708 compound of catalase with, 788 of ferricytochrome c with, 754-755 of peroxidase with, 812 and cytochromc b, lack of reaction between, 745 effect of on activity and spectrum of myeloperoxidase, 799 on metaphosphatase, 579 on 3'-nucleotidase, 554 inhibition of alkaline phosphatases by, 527, 533

of amine oxidase by, 393 of apparent L-amino acid oxidase by, 211 of ascorbic acid oxidase by, 835 of aspartase by, 388 of bacterial luciferase by, 860 of carbonic anhydrase by, 844, 845 of carnosinase by, 96 of catalase by, 712, 718, 790 of cysteine desulfhydrase by, 317 of cytochrome bl of E. coli by, 748 distinction from diphtherial cytochrome bl, 748 of cytochrome c degradation by, 169 of cytochrome c peroxidase by, 763 of 5-dehydroquinase by, 307 of diamine oxidase by, 396 of DPNH oxidase by, 308, 683 of enzymatic nitrogen gas formation by, 423 of histidase by, 231 of homogentisate oxidase by, 292 of hydrogenases by, 866, 868, 870 dependence on aerobic conditions in P. vulgaris, 866 on anaerobic conditions in E. coli, 868 of hydroxylamine reductase by, 419 of kynureninase by, 253 of nitrate reductase by, 415 of nitroaryl reductase by, 410 of nitroethane oxidase by, 402 of rhodanese by, 337 of D-serine (D-threonine) dehydrase by, 324 of tryptophanase by, 241 of tryptophan peroxidase by, 246 of tryptophan synthetase by, 237 of tyrosinase by, 826 of uricase by, 489 of xanthine oxidase by, 484 relation to metal content, 484-485 removal of molybdenum from nitrate reductase with, 415 as substrate for rhodanese, 334-337 Cyanide-C 14, in preparation of radiobiotin, 631 Cyclic nucleotides, see under Mononucleotides, Dinucleotides, Pyrimidine ribose nucleotidcs

SUBJECT INDEX

Cypridina hilgendorfii, luciferase and luciferin from, 851-853 Cyst, athionine, preparation of L- and D-allo-isomers of from ~-homocysteine, 311,312 Cystathionine cleavage enzyme(s), 311314 from bacteria, 314 from liver, 311-314 Cysteic acid, decarboxylation in mammalian tissues, 199 Cysteine, see also Thiol compounds, Mercapto compounds, activation of adenylate kinase by, 603 of 5'-AMP deaminase by, 472 of eathepsin C by, 68 of dephospho-CoA kinase by, 649, 651 of dihydroSrotic dehydrogenase by, 496 of fructosediphosphatase (purified) by, 546 of guanidinoacetate methylpherase by, 263 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 of myosin ATPase by, 587 of nitrate reductase by, 415 of papain by, 59 of phosphoglucomutase by, 676677 activation and stabilization of tryptophanase by, 241 in assay system for hippuric acid synthesis, 349 effect on nitroaryl reductase, 410 on 3'-nucleotidase, 554 formation by cleavage of cystathionine, 311, 312 glutathione synthesis from, 342 inhibition of alkaline phosphatases by, 527, 538 of aminotripeptidase of thymus by, 87 of carnosinase by, 96 of fructose diphosphatase by, 546 of hippuric acid formation by, 350 of n-serine (D-threonine) dehydrase by, 324

919

of tryptophan synthetase by, 237 of tyrosinase by, 826 phosphopantetheine maintenance by, 669 presence in myosin, 587 protection of carbonic anhydrase (plant) with, 8A2, 843, 846 of hydrogenase by, 218 of nitrate reductase by, 414 reduction of dehydroascorbie acid by, 850 of, nitrate esters by, 405 of p-quinone by, 728-729 as substrate for L-amino acid oxidase, 2O8 Cysteine desulfhydrase, distribution of, 318 from liver, 315-318 Cysteine sulfinate--glutamate transaminase, paper chromatography for detection of, 172 Cysteine sulfinic acid, conversion to ~-suliinyl pyruvic acid by transaminase, 333 Cystine, action of cysteine desulfhydrase on, 317 inhibition of homogentisate oxidase by, 295 reversal by mercaptoacetie acid, 295 Cytidine (cytosine riboside) (CR), deamination of, 478 molecular extinction of, 478 nueleosidase action on, 459 R/values for, 466 Cytidine-3'-benzylphosphate, 3'-cytidylic acid formation from by spleen diesterase, 568 Cytidine triphosphate (CTP), as phosphate donor to AMP, 603 3'-Cytidylic acid (3'-CMP), 3r-nucleotidase action on, 553 5'-Cytidylic acid (5'-CMP), as acceptor in nucleoside monophosphate kinase reaction, 603 "5" nucleotidase action on, 549 Cytidylic acid derivatives, 2'- and 3'-cytidylic acid formation from, 568

920

SUBJECT INDEX

Cytochrome a, assay of with CO, 733 extinction coefficient for, 733 in heart particles, 737, 739 occurrence with cytochrome a3, 733 in oxidation of hematin peptide, 169 in purified cytochrome oxidase preparation from heart particles, 739 Cytochrome al, in bacteria, 732 particulate nature of, 737 as terminal respiratory enzyme of Acetobacter pasteurianum, 732733, 734 extinction coefficients for CO compound and reduced form, 734 Cytochrome a2, in bacteria, 732, 735 occurrence with cytochrome al, 733 particulate nature of, 737 Cytochrome a.~ (cytochrome oxidase), assay of, 735-737 by cytochrome oxidase activity, 735-737 by spectral properties, 735 i~ B. subtilis, 734, 735 failure of to oxidize mammalian cytochrome c, 735 in cytochrome c assay, 750 distribution of, 732 extinction coefficients for CO compound and reduced form in different sources, 734 in heart particles, 734, 737-740 oxidation by molecular oxygen, 732 oxidation of bacterial hemochromogen by mammalian, 699 of cytochrome b by~ 740 of reduced cytochrome c by, 732 in purified preparation from heart particles, 739 as terminal respiratory enzyme, 732 Cytochrome a group (a, al, a2, a3), 732740 absorption spectra of reduced and CO compounds, 733 assay methods for, 733-737 preparations containing, 737-740 heart muscle particle suspension, 738-740

special aPparatus for distinguishing CO compounds of reduced components, 734, 735 Cytochrome b, in baker's yeast and bacteria, 744, 745 in oxidation of hematin pcptide, 169 from pig heart, 740-744 absorption bands of ferrous form, 740, 744 comparison with bands of cytochrome 5532 744 assay of, 741-742 as link between succinate and cytochrome c (SC activity), 740741,742, 745 oxidation by cytochrome oxidase, 740 oxidation-reduction potential of, 740 purification of, 741, 742-744 Cytochrome bl, 744, 745, 746-748 from Corynebacterium diphtheriae, 746-748 absorption bands of, 747 assay of enzyme activity of, 747748 autoxidation of, 748 hemin content of, 747 methylene blue or ferricyanide oxidation of, 748 reduction of, 747 nitrate reduction and, 746 in succinate oxidation, 745, 746 Cytoehrome b2, in yeast, 744, 745, 746 association with lactic dehydrogenase activity, 746 Cytochrome b group (b, bl, b2), in bacteria, 744-748 absorption bands of, 744, 745 variation with species and strain, 744, 745 autoxidizability of, 745 non-reactivity with iron reagents, 745 oxidation potential of, 745 position in respiratory'chain, 745 protoheme as prosthetic moiety of, 745

SUBJECT INDEX Cytochrome c (Ferri- and Ferrocytochrome e), absence from purified cytochrome oxidase preparations, 739 bacterial sources of, 759 from heart, 749-755 assay methods for, 749-750 manometric, 750 spectrophotometric, 749 bound form of in particulate preparations, 710, 738 combination of oxidized form with azide, 755 with cyanide, 754-755 with cytochrome c reductase, 692 of reduced form with CO, 754 competition with citrate and pyrophosphate for cytochrome reductase, 692 cytochrome b as possible link between succinate and, 740-741, 742 cytochrome b assay by reduction of, 741-742 difference in activity of added and endogenous, 750 electrophoretically purified preparations of, 754 enzymatic degradation of, 167-169 enzymatic reduction by L-a-glycerophosphate, 559 equation for degree of reduction of, 736 extinction coefficients for oxidized and reduced, 700 failure of bacterial eytochromes al and as to oxidize mammalian, 733 fractionation by chromatography, 752 histidine residue involved in iron binding by, 753 inactive autoxidizable form of, 749 detection by CO method, 749 iron content of, 751, 752 iron in prosthetic group of, 753 isoelectric point of, 750-751 isolation of oxidized form by TCA method, 751-752 of reduced form by resin method, 752-753

921

microscale application of, 753 magnetic properties of, 754 molar absorption coefficients for, 749, 753 myoglobin as contaminant of, 751752 oxidation by acidification, 751, 754 by alkalinization, 754 by ferricyanide, 737, 754 by H~O2, 736 oxidation reduction potential of, 754 in oxidative phosphorylation assay, 611, 615, 616 peroxidase assay with, 774 preparation of H~O2-free, 736 presence in various species, 751 reduction, by chemical agents, 754 by D P N H cytochrome reductases, 692, 698-699 by T P N H cytochrome c reductase system, 703 by hydrogenase system, Mo, FAD and phosphate requirements for, 869-870 by succinate and cytochrome b, 744 by xanthine oxidase, 482 requirement by modified mitochondria, 615 spectrophotometrie measurement of reduction, 688 spectrum of reduced, 749 stability of, 754 structure compared with Ustilago cytochrome c, 758 from Ps. fluorescens, 758-760 absorption bands for oxidized and reduced, 758 adaptive formation of, 760 association of pigment with cytochrome c peroxidase, 758, 759 comparison with animal pigment, 758 inactivity with liver TPN cytochrome e reductase, 760 red fluorescence of reduced form, 760 from Ustilago sphaerogena, 755-758 absorption spectrum of, 757

922

SUBJECT INDEX

assay with Zeiss band spectroscope, 755 autoxidizability of, 757 chromatography of, 756--757 iron content of, 757 isoelectric point of, 758 prosthetic group of, 757-758 purity of, 757 Cytoehrome c oxidase, see Cytochrome aa

Cytochrome c peroxidase, distribution of, 764 effect on assay of cytochrome a~, 737 from Ps. fluorescens, 760-764 Cytochrome c reductase, see also DPNH and TPNH eytochrome c reductases, effect on assay of cytoehrome a3, 737 Cytochrome e, distribution of, 745 as member of cytochrome b group, 745 Cytochrome component, absorbing at 554 m# in Acetobacter, 744, 745 at 553 m~ in eytochrome b preparations from heart, 744 Cytochrmne o×idase, see Cytoehrome a3 Cytochrome system, 732-764, see also under names of individual components, dopa oxidation to melanin by, 828 in heart particles, 737-740 role in oxidation of mandelic acid, 277 Cytosine, as acceptor of deoxyriboside group, 468 Rs values for, 466 Cytosine compounds, absorption change on deamination of, 478 Cytosine deaminase, spectrophotometric determination of cytosine with, 458 Cytosine deoxyriboside (CDR), bioassay of, 464 comparison with cytidine in rates of deamination, 479 deamination of, 478 RI values for, 466 in transdeoxyribosidase reaction, 464468

Cytosine nucleoside deaminase, from E. coli, 478-480 D

Deaminodiphosphopyridine nucleotide (Deamino-DPN), as coenzyme for L-glutamic dehydrogenase, 224 lack of DPN kinase inhibition by, 654 Neurospora DPNase action on, 666 transhydrogenase reaction with DPNH, 681-687 Deaminotriphosphepyridine nucleotide (Deamino-TPN), transhydrogenase reaction with TPNH, 685 Deeamethylenediamine, amine oxidase action on, 393 Dehydrases, 319-324 D-serine (D-threonine) dehydrase, 322324 n-serine (5-thrconine) dehydrase, 319322 Dehydroalanine, peptides of, specific absorption bands of, 110 Dehydro-D-araboaseorbic acid, as substrate for dehydroascorbic reductase, 850 Dehydro->aseorbic acid, preparation of, 847 as product of aseorbie acid oxidase, 831 reduction by thiol compounds, 847-85C enzymatic, 847-850 non-enzymatic, 848-849, 850 Dehydroaseordic reductase, in cabbage juice, 848, 850 m cauliflower, 849, 850 distribution of, 84 from pea plant, 847-850 Dehydropeptidase (s), assay by ammonia determination, 110 111 of kidney, 109-114 of liver, 114 possible role in exocystine desulfhydrase action, 319 preparation of substrates for, 110 terminology of, 114 Dehydropeptidase I (solubilized aminopeptidase),

SUBJECT INDEX from particulate fraction of hog kidney, 107, 108, 109-114 action on glyeyMeucine isomers, 107 comparison with glycylglycine dipeptidase of muscle, 108 purification of, 112-113 Dehydropeptidase II (soluble acylase I), see Amino acid acylase I Dehydropeptide(s), amino acyl, as substrates for dehydropeptidase I, 109 dehydroalanyl, as specific substrates for dehydropeptidase II, 113 as probable product of exocystine desulfhydrase action, 319 resistance to prolidase, 105 5-Dehydroquinase, distribution of, 307 from E. coli, 305-307 effect on quinic dehydrogenase assay, 308 separation from 5-dehydroshikimie reductase of E. coli, 306 5-Dehydroquinic acid, formation of by quinie dehydrogenase, 307-311 as intermediate in aromatic biosynthesis, 300, 301 5-Dehydroshikimie acid, accumulation of in E. coli mutant, 307 bioassay of, 308, 309 as intermediate in aromatic biosynthesis, 300, 301 molar extinction coefficient of, 305 as product of 5-dehydroquinase action, 305-307 5-Dehydroshikimic reductase, in A. aerogenes, 308 distribution of, 304 from E. coli, 301-304 Deoxycholate, extraction of cytochrome b with, 74 Deoxycorticosterone, inhibition of D P N H cytochrome e reductase by, 693 Deoxygenase, see Glucose oxidase Deoxyoligonucleotide, as substrate for phosphodiesterase and 5'-nucleotidase, 561

923

Deoxyribonuclease(s) (DNase), from baker's yeast, 445-446 chymotrypsinogen B and, 10, 17 from group A hemolytic streptococci, 446-447 inhibition of pancreatic and streptococcal, by respective antisera, 447 in mouse leukemic tissues, 447 from pancreas, 438-443 crystallization of, 440-441 denaturation by salt, 443 physical-chemical properties of, 443 in serum, 447 from spleen, 444-445 from thymus, 443-444 activation by acid treatment, 444 Deoxyribonuclease inhibitor, as byproduct of DNase preparation from yeast, 446 in tissues, 443 Deoxyribonucleic acid (DNA), deoxyoligonucleotide formation from by DNase, 561 denatured, hydrolysis by DNase, 441 depolymerization of by sonic treatment, 274 DNase for removal of from Pseudomonas extracts, 274 inhibition of RNase by,'434 hydrolysis of by~DNase,r 437, 441, 442 physical and chemical changes during, 437, 442 products of, 441 resistance to prostatic phosphatase, 526 source of, 437 Deoxyribose-l-phosphate, elimination as intermediate in transdeoxyribosidase reaction, 468 preparation from guanine deoxyriboside, 449 Deoxyriboside (s), of 8-azaguanine, enzymatic synthesis of, 448 chromatography of and RI values for, 465-466 of hypoxanthine and guanine, action of phosphorylase on~ 448 quantitative bioassay of with thermobacterium acidophilus, 464

924

SUBJECT I N D E X

Deoxyxanthosine, phosphorolysis of, 448 Dephospho-CoA, formation from ATP and phosphopantetheine, 667, 669 Dephospho-CoA kinase, assay by firefly bioluminescence system, 651 in dephospho-CoA pyrophosphorylase preparations, 667, 669 from pigeon liver, 649-651 Dephospho-CoA pyrophosphorylase, from hog liver, 667-669 Desthiobiotin, inhibition of d-biotin oxidation by, 632 Desulfhydrase(s), activity in liver tissue, 313 cysteine desulfhydrase, 315-318 exocystine desulfhydrase, 319 homocysteine desulfhydrase, 318 from P. morgani,.318 Desulfinases, in animal tissues, 333 in bacteria, 333

Desulfovibrio desulfuricans, hydrogenase from, 869, 870 Deuterium oxide, see Heavy water Deuteron and electron bombardment, for molecular weight of DNase, 443 Diacetylmonoxime, in colorimetrie determination of citrulline, 351, 359 Dialyzer, Kunitz and Simms, 431 Dialyzing membranes, treatment of with glutathione, 414 Diamine oxidase (histaminase), from hog kidney, 394-396 Diamines, oxidation by peroxidase complex II, 8O8 Diaminopimelic acid, D-amino acid oxidase and, 202 Diamino purine, see 2-Aminoadenosine Diamox 6063 (2-acetylamino-l,3,4thiodiazole-5-sulfonamide), inhibition of erythrocyte carbonic anhydrase by, 845 Diaphorase, action of D P N H cytoehrome c reductases as, 692, 697

DPNH-linked, from pig heart, 707711 TPNH-linked, questionable existence of, 711 Diazo-coupling method, nitroaniline assay by, 406 Diazomethane (CH2N2), compound with horseradish peroxidase, 812 Dibenamine, inhibition of amine oxidase by, 393 Dichloroflavin, phosphorylation by flavokinase, 644 2,6-Dichlorobenzenoneindo-3'-chlorophenol, in assay of cytochrome c peroxidase, 761-762 preparation of reduced form, 761 cytochrome c reduction by reduced form of, 761 2,6-Dichloro-4-nitrophenol, inhibition of quinone reductase by, 729 2,6-Dichlorophenolindophenol, in diaphorase assay, 707 measurement of ascorbic acid with, 847, 848 reduction of by L(-k)-mandelie acid dehydrogenase, 278 succinate oxidation measured with, 748 2,4-Dichlorophenoxyacetic acid, as uncoupling agent, 615 Dichloroquinone chlorimide, color reaction for uric acid, 486 Dicoumarol, effect of on clotting mechanism, 140, 154 Di (dinitrophenyl) phosphate, hydrolysis of by 3'-nucleotide phosphatase of barley, 524 Diethyldithiocarbamate, activation of uricase by, 489 inhibition of ascorbic acid oxidase by, 835 of hydroxylamine reductase by, 419 of tyrosinase by, 826 Digitonin, solubilization of beef heart transhydrogenase by, 686-687 DihydroSrotic acid, hydrolysis by dihydroSrotase, 496

SUBJECT INDEX DihydroSrotic dehydrogenase, from Zymobacterium oroticum, 493-496 Dihydroxymaleic acid, oxidation by peroxidase complex II,, 810 o-Dihydroxyphenyl compounds, oxidation by plant versus mammalian tyrosinase, 830 3, 4-Dihydroxyphenyl-L-alanine (dopa), decarboxylation in higher plants, 194 hydrogen peroxide inhibition of peroxidation of, 816 oxidation by mammalian tyrosinase, 827-831 plant tyrosinase compared with, 830 oxidation to melanin by cytochrome system, 828 peroxidase assay with, 774, 813 3,4-Dihydroxyphenylalanine (dopa) decarboxylase from guinea pig or rabbit kidney, 195, 199 from liver, 199 2,5-Dihydroxyphenylpyruvate, oxidation of, 288 Diisopropyl fluorophosphate (DFP), reaction of with a-chymotrypsin, 13 with ~-chymotrypsin, 16 with trypsin and chymotrypsins, 14 Diisopropyl phosphate a-chymotrypsin (DP-a-chymotrypsin), preparation of, 13-14 crystallization of, 14 Diisopropyl phosphate ~-chymotrypsin (DP-~-chymotrypsin), crystallization of, 8, 16 Diisopropyl phosphate trypsin (DPtrypsin), molecular weight of, 35 optical factor for, 33 Diketo acids, action of hydrolase on, 298-299 2,3-Dimercaptopropanol, see British anti-lewisite p-Dimethylaminobenzaldehyde, determination of indole with, 234 Dimethylg]ycine, as buffer, 226 3,4-Dimethylphenol, oxidation by tyrosinase, 826

925

4,6-Dimethyltryptophan, as substrate for tryptophanase, 242 Dinicotinamide ribose-5'-pyrophosphate, cleavage by Neurospora DPNase, 666 m-Dinitrobenzene, as substrate for nitroaryl reductase, 406-411 p-Dinitrobenzene, reduction of by Neurospora, 410 3,5-Dinitrobenzoic acid, reduction by Neurospora, 410 Dinitrocresol, as uncoupling agent, 615 2,4-Dinitrophenol and related compounds, inhibition of quinone reductase by, 729 reduction by Neurospora, 410 as uncoupling agent, 615 Dinitrophenylhydrazine, in colorimetric measurement of pyrurate, 174-175 2,4-Dinitrophenylhydrazone, of a-ketoglutarate, 170 Dinucleotides and related compounds, as substrates for spleen pbosphodiesterase, 568 Diol (2-amino-2-m ethyl- 1,3-propanediol) buffer, 688 Dipeptidases, carnosinase, 93-96 glycylglycine dipeptidase, 107-109 glycyl-I~leucine dipeptidase, 105-107 iminodipeptidase (prolinase), 97-100 prolidase (imidodipeptidase), 100-105 Dipeptidases, metal activated, inhibition by Versene, 87 Dipeptide(s), containing normal peptide bond, resistance to prolidase, 104 I~-histidine-containing, as substrates for carnosinase, 93, 96 lacking peptide hydrogen, as substrates for prolidase, 104 as product of aminotripeptidase action, 83 z-proline or hydroxy-L-proline-containing, as substrates for iminodipeptidase, 97 as substrates for dehydropeptidase I (solubilized aminopeptidase), 113 for leucine aminopeptidase, 91

026

SUBJECT INDEX

Dipeptide amides, as substrates for leucine aminopeptidase, 92 Diphenylamine procedure, for deoxypentose, in DNase assay, 438 Diphenylphosphate, resistance to bone phosphatase activity, 539-541 as substrate for phosphodiesterase, 561, 564

Sym-Diphenylpyrophosphate, conversion to monophenylphosphate by venom diesterase fractions, 565 3",5'-Diphosphoadenosine, action of nonspecifie deaminase on, 477, 478 Diphosphopyridine nucleotidase (DPN nucleosidase) (DPNase), from animal tissues (pyridine transglycosidase), 653, 660-663, 683 from brain, 662 inhibition of by nicotinamide, 662, 670, 683 in pigeon liver, 653 species variation in sensitivity to INH, 663 from spleen, 661-662 from Neurospora, 664-666 in potato, 655 Diphosphopyridine nucleotide (DPN, cozymase, coenzyme I), see also under DPN, action of nonspecific deaminase on, 477, 478 analogs of formed by brain DPNase, 663 cleavage by Neurospora DPNase, 664, 666 by nucleotide pyrophosphatase, 655, 659 as coenzyme for amino acid reductases, 220 for benzaldehyde dehydrogenase, 280-281 for dihydro5rotie dehydrogenase, 493, 496 for I,-glutamic dehydrogenase, 220 for quinic dehydrogenase, 307-311 for quinone reductase, 725

measurement by alcohol dehydrogenase, 660, 670 by cyanide reactivn, 660 measurement of pyridinium linkage in, 655 in nitroaryl reductase assay, 407 in oxidative phosphorylation assay, 612, 615, 616 reduction by molecular hydrogen, 729, 732 as substrate for cleavage and group transfer by animal DPNase, 660 synthesis of from NMN and ATP, 670 transhydrogenase reaction of with deamino-DPNH or TPNH, 681687 with NMNH, 685 Diphosphopyridine nucleotide, C l~-nicotinamide-labeled, Pseudomonus transhydrogenase exchange reaction studied with, 686 Diphosphopyridine nueleotide, reduced (DPNH), acid destruction of, 729 cleavage by potato nucleotide pyrophosphatase, 659 by snake venom nucleotide pyrophosphatase, 654 compared with TPNH in GSSG reductase system of liver, 725 of yeast, 724 dehydroascorbic reductase and, 850 extinction coefficient of, 670 as hydrogen donor in bacterial luciferase system, 857, 860, 861 inhibition of DPN kinase by, 654 Km value for combination with cytochrome c reductase, 692 methods for preparation of, 694 oxidation of by old yellow enzyme, 715 by peroxidase complex II, 810 P:O ratio during oxidation of, 613, 616 reduction of flavin component of cytochrome c reductase by, 691 of hydroxylamine to NH3 by, 416, 418 of iron component of cytochrome c reductase by, 692

SUBJECT INDEX of nitroaryl compounds by, 406, 408, 410 of quinones by, 725-729 role in nitrogen gas formation from nitrite, 422 as substrate for diaphorase, 707 synthesis from N M N H and ATP, 672 transhydrogenase reaction with deamino-DPN or TPN, 681-687 with NMN, 685 Diphosphopyridine nucleotide'analogs, cleavage by animal DPNase, 662 failure of Neurospora DPNase to form or cleave, 666 Diphosphopyridine nucleotide kinase (DPN kinase), from pigeon liver, 652-655 Diphtheria toxin, detoxication of by myeloperoxidase plus donor substances, 801 Diplococcus pneumoniae, aspartase in, 388 Dipotassium-2-hydroxy-5-nitrophenylsulfate, as substrate for arylsulfatascs, 328 a,a'-Dipyridyl, activation of uricase by, 489 inhibition of homogentisate oxidase by, 295 of hydroxylamine reductase by, 419 Disulfide bond, as possible active group in rhodanese, 337 Dithionite (hydrosulfite), activation of hydrogenases by, 866 decomposition of hematin peptide by, 169 deoxygenation with, for hydrogenase assays, 863, 866, 868 effect on absorption bands of cytochrome b, 744 inhibition of heparin cofactor by, 163 reduction of cytochrome bl by, 747 of cytochrome c by, 483, 749, 759 of flavin component of cytochrome e reductase by, 691 of horseradish peroxidase by, 812 Dithizone, activation of uricase by, 489

927

I.-Djenkolic acid, cleavage of, 313 Dodecyl aldehyde, as component of bacterial luciferase system, 857, 861 Dodecylamine, amine oxidase action on, 393 Dopa, see 3,4-Dihydroxyphenyl-Lalanine Dowex-1, removal of CoA with, 349, 633 DPN nucleosidase, see Diphosphopyridine nucleotidase DPN pyrophosphorylase, in brain, 671 from. hog liver, 671-672 rates of DPN synthesis and breakdown by, 612 from yeast, 671 D P N H cytochrome e reductase, from heart, 688-693 diaphorase activity of, 708, 711 from E. coli, 693-699 rate constants compared with yeast T P N H enzyme and old yellow enzyme, 697 in mitochondria, BAL and antimycin sensitivity of, 693 in particulate preparations, 710 separation from T P N H enzyme of liver, 706 D P N H oxidase, in A. aerogenes, 308 DP-derivatives, see under Diisopropyl phosphate Dyes (oxidation-reduction indicators), reduction by l)-amino acid oxidase, 204 by bacterial luciferasc system, 857, 86O by cytochrome c reductase, 692 by hydrogenase system, 862, 868 Dyes, reduced, as electron donors to F M N in bacterial luciferase system, 861 E

Egg white, trypsin inhibitor from, 37, 49 Electrophoresis, purification of L-amino acid oxidase by, 207

928

SUBJECT INDEX

of horseradish peroxidase by, 805 of myeloperoxidase by, 797-798 separation of polysaccharide from old yellow enzyme by, 714 Empyema, myeloperoxidase from, 797-798 Ene-diols, oxidation by ascorbic acid oxidase, 834 by peroxidase complex II, 810 Enol-keto tautomerase, for p-hydroxyphenylpyruvate from liver, 289-292 Enterokinase, from swine duodenum, 31-32 determination of activity of, 32 Entropy of activation, for DNase, 443 Enzyme-substrate complexes, in carbonic anhydrase reaction, 844 rate constant for formation of, 844 in peroxidase reaction, 770, 801-813 magnetic susceptibility of, 811, 813 oxidation of various hydrogen donors by, 808 rate and equilibrium constants for formation of, 810 spectra of, 811 Epinephrine (adrenaline), cytochrome c reduction by, 754 oxidation of by tyrosinase, 826 Erythritol tetranitrate, as substrate for organic nitrate reductase, 405 Erythrocytes, acid phosphatase in, 527 aminotripeptidase from, 84-85 carbonic anhydrase from, 838, 841842 catalase from, 209, 765, 775, 781-784 prolidase from, 101, 102-103, 105 RNase in, 436 uric acid riboside (UAR) in, 459 Escherichia coli, acetyl phosphatase from, 556 adenylate kinase in, 619 arginine decarboxylase from, 187 aspartase in, 388 ATPase in, 619 eytochrome bl in, 745, 746

in suecinate oxidation and nitrate reduction, 746 cytochrome c peroxidase in, 764 cytosine nucleoside deaminase from, 478, 480 5-dehydroquinase from, 305-307 5-dehydroshikimie reductase from, 301-304 D P N H cytochrome e reductase from, 693-699 glutamic acid decarboxylase from, 182, 183, 186, 187-216 growth medium for, 183 glutamic-oxalacetic transaminase activity of, 184 L-glutaminase from, 380, 382 7-glutamyltransferase (GTF) in, 269 glutathione-synthesizing system in, 342 growth of, 186, 187, 239, 380-381, 479, 512, 695, 867 hydrogenase from, 867, 868 inorganic pyrophosphatase in, 619 isolation of 5-amino-4-imidazolecarboxamide riboside from cultures of, 505, 512, 514 lysine decarboxylase from, 186-187 pantothenate auxothroph of, 619 pantothenate-synthesizing enzyme from, 619, 622 pyrimidine nucleoside phosphorylase in, 480 D-serine (D-threonine) dehydrase from, 322, 323 L-serine (L-threonine) dehydrase in, 320 transaminase from, 172, 173, 174, 176, 177 transhydrogenase in, 686 tryptophanase from, 238-242 tryptophan synthetase in, 234 Esterase activity, of cathespin C, 68 of trypsin, 36 of trypsin and chymotrypsins, 23 Ethanol, activation of ,-glutamyltransferase (bacterial) by, 269 solubilization of diaphorase by, 708 of DPNH cytoehrome c reductase of heart by, 689

SUBJECT INDEX

929

of DNase by, 442 of glutamic dehydrogenase by, 224 of penicillinase by, 123 of renin by, 134 of splitting enzyme for arginine synthesis by, 367 of thiaminase by, 625 reduction by hydroquinone, 726 Ferricyanide, effect of on induction period of monopheno!ase, 825 inhibition of aspartase by, 388 oxidation of cytoehrome b by, 748 of cytochrome e by, 737, 759 reduction of by bacterial luciferase system, 857, 860 by hydrogenase system, 862 Ferricytochrome c (CyFe+++), see Cytochrome c Ferrous ion, see also Iron, activation of fructose diphosphatase by, 546 of homogentisate oxidase by, 294, 295 of hydrogenases by, 868, 870 of p-hydroxyphenylpyruvate enolketo tautomerase by, 291 of metaphosphatase by, 579 of pyrocatechase by, 282 of triphosphatases by, 581 inhibition of 5-dehydroquinase by, 307 of dissociable amino acid decarboxylases by, 189 of DNase by, 442 of leucine aminopeptidase by, 93 of splitting enzyme for arginine synthesis by, 367 of thiaminase by, 625 Fibrin, mechanism of formation of, 140 urea resistant clot of, 158, 160 Fibrinogen, Fat, neutral, changes in end groups on conversion to presence of in heparin eofactor, 163 fibrin, 160 in thromboplastin, 150 concentration of in oxalated horse F a t t y acids, plasma, 147 inhibition of D-amino acid oxidase by, purification of, from blood, 158, 159 203 from fraction I (Armour), 159 of d-biotin oxidation by, 632 role in clotting mechanism, 140, 158Ferric ion, see also Iron, 160 inhibition of 5-dehydroquinase by, 307

Ether, effect on renin action, 134 plasmolysis of top yeast by, 581 Ethylenediamine, action of diamine oxidase on, 396 inhibition of histidase by, 231 Ethylenediaminet etraacetate (Versene), activation of cystathionine cleavage enzyme by, 313 of uricase by, 489 effect on myosin ATPase, 587 inhibition of aspartase by, 388 of bacterial luciferase by, 860 of histidase by, 231 of leucine aminopeptidase by, 87, 93 of metal-activated dipeptidases by, 87 of prolidase by, 105 of tryptophan synthetase by, 237 protection of hydroxylamine reductase by, 419 of nitrate reductase by, 414 Ethyl nicotinate, conversion to and release from DPN analog by brain DPNase, 662, 663 Ethyl-5-phenyl-3-ketovalerate, hydrolysis of C - - C bond of by a-chymotrypsin, 21 Euphorbain, protease from latex of caper spurge, 57, 64 Euphorbia lathyris, cerifera, see Caper spurge Exocystine desulfhydrase, from liver, 319 Eye tissue, pigmented, tyrosinase from, 828

930

SUBJECT INDEX

inhibition of DPNH eytochrome c two-stage prothrombin assay with, 141, reductase by, 693 142 as prosthetic group of D-amino acid Fibrinolysin (plasmin), oxidase, 199, 200, 227 activators and inhibitors of, 165 separation of from, 200 formation of by activators of proof L-amino acid oxidase of N. crassa, fibrinolysin, 165 211 role in clot-dissolving, 140 of L-amino acid oxidase of snake streptokinase-activated, 165 venom, 205 Fibrinolytic activators, of diaphorase, 710 fibrinokinase from heart, 165 reduction of by DPNH, 710 fibrinolysokinase from lung, 165 of DPNH cytochrome c reductase of urine activator, 165 bacteria, 698 Fibrinoplastic substances, of glycine oxidase, 227 acacia as example of, 159 of hydrogenase, 869, 870 Ficin, of new yellow enzyme, 718 protease from latex of fig, 56, 61 reversible removal of, 718 crystallization of, 61 of nitrate reductase, 415 Fieser's solution, preparation of, 865 of nitroaryl reductase, 407, 410 Fig (Ficus carica, glabrata, doliara), of TPNH cytochrome c reductase crystalline protease (ficin) from, 56, 61 of liver, 706 peroxidase in, 801-802 of xanthine oxidase, 485 Firefly (Photinus pyralis), protection of L-amino acid oxidase by, luciferase and luciferin from t 851, 8542O8 856 source of, 201 Firefly bioluminescence system, see also substance competing with in D-amino Luciferase, acid oxidase reaction, 203 CoA effect on, 651 synthesis of from FMN and ATP, 673 Flavin(s), see also under Alloxazine and Flavin adenine dinucleotide pyrophosRiboflavin, phorylase, from brewer's yeast, in boiled juice of Cl. kluyveri, 731 673-675 from boiled pig heart extract, 407, 416 Flavin adenine nucleotide, yeast lactic dehydrogenase containing as component of DPNH cytochrome e cytochrome b2 and, 746 reductase of heart, 691-692 Flavin adenine dinucleotide (FAD), involvementin diaphorase action, 692 activation of hydroxylamine reductase non-identity with FAD, 691 by, 419 Flavin mononucleotide (FMN, riboof nitrogen gas-forming enzyme by, flavin-5'-phosphate), 423 as absolute requirement for bacterial boiled pig heart as source of, 408 luciferase system, 861 cleavage of by pyrophosphorylase, 673in assay of nitrate reductase, 412, 415 675 complex with protein moiety of old complex of with quinine, 203 yellow enzyme, 715 determination of by D-amino acid free radical of, 715 oxidase, 673, 698 distribution coefficient (benzyl alcohol: water) for, 642 hydrolysis of by boiling, 641 by nucleotide pyrophosphatase, 655, FAD synthesis from ATP and, 673 formation of by flavokinase, 640, 641 659 ATP as competitive inhibitor of, inhibition of DPNH cytochrome c reduetase by, 693 673

SUBJECT INDEX

as prosthetic group of mammalian L-amino acid oxidase, 210 of old yellow enzyme, reversible removal of, 715 of TPNH cytochrome c reductase of yeast, 703 reduction by DPNIt in bacterial luciferase system, 857, 860 by hydrogenase system, 869 stimulation of nitrogen gas formation from nitric oxide by, 423 Flavin mononucleotide, reduced (FMNH~), autoxidation of, 861 as hydrogen donor in bacterial lueiferase system, 857 Flavokinase, from brewer's yeast, 640-645 in intestinal mucosa, 642 Flavoproteins, absorption maxima of, 691, 703, 710, 715, 719 I~-amino acid oxidases as, 208, 210, 211 atabrine and aeriflavine as specific inhibitors of, 698 DPNH- or TPNH-linked, assay by means of T2Z or NTZ, 695 Fluoride, compound with horseradish peroxidase, 812 effect on activity and spectrum of myeloperoxidase, 799 on glueose-6-phosphatase, 542 inhibition of aeetyl phosphatase by, 556 of acid prostatic phosphatase by, 527 of adenylate kinase by, 603 of aminodipeptidase, 100 of Y-AMP deaminase by, 472 effect of 5'-AMP concentration on, 472 of pH on, 473 of arginine-synthesizingsystem by, 359, 364 of citrullinase by, 378 independence of Mg ++, 378 of DNase by, 442, 447 of fructose diphosphatase by, 546 of glutamine synthetase by, 342 of -/-glutamyltransferase by, 267 of metaphosphatase by, 579

931

of methionine-activating enzyme by, 256 of Mg-activated ATPase by, 590 of nitroethane oxidase by, 402 of "5" nucleotidase of seminal plasma by, 549 of nucleotide pyrophosphatase by, phosphate requirement for, 659 of prolidase by, 105 of pyridoxal kinase by, 649 Fluoroaeetyl amino acids, action of acylase I on, 118 Folio acid eoniugase, from chicken pancreas, 629-630 in hog kidney, 629, 630 sources of, 629 in takadiastase, 630, 631 Folio acid de ivative(s), see also Leueovorin, Tetrahydrofolie acid, Anhydroleucovorin, removal by Dowex-l, 509 as requirement foJ~fo:n~ylglycinamide ribotide synthesis, 509 Folio acid heptaglutamate, effect of folio acid conjugase on apparent CF content of, 629 Folin-Cioealteu phenol reagent, in assay of hydroxytyramine, 195 of pepsin, 3-4 of proteinases, 55 Formaldehyde, inhibition of ItNase by, 434 Formamido compounds, aromatic, as substrates for kynurenine fo:mamidase, 2=19 Formate, activation of hydrogenase system by, 866 formation from formylkynurenine, 246 C 14-Formate, in measurement of formylation of 5-IRMP, 519 of formylglycinamide ribotide synthesis, 509-510 of purine synthesis, 505, 507 of tetrahydrofolate formylase reaction, 517 formazan, as insoluble product of reduction of TTZ or NTZ, 695

932

SUBJECT INDEX

Formyl amino acids, action of acylase I on, 118 Formylanthranilic acid, as substrate for kynurenine formamidase, 246, 249 Formylase, see Kynurenine formamidase Formylation, stable cofactor(s) for, 517 tetrahydrofolic acid as cofactor for, 516, 517 Nl°-Formylfolic acid, as intermediate in conversion of bound folic acid to CF, 629 as product of oxidation of tetrahydro compound, 518 Formyl-~glutamate, role in citrulline synthesis, 355 Formylglycinamide ribotide, biosynthesis of, 509-512 requirement of folic acid derivative for, 509 conversion to 5-amino imidazole ribotide, 505 isolation of, 511, 512 Formylkynurenine, hydrolysis of~ 246 as product of tryptophan peroxidase reaction, 242 N 10-Formyltetrahydrofolic acid (CHOFAH4), chemical synthesis of from N ~°formylfolie acid, 519 conversion to NS-N~°-imidazolium derivative (ACF), 518 as formyl donor to 5-IRMP, 518, 519 to glycinamide ribotide, 518 oxidation of to Nl°-formylfolic acid, 518 as product of reaction of FAH4 with formate, 517 with serine, 518 spectrophotometrie measurement of, 518 Fraction I (Armour), purification of fibrinogen from, 159 Fraction I I I of plasma, profibrinolysin from, 164-165 Fraction IV (Armour), as source of antihemophilic factor, 149

Free radical, yellow enzyme in form of, 712 Fructose-l,6-diphosphatase, from rabbit liver, 543-546 Fructose-l,6-diphosphate (FDP), resistance toward prostatic phosphatase, 525 Fructose-l-phosphate (F-I-P), hydrolysis by fructose diphosphatase, 545-546 Fructose-6-phosphate, formation of by fructose-l,6-diphosphatase, 543, 546 unknown product accompanying, 546 Fuller's earth, see Kaolin *Fumarase, in assay of splitting enzyme for arginine synthesis, 364-365, 367 Fumarate, activation of d-biotin oxidation by, 632 inhibition of aspartase by, 390 of cytochrome c degradation by, 169 of hydrogenase exchange reaction by, 867 of splitting enzyme for arginine synthesis by, 367 as product of arginine-synthesizing system, 356 of aspartase action, 386 of fumarylacetoacetate hydrolase, 298-300 of tyrosine oxidation, 287 reduction of by hydrogcnase system, R62, 867 effect of fumarate in growth medium on, 867 spectrophotometric measurement of~ 739 Fumarylacetoacetate, conversion to fumarate and aceto° acetate, 298-300 conversion of maleylacetoacetate to, 295-298 molar extinction coefficient of, 296, 299 Fumarylacetoacetate hydrolase, in assay of maleylacetoacetate isomerase, 296

SUBJECT INDEX from liver, 293, 294, 297, 298, 300 separation from homogentisate oxidase, 293, 294 from maleylacetoacetate isomerase, 297-298 Furfurylamine, amine oxidase action on, 393 G Galactose, presence of in fibrinogen and fibrin, 160 *Galactowaldenase, UDPG determination with, 677 Gastric mucosa, carbonic anhydrase in, 841 pepsin and pepsinogen from swine, 3-7 from various species, 7 Gelatin, protective action on pcnicillinase, 123 stabilization of carbonic anhydrase by, 846

Glomerella cingulata, tryptophan synthetase in, 234 Glucoascorbic acid, in oxidation of p-hydroxyphenyl pyruvate, 289 Glucosamine, presence in fibrinogen and fibrin, 160 Glucose, as acceptor for transphosphorylation by phosphatases, 533, 556 activation of -y-glutamyltransferase (bacterial) by, 269 of hydrogenase system by, 866 determination of by yeast fermentation, 330 formation of by glucose-6-phosphatase, 541-543 kinase for in pyridoxal kinase preparations, 648-649, see also Hexokinase, in oxidative phosphorylation assay, 611 *Glucose dehydrogenase, generation of D P N H by, 493, 494 Glucose-l,6-diphosphate (G-1,6-P2), ribose- 1,5-diphosphate formation from, 503

933

*Glucose oxidase (notatin, deoxygenase), deoxygenation for hydrogenase assays by use of glucose and, 866 inhibition by nitrate, 579 peroxide generation by, 244 Glucose-6-phosphatase, affinity for G-6-P, 560 from rat liver, 541-543 sources of, 542 Glucose-l-phosphate (G-I-P), enzymatic assay of, 675 UDPG synthesis from UTP and, 675 Glucose-6-phosphate (G-6-P, Robison ester), determination of formation during oxidative phosphorylation, 613 enzymatic assay of, 675 as hydrogen donor in nitrogen gas formation, 420, 422 TPN as cofactor for, 422 preparation of crystalline calcium salt, 699 as product of transphosphorylation by phosphatases, 556 enzymatic measurement of, 560 prostatic phosphatase action on, 525 as substrate for alkaline phosphatase, 533 *Glucose-6-phosphate dehydrogenase (Zwischenferment), assay of ATP with, 497 of glucose 1-P with phosphoglucomutase and, 675, 676, 677 cytochrome reductase-free preparations of, 699-700 lyophilized preparation of, 720 in preparation of ribose-l,5-diphosphate, 504 removal by ethanol fractionation, 677 stabilization of, 713 TPNH generation by, 699, 712, 715, 719 Glucose-6-sulfate, as substrate for glucosulfatases 330 Glucosulfatases, 324, 330 Glutamate, action of D-serine (D-threonine dehydrase on, 324 chromatographic determination of, 289

934

SUBJECT INDEX

as component of transaminase system, 176, 180 determination of in protein hydrolysates, 192 in fibrinogen, 160 formation of by glutaminase, 380 formation of in tyrosine-glutamic acid transamination, 289 incorporation into glutathione, 342, 343 manometric estimation of, 182 P:O ratio during oxidation of, 613 requirement for in citrulline synthesis, 355 role in tyrosine-oxidizing system, 287 as substrate for L-glutamic dehydrogenase, 220-225 as substrate for glutamine synthetase, 337 as C-terminal residue, effect on iminodipeptidase, 100 transaminations involving, 170-177 with aspartic acid, 170, 171, 172, 174, 175 with cysteine-sulfinate, 172 D-Glutamate, assay of in presence of L-isomer, 175176 inhibition of glutamic dehydrogenase by, 224 resistance to D-amino acid oxidase, 2O2 as substrate for glutamo-transferasc, 341 Glutamic acid decarboxylase, assay of ~glutamic acid with, in glutamic acid raeemase system, 215 in protein hydrolyzates, 192 in transaminase tests, 170, 173-174, 176, 182-183, 289 bacterial sources of, C1. welchii, 182 E. coli, 182-183, 186-187, 216 in brain, 199 plant sources of, 182, 190-194 carrots, 194 squash, 182, 190-194 unsuitability in transaminase assay, 182

product of, C02 versus HCO3- as, 841 Glutamic acid racemase, from L. arabinosus, 215-217 L-Glutamic dehydrogenase, from liver, 220-225 i~Glutaminase, from animal tissues (glutaminase I), 382 distribution of, 382 from E. coli, 380-382 Glutamine, deamidation of in presence of a-keto acids, 382 formation from glutamic acid and ammonia, 337 formation of glutamyl hydroxamic acid from, 263, 267 hydrolysis of by glutaminase, 380 inhibition of glutamic dehydrogenase by, 224 in synthesis of 5-amino imidazole ribotide, 505 of glycinamide ribotide, 504, 509512 of inosinic acid, 506 transamination with aliphatic keto acids in animal tissues, 172 Glutamine (CONI~H2), ~.-glutamyltransferase reaction for preparation of, 267 Glutamine synthetase, association with ~,-glutamyltransferases, 264, 267, 272, 341 in liver, 339, 341-342 from pea seeds, 337-342 from sheep brain, 339, 341, 342 in Staph. aureus, 342 Glutamine transaminase, 382 Glutamohydroxamic acid, see 7-Glutamylhydroxamic acid Glutamotransferase, see -y-Glutamyltransferase L--/-Glutamyl-~-cysteine, as intermediate in formation of glutathione, 342 v-Glutamylhydroxamic acid (glutamohydroxamic acid, GHA), assay of glutamine synthetase by formation of, 337-338 colorimetrie determination of, 267, 268

SUBJECT INDEX

as product of 3,-glutamyltransferase reaction, 263, 267 synthetic, 268 3,-Glutamyltransferases (glutamotransferases, GTF), metal and nucleotide dependent, association with glutamine synthetase activity, 264, 267, 272, 341 from brain, 270-271 from liver, 271 from peas, 263-266 from P. vulgaris, 271-272 from pumpkin (PGT), 263-264, 266-267 metal and nucleotide independent, occurrence in bacteria, 269 preparation from P. vulgaris, 268269 Glutathione (GSH), see also Mercapto compounds, Thiol compounds, activation of adenylate kinase by, 603 of alanine racemase by, 213, 215 of amino acid reduetases by, 220 of cathepsin C by, 68 of eitrullinase by, 378 of guanidinoacetate methylpherase by, 260, 263 of histidase by, 231 of hydrogenase by, 868 of insect muscle ATPase by, 598 of lucifcrase (bacterial) by, 860 of nitrate reductase by, 415 of pyrocatechase by, 282 of thio ether cleavage enzyme by, 314 of tryptophanase by, 241 of tryptophan synthetase by, 237 as coenzyme for maleylaeetoacetate isomcrase, 295, 296, 298 effect on 3'-nucleotidase, 554 formation by reduction of GSSG, 719 hydrolytic enzymes for in liver, 343 inhibition of hippuric acid formation by, 350 of metaphosphatase by, 579 of tyrosinase by, 826 measurement by various methods, 719 reduction of dehydroascorbic acid by, 849, 850 of nitrate esters by, 403-406

935

enzymatic, 403-406 non-enzymatic, 405, 406 of p-quinone by, 728-729 requirement for, in methionineo activating reaction, 254, 255, 256 role in blood clotting mechanism, 160 stabilization of carbonic anhydrase by, 846 of nitrate reductase by, 414 of tryptophanase by, 241 of tryptophan synthetase by, 237 treatment of dialyzing membranes with, 414 Glutathione, oxidized (GSSG), reduction of by molecular hydrogen, 732 by TPNH, 719, 722 Glutathione reductase, in assay of 5-dehydroshikimic reductase, 301-302 from beef liver, 724-725 evidence for pgrphyrin in, 725 from higher plants, 719-722 distribution of, 721 in Itill reaction, 721 reduction of GSSG with hydrogenase and, 732 from yeast, 723-724 crystallization of, 723 Glutathione-synthesizing system, in E. coli extracts, 342 from pigeon liver, 342-346 L--y-glutamyl-L-cysteineas intermediate in, 342 enzyme for formation of, 342 glutathione synthetase for utilization of, 342-346 Glutathione synthetase, from brewer's yeast, 345-346 distribution of, 343 from pigeon liver, 344-345 Glycerol, as acceptor for transphosphorylation by phosphatases, 556 activation of ~-glutamyltransferase (bacterial) by, 269 extraction of brewer's yeast with, 581 Glycerol trinitrate, see Nitroglycerine

936

SUBJECT I N D E X

Glycerophosphatase, in microsomes, 542 Glycerophosphates, prostatic phosphatase action on aand ~-, 525 fl-Glycerophosphate, as substrate for alkaline phospharases, 530-533, 538, 539 DL-a-Glycerophosphate, as product of transphosphorylation by phosphatases, 556 L-a-Glycerophosphate, enzymatic measurement by reduction of ferricytochrome c, 559 *L-a-Glycerophosphate dehydrogenase, in insoluble particles of rabbit muscle, 559 Glycinamide, hydrolysis by leucine aminopeptidase, 92 Glycinamide ribotide, formylation of, 505, 509-512 isolation of, 511-512 Glycine, determination of, 347 in fibrin, 160 inhibition of alkaline phosphatase by, 538 of histidase by, 231 oxidation of, 225-227 as precursor of 5-amino-4-imidazolecarboxamide ribotide, 512 as product of aminotripeptidase action, 87 of formylation of FAH4 by serine, 518 reduction of, 217 resistance to D-amino acid oxidase, 202 stabilization of arginase by, 373 of renin by, 134 in synthesis of glutathione, 342, 343 of glycinamide ribotide, 504, 509-512 estimation with glycine-l-C TM, 509-511 of hippuric acid, 346 of inosinie acid, 506 Glycine buffer, high values for ATPase in, 587 Glycine oxidase, from pig kidney, 225-227

*Glycolytic enzymes, in liver, 343 from rabbit muscle, 358 regeneration of ATP with, 351, 356, 357 Glyeylallohydroxy-L-proline, as substrate for prolidase, 104 Glycyl-amino acids, action of acylases on, 118, 119 of dehydropeptidases I and I I on, 113 Glyeyldehydroamino acids, action of dehydropeptidases on, 109, 110, 113, l l 4 Glycylglyeine, action of acylase I on, 118 derivatives of, resistance to glycylglycine dipeptidase, 109 probable reaction with quinone, 729 as product of aminotripeptidase action, 83 Glycylglycine dipeptidase, 107-109 from human uterine tissue, 108-109 presence in purified iminodipeptidase, 99 from rat muscle, 108 comparison with dehydropeptidase I of swine kidney, 108 sources of, 108 stability of, 109 from swine kidney, 108, 109 Glycylglycylglycine (triglycine), as substrate for aminotripeptidase, 83-84, 87 Glycylglycyl-L-leucine, as substrate for aminotripeptidase, 83, 87 comparison with D-form, 87 Glycylglyeyl-~-proline, as substrate for aminotripeptidase, 87 Glycyl-L-histidinamide, activity of carnosinase on, 96 Glycyl-L-histidine, activity of carnosinase on, 96 Glycylhydroxy-L-proline, as substrate for prolidase, 104 Glycyl-L-leucinamide, and carbobcnzoxy derivative of, resistance to glycyl-L-leucine dipeptidase, 107 as substrate for leucine aminopeptidase, 92

SUBJECT INDEX Glycyl-n-leucine, carbobenzoxy derivative of, resistance to glycyl-~leucine dipeptidase, 107 as product of aminotripeptidase action, 83 as substrate for dehydropeptidase I, 107 comparison with D-isomer, 107, 113 Glyeyl-L-leucine dipeptidase, from human uterine tissue and swine intestinal mucosa, 105-107 specificity of, comparison with dehydropeptidase I, 107 Glycyl-L-leucylglycine, as substrate for aminotripeptidase, 87 comparison with D4orm, 87 Glycyl-L-phenylalaninamide (GPA), action of amidase on, 399 as substrate for cathepsin C, 64-65, 68 hydrolysis reaction, 64 transamidation reaction with hydroxylamine, 65 Glycyl-L-phenylalaninamide acetate, preparation of, 66 Glycyl-L-phenylalanine ethyl ester, as substrate for cathepsin C, 68 Glycyl-L-proline, resistance of carbobenzoxy derivative of to prolidase, 104-105 as substrate for prolidase, 100, 104 Glycyl-u-prolylglycine, comparison with other tripeptides in aminotripeptidase reaction, 87 Glycylsarcosine, as substrate for prolidase, 104 Glycyl-I,-tyrosinamide, as substrate for cathepsin C, 68 Glycyl-L-tyrosine ethyl ester, as substrate for cathepsin C, 68 *Glyoxalase, assay of glutathione with, 343, 719 Glyoxylic acid, as product of glycine oxidase reaction, 225 reaction with hydrogen peroxide, 225 Gramieidin, as uncoupling agent, 615 Grasshopper eggs, tyrosinase occurrence as protyrosinase in, 831

937

Grassmann and Heyde procedure, assay of peptidases by, 83, 88, 93-94, 97, 100, 105-106, 107-108 Guaiacol, see also Catechol monomethyl ether, assay of peroxidase with, 770-773, 792 Guanase, from rat liver, 480-482 removal of guanine with, 449 spectrophotometric determination of guanine with, 458, 481-482 Guanidines, inhibition of diamine oxidase by, 396 Guanidinoacetic acid (GA), methylation of, 260, 263 stoichiometry of reaction, 263 Guanidinoacetate methylpherase, from pig liver, 260-263 Guanine, as acceptor of deoxyriboside group, 468 conversion to xanthine, 480-482 shift in spectrum during, 480 RI values for, 466 spectrophotometric assay of, 458, 481482 Guanine deoxyriboside, bioassay of, 464 phosphorolysis of, 448 R/values for, 466 in transdeoxyribosidase reaction, 464468 Guanosine (guanine riboside) (GR), inhibition of inosine cleavage by, 463 nucleosidase action on, 459, 463 phosphorolysis of by spleen enzyme, 448 by yeast enzyme, 453 Guanosine triphosphate (GTP), as phosphate donor to AMP, 603 3'-Guanylic acid (3'-GMP), 3'-nucleotidase action on, 553 5'-Guanylie acid (GMP), as acceptor in nucleoside monophosphate kinase reaction, 603 H

Heart, adenylate kinase (myokinase) in, 602 cytochrome a in particles from, 737, 739

938

SUBJECT INDEX

cytochrome a3 in particles from, 734, 737, 740 cytochrome b from, 740-744 cytochrome c from, 749-755 in particles from, 738 cytochrome c oxidase activity in particles from, 738 cytochrome component absorbing at 553 m~ in, 744 diaphorase (DPNH-linked) from, 707711 D P N H cytochrome c reductase from, 688, 693 fibrinokinase from, 165 glutamic-oxalacetic transaminase activity of, 184 isocitric dehydrogenase in, 681 nitroaryl reductase in, 406 oxidative phosphorylation in mitochondria from, 614 preparation of particle suspensions from, 737, 740 hemoglobin removal from, 738-739 pyridine nucleotide transhydrogenase from, 686, 687 species variation in, 687 succinie dehydrogenase in, 737 succinic oxidase system in, 738, 750 Heat of activation, for DNase, 443 Heavy metals, inhibition of D-amino acid oxidase by, 202 of bacterial luciferase by, 860 Heavy water (D20), in hydrogenase assay by exchange reaction, 863 Helvolic acid, inactivation by crude penicillinase, 123 Hematin, content in bacterial catalase, 788 Hematin compounds, removal from cytoehrome c reductase by adsorption with gels, 690, 691 Hematin peptide, formation from cytochrome c, 167169 Hematohemin, formation of from cytoehrome c, 753

Iteme, spectrophotometric assay of, 791 Heme-protein, as byproduct of catalase crystallization from liver, 794 Hemin, see also Protoporphyrin, content of in cytochrome bl, 747 content of in horseradish peroxidase, 809 green, as prosthetic group of myeloperoxidase, 799 as impurity in D P N H cytochrome c reductase of heart, 691 Hemochromogen, from E. coli, as acceptor for D P N H cytochrome reductase of bacteria, 698-699 reoxidation by mammalian cytochrome oxidase, 699 extinction coefficient for pyridine ferroprotoporphyrin, 809 preparation of from catalase, 788, 790 Hemoglobin, in assay of pepsin, 3-4 denaturation by Tsuchihashi procedure, 102 proteolysis by rennin, 77 removal by alcohol-chloroform treatment, 783 removal of from heart particles1 738-739 solubility of in ammonium sulfate solutions, 84 Hemoglobin digestion method, for determination of trypsin, 34 Hemophilic plasma reagent, in assay of antihemophilic factor, 148 Hemoproteins, see Cytochromes Heparin, cofactor of from mast cells, 163 inhibition of RNase by, 434 of thrombin by, 158 precipitation of by protamine, 163 Heptylamine, amine oxidase action on, 393 ttexametaphosphate, as substrate for metaphosphatase, 577-578

SUBJECT INDEX Hexamethylenediamine, action of diamine oxidase on, 396 *Hexokinase, in assay of adenylate kinase, 598-599, 6O0 of ATP, 497 coupling of citrullinase reaction with, 377 in oxidative phosphorylation assay, 611 *Hexose isomerase, see Phosphohexoisomerase Hexosephosphate, inhibition of acetyl phosphatase by, 556 Hexylamine: amine oxidase action on, 393 Hill reaction, glutathione reductase in, 721 Hippuric acid, enzymatic synthesis of in kidney cortex extracts, 348-349 in liver, 346-350 Hippuricase, see also Amino acid acylase I, probable identity with acylase I, 115 a-Hippuryl-L-lysinamide (HLA), as substrate for trypsin, 36 Histaminase, see Diamine oxidase Histamine, formation of from histidine in higher plants, 194 oxidative deamination of, 394 Histidase, in liver, 229 from Ps. fluoresceus, 228-231 Histidine, activation of DNase by, 442 as component of dipeptides hydrolyzed by carnosinase, 93, 96 content of in horseradish peroxidase, 8O9 conversion to urocanic acid by histidase, 228 decarboxylation of in higher plants, 194 in mammalian tissues, 199 as substrate for L-amino acid oxidase, 2O8

939

Histidine decarboxylase, from Cl. welchii, 187 in measurement of transaminase reactions, 171 Histozyme, see also Amino acid acylase I, probable identity with acylase I, 115

bis-Homobiotin, inhibition of d-biotin oxidation by, 632 Homocysteine~ action of cysteine desulfhydrase on, 317 determination by modification of Brand method for, 314 formation from eystathionine, 314 in over-all system for I M P synthesis, 5O6 Homocysteine desulfhydrase, from liver, 318 Homogenates, tissue, oxidative phosphorylation in, 610-616 Homogentisate, conversion of to maleylacetoacetate, 292-295 oxidation of, 288 Homogentisate oxidase, distribution of, 294 from liver, 292-295 spectrophotometric method for assay of, 295 Homosulfanilamide, activation of bacterial thiaminase by, 628 amine oxidase action on, 393 Homotryptophan, inhibition of tryptophanase by, 241 Horse nettle (Solanum elaeagnifolium), protease (solanain) from, 57, 63-64 Horseradish, allylisothiocyanate from, 804 peroxidase from, 801-813, see also under Peroxidase Hura crepitans, see Jabillo plant Hurain, protease from sap of jabillo plant, 57, 64 Hyaluronic acid, inhibition of acetyl phosphatase by, 556 Hydrazine, in glutamine synthetase reaction, 341 inhibition of bacterial transaminases by, 177

940

SUBJECT I N D E X

of tryptophanase by, 241 as substrate for ~.-glutamyltra~lsferase, 269 Hydrazoie acid, see Azide Hydrochloric acid, compound with horseradish peroxidase, 812 Hydrocyanic acid, see Cyanide Hydrogen gas, absorption of in amino acid reduetase reaction, 217 role of hydrogenase in, 218 activation of hydrogenases by, 866, 868 O~-free, 863-865 ortho and parahydrogen conten~ of at equilibrium, 864 different thermal conduetivities of isomers, 864 reduction of pyridine nucleotides by, 729 of various substances by, 861 reversible combination of with flavoproteln, 870 Hydrogenase(s), 729-732, 861-870 in amino acid reductase reaction, prevention of air inactivation of, 218 assay of, 729-730, 862-865 by conversion of parahydrogen, 864-865 by evolution of hydrogen from substrates, 862 by exchange reaction, 862, 864 by reduction of methylene blue (manometric method), 865 of pyruvate via DPN, 729-730 preparation and properties of, 730-732, 865-870 from Cl. kluyveri, 730-732 cofactor for, 730, 731 purification of, 731, 732 from Cl. pasteurianum, 869, 870 characterization as Mo-FADprotein, 869, 870 spectrum of, 870 from Desulfovibrio sulfuricans, 869, 87O from E. coli, 867, 868 from Proteus vulgaris, 865, 867 characterization as iron-porphyrin-protein complex, 866

variation in inhibitor effects with nature of assay, 867 sources of, 870 specific activity of preparations of, 868

Hydrogenomonas facilis, hydrogenase in, 870

Hydrogenomonas ruhlandii, hydrogenase in, 870 Hydrogen peroxide, formation of by amine oxidase, 390 by amino acid oxidases, 199, 204 by diamine oxidase, 394 by glycine oxidase reaction, 225 by leuco methylene blue oxidation, 715 by old yellow enzyme, 712 by uricase, 485 by xanthine oxidase, 482 inhibition of adenylate kinase by, 603 of amino acid reductase by, 219 of myosin ATFase by, 587 oxidation of cytochrome c (mammalian) by, 736 of cytochrome e (Ps. fluorescens) by, 759, 761 of a-keto acids by, 204 of tryptophan by, 242, 246 as substrate for horseradish peroxidase, 807, 810, 811 see also Enzyme-substrate complexes for lactoperoxidase, 816 Hydrogen sulfide, see also Sulfide, determination of, 315-316 formation by eysteine desulfhydrase, 315-319 inhibition of alkaline phosphatase by, 527 of D-amino acid oxidase by, 203 phosphopantetheine maintenance by, 669 Hydroquinone, determination of, 726 formation of by quinone reductase, 725 reduction of cytochrome c by, 754 of peroxidase complex I I by, 810 Hydroquinone monomethyl ether, oxidation of by peroxidase complex II, 810

SUBJECT INDEX Hydrosulfite, see Dithionite Hydroxamic acid method, acetyl phosphate analysis by, 650 p-Hydroxyaeetophenone, assay as organic product of arylsulfatases, 327-328 a-Hydroxy acids, complex formation with borate, 290 L-a-Hydroxy acids, as substrates for L-amino acid oxidase, 211 Hydroxyanthranilic acid, tryptophan peroxidase and, 244 p-Hydroxybenzoic acid, as precursor of protocatechuic acid, 273 as product of aromatic biosynthesis, 300 p-Hydroxybenzoyl formic acid, decarboxylation of, 280 /~-Hydroxybutyrate, P:O ratio during oxidation of, 613, 616 p-Hydroxydiphenyl, oxidation by peroxidase complex II, 810 fl-Hydroxyglutamic acid, as substrate for glutamic acid decarboxylase of plants, 192 7-Hydroxyindole, as substrate for tryptophan synthetase, 236 L-Hydroxykynurenine, as substrate for liver kynureninase, 253 Hydroxylamine, effect on activity and spectrum of myeloperoxidase, 799 inhibition of amine oxidase by, 393 of arginine desimidase by, 376 of bacterial transaminase by, 177 of cytochrome c peroxidase by, 763 of 5-dehydroquinase by, 307 of dopa decarboxylase by, 198 of glutamic acid decarboxylase by, 193 of glutamic dehydrogenase by, 224 of -y-glutamyltransferase (brain) by, 272 of kynureninase by, 253 of nitroethane oxidase by, 402

941

of D-serine (D-threonine) dehydrase by, 324 of tryptophanase by, 241 of tryptophan synthetase by, 237 replacement of NH3 in glutamine synthetase reaction by, 338 role in ~-glutamyltransferase reaction, 263, 267, 269 in transamidation by eathepsin C, 65 Hydroxylamine reductase, as adaptive enzyme, 411, 417 in Bacillus organisms, 417 from Neurospora crassa, 416-419 presence in nitroaryl reduetase preparations, 410, 411 turnover number of, 418 Hydroxylaminodinitrotoluene, reduction of trinitrotoluene to, 406 Hydroxyl radicals, inhibition of RNase by, 434 p-Hydroxyphenylpyruvate, ascorbate requirement for oxidation of, 288 determination of, 289 enol form of, light absorption of, 289 stabilization of, 290 keto form as product of tyrosineglutamic acid transamination, 289 m-Hydroxyphenylpyruvate, enol-keto tautomerase action on, 291 p-Hydroxyphenylpyruvate enol-keto tautomerase, distribution of, 291 from liver, 289-292 Hydroxyphenylserine, decarboxylation of in mammalian tissues, 199 Hydroxy-L-prolinamide, hydrolysis by leucine aminopeptidase, 92 Hydroxy-L-prolylglycine, as substrate for iminodipeptidase, 97 Hydroxy-L-prolylglycylglycine, as substrate for aminotripeptidase, 87 8-Hydroxyquinoline, inhibition of ascorbic acid oxidase by, 835 of D P N H cytochrome c reductase by, 693

942

SUBJECT INDEX

of nitrate reductase by, 415 of D-serine (D-threonine) dehydrase by, 324 of tryptophan synthetase by, 237 Hydroxytyramine, biological and colorimetric determinations of, 195 formation of from 3,4-dihydroxyphenyl-L-alanine in higher plants, 194 Hypertensin, see Angiotonin Hypertensinogen, see Renin substrate Hypoxanthine, as acceptor of deoxyriboside group, 468 incorporation of C ~4formate into, 505-509 isolation and degradation of, 507-508 apparatus for, 508 molecular extinction coefficient of, 507 oxidation to uric acid, 448, 449, 482, 484 RI values for, 466 spectrophotometric assay of, 458 as substrate for nucleoside phosphorylase, 448 synthesis of IMP from R-5-P and, 503 Hypoxanthine deoxyriboside, phosphorolysis of, 448 R! values for, 466 in transdeoxyribosidase reaction, 464468 bioassay of, 464 Hypoxanthine riboside, see Inosine

Imidazole, inhibition of xanthine oxidase by, 485 Imidodipeptidase, see Prolidase Iminodipeptidase (prolinase), from swine kidney, 97-100 pH optimum of, dependence of on purity, 97 Indigodisulfonate, in assay of diamine oxidase, 394 Indigotrisulfonate, as electron donor to FMN in bacterial luciferase system, 861

Indole (benzopyrrol), colorimetric measurement of, 233-234, 238-239 detoxication of diphtheria toxin by myeloperoxidase and, 800 formation of by tryptophanase in E. coli, 238 inhibition of tryptophanase by, 241 of tryptophan synthetase formation by, 237 L-tryptophan synthesis from, 233 Indophenols, oxidation by peroxidase complex II, 8O8 Induced enzymes, see Adaptive enzymes Induction, see Sequential induction Infusion broth, protective action on penicillinase, 123 Inorganic phosphate, see Phosphate, inorganic Inorganic pyrophosphatase, see Pyrophosphatase, inorganic Inosine (Hypoxanthine riboside, HxR), formation from adenosine, 473 inhibition of nicotinamide riboside phosphorolysis by, 453, 455, 456 nucleosidase action on, 459, 463 phosphorolysis of, by spleen enzyme, 448 by yeast enzyme, 453 RI values for, 466, 513 Inosine compounds, formation of by deaminase, 475 Inosine diphosphate (IDP), action of insect muscle ATPase on, 597 of liver mitochondrial ATPase on, 594 Inosine-5'-phosphate, see 5'-Inosinic acid Inosine triphosphate (ITP), action of insect muscle ATPase on, 597 of liver mitochondrial ATPase on, 594 of myosin on, 586 3'-Inosinic acid (3'-IMP), 3'-nucleotidase action on, 553 5'-Inosinic acid (inosine-5'-monophosphate, Y-IMP), formation of from 5'-AMP, 469

SUBJECT INDEX

molecular extinction coefficient of, 469 " 5 " nucleotidase action on, 549 paper chromatography of, 516 preparation of aminoimidazolecarboxamide ribo~ide from, 514 scheme for de novo biosynthesis of, 504-5O5 synthesis of from R-5-P and hypoxanthine, 503 Insect muscle, see Muscle Insect tissues, see also under names of individual species, cytochrome e in, 745 Intestinal mucosa, adenosine deaminase from, 473-475 aminotripeptidase in, 84 flavokinase in, 642 glycyl-L-leucine dipeptidase from, 105107 leucine aminopeptidase in, 89 phosphodiesterase from, 569-570 phosphomonoesterase from, 473-475, 530 533, 539 prolidase in, 102, 105 pyrophosphatase in, 475 Intestine, glucose-6-phosphatase in, 542 kynurenine formamidase in, 248 Iodine, inhibition of alkaline phosphatase by, 538 of aspartase by, 388 of erythrocyte carbonic anhydrase by, 845 of myosin ATPase by, 587 Iodoacetamide, inhibition of aspartase by, 388 of 5-dehydroquinase by, 307 of myosin ATPase by, 587 of prolidase by, 105 protection against by manganese ion, 105 of RNase by, 434 resistance of leucine aminopeptidase to, 93 Iodoacetate, inhibition of L-amino acid oxidases by, 208, 211 of DPNH cytochrome c reductase by, 693

943

of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 of insect muscle ATPase by, 598 of myosin ATPase by, 587 of RNase by, 434 Iodoacetyl amino acids, action of acylase I on, 118 Iodosobenzoate, inhibition of aspartase by, 388 of DPNH cytochromc c reductase by, 693 of myosin ATPase by, 587 Ionophoresis, of purine and pyrimidine bases and nucleosides, 457 Iron, see also under Ferric ion and Ferrous ion, as component of DPNH cytochrome c reductase of heart, 691, 692 valence state of, 692 effect of DPNH and ferricytochrome c on, 692 content of in catalase from blood, 784 from spinach, 790 content of in lactoperoxidase A, 817 in myeloperoxidase, 798 in uriease preparations, 489 firefly luciferase system and, 856 inhibition of bacterial luciferase by, 860

of uricase by, 489 in prosthetic group of cytochrome c, 753 Iron enzymes, derivatives of cytochrome c as, 167-169 Iron-porphyrin-protein, hydrogenase of P. vulgaris as, 866 Iron protoporphyrin, as prosthetic group of Ustilago cytochrome c, 757-758 Isoalloxazine," derivatives of, as substrates for flavokinase, 644 D-Isoascorbic acid, in oxidation of p-hydroxyphenylpyruvate, 289 Isobutyrate, inhibition of d-biotin oxidation by, 632 Isocitrate, P:O ratio during oxidation of, 616

944

SUBJECT INDEX

*Isocitric dehydrogenase, DPN-linked, 'in purified yeast fraction, transhydrogenase assayed with, 682 *Isocitric dehydrogenase, TPN-linked, from pig heart, TPN assay with, 652 TPNH generation with, 681 TPNH preparation with, 411 Isocitric lactone, as source of isocitrate, 652 L-Isoglutamine, hydrolysis by leucine aminopeptidase, 92 L-Isoleucinamide, hydrolysis by leucine aminopeptidase, 92 L-Isoleucine, as substrate for L-amino acid oxidase, 211 transamination with valine in microorganisms and animal tissues, 171, 176 Isonicotinamide, reversible conversion to D P N analog by animal DPNases, 662, 663 1-Isonicotinyl-hydrazine (isoniazid) (isonicotinic acid hydrazide) (INH), inhibition of amine oxidase by, 393 of diamine oxidase by, 396 reversible conversion of to DPN analog by animal DPNases, 662, 663 species variation in sensitivity of animal DPNases to, 663 1-Isonicotinyl-2-isopropylhydrazine (iproniazid) (marsilid) (isopropyl INH), inhibition of amine oxidase by, 393 reversible conversion of to D P N analog by animal DPNases, 662, 663

Jabillo plant (Hura crepitans), protease (hurain) from sap of, 57, 64 Jack bean meal, urease from, 378-379

Janus Green B, as electron acceptor for D P N H eytochrome c reductase of bacteria, 697 K

Kaolin, cytochrome c (bacterial) purification by chromatography on, 762 preparation of acid-treated, 762 Kat. f . ( Katalasef dhigkeit) ,

catalase activity and purity expressed as, 767, 779-780, 782, 788, 789, 790, 791 a-Keto acid(s), activation of aspartic acid decarboxylase by, 188, 189 measurement of, for assay of L-amino acid oxidase, 204 oxidation by hydrogen peroxide, 204 a-Keto acid-~-amidase, from rat liver, 382, 384-385 *a-Keto acid decarboxylases, see Carboxylase f~-Ketoadipic acid, aromatic ring cleavage and, 273 formation of from t~-carboxymuconic acid, 273, 285 from ~-carboxymethyl-A=-butenolide, 273, 282-284 ~-Ketobutyrate, formation by cleavage of cystathionine, 311 by dehydrase action on threonine, 319-322 by desulfhydrase action on homocysteine, 318 ~-Ketoglutaramate, as intermediate in deamidation of glutamine, 382 as substrate for a-keto aeid-~-amidase, 384 a-Ketoglutarate, in glutamic dehydrogenase reaction, 220 measurement of as succinate with succinoxidase, 170 as 2,4-dinitrophenylhydrazone, 170 P: O ratio during oxidation of, 613, 616 requirement for in tyrosine-oxidizing system, 287, 288

SUBJECT INDEX

transaminase measurement with, 170, 172-175, 179-182 transamination with L-tyrosine, 289 with various amino donors, false glutamate values due to, 193 *a-Ketoglutaric decarboxylation system, COs versus HC0s as product of, 841 Ketone reagents, inhibition of cysteine desulfhydrase by, 317 Ketosteroid, as product of steroid sulfatase, 330 a-Ketosuccinamate, as substrate for a-keto acid-~-amidase, 384 Kidney, acetyl phosphatase from, 556 adenosine kinase in, 499 amino acid amidase from, 397-400 D-amino acid oxidase from, 171, 199204, 212 L-amino acid oxidase from, 209-211 arginine-synthesizing system from, 357, 358-359, 365-367 argininosuccinate-splitting enzyme as component of, 365-367 d-biotin oxidase in slices of, 631-632 carbonic anhydrase in, 841 catalase in, 765, 775 cysteine desulfhydrase in, 381 decarboxylases for various amino acids in, 199 diamine oxidase (histaminase) from, 394-396 3,4-dihydroxyphenylalanine (dopa) decarboxylase from, 195-199 folio acid conjugase in, 629-630 glucose-6-phosphatase in, 542 glutamic-oxalacetic transaminase activity of, 181 L-glutaminase in, 382 glycine oxidase from, 225-227 hippuric acid synthesis in, 348-349 kynurenine formamidase in, 248 oxidative phosphorylation in mitochondria from, 614 peptidases from, amino acid acylase I (soluble acylase I, dehydropeptidase II), 109-114, 116-119

945

amino acid acylase II, 117, 119 carnosinase, 93-96 dehydropeptidase I (solubilized aminopeptidase), 109-114 glycylglycine dipeptidase, 108, 109 iminodipeptidase, 97-100 leucine aminopeptidase, 88-93, 98 prolidase, 98, 101, 103-104, 105 purification of by common initial procedure, 98 renin from, 124-135 RNase in, 436 succinic oxidase in cortex of, 750 transhydrogenase in, 687 triphosphatase in, 580, 581 uricase from, 485-489 King-Armstrong unit, for phosphatase values in serum, 528 Kochsaft, see Boiled juice Kojic acid, inhibition of D-amino acid oxidase by, 203 Kynurenic acid, absorption maximum for, 250 tryptophan peroxidase and, 244 Kynureninase, distribution of, 253 from Ps. fluorescens, 249-253 from rat liver, 244, 249-253 L-Kynurenine, conversion to anthranilic acid and L-alanine, 249 determination of, 242 extinction coefficients of, 250 formation by formamidase, 242, 246 Kynurenine formamidase (formylase), distribution of, 248 in Neurospora crassa, 253 from rat liver, 246-249 Kynurenine transaminase, formation of kynurenic acid by, 250 in liver homogenates, 244 L Laccase, effect on induction period of monophenolase, 825 Lactate, activation of 5'-AMP deaminase by, 472

946

SUBJECT INDEX

L-Lactic acid, as substrate for L-amino acid oxidase, 211 *Lactic dehydrogenase, pyruvate reduction with hydrogenase and, 729-730 in yeast, as crystalline hemoprotein (cytochrome b2) containing flavin, 746 LactobaciUus arabinosus, gtutamic acid racemase from, 215-217 growth medium for, 216 Lactobacillus casei, in assay for "folic acid activity," 629 Lactobacillus delbriickii, acetyl phosphatase from, 556 nucleoside transdeoxyribosidase in, 467 LactobaciUus fermenti, arginine desimidase in, 376 Lactobacillus helz,eticus, aspartase in, 388 growth of, 467-468 nucleoside transdeoxyribosidase from, 467-468 in preparation of medium for propionic acid bacteria, 386 Laclobacillus ~rlesenteroides, arginine desimidase in, 376 Lactobacillus pentosus, growth of, 459 hydrolytic nucleosidases from, 456-461 Lactone-splitting enzyme, from Ps. fluorescens, 273, 282-284 Lactonizing enzyme, for cis,cis-muconic acid from Ps. fluorescens, 273, 282-284 Lactoperoxidase A, see also Peroxidase from milk, chemical and physical properties of, 817 nitrogen content of, 799 preparation of crystalline, 813-817 Lactoperoxidase B, from spring milk, 816-817 turnover number of, 817 Lamb's quarters (Chenopodium album), carbonic anhydrase in leaf of, 842 Lanthionine, meso- and L-forms as substrates for cleavage enzyme, 312, 313

Latex, proteolytic enzymes from, see Proteolytic enzymes, plant Lead ion, activation of metaphosphatase by, 579 inhibition of aspartase by, 388 of leueine aminopeptidase by, 93 of prolidase by, 105 Lead subacetate reagent, preparation of, 824 Lead sulfide, colloidal, measurement of light scattered by, 315 Leaf tissue, carbonic anhydrase in, 842 Lebedew juice, phosphorylation of thiamine by protein from, 636 as source of old yellow enzyme, 713 Lecithin, presence of in heparin cofactor, 163 *Lecithinase, of Cl. welchii, inactivation of Mgactivated ATPase by, 590 Leguminosae, dehydroascorbic reductase in, 849 L-Leucinamide, action of dehydropeptidases I and II on, 113 as substrate for amidase, 399 for leucine aminopeptidase, 88, 89, 91, 92 comparison with D-isomer, 91 L-Leucine, inhibition of D-amino acid oxidase by, 203 as product of aminotripeptidase action, 87 as substrate for L-amino acid oxidases, 205, 208, 209, 211 transaminase for, 176 Leucine aminopeptidase (leucylpeptidase), action on amides and peptides~ 92 distribution of, 93 inhibition of by Versene, 87 substrates for, 89, 91-92 in swine intestinal mucosa, 89 from swine kidney, 88-93, 98 Leucobenzylviologen,see Benzylviologen, reduced

SUBJECT INDEX

Leucocytes (W.B.C.), myeloperoxidase from, 245, 794-800 Leucodyes~ oxidation by amino acids in Cl. sporogenes, 218 by peroxidase complex II, 808, 810 Leucovorin, see also Citrovorum factor, in formylation of glycinamide ribotide, 510 I,-Leucyl-D-alanine, comparison with I~form in leucine aminopeptidase reaction, 92 L-Leucylglycine, as substrate for leucine aminopeptidase and other peptidases, 89, 91 comparison of with D-isomer, 91 L-Leucylglycylglycine, as substrate for aminotripeptidase, 83, 87 comparison with D-form, 87 as substrate for leucine aminopeptidase and other peptidases, 83, 89 L-Leucyl-D-amino acids, hindrance of leucine aminopeptidase action by D-residues of, 92 Leucyl peptidase, see Leucine aminopeptidase Leukemic tissues, mouse, DNase in, 447 Lima bean, protease from, 63 trypsin inhibitor from, 37, 48-49, 51, 53 Lipoprotein~ nucleoside phosphorylase as f~-, 452 thromboplastin as, 150 Littorina littorea (mollusk), arylsulfatases in, 332 Liver, acetone powder preparation from, 509, 704 activation of "1-C unit" as folic acid derivative in extracts of, 505, 516518 adenosine kinase in, 499 adenylate kinase (myokinase) in, 602, 603, 604 amine oxidase from, 393 L-amino acid oxidase in, 209, 213 arginase from, 357-358, 368-374

947

arginine-synthesizing system from, 357, 358-359 condensing enzyme, 360-362 splitting enzyme, 365-367 ATPase in extract of, 653 from mitochondria of, 593-595 d-biotin oxidase in slices of, 631-632 boiled extract of, 506 carnosinase in, 94 catalase from, 765, 775-781 citrovorum factor formation from 10formylfolic acid by enzyme from, 629 citrulline-synthesizing system from, 350-355 CoA formation in extract of, 633 cystathionine cleavage enzyme from, 311-314 cysteine desulfhydrase from, 315-318 decarboxylases for various amino acids in, 199 dehydropeptidases in, 114 changes of in malignancy, 114 dephospho-CoA kinase from, 649-651 dephospho-CoA pyrophosphorylase from, 667-669 desulfhydrase activity of, 313 dopa decarboxylase from, 199 DPN kinase from, 652-655 DPN pyrophosphorylase from, 671672 DPNasc in, 653 exoeysiine desulfhydrase from, 319 folio acid eonjugase in, 629 fruetose-l,6-diphosphatase from, 543546 fumarylaeetoaeetate hydrolasc from, 293-294, 297, 298-300 glueose-6-phosphatase from, 541-543 L-glutamie dehydrogenase from, 220225 glutamic-oxalacetic transaminase activity of, 184 L-glutaminase in, 382 glutamine synthetase from, 339, 341342 7-glutamyltransferase (Mn-dependent) from, 271 glutathione reduetase from, 724, 725

948

SUBJECT I N D E X

glutathione-synthesizing system from, 342-346 glycolytic enzymes in, 343 guanase from, 480-482 guanidinoacetate methylpherase from, 260-263 hippuric acid synthesis by enzyme from, 346-350 histidase in, 229 homocysteine desulfhydrase from, 318 homogeniisate oxidase from, 292-295 inorganic pyrophosphatase in, 362 a-keto acid-a-amidase from, 382, 384385 kynureninase from, 249-253 kynurcnine formamidase (formylase) from, 246-249 maleylaeetoacetate isomerase from, 293-294, 295-298 metaphosphatases from, 579 methionine-activating enzyme (MAE) from, 254-256 myokinase in, 362 nicotinamide methylpherase from, 257260 nicotinamide riboside phosphorylase from, 453, 454-456 nitroaryl reductase in, 406 nucleoside phosphorylase from, 453 nucleotide pyrophosphatase in, 653 nucleotide synthesis from R-5-P and adenine in extracts of, 501-504 nucleotide synthesis de novo in extracts of, 504-519 organic nitrate reductase from, 403-406 oxidative phosphorylation in mitochondria from, 614 pantetheine kinase from, 633-636 peroxidase from, 791-794 rhodancse from, 334-337 RNase in, 436 synthesis of formylglycinamide ribotide and glycinamide ribotide in extracts of, 505, 509-512 tetrahydrofolate formylase in, 517 thymidine phosphorylase from, 453 as TPN source, 700 TPNH cytochrome c reductase from, 704-706

transformylation of active "1-C u n i t " to form I M P in extracts of, 505 transhydrogenase in, 687 L-tryptophan peroxidase from, 242-246 uricase from, 489 urocanase in, 232 Liver fraction L, folic acid and CF content of, 630 effect of folic acid conjugase on, 630 Luciferase, bacterial (Achromobacter flscheri), 857861 identification as flavoprotein, 857, 860-861 kinetics of, 861 from Cypridina, 851-853 from firefly, 851, 854-856 assay of, 851 CoA assay with, 651 crystallization of, 856 Luciferin (LH~), from Cypridina, preparation of, 853 from firefly, absorption maxima of, 855 CoA assay with, 651 competition of with bcnzimidazole and benztriazole, 856 fluorescence of, exciting wave length for, 855 preparation of, 854-855 role in luciferase system, 856 Lumiflavin, inhibition of flavokinase by, 645 molecular weight of new yellow enzyme by determination of, 718 Lung, antifibrinolysin from, 165 fibrinolysokinase from, 166 prolidase in, 105 thromboplastin from, 139, 148-149 Lymphosarcoma, I N H sensitivity of DPNase from mouse, 663

L-Lysinamide, hydrolysis by leucine aminopeptidase, 92

Lysine, activation of DNase by, 442 content in horseradish pcroxidase, 809

SUBJECT INDEX

Lysine decarboxylase, from Bact. cadaveris or E. coli, 188189 resolution of, 189 in measurement of transaminase reactions, 171 L-Lysine ethyl ester, as substrate for trypsin, 36 *Lysozyme, extraction of B. subtilis with, 421 of M. Lysodeikticus with, 786 M

MacFadyen's reagent, use of in RNase assay, 428 Maclura pomifera Raf., see Osage orange Magnesium chloride, activation of RNase by high concentrations of, 433-434 in conversion of aminoimidazolecarboxamide riboside to ribotide, 515 in enzymatic assay of UDPG, 676 in isocitric dehydrogenase system, 682 in oxidative phosphorylation, 611 in synthesis of glycinamide ribotide, 510 of inosinic acid (IMP), 506 Magnesium hydroxide, preparation of and use in purification of prothrombin, 143, 144 Magnesium ion, activation of acetyl phosphatase by, 556 of adenosine kinase by, 500 of adenylate kinase by, 599, 603 of alkaline phosphatase by, 533, 538 of amino acid amidase by, 399 of ATPase of actomyosin by, 587,589 of insect muscle by, 598 of liver mitochondria by: 595 in muscle particles by, 588-591 of ATP-creatine transphosphorylase by, 610 relation of amount required ~o nucleotide concentrations, 610 of bone phosphatase by, 540 of eitrullinase by, 377, 378

949

of citrulline synthesis by, 355 of dephospho-CoA kinase by, 649, 651 of dephospho-CoA pyrophosphorylase by, 667, 669 of dihydroSrotic dehydrogenase by, 496 of DNase (pancreatic) by, 442 relation to DNA concentration, 442 of DPN kinase by, 652, 654 of DPN pyrophosphorylase by~ 672 of FAD pyrophosphorylase by, 675 of firefly luciferase by, 651,856 of flavokinase by, 645 of fructose diphosphatase by, 546 of glutamine synthetase by, 341 of 7-glutamyltransferases by, 266, 272 of inorganic pyrophosphatase by, 575 antagonism by calcium, eobaltous, and manganous ions, 575 of lactonizing enzyme for cis,cismuconic acid by, 284 of latent inorganic pyrophosphatase by, 669 of leucine aminopeptidase by, 88, 93 time required fol:, 88, 93 of metaphosphatase by, 579 of " 5 " nucleotidase of seminal plasma by, 549 of oxidative phosphorylation system by, 615 of pantetheine kinase by, 633, 635 of pantothenate-synthesizing enzyme by, 621 of system for nucleotide synthesis by, 504 of tetrahydrofolate formylase by, 517 of thiaminokinase by, 636, 640 in assay system for hippuric acid synthesis, 348, 349 complex of with ATP, 604 effect on adenylate kinase equilibrium, 604 on apurinic acid, 441

950

SLrBJECT I N D E X

on triphosphatases, 581 inhibition of alkaline phosphatase (crude) by, 530, 535 of arginine desimidase by, 376 of ATPase (myosin) by, 587 of DNase (pancreatic) by high concentrations of, 442 of D P N H cytochrome c reductase by, 692 of RNase by low concentrations of, 433, 434 requirement for in methionine-activating reaction, 254, 256 Magnesium oligonucleotide, as substrate for phosphodiesterase and 5r-nucleotidase, 561 Magnesium sulfate, in pyridoxal kinase assay, 646 Magnetic susceptibility, of horseradish peroxidase compounds, 811, 813 Malate, as hydrogen donor in nitrogen gas formation, 420, 422 DPN as cofactor for, 422 1): 0 ratio during oxidation of, 613, 616 Maleate, solubilizing effect of on manganous ion, 368, 371-372 Maleic acid, dissociation constant of, 372 Maleylacetoacetate, conversion to fumarylacetoacetate, 295-298 extinction coefficients of, 296 oxidation of homogentisic acid to, 292-295 Maleylacetoacetate isomerase, from liver, 293-294, 295-298 separation from fumarylacetoacetate hydrolase, 297 from homogentisate oxidase, 293294 Mal0nate, blocking of succinate step in oxidative phosphorylation by, 511, 616 inhibition of aspartase by, 390 of d-biotin oxidation by, 632 of cytochrome c degradation by, 169

as product of barbiturase action, 492 Mandelic acid, inhibition of L-amino acid oxidase by, 2O8 Mandelic acid-C140OH, racemization of, 277 L(4-)-Mandelic acid dehydrogenase, from Ps. fluorescens, 273, 274, 277-278 assay of mandelic acid racemase with, 276 Mandelic acid racemase, from Ps. fluorescens, 273, 274, 276-277 Manganous chloride, removal of nucleic acid by, 303 Manganous ion, activation of adenosine kinase by, 500 of alkaline phosphatase by, 538 of amino acid amidase by, 397, 399 of arginase by preincubation with, 369 of ATPase of muscle particles by, 590 of ATP-creatine transphosphorylase by, 610 of carnosinase by, 94, 95, 96 time required for, 94, 95 of citrullinase by, 378 of DPN kinase by, 654 of DPN pyrophosphorylase by, 672 of flavokinase by, 645 of fructose diphosphatase (purified) by, 546 of glutamine synthetase by, 341 of ~,-glutamyltransferases by, 266, 272 of glycylglycine dipeptidase by, 107, 109 of glycyl-L-leucine dipeptidase by, 105, 106, 107 time course of, 106, 107 of iminodipeptidase by, 100 of inorganic pyrophosphatase by, 575 of lactonizing enzyme for cis,cismuconic acid by, 283, 284 of leucine aminopeptidase by, 88, 93 time required for, 88, 93 of luciferase (firefly) by, 856 of metaphosphatase by, 579

SUBJECT INDEX

of pantetheine kinase by, 635 of pantothenate-synthesizing enzyme by, 621 of prolidase by, 100-101, 103, 105 mechanism for, 105 time required for, 101, 103 of thiaminokinase by, 640 binding agents for, 100 effect on amino acid amides, 398 on pH optimum of carnosinase, 96 inhibition of condensing system for arginine synthesis by, 364 of DPNH cytoehrome c reductase by, 692 of fructose diphosphatase (crude) by, 546 of RNase by, 433 of thiaminase by, 625 of uricase by, 489 solubilizing effect of maleate on, 368, 371-372 stabilization of carnosinase by, 94, 96 of iminodipeptidase by, 98, 100 Mannitol hexanitrate, as uncoupling agent, 615 Mannose, presence in fibrinogen and fibrin, 160 Marsilid, see 1-Isonicotinyl-2-isopropylhydrazine Mast cells, heparin cofactor from, 163 Maya plant (Bromelia pinguin), protease (pinguinain) from fruit of, 56, 63 Melanin, formation of by cytochrome system, 828 by mammalian tyrosinase, 827-831 Melanocytes, histochemical assay of tyrosinase in, 828-829, 830 cytoplasmic location of, 830 radioactive tyrosine method for assay of tyrosinase in, 829-830 Melanoma, tyrosinase from, 828-831 Menadione reductase, 2-methyl-l,4-naphthoquinoneas electron acceptor for, 728

951

Mercapto compounds, see also under names of individual compounds, activation and stabilization of tryptophanase by, 241 ~-Mercaptoethanol, activation of cathepsin C by, 68 ~-Mercaptovaline, see Penicillam~ne Mercurials, organic, inhibition of citrullinase by, 378 p-Mercurichlorobenzoate, see p-Chloromercuribenzoate Mercuric ion, inhibition of aminotripeptidase by, 87 of 5'-AMP deaminase by, 472 of arginine desimidase by, 376 of aspartase by, 388 of citrullinase by, 378 of DPNH cytochrome c reductase by 692 of glutanfic dehydrogenase by, 224 of homogentisate oxidase by, 2.(t5 reversal by mercaptoacetic acid, 295 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 reversal by cysteine, 292 of leucine aminopeptidase by, 93 of metaphosphatase by, 579 of myosin ATPase by 587 of prolidase by, 105 of RNase by, 434 Mercuripapain, crystallization of, 59-61 Merelrix meretrix (clam), thiaminase from viscera of, 622-626 Merthiolate, as preservative for phosphodiesterase, 564 Mescaline, amine oxidase action on, 393 Mesidine, peroxidase assay with, 774 Mesoporphyrin, formation of from cytochrome c, 753 Metal-chelating substances, activation of D-amino acid oxidase by, 202 effect of on ascorbic acid oxidase, 835 inhibition of alkaline phosphatase by, 538

952

SUBJECT INDEX

Metallic ions, see also under names of individual metals, inhibition of dehydroascorbic reductase by, 849 Metalloenzymes, see under names of individual metals Metals.heavy, see also under names of individual metals, in colorimetric determination of citrulline, 351, 359 Metaphosphatase (s), 577-580 assay procedure for low molecular weight substrates, 577 for high molecular weight substrates, 578 change in pH optimum with source and substrate, 579-580 molecular weight of A. niger enzyme, 580 occurrence of, 577, 579 Metaphosphates, inhibition of ~-glutamyltransferase (brain) by, 272 Metaphosphates, high molecular weight, depolymerization of by metaphosphatase, 577, 578 assay by viscosity change, 578 Metaphosphates, low molecular weight, orthophosphate formation from by metaphosphatase, 577-578 Methanol, activation of ~-glutamyltransferase (bacterial) by, 269 Methemoglobin, hemoglobin conversion to, 739 Methionine, cleavage of, 313 conversion to S-adenosylmethionine, 254 stimulation of tryptophan synthetase formation by, 237 as substrate for L-amino acid oxidase, 208, 211 Methionine-activating enzyme, from rabbit liver, 254-256 separation of into two protein fractions, 256 Methionine sulfoxide, inhibition of glutamine synthetase by, 342

Methyl acceptor systems, 257-263 guanidinoacetate methylpherase, 260-263 nicotinamide methylpherase, 257-260 S-Methylcysteine, cleavage of, 313 5-Methylcytosine, as acceptor of deoxyriboside group, 468 RI values for, 466 Methyl hydrogen peroxide (MeOOH), for peroxidase assay (direct) in yeast cells, 769 for peroxidase assay with guaiacol, 792 as substrate for horseradish peroxidase, 897, 810, 811 see also Enzyme-substrate complexes 2-Methyl-l,4-napthoquinone, as electron acceptor for menadione reductase, 728 Methylene blue (MB), copper salts in commercial preparations of, 716 as electron acceptor for D-amino acid oxidase, 204 for bacterial luciferase system, 860 for cytochrome bl, 746-748 for diaphorase system, 707 for hydrogenase systems, 862, 865, 868, 869 FAD requirement for, 869 for new yellow enzyme, 715, 716, 718 for old yellow enzyme, 715 for xanthine oxidase, 482 formation of from sulfide and p-aminodimethylaniline, 315 Methylene blue, leuco form, oxidation of by molecular oxygen, 715, 716, 748 catalysis by copper salts, 716 Methylene blue-sulfate ester, in assay of alkylsulfatases, 330 a-Methylglutamic acid, as substrate for glutamine synthetase, 341 a-Methylhistamine (imidazoleisopropylamine), resistance to histaminase, 396

SUBJECT INDEX

6-Methylindole, as substrate for tryptophan synthetase, 236 N-Methyl-L-leucine, as substrate for ~-amino acid oxidase, 211 N 1-Methylnicotinamide (NMeN), determination of, 257 synthesis of by nicotinamide methylpherase, 257 3-Methyl-4,6,4'-triaminodiphenyl sulfone, use in preparation of thrombin, 157 6-Methyltryptophan, as substrate for tryptophanase, 242 Mexicain, protease from latex of cuaguayote, 56, 62 crystallization of, 62 Mickle electric shaker, 468, 495 Micrococcus spp., hydrogenase in, 870 Micrococcus aureus, aspartase in, 388 Micrococcus lysodeikticus, catalase from, 775, 784-788 crystallization of, 787 growth and lysis of, 786 Microsomes, AMPase in, 542 ATPase in, 542 glucose-6-phosphatase in, 542 glucose-6-phosphatase as indicator for presence of, 542-543 glycerophosphatase in, 542 hexose isomerase in, 542 Milk, alkaline phosphatase from~ 533-539 peroxidase (lactoperoxidase) from, 245~ 813-817 xanthine oxidase from, 482-485 Milk clotting, assay of rennin by, 69 effect of CaCI~ in, 69 assay of various proteinases by, 56-59 Milkweed (Asclepias speciosa, mexicana, syriaca), crystalline protease (asclepain) from latex of, 56, 61-62

953

Mitochondria, adenylate kinase (myokinase) in muscle and liver, 602 ATPase in insect muscle, 595 ATPase from mouse liver, 593-595 low activity of in intact, 615 disintegration of, 594 DPNH cytochrome c reductase activity in, 693 glucose-6-phosphatase content of, variation with method of preparation, 543 hippuric acid-forming enzymes in, 348 oxidative phosphorylation in, 610-616 oxidative phosphorylation in modified, 615, 616 from poky strain of Neurospora, 168 uricase from liver, 489 Moccasin venom (A gkistrodon piscivorus ) , ~amino acid oxidasc from, 205-209 Molds, metaphosphatase in, 577 Molecular weight determinations, deviations due to use of different ultracentrifuges, 808 Mollusks, see Patella vulgata and Littorina littorea Molybdate, inhibition of glueose-6-phosphatase by, 542 Molybdenum, as constituent of nitrate reductase, 415 cyanide in removal of, 415 requirement of for reduction of cytochrome c by hydrogenase system, 869 Monoamine oxidase, from liver, 393 Monoesterase, see Phosphatase Monomethylamine, as product of ssrcosine oxidation, 225 N-Monomethylglycine, see Sarcosine Mononucleotides, see also Nucleotides, and under names of individual nucleotides, alkyl esters of as substrates for intestinal di'esterase, 570 for spleen phosphodiesterase, 568 inhibition of RNase by, 428, 434

954

SUBJECT INDEX

2'-Mononucleotides, formation from cyclic structures by spleen phosphodiesterase, 568 3'-Mononucleotides, formation from diesters by spleen enzyme, 568 from diesters or cyclic structures by intestinal diesterase, 570 5'-Mononucleotides, formation from ribo- and deoxyribopolynucleotides by venom diesterase, 561 Monophenolase (cresolase), induction period for, 825, 827 effects of various agents on length of, 825 Monophenols, see Phenols Monophenylphosphate, alkaline phosphatase action on, 533, 534, 538, 539 as donor for transphosphorylation, 539, 556 formation of from sym-diphenylpyrophosphate, 565 prostatic phosphatase action on, 524 kinetics of, 525 Morphine, effect of on renin action, 134 cic,cis-Muconic acid, absorption spectrum of, 282 formation of by pyrocatechase, 281282 lactonizing enzyme for, 273, 282-284 Muscle, acetyl phosphatase from, 555-556 adenylate kinase (myokinase) from, 598-604 5'-adenylic acid deaminase from, 469473 apyrase (ATPase), Mg-activated, from insect, 590-591 ATPase from insect, 595-598 ATPase, Mg-activated, from particles of, 588-591 ATPase (myosin) from, 582-588 ATP-creatine transphosphorylase from, 605-610 L-a-glycerophosphate dehydrogenase in insoluble particles from, 559 glycolyzing enzymes from, 351, 358

glycylglycine dipeptidase from, 108 nucleotide synthesis from R-5-P and adenine in extracts of, 501-504 prolidase in, 105 protein antithromboplastin from, 161-162 transhydrogenase in pigeon breast and rabbit, 687 triphosphatase in, 580, 581 "Muscle adenylic acid," see 5'Adenylic acid Muscle enzyme fraction, ATP generation with phosphoglyccrate and, 515 Mushroom, tyrosinase (polyphenol oxidase) from, 822-827 Mussel, thiaminase in, 625

Mycobacterium, aromatic oxidations in, 273 barbiturase from, 492-493 uracil-thymine oxidase from, 490-491 growth of cells for, 490-491 Mycobacterium tuberculosis, strain BCG, eytochrome e peroxidase in, 764 Myeloperoxidase (verdoperoxidase), see Peroxidase from leucocytes Myoglobin, test for in cytochrome c preparations, 751-752 Myokinase, see also Adenylate kinase, in adenosine kinase assay, 497 in arginine condensing system from liver, 362 assay of ADP with hexokinase and, 497 Myosin, ATPase activity of, 582 as specialized function of myosin molecule, 586 triphosphatase in, 580 L-Myosin, impurities in "crystalline" myosin and, 583-584 Myrosulfatases, 324 N

Naphthoquinones, reduction of by DPNH, 728

SUBJECT INDEX

a-Naphthylamine, nitrite assay with, 400 a-Naphthylethylenediamine, nitroaniline assay with, 407 N-(1-Naphthyl)ethylenediamine, nitrite determination with, 403, 412 Narcotics, inhibition of prostatic phosphatase by, 527 Nasturtium (Tropaeolum majus), carbonic anhydrase in leaf of, 842 Nembutal, effect on renin action, 134 Neotetrazolium, in assay of flavoproteins, 695 Nessler method, interference with by caprylic alcohol, 111 Nessler reagent, of Vanselow, 596

Neurospora, L-amino acid oxidase in, 211 cytochrome c destroying system in poky strain of, 168 cytochrome e peroxidase in poky strain of, 764 DPNase from, 664-666 effect of deficiencies on enzyme concentration, 666 growth medium for, Zn-deficient, 664 growth of, 168, 235, 401,417 hydroxylamine reductase from, 416419 kynureninase in, 253 kynurenine formamidase in, 253 nitrate reductase from, 411-415 nitroaryl reductase from, 406-411 independence of nitrogen source, 409, 411 nitroethane oxidase from, 400-402 D-serine (D-threonine) dehydrase from, 322-324 L-serine (L-threonine) dehydrase from, 319-322 tryptophan synthetase from, 233-238 Nickel(ous) ion, activation of arginine desimidase by, 376 inhibition of aspartase by, 388 of DNase by, 442

955

of fructose diphosphatase by, 546 of RNase by, 434 of uricase by, 489 Nicotinamide, formation of by animal tissue DPNase, 660 by Neurospora DPNase, 664 by nicotinamide riboside phosphorylase, 454 inhibition of animal tissue DPNase (DPN nucleosidase) by, 662, 670, 683 of Neurospora DPNase by, 666 methylation of, 257 Nicotinamide, C14-1abeled, exchange with bound nicotinamide of DPN, 662 Nicotinamide methylpherase, from rat liver, 257-260 5'-Nicotinamide mononucleotide (NMN), DPN formation from, 670 formation by nucleotide pyrophosphatase, 655 "5" nucleotidase action on, 549, 550 transhydrogenase reaction with DPNH or TPNH, 685 Nieotinamide mononucleotide, reduced (NMNH), DPNH synthesis from, 672 formation from DPNH by snake venom enzyme, 654 Nicotinamide riboside (NR), fluorimetric method for, 454 nucleosidase action on, 463 synthesis of by purine nucleoside phosphorylase, 448 Nicotinamide riboside phosphorylase, from beef liver, 453, 454-456 possible identity with nucleoside phosphorylase, 448, 453, 455 Ninhydrin, inhibition of insect muscle ATPase by, 598 of RNase by, 434 Ninhydrin-CO~ analysis, Van Slyke, assay of aminoacylases by, 115 Ninhydrin method, colorimetric, assay of carboxypeptidase by, 79 of glycylglyeine dipeptidase by, 107

956

SUBJECT I N D E X

Nitrate, inhibition of erythrocyte carbonic anhydrase by, 845 of glucose oxidase of A. niger by, 579 of hydrogenase exchange reaction by, 867 reduction of by hydrogenase system, 862, 867 stoichiometry of, 867 requirement of in growth medium for production of nitrogen gas-forming enzymes, 423 Nitrate esters, reduction of by eysteine, non-enzymatic, 405 by glutathione, 403-406 enzymatic, 403-406 non-enzymatic, 405, 406 Nitrate reductase, as adaptive enzyme, 415 mechanism of action of, 415 from Neurospora, 411-415 in soybean leaves, 414-415 comparison of TPNH and DPNH in, 414, 415 turnover number of, 413 Nitrate reductase, organic, from hog liver, 403-406 Nitric oxide gas, alkaline sulfite as trapping agent for, 420, 422 conversion to nitrogen dioxide by oxygen, 421 formation from nitrite and conversion to nitrogen gas, 420-423 inhibition of hydrogenase by, 867 Nitrite, determination of, 403 enzymatic conversion to nitric oxide and to nitrogen gas, 420-423 formation of by nitrate reductase, 411 by nitroethane oxidase, 400 measurement of, 400 by organic nitrate reductase, 403, 405 hemoglobin oxidation by, 739 inhibition of hydrogenase exchange reaction by, 867 oxidation of by peroxidase complex II, 810

as uncoupling agent, 615 Nitrite reductase, as adaptive enzyme, 411 presence in hydroxylamine reductase preparations, 418 in nitrate reductase preparations, 412 cyanide inhibition of, 412 in nitroaryl reductase preparations, 410, 411 m-Nitroaniline, formation from m-dinitrobenzene, 406 Nitroaryl reductase, distribution of, 409 in liver, 406 from Neurospora crassa, 406-411 in pig heart, 406 p-Nitrobenzoic acid, reduction by Neurospora, 410 4-Nitrocatechol, competitive inhibition of tyrosinase by, 826 Nitroethane oxidase, from Neurospora crassa, 400-402 Nitrofurans, as electron acceptors for DPNH cytochrome c reductase of bacteria, 697 Nitrogen, effect of deficiency of on Neurospora DPNase content, 666 Nitrogen dioxide, formation from nitrogen oxide, 421 Nitrogen gas, enzymatic formation of from nitrite and nitric oxide, 420-423 adaptive nature of enzymes for, 423 particulate nature of reducing system for, 422 Nitroglycerine (glycerol trinitrate), as substrate for organic nitrate reductase, 403, 405 Nitrophenol(s), inhibition of quinone reductase by, 729 p-Nitrophenol (4-Nitrophenol), competitive inhibition of tyrosinase by, 826 liberation from bis (p-nitrophenyl) phosphate by phosphodiesterase, 561

SUBJECT INDEX

m-Nitrophenylhydroxylamine, formation from m-dinitrobenzene, 406 p-Nitrophenyl phosphate, as substrate for alkaline phosphatase, 533 Nitrophenylphosphates, as possible substrates for clinical determination of phosphatase, 528 bis(p-Nitrophenyl) phosphate, as substrate for phosphodiesterase, 561 Nitropropane, action of nitroethane oxidase on 1- and 2-isomers of, 402 f3-Nitropropionic acid, action of nitroethane oxidase on, 402 Nitroprusside reaction, g|utathione measurement with, 719 Nonprotein nitrogen (NPN), method for assay of proteinases, 58 Norbiotin, inhibition of d-biotin oxidation by, 632 DL-Norleucinamide, hydrolysis by leucine aminopeptidase, 92 L-Norleucine, as substrate for L-amino acid oxidase, 208 transaminase for, 176 DL-Norvalinamide, hydrolysis by leucine aminopeptidase, 92 L-Norvaline, as substrate for x-amino acid oxidase, 208 transaminase for, 176 *Notatin, see Glucose oxidase Nucleic acid(s), enzymes for metabolism of, 427-519 inhibition of acetyl phosphatase by, 556 precipitation of aspartase as complex with, 387 protein fractionation with, 340, 498 Nucleosidases, hydrolytic, from baker's yeast, 461-464 purine nucleosidase, 462-464 uridine nucleosidase, 461-462 evidence for separate purine and pyrimidine nucleosedases, 460 from L. pentosus, 456-461

957

mechanism of action of, 460 Nucleoside monophosphate kinases, in yeast and animal tissues, 603 Nucleoside phosphorylase, purine, see also Thymidine phosphorylase, from beef liver, 453 from calf spleen, 448-453 differential spectrophotometry of purine compounds with, 449 from yeast, 453 Nucleoside transdeoxyribosidase, from bacteria, 464-468 from Lactobacillus helveticus, 467, 468 sources of, 467 Nucleoside triphosphates, hydrolysis of by myosin, 586 3'-Nucleotidase (3'-nucleotide phosphatase), hydrolysis of di(dinitrophenyl) phosphate by barley enzyme, 524 from rye grass, 551-555 factor responsible for heat lability of, 554-555 5'-Nucleotidase, see also Adenosine-5phosphatase, apparent activation of diesterase by, 561 from potato, 550 from seminal plasma, 547-549 from snake venom, 549-550, 561 Nucleotide(s), see also Mononucleotides and Dinucleotides, bone phosphatase action on, 539 inhibition of 3'-nucleotidase by 2'- and 5'-, 554 as substrate for prostatic phosphatase, 524 synthesis from R-5-P and adenine, 501504 enzyme for phosphorylating R-5-P, 502 heat lability of, 504 nucleotide-forming enzyme, 502 heat stability of, 504 Nucleotide (s), purine, de novo synthesis of, 504-519 activation of "1-carbon unit" as folic acid derivative in pigeon liver extract, 516-518

958

SUBJECT INDEX

conversion of 5-amino-4-imidazoleearboxamide riboside to ribotide by yeast enzyme, 514-516 isolation of 5-amino-4-imidazoleearboxamide from E. coli cultures, 512-514 over-all system for in pigeon liver extracts, 505-509 synthesis of formylglycinamide ribotide and glycinamide ribotide in pigeon liver extracts, 509-512 tentative scheme for, 504-505 transformylation of "1-carbon u n i t " to form IMP, 518-519 Nucleotide pyrophosphatase, ATP inhibition of FAD hydrolysis by, 673 as contaminant of FAD pyrophosphorylase, 675 D P N H cleavage by snake venom enzyme, 654 in pigeon liver, 653 from potato, 656-659 other phosphatase activities in, 659 variation with age and variety, 658 Nncleotide transhydrogenase, see also Pyridine nucleotide transhydrogenase, in A. aerogenes, 308 role in coupling shikimic acid reduction with quinic acid oxidation, 309, 310 equilibrium constant for, 311 O Oat seedlings, glutamic-oxalacetic transaminase activity of, 184 Octanoyl phosphate, hydrolysis of, 556 Oligonucleetide, see Deoxyoligonucleotide, Magnesium oligonucleotide "One-carbon unit" (" 1-C unit"), in final step of I M P synthesis, 505, 518-519 in formylation of glycinamide ribotide, 505 Organic nitrate reductase, see Nitrate reductase, organic

~-Ornithine, citrulline synthesis from, 350-355 colorimetric assay for p-amino benzoyl derivatives of, 350 colorimetric method for, 376 formation of by arginase, 356, 368 by citrullinase, 374 inhibition of citrullinase by, 378 oxidation of by L-amino acid oxidase, 208 reduction of, 217, 219, 220 stabilization of arginase by, 373 Ornithine decarboxylase, citrullinase and, 378 from Cl. septicum, 189 resolution of, 189 measurement of transaminase reactions with, 171 Orotic acid, incorporation of into nucleotides, 504 reduction of by DPNH, 493 loss of ultraviolet absorption during, 493 Ortho-para hydrogen conversion, catalysis by hydrogenase, 862, 864865 Orthophosphate, see Phosphate, inorganic Orthophosphomonoesters, as substrates for alkaline phosphatase, 533, 538 Osage orange (Maclura pomifera Raf.), protease (pomiferin) from latex of, 57, 64 Ovalbumin, conversion of component A1 to As by prostatic phosphatase, 526 Ovomucoid, as trypsin inhibitor, 37, 49, 51, 53 Oxalacetate, as component of transaminase system, 170-171 measurement of formation of, 171, 174-175, 179-182 by decarboxylation with aniline citrate, 171, 174-175 by determination of pyruvic acid after decarboxy|atiou, 171, 175 by spectrophotometric method, 171, 175, 179-182

SUBJECT INDEX

non-enzymatic decarboxylation of, 180 P: 0 ratio during oxidation of, 613 *0xalaeetic decarboxylation system, COs versus ttCOa- as product of, 841 Oxalate, activation of prothrombin by, 145 inhibition of acetyl phosphatase by, 556 of aspartase by, 388, 390 Oxidases, see under names of substrates for individual oxidases Oxidation-reduction indicators, see Dyes Oxidative phosphorylation, see Phosphorylation, oxidative Oxy-acid buffers, catalysis of COs hydration by, 836 Oxygen, catalase assay by manometric determination of, 769 as electron acceptor for DPNH cytochrome c reductase of bacteria, 697 for new yellow enzyme, 716, 718 for old yellow enzyme, 715, 716 for TPNH cytochrome c reductase system of yeast, 703 inhibition of amino acid reductase by, 219 of hydrogenases by, 866, 868, 869 mechanism of, 866 reactivation by hydrogen or other reducing substances, 866, 868 reduction by hydrogenase system, 862 requirement for, in firefly luciferase system, 651 1a Palmityl phosphate, hydrolysis of; 556 Pancreas,

carbonic anhydrase in, 841 carboxypeptidase and procarboxypeptidase from, 77-83 chymotrypsinogen and chymotrypsins from, 8-26 cysteine desulfhydrase in, 318 DNase from, 438-443 folic acid conjugase from, 629-630 RNase from, 427-436

959

trypsin and trypsinogen from, 26-36 trypsin inhibitor of Kazal from, 36, 40, 50, 51, 52 of Kunitz and Northrop from, 36, 38-40, 50, 51, 52 Pantetheine kinase, from pigeon liver, 633-636 Pantoate, enzymatic conversion to pantothenate, 619 Pantothenate-synthesizing enzyme, from E. coli, 619-622 assay of by chemical method, 622 by manometric method, 622 by microbiological method, 619 Papa[n, protease from latex of papaya, 56, 59, 60 activation of by cyanide, 59 by cysteine, 59 crystallization of, 59, 60 inactivation of penicillinase by, 123 use as meat tenderizer, 55 Papaya (Carica papaya), latex of as source of chymopapain, 56, 61 of papain, 56, 59, 60

Paphia philippinarum, thiaminase in, 625 Parahydrogen, conversion to normal hydrogen by hydrogenase, 864-865 Parsley root, glutathione reduetase in, 721 Patella vulgata (mollusk), arylsulfatase in, 332 steroid sulfatase in, 332 Peanut (Arachis hypogen), protease (arachain) from, 57, 63 Pea plant (Pisum sativum), dehydroaseorbic reductase from, 847850 distribution in plant and changes in development, 849 Peas (Pea seeds), 5-dehydroshikimie reductase and 5-dehydroquinase in, 304, 307 glutamine synthetase from, 337-342 ~-glutamyltransferase from, 263-266 pyridine nucleotide quinone reductase from, 725-729

960

SUBJECT INDEX

Penicillamine (/~-mercaptovaline), inhibition of penicillinase by, 123 Penicillin, as antibiotic in tyrosinase assay, 830 assay of, 120-121 Staph. aureus in, 121 inhibition of RNase by, 434 Penicillinase, from B. cereus, 120-124 as antigen in rabbits, 123 heat stability of, 123 Penicillium notatum,

aspartase in, 388 Penicillium sp.,

metaphosphatasc in, 577, 579 Penicilloic acid, as product of penicillinase reaction, 120 Pentacyanoammine ferroate, assay of m-nitrophenylhydroxylamine with, 406, 407 Pentothal, see Thiopental Pepsin, from commercial preparations of swine pepsin, 3-7 crystallization of, 5 inactivation of adenylate kinase by, 603 of RNase by, 434 inhibitor of from pepsinogen, 7 sources of, 7 Pepsinogen, from swine mucosa, 3-7 crystallization of, 6 from various species, 7 Peptidases, see Aminoacylases, Aminopeptidases, Carboxypeptidases, Dehydropeptidases, Dipeptidases Peptides, formation of by cathepsin C, 68 formation from fibrinogcn, 160 hydrolysis of, Grassmann and Heyde procedure for estimation of, 83, 88, 93-94, 97, 100, 105-106, 107-108 Peptides, synthetic, as substrates for proteolytic enzymes, 21 Peptone, protection of carbonic anhydrase by, 838, 846

Perborate, as oxidizing agent in catalase reaction, 78O Perchlorate, inhibition of D P N H cytochrome c reductase by, 692 Periodic acid, inhibition of RNase by, 434 Permanganate, assay of catalase by titration of H202 with, 768, 779, 781-782, 791-792 oxidation of a-ketoglutarate with, 170 Permutit, deaminase purification with, 476 separation of hydroxytyramine from dopa with, 195 Peroxidase(s), assay of, 769-775 by direct measurement of enzymesubstrate compound in intact cells, 769 by guaiacol test, 770-773, 792 by pyrogallol test, 773-775 by various tests, 774, 794 interpretation in terms of rate constants for formation and utilization of enzyme-substrate complex, 770, 775 from beef liver, 791-794 catalase association with, 793-794 conversion to inactive heme-protein, 794 distribution in plants, 801-802 from horseradish, 770, 801-813 absorption bands of, 803 activity, dissociation constants and spectra of derivatives of, 812 assay of, 803 chemical analysis of, 809, 811 compounds (complexes) formed with oxidizing agents~ 807 with substrates, 770, 801-813 see also Enzyme-substrate complexes with various reagents, 812 crystallization of, 806-807 nitrogen content of, 799 oxidation reduction potential of, 809 physical properties of, 808-809

SUBJECT INDEX

rate constant for formation of complex I, 773 reaction mechanism for, 770, 802 formation of complexes I and II, 8O2 of complex III, 803 RZ unit as measure of purity of, 803 solubility curve described for, 806 specificity of, 807-808 spectroscopic, magnetic and kinetic data on, 810, 811-813 titration curves for, 809 from leucocytes (myeloperoxidase, MyPO) (verdoperoxidase), 794801 assay of purity by activity measurements (uric acid method), 794796 by spectrophotometric measurements, 801 physiological role of in detoxication, 800, 801 preparation from empyema, 797-798 from ox leucocytes, 796-797 properties of, 798-799, 801 rate constant for formation of complex I, 773 sources of, 794 from milk (lactoperoxidase), 813-817 assay of, 813 crystallization of, 816-817 extinction coefficients for, 817 isoelectric point of, 817 phosphate effect on, 816, 817 optical density ratios for assay of, 813 rate constant for formation of complex I, 773 turnover number of, 817 from yeast, rate constant for formation of complex I, 773 Peroxide, see also Hydrogen peroxide, Methyl peroxide, Ethyl peroxide, inhibition of homogentisate oxidase by, 295 o-Phenanthroline, activation of uricase by, 489 complex of with ferrous ions, 726

961

inhibition of DPNH cytochrome c reductase by, 693 of hydroxylamine reductase by, 419 of nitrate reductase by, 415 Phenol(s), detoxication of diphtheria toxin by myeloperoxidase and, 800 inhibition of squash carboxylase (a-keto acid) by, 192 liberation of from diphenyl phosphate by phosphodiesterase, 561,564 oxidation of by lactoperoxidase, 816 by peroxidase complex II, 808, 810 by tyrosinase, 825-826, 830 comparison of plant and mammalian enzymes in, 830 as precursor of catechol, 273 as product of transphosphorylation by phosphatases, 556 measurement of, 559 substituted, inhibition of TPNH cytochrome c reductase of yeast by, 703 Phenol color method, for assay of proteinases, 55-58 Phenolic ethereal sulfates, as substrates for arylsulfatases, 328 Phenolphthalein phosphate, in assay of serum phosphatase, 528 inhibitory effect of, 528 L-Phenylalaninamide, action of amidase on, 399 hydrolysis of by leucine aminopeptidase, 92 L-Phenylalanine, decarboxylase action on various hydroxy derivatives of, i97 decarboxylation of in mammalian tissues, 199 inhibition of D-amino acid oxidase by, 203 as product of aromatic biosynthesis, 300 stimulation of tryptophan synthetase formation by, 237 as substrate for L-amino acid oxidase, 208 transaminase for, 176 L-Phenylalanine ethyl ester, as substrate for trypsin and chymotrypsin, 23, 25

962

SUBJECT INDEX

use in manometric assay of chymotrypsin, 25 ~Phenylalanylhydroxy-L-proline, as substrate for prolidase, 104 Phenylarsine oxide, inhibition of aspartase by, 388 Phenylenediamine, cytochrome c reduction by p-form of, 754 oxidation of o- and p-forms of by peroxidase complex II, 810 L-Phenylglycolie acid, as substrate for i-amino acid oxidase, 211 Phenylhydrazine, inhibition of cysteine desulfhydrase by, 317 Phenylisocyanate, inhibition of RNase by, 434 L-Phenyllactic acid, as substrate for L-amino acid oxidase, 211 Phenyl phosphate, see Monophenyl phosphate f~-Phenylpropionic acid, inhibition of carboxypeptidase by, 79 Phenylpyruvate, enol-keto tautomerase action on, 291 Phosphatase, see also Phosphomonoesterase and Phosphodiesterase, effect on oxidative phosphorylation in tissue homogenates, 614 inhibition of by citrate and Versene, 313 of yeast enzyme by "pyrimidyl" and thiamine, 638 lung thromboplastin and, 150 rennin as, 77 Phosphate, inorganic (orthophosphate), activation of dehydroascorbic reductase by~ 849 of dopa peroxidation by, 816 competitive inhibition of by H20~, 816 of ~-glutamyltransferase by, 267, 272 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 of pantetheine kinase by, 635 of prothrombin by, 145 binding of by lactoperoxidase, 816

catalysis of COs hydration by, 836, 838 effect on thiaminokinase, 640 enzymes for metabolism of, 523-616 formation of by acid and alkaline phosphatases, 523-541 by apyrsse, 591 by ATPases, 582, 588, 593, 595 by fructose-l,6-diphosphatase, 543 by glucose-6-phosphatase, 541-543 by inorganic pyrophosphatase, 570 by metaphosphatase, 577, 578 by methionine-activating system, 254 by "5" nucleotidases, 546-550 by nucleotide pyrophosphatase action on ATP, 655 by nucleotide synthesizing system, 501 by triphosphatase, 580 inhibition of acetyl phosphatase by, 556 of alkaline phosphatase by, 473-475, 533, 538 of L-amino acid oxidase by, 208 of 5'-AMP deaminase by, 472 effect of 5'-AMP concentration on, 472 of arsenate-catalyzed citrullinase reaction by, 378 of arylsulfatases by, 328 of carnosinase by Mn ++ plus, 96 of D P N H cytochrome c reductase by, 692 of iminodipeptidase by, 100 of purine nucleosidases by, 460, 463-464 of xanthine oxidase by, 485 isobutanol extraction method in measurement of, 592 requirement of, for activation of glycyl-L-leucine dipeptidase by zinc, 107 for fluoride inhibition of nucleotide pyrophosphatase, 659 for reduction of cytochrome e by hydrogenase system, 870 role in nicotinamide riboside phosphorylase reaction, 454

SUBJECT X~DEX

in nueleoside phosphorylase reaction, 448 in Pseudomonas transhydrogenase reaction, 685 stabilization of pyrimidine nucleosidase by, 459 uptake of during citrullinase reaction, 376, 378 replacement of by arsenate, 377, 378 during oxidative phosphorylation, 610 Phosphatides, presence of in thromboplastin, 150 Phosphoamidase, rennin as, 77 Phosphoamides, as substrates for alkaline phosphatase, 533, 538 Phosphocholine, see Phosphorylcholine Phosphocreatine, determination of, 605 formation during oxidative phosphorylation, 613 phosphomonoesterase-eatalyzed transfer of phosphate to glucose from, 533 phosphorylation of ADP by, 605 as substrate for alkaline phosphatase, 533, 538 Phosphodiesterase (s), in bone, 540 separation from monoesterase, 540 from calf intestine, 569-570 RNase as, 433 from snake venom, 561-565 from spleen, 565-569 fractions free of activity against cyclic nueleotides, 568 Phosphoenolpyruvate, as substrate for alkaline phosphatase, 538 *Phosphoglucomutase, in assay of glucose-l-P, 675, 676 formation of R-I,5-P2 with, 503 *6-Phosphogluconate dehydrogenase, in glucose-6-phosphate assay, 676 removal by ethanol fractionation, 677 6-Phosphoglucose, see Glucose-6phosphate

963

3-Phosphoglyceric acid (3-PGA), regeneration of ATP by, in various biosynthetic processes, 343, 351, 356, 357, 506, 510, 5157 517 *Phosphohexoisomerase (hexose isomerase), in microsomes, 542 Phosphomonoesterases, group-specific, 523-541 sources of, 540 comparison with activity of bone enzyme, 540 substrate-specific, 541-555 transphosphorylation by acid and alkaline, 556-561 Phosphomonoesterase, acid, distribution of in animal tissues, 523 in erythrocytes, 527 differentiation from serum phosphatase of prostatic origin, 527528 in preputial glands of rats, 523 from prostate gland, 523-530 action of on high-molecular phosphorus compounds, 525-527 nucleic acids, 526 phosphoproteins, 526-527 dependence of pH optimum on substrate, 527 effect of hormones on enzyme concentration, 523 inhibitors of, 527-528 K~ values for, 527 limitations as analytical tool, 525, 526 purification of, 529-530 species variation in, 523 specificity of, 524-525 titration method for detecting diesterase action of, 525 in seminal vesicles of guinea pigs, 523 in serum, 524, 527, 528 ~tartrate inhibition as test for prostatic origin of, 528 Phosphomonoesterase, alkaline, from bone, 539-541 from intestine, 473-475, 530-533, 539 inhibition of by phosphate, 473-475 separation of from adenosine deaminase, 473-474

964

SUBJECT

from diesterase, 569-570 transferase activity of, 533 from milk, 533-539 transphosphorylation activity of, 539 from various sources, 539, 560 comparison of, 539 extraction of with n-butanol, 560 transphosphorylation by, 561 4~-Phosphopantetheine, conversion to CoA by pigeon liver extract, 633 to dephospho-CoA, 667, 669 formation by pantetheine kinase, 633 maintenance of in reduced form, 669 Phosphopeptone, as substrate for phosphatase action of rennin, 77 Phosphoproteins, see Casein, Ovalbumin Phosphopyruvate, role in adenosine kinase assay, 497 5-Phosphoribosyl pyrophosphate, as intermediate in nucleotide synthesis from adenine and R-5-P, 501 in nucleotide synthesis de nova, 504 Phosphorus 82, incorporation into ATP as measure of oxidative phosphorylation, 614 Phosphorus: oxygen ratio (P: O ratio), definition of, 612 table for value with different substrates, 613 Phosphorylation, oxidative, in homogenates and mitochondria, 610-616 assay systems for, 610-614 direct, 610-613 indirect, 614 protective effects of substrate, ATP and oxidative activity on, 612 P:O ratios with different substrates, 613 requirements for and lability of, 615 uncoupling phenomenon in, 615-616 in tissue slices, 614 Phosphoryl choline (phosphocholine), a s product of Cl. welchii lecithinase action, 590

INDEX

prostatic phosphatase action on, 525 high pH optimum for, 527 Pho sphorylethanolamine, prostatic phosphatase action on, 525 Phosphorylserine, prostatic phosphatase action on, 525 Phosphotransacetylase, in assay of dephospho-CoA kinase, 649 Phosphotungstic acid, color reaction for uric acid, 486 Photinus pyralis, see Firefly Pileus mexicanus, see Cuaguayote Pineapple (Anana sativa), protease (bromelin) from, 56, 62-63 Pinguinain, protease from fruit of maya plant, 56, 63 Pisum sativum, see Pea plant Pituitary, prolidase in, 105 Plants, see also under names of individual species, cytochrome a3 in, 732 triphosphatase in, 580 Plasma, Ac-globulin from, 151-152 amine oxidase from, 390-393 antihemophilic factor from, 149 antithrombin in defibrinated, 162 convertin from, 155-156 fibrinogen from, 158-160 preparation of from horse blood for assay of antihemophilic factor, 147 proconvertin from, 153-154 prothrombin-free, 140 use in prothrombin assay~ 140 prothrombin from~ 140-146 stabilization of carbonic anhydrase by, 846 thrombin from, 156-158 thromboplastin, heat-labile component of (AHF, PTC), in, 139 trypsin inhibitor from, 37, 49-54 Plasma Ac-globulin (proaccelerin, Factor V), activation of by thrombin, 157 complexes of with thromboplastin and convertin, 151

SUBJECT INDEX concentration of in bovine and human plasma, 152 in oxalated horse plasma, 147 preparation of serum Ac-globulin from, 153 use of thrombin for, 152, 153 purification of from ox blood, 151-152 role in clotting mechanism, 139, 150, 152 Plasma thromboplastin antecedent (PTA), adsorption of by BaS04, 150 role in clotting mechanism, 139 Plasma thromboplastin component (PTC), see Antihemophilic factor Plasmin, see Fibrinolysin Plasminogen, see Profibrinolysin Plastic matting (Neotex), use in collection o[ pancreatic juice, 81 Platelet factor (heat stable factor of thromboplastin), assay of, 147-148 purification of, 150 Platelet reagent, use in assay of antihemophilic factor, 148 Platelets, accelerin in, 139 lysis of by thrombin, 157 thromboplastin in, 139 Platinic salts, inactivation of renin by, 134 P: 0 ratio, see Phosphorous: oxygen ratio Poky mutant, see Neurospora Polarograph, catalase assay by use of, 780 Polynucleotides, hydrolysis of by spleen phosphodiesterase, 555-556 by venom phosphodiesterase, 561,564 Polypeptide(s), as component of heparin cofactor, 163 as substrates for leucine aminopeptidase, 91 Polyphenoloxidase, see Tyrosinase Polyribopho sphate, degradation of by RNase, 433 Polysaccharides, separation from old yellow enzyme by electrophoresis, 714

965

Pomiferin, protease from latex of Osage orange, 57, 64 Porphyrin, see also Hemin, Hematin, Protoporphyrin, possible presence in liver glutathione reductase, 725 Potassium chloride, effect of on myosin ATPase, 587 inhibition of nitroethane oxidase by, 402 of xanthine oxidase by, 485 in oxidative phosphorylation assays, 612 Potassium ethyl xanthate, inhibition of ascorbic acid oxidase by, 835 of hydroxylamine reductase by, 419 of nitrate reductase by, 415 of tyrosinase by, 826 Potassium ion, activation of pantothenate-synthesizing enzyme by, 621 of tryptophanase by, 242 inhibition of DPNH cytochrome c reductase by; 692 Potassium nitrate, inhibitionof nitroethane oxidase by, 402 Potato, adenosine-5-phosphatase from, 550 apyrase from, 591-593, 646 diphosphopyridine nucleotidases in, 655 glutamic-oxalacetic transaminase activity of root, stem and leaf from, 184 nucleotide pyrophosphatase from, 655659 phosphatase in, 540 triphosphatase from, 581, 582 Preputial glands, acid phosphatase in rat, 523 Pressor substance, see Angiotonin Proaccelerin, see Plasma Ac-globulin Procarboxypeptidase, from beef pancreas, 77-83 activation of by trypsin, 80 electrophoretic mobility of, 79-80 purification of, 79-80 resistance to activation by other proteases, 80

966

SUBJECT I N D E X

Proconvertin (SPCA precursor), concentration of in oxalated horse plasma, 147 effect of dicoumarol on synthesis of, 140, 154 purification of from human plasma, 153-154 role in clotting mechanism, 140, 154 Profibrin, in fibrinogen solutions, 160 Profibrinolysin (plasminogen), 140, 163-165 from Fraction I I I of plasma, 164-165 role in clot dissolving, 140 Prolidase (imidodipeptidase), from equine erythrocytes, 101, 102103, 105 in intestinal mucosa, 102, 105 from kidney, 98, 101, 103-104, 105 mechanism cf action of, 105 presence of in purified iminodipeptidase, 99 L-Prolinamide, amidase action on, 399 leucine aminopeptidase action on, 92 Prolinase, see Iminodipeptidase ~Proline, P:O ratio during oxidation of, 613 as product of aminotripeptidase action: 87 reduction of D- or, 217, 219, 220 as substrate for L-amino acid oxidase, 211 ~-Prolylglycine, as substrate for iminodipeptidase, 97 ~Prolylglycylglyeine, as substrate for aminotripeptidase, 83, 87, 97 in crude prolinase preparations, 97 L-Prolyl-L-proline, as substrate for prolidase, 104 Propionic acid bacteria, aspartase from, 386-387, 388 growth of, 386--387 Propionyl amino acids, action of acylase I on, 118 Propionyl-L-glutamate, role in citrulline synthesis, 355 Propionyl phosphate, hydrolysis of, 556

Propylamine, amine oxidase action on, 393 Propylenediamine, action of diamine oxldase on, 396 Prorennin, activation of to rennin, 72, 73 Prostate gland, acid phosphomonoesterase from, 523530, 540 elevated acid phosphomonoesterase in serum following carcinoma of, 524 INH insensitivity of DPNase from human, 663 ribonuclease in, 526, 529 Protamine, activation of profibrinolysin by, 165 fractionation of barbiturase with, 492-493 of dihydro6rotic dehydrogenase with, 495 of protein with, 344 inhibition of enzymatic degradation of cytochrome c by, 169 precipitation of heparin by, 163 of nucleic acid by, 213, 214, 275, etc. preparation of solution for, 214 variability in effectiveness for, 275 of pantethcine kinase from liver extract by, 633 Protease, see Proteolytic enzymes Proteinases, see Proteolytie enzymes Protein(s), determination of concentration of by optical method, 19 by turbidimetric method, 743 enzymes for metabolism of, 3-423 oxidation of by tyrosinase, 826 Proteolysis, by mitochondria from poky strain of Neurospora, 168 Proteolytic activity, removal of during RNase purification, 430 Proteolytic enzymes, animal, see under names of individual enzymes Proteolytic enzymes, plant, 54-64, see also under names of individual enzymes,

SUBJECT INDEX

determination of activity of, 55-59 milk clotting method, 58-59 nonprotein nitrogen method, 58 phenol color method, 55-58 heat resistance of, 54 isolation of arachain from peanut, 57, 63 of asclepain from milkweed, 56, 61-62 of bromelin from pineapple, 56~ 62-63 of chymopapain from papaya, 56, 61 of euphorbain from caper spurge, 57, 64 of ficin from fig, 56, 61 of hurain from jabillo, 57, 64 of mexicain from cuaguayote, 56, 62 of papain from papaya, 56, 59, 60 of pinguinain from maya, 56, 63 of pomiferin from osage orange, 57, 64 of solanain from horsenettle, 57, 63-64 of soyin from soya bean, 56, 63 of tabernamontain from

Tabernamontana grandiflora, 56, 63 milk clotting activity of, 54 non-SH enzymes, 54, 63-64 optimum pH for, 54 SH enzymes, 54, 59-63 table of properties of, 56-57 technological and medical uses of, 55 various other sources of, 63, 64

Proteus, transaminase in, 173-174

Proteus morganii, cystathionine cleavage enzyme from, 314 desulfhydrases from, 318

Proteus vulgaris, aspartase in, 388 -r-glutamyltransferase from, 268-269 -r-glutamyltransfer~se (Mn-dependent) from, 271-272 growth medium for, 268, 865-866 hydrogenase from, 865-866 hydrolysis of ~-glutamyl hydroxamic acid by extracts of, 339

967

metaphosphatase in, 577, 579 Prothrombin, 140-146 activation of, 146 assay of, one-stage, 140-141 two-stage, 141-143 concentration of in oxalated horse plasma, 147 as contaminant of proconvertin, 154 preparation of thrombin from, 157 purification of from plasma, 143-145 simplified method for, 145 role in clotting mechanism, 139, 152, 156 Protocatechuic acid, molar extinction coefficients for, 285 spontaneous oxidation of, 287 Protocatechuic acid oxidase, from Ps. fluorescens, 273, 284-287 Protohematin, as prosthetic group of spinach catalase, 790 Protohematin IX, as prosthetic group of catalase from M. lysodeikticus, 788 Protohemin, see also Hemin, content of in horseradish peroxidase, 809 in catalase, 784 Protoporphyrin, extinction coefficient for pyridine hemochromogen of ferro-, 809 Protyrosinase, activating agents for in grasshopper eggs, 831 Psalliota compestris, see Mushroom

Pseudomonas, citrullinase in, 378 tryptophan peroxidase in, 245

Pseudomonas aeruginosa, arginine desimidase in, 376 aspartase in, 388 transhydrogenase in, 686

Pseudomonas fluorescens, aspartase from, 387, 388 determination of L-aspartic acid with, 389 benzaldehyde dehydrogenase from, 273, 280-281 benzoylformic carboxylase from, 273, 278-280

968

SUBJECT I N D E X

cytochrome c from, 758-760 cytochrome c peroxidase from, 761-764 cytochrome system in particulate fraction from, 275, 277 growth of, 229, 251,387, 762 histidase from, 228-231 kynureninase from, 249-253 lactone-splitting enzyme from, 273, 282-284 lactonizing enzyme for cis,cismuconic acid from, 273, 282-284 L(W)-mandelic acid dehydrogenase from, 273, 274, 277-278 mandelic acid racemase from, 273, 274, 276-277 particulate fraction of, electron transport system of, 275 L(~)-mandelic dehydrogenase in, 275 protocatechuic acid oxidase from, 273, 284-287 pyridine nucleotide transhydrogenase from, 681-686 pyrocatechase from, 273, 274, 281-282 strain differences in aromatic oxidations by, 273 transaminase in, 172-173 urocanase from, 231-233 Pseudomonas pyocyaneus, see also Pseudomonas aeruginosa, aspartase in, 388 Pseudomonas stutzeri, enzymes for nitrogen gas formation from, 420-423 growth of, 421 Pteroyglutamic acid, see Folic acid Pumpkin, 7-glutamyltransferase (PGT) from, 263-264, 266-267 protease in, 64 Purines, as aeeeptors of deoxyriboside group, 468 chromatographic and ionophoretic separation from nucleosides, 457 as products of nucleosidase action, 456 spectrophotometric methods for determination of, 457-458 synthesis of, method for determining with C14-formate, 505-507

Purine nucleosidase, see Nucleosidases, hydrolytic Purine nucleoside phosphorylase, see Nucleoside phosphorylase, purine Purine ribosides (PuR), synthetic, nucleosidase action on, 463 Purpurogallin, extinction coefficient for, 773 formation of from pyrogallol by peroxidase, 773-775 Purpurogallin number, peroxidase activity exprcssed by, 774 Purpurogallin test, in assay of peroxidase, 813, 816 Putrescir~e (1,4-diaminobutane), as substrate for diamine oxidase, 396 Pyribenzamine, inhibition of amine oxidase by, 393 Pyridine, activation of thiaminase by, 625 Pyridine hemochromogen, see also Itemochromogen, hematin peptide and, 169 Pyridine nucleotide oxidases, 712-719 new yellow enzyme, 715-719 old yellow enzyme, 712-715 Pyridine nucleotide quinone reductase, from pea seeds, 725-729 Pyridine nucleotide transhydrogenase, 681-687, see also Nucleotide transhydrogenase, in brain, 681-687 specificity of hog enzyme for exchange reaction, 681 distribution of, 686, 687 in animal tissues, 687 in bacteria, 686 from heart, 686-687 from Ps. fluorescens, 681-686 role in apparent reaction of TPNH with diaphorase, 710, 711 in yeast, 682 Pyridine nueleotides, see also Di- and Triphosphopyridine nucleotide, discovery of, 712 reduction of by molecular hydrogen, 729 requirement for benzaldehyde oxidation, 277

SUBJECT INDEX

Pyridine transglycosidase, see Diphosphopyridine nucleotidase Pyridoxal, phosphorylation of by A.TP, 646 stabilization of pyridoxal kinase by, 648 Pyridoxal kinase, from brewer's yeast, 646-649 Pyridoxal phosphate (pyridoxal-5phosphate) (B6alP), ammonium salt of, 233 assay of, 647 calcium salt of, 212 as coenzyme for various systems, amino acid dcearboxylases, 189, 193, 198 effect of coenzyme on kinetics, 193, 198 cysteine and homocysteine desulfhydrases, 318 kynureninase, 250, 253 racemases, 213, 217 alanine racemase, 213 glutamic racemase, 217 D-serine (D-threonine) dehydrase, 323-324 L-serine (L-threonine) dehydrase, 320, 322 transaminases, 170, 172-175, 177, 179-182, 289 tryptophanase, 238, 241 demonstration of with pyridoxinerequiring mutant, 237 stabilizing effect of coenzyme, 237 tryptophan synthetase, 236-237 combination of with carbonyl reagents, 241 content of in D-amino acid oxidase preparation used for racemase assays, 213 formation of, 646 protection of by citrate and Versene, 313 Pyridoxamine, inhibition of amine oxidase by, 393 pyridoxal kinase action on, 648 Pyridoxamine phosphate, transamination with cysteine-sulfinatc in Cl. welchii, 173

969

Pyridoxine, pyridoxal kinase action on, 648 Pyrimidine(s), as aceeptors of deoxyriboside group, 468 chromatographic and ionophoretic separation from nucleosides, 457 distinction from nucleosides by light absorption in alkali, 458, 461 enzymatic methods for, 458 as products of nucleosidase action, 456 Pyrimidine derivatives, activation of thiaminase by, 627-628 inhibition of thiaminase by, 625 Pyrimidine nucleoside phosphorylase, see also Thymidine phosphorylase, distinction from purine nucleoside phosphorylase, 448 in E. coli, 480 Pyrimidine oxidase, see Uracil-thymine oxidase Pyrimidine ribose nucleotides, cyclic (2/3'-monohydrogen phosphate esters of nucleosides), digestion by RNase, 433 Pyrimidine riboside 3'-phosphates, secondary phosphate esters of as substrates for RNase, 433 "Pyrimidyl" (2-methyl-4-amino-5ethoxymethylpyrimidine), in assay of thiaminokinase, 636 inhibition of yeast phosphatase by, 638 Pyrocatcchase, from Ps. fluorescens, 273, 281-282 Pyrocatechol (1,2-bcnzenediol), detoxication of diphtheria toxin by myeloperoxidase and, 800 Pyrogallol, in assay of peroxidase, 773-775 oxidation of by peroxidase complex II, 810 by tyrosinase, 826 Pyrogallol test, see Purpurogallin test Pyrophosphatasej inorganic, as contaminant of FAD pyrophosphorylase, 675 distinction from metaphosphatase by heat, 579 from triphosphatase by heat, 581

970

SUBJECT INDEX

in E. coli, 619 in liver, 362, 667, 669 activation of latent enzyme by magnesium ion, 669 in methionine-activating reaction, role of, 256 from yeast, 570-576 purification by method of Kunitz, 571-575 alternative procedure, 576 crystallization of, 573-574 Pyrophosphatase, organic, in intestinal mucosa, 475 role in determination of bound adenosine, 475 in snake venom, relation to diesterase, 565 Pyrophosphate, inorganic, activation of D-amino acid oxidase by, 202 formation and utilization of by reversible pyrophosphorolysis of dephospho-CoA, 667, 669 of DPN, 670 of DPNH, 672 of FAD, 673 of UDPG, 675, 676 formation of by mononucleotide synthesizing system, 501 by oxidative phosphorylation, 613 by pantothenate-synthesizing system, 619, 622 by triphosphatase, 580, 582 inhibition of aeetyl phosphatase by, 556 of alkaline phosphatase by, 538 of 5'-AMP deaminase by, 472 of aspartase by, 388 of D P N H cytochrome c reductase by, 692 competition with cytochrome c, 692 of ~-glutamyltransferase (brain) by, 272 of homogentisate oxidase by, 295 of iminodipeptidase by, 100 of leucine aminopeptidase by, 93 of luciferase (firefly) by, 856 of prolidase by, 105

protection of hydroxylamine reductase by, 419 of nitrate reductase by, 414 of renin by, 129, 133, 134 Pyruvate, activation of hydrogenase system by, 866 decarboxylation of as TPP assay, 636 determination of, 316, 730 formation of by cysteine desulfhydrase, 315-318 by cystathionine cleavage enzyme, 314 by exocystine desulfhydrase, 319 by pyruvate phosphokinase in test for adenosine kinase, 497 by L-serine dehydrase, 319-322 by tryptophanase in E. coli, 238 P: O ratio during oxidation of, 613, 616 protection of hydrogenase by, 218 reduction by molecular hydrogen, 729-730 *Pyruvate phosphokinase, role in adenosine kinase assay, 497 *Pyruvic decarboxylation system, CO2 versus tICO3- as product of, 841

Q Quinaerine (atabrine), inhibition of amine oxidase by, 393 of D P N H cytochrome c reductase by, 693, 698 of TPNH cytoehrome c reductase of yeast by, 703 as uncoupling agent, 615 Quinic acid, over-all equilibrium constant for conversion to shikimic acid, 311 in scheme for aromatic biosynthesis, 300, 301 Quinic dehydrogenase, from A. aerogenes, 307-311 Quinine, inhibition of n-amino acid oxidase by, 203 Quinoline, activation of thiaminase by, 625

SUBJECT INDEX

p-Quinone, see p-Benzoquinone Quinones, as electron acceptors in bacterial luciferase system, 857, 860 apparent inhibition by, 860 inhibition of homogentisate oxidase by, 295 R

Rapid-flow methods, for carbonic anhydrase, 840 Rattlesnake, diamond, 5'-nueleotidase in venom from, 561 phosphodiesterase in venom from, 564, 565 Reaction inactivation, as characteristic of ascorbic acid oxidase, 835 of plant tyrosinase, 826 Red blood cells, see Erythrocytes Reductic acid (1,2,3-triketocyclopentane), as substrate for dehydroascorbic reductase, 850 Reduetone, oxidation of by peroxidase complex II, 810 Reinheitszahl, see RZ Renin, from kidney, 124-135 as antigen, 129-130, 133 a-, f~- and w-forms of, 135 methods of stabilization of, 129, 133, 134 presser effect on dog, 124-125 purification procedures, 125-133 resistance to acid, 134 ultraviolet spectroscopy of, 135 Renin-protein complex, 135 Renin substrate (hypertensinogen), 124, 135-139 from hog serum, 135-139 electrophoretic analysis of, 138-139 identification in as fraction, 139 Rennet, commercial, as source of rennin, 73, 74 Rennin, from calf stomach, 69-77 amino acid composition of, 77

971

crystallization of, 74-76 formation from prorennin, 72, 73 peptone formation from a-casein by, 77 phosphatase activity of on phosphopeptone, thermostable activator for, 77 phosphoamidase activity of, 77 physical properties of, 77 proteolytic action on casein, 77 on hemoglobin, 77 in preparation of lactoperoxidase, 813 of medium for propionic acid bacteria, 388 Resolution of a-amino acids, use of aminoacylases for, 119 Resorcinol (1,3-benzenediol), detoxication of diphtheria toxin by myeloperoxidase and, 800 oxidation of by peroxidase complex II, 810 Respiratory enzymes, 681-870 Rhodanese, from beef liver, 334-337

Rhodopseudomonas fluorescens, cytochrome c in, 759

Rhodospirillum rubrum, hydrogenase in, 870 F t k f i a v i n (vitamin B2), distribution coefficient (benzyl alcohol: water) for, 642 inhibition of L-amino acid oxidase by riboflavin analogs and, 208 of bacterial luciferase by, 860 of DPNH cytochrome e reductase by, 693 kinase for in pyridoxal kinase preparations, 648-649, see also Flavokinase molecular extinction coefficient for, 641 phosphorylation of, 640, 644 relationship to old yellow enzyme, 712 spectrophotometric determination of in flavoproteins, 709 Riboflavin kinase, in FAD synthesizing system, 675 Riboflavin-5'-phosphate, see also Flavin mononucleotide (FMN), formation from FAD by nucleotide pyrophosphatase, 655

972

SUBJECT INDEX

Riboflavin, reduced, as electron donor in bacterial luciferase system, 861 Ribonuclease, amino acid composition of, 434 as antigen, 435 assay in tissues, unreliability of, 436 of beef spleen, 436 action of on product of pancreatic RNase action, 436 from bovine pancreas, 427-436 crystallization of, 430-432 as byproduct of DNase preparations, 439 chromatographic fractionation of into two components, 435-436 elementary composition of, 434 physical constants for, 435 preparation of radioactive, 429 in prostatic phosphatase preparations, 526, 529 in rye grass 3'-nucleotidase preparations, 553-554 various sources of, 427, 436 Ribonucleic acid, enzymatic hydrolysis of, 427-436 changes in solubility, diffusibility and ultraviolet absorption during, 427 extent of hydrolysis by prostatic phosphatase, 526 inhibition of streptococcal DNase by bacterial, 447 3'-nucleotidase action on, 553-554 preparation of solution of, 428 presence of in thromboplastin, 150 purification of commercial samples of, 428 resistance to DNase action, 441 ribonuclease-resistant "core" from, 566, 568-570 resistance to intestinal phosphodiesterase, 569, 570 as substrate for spleen phosphodiesterase, 566, 568 spontaneous hydrolysis of, 433 as substrate for intestinal phosphodiesterase, 569 for spleen phosphodiesterase, 565, 568

Ribonucleic acid, deaminated, degradation of by RNase, 432 Ribose, inhibition of uridine nucleosidase by, 462 as product of nucleosidase action, 456, 460-461 determination of, 457 D-Ribose, paper chromatography of, 513 Ribose-l,5-diphosphate (R-1,5-P2), formation by phosphoglucomutase, 503 as intermediate in nueleotide synthesis, 501 Ribose-l-phosphate (R-l-P), conversion to R-1,5-P:, 503 elimination as intermediate in nucleosidase action, 460, 461 formation by nicotinamide riboside phosphorylase, 454 preparation of, 448-449 as substrate for nucleoside phosphorylase, 448 Ribose-5-phosphate (R-5-P), conversion to 5-phosphoribosyl pyrophosphate, 504 degradation in cells grown on xylose, 460-461 in de novo purine nucleotide synthesis, 506 elimination of as intermediate in nucleosidase action, 460, 461 in glycinamide ribotide synthesis, 510 " 5 " nucleotidase action on, 550 phosphorylation of, 502 heat-lability of enzyme for, 504 prostatic phosphatase action on, 525 synthesis of IMP from hypoxanthine and, 503 of nucleotides from, 501-504 Riboside (s), of 8-azaguanine, enzymatic synthesis of, 448 of hypoxanthine and guanine, action of phosphorylase on, 448 Rice bran, metaphosphatase from, 579 Robison ester, see Glueose-6-phosphate

SUBJECT INDEX

Rosindulin GG, as electron donor to FMN in bacterial luciferase system, 861 Rubidium ion, activation of tryptophanase by, 242 Rye grass, 3'-nucleotidase from, 551-555 RZ (Reinheitszahl) unit, purity of horseradish peroxidase measured by, 803 of myeloperoxidase measured by, 798, 801

Saccharomyces cerevisiae, see Yeast Safranin T, as electron donor to FMN in bacterial luciferase system, 861 Salicylaldoxime, inhibition of hydroxylamine reductase by, 419 of tyrosinase by, 826 Salicylic acid, inhibition of L-amino acid oxidase by, 2O8 Salmine sulfate, see also Protamine, precipitation of nucleic acids with, 499 Salmonella enteritidis, aspartase in, 388 Salts, inhibition of 7-glutamyltransferase (brain) by, 272 Sarcina spp., aspartase in, 388 Sarcina lutea, cytochrome b in, 745 Sarcosine (N-monomethylglycine), as substrate for glycine oxidase, 225~ 227 Sarcosylglyeine, as substrate for glycylglycine dipeptidase, 107 Sarcosyl-L-leucine, hydrolysis by glycyl-L-leucine dipeptidase, 107 Schmidt's deaminase, see 5'-Adenylie acid deaminase Selenite, catalysis of COs hydration by, 836 inhibition of DNase by, 442

973

Semen, acid phosphatase of prostatic origin in, 523, 540 Semicarbazide, inhibition of amine oxidase by, 393 of arginine desimidase by, 376 of aspartic acid decarboxylase by, 188 of kynureninase by, 253 Seminal vesicles, acid phosphatase in, 523 Seminal plasma, " 5 " nucleotidase from, 547-549 Sequential induction, in aromatic oxidations by Ps. fluorescens, 274 L-Serinamide, hydrolysis by leucine aminopeptidase, 92 Serine, conversion of/3-carbon of to formyl group, 518 in peptide A from fibrinogen, 160 >tryptophan synthesis from, 233 D-Serine (D-threonine) dehydrase, in E. coli, 322, 323 from Neurospora crassa, 322-324 L-Serine (L-threonine) dehydrase, distribution of, 320 from Neurospora crassa, 319-322 Serratia marcescens, aspartase in, 388 Serum, acid phosphatase of prostatic origin in, 524 L-tartrate inhibition as criterion for, 528 value in diagnosis of prostate carcinoma, 524 L-asparaginase from, 383-384 convertin from, 156 DNase in, 447 as-globulin fraction of, renin substrate in, 139 prolidase in, 105 prothrombin-free, Ac-globulin activity of, 141 renin substrate from, 135-139 stabilization of carbonic anhydrase by, 846

974

SUBJECT I N D E X

Serum At-globulin (accelerin), preparation of from plasma Acglobulin, 153 use of thrombin for, 152, 153 in prothrombin-free serum, 141 role in clotting mechanism, 139, 156 Serum prothrombin conversion accelerator (SPCA), see Convertin SH compounds, see Sulfhydryl Compounds Shellfish, thiaminase from, 622-626, 627-628 Shikimic acid, as intermediate in aromatic biosynthesis, 300, 301 over-all equilibrium constant for conversion to quinic acid, 311 as product of 5-dehydroshikimic reductase action, 301-304 Silica-celite, lactoperoxidase purification by chromatography on, 815 Silicone technique, for collection of blood, 149 Silver ion, inhibition of aspartase by, 388 of glutamic dehydrogenase by, 224 of hydrogenase exchange reaction by, 867 of metaphosphatase by, 579 of renin by, 134 of RNase by, 434 Skim milk powder, as substrate for rennin assay, 70 Skin, tyrosinase from, 829, 831 Snake venom(s), L-amino acid oxidase from, 205-209 DPNH cleavage by nucleotide pyrophosphatase from, 654 5'-nucleotidase from, 549-550, 561 phosphodiesterases from, 561-565 relative activities of, 565 pyrophosphatase accompanying phosphodiesterase activity in, 565 source of dried, 563 Sodium azide, see Azide Sodium chloride, effect on Cypridina luciferase, 853 on RNase, 433-434

inhibition of DNase (pancreatic) by, 442 of glutathione reductase (yeast) by, 725 of nitroethane oxidase by, 402 of xanthine oxidase by, 485 stabilization of renin by, 129 Sodium dehydroepiandrosterone sulfate, as substrate for steroid sulfatases, 330 Sodium dichlorophenoxyethylsulfate, as substrate for alkylsulfatases, 330 Sodium fluoride, in oxidative phosphorylation assay, 611 Sodium hydrosulfide, see Sulfide Sodium hydrosulfite, see Dithionite Sodium ion, effect on RNase, 433 inhibition of DPNH cytochrome c reductase by, 692 of pantothenate-synthesizing enzyme by, 621 Sodium nitrate, inhibition of nitroethane oxidase by, 402 Sodium sulfide, see Sulfide Sodium usnate, inhibition of DNase by, 442--443 dependence on cobaltous ion, 443 Soja hispidus, see Soybean Solanain, protease from horsenettle, 57, 63-64 Solanum elaeagnifolium, see Horsenettle Solubilization, of particulate enzymes, with n-butanol, 112 by cholate, 743 by cholate plus trypsin, 739 by deoxyeholate, 741 with digitonin, 686-687 with ethanol, 689, 708 Solubilized aminopeptidase, see Dehydropeptidase I Soluble acylase I, see Amino acid acylase I L-Sorbose-l,6-diphosphate, hydrolysis by fructose diphosphatase, 545-546 Soybean (Soja hispidus), protease (soyin) from, 56, 63 ribonuclease in, 427

SUBJECT INDEX

trypsin inhibitor from, 36-37, 40-44, 50, 51, 52 Soybean leaves, cytochrome c peroxidase in, 764 nitrate reductase in, 414 Soyin, protease from soybean, 56, 63 Spermidine, amine oxidase action on, 393 Spermine, amine oxidase action on, 393 Spinach leaf (Spinacea oleracea, Tetragonia expansa ) , acetone powder preparation of, 789 carbonic anhydrase from, 842-843 catalase from, 789-791 5-dehydroshikimic reduetase and 5-dehydroquinase in, 304, 307 glutathione reductase in, 721 Spleen, adenylate kinase (myokinase) in, 602 carnosinase in, 94 cathepsin C from, 64-68 DNase from, 444-445 DPNase (pyridine transglycosidase) from, 660-663 L-glutaminase in, 382 kynurenine formamidase in, 248 nueleoside phosphorylase from, 448453 phosphodiesterase from, 565-569 RNase in, 436 Spores, Bacillus, alanine racemase in, 215 Squash, glutamie acid decarboxylase from, 190-194 keto acid decarboxylases in, 182, 192, 193 transaminases in, 182 Squash, yellow (Cucurbita pepo condensa), ascorbie acid oxidase from, 831-835 Stainless steel, adverse effect on renin, 130 Staphylococcus albus, cytochrome b in, 745 Staphylococcus aureus, arginine desimidase in, 376 glutamine synthetase in, 342

975

penicillin assay with, 121 Staphylokinase, activation of profibrinolysin by, 165 Steroid sulfatases, 324, 330, 332 in Patella vulgata (mollusk) , 332 Stomach, see also Gastric mucosa, rennin from calf, 69-77 Streptococcii group A hemolytic, DNase from, 446-447 Streptococcus faecalis, alanine raeemase from, 212-215 arginine desimidase in, 375, 376 in assay for "folic acid activity," 629 citrullinase in, 378 growth of, 188, 213, 647 transaminase in, 176 tyrosine decarboxylase from, 188, 646, 647-649 preparation of apoenzyme, 646, 647648 Streptococcus lactis, arginine desimidase in, 376 citrullinase in, 378 Streptokinase, activation of profibrinolysin by, 165 Streptomycin, effect of on RNase, 434 Succinate, activation of hydrogenase system by, 866 in assay of cytochrome c, 750 oxidation of, blocking by malonate of, 616 measurement of rate of, 748 P:O ratio during, 613, 616 role of cytochrome b in, 744, 745 of cytochrome bl in, 745-748 reduction of hematin peptide by, 167-169 stabilization of pyrimidine nucleoside by, 459 Suecinate-fumarate system, oxidation-reduction potential of, 740 *Sueeinic dehydrogenase, in assay of cytochrome b, 740, 741742 of cytochrome c, 750, 758 comparison of heart and Ustilago cytochrome c preparations, 758

976

SUBJECT I N D E X

cytochrome b preparations containing, 744 eytoehrome bl (diphtherial) and, 748 in heart particles, 737 manometric methods for, 748 Succinie dehydrogenase-cytochrome e linking activity (SC activity), of cytochrome b from heart, 742 *Succinic oxidase, cytochrome bl (diphtherial) and, 748 in heart particles, 738, 739, 750 assay methods for, 739 reduction of endogenous cytochrome c in preparations of, 75O from kidney cortex, 750 cytochrome c assay with, 750 Succinylacetoacetate, action of hydrolase on, 299 Succinyl phosphate, hydrolysis of, 556 Sucrose, in oxidative phosphorylation assay, 612 Sulfadiazine, see Sulfonamides Sulfa drugs, see also Sulfonamides, activation of bacterial thiaminase by, 628 Sulfanilamide, acetylation of in liver extract, 633 determination of, 634 inhibition of erythrocyte carbonic anhydrase by, 845 nitrite determination with, 403, 411 Sulfanilic acid, nitrite assay with, 400 Sulfatases, 324-332, see also Arylsulfatases, alkylsulfatases, 324, 330 arylsulfatases, 324, 327-332 assay of, 324-330 by determination of organic part, 327-330 by determination of sulfuric acid formed, 324-327 in aqueous solution, 325-326 in tissue suspension, 324, 326-327 chondrosulfatases, 324 glucosulfatases, 324, 330 myrosulfatases, 324

steroid sulfatases, 324, 330 Sulfate, activation of prothrombin by, 145 inhibition of acetyl phosphatase by, 556 of carbonic anhydrase (erythrocyte) by, 845 of D P N H cytochrome c reductase by, 692 of sulfatases by, 326 stabilization of pyrimidine nucleosidase by, 459 Sulfathiazole, see also Sulfonamides~ inhibition of tyrosinase by, 826 Sulfhydryl (SH) compounds (Thiol compounds), see also Cysteine, Glutathione, Mercapto compounds, activation of amino acid reductases by, 220 of homogentisate oxidase by, 295 of hydrogenase by, 869 of guanidinoacetate methylpherase by, 260, 263 of thiaminase by, 625, 627 of thio ether cleavage enzyme by, 314 of xanthine oxidase by, 485 cytochrome c reduction by, 754 effect of on 3'-nucleotidase, 554 inhibition of cysteine desulfhydrase by, 317 of hippuric acid formation by, 350 Sulfhydryl (SH) groups, presence in myosin, 587 requirement for methionine-activating enzyme, 256 for methylation of guanidinoacetic acid, 260 Sulfhydryl reagents, see also under names of individual reagents, e.g. p-Chloromereuribenzoate, Iodoacetate, etc., inhibition of D-amino acid oxidase by, 2O3 of ATPase (insect muscle) by, 598 of ATPase (myosin) by, 587 of luciferase (bacterial) by, 860 of rhodanese by, 337 Sulfide, activation of hydrogenase by, 868

SUBJECT INDEX compound of with horseradish peroxidase, 812 determination of, 315-316 inhibition of ascorbic acid oxidase by, 835 of carbonic anhydrase by, 845 of carnosinase by, 96 of DNase by, 442 of tryptophan peroxidase by, 246 of tyrosinase by, 826 ~-Sulfinyl pyruvic acid, desulfination of, 333 formation from cysteinesulfinic acid, 333 Sulfite, activation of amorphous form of asclepain (protease) by, 62 inhibition of rhodanese by, 337 Sulfonamides, accumulation of aminoimidazolecarboxamide riboside in E. coli cultures containing, 512-514 inhibition of L-amino acid oxidase by, 208 of carbonic anhydrase (erythrocyte) by, 845 p-Sulfonamidobenzoic acid, inhibition of erythrocyte carbonic anhydrase by, 845 Sulfonic acids, aromatic, inhibition of L-amino acid oxidase by, 2O8 Surface denaturation, serum albumin as protecting agent against, 724 T

Tabernamontana grandiflora, protease (tabernamontanain) from fruit of, 56, 63 Takadiastase, adenosine deaminase (nonspecific) from, 475-478 arylsulfatase from, 328 folic acid conjugase in, 630-631 metaphosphatase in, 579 source of, 476 triphosphatase ill, 580, 582

977

L-Tartrate, inhibition of prostatic phosphatase by, 528 clinical use of, 528 Tea tannins, oxidation by tyrosinase, 826 Tenbroeck homogenizer, extraction of liver acetone powder with, 653 L-Tertiary leueinamide, action of amidase on, 399 Testosterone, increased acid phosphatase in prostate gland after injections of, 523 Tetradecyl aldehyde, as component of bacterial luciferase system, 860 Tetraethyl ammonium chloride, in angiotonin assay, 136 Tetragonia expansa, see Spinach Tetraguaiacol, formation from guaiacol in peroxidase assay, 772 Tetrahydrofolic acid (FAH4), as cofactor in formylation of 5-IRMP, 519 of glycinamide ribotide, 510 formylation of, 516-518 by reaction with formate, 517-518 with serine, 518 Tetrazolium salts, see also Triphenyltetrazolium and Neotetrazolium, as electron acceptors for DPNHcytochrome c reductase of bacteria, 697 Thermal conductivity cell, for analysis of ortho-parahydrogen mixtures, 864 Thermobacterium acidophilus, growth medium for, 467 nucleoside transdeoxyribosidase in, 467 Thiaminase, 622-628 from Bacillus thiaminolyticus culture medium, 626-628 activation by sulfa drugs, 628 in Carassius carassius, 625 in carp, 625 in clam viscera (Meretrix meretrix), 622-626

978

SUBJECT I N D E X

kinetics of base exchange versus hydrolytic actions of, 626 sources of, 625 substrate specificity of shellfish and bacterial enzymes, 628 Thiamine, exchange of thiazole moiety of, for other bases by thiaminase, 622 inhibition of yeast phosphatase by, 638 phosphorylation of, 636 Thiamine derivatives, thiaminase action on, 624, 628 table for, 628 Thiamine pyrophosphate (TPP), cleavage by nucleotide pyrophosphatase, 659 as coenzyme for benzoylformic carboxylase, 279-280 determination of, 636-638 formation by thiaminokinase action, 636 thiaminase action on, 624 Thiaminokinase, 636-640 from baker's yeast, 639-640 from brewer's yeast, 638 Thiazole, as product of thiaminase action, 622 Thiobenzoic acid, inhibition of heparin cofactor by, 163 Thiochrome method, for thiamine determination, 622-623 Thiocyanate, colorimetric determination in rhodanese reaction, 334 Thio ethers, cleavage of, 313 Thioglycolate, activation of histidase by, 231 of hydrogenase by, 868 inhibition of DNase by, 442 of heparin cofactor by, 163 reduction of dehydroaseorbio acid by, 85O role of in blood clotting mechanism, 160 Thiolactic acid, reduction of dehydroascorbic acid by, 850

Thiol compounds, see Sulfhydryl compounds Thiopental (Pentothal), as uncoupling agent, 615 Thiophene-2-sulfonamide, inhibition of erythrocyte carbonic anhydrase by, 845 Thiophosphates, as probable substrates for alkaline phosphatase, 538 Thiosulfate, as sulfur donor for rhodanese, 334-337 Thiosulfonates, as sulfur donors in rhodanese reaction, 336 Thiouracils, inhibition of tyrosinase by, 826 Thiourea(s), activation of uricase by, 489 inhibition of nitrate reductase by, 415 of tyrosinase by, 826 Thrombin, 156-158 adsorption of during clotting, 158 preparation from prothrombin, 156, 157 role in clotting mechanism, 139, 140, 152, 156-158, 159 use in two-stage prothrombin assay, 141 Thromboplastin (thrombokinase), 139, 140, 146-151, 154 activation of proconvertin by, 154 of prothrombin by, 145 adsorption on calcium oxalate, 147 assay by one-stage method, 146 by two-stage method, 147 from brain, 140, 149 use in prothrombin assay, 140, 141 complexes with convertin and plasma Ac-globulin, 151 components of, 139 concentration in oxalated horse plasma, 147 heat-labile factor of (antihemophilic factor, AHF), 139, 147-148 occurrence in plasma, 139 from lung, 148-149 role in clotting mechanism, 139, 152, 154

SUBJECT INDEX

Thymic acid, degradation by RNase, 433 Thymidine (thyminedeoxyriboside), bioassay of, 464 R! values for, 466 in transdeoxyribosidase reaction, 464-468 Thymidine phosphorylase, from horse liver, 453 Thymine, as acceptor of deoxyriboside group, 468 oxidation to 5-methylbarbituric acid, 490 increase in optical density during t 490 Rs values for, 466 Thymine deoxyriboside, see Thymidine Thymine oxidase, identity with uracil oxidase, 491 Thymus, aminotripeptidase from, 84, 85-86 DNase from, 443-444 RNase in, 436 Tissue slices, assay of oxidative phosphorylation in, 614 d-biotin oxidase in, 631-632 a-p-Toluenesulfonyl derivatives of arginine and lysine, as substrates for trypsin, 36 Toluidine (o or p), detoxication of diphtheria toxin by myeloperoxidase and, 800 Toluquinone, reduction of by DPNH, 728 Top yeast, see Yeast, baker's Toxins, bacterial, detoxication by myeloperoxidase plus donor substances, 801 TPNH cytochrome c reductase, from liver, 704-706 inactivity with bacterial cytochrome c, 760 from yeast, 697, 699-703 rate constants for reduction and oxidation of, 697, 703 comparison with related enzymes~ 697

979

T P N H cytochrome reductases, TPNH-diaphorase activity of, 711

Tradescantia fluminensis, cyanide sensitivity of carbonic anhydrase from, 845 Transamidation, by cathepsin C, 65, 68 Transaminase (s), from bacteria, 170-177, 182, 184 for aliphatic amino acids (transaminase B), 176 for D-amino acids, 171-172 for aromatic amino acids (transaminase A), 176 for pyridoxamine-alanine, ]73 for valine-alanine and valine-aaminobutyrate, 176 distribution of (glutamic-oxalacetic enzyme), 184 estimation of, 172-176, 178-184 manometric, 182-184 quantitative filter paper chromatography, 178 speetrophotometric, 179-182 from liver, 289 role of in dcsulfinase system, 333 in oxidation of L-amino acids, 211 in squash, 182 Transhydrogenase, see Pyridine nucleetide transhydrogenase Transpeptidation, a-chymotrypsin and, 21 Transphosphorylation, by phosphatases, 556-561 efficiency of, 560 Treburon (synthetic sulfated polygalacturonic acid), inhibition of RNase by, 434 Tri-acetic acid-hydrolyzing enzyme, probable identity with fumarylacetoacetate hydrolase, 298 Triaminodiphenyl sulfones~ effects on prothrombin activation, 145 digestion by plant proteinases, 55 Trichloroacetic acid, use in purification of trypsin, 30 2,3',6-Trichloroindophenol, in nitrate reductase system, 415 Trichuris, digestion of, 55 Triethanolamine buffer, 676

980

SUBJECT INDEX

Trifluoroacetyl amino acids~ action of acylase I on, 118 q'rimetaphosphate, es substrate for metaphosphatase, 577-578 1,3,5-Trinitrobenzene, reduction of by Neurospora, 410

2,4,6-Trinitrophenol, inhibition of quinone reductase by, 729 2,4, 6-Trinitrotoluene (TNT), enzymatic reduction of, 406, 410 Tripeptidase, see Aminotripeptidase Tripeptides, resistance of to iminodipeptidase, 100 to prolidase, 105 as substrates for leucine aminopeptidase, 91 2, 3, 5-Triphenyltetrazolium, in assay of flavoproteins, 695 Triphosphatase, 580-582 orthophosphate as sole product of in yeast, 580 pyrophosphate as product of cleavage by A. oryzae enzyme, 580, 582 sources of, 580 Triphosphate, preparation of, 580-581 protection of nitrate reductase by, 414 Triphosphopyridine nucleotide (TPN), assay of, 652 of ATP and ADP by reduction of, 497 of glucose-6-phosphate by reduction of, 675, 676, 677 cleavage by DPNase (animal), 662 by DPNase (Neurospora), 666 by nucleotide pyrophosphatase, 655, 659 by plant enzymes, 720 as eoenzyme for benzaldehyde dehydrogenase, 280-281 for 5-dehydroshikimic reductase, 301 for L-glutamic dehydrogenase, 224 complex of with protein moiety of old yellow enzyme, 715 formation of by DPN kinase, 652-655 large-scale preparation, 655 liver (horse) as source of, 700

in oxidative phosphorylation assay, 612, 615, 616 reduction of by molecular hydrogen, 732 role of in formylation of FAH4 by serine, 518 in transhydrogenase reaction, 681-687 Triphosphopyridine nucleotide, reduced (TPNH), see also under TPNH, extinction coefficient for, 497 generation of by glucose-6-phosphate dehydrogenase, 699 as hydrogen donor for GSSG reduetase, 301, 719, 722, 724 for hydroxylamine reductase, 418 for luciferase (bacterial), 861 for new yellow enzyme, 715 rate constant for, 716 for nitrate reductase, 411, 414, 415 for nitroaryl reductase, 408, 410 for nitrogen gas formation from nitrite, 422 for old yellow enzyme, 712 rate constant for reaction, 716 for quinone reductase, 728 for transhydrogenase, 681-687 P: O ratio during oxidation, 616 preparation of, 411 Triphosphopyridine nucleotide, 3'isomer, action of nonspecific deaminase on, 477, 478 Triphosphopyridine nucleotide-linked dehydrogenases, determination with glutathione reductase, 721 Tris (hydroxymethyl)aminomethane (Tris buffer, THAIV[ buffer), L-glutamic dehydrogenase and, 224 low affinity of for zinc, 79 protection of hydroxylamine reductase by, 419 Tropaeolum majus, see Nasturtium Trypsin, activation of a-chymotrypsinogen to a-chymotrypsin by, 8, 12 of a-chymotrypsinogen to ~r- and ~chymotrypsins by, 8, 15-16 of procarboxypeptidase by, 80

S ~ J E C T INDEX cross reactivity with chymotrypsins on a synthetic substrate, 21 determination of activity of, 32-36 casein digestion method fo~, 33-34 hemoglobin digestion method for, 34 with synthetic arginine derivatives, amidase activity, 34-36 esterase activity, 23, 36 isoelectric point of, 35 effect of calcium on, 35 isolation of from beef pancreas, 26-36 crystallization of, 26, 29-31 improved method for, 26, 29 preparation as byproduct of DNase isolation, 439 triehloroacetic acid for purification of, 30 molecular weight of, 35 nitrogen content of, 32 optical factor for, 32 proteolytie action of, use in purification of cytochrome oxidase, 739 of myeloperoxidase, 796 of TPNH eytochrome c reductase, 7O6 of xanthine oxidase, 706 stability of, 32 effect of calcium and other cations on, 32 standard activity curve for, 33 Trypsin inhibitor(s), antifibrinolysin and, 165 determination of, 37-38 standard preparation of trypsin for, 38 general properties of, 37 isoelectric points of, 51 molecular weights of, 52 optical factors for, 50 trypsin complexes with, general properties of, 37 isoelectric points of, 51 molecular weights of, 52-53 optical factors for, 50 from various sources, Ascaris, 37, 54 blood plasma, 37, 49-54 multiplicity of inhibitors in, 49 colostrum, 37, 46-48, 50, 51, 52 crystallization of, 48

981

trypsin complex with, 47-48, 50, 51, 52 crystallization of, 47-48 lima bean, 37, 48-49, 51, 53 potency of amorphous and crystalline forms, 48-49 ovomucoid, 37, 49, 51, 53 pancreas~ as byproduct of insulin preparation, 40 removal from trypsin by trichloroacetic acid, 30 use in recrystallization of trypsinogen, 28 pancreas (Kazal preparation), 36, 40, 50, 51, 52 pancreas (Kunitz and Northrop preparation), 36, 38-40, 50, 51, 52 crystallization of, 39-40 trypsin complex with, 38-39, 50, 51, 52 crystallization of, 38-39 preparation as byproduct of DNase isolation, 439 soybean, 36-37, 40-44, 50, 51, 52 crystallization of, 42-44 trypsin complex with, 44-46, 50, 51, 52 crystallization of, 44-46 Trypsinogen, from beef pancreas, 26-36 as byproduct of DNase preparation, 439 crystallization of, 26, 27-29 molecular weight of, 35 recrystallization of, 28 use of diisopropyl fluorophosphate (DFP) during, 28 of pancreatic trypsin inhibitor during, 28 transformation into trypsin, 26, 29, 32 by autocatalysis, 26, 29 by enterokinase, 26, 29, 32 "inert protein" formation during, 26, 29 suppression by calcium ions, 26, 29 by kinase from Penicillium, 26

982

SUBJECT INDEX

mechanism of, 26 peptide formed during, 26-27 aspartic acid content of, 27 Tryptazan, inhibition of tryptophanase by, 241 Tryptophan, colorimetric determination of, 233 decarboxylation in mammalian tissues, 199 inhibition of tryptophanase by D-isomer of, 241 of tryptophan synthetase by, 237 of tryptophan synthetase formation by, 237, 238 oxidation of, enzymes for, 242-253 kynureninases, 249-253 kynurenine formamidase (formylase), 246-249 L-tryptophan peroxidase, 242-246 as precursor of authranilic acid and catechol, 273 as product of aromatic biosynthesis, 3OO L-serine (L-threonine) dehydrase action on, 322 as source of indole in E. coli, 238 as substrate for L-amino acid oxidases, 208, 211 for transaminase, 176 L-Tryptophanamide, hydrolysis by leucine aminopeptidase, 92 Tryptophanase, from E. coli, 238-242 kynureninase and, comparison of mechanisms, 253 Tryptophan desmase (Tryptophan desmolase), see Tryptophan synthetase L-Tryptophan peroxidase, adaptive increase by tryptophan administration, 244, 246, 253 differentiation from other peroxidases, 245 distribution of, 245 from liver, 242-246 in tryptophan-adapted Pseudomonas, 245 Tryptophan synthetase (tryptophan desmase, tryptophan desmolase),

in g. coli, 234 formation of in microorganisms, 237238 effect of nutrients on, 237 genetic control of~ 238 in Glomerella cingulata, 234 from Neurospora crassa, 233-238 Tsuchihashi procedure, for denaturation of hemoglobin, 102 use in prolidase purification, 102 Tumors, absence of cysteine desulfhydrase from, 318 Tungstate, use in purification of renin, 127-128 Turnover numbers, for carbonic anhydrase and catalase compared, 844 Tyramine [p-(~-aminoethyl) phenoll, amine oxidase action on, 393 detoxication of diphtheria toxin by myeloperoxidase and, 800 Tyramine phosphate, as standard for angiotonin assay, 136 L-Tyrosinamide, hydrolysis by leucine aminopeptidase, 92 Tyrosinase, insect, from grasshopper eggs, 831 activating agents for protyrosinase in, 831 Tyrosinase, mammalian, 827-831 assay methods for, 827-830 choice of method, 830 histochemical, 828-829 isotopic tyrosine method for pigmented tissue, 829-830 manometric, 827-828 from ciliary bodies, 829, 830 cytoplasmic particles as site of, 830 in melanomas, 828-831 existence in active state, 831 from pigmented eye tissue, 828 from skin, 829, 831 activation by ultraviolet irradiation, 831 distinction from tyrosinase of melanomas, 831 Tyrosinase, plant (polyphenol oxidase), 817-827, 830

S~ECT

comparison with mammalian tyrosinase, 830 distribution of, 817 from mushroom (Psalliota campestris), 822-827 chronometrie assay of catecholase activity, of, 819-821 diagram of apparatus for, 820 manometric assay of cresolase activity of, 818-819 properties of, 825-827 purification procedure for, 822-825 high catecholase preparation, 822-824 high cresolase preparation, 824825 reaction inactivation as characteristic of, 826 distinction from mammalian enzyme, 830-831 Tyrosine, standard solution of for proteinase assay, 55 Tyrosine, Cl 4 labeled, tyrosinase assay in melanoeytes with, 829 L-Tyrosine• conversion to acetoacetate, 287-300 decarboxylation in mammalian tissues, 199 in fibrin and fibrinogen, 160 oxidation by mammalian tyrosinase, 827-831 induction period preceding, 827 oxidation by plant tyrosinase, 826, 830 in peptide B from fibrinogen, 160 preparation of from DL mixture, 829 as product of aromatic biosynthesis, 3OO as substrate for ~amino acid oxidases, 208, 211 for transaminases, 176, 289 Tyrosine apodecarboxylase, from Strep. faecalis, 646, 647-648 activation of as measure of pyridoxal kinase action, 646 Tyrosine decarboxylase, in measurement of transaminase reactions, 171 from Strep. faecalis, 188

INDr.X

983

resolution of, 189 L-Tyrosine ethyl ester, as substrate for trypsin and chymotrypsin, 23 Tyrosine-glutamic acid transaminase, from liver, 289 L-Tyrosine-oxidizingsystem, distribution of, 289 from liver, 287-300 fumarylacetoaeetate hydrolase, 298-300 homogentisate oxidase, 292-295 p-hydroxyphenylpyruvate enol-keto tautomerase, 289-292 maleylacetoacetate isomerase, 295298 over-all system, 287-289 tyrosine-glutamic acid transaminase, 289 U

Ultracentrifuges, comparison of molecular weight values obtained with different, 808 Uncoupling phenomenon, agents for in oxidative phosphorylation, 615 Uracil, as acceptor of deoxyriboside group, 468 inhibition of uridine nucleosidase by, 462 oxidation of to barbituric acid, 490 • increase in optical density during, 490 Rt values for, 466 Uracil deoxyriboside (UDR), formation from cytosine deoxyriboside, 478 Rt values for, 466 in transdeoxyribosidase reaction, 464-468 bioassay of, 464 Uracil nucleosides, identification of, 479, 480 interference of pyrimidine nucleoside phosphorylase with, 480 Uracil oxidase, manometric determination of uracil with, 458

984

SUBJECT I N D E X

from Mycobacterium, 490-491 identity with thymine oxidase, 491 Uranium acetate, use in RNase assay, 427 "Uranium-soluble" phosphate, as measure of phosphodiesterase, 561 Uranyl acetate-perchloric acid reagent, in assay of phosphodiesterase, 565-566 Uranyl aeetate-trichloroacetic acid reagent, in assay of phosphodiesterase, 562 Urea, determination of~ 356, 364-365, 370371, 379 in arginine-synthesizing system, 356, 364-365 in blood with urease paper, 379 formation by arginase, 368 by barbiturase, 492 Urease, arginoly~ic activity of, 370 CO2 versus HC03- as product of, 841 from jack bean meal, 378-379 Ureidosuccinase, irreversibility of, 497 Ureidosuccinie acid, conversion to aspartic acid, NH3 and C02, 497 to 5-(acetic acid)-hydantoin, 496497 formation from dihydroSrotic acid, 496 Urethan(s), inhibition of enzymes by, 527, 867 hydrogenase, dependence on assay method, 867 prostatic phosphatase, 527 Uric acid, assay of, colorimetric, 486 spectrophotornetric, 458 cytochrome c reduction by, 754 formation by xanthine oxidase, 480, 481, 482 increased absorption during, 480 oxidation of, 485, 794, 810 in myeloperoxidase assay, 794 by peroxidase complex II, 810 peroxidase assay with, 774 preparation of lithium salt of, 485 RS values for, 466

Uric acid riboside (UAR), in beef erythrocytes, 459 nucleosidase action on, 459 Uricase, evidence for metal component in, 489 interference with guanase assay, 481 from kidney, 485-489 from liver mitochondria, 489 new procedure for purification of, 489 spectrophotometric method for, 458, 486 Uridine (UR), formation from cytidine, 478 molecular extinction coefficient, 478 nucleosidase action on, 459 Uridine nucleosidase, absorption changes during action of, 461 from baker's yeast, 461-462 Uridine triphosphate (UTP), hydrolysis by myosin, 586 as phosphate donor to AMP, 603 UDPG synthesis from glucose-l-P and, 675 Uridin ediphosphoglucose (UDPG), cleavage of by nueleotide pyrophosphatase, 659 determination of by galactowaldenase, 677 by a specific pyrophosphorylase, 676 by UDPG dehydrogenase, 677 synthesis from UTP and glucose-l-P, 675 Uridinediphosphoglucose dehydrogenase, UDPG determination with, 677 Uridinediphosphogluce se pyrophosphorylase, purification procedure for, 677 UDPG determination with, 676-677 from yeast (brewer's), 675-677 3'-Uridylic acid (3'-UMP), 3'-nucleotidase action on, 553 5'-Uridylic acid (5'-UMP), as acceptor in nucleoside monophosphate kinase reaction, 603 "5" nucleotidase action on, 549 Uridylic acids, prostatic phosphatase action on 2' and 3'-, 524

SUBJECT INDEX Urine, acid phosphatase of prostatic origin in, 523 fibrinolytic activator from, 165 Urocanase, in liver, 232 from Ps. fluoreseens, 231-233 Urocanic acid, degradation by urocanase, 231 formation from L-histidine by histidase, 228 molar extinction coefficient of, 228 Usnic acid, as uncoupling agent, 615

Ustilago sphaerogena, cytochrome c from, 755-758 content in dried cells, 757 growth of, 755-756 Uterine tissue, glycylglycine dipeptidase from, 108109 glycyl-L-leucine dipeptidase from, 105-107 V L-Valinamide, hydrolysis by leucine aminopeptidase, 92 L-Valine, as substrate for L-amino acid oxidase, 211 transaminase for alanine or a-amino butyric acid with, 176 transaminatiou with isoleucine in microorganisms and animal tissues, 171 Vanadate, inhibition of DPNH cytochrome c reductase by, 692 Vasoconstrictor substance, see Angiotonin Venom, see Snake venom Venus mercuriana (Quahog clam), thiaminase in, 625 Verdohemachrome peptide, formation from cytochrome c, 167-169 Verdoperoxidase (myeloperoxidase), see Peroxidase from leucocytes

985

Veronal (Barbital), catalysis of C02 hydration by, 836 in colorimetric assay of carbonic anhydrase, 839 inhibition of glycine oxidase by, 227 low affinity for zinc, 79 Versene, see Ethylenediamine tetraacetate

Vibrio, aromatic oxidations in, 273 Viper, Russell's, 5'-nucleotidase in venom of, 561 phosphodiesterase from venom of, 564, 565 Vitamin B~, see Riboflavin Vitamins, metabolism of coenzymes and, 619-677 W

Wheat, as source of protease, 63 Wheat germ, glutathione reductase in, 721 White blood cells, see Leucocytes X

XR, see Xanthosine Xanthine, as aceeptor of deoxyriboside group, 468 formation by guanase, 480 oxidation to uric acid, 484 as source of peroxide, 244 spectrophotometric assay of, 458, 480, 481 Xanthine oxidase, diaphorase activity of, 711 from milk, 482-485 reduction of trinitrotoluene by, 406 of dinitrobenzene by, 409 removal of product of nucleoside phosphorylase by, 448, 449 spectrophotometrie determination of adenine, hypoxanthine, and xanthine with, 458 Xanthosine (XR), nucleosidase action on, 459, 463 phosphorolysis of, 448

986

SUBJECT INDEX

Xanthydrol reagent, preparation for urea determination, 368-369 X-ray analysis, molecular weight of RNase by, 435 X-rays, inactivation of RNase by, 434 p-Xyloquinone, reduction of by DPNH, 728 Xylose, degradation of R-5-P in cells grown on, 460-461 Y Yeast, adenylate kinase (myokinase) in, 602 arginine-synthesizing system in, 357, 360, 362-364 asparaginase in, 384 cytochrome as in, 732, 734 cytochrome b~ (lactic dehydrogenase) in, 745 cytochrome e in, 745 5-dehydroshikimic reductase and 5-dehydroquinase in, 304, 307 DPN pyrophosphorylase from, 671 lactic dehydrogenase (cytochrome b2) in, 746 metaphosphatase in, 577, 578 phosphatase in, 638 inhibition by "pyrimidyl" and thiamine, 638 purina~cleeside phosphorylase from, 453 TPNH cytochrome c reductase from, 697 transhydrogenase in, 682 Yeast, baker's (top yeast), adenosine phosphokinase in, 498 alcohol dehydrogenase from, 660 I)PN assay with, 660 arginine desimidase from, 375-376 component of arginine condensing system from, 362-364 cytochrome b in, 744, 745 DNase from, 445-446 glutathione reductase from, 723-724 inhibitors of thaminokinase in, 640 inorganic pyrophosphatase from, 364, 570-576

plasmolysis of, 375, 376, 581,639 purine nucleosidase from, 462-464 thiaminokinase from, 639-640 triphosphatase in, 580, 581, 582 uridine nucleosidase from, 461-462 Yeast, brewer's (bottom yeast), adenosine phosphokinase from, 4975OO alkaline washed, preparation of, 637 thiamine pyrophosphate assay by carboxylase from, 636-637 aspartase in, 388 conversion of 5-amino-4-imidazolecarboxamide riboside to ribotide by enzyme from, 505, 514-516 FAD pyrophosphorylase from. 673675 flavokinase from, 640-645 glutathione synthetase from, 345-346 glycerol for extraction of, 581 new yellow enzyme from, 715-719 old yellow enzyme from, 712-715 plasmolysis by sodium chloride, 638 pyridoxal kinase from, 646-649 thaminokinase from, 638 triphosphatase in, 580, 581, 582 UDPG pyrophosphorylase from, 675677 Yeast, top ale, phosphorylating enzymes for glucose6-P preparation in, 699 T P N H cytochrome c reductase from, 699-703 "Yeast adenylic acid" (mixture of 2'and 3'-adenylic acids), prostatic phosphatase action on, 527, 528 Yeast extract (s), folic acid and CF content of, 630 effect of folic acid conjugase on, 630 as hydrogen donor for nitrogen gas formation, 422 Yeast protein, inhibition of yeast DNase by, 447 Yeast sodium nucleate, see Ribonucleic acid "Yeast uridylic acid" (mixture of 2'and 3'-uridylic acids), K,~ value for prostatic phosphatase action on, 527

SUBJECT INDEX

Yellow enzyme, new, from bottom yeast, 715-719 Yellow enzyme, old, from bottom yeast, 712-715 comparison with new yellow enzyme, 716 crystallization of, 714 non-identity of protein moieties of new yellow enzyme and, 718 rate constant for reaction with cytochrome e, 697 with oxygen, 697, 715 with TPNH, 715 Z

Zinc, as component of earboxypeptidase, 78 content in carbonic anhydrase from erythrocytes, 842, 843 from spinach, 843 content in uricase preparations, 489 deficiency, effect on Neurospora DPNase content, 666 purification of aminotripeptidase of thymus with, 85-86 requirement for formation of tryptophan synthetase, 237 Zinc ion, activation of alkaline phosphatase by, 538 of carnosinase by, 94, 96 effect of on pH optimum, 96 of citrullinase by, 378 of flavokinase by, 645 of glycyl-L-leucine dipeptidase by, 105, 106, 107 requirement of phosphate for, 107 time required for, 106 of metaphosphatase by, 579

987

effect on metaphosphatase content of A. niger, 579 inhibition of alkaline phosphatase by, 538 of amino acid amidase by, 399 of arginine desimidase by, 376 of aspartase by, 388 of ATP-creatine transphosphorylase by, 610 of 5-dehydroquinase by, 307 of DNase by, 442 of DPNH cytochrome c reductase by, 692 of fructose diphosphatase by, 546 of glutamic dehydrogenase by; 224 of glycylglycine dipeptidase by, 109 of leucine aminopeptidase by, 93 of pantothenate-synthesizing enzyme by, 621 of prolidase by, 105 of RNase by, 434 of D-serine (D-threonine) dehydrase by, 324 of splitting enzyme for arginine synthesis by, 367 of tryptophan synthetase by, 237 of uricase by, 489 low affinity of for Veronal and Tris buffers, 79 Zince~ ions, nonexchangeability with zinc of carbonic anhydrase, 843-844 *Zwischenferment, see Glueose-6-phosphate dehydrogenase

Zymobacterium oroticum, dihydroSrotic dchydrogenase from, 493-496 growth of, 494 Zymogen, 79, see also under names of individual proenzymes

Methods in Enzymology, Volume 2: Preparation and Assay of Enzymes

Methods In Enzymology, Volume 1: Preparation and Assay of Enzymes

Methods in Enzymology, Volume 3: Preparation and Assay of Substrates

Methods in Enzymology Vol 19: Proteolytic Enzymes

Methods in Enzymology Vol.334, Hyperthermophilic Enzymes (Part C)

Nuclear Receptors, Volume 364 (Methods in Enzymology)

Methods in Enzymology. Liposomes

Methods in enzymology, Volume 167 - Cyanobacteria

DNA Replication, Volume 262 (Methods in Enzymology)

Methods in Enzymology. Liposomes

Biochemic drug assay methods

Methods in Enzymology (Liposomes )