Methods in Enzymology, Volume 4: Special Techniques for the Enzymologist

PREFACE The first three volumes of this series have dealt with the commonly employed methods for the preparation and ass...

Author: Sidney P. Colowick | Nathan P. Kaplan

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PREFACE The first three volumes of this series have dealt with the commonly employed methods for the preparation and assay of enzymes, coenzymes, and substrates. The present volume deals with certain special techniques which are considered to be of interest to those in the field of enzymology. Descriptions of many of these procedures are available in monographs dealing respectively with the physical chemistry of the proteins, with specialized methods for the measurement of metabolism, or with procedures for the synthesis, degradation, and measurement of isotopically labeled compounds. It nevertheless appeared worthwhile to have these descriptions written with the special problems of the enzymologist in mind and to have them brought together in a single volume. With the publication of this volume we bring to completion our original plans for a treatise on Methods in Enzymology. It has been our policy to select a recognized authority for the description of each laboratory procedure, with the aim of obtaining a reliable collection of reproducible methods. For the extent to which we have succeeded in this aim, ,we are grateful to all those investigators who have contributed. It would of course have been desirable that all procedures described in these four volumes be checked in an independent laboratory prior to publication. However, it is obvious that with such a system of checking, the publication of an organized treatise of this scope would have been impossible within any reasonable time period. Users of these volumes will undoubtedly find that certain articles are already in need of revision and that certain important new subjects are not covered at all. In an attempt to correct these unavoidable deficiencies, an organized single supplementary volume is planned, which will include material not heretofore covered, as well as revised procedures, to supplement all four of the published volumes. We welcome any suggestions concerning the content of this supplementary volume, which will be published approximately two years from this date.

Baltimore, June 1~, 1957

SIDNEY P. COLOWlCK NATHAN O. KAPLAN

Contributors to Volume IV Article numbers are

s h o w n in parentheses following the n a m e s Affiliations Listed are current.

S. ABRAHAM(22), University of California,

Berkeley, California I-I. S. ANKER (32), University of Chicago, Chicago, Illinois CAMILLO ARTOM (33), Bowman-Gray School of Medicine, Wake Forest College, Winston-Salem, North Carolina A. A. BENSON (36), Pennsylvania State University, State College, Pennsylvania I. A. BERNSTEIN (23), University of Michigan, Ann Arbor, Michigan M. BIER (5), Fordham University, New York, New York ](. BLOCH (29), Harvard University, Cambridge, Massachusetts R. It. BVRRIS (16), University of Wisconsin, Madison, Wisconsin M. CALVIN (36), University of California, Berkeley, California I. L. CHAIKOFF (35), University of California, Berkeley, California BRITTON CHANCE (12), University of Pennsylvania, Philadelphia, Pennsylvania MILDRED COHN (37), Washington University, St. Louis, Missouri S. P. COLOWICX (34), The Johns Hopkins University, Baltimore, Maryland F. H. C. CRICK (4), Cambridge University, Cambridge, England H. FRAENKEL-CONRAT (ll), University of California, Berkeley, California GEORGE GOMORI* (18), Palo Alto Medical Research Foundation, Palo Alto, California ])AVID M. GREENBERG (28), University of California, Berkeley, California W. Z. HASSID (22), University of California, Berkeley, California ROGER ~ . ]-~ERRIOTT (8), The Johns Hopkins University, Baltimore, Maryland N. O ]~-APLAN (34), Brandeis University, Waltham, Massachusetts

of contributors.

EDWARD D. KORN (26), National Insti-

tutes of Health, Bethesda, Maryland A. HELGE F. LAURELL (1A), University of

Uppsala, Uppsala, Sweden DONALD J. R. LAURENCE (7), Chester

Beatty Research Institute, London, England M. LEvY (10), New York University, New York, New York KATHARINE F. LEWIS (25), Lankenau Research Institute, Philadelphia, Pennsylvania OLIVER H. LOWRY (17), Washington University, St. Louis, Missouri WILFRIED F. H. M. MOMMAERTS (6),

School of Medicine, University of California, Los Angeles, California R. R. PORTER (9), The National Institute for Medical Research, London, England J. H. QUASTEL (13, 14), McGill-Montreal General Hospital Research Institute, Montreal, Canada MORTON ROTHSTEIN (28), University of California, Berkeley, California ANTHONY SAN PIETRO (21), The Johns Hopkins University, Baltimore, Maryland ]~. K. SCHACHMAN (2}, University of California, Berkeley, California P. G. SCHOLEFIELD (14), McGill-Montreal General Hospital Research Institute, Montreal, Canada DAVID SHEMIN (27), Columbia University, New York, New York FRITIOF S. SJOSTRAND (19), Karolinska Institute, Stockholm, Sweden DANIEL STEINBERG (20), National Institutes of Health, Bethesda, Maryland J. A. STEKOL (30), Lankenau Hospital Research Institute, Philadelphia, Pennsylvania It. E. SwIM (24), Western Reserve University, Cleveland, Ohio

* Deceased. vii

° . .

Vlll

CONTRIBUTORS TO VOLUME IV

University of California, Berkeley, California ALVIN TAUROG (35), University of California, Berkeley, California ARNE TISELIUS (1), University of Uppsala, Uppsala, Sweden SIDNEY UDENFRIEND (20), National Institutes of Health, Bethesda, Maryland M. F. UTTER (24), Western Reserve University, Cleveland, Ohio HAROLD TARVER (31),

WOLF VISHNIAC (15), Yale University,

New Haven, Connecticut Lankenau Research Institute, Philadelphia, Pennsylvania P. W. WILSON (16), University of Wisconsin, Madison, Wisconsin D. L. WOOD (3), Bell Telephone Laboratories Inc., Murray Hill, New Jersey HARLAND G. WOOD (23), Western Reserve University, Cleveland, Ohio SIDNEY WEINHOUSE (25),

Outline

of Volumes

I, II, III,

VOLUME PREPARATION Section I. General Preparative

AND

IV, and V

I

ASSAY

OF ENZYMES

Procedures

A. TissueSliceTechnique. B. TissueHomogenates.

C. Fractionation of Cellular Components. D. Methods of Extraction of Enzymes. E. Protein Fractionation. F. Preparation of Buffers.

Section II. Enzymes of Carbohydrate

Metabolism

A. Polysaccharide Cleavage and Synthesis. B. Disaccharide, Hexozide, and Glucuronide 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 D. Phospholipid and Steroid Enzymes.

and Transfer. C. fipases and E&erases.

Section IV. Enzymes of Citric Acid Cycle VOLUME PREPARATION

AND

II

ASSAY

OF ENZYMES

Section I. Enzymes of Protein Metabolism A. Protein Hydrolyzing Enzymes. B. Enzymes in Amino Acid Metabolism (General). C. Specific Amino Acid Enzymes. D. Peptide Bond Synthesis. E. Enzymes in Urea Synthesis. F. Ammonia Liberating Enzymes. G. Nitrate Metabolism.

Section II. Enzymes of Nucleic Acid Metabolism A. Nucleases. B. Nucleosidases. C. Deaminazes. D. Oxidases. E. Nucleotide Synthesis.

Section III. Enzymes in Phosphate Metabolism A. Phozphomonoesterases. B. Phosphodiesteraaes. C. Inorganic Pyro- and Poly-phosphatazes. D. ATPases. E. Phosphate-Transferring Systems.

Section IV. Enzymes in Coenzyme and Vitamin

Metabolism

A. Synthesis and Degradation of Vitamins. B. Phozphorylation zyme Synthesis and Breakdown.

Section V. Respiratory

of Vitamins. C. Coen-

Enzymes

A. Pyridine Nucleotide-Linked, Enzymes. D. Unclassified.

Including Flavoproteina. B. Iron-Porphyrins. xi

C. Copper

xii

OUTLINE

OF

VOLUMES

I,

II,

VOLUME

PREPARATION AND

III,

IV,

AND

V

III

ASSAY

OF SUBSTRATES

Section I. Carbohydrates A. Polysaccharides. B. Monosaccharides. C. Sugar Phosphates and Related Compounds. D. Unphosphorylated Intermediates and Products of Fermentation and Respiration.

Section II. Lipids and Steroids A, Isolation and Determination of Lipids and Higher Fatty Acids. B. Preparation and Analysis of Phospholipids and Derivatives. C. Fractionation Procedures for Higher and Lower Fatty Acids. D. Preparation and Assay of Cholesterol and Ergosterol.

Section III.

Citric Acid Cycle Components

A. Chromatographic Compounds.

Analyses of Organic Acids. B. Specific Procedures for Individual

Section IV. Proteins and Derivatives A. General Procedures for Determination of Proteins and Amino Acids. B. General Procedures for Preparation of Peptides and Amino Acids. C. Specific Procedures for Isolation and Determination of Individual Amino Acids.

Section V. Nucleic Acids and Derivatives A. Determination, Isolation, and Characterization of Nucleic Acids. B. Determination, Isolation, Characterization, and Synthesis of Nucleotides and Nucleosides.

Section VI. Coenzymes and Related Phosphate Compounds A. General Procedures for Isolation, Determination, and Characterization of Phosphorus Compounds. B. Specific Procedures for N-Phosphates and Individual Coenzymes.

Section VII. Determination

of Inorganic Compounds

VOLUME SPECIAL

TECHNIQUES

IV

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 Soluhility Method for Protein Purity. I. Determination of Amino Acid Sequence in Proteins. J. Determination of Essential Groups for Enzyme Activity.

OUTLINE

OF

VOLUMES

Section II. Techniques for Metabolic

I,

II,

III,

IV,

AND

V

... x111

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 Acceptors 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) : Monosaccharides and Polysaccharides; Citric Acid Cycle Intermediates; Glycolic, Glyoxylic and Oxalic Acids; Purines and Pyrimidines; Porphyrins; Amino Acids and Proteins; Steroids; Methylated Compounds and Derivatives; Sulfur Compounds; Fatty Acids; Phospholipids; &enzymes; Iodinated Compounds; Intermediates of Photosynthesis; On-Labeled Phosphorus Compounds.

VOLUME PREPARATION

Section I. General Preparative

AND

V

ASSAY

OF ENZYMES

Procedures

A. Column Chromatography of Proteins. B. Preparative Electrophoresis. C. Preparation and Solubilization of Particles (Bacterial, Mammalian, and Higher plant). D. Mammalian Cell Culture. E. Protoplasm.

Section II. Enzymes of Carbohydrate

Metabolism

A. Polysaccharide Cleavage and Synthesis. B. Disaccharide, Hexoside and Glucuronide Metabolism. C. Metabolism of Hexoses, Pentoses and 3-Carbon Compounds. D. Hexosamine and Sialic Acid Metabolism. E. Aromatic Ring Synthesis.

Section III. Enzymes of Lipid Metabolism A. Fatty Acid Synthesis and Breakdown. B. Acid Activating Enzymes. C. Phospholipid Synthesis and Breakdown. D. Steroid Metabolism.

Section IV. Enzymes of Citric Acid Cycle A. Krebs Cycle. B. Krebs-Kornberg Cycle. C. Related Enzymes.

Section V. Enzymes of Protein Metabolism Proteolytic Enzymes. B. Amino Acid Dehydrogenases and Transaminases. C. Amino Acid Activating Enzymes. D. Other Enzymes of Amino Acid Breakdown and Synthesis. E. Enzymes of Sulfur Metabolism. A.

xiv

Erratum P. 140, line 3: should read “19.21

for Volume I

g.” instead of “21.01

Erratum

g.”

for Volume II

P. 432, line 2: should read “6.0 ml.” instead of “60 ml.”

Errata for Volume III P. P. P. P.

663, line 3: should read “0.35 y” instead of “35 7.” 843, line 2 from bottom: should read “KH2POr” instead of “K2HPOd.” 948, line 6: should read “31” instead of “0.031.” 1055, entry in author index: Abraham, S., 64.

[1]

ELECTROPHORESIS

[1]

3

Electrophoresis

By ARNE TISELIUS Enzymes, as well as proteins in general, are ampholytes, and therefore their electrochemical properties in relation to the pH of the medium are useful for their characterization. The mobility in an electric field at defined pH values and particularly the pH value at which the migration is zero (the isoelectric point) are widely used for this purpose, as these properties can be determined accurately and conveniently with the methods now available. The name electrophoresis for such phenomena used to refer chiefly to colloids and substances of large molecular weight. However, as the methods gradually have been extended also to low molecular weight material, the new term of "ionophoresis" has been introduced for this case. As it is very difficult to draw a limit here, and as the phenomena involved and the experimental methods applied are practically the same, the author has preferred to use the term electrophoresis throughout this paper. A particularly interesting and valuable feature of electrophoresis is that the differences in rates of migration for the various constituents of a mixture lead to a certain separation, each component migrating at its own characteristic rate. Thus an electrophoretic analysis is possible which can give valuable information concerning the composition of complex mixtures in solution and the homogeneity or purity of a preparation. Another advantage is that this separation is such a gentle procedure, as it involves only the migration in a medium of approximately constant composition. For preparative purposes this may be very important, especially the recent developments in this field which have provided some very useful methods with many possible applications in enzymology as well in protein chemistry in general. A considerable experience collected during the past twenty years shows that the electrophoretic mobility is a highly specific property, and thus even closely similar proteins often may be differentiated by electrophoresis. This is true, however, only if a not too narrow range of pH is investigated, as in certain pH regions mobilities of different substances may differ very little. It should also be noted that the method, on account of its gentleness, will not resolve complexes of different substances unless they are extensively dissociated in solution under the conditions of the experiment. Many cases are known in which enzymes or other proteins, even after repeated crystallization, give inhomogeneous electrophoretic diagrams. Protein crystals tend to adsorb foreign material, but this phenomenon is probably less marked in electro-

4

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[1]

phoretic separation where all components are in solution. Thus positively and negatively charged proteins may migrate almost independently of each other in buffered solutions. This is by no means universally true, however, and cases are known where interaction occurs. 1-3 Fairly stable complexes may be formed between dyes and proteins in solution, and these are not separated by electrophoresis. The demonstration of the existence of such complexes may, however, be of great interest from an entirely different point of view, if they are found in extracts of biological material which have not been subject to drastic operations. They give an indication of the presence of such associates in the original material which for many problems in enzyme chemistry may be significant. Complex formation may also be utilized to aid in the separation. The bestknown example is perhaps the electrophoretic fractionation of uncharged carbohydrates which have been complexed with boric acid (Consden and Stanier4). No doubt there are many interesting possibilities of increasing the specificity of electrophoretic analysis by such methods (see also Cann et al.4~). With methods which depend on differBOUNDARY ZONE ential migration through a medium it is SEPARATION SEPARATION convenient to distinguish between boundFIG. 1. Boundary and z o n e ary methods and zone methods, as entirely separation. different techniques are used (Fig. 1). In boundary separation the migration of the boundaries is observed, and the experiment is usually performed in a U-shaped tube where the protein solution is layered under a buffer solution. Thus the system is stabilized by the density differences, and the migration can take place in free solution without the addition of a stabilizing medium. In this case, however, only the fastest and slowest components can be isolated in substance. Intermediate components overlap, but although their separation is observed it cannot easily be utilized for a real isolation. In this respect zone separation is superior. A zone of the sample will, during its migration, split up into as many different zones as it contains differently migrating components, each zone consisting of a single component, which is easily isolated. As the zones have a higher density than the medium, however, 1L. G. Longsworthand D. A. MaeInnes, J. Gen. Physiol. 25, 507 (1942). W. Grassmann, in "The Chemical Structure of Proteins" (Wolstenholme and Cameron, eds.), pp. 55, 195. Churchill, London, 1953. s IV[.Lautout and M. de Serge, Compt. rend. 240, 1282 (1955). R. Consden and W. M. Stanier, Nature 169, 783 (1952). ~"J. R. Cann, J. G. Kirkwood, R. A. Brown, and O. J. Plescia, J. Am. Chem. Soc. 71, 1603_(1949).

/[:

[1]

ELECTROPHORESIS

5

they are not stable, and the important advantage of zone methods has to be gained at the price of stabilizing the system by using a suitable filling material, such as cellulose, starch powder, filter paper, or a density gradient. This introduces a new factor which may influence mobilities and isoelectric points in a way which is difficult to predict. But if the separation is the main purpose, these methods are very useful and have found many applications during the last years. Paper strip electrophoresis has become particularly popular as a micro-method and will be discussed in the following section by Dr. H. Laurell (Vol. IV [1A]). Zone electrophoresis in packed columns has recently been developed to a high degree of perfection. It bears the same relation to paper strip electrophoresis as column chromatography does to paper chromatography, and it allows the separation of considerable quantities of material. This method will therefore also be discussed in some detail. There are some special modifications of electrophoretic analysis without a clear-cut zone or boundary separation which have gained preparative importance. This is particularly true of the electrophoresis convection method and of methods depending on electrophoresis through membranes. The literature on electrophoresis is very extensive. A selection of references to books or articles of a more general interest is given below. 1. General principles and boundary methods. 5-~9 2. Theory. ~4,2° 5 H. A. Abramson, L. S. Moyer, and M. H. Gorin, "Electrophoresis of Proteins." Reinhold Publishing Corp., New York, 1942. 5 H. A. Abramson, E. J. Cohn, B. D. Davis, F. L. Horsfall, L. G. Longsworth, D. A. MacInnes, H. Mueller, and K. Stein, Ann. N . Y . Acad. Sei. 39, 105 (1939). 7 H. J. Antweiler, " D i e quantitative Elektrophorese in der Medizin." SpringerVerlag, Berlin, 1952. 8 D. R. Briggs, in "Biophysical Research M e t h o d s " (F. M. Uber, ed.), pp. 271-300. Interscience Publishers, New York, 1950. 9 H. B. Bull, "Physical Biochemistry," 2nd ed., pp. 161-190. John Wiley & Sons, New York, 1951. 10 L. G. Longsworth and D. A. MacInnes, Chem. Revs. 24, 271 (1939). n L. G. Longsworth, Chem. Revs. 30, 323 (1942). 1, j . A. Luetscher, Physiol. Revs. 27, 621 (1947). 13 K. G. Stern and M. Reiner, Yale J. Biol. and Med. 19, 67 (1946). 14 It. Svensson, Arkiv Kemi Mineral. Geol. 22A (1946). 15 E. Wiedemann, Scientia Pharm. 17, 45 (1949). 16 A. Tiselius, Scientia 45, 163 (1951). 17 A. Tiselius, Naturwissenschaften 37, 25 (1950). Is A. Tiselius, Trans. Faraday Soc. 35, 524 (1937). 19 A. Tiselius, Nova Acta Regiae Soc. Sci. Upsaliensis [4]7, No. 4 (1930). 25 L. G. Longsworth and D. A. MacInnes, J. Am. Chem. Soc. 62, 705 (1940).

6


[l]

3. Zone methods in general and preparative electrophoresis. 21-25 4. Paper electrophoresis (see Vol. IV [1A]).

Boundary Electrophoresis For boundary electrophoresis experiments a U-shaped tube is generally used in which the sample, dissolved in a suitable buffer solution, is allowed to form a layer under a buffer solution of the same composition, so that the boundaries will migrate in a medium of approximately constant composition. The electric current is introduced with reversible electrodes, separated from the boundaries by a sufficient volume of buffer solution to prevent changes in the composition produced at the electrodes to reach the boundaries. The boundaries are stabilized by the differences in density, and thus no special stabilizing medium is required as in zone electrophoresis. Consequently the " f r e e " electrophoretic migration of the components can be measured, and the true mobilities can be calculated directly from the experiments. To secure a sufficient stability the concentrations should not be too low, and it is generally difficult to obtain reliable results at concentrations below 0.01% (protein) unless special precautions are taken (low current). The heat produced by the current may cause convections, and when studying systems of high conductivity (e.g., serum) or low protein concentration it is essential to minimize this disturbance by running the experiment at a temperature just above 0 °. 18As water has a density maximum around -~4 °, the density differences produced by temperature changes in this region are very much smaller than at room temperature, for example. As low temperature is of advantage for other reasons in protein and enzyme work, most experiments of this type are nowadays being performed in refrigerated baths. To facilitate optical observation of the boundaries by the methods described below, the electrophoresis cell is given a rectangular cross section. The U-tube is assembled by one or several such cells, mounted with plane-parallel plates which are greased so that they can slide horizontally a sufficient distance to cut the contents of the tube into a number of fractions. This sliding arrangement is also used when the boundaries 21 H. G. Kunkel, in " M e t h o d s of Biochemical Analysis," Vol. I, pp. 141-170. Interscience Publishers, New York, 1954. 22 H. J. McDonald, "Ionography. Electrophoresis in Stabilized Media." Year Book

Publishers, Chicago, 1955. 23H. Svensson, Advances in Protein Chem. 4, 252 (1948). ~4R. L. M. Synge, in "General Methods of Separation: Electrical Transport Methods in Modern Methods of Plant Analysis, I" (Paech and Tracey, eds.), pp. 55-65. Springer-Verlag, Berlin, 1956. 25A. Tiselius and P. Flodin, Advances in Protein Chem. 8~ 461 (1955).

[1]

ELECTROPHORESIS

7

are to be formed at the beginning of the experiment. Most modern equipment of this type is provided with a "compensation device" which allows a slow and accurately controlled injection of buffer solution into one or the other of the electrode tubes during the experiment. Thus the boundaries may be displaced to any desired position in the tube, and fast-moving boundaries may be kept in the observation field much longer than would otherwise be possible.

FIG. 2. Schematic drawing of an apparatus (manufactured by LKB-Produkter Fabriks AB, Stockholm) for boundary electrophoresis, showing the composite U-tube and the electrodes. Cells 2, 3, and 4 are filled with the sample, and ceils 1 and 5 as well as the electrode vessels with buffer solution. Figure 2 shows a diagrammatic representation of a boundary electrophoresis apparatus with a composite U-tube. Although optical observation of the boundary migration is usually preferred in this type of work, it should be noted that electrophoretic mobilities may also be determined analytically in this apparatus ("transference method"). If cells 2, 3, and 4 in Fig. 2 at the start contain the sample, of known concentration (for example, nitrogen, or any enzymatic activity), and cells 1 and 5 contain only buffer, the experiment is allowed to run until cell 1 is partially filled with migrating material and cell 4 partially emptied. If the bottom cell (cell 3) composition does not change, the increase on one side must

8


[1]

be equal to the decrease on the other. If this difference is Am, the original concentration is c (in the same unit), and the cross-section area of the tube is q, the distance of migration is hm/cq. The quantity Am is determined by taking out the contents from each cell and analyzing at the end of the experiment. It should be noted that this method is applicable also when c and m refer to enzymatic activities and thus may be useful when the actual enzyme concentration in the sample is too low to make optical observation possible. In complicated systems valuable information may also be obtained by combining this procedure with optical observation of the boundaries, and it may be possible this way to determine which of the observed boundaries refers to the enzyme or other material with measurable biological activity which is being studied. Direct optical observation (by photography) of the migration of the boundaries is most commonly used, however, and has the great advantage that it offers a convenient way of studying the heterogeneity of the sample, as each differently migrating component usually will give rise to a separate boundary. The optical methods also allow a quantitative estimation of the relative amount of the different components present (electrophoretic analysis). As substances to be studied generally are colorless, refractive index or ultraviolet light absorption methods are usually preferred. One may distinguish between concentration and concentration-gradient methods, the latter being most suitable for heterogeneity studies. In the author's early experiments 19 the ultraviolet absorption method introduced by Svedberg and Nichols :6 for ultracentrifugation measurements was used. The electrophoresis U-tube is made of quartz and is photographed in transmitted ultraviolet light from a quartz mercury vapor lamp, the light being filtered through quartz cells containing bromine and chlorine. In later work when this method has been used for studying protein chromatography we have found that the somewhat troublesome chlorine-bromine filters may be replaced by a solution of cobalt and nickel sulfate, according to Kasha. 2~ As this method is not sensitive enough to analyze composite boundaries, the author 28 in 1937 introduced refractive index observation, based on the well-known Toepler schlieren method, which depends on gradients rather than on concentrations. The ultraviolet method has the advantage of being more specific than refractive index methods, however, and may perhaps deserve renewed attention, even though it requires the introduction of an all quartz system. Interferometric observation also records concentrations and has been introduced lately by several authors (see, ~6 T. Svedberg and J. B. Nichols, J. Am. Chem. Soc. 48, 3081 (1926). ~ M. Kasha, J. Opt. Soc. Amer. 88, 929 (1948). 28 A. Tiselius, Trans. Faraday Soc. 35, 524 (1937).

[1]

ELECTROPHORESIS

9

for example, Svensson 29 and Svensson and Forsberg~°). These methods are highly sensitive and are often useful, but the evaluation of the diagrams is perhaps somewhat more laborious than with the gradient methods. The former are particularly useful for determinations of relative amounts of the components, the latter for high resolution of boundary composition. A combination of both is probably most valuable. So far most results published have been obtained by gradient methods, and these will therefore be discussed in more detail. They are based on the deviation which a light beam suffers if it passes through a refractive index (that is, a concentration) gradient. Thus if the beam is made to pass horizontally through an electrophoresis tube containing a boundary, it will be deviated downward in those parts of the tube where the boundary is located. If a camera is focused on the tube and if the camera lens is provided with a horizontal edge diaphragm, light from all parts of the tube will be able to pass into the camera except from those levels where the boundary is situated, the light from these parts being deviated below the diaphragm edge. Thus on the image the boundary will come out as a black band. Each setting of the diaphragm corresponds to a certain deviation--that is, to a certain value of the refractive index gradient. As Longsworth ~1 first showed, a complete recording of the gradient throughout the boundary region as a function of the vertical distance in the electrophoresis tube may be obtained by coupling the movement of the edge with a movement of the photographic plate. The same result may also be obtained by a purely optical arrangement introduced by Philpot 32 and further developed by Svensson, ~3 where no movable parts are required and the recording is achieved by the aid of a cylindrical lens combined with a special diaphragm (Fig. 3). For details the reader is referred to the literature recommended in footnotes 5 to 15. The diagrams obtained give the refractive index gradient, d n / d x , as a function of the distance of migrating in the cell. Thus, by integration, the total n v a l u e - - t h a t is, the concentration--can be computed for each sufficiently well-separated component. Typical gradient diagrams are shown in Fig. 4. Both these procedures work satisfactorily and have found extensive use. In addition, mention should be made of the highly accurate "scale m e t h o d " by Lamm 34 which has found wide application especially in 59H. Svensson, Acta Chem. Scan& 3, 1170 (1949). 30H. Svensson and R. Forsberg, J. Opt. Soc. Amer. 44, 414 (1954). al L. G. Longsworth, J. Am. Chem. Soc. 61, 529 (1939). 3~j. S. L. Philpot, Nature 141, 283 (1938). a3H. Svensson, Kolloid-Z. 87, 181 (1939); see also ref. 14. 34O. Lamm, Z. physik. Chem. A138, 313 (1928).

10


Ill

ultracentrifugation and diffusion work but does not seem to have come into general use in electrophoresis, probably because the evaluation of the diagrams is somewhat laborious. Detailed instructions for working with the different types of commercially available electrophoresis apparatus are provided with the instruments and will not be discussed further here. Some general remarks about the procedure may, however, be useful to readers who have no previous experience in electrophoresis work. Boundary electrophoresis, including transference measurements (see p. 7), is so far the most accurate and Op//CO/ S~/s/ern

A

B

C

D

E

f

G

H

Fro. 3. Optical system for boundary observation in eleetrophoresis. The dashed beams . . . . and pass through the cell, C, outside the boundary region and are therefore not deviated but are focused at the upper end of the inclined slit, E. They pass through the cylindrical lens, G, without horizontal deviation and give rise to the "base line" in the curve on the film, H. The beam passes through the boundary in t h e cell, C, and is deviated to the lower end of the inclined slit, E, which gives rise to a horizontal deviation in the cylindric lens, G, resulting in an image point near the peak of the curve on the film, H. [From H. Svensson, Tek. Tidskr, $7, 1952.]

least objectionable method of determining mobilities and isoelectric points for proteins and other large molecular weight substances with a pronounced electrochemical character. It should always be observed that, even if these characteristics are sharply defined and very useful for the characterization of such substances, they depend markedly on other ions present, so that the buffer used and its concentration always should be stated. The other application is the study of homogeneity, and electrophoretic analysis is often used as a sensitive criterion on purity. One must remember, however, that differences may exist which are not reflected in the electrophoretic behavior, and under all conditions the analysis should be made at a number of different pH values before any conclusions are drawn. The possibility of complexes which are too weakly dissociated to be separated into their constituents by electrophoresis should also be kept in mind (cf. p. 4). There are some complications inherent in the method which often

[1]

ET.ECTI~OPHORESm

11

A scend;n9

J

j

~

AIo.

Descer~/n 9

t . 6lob. A

B FIG. 4. Typical boundary electrophoresis diagrams, as obtained by the apparatus and optical arrangement in Figs. 2 and 3. (A) Human pathological serum, showing the main protein components: albumin and a-, f~-, and -y-globulins. (B) Crystalline lactic dehydrogenase, containing two components. Descending pattern in phosphate buffer, pH 5.7, ionic strength 0.1. [According to J. B. Neilands, S c i e n c e 115, 143 (1952).]

12


[1]

puzzle research workers in this field and which have been given the common name of "boundary anomalies." Thus, one finds frequently that descending and ascending boundaries of the same substance and in the same experiment move at somewhat different rates. "False" boundaries, which are due to discontinuities in the buffer medium and thus do not represent new components, are sometimes observed, especially in concentrated solutions, as the well-known ~-boundary in serum experiments. As a rule these disturbances are much reduced if the protein concentration is lowered. The anomalies are more marked, however, with substances of lower molecular weight, and they make it generally impossible to study by this method the migration of amino acids, for example. The general problem of the migration boundaries in electrolyte solutions is a very complex one, but the theory has been worked out in detail (see, for example, Longsworth and MacInnes, 2° Dole, 35 and Svensson~4). Moving boundary electrophoresis of proteins and similar substances is to be regarded as a limiting case, in which one component (the protein) contributes relatively little to the electrolytic conductivity of the system, although it is present in an appreciable concentration. Thus the migration of the protein does not change appreciably the distribution of the potential gradient, and it moves in a medium of practically constant composition and therefore in a constant electric field. This is generally only approximately true, however. If substances are studied for which the ratio conductivity:weight is higher and approaches that value for the buffer ions used, the anomalies dominate the phenomenon and make the interpretation of the results very difficult The decisive factor is thus not the molecular size but the conductivity per unit weight of the substance to be studied The methods of zone electrophoresis to be described below have the advantage of showing a much greater latitude toward boundary anomalies and thus permit the study of substances which previously could not be subject to electrophoresis.

Zone Electrophoresis in Packed Columns and Similar Media (Except Paper Strip Electrophoresis) The development in this field has been particularly rapid during the last few years, and no attempt will be made here to present the historical development. The author has preferred to review the present situation with particular emphasis on procedures of which he has had personal experience. The apparatus required for zone electrophoresis is comparatively simple. Optical observation is not necessary (although in some cases it a~ V. P. Dole, J. Am. Chem. Soc. 67, 1119 (1945).

[l]

ELECTROPtIORESIS

13

would no doubt be of advantage). The zones are localized by analytical or by staining methods. The crucial problem is the filling material necessary for the stabilization of the zones. Proteins and other substances to be studied are often adsorbed to finely divided powders. This tends to give rise to tailing phenomena which greatly interfere with the separation. Thus sometimes the mobility may be reduced to practically zero. The stabilizer should be hydrophilic but insoluble. If it is a powder it should be easy to pack into homogeneous columns which contain a sufficient amount of the solvent medium. There will always be some electrical transport of the solvent through the stabilizer during the experiment (electroendosmosis). This will not interfere with the separation as it only moves the system of zones in the direction of electroosmotic flow, but experience shows that it is important to keep this flow as low as possible. Among powdered materials cellulose and starch have been most widely used so far. In the author's laboratory Flodin and Kupke 3~have found that cellulose powder prepared from cotton by alcoholysis is superior to ordinary cellulose, and this material is now preferred in all our zone electrophoresis investigations. It is advisable, however, to test the material for its adsorption properties on the system to be studied by running a zone through a small column without c u r r e n t - - t h a t is a chromatographic experiment. Gels are often permeable enough also to large protein molecules to make them suitable as stabilizing media for zone electrophoresis. They have the advantage of giving sufficient stabilization at low dry weight, and thus adsorption phenomena are less disturbing. They often give beautiful zones which may be observed optically if the gel is translucent. Silica gel was used by Consden et al.'~7 in their " i o n o p h o r e t i c " separations of peptides and amino acids, but this gel appears to be less suitable for proteins. Agar gel has been used with success by Gordon et al., 3~ and recently Grabar et al. '39 have made extensive electrophoretic investigations by this technique, also combining it with the Oudin serological precipitation method which makes possible a very sensitive identification of different antigens (immuno-clectrophoresis). Smithies t° has obtained very beautiful separations of proteins in starch gels. A disadvantage of the gels is that it is difficult to elute the zones quantitatively without contamination. In this respect powdered stabi36p. Flodin and D. W. Kupke, Biochim. et Biophys. Acta 21, 368 (1956). 37R. Consden, A. H. Gordon, and A. J. P. Martin, Bioehem. J. 40, 33 (1946). a8A. H. Gordon, B. Keil,' and K. Sebesta, Nature 164, 418 (1949). ~9p. Grabar, C. A. Williams, Jr., and J. Courcon, Biochim. et Biophys. Acta 17, 67 (1955). 400. Smithies, Biochem. J. 61, 629 (1955).

[1]

ELECTROPHORESIS

15

-:-4 D4

---2 -_w

-----

--A,

z----.--

----AJ

~_w_

0

_.--__ -

--2-

~.-

--_--.

--_--.

Fu~. 5. Large t y p e of zone c o l u m n electrophoresis a p p a r a t u s according to J. P o r a t h

[Biochim. et Biophys. Acta 22~ 151 (1956)]. Details in the text.

16


[1]

.c. .j

~5

!1 ~o

2b

~o

~0

~

60

7o

'

~o

9o

Tube r~m~er

~o

A Fro. 6A. Mixture of "neutral" amino acids in I N acetic acid resolved by 61 hours of electrophoresis at 30 ma. (2300 v.) in a column 2 X 250 cm. packed with cellulose powder. E is the extinction as determined by the ninhydrin reaction. Each amino acid, 2 mg. [J. Porath, Biochim. et Biophys. Acta 22, 151 (1956).]

.~

~70

~,a.~

~

0

,~ I I

~

~ 11

~0

~00

I,/

//

thO

"~"

I1

H

200 250 [itlcft~e, rd vol~jrn~,ml

B FTO. 6B. Mixture of vitamin B~ components in acetate buffer, p H 5,1, ionic strength 0.05, resolved by 14 hours of electrophoresisat 18 ma. in a column 3 X 50 cm. packed with starch. Each component 2.6 mg. [From N. Siliprandi, D. Siliprandi, and H. Lis, Biochim. et Biophys. Acta 14, 212 (1954).]

[1]

ELECTROPHORESIS

17

l a r g e c o l u m n s t h e c a p a c i t y is c o n s i d e r a b l e . T h u s on c o l u m n s of 150-cm. l e n g t h a n d 6.5-cm. d i a m e t e r as m u c h as 20 to 30 g. of p r o t e i n c a n be s e p a r a t e d a n d s e r u m s a m p l e s c o r r e s p o n d i n g t o 200 ml. u n d i l u t e d s e r u m h a v e b e e n f r a c t i o n a t e d . P e p t i d e s , a m i n o acids, a n d o t h e r low m o l e c u l a r w e i g h t m a t e r i a l l e n d t h e m s e l v e s v e r y well to t h i s k i n d of s e p a r a t i o n . F i g u r e s 6 A - C s h o w s o m e e x a m p l e s of r e s u l t s o b t a i n e d w i t h y e a s t enolase, 2.0

2.0

o

~I,5

1.5

2

~ I.0

1.0

O

~0.5

0.5 5"

20

30

40

FRACTION NUMBER

C Fro. 6C. Cellulose column electrophoresis of yeast enolase. Column dimensions: 1.8 cm., diameter; 70 cm., length. Buffer: 0.0154 M NaH~P04, 0.0115 M Na:B4OT, pH 8.2, # = 0.05. Current: 15 ma. Time: 45 hr. The experiment was performed in a cold room (4°). The protein concentration (O) of the fractions was determined by measuring the optical density at 280 m# in a Beckman DU spectrophotometer using 1-cm. cells, 1 rag. per milliliter giving a value of 0.895. The activity (A) was estimated as the change in optical density at 240 m~ per minute (&D.~40/min.) at 22 ° when 10 p]. of a fraction was added to I ml. of substrate solution (2.4 X 10-3 M DL-2-phosphoglycerate and l0 -3 M mg. ++ in tris(hydroxymethyl)aminomethane-HC1 buffer, pH 7.4, ~ = 0.05) in a 1-cm. cell. [Experiments by Dr. B. Malmstr6m, Uppsala, to be published.[ t h e d a t a b e i n g g i v e n in t h e figure legend. W h e n s e p a r a t i n g low m o l e c u l a r w e i g h t s u b s t a n c e s v o l a t i l e buffers as r e c o m m e n d e d b y P o r a t h 48 a r e v e r y useful, as d i a l y s i s c a n n o t be u s e d in t h i s case to g e t r i d of t h e e l e c t r o l y t e s c o n t a i n e d in t h e f r a c t i o n s i s o l a t e d . P r e c i s e m o b i l i t y m e a s u r e m e n t s c a n n o t be m a d e in zone m e t h o d s w h e r e s t a b i l i z i n g m e d i a a r e used. I t a p p e a r s , h o w e v e r , t h a t t h e d i f f e r e n t c o m p o n e n t s e m e r g e f r o m t h e c o l u m n in t h e o r d e r t o be e x p e c t e d f r o m t h e i r m o b i l i t i e s in free s o l u t i o n . K o l i n , 49 S v e n s s o n a n d V a l m e t , 5° a n d 48j. Porath, Nature 174, 478 (1955). 49 A. Kolin, Proc. Natl. Acad. Sci. U.S. 41, 101 (1955). 5o H. Svensson and E. Vahnet, Science Tools 2, 11 (1955).

[1]

ELECTROPHORESIS

19

vessel and will therefore flow as a narrow vertical zone through the medium and leave the cell through one or a few of the bottom outlets. If a horizontal electric field is applied, however, the zone will split up as shown in the figure, and separated fractions may be collected at suitable outlets. In principle, it should be possible to fractionate very large quantities by such procedures, and the method may even gain industrial application. Maintaining constant conditions of flow and a constant current is very essential for obtaining good resolution. Brattsten 56-5~ has recently published a detailed study of the method and has described its application to serum protein fractionation. For smaller quantities sheets of filter paper may be used which simplifies the apparatus. Further information about this method may also be found in a review by Durrum et al. 59 So far, the capacity does not seem to surpass what can be conveniently handled in a zone column apparatus of the type described above (pp. 14-15), which also has the advantage of simpler construction and more easily controlled manipulation. For still larger capacities the continuous zone electrophoresis no doubt offers promise.

Other Methods of Preparative Electrophoretic Separation The above methods have in common that they allow a control of the separation by observation of boundaries or zones either in the electrophoresis tube itself or in the elution diagrams from the columns. There are some other methods where this is not possible (at least not directly) but which have proved to be quite useful in preparative work. This is particularly true of the "electrophoresis convection" method first proposed by Kirkwood 6° and experimentally realized by Nielsen and Kirkwood. 6~ A description of the apparatus is given in papers by Cann el al. 4~ and Timasheff et al. 82 The latter paper describes a semicontimlou~ modification of the method and gives further references. The method is based on the layering phenomenon sometimes observed in electrodialysis of proteins between vertical membranes. The proteins are concentrated by migration toward one membrane and tend to form a layer, which on account of its higher density will be transported by 5~ I. Brattsten, Arkiv Kemi 4~ 503 (1952). 57 I. Brattsten, Arkiv Kemi 8, 205, 227, 347 (1955). 5a I. Brattsten, Continuous Zone Electrophoresis by Crossed Velocity Fields in a Supporting Medium, Dissertation, Uppsala, 1955. ~ R. J. Block, E. L. Durrum, and G. Zweig, " A Manual of Paper Chromatography and Paper Electrophoresis," pp. 380-390. Academic Press, New York, 1955. s 0 j. E. Kirkwood, J. Chem. Phys. 9, 878 (1941). e~L. E. Nielsen and J. G. Kirkwood, J. Am. Chem. Soc. 68, 181 (1946). 6~ S. N. Timasheff, J. B. Shumaker, Jr., and J. G. Kirkwood, Arch. Biochem. and Biophys. 47, 455 (1953).

20


[l]

convection to the bottom of the cell. Several sharp horizontal boundaries, separating layers of different composition, may thus often be obtained. The Kirkwood apparatus consists essentially of a flat rectangular cell, separated by large surface membranes from the electrode compartments, through which a suitable buffer solution is circulated. The membranes should be semipermeable, thus allowing the buffer ions but not the protein material to pass freely. In the course of the experiment the fastest migrating substances are collected toward the bottom and the slowest at the top. The method is in principle analogous to the well-known Clusius thermodiffusion method for separation of gases. It has been applied with notable success to the fractionation of serum proteins and insulin, for example, and should be easy to adopt for large-scale work. Multimembrane apparatus where the material to be separated is migrating through the membranes has found some use for the electrophoretic separation of peptides and amino acids. With suitable membranes proteins and other large molecular weight material also may be fractionated. The apparatus required is of the same type as a common electrodialyzer and thus quite simple. The membranes serve to keep the separated components apart, but also have a very marked effect on the course of the separation. The result depends largely on the rates of migration within the membranes, which may differ quite considerably both absolutely and relatively from m0bilities in free solution, as the membrane charge and other specific effects have a decisive influence. Thus it is usually necessary to combine membranes of different kinds. Specific effects can naturally be utilized to improve the separation. A detailed discussion of methods of this type has been given by Svensson. 28 Mould and Synge 63 have had some success in attempts to separate substances according to molecular size by migrating them in collodion strips with the aid of electroosmotic transport of the solvent (electrokinetic ultrafiltration). In part the effects obtained seem to be due to adsorption , but the method appears to have interesting possibilities, also in zone separation. t3 D. L. Mould and R. L. M. Synge, Biochem. J. 58, 571 (1954).

32


[2]

[2] Ultracentrifugation, Diffusion, and Viscometry B y H. K. SCHACHMAN

I. Introduction Theoretical and experimental advances which have been made in recent years in ultraeentrifugation, diffusion, and viscometry have resulted in the availability of powerful methods for the determination both of the degree of purity of a given preparation of a protein and of the size and shape of the macromolecules. It is of importance, therefore, that enzymologists be aware of these methods, their potentialities, and their limitations. In addition to considerations of purity and molecular size we will also discuss in this chapter some applications of diffusion and sedimentation methods which possess special appeal to the enzymologist. Techniques are available, although infrequently used, whereby the sedimentation and diffusion of a biologically active material can be studied, even in impure preparations and with only microgram quantities of material, as long as there is a sensitive and accurate biological assay specific for the entity under investigation. With these methods, reasonably accurate values of the molecular weight of an enzyme can be obtained even before purification of the enzyme is achieved. Some of the methods we will discuss are rapid, and the techniques can be performed at low temperature so that labile enzymes can be studied without danger of inactivation. In most cases, a fairly complete study can be performed with as little as 10 to 20 mg. of material, and in special circumstances microgram quantities will suffice. The same methods are equally applicable to the examination of molecules smaller than most proteins and can be used therefore in studies of coenzymes and substrates or the products of proteolytic enzymes or nueleases. In this way, these physical methods can be used to study the mechanism of enzymatic action on large substrate molecules. Ultracentrifugation, in particular, is of use in the study of interaction of enzyme molecules with themselves as in association equilibria or in interaction of enzyme molecules with other molecules such as DPN. For all the work described in this chapter there is now available commercial apparatus which, though expensive and complicated, is generally installed by the manufacturer ready for operation. The detailed description and explanation of the working parts of this equipment will be omitted from our discussion as will be those operating procedures generally supplied by the manufacturer in technical manuals. Only special

[9.]

ULTRACENTRIFUG&TION'~ DIFFUSION, AND VISCOMETRY

33

parts of the experimental procedures common to all different makes of apparatus and requiring techniques developed through experience will be elaborated upon in this discussion.

II. Ultracentrifugation Introduction Ultracentrifuges can be used either to study the velocity of sedimentation of macromolecules through a solution under the influence of a centrifugal field or to study the distribution of the macromolecules in a centrifuge cell which is rotating at much lower speeds so that the molecules obtain equilibrium positions throughout the cell. The former method is known as the sedimentation velocity method and has to date been the more widely used. In a sedimentation velocity experiment the ultracentrifuge is operated at speeds up to 60,000 r.p.m. so that the solute molecules which initially were uniformly distributed throughout the solution in the ultracentrifuge cell are caused to settle toward the periphery of the cell. This migration of the solute molecules leaves a region containing only solvent molecules (if the solute molecules are more dense than the solvent, then the newly created solvent region will be near the meniscus; for solutes which are less dense than the solvent, such as lipoproteins in concentrated salt solutions, the solute will float, leading to a region of pure solvent at the bottom of the cell) in addition to the region in the cell where the concentration of solute is uniform. Between the supernatant and the solution of uniform concentration, known as the plateau region, there is a transition zone in which the co~centration varies with distance from the axis of rotation. This transition zone is called the boundary; and the sedimentation velocity method is based on observations, by optical methods, of the movement of the boundary which in turn is a measure of the movement of the solute molecules in the plateau region. Figure 1A shows schematic drawings of the concentration of solute as a function of distance in the cell at different times. Since the more commonly used optical systems do not record concentration, but rather the change in concentration with distance, the corresponding curves are drawn in Fig. lB. It is such diagrams, as illustrated in Fig. 1B, which are used to study the purity of a given preparation and to determine the sedimentation coefficient, s, of the sedimenting material. It should be noted that the sedimentation velocity method permits the direct measurement of sedimentation coefficients which are related to the size and shape of the sedimenting molecules, but other independent data are needed for the evaluation of molecular weights. In the second application of the ultracentrifuge, known as the sedimen-

34

T E C H N I Q U E S FOR CHARACTERIZATION OF P R O T E I N S

[2]

tation equilibrium method, the ultracentrifuge is operated at relatively low speeds (about 8000 r.p.m, for a protein with a molecular weight of 69,000). Under these conditions the transport of solute in a centrifugal direction due to sedimentation is sufficiently slow as to be counterbalanced by transport in a centripetal direction by the diffusion resulting FROM M E N I S C U S ( C M )

DISTANCE 0

I

I00 Z 0

oi8

I

I

I

,.2

S

1-.IZ w ¢_) z

o ¢3

oi4

50-

J z (.9 ¢e o

/ 0 '

I

B

I-Z tl.I

£9, Z 0 I.n,F.z hi (...) Z 0 U I

I

6.O 6,4 6~8 L2 DISTANCE FROM AXIS OF ROTATION (CM)

Fro. 1. Plot of concentration (A) and concentration gradient (B) versus distance from the axis of rotation during a sedimentation velocity experiment. Sedimentation is to the right.

from the concentration gradient created by the partial sedimentation of the macromolecules. During the first stages of a sedimentation equilibrium run, the concentration decreases at the meniscus and increases at the bottom of the cell, owing to sedimentation. As a consequence of back diffusion, however, a region devoid of solute is not created as in the sedimentation velocity method. Instead the concentration will remain finite at the meniscus as long as the centrifuge is not operated at too high a speed. Under ideal circumstances the concentration at the meniscus

[9.]

ULTRA.CENTRIFUGATION',

DIFFUSION,

AND

VISCOMETRY

35

will approach a value about one-half its initial value. Similarly, sedimentation plus back diffusion at the bottom of the cell leads to a region in which the concentration is higher than the original concentration. In the sedimentation velocity method, where the role of diffusion is much less important, this region of high concentration is restricted to a very thill

O z

DISTANCE FROMMENISCUS(CM) 0.4 0.8 1.2 i ~

o_ I-< n-I-z ~A Z 0°

IO0

Z 0 ~

A

kz hi

B

Z

_o i.n,i-.z

~

4j

Z 0u

g i

i

6.0 6.4 6.8 7.2 DISTANCE FROMA X I S O F R O T A T I O N ( C M ) Fla. 2. Plot of concentration (A) and concentration gradient (B) versus distance from the axis of rotation during a sedimentation equilibrium experiment. Centrifugal direction is toward the right. layer on the cell bottom where the sedimented material is packed as a gel-like pellet. In the center of the cell, during the early stages of a sedimentation equilibrium study, the concentration is independent of position in the cell and practically the same as the initial concentration. As the run proceeds, the plateau region disappears and there is only one position in the cell having a concentration equal to the initial concentration. Finally, after a considerable period of time an equilibrium state is reached after which no further changes in concentration occur with time. These changes in concentration with distance as a function of time are

36

TECHNIQUES FOR CHARA.CTERIZA.TION" OF PROTEIN'S

[2]

visualized in Fig. 2A, and the corresponding gradient curves are illustrated in Fig. 2B. In cont-rast to the sedimentation velocity method, measurements of the concentration distribution at sedimentation equilibrium give directly the molecular weight of the sedimenting macromolecules (both methods require knowledge of the partial specific volume of the macromolecule, and this will be discussed later). Despite this obvious advantage and the additional factor that the theoretical foundations for the equilibrium method are more firm than those for the sedimentation velocity method, there are few recorded examples of the application of the sedimentation equilibrium method to the study of enzymes. This seemingly anomalous situation has arisen both because of the lack of apparatus capable of sustained, continuous use for the long periods (days in the case of proteins) required before equilibrium is reached, and because many proteins are not sufficiently stable to withstand such experiments. A commercial apparatus is now available which is capable of continuous operation even at low temperatures, but it is of greater importance to the enzymologist that considerable theoretical advances have been made which enable us now to determine molecular weights in times as short as one hour. Owing largely to the theoretical work of Archibald I and certain technical improvements especially with regard to optical methods, it seems safe to predict that measurements during the approach to sedimentation equilibrium will become as routine as those by the currently more popular sedimentation velocity method. Because of the importance of these developments we will present in this chapter the method involving the approach to sedimentation equilibrium despite the knowledge that the method is still in its infancy and further refinements both in theory and technique may render the discussion obsolete. We are here using the term "approach to sedimentation equilibrium" rather loosely, since this title leads to the implication that the experiments are conducted for times almost sufficient to attain complete equilibrium. Actually for the use of the equations of Archibald there are no restrictions that the system be near equilibrium. There might be serious objection then to our inclusion of this method under the section entitled "sedimentation equilibrium," especially since this method is clearly not a thermodynamic one. To some extent our classification is arbitrary although it can be justified operationally. If the experiments are designed so as to measure most accurately the velocity of transport of material through the cell as by the proper choice of the centrifugal field for the system under investigation, then the experiments can be classified as sedimentation velocity studies. If, alternatively, the experimental condi1 W. J. Archibald, J. Phys. & Colloid Chem. 51, 1204 (1947).

[9.]

ULTRACENTRIFUGATION, DIFFUSION~ AND VISCOMETRY

37

tions are selected so as to measure precisely the distribution of matter throughout the cell, then the experiments are of the sedimentation equilibrium type. Most ultracentrifuge work done at present is performed in the Model E ultracentrifuge manufactured by the Spinco Division, Beckman Instruments, Inc., Belmont, California. This instrument is completely equipped with the necessary accessories for a variety of ultracentrifuge studies. The majority of the work is performed with the schlieren optical system for viewing and photographing the movement of sedimenting materials. Some special applications, however, can be better performed by absorption techniques using either ultraviolet light or visible light of the appropriate wavelength. A brief description of applications using light absorption techniques will be presented in a later section. A variety of ultracentrifuge cells in optical thicknesses from 1.5 ram. to 30 ram. are available, and the choice of cell depends to some extent on the type of study. For very dilute solutions the cells with the longer optical path are desirable, even though they require larger volumes of solution and even a special rotor. The smaller cells require even less than 0.2 ml. of solution; but, of course, optical registration of the boundary suffers unless high concentrations are used. Cells can be either 4 ° sectors or 2 ° sectors; the latter require longer exposure times and more care in alignment of the cell in the rotor, but they have the decided advantage of requiring smaller volumes of solution. Cells should always be cleaned and dried at the conclusion of the run because salt solutions can cause etching of the cells. Filling needles should have their tips ground flat so as to remove sharp points, and special caution is needed to avoid scratching of the cells. For alkaline or acid solutions, cells with a plastic centerpiece made of Kel-F or epoxy are recommended, and the quality of the latter is so high that they may prove to be the cell of greatest practicality and economy. With plastic cells, no gaskets are required between the centerpiece and the windows, so that assembly of the cells is facilitated. Doublesector cells are available in which one section can be filled with the solution and the other with the solvent. Although the patterns are not so distinct with these double cells, they furnish a base line superimposed on the pattern containing the sedimenting protein, and they can be of use in sedimentation velocity studies. Also special optical selectors facilitate the simultaneous recording of more than one ultracentrifugal analysis in a special rotor containing two or more cells. For equilibrium runs the special one-third speed attachment for the ultracentrifuge is necessary. The optical systems have for years had a bar as the schlieren diaphragm, but it is clear that a phase plate, now commercially available,

38

T E C H N I Q U E S FOR CHARkCTERIZATION OF PROTEINS

[2]

gives much better results and is strongly recommended for all types of work and especially for sedimentation equilibrium. The phase plate can be mounted on the optical track in place of the bar. Since it has been found in recent years that the rotor cools about 0.8 ° on stretching, owing to the high speeds, there has been a growing need for a continuously reading indicator of the temperature of the rotor during the run. This is now available, and the same unit can also control the temperature by activating a heater in the vacuum chamber. For long runs, especially at low temperatures, this is a valuable addition to the ultracentrifuge. Most of the patterns are photographed with Kodak spectroscopic plates, Type I-D(2), with a yellow filter in the optical system, but higher resolution photographs can be obtained with either Kodak M or Kodak Metallographic plates. These are slower plates and longer exposure times are needed, but this disadvantage is largely offset by the replacement of the half-surfaced viewing mirror with the newly developed swing-out mirror. For colored materials like hemoglobin, the yellow filter is replaced by a red filter, and red-sensitive I-N plates are used.

Sedimentation Equilibrium Principle. Although the time required for attainment of equilibrium may be as long as 4 days for a protein of molecular weight about 60,000, Archibald has shown how molecular weights can be calculated from data obtained during the early stages of the run. He pointed out that solute does not leave the centrifuge cell either at the meniscus or the bottom of the cell and that, therefore, the conditions for equilibrium (transport of solute by sedimentation equals transport by diffusion) are fulfilled at these two places in the cell at all times of the run. The equation describing this condition is identical to that which holds elsewhere in the cell when equilibrium is attained, and we can write for a homogeneous material M -

RT (1 -

?p)~2

(dc/dx)m _ x~cm

RT (1 -

~p)~2

(dc/dX)b

(1)

XbCb

In equation 1, M and I~ are the anhydrous molecular weight and the partial specific volume of the solute, R is the gas constant, 8.314 X 107 ergs/mole/degree, T is the absolute temperature, p is the density of the solution, (o is the angular velocity of the centrifuge rotor in radians per second, (r.p.m.) (27r)/60, and cm and (dc/dx)m are the concentration and concentration gradient at the position x~, which is the distance from the axis of rotation to the meniscus. The corresponding quantities with the subscript b refer to the bottom of the cell. The procedure requires knowledge of the concentration gradients, (dc/dx)m and (dc/dX)b, and the concentrations, c~ and Cb. The former can

[2]

ULTRACENTRIFUGATION, DIFFUSION~ AND VISCOMETRY

39

be obtained directly from the photographic plates after suitable corrections for optical constants or with the aid of an auxiliary sedimentation run, data from which obviate measurement of the optical constants both of the apparatus and the solution. As shown in Fig. 2A, cm and cb are, respectively, less and greater than Co, the initial concentration; if the measurements are made early in the run, cm and cb are almost equal to Co. Corrections for the change in concentration at the meniscus and bottom of the cell are made with the equations derived by Klainer and Kegeles: ~ em ~

Co

- -m 2 X

and cb = co d- xb ~1

x2(dc/dx)dx

//

(2) x2(de/dx)d x

where X refers to a position in the plateau region ( d c / d x equals zero). It should be emphasized that the method as presented here, employing equation 2, is that proposed by Klainer and Kegeles, and it requires that the ultracentrifuge run be of limited duration such that there is still a region in the cell in which the concentration is independent of position, i.e., some plateau region still exists. This is clearly evident from the photographic plates, since the plateau region produces the fiat portion of the curve (usually known as the base line) in the optical system commonly employed. If the run is continued for longer times so that there is no longer a fiat portion of the curve seen on the photographic plates, then equation 2 is not applicable and cm and Cb must be evaluated in a manner different from that proposed by Klainer and Kegeles. Since accurate results can be obtained in the shorter runs, we will emphasize in our discussion the evaluation of molecular weights from equations 1 and 2. When the greatest accuracy is desired and experimental factors such as the lability of the enzyme do not preclude runs of long duration, it is advantageous to continue the ultracentrifuge runs for longer times, even after the plateau region is lost. The calculations then become laborious unless complete equilibrium throughout the cell is attained, in which case there is a minimum of calculations. In order to demonstrate the procedures, a sample calculation is included. Whenever such long runs are contemplated it is advisable to fill the ultracentrifuge cell to only about one third of its normal capacity. This reduction in the height of the liquid column, in a radial direction, will materially reduce the length of time required for attainment of equilibrium. E x p e r i m e n t a l Procedure. About 0.8 ml. of solution of enzyme at a concentration between 0.5 and 1.0% is required for the measurements. 2 S. M, Klainer and G, Kegeles, J. Phys. Chem. 59, 952 (1955).

40


[2]

Knowledge of the exact concentration of the protein is not necessary despite the fact that co appears in equation 2. If the protein is available in a dried form devoid of impurities, then dissolution of the protein in the desired buffer will suffice; otherwise it is necessary to dialyze the protein solution against the buffer to equilibrate the two liquids. An additional small amount of the dialyzate or buffer used to dissolve the enzyme is also necessary. Buffers with ionic strengths of 0.1 are desired in order to minimize electrostatic effects caused by the sedimentation of charged macromolecules. Two and possibly three ultracentrifuge runs are required, and these will now be discussed in the order in which they are customarily performed. For the first, the sedimentation equilibrium run, a conventional 12-mm. ultracentrifuge cell is required. In general, 4 ° sector centerpieces are used, but if material is in short supply a 2° sector cell will work well and some saving of material can be effected by its use. Before filling the cell with the solution, 0.1 ml. of Dow-Corning No. 555 silicone fluid is introduced into the cell by means of a 0.25-ml. syringe with a No. 22 needle. The solution of protein is then added to the cell, which will hold about 0.6 ml. if the 4 ° centerpiece is used. The layer of silicone fluid serves a double purpose. First, it creates a base for the aqueous solution which has the contour of an arc drawn from the center of rotation. This eliminates the convective disturbances which result when the aqueous solution is placed directly in the cell, the bottom of which does not have the proper shape. Second, it provides a dense, transparent medium for the bottom so that the bottom of the aqueous phase is clearly visible on the photographic plate. Thickening of the line between the two phases during the run is a good indication of the presence of aggregated or denatured material. Without this dense, transparent layer in the bottom of the cell, the values of xb, cb, and (dc/dx)b are not so readily measurable because of the arrangement of the optical system in existing equipment and the deviation of light due to the concentration gradient at the bottom of the cell. The use of silicone fluids or any noninteracting dense liquid effects a considerable saving in calculations plus increased precision in the evaluation of cb and (dc/dx)b. After the cell is sealed in the conventional manner and aligned properly in the rotor, the rotor is fastened onto the coupling of the drive shaft and the temperature of the rotor is measured by means of the external thermocouple supplied with the instrument. The vacuum chamber is then closed, the vacuum pumps turned on, and after a vacuum of about 1.0 ~ is reached the centrifuge is accelerated to the desired speed. For satisfactory work the ultracentrifuge rotor should be operated at a speed such that the concentration at the bottom of the cell, at equilibrium, is about five times that at the meniscus. If the

[9.]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

41

molecular weight is known approximately, then a satisfactory speed in revolutions per minute can be calculated from [5 X 1 0 n / M ( 1 - Vp)]~, but it should be emphasized t h a t good results can be obtained even at speeds quite far removed from such calculated speeds. In the case of proteins varying in molecular weight from 10,000 to 200,000, speeds from 15,000 to 3000 r.p.m, will be ideal. As soon as the centrifuge rotor attains the desired operating speed, pictures are taken at various angles of the schlieren diaphragm. If the concentration of enzyme is in the neighborhood of 1%, then angles of 80 °, 70 °, and 60 ° are recommended. For more dilute solutions, 70 °, 60 °, and 50 ° are preferred; and for very dilute solutions an early picture at 40 ° is desirable. The optical system of the ultracentrifuge requires only the modification t h a t the bar in the standard instrument be replaced b y a phase plate. This replacement of the bar by a phase plate effects a considerable sharpening of the curve of concentration gradient versus distance on the photographic plates. As a consequence, values proportional to dc/dx can be obtained with high precision. The early photographs are obtained so as to have available a base line for later measurements. After experience with the method is gained, some of the initial photographs at various angles (optical sensitivity) can be omitted, and only those taken which correspond to the angles used later in the run. Pictures during the run should be taken every 8 to 16 minutes at angles of the schlieren diaphragm such that, at the meniscus and b o t t o m of the cell, the deviation of the curve above the base line is 2 to 3 ram. on the photographic plate. Deviations much greater than that are difficult to measure because of the steepness of the curves at the meniscus and b o t t o m of the cell; deviations much less than about 2 ram. are too small for precise measurements (unless plates are to be read on a microcomparator). Operation of the ultracentrifuge for 1 or 2 hours (five to ten pictures at 16-minute intervals) will yield sufficient data for the precise determination of molecular weights. If the material is unstable, however, the length of the run can be reduced. F r o m longer runs additional data can be obtained which will yield greater precision especially in the detection of inhomogeneity. After the requisite number of pictures are taken, the centrifuge rotor is decelerated, the vacuum chamber opened, and the temperature measured. The contents of the cell can be recovered by means of a syringe, and the cell should then be dismantled and cleaned with detergent solution, rinsed with distilled water, dried, and reassembled for subsequent runs. The second ultracentrifuge run performed in a synthetic boundary cell 3 provides the value of co, in equation 2, in arbitrary units, and it also 3 E. G. Pickels, W. F. Harrington, and H. K. Schachman, Proc. Natl. Acad. Sci. U.S. 38, 943 (1952).

42


[2]

furnishes a calibration of the optical system so that the values of (dc/dx) can be determined readily. Cells of various designs and sector angles (2 ° , 3° or 4 °) can be used for this run, but the optical path or centerpiece thickness must be the same as in the cell used for the sedimentation equilibrium run. Almost invariably this will be 12 ram. Another basic requirement is that there should be no mixing as the contents from the upper reservoir or cup empty into the sectoral cavity to form the boundary. About 0.3 ml. of the same enzyme solution used in the previous run is introduced into the sectoral cavity. This is the bottom solution. If the synthetic boundary cell manufactured by the Spinco Division, Beckman Instruments, Inc., is used, the cup is placed into the cell with an orientation such that the pinhole opening in the bottom of the cup is superimposed over the rubber plug which is in the hole drilled into the shoulder of the centerpiece. Then the trap door gasket is placed around the cup. By means of a 1-ml. pipet or a syringe, about 0.2 ml. of solvent is placed in the cup, and then the trap door is placed on the cell which is then ready for insertion into the rotor. If the cell designed by Kegeles4 is used, the solvent is placed into each of the upper reservoir holes before the window is placed on the centerpiece. Then the cell is assembled in the conventional way, and the enzyme solution (bottom solution) is introduced with a syringe as in the conventional cell. The cell is then sealed just as in the sedimentation equilibrium run. After the cell is placed in the rotor and the rotor coupled to the drive shaft, the vacuum chamber is closed without measurement of temperature which is unnecessary for this run. It is advisable, especially in the case of the synthetic boundary cell containing a cup, to start the rotor spinning at low speed (with about 3 to 6 amperes of driving current) without waiting for the chamber to be evacuated. This forces the solution to the bottom of the cell and minimizes possibilities of mixing of solution and solvent prior to the actual formation of the boundary. After the rotor is spun for about 5 minutes with the low driving current, the drive voltage is raised and maintained at a value necessary to provide about 6 to 8 amperes of drive current. This produces a slow acceleration of the rotor, leading to sharp, symmetrical boundaries. In the synthetic boundary cell with the cup, the contents of the cup begin to empty at about 5000 r.p.m., but slight variations in this break-through speed do occur, depending on the age of the rubber plug. The rotor should be accelerated slowly with the drive current raised to about 10 amperes. Frequently, the cup is not entirely emptied before reaching a speed equal to that utilized in the equilibrium run, and it is therefore advisable to use a higher speed setting so that the braking mechanism will not be brought into operation. 4 G. Kegeles, J. Am. Chem. Soc. 74, 5532 (1952).

[9.1


43

It is not necessary that this run be at the exact speed of the equilibrium run, since the run is used only to provide area measurements which serve as a calibration of the optical system and a measure of the concentration and refractive index increment of the solution. After the boundary has spread sufficiently so t ha t the entire curve is visible, which requires about 5 minutes for proteins, pictures are taken at different angles of the schlieren diaphragm corresponding to those used in the sedimentation equilibrium run. Caution should be exercised that the angles in the second run reproduce faithfully those of the first run. To avoid errors due to backlash in the remote control mechanism for adjusting the angle of the schlieren diaphragm, the desired angular setting should always be approached from the same direction, i.e., clockwise or counterclockwise. This run with the synthetic boundary cell should only co~lsume a shor~ time, sufficient for photographs at each angle used in the sedimentatioH equilibrium run. A period of 20 minutes allows the boundary to spread sufficiently by diffusion for accurate measurements of the area. The centrifuge can then be stopped and the sample recovered, if desired. These two runs will generally suffice for the determination of the molecular weight of enzymes, but for small molecules in salt solutions it is necessary to perform still a third run. Before describing this run, it should be pointed out that the second run (in the synthetic boundary cell) may be omitted if it is certain that the sedimenting material is homogeneous with respect to molecular weight. This will be illustrated later when the calculations are presented. The third run performed with the pure solvent provides the base line necessary for the calculations made from the sedimentation equilibrium run. If one is dealing with a coenzyme, a substrate, or even a protein of molecular weight much below 10,000, then the centrifugal force required for the equilibrium study will be sufficient to cause some redistribution of the buffer ions in the solution. Thus the curvature of the (dc/dx) versus x curve in the sedimentation equilibrium run would be due in part to changes in the salt concentration, and corrections must be made for this effect. At speeds of about 10,000 r.p.m, the sedimentation of the salt is so little and its diffusion so great that the salt concentration is uniform throughout the cell. Under those conditions the base line is obtained as already indicated in the discussion of the sedimentation equilibrimn run. If the centrifuge is run at higher speeds, then a separate run of the solvent (or dialyzate) is necessary, and this should be performed in a manner identical to that used for the solution. This requires not only that the speed and bar angles of the pictures be the same, but also that the same cell should be used with extra precautions to ensure filling to the same extent. Reproducing the volumes in the cell can be performed most conveniently by weighing

[9.]

ULTRACENTRIFUGATION, DIFFUSIOST, AND VISCOMETRY

45

run in the ultracentrifuge and in the calibration run with the synthetic boundary cell. The sample was ribonuclease at a concentration of 1%, and the centrifuge was operating at 11,150 r.p.m. Shortly after reaching speed, the first picture was taken in order to give a base line. The second, third and fourth pictures show how the picture changes during the first 3 hours. In order to show the pattern after the base line (plateau region) is lost, the last pictures were taken after 7 and 26 hours of operation. These pictures are photographic prints of the original centrifuge plates, with the white vertical bands at each side of the pictures corresponding to the holes in the reference cell; thus on each photograph there are two points whose distances from the axis of rotation are readily determined from the construction of the rotor and reference cell. The right-hand

~: ~

i

.... :i

FIG. 4. Ultracentrifuge patterns from a run in the synthetic boundary cell for the determination of the concentration required for a study during the approach to equilibrium.

edge of the black band on the left part of each photograph from most ultraeentrifuges lies at a distance 5.70 em. from the axis of rotation. This should be determined for each instrument. Acceleration of the rotor produces some stretching, and this line then corresponds to a distance 5.72 em. from the axis of rotation at speeds of 60,000 r.p.m. Once this position is known, all measurements on the photographic plate, or an enlargement of it, can be related directly to distances from the axis of rotation. The photographic plates are placed in an enlarger, emulsion side up, and the. curves such as those shown in Fig. 3 for ribonuclease are traced at a magnification of about fivefold. It has been found that tracing the curves on thin, unruled paper is most desirable. To facilitate tracing the necessary parts of the photograph, the vertical lines corresponding to the inner edges of the two reference cell holes, the air-solution meniscus, and the solution-silicone fluid interface are drawn first with the aid of a ruler. Then the horizontal lines through the reference holes, the lines through the air phase and the silicone solution, and finally that through the solution are drawn. In drawing the curved line corresponding to the concentration gradient near the meniscus and the bottom of the cell, it is advisable to draw those regions as if the vertical lines were not present, almost as if one could imagine those curves pro-

46


[2]

jected a slight distance beyond each of the vertical lines. This represents the most difficult part of the tracing, and it is especially difficult when low angles of the schlieren diaphragm are used so that the curves are very steep at the ends of the cell. It is for that reason that angles of 80 ° and 70 ° are preferred, as long as there is sufficient gradient to allow measurements. As the run proceeds, the curvature extends over a greater region in the cell and, as a consequence, tracing is somewhat easier. After the curves are drawn, the tracings are placed on a light box, and graph paper with centimeter and millimeter divisions is superimposed on the original tracings in such a manner that the vertical line corresponding to the air-solution meniscus lies on one of the heavy centimeter divisions. If possible, the flat part of the curve corresponding to the plateau region should also be placed under one of the heavy ruled divisions of the graph paper. In this way, the tracing can be transferred to graph paper. For experiments in which the centrifugal force is sufficiently low that no redistribution of electrolyte occurs and a separate ultracentrifuge run of the buffer can be neglected, it is sufficient to continue the straightline portion of the curve in both directions (toward top and bottom of the cell) until it intersects the vertical lines corresponding to the liquidair and solution-silicone fluid interfaces. This is illustrated in Fig. 5, which also shows some of the symbols used in the detailed calculations. For runs in which the speed is so great that salt redistribution occurs, then the corrections must be made as discussed later. At millimeter intervals starting with the meniscus, called n = 0, the vertical distances between the curved and the extrapolated straight-line portions are recorded. These distances, labeled z~, are proportional to dc/dx with the proportionality constant including optical constants of the apparatus, the concentration of the solute, the refractive index increment of the solute, and the magnification factor of the enlarger. In view of the fact that the same factors are involved in the last picture of Fig. 4, which is a representative photograph from the ultracentrifuge run with the synthetic boundary cell, their evaluation is unnecessary. Tables IA and IB show a sample calculation from the sedimentation equilibrium run. In column 1 are listed the values of n, i.e., the number of the interval. Column 2, labeled R~, gives the distance in centimeters from the reference mark to the interval in question. This distance is next converted into a real distance within the centrifuge cell, and the values are listed in column 3 under R,,/F, where F is the magnification factor relating distances in the cell to distances on the enlargement. This is readily evaluated, since the tracing includes the inner edges of the reference holes in the counterbalance cell. These edges are 1.60 cm. apart in the cell, and F is, therefore, RR/1.60. In the next column the distances R,,/F are con-

[2]

ULTRKCEINTTRIFUGATION~ DIFFUSION~ AND VISCOMETRY

47

verted to values of x,, which are the distances from the axis of rotation (xn = R , / F + 5.70). Column 5 contains the square of these values, and column 6 contains the z~ values. In column 7 are listed the values of .~.r,~z~, and the sum of these values, at the b o t t o m of the column, gives a

RTR

B

M

RBR

iJ-'F m"*l

x\\

Rb \\\

\\\

Rn In: tOO)

x\\

\\\

\\\

(n:lOOl /Z b

Z n (n:3)

\\\

Zn " ~ - ~

m~/

9'0 1001104

,\\\

I \\\

RTR RBR

,k\\ REE AIR REE SILICONE HOLE LAYER LAYER HOLE FIG. 5. Schematic diagram for illustrating the calculations of molecular weight from run during the approach to equilibrium. This corresponds to a print of the original ultracentrifuge pattern. The cross hatched regions and all black lines would be white on the original plates. Also on the original plate would be two horizontal lines corresponding to the image of the phase plate border at the reference holes. These are omitted in the above sketch but should be used in tracing of the curves and in the microcomparator since they furnish a good reference for all measurements in the vertical direction. measure of the integrals in equation 2. To obtain the exact value of the integral in the proper units, this sum must be multiplied by O.1/F, which is the horizontal width of each segment of the summation. The first part of the table contains the data obtained at the top of the cell and, according to equation 2, the value of the summation must be multiplied

48


[2]

b y 1 / x , , 2. T h i s n u m b e r is t h e n s u b t r a c t e d f r o m co t o g i v e cm. T a b l e I B g i v e s t h e d a t a f r o m t h e b o t t o m r e g i o n of t h e cell; t h e s u m m a t i o n h e r e is m u l t i p l i e d b y 1 / x b ~, a n d t h e r e s u l t a n t is a d d e d t o co t o g i v e cb. T h e v a l u e T A B L E IA SEDIMENTATION EQUILIBRIUM OF RIBONUCLEASE

Calculations at the Meniscus (Time = 38 minutes, F = 12.19, 17 = 0.709 cc./g., T = 298.9 °, ~2 = 1.3633 X 106) 1

2

3

4

5

n

R~ cm.

R, F cm.

x~ (5.70 + R , , / F ) cm.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0

0.181 0.189 0.197 0.205 0.213 0.222 0.230 0.238 0.246 0.254 0.263 0.271 0.279 0.287 0.295 0.304 0.312 0.320 0.328

5.881 5.889 5.897 5.905 5.913 5.922 5.930 5.938 5.946 5.954 5.963 5.971 5.979 5.987 5.995 6.004 6.012 6.020 6.028

6

7

x,, "~

z,~

x,,2z,~

cm. ~

em.

em. 8

1.41 1.30 1.20 1.08 0.99 0.88 0.77 0.66 0.57 0.50 0.42 0.35 0~28 0.19 0.13 0.10 0.08 0.04 0.02

48.766 45.084 41.730 37.659 34.614 30.862 27.077 23.272 20.152 17.725 14.934 12.479 10.009 6.810 4.672 3.605 2.892 1.450 0.727

34.586 34.680 34.775 34.869 34.964 35.070 35.165 35.260 35.355 35.450 35.557 35.653 35.748 35.844 35.940 36.048 36.144 36.240 36.337

384.519 nx

cm = co

= 18

x,,, ~ F

x"~zn;

"dx ,, =

1.41

n0~0

1 0.1 c~ = 1.2167 -- 34.58~ ' 19.5/1.6 (384.519) = 1.1255 Mr, _

RT

(1

--

F'p)~

(dc/dx),~

_

6.3636 • 104 ' (0 lz~)"-.---"-.~---" ~ l ) 1.41 (l

13,600

x,,,c,,,

of Co is o b t a i n e d f r o m a n e n l a r g e m e n t of t h e p a t t e r n o b t a i n e d i n t h e r u n u t i l i z i n g t h e s y n t h e t i ~ b o u n d a r y cell. T h e m a g n i f i c a t i o n of t h e e n l a r g e r m u s t , of c o u r s e , b e t h e s a m e i n b o t h t r a c i n g s . F o r t h e c o m p u t a t i o n of co t h e b a s e l i n e is d r a w n u n d e r t h e p e a k f r o m t h e s t r a i g h t - l i n e r e g i o n s o n

[2]

49

ULTRACENTRIFUGATION'j DIFFUSION~ AND VISCOMETR¥

e i t h e r side of t h e p e a k a n d t h e a r e a e v a l u a t e d . T h i s c a n b e d o n e e i t h e r b y m e a n s of a p l a n i m e t e r g i v i n g r e s u l t s in s q u a r e c e n t i m e t e r s or b y s u m m i n g t h e o r d i n a t e s , z~, a t 0.1-cm. i n t e r v a l s a n d t h e n m u l t i p l y i n g t h e r e s u l t a n t b y 0.1. I t h a s b e e n f o u n d t h a t t h e p l a n i m e t e r o p e r a t i o n is less tedious, but both methods give comparable accuracy. The value obtained TABLE IB SEDIMENTATION EQUILIBRUM OF RIBONUCLEASE

Calculations at the Cell Bottom 1

2

3

4

Rn

xn

n

R, cm.

F cm.

(5.70 -t- R,,/F) cm.

116 118 120 122 124 126 128 130 132 134 136 138 140 142

13.8 14.0 14.2 14.4 14.6 14.8 15.0 15.2 15.4 15.6 15.8 16.0 16.2 16.4

1.132 1.149 1.165 1.182 1.198 1.214 1.231 1.247 1.264 1.280 1.296 1.313 1.329 1.346

6.832 6.849 6.865 6.882 6.898 6.914 6.931 6.947 6.964 6.980 6.996 7.013 7.029 7.046

5

6

7

x,~2

z,~

x,,2z,,

cm. ~

cm.

cm?

46.676 4fi.909 47.128 47.362 47.582 47.803 48.039 48.261 48.497 48.720 48.944 49.182 49.407 49.646

0.01 0.03 0.06 0.09 0.12 0.18 0.26 0.42 0.60 0.82 1.05 1.35 1.68 2.06

0.467 1.407 2.828 4.263 5.710 8.605 12.490 20.270 29.098 39.950 51.391 66.396 83.004 102.271 428.150

nb= 142 cb = co + zb---1 2 dX.F ~

x,Yz.; ( d=e2)"b0 6 d x x

nx=ll6 1 0.2 cb = 1.2167 + 49.64-~6 " 19.5/1.6 (428.150) = 1.3582

Mb -

I?T

(1 - ~p)~2

(dc/dx)b _ 6.3636. 104. 2.06 Xbcb (7.046)(1.3582)

13,700

b y e i t h e r of t h e s e t w o m e t h o d s is t h e n m u l t i p l i e d b y 1 / F t o c o r r e c t for m a g n i f i c a t i o n in t h e x d i r e c t i o n , a n d t h e r e s u l t a n t v a l u e is Co. A t t h e b o t t o m of T a b l e s I A a n d I B a r e s h o w n t h e final c a l c u l a t i o n s . Since t h e t e m p e r a t u r e of t h e , r o t o r is k n o w n , i t is c o n v e r t e d i n t o a b s o l u t e t e m p e r a ture, a n d t h e m o l e c u l a r w e i g h t is c a l c u l a t e d f r o m e q u a t i o n 1. W h e n one is d e a l i n g w i t h s m a l l e r m o l e c u l e s a n d u t i l i z i n g h i g h e r c e n t r i f u g a l fields, t h e s o l v e n t is r u n s e p a r a t e l y a n d p i c t u r e s a t t i m e s a n d

[2]

51

ULTRACENTRIFUGATION~ DIFFUSION~ AND VISCOMETRY

T A B L E II SEDIMENTATION EQUILIBRIUM OF RIBONUCLEASE Calculations throughout the entire cell after loss of plateau region* (Time = 63 hours, F = 1.882, V = 0.0709 cc./g., 7' = 296 °, o~2 = 1.3633 × 10 ~) 1

2

n

n ~°

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 100 104 105.7

3

5

4

R,~"°

x,~~

cm.

cm.

6

(z,~2)~'o cm.

2

z,, ~ ° ell1.

2 6 10 14

1.447 1.487 1.527 1.567

6.469 6.490 6.512 61533

41.8415 42.1200 42.3995 42.6735

0.0774 0.0794 0.0817 0.0838

18 22 26 30 34 38 42 46 50 54 58 62 66 70 74 78 82 86 90 94 98 102 104.8

1.607 1.647 1.687 1.727 1.767 1.807 1.847 1.887 1.927 1.967 2.007 2.047 2.087 2.127 2.167 2.207 2.247 2.287 2.327 2.367 2.407 2.447 2.475

6.554 6.576 6.597 6.618 6.639 6.661 6.682 6.703 6.724 6.746 6.767 6.788 6.809 6.831 6.852 6.873 6.894 6.916 6.937 6.958 6.979 7.001 7.016

42.9550 43.2370 43.5135 43.7910 44.0765 44.3625 44.6425 44.9235 45.2120 45.5020 45.7860 46.0705 46.3625 46.6555 46.9430 47.2315 47.5275 47.8240 48.1150 48.4070 48.7065 49.0070 49.2240

0.0861 0.0889 0.092l 0.0957 0.0997 0.1039 0.1080 0.1121 0.1164 0.1209 0.1259 0.1307 0.1354 0.1410 0.1471 0.1527 0.1584 0.1646 0.1706 0.1771 0.1836 0.1901 0.1959

c,a =

Co -~" Xb 2

-- X,n~ L J x ~

1

Ff

dx

xz dc

7

8

c~ cm.

x 2

cHI.

9

z,,

10 I dc

xc dx

cm.

0.05158 0.05313 0.05491

6.458 6.479 6.501

0.0766 0.230 0.0782 0.227 0.0806 0.226

0.05665 0.05843 0.06026 0.06215 0.06411 0.06614 0.06826 0.07047 0.07277 0.07515 0.07762 0.08019 0.08287 0.0~565 0.08853 0.09153 0.09465 0.09790 0.1013 0.1048 0.1084 0.1122 0.1161 0.1201 0.1219

6.522 6.543 6.565 6.586 6.607 6.628 6.650 6.671 6.692 6.713 6.735 6.756 6.777 6.798 6.820 6.841 6.862 6.883 6.905 6.926 6.947 6.968 6.990 7.011 7.020

0.0828 0.0847 0.0874 0.0904 0.0938 0.0976 0.1018 0.1060 0.1100 0.1141 0.1186 0.1232 0.1286 0.1328 0.1380 0.1440 0.1502 0.1552 0.1616 0.1676 0.1736 0.1806 0.1866 0.1936 0.1981

-zb2

x~ dc

fx

m dxx

0.223 0.222 0.221 0.221 0.222 0.223 0.224 0.226 0.226 0.226 0.227 0.227 0.229 0.228 0.229 0.230 0.231 0.230 0.231 0.231 0.231 0.231 0.230 0.230 0.231

dx]

n = 105.7

fxz~ dC dx = dx m dxx ~-

~ n=O

z~ -- (2.126 × 10-2)(3.223) + (0.925 × 10-2)(0.1959) = 7.033 X 10 -2

n = 105.7

~xb m

dc _ dx x ~ --,- clx =

~

xn2z,, = (2.126 X 10-2)(148.19) -~ (0.925 X 10-2)(9.641) = 3.240 (3.240 -- (49.280)(7.033 X 10 -2) c,, = 0.08148 -b 49.280 -- 41.706 = 0.05158 * The centrifuge plate for this calculation was measured with a microcomparator, and the n intervals are in t e n t h s of millimeters. ax

n=O

52


indeed a plot of this data as log ( 1 / x ) ( d c / d x ) pletely straight.

[2]

versus x 2 is almost com-

Sedimentation Velocity I n t r o d u c t i o n . The primary function of this method is the determina* tion of the sedimentation rate of molecules, but it should be stressed that another powerful application of the method lies in its ability to furnish, often in a simple experiment of short duration, information about the purity of the material under investigation. In this latter regard, sedimentation velocity studies are like electrophoresis experiments, since each of the techniques causes the partial separation of different types of molecules, depending on their physical chemical properties. As a result several boundaries may be observed, or one broad boundary which clearly indicates inhomogeneity of the particles. Thus the sedimentation velocity method can indicate, within limits, of course, whether one or more molecular species is present in solution, provided these different molecules show variations in molecular size and shape. With sufficient research, precise information can be obtained about the amount of each species present in a solution, and thus the ultracentrifuge is an extremely valuable guide in studies aimed at the isolation and purification of an enzyme. Solute molecules in a solvent of different density are caused to move under the influence of a high centrifugal field, and the rate at which they move is a function both of the molecular weight of the solute and of the frictional resistance which the molecules or particles experience as they move through the solvent. Sedimentation velocity measurements of themselves do not yield molecular weights without recourse to other data, such as diffusion coefficients. The equation describing the relationship between the sedimentation rate and the properties of the molecule is

M(1 s -

Nf

Cp)

(4)

where s is the sedimentation coefficient, M is the anhydrous molecular weight of the sedimenting substance, l? is the partial specific volume of the solute, p is the density of the solution, N is Avogadro's number, 6.02 X 1038, and f is the frictional factor which is a function of the size, shape, and hydration of the sedimenting molecules. Movement of individual molecules is followed by examination of the boundary between solvent and solution, as illustrated in Fig. 1. For most purposes, if the boundary is symmetrical, movement of the maximum ordinate of the peak is a sufficiently accurate indicator of the movement of individual molecules. Actually it is the second moment of the gradient curve which

[2]


53

gives a true picture of the movement of the molecules, but calculations of this second moment are very laborious and often unnecessary. The sedimentation coefficient is defined as the velocity of the sedimenting molecules per unit field, as shown in equation 5, s

-

1 dx ~ x dt

(5)

where x is the distance of the boundary in centimeters from the axis of rotation, t is the time in seconds, and ~ is the angular velocity in radians per second. Generally, sedimentation coefficients lie in the range of 0.25 to 500 X 10-13 cm./sec./dyne/g., or 0.25 to 500 X 10-'a second, and the unit 1 X 10-'3 second has been termed 1 S. where S. is the Svedberg. Determination of Sedimentation Coe~icients. For proteins of molecular weight below about 250,000 the ultracentrifuge should be operated at the top speed of 59,780 r.p.m. ; and for larger molecules the speed should be adjusted so that the boundary moves across the cell in a period of several hours. Photographs are taken at periodic intervals, depending oil the rate of movement of the boundary. It is always advisable to examine the pattern through the viewing screen during the acceleration of the rotor in case material present in the solution sediments across the cell before the rotor attains the desired operating speed. If the substance under investigation has a sedimentation coefficient below 3 S., the use of the synthetic boundary cell is recommended. To damp out charge effects with proteins, ultracentrifuge runs are generally made in salt solutions at an ionic strength of 0.1 or greater. The temperature before and after the run is recorded if the external contact thermocouple is still in use; if the new temperature indicator is available, then the temperature can be recorded during the run. It should be stressed, for the former case, that the temperature of the rotor during a run at 60,000 r.p.m, is about 0.8 ° less than the average of the temperatures obtained from the contact thermocouple. Photographs can be read in a variety of ways, depending on the accuracy desired. For routine work with materials which sediment as relatively sharp boundaries, we have employed a simple light box containing a calibration plate which permits direct reading of boundary positions in centimeters from the axis of rotation. The calibration plate is made by photographing the ruled glass disk supplied by the manufacturer for the determination of the magnification factor of the different lenses. The lines on the resulting photograph will be from 1.8 to 2.2 mm. apart, depending on the particular instrument. With this plate fastened onto the frosted-glass top of a light box, a grid is available whose lines

[9.]


55

sedimentation coefficients for each time interval and correct them indep e n d e n t l y for temperature, assuming a linear change in temperature. For m a x i m u m use of the data, the averaging of the resulting sedimentation coefficients should be performed in the manner suggested b y Kegeles and G u t t e r ! Alternatively, the times between photographs can be corrected for the variation of the viscosity of water with temperature, as suggested b y Oncley, 9 and then log x can be plotted against the corrected times. log x

x (cml~

~t

Temo:23.7" c,( 20) :0 0.82600

(-~o)

6.700

=1.0065, ~] =0.709CC

solv

/ 0.81800

o/° D/" S = 2.503d

Iogx/dt (60)( ,,02)

6.580 =

(slope)(2.3 0 3 / 6 0 [( 2"rr)(, ,j59780 . . ~ - - - ~]2

)

S = 1.98 x 10"13sec. = 1.98 S 6.520 t , I t o t6 52 Time in m{nutes FIG. 6. D e t e r m i n a t i o n of sedimentation coefficient of ribonuelease from a plot of log x (x is the distance of b o u n d a r y to axis of rotation) versus t (t is the time in minutes). 0.81400 .....

,

Sedimentation coefficients are generally reported as s20.~, the value the material would have in a solvent with the density and viscosity of water at 20 ° . Corrections of the observed sedimentation coefficient, sob,., to this standard state are made according to the equation

where (nt/n20) is the principal correction factor corresponding to the viscosity of water at t ° relative to t h a t at 20 ° , (n/no) is the relative viscosity of the solvent to t h a t of water, and p20.~ and pt are the densities of water at 20 ° and the solvent at t °, respectively. The term pt can be calculated readily from (P/Po)Pt,w. I t is implicitly assumed t h a t V in the reference solvent is the same as in the actual solvent used in the experi9 j . L. Oncley, Ann. N. Y. Acad. ~ci. 41, 121 (1941).

56


[9.]

ment. For the experimental d a t a presented in Fig. 6, calculation of s20.w according to equation 6 leads to the result 820.~ = (1.98)(0.9147)(1.024)(1.0139) = 1.88 S. We have used p, as the density of the solvent, although some workers recommend use of the density of the solution. I t is clear t h a t the density of the solution is correct for sedimentation equilibrium; but there is no corresponding theoretical basis for the density t e r m in sedimentation velocity. In a n y case the difference between solution and solvent density is generally very small, and further, data should be extrapolated to infinite dilution so t h a t no significant error can result from the use of either density. For slowly sedimenting materials in high salt concentrations, formation of the b o u n d a r y between solvent and solution near the center of the cell is especially useful, since it virtually eliminates the need for a reference run of solvent alone. This run is ordinarily necessary as a " b a s e l i n e " run, since the sedimentation of salts causes refractive index gradients at the meniscus and b o t t o m of the cell. The observed p a t t e r n would then be the sum of t h a t due to the material of interest plus t h a t due to the salt. If a conventional cell is used, then a reference run of solvent is made under identical conditions, and the patterns from this run are subtracted from the corresponding patterns in the run of the solution. When the synthetic b o u n d a r y cell is used, the rate of acceleration of the rotor should not be so great as to cause convection which leads to poor boundaries. After the cup containing the solvent is completely emptied, the drive current should be adjusted to about 10 amperes until the operating speed is achieved. For m a n y substances the sedimentation coefficient depends on concentration, and runs at different concentrations are necessary to enable extrapolation of the data to infinite dilution. T h e data can be plotted in a variety of ways, but it is preferable to plot 1Is versus concentration. If viscosity data for the solutions at different concentrations are available, then a plot of s versus c and (v/~o)s versus c (c is concentration) on the same scale provides two curves which should extrapolate to the same value. This greatly facilitates the determination of s at infinite dilution. Measurement of Concentration. The area under the curves shown in Fig. 1 gives a measure of the concentration of the sedimenting material, and the m e t h o d of evaluating the area from synthetic b o u n d a r y cell runs has already been discussed. With a microcomparator and patterns obtained with the phase plate as the schlieren diaphragm these areas can be reproduced with a precision of b e t t e r t h a n 1%. Areas from runs at high speeds in a conventional cell cannot be obtained so accurately. For the

[2]

ULTR&CENTRIFUGATION, DIFFUSION, AND VISCOMETRY

57

latter, it is essential that a "base-line" run be made with pure solvent. This corrects not only for the gradients of refractive index due to sedimentation of salts but also for cell distortion. If possible, this "base-line" run should be made without taking the cell apart, and the volume of solvent in the cell should be the same as in the run with solution. The plastic, double sector ultracentrifuge cell permits the superposition on the photographic plate of the pattern due to the solution and the solvent. With this cell both solvent and solution are run simultaneously and the area of a sedimenting boundary can be determined with great precision since the superposition of the base line on the pattern of the moving boundary eliminates the uncertainties in construction of the base line from separate ultracentrifuge runs. Small amounts of both more rapidly and more slowly sedimenting material can be readily detected by the deviation of the solvent pattern from that of the solution in the region of the cell in front of or behind the principal boundary. In using this cell special efforts should be made to fill both halves of the cell equally so that the menisci are at the same distance from the axis of rotation. The syringe, 0.5 ml., which is first used for transferring the solution into the cell can then be rinsed and used for the solvent. Before using the cell for concentration determinations it should be tested by running solvent in both halves to be certain that cell distortion does not preclude superposition of the base lines from each sector. Since there is some loss of contrast on the photographic plates due to overlapping of the patterns from the two halves of the cell, high contrast photographic plates, such as the 5~etallographic plates, should be used. Whenever concentration determinations are to be made, either on purified components or in the analysis of mixtures, it is recommended that the double sector cell be employed. The solvent and solution patterns should be superimposed either from tracings of enlarged patterns (5X) made with a photographic enlarger or arithmetically if the areas are obtained by summing ordinates measured with a two-dimensional comparator. For this superposition of the patterns, use is made of the images corresponding to the reference holes in the counterbalance. After the base line is drawn under the peak, the area can be measured. The concentration of sedimenting material ill per cent, co, is obtained from the equation Atan~

(x) 2

co = a b m x m ~ A n E ~

where A is the measured area, a is the cell thickness in centimeters (along the optical path), b is the optical lever arm in centimeters (this value is supplied by the manufacturer and is equal to the focal length of the upper

58


[9.]

collimating lens or the distance from this lens to the plane of the schlieren diaphragm), Ois the angle of the schlieren diaphragm as read directly on the ultracentrifuge, mx and mc are the magnification factors of the camera and cylindrical lenses, respectively (these are evaluated according to the manufacturer's instructions with the aid of the ruled glass disk supplied with the ultracentrifuge), and E is the magnification of the enlarger (this is unity if a comparator is used). The term (X/Xo) 2, where x0 is the position of the boundary at zero time and x is the position at a time corresponding to the pattern subject to analysis, is the correction factor to account for the dilution of the contents of the plateau region as a result of the sector shape of the cell and the variation of centrifugal field with distance. To obtain the concentration in weight per cent, An, which is the specific refractive increment, must also correspond to weight per cent. It is equal to the difference in refractive index between a solvent and a solution at a concentration of 1%. For many proteins, An is about 0.00186. If a pure protein is available, the synthetic boundary cell can be used to calibrate the optical system by forming a boundary between solvent and a solution of known concentration. Also, there is available from the manufacturer a special calibration cell which is very useful for checking or determining the optical constants of the ultracentrifuge. Determination of Homogeneity. The minimum requirement for homogeneity of a given sedimenting substance is that the material sediment across the cell as a single, symmetrical boundary without too much broadening of the boundary. This is, of course, a qualitative statement; detailed quantitative measurements can be and, indeed, must be made if homogeneity is to be demonstrated. It should be emphasized that it is extremely dangerous and often incorrect to conclude that a material is homogeneous just because the boundary remains symmetrical and sharp. Frequently boundaries are artificially sharp because of the dependence of sedimentation coefficient on concentration, and tests for homogeneity should be made on dilute solutions and, if possible, as a function of concentration to enable extrapolation to infinite dilution. This is also important in the analyses of mixtures in terms of the relative concentrations of the different components, because of the ultracentrifuge anomaly which causes the slower component to appear to be present in greater concentration than the true concentration. 1°,11 For materials with a sedimentation coefficient below about 1 S., the boundary cannot traverse the whole cell becatlse of the present limitations in rotor speed. Thus the great potential of the ultracentrifuge in resolving different molecular 10 j. p. Johnston and A. G. Peston, Trans. Faraday Soc. 42, 789 (1946). 11 W. F. Harrington and H. K. Schachman, J. Am. Chem. Soc. 75, 3533 (1953).

60


[2]

employing conventional optical techniques, and recourse is made to the so-called analytical method which is based on the determination of the quantity of substance sedimenting across a fixed plane in a centrifuge cell. This method requires a special ultracentrifuge cell called the separation or partition cell which is so constructed as to effect a separation of the contents of the cell into two parts at the conclusion of the run. The amount sedimenting across the partition is determined by the difference between the total amount of material originally in the cell and that which is left above (or centripetal to) the separating plate at the conclusion of the ultracentrifuge experiment. Also required for the method is, of course, a specific and sensitive analysis for the material in question. This may be a bioassay, a chemical analysis, or a physical-chemical measurement such as radioactivity or ultraviolet absorption. As will be seen shortly, the analyses need be only relative so that measurement at the conclusion of the run of the number of units of activity in the upper solution relative to the number in the original solution gives a value of the sedimentation coefficient. The separation cell affords still another important advantage to biochemists interested in a specific biologically active substance. Frequently in the isolation and purification of a given substance two or more boundaries are observed optically in the ultracentrifuge, and it is important to determine which, if either of these corresponds to the material of interest. As long as there is a specific biological test available, the sedimentation coefficient of the substance can be determined even in the presence of much larger quantities of other components. Even when only one component is observed optically and the material is thought to be purified, it may be profitable to measure the sedimentation rate by activity measurements to see if there is a correlation between the physical property of the bulk constituent and the component with the biological activity. For some time there has been available only the partition cell of Tiselius et al., 12 but very recently a cell designed from quite a different point of view has been introduced by Yphantis and Waugh. 1~ Both of these cells are now available commercially from the Spinco Division, Beckman Instruments Inc., Belmont, California, and they are both made by modification of standard 12-mm. 4 ° centerpieces. In the cell of the type proposed by Tiselius et al., slots are made in the walls of the centerpiece at a position approximately two-thirds of the distance from the top. Into these slots fits a thin perforated plate containing about twenty three holes drilled through it, each hole being about 0.28 mm. in 1, A. Tiselius, K. O. Pedersen, and T. Svedberg, Nature 140, 848 (1937). 13 D. A. Yphantis and D. F. Waugh, J. Phys. Chem. 60, 623, 630 (1956).

[2]

ULTRACENTRIFUGATION, DIFF~ISION, AND VISCOMETRY

(}l

diameter. In operation, a piece of Whatman No. 50 filter paper, 12 mm. long and about 0.5 mm. narrower than the porous plate, is overlaid on the plate and they are inserted into the centerpiece. It is implicitly assumed that neither the filter paper nor the perforated plate interferes with the process of sedimentation and, further, that the sedimentation process is convection-free as in the standard ultracentrifuge cells. The function of the barrier is to prevent mixing of the upper and lower solutions at the conclusion of the experiment. In the cell designed by Yphantis and Waugh the separating plate is actually movable and rests on two thin synthetic rubber strips which serve as a spring. Since the plate and rubber are more dense than the liquids customarily employed for analysis, the plate sinks to the bottom of the cell during acceleration of the rotor and the cell simulates a conventional ultracentrifuge cell. On deceleration, at 2000 to 3000 r.p.m., the centrifugal force on the plate is no longer sufficient to counteract the lifting action of the rubber springs, and the plate rises very slowly until it rests against specially constructed stops in the centerpiece. If the cell and movable partition are made and assembled correctly so that there is a precise fit when the plate is in the rest position, the solutions in the two compartments can be effectively separated from each other with little risk of mixing. Surprising as it may seem, the slow movement of the partition from the bottom of the cell to its rest position causes little or no generalized stirring of the contents of the cell. This has been the subject of careful experimentation by Yphantis and Waugh, 13 who, in experiments in which the rotor was decelerated and accelerated, showed that the movement of the partition back and forth through a boundary caused only a little distortion of the boundary. This is true even with solid partitions, so that the liquid must flow around the partition when it is Caused to move in the cell. The success of this technique depends, as in the case of the synthetic boundary cell, on the stability resulting from magnification of small density gradients under the influence of a centrifugal field. Whereas stirring of stratified layers of solution differing in density by only 0.001 g./ml, will occur readily at one gravity, such stirring will not take place in a centrifugal field in which the force on the liquid is many hundreds of times that of gravity. Static experiments with a dye indicator present in the centrifugal compartment and absent in the centripetal part showed that there was little mixing of the liquids when the cell was handled in a fashion simulating a typical ultracentrifuge experiment. Thus the two separation cells functioning in quite different ways achieve, to more or less the desired extent, the same purpose. There is not as yet sufficient experience with the movable partition cell for a detailed comparison with the older cell, but it appears that the

62

TECHNIQUES FOR CHARACTERIZATION OF PROTEIN'S

[2]

newer cell will function better for proteins and low molecular weight materials, whereas the partition cell of Tiselius et al. does interfere with sedimentation and there is a partial disintegration of the sedimenting boundary as it approaches the partition from above. Furthermore, a second boundary forms just centrifugal to the barrier. This probably results from a temporary pile-up of some sedimenting material on the filter paper. Whatever the cause, convection does occur and this may lead to erroneous results. Epstein and Lauffer14 circumvented this effect of the barrier by adding a small amount of sucrose to the solution below the barrier, thereby causing this solution, even if there is some removal of solute by sedimentation, to be heavier than the solution centripetal to the partition. In this manner the disturbance in the upper liquid can be effectively eliminated. In terms of operating procedure there are only minor differences, depending on which cell is used. With the old cell, complete filling in one step is almost impossible. The top portion of the cell is filled by means of a 1-ml. syringe equipped with a No. 22 stainless steel needle the tip of which is ground fiat. This liquid is forced into the bottom compartment by centrifuging at a speed of about 2000 to 3000 r.p.m, for about 1 minute. Then the upper compartment can be filled. This intermediate step can be eliminated in the use of the Yphantis-Waugh cell, since the tip of the needle can be used to depress the platform during the filling of the entire cell. It is a good idea to weigh the filled cell before the ultracentrifuge run and then again at the conclusion of the run, before and after removing the contents of the upper compartment. This is useful, since small leaks in the cell can be detected, and also the weight of the sample removed is determined readily. For this weighing, a small plastic block with a hemicylindrical cavity in the top serves as a stand for proper orientation of the cell with the filling hole pointing upward. Unlike most ultracentrifuge studies, the exact time of the run is important, and it is therefore necessary to record the speed of the rotor at different times on the way up to speed and then at the conclusion of the run during the deceleration period. Generally it will be found that acceleration of the rotor under the influence of 12 to 14 amperes will be linear and, to a very good approximation, the time of acceleration to the desired speed is equivalent to one-third of that period at the final operating speed. Thus one-third of the acceleration and deceleration times must be added to the time that the rotor is at speed for the computation of the sedimentation time. Special precautions are needed in the deceleration of the rotor, since rapid deceleration leads to convective disturbances, as evidenced by the formation of hypersharp spurious boundaries. Yphantis 14 H. T. Epstein and M. A. Lauffer, Arch. Biochem. and Biophys. 35, 371 (1952).

64


[2]

Several runs should be made for different periods of time such that

ct/co varies from about 0.8 to about 0.1. For most enzymes, runs of a duration sufficient to produce a lowering of the average concentration in the upper solution to about 10% of the original will take only 2 or 3 hours at top speed, and the equation presented above is valid. If the molecules being studied are smaller, say about 2000 in molecular weight, then the plateau region will vanish in a 3-hour period and equation 9 is no longer applicable. Yphantis and Waugh 13 have shown, however, that it is possible to obtain both sedimentation coefficients and diffusion coefficients from experiments in which the plateau region is lost and the reader is directed to their papers for this important new development. Application of Absorption Optics to Ultracentrifugation. Many substances of interest to enzymologists have such high extinction coefficients either in a particular region of the visible spectrum or in the ultraviolet that the sedimentation of these materials is more readily examined by absorption techniques than by customary schlieren methods. For example, the amount of ultraviolet light transmitted through a solution of deoxyribonucleic acid at a concentration of 0.001% is only about half the light transmission of the solvent. This difference is readily recorded as variations in the degree of blackening of photographic plates. In contrast, the difference in refractive index between such a solution and the solvent is so small as to be virtually indetectahle by present schlieren methods. Thus ultracentrifugal analysis of materials at such great dilutions is dependent on the availability of absorption methods. Also many substances, the main features of whose absorption spectra are known, such as the cytochromes or hemoglobin, can be studied specifically, even in crude mixtures, by the use of light of the appropriate wavelength. Therefore, it seems likely that there will be a return to the absorption method originally developed and employed by Svedberg and his co-workers. 15 Techniques at present are still in a state of flux, and only very general comments will be made here. A special ultraviolet absorption optical system is now available commercially and can be incorporated in existing ultracentrifuges. This employs light of 254 m~ and can be used for materials which, at that wavelength, have an optical density of 0.2 (in a 1-cm. cuvette). For absorption work at wavelengths in the visible, the schlieren system can be used as an absorption system through the use of appropriate filters to isolate the desired wavelengths. Interference filters are very useful for this purpose. If the absorption method is to give accurate information concerning 15T. Svedberg and K. O. Pedersen, " T h e Ultracentrifuge," Oxford University Press, New York, 1940.

[9.]

ULTRACENTRIFUGATION', DIFFUSION, AN'D VISCOMETRY

65

the change in concentration of the absorbing material as a function of the position in the ultracentrifuge cell, the blackening of the photographic plate must give a faithful record of the intensity of light striking it. Thus, the blackening of the plate must be inversely related to the optical density of the solution in the cell. Further, this blackening must be measured in some type of photodensitometer. For this purpose use is made of the modified Analytrol, manufactured by the Spinco Division, Beckman Instruments, Inc. With suitable precautions and some calculations this instrument in conjunction with the photographic plates gives a curve relating concentration of the absorbing substance as a function of distance from the axis of rotation. This will be recognized as the integral of the patterns given by schlieren methods. From these curves at different times, sedimentation coefficients are readily calculated. Also analysis of the boundary spreading to determine the diffusion coefficient and polydispersity of the sedimenting material is readily achieved by the use of absorption patterns. For strongly absorbing materials like cytochromes or nucleic acids, a detailed analysis can be performed, with the aid of the synthetic boundary cell, on only about 10 ~ of material.

Partial Specific Volume The use of ultracentrifuge data from either sedimentation equilibrium or sedimentation velocity studies requires the evaluation of the term (1 - Vp). Since the partial specific volume, V, of proteins is generally between 0.70 and 0.75 ml./g., and the density, p, is normally close to unity, errors in the determination of 17 are essentially multiplied by a factor of 3 in the final calculation of molecular weights. As a consequence it is imperative that accurate values of V be obtained, and it is just in this area that detailed experimental studies are lacking. The classical method for determining partial specific volumes involves density measurements both on solutions of known concentrations and oll the solvent. From each solution density, p, paired with the density of the solvent, p0, an apparent specific volume is calculated. It has been found almost invariably for proteins that the apparent specific volume is independent of concentration of the protein, and thus the average value for different concentrations is equal to the partial specific volume. In addition to the classical method of determining partial specific volumes there are two other methods which give a reasonably reliable value for 17, and a brief discussion of them will follow a treatment of the more popular method. There are a variety of procedures for determining densities of solutions, and all of them have been used at one time or another. The most

[9.]

ULTRACENTRIFUGATION~ DIFFUSION, AND VISCOMETRY

67

pycnometers of only 2-ml. capacity can be used although greater precision is attainable with larger volumes. Greater precision in measuring the density of solutions can be obtained either through the use of the density gradient column of Linderstr0mLang and Lanz 16 or by means of the magnetic float method developed by MacInnes et al. 17 The former method requires only very small volumes of solution, whereas the latter requires volumes that in many cases are almost prohibitively large. For the use of the magnetic float method which requires apparatus that is not as yet commercially available, the reader should consult the paper by MacInnes and co-workers, is Density measurements with the gradient column require only simple equipment consisting of a constant-temperature bath, a column of liquid in which the density increases, preferably in a linear manner, from top to bottom and in which aqueous solutions are insoluble, a capillary pipet for the delivery of small drops of protein solution into the column, a method for the precise measurement of the height of the drops in the column, and, finally, a set of reference liquids whose densities are accurately known. In the use of the gradient column, mixtures of c.p. bromobenzene and rectified white kerosene are employed. Two mixtures of these liquids are prepared so that they differ in density by about 0.02 g./ml., and they encompass the density range of the solvent and protein solutions. Each of the bromobenzene-kerosene solutions must be saturated with water by shaking with a dilute solution (about 0.2 M) of potassium bromide fo}lowed by storage overnight. This saturation of the liquids for the gradient column is necessary so that there will be no evaporation of water from the drops of protein solution; otherwise, there would be an increase in concentration of protein in the drops, leading to erroneous results. After separation of the individual solutions from the potassium bromide solution, the gradient column is prepared by the method of Linderstr0m-Lang and Lanz. 16 This involves careful layering of the lighter solution over the heavier one in a long cylindrical column held in the well-controlled constant-temperature bath. The two solutions are then mixed partially by gentle stirring so as to produce a gradual change in composition, and hence in density, from the top to the bottom of the column. The stirring operation is an art, and it takes considerable practice before this operation can be performed in a satisfactory manner. 1~K. LinderstrOm-Langand H. Lanz, Compt. rend. trav. lab. Carlsberg S$r. chim. 21, 315 (1938). 17D. A. MacInnes, M. O. Dayhoff, and B. R. Ray, Rev. Sci. Instr. 9.2, 642 (1951). is M. O. Dayhoff, G. E. Perlmann, and D. A. MacInnes, J. Ant. Chem. Soc. 74, 2515 (1952).

68

TECHNIQUES FOR CHARACTERIZ.~TION OF PROTEINS

[2]

Too little stirring leads to a density gradient of only limited extent with uniform regions above and below the actual density gradient, whereas too much stirring produces a column equally unsatisfactory, since the column is almost uniform in density. The density gradient column is allowed to stand undisturbed for a period of 24 hours so that diffusion can smooth out any abrupt density gradients. Then the appropriate standards made from solutions of analytical-grade recrystallized potassium chloride are added to check the gradient column. If the positions of the drops show that the density gradient is satisfactory, then the column is ready for use. Drops are added by means of a capillary pipet delivering about 0.2 mm. 3, and there should be sufficient standard drops of known density to bracket closely the solutions whose densities are to be determined. Several drops can be measured along with several standards almost simultaneously, and the drops should be added with the most dense being first, then the next dense, and so on. The positions of the drops after they reach equilibrium positions, which requires only about 15 minutes, are most readily determined by a cathetometer, although a scale mounted alongside of the column may suffice, depending on the accuracy desired. The density of a given unknown solution, d, is calculated by interpolation according to the equation h - h, (d2 - dl) d = dl T h~ hi

(10)

where h, hi, and h~ are, respectively, the positions measured from some reference mark near the top of the column to the center of the unknown drop, the lower density standard, dl, and the higher density standard, d2. After each experiment the drops are removed from the column which is then ready for use after several hours of standing. Removal of the drops is achieved either by touching the drops with a thin glass rod onto which the drops will adhere so that they can be transferred to the wall of the column at the top of the liquid or by pouring a small amount of sea sand (obtainable from Merck) through the column. Density measurements can also be achieved by hydrostatic weighing in a commercial apparatus such as a Westphal balance or in a modified analytical balance. Accurate densities can also be achieved by differential density measurements using a pair of similar pycnometers one of which is filled with solution and the other with solvent, and then the solutions are exchanged. 19 After the densities of the solutions and solvent are determined, the 19 For a review see chapter on determination of density by N. Bauer, in "Physical Methods of Organic C h e m i s t r y " (Weissberger, ed.), 2nd ed., Part I, p. 253. Interscience Publishers, New York, 1949.

[9"]


69

apparent specific volume is determined by substituting in the equations Va,p. = 100/d - (100 - n)/do

(11a)

n

or 1

V~,,. = d0

1 (~odO) x

(llb)

where d and do are the densities of solution and solvent, respectively, n is the percentage concentration of solute by weight, and x is the concentration of protein in grams per milliliter of solution. The concentration (tan be determined in a variety of ways. If the protein is in distilled water, then a weighed aliquot is lyophilized followed by drying in vacuo over P~05 at about 110° until constant weight is achieved. Alternatively, the dry weight can be determined by micro-Kjeldahl analysis for nitrogen if the conversion factor relating nitrogen to protein is known. For proteins which must be maintained in a buffer, it is important to perform a dialysis and then the composition can be determined by the difference in dry weights between solution and dialyzate. Frequently the amount of protein is so small as to preclude measurements both of concentration and of density, and recourse can be made to a method employing the ultracentrifuge. As shown in equation 4, the sedimentation coefficient is proportional to (1 - ITp). Thus determination of the density of a solution in which the sedimentation coefficient is equal to zero gives a measure of l?. Accordingly the sedimentation rate of the protein is measured in a series of solutions of increasing density and the resulting data plotted in a manner to enable extrapolation to the value of p corresponding to zero sedimentation rate. This value for the density of the solution is equal to l/i?. Since the equation is restricted to two component systems, it is not valid to employ any material such as sucrose to increase the density of the solution. It appears, however, that mixtures of D~O and H20 act as a one-component solvent and therefore they can be used for these experiments. Unfortunately, D20 solutions are not sufficiently dense to reduce the sedimentation rate of proteins to zero, and a long extrapolation of the data is necessary. With accurate data, however, this can be performed to yield reliable results, and the method has, therefore, much to offer. The data are plotted as ~s versus p, where v is the viscosity of the solvent in poises, i.e., including the buffer contribution and the D20, s is the measured sedimentation coefficient, and p is the density of the solvent. The best-fitting straight line is obtained by the least squares method, and the equation of this line is then used to calculate the value of p corresponding to zero sedimentation rates. Even if the data are obtained with high precision, there is some

70


[9.]

u n c e r t a i n t y i n t h e final result, n o t o n l y b e c a u s e of t h e h a z a r d o u s e x t r a p o l a t i o n , b u t also b e c a u s e of e x c h a n g e of some of t h e h y d r o g e n s of t h e p r o t e i n m o l e c u l e w i t h d e u t e r i u m i n t h e water. T h i s l a t t e r effect is, however, likely to be a s m a l l one a n d for t h e p r e s e n t c a n be neglected. TABLE I I I CALCULATION OF THE APPARENT SPECIFIC VOLUME OF RIBONUCLEASE FROM ITS AMINO ACID COMPOSITION

Amino acid residue

Number of residues/ molecule~

W~

(% by weight a of residue)

Vi

ViWi

(specific volume of residue)

(% by volume of residue)

Aspartic acid Asparagineb

7 9b

5.69 7.31 b

0.59 0.60

3.36 4.39

Glutamie acid Glutamineb

4 8b

3.63 7.27 b

0.66 0.67

2.40 4.87

1.25 6.12 6.34 1.74 2.30 9.44 7.56 5.95 3.52 3.32 3.13 6.84 3.73 9.22 4.43

0.64 0.74 0.86 0.90 0.90 0.63 0.70 0.63 0.75 0.76 0.77 0.71 0.67 0.82 0.70

0.80 4.53 5.45 1.57 2.07 5.95 5.29 3.75 2.64 2.52 2.41 4.86 2.50 7.56 3.10

Glycine Alanine Valine Leucine Isoleucine Serine Threonine Half cystine Methionine Proline Phenylalanine Tyrosine Histidine Lysine Arginine

3 12 9 2 3 15 10 8 4 5 3 6 4 10 4

Total = 126

NW~ = 98.79

~,W~VI = 70.02

Y,W~V~/F,W~ = V~ = 0.709 cc./g.

a The number of each amino acid residue per molecule of ribonuclease, which gives a molecular weight of 13,895 for this enzyme, was obtained from the data of C. H. W. HiTs, W. H. Stein, and S. Moore, J. Biol. Chem. 211, 94i (1954). b To take into account the seventeen amide groups per molecule of RNase, it was assumed that these were approximately equally distributed between the glutamic and aspartic residues. A p p a r e n t specific v o l u m e s of p r o t e i n s c a n be e s t i m a t e d q u i t e r e l i a b l y f r o m t h e a m i n o acid c o m p o s i t i o n , since it has b e e n f o u n d t h a t t h e v o l u m e s of t h e m a c r o m o l e c u l e s are closely e q u a l t o t h e s u m of t h e i n d i v i d u a l a m i n o acid residues. T h i s p o i n t has b e e n e m p h a s i z e d b y M c M e e k i n a n d

72


[2]

concept, formulated by Fick in 1855, can be written dc

dm = - D A ~ dt

(12)

where dm is the mass of material transported in the time, dr, and A is the cross-sectional area of the diffusion cell. The negative sign is present in this equation because the transport of solute is in the direction of decreasing concentration. Thus, if we think of a vertical cell in which the bottom solution is the more concentrated and the positive direction is downward, then mass is transported in the negative direction. Diffusion coefficients can be measured, by direct application of equation 12, from determinations of the amount of material diffusing across a porous membrane separating a pure solvent from a homogeneous solution of some solute. Such diffusion studies, referred to as diffusion through a porous disk, are dependent on the establishment of a steady state in which the amount of solute diffusing per unit time is constant. This method is particularly useful for biological materials for which a specific bioassay is available. Though the method does not give absolute values for the diffusion coefficient, it is particularly valuable for the enzymologist who can use the method for studies of enzymes which have not been purified. The second, more popular method of studying diffusion, known as the free diffusion method, is based on another equation derived by Fick. This relates the rate of change of concentration with time to the rate at which the concentration gradient is changing with position, x, in the cell, as shown in equation 13. dc d2c d--{ = D ~dx (13) In performing measurements by this method, a sharp boundary is created between the solvent and the solution, and the change in concentration of the solute as a function both of distance and of time is followed by any one of a variety of optical procedures. With modern procedures and in apparatus now commercially available it is possible to obtain absolute diffusion coefficients with an accuracy exceeding 1%. Measurements by this method are also sensitive in the detection of impurities either much larger or smaller than the main component or in the determination of the homogeneity with respect to size of the principal diffusing species. In the free diffusion method great care is required in the design and use of the apparatus to ensure the formation of almost infinitely sharp boundaries and to eliminate any vibrations and resultant convection stemming from inadequacies in the mechanical construction or temperature control. For porous disk diffusion experiments, however, most of

[2]


73

these difficulties are nonexistent because the diaphragm with its extremely fine pores effectively prevents convective transport of liquid from the upper solution to the lower. Meanwhile independent stirring above and below the diaphragm can be used to maintain the homogeneity of each of the two solutions. Solute molecules in a homogeneous solution are in constant motion due to thermal energy, and such motion, known as Brownian movement, has been related to diffusion by Einstein and Smoluchowski. In fact, if we were able to measure precisely the random movements of a given macromolecule in solution over a long period of time (or, alternatively, the motions of many particles at a specific time), we could calculate its diffusion coefficient from an equation derived by these workers. The driving force for such movement is the thermal energy, kT, where k is the Boltzmann constant, 1.38 X 10- ~ erg/degree, and T is the absolute temperature. These randomly moving macromolecules experience a resistance to motion caused by the viscosity of the medium, and this resistance is characterized by a frictional coefficient, f, which is related to the size and shape of the kinetic unit. For particles of any shape which are large compared to the solvent molecules, D-

kT f

(14)

and therefore determination of the diffusion coefficient gives a direct measure of the frictional coefficient which also appears in the equation relating the sedimentation coefficient to molecular weight. Diffusion Through a Porous Disk

This method, introduced by Northrup and Anson 2~ and applied by them and others to the measurement of the diffusion of enzymes, is elegantly simple. Despite numerous limitations it should be employed whenever approximate results are desired for a specific enzyme, whether the enzyme is pure or not. The method is ideal in the latter situation as long as a sensitive and specific assay is available. Figure 7 shows a bell-shaped glass diffusion cell fitted with a stopcock at the top and with a fiat sintered-glass disk (or diaphragm) at the base. In practice, the cell is filled with the solution containing the diffusing substance and is carefully lowered, by means of an adjustable suspension on a rack and pinion device, until the lower edge of the sintered-glass disk just contacts the upper surface of the solvent contained in the outer vessel. Special efforts are necessary so that the glass diaphragm touches the lower liquid over its entire area. Also, caution should be exercised so ~2j. H. Northrop and M. L. Anson, J. Gen. Physiol. 12, 543 (1929).

74


[2]

that the diaphragm is not below the surface of the outer liquid; otherwise a stagnant layer will develop. The technique then consists in measuring the amount of solute appearing in the outer liquid as a function of time. This gives dm/dt of equation 12. Initially the difference in concentration between the inner and outer solutions is known, and for most experiments the time of diffusion is so short that there is no significant change in these concentrations. For the use of equation 12 we must know the concentration gradient which involves dx, the effective thickness of the diaphragm, and al~o A, the cross-sectional area of the diaphragm. Neither of these can be determined readily, since the diameter and length of the pores in the diaphragm no doubt vary from region to region. This

1

-I---"SOLUTION

FIG. 7. Porous disk diffusioncell. difficulty can be obviated through the determination of the "cell cons t a n t " by studies with a substance of known diffusion coefficient. As the technique was originally devised by Northrup and Anson, the solution, or more dense liquid, is on the top, but the pores of the diaphragm are sufficiently narrow to prevent mass flow of liquid despite the obvious gravitational instability. Thus diffusion of solute molecules through the pores of the sintered-glass disk into the solvent below creates a region just below the diaphragm which is more dense than the solvent further down in the cell. Convective stirring results, and the heavier liquid sinks to the bottom being replaced by fresh solvent. Similarly, just above the diaphragm there is a temporary diminution in concentration which also produces instability so that stirring results. The working basis of the method then rests on the hypothesis that the concentration above the membrane remains the same as the original concentration and that in the lower solution is similarly equal to the original concentration of that liquid. Of cours% after a long period of diffusion both of these concentra-

7(]

TECHNIQUES FOR CttARACTERIZATION OF PROTEINS

[2]

open or single cell to a double cell in which the volumes of both compartments are fixed and neither of the solutions is in contact with air, thereby minimizing difficulties due to evaporation. Several different cells have been suggested, and one which seems to be simple to use and free of some of the disadvantages of the open cell is shown in Fig. 8. 23 This cell also has provision for mechanical stirring of the liquid on either side of the porous membrane, since evidence has been presented showing that the density stirring in the Northrup-Anson cell is not completely effective and that stagnant layers exist in the vicinity of the diaphragm. In the

J

~.

ISOLVENT

--SINTERED GLASS

~

--SOLUTION

FIG. 8. Closed porous disk diffusioncell. cell shown in Fig. 8, the more dense solution is placed in the bottom so that gravity is a stabilizing rather than a disturbing force, thereby lessening bulk flow of liquid because of either a faulty membrane or gravitational instability when high concentrations were used in the upper solution. The cell is made of Pyrex glass with each end having a volume of about 50 ml. The diaphragms are Corning medium-grade or Jena G-4, fritted-glass disks (average pore size 15 #), of about 5-cm. diameter and 2- or 3-mm. thickness. In each compartment is placed a glass-enclosed iron wire about 3 mm. in diameter and of length slightly less than the diameter of the diaphragm. These glass stirrers are so constructed with respect to length of wire and thickness of glass that the one in the upper compartment sinks in the liquid and that in the lower one floats. The stirrers can be made to rotate by surrounding the cell with a rotating ~a R. H. Stokes, J. Am. Chem. Soc. 72, 763, 2243 (1950).

[2]

ULTR/kCElgTRIFUGATIOI% DIFFUSION~ AND VISCOMETRY

77

permanent magnet. With a stirring rate of about 20 r.p.m, it is found that no stagnant layers exist and that the solutions in each compartment are uniform in composition. To reduce difficulties from contamination of the porous disk by stopcock grease, Stokes uses rubber stoppers to seal each compartment. The rubber stoppers are equipped with small capillary tubes so that the liquid in the diaphragm is not disturbed when the stoppers are inserted or withdrawn. The capillary in the upper rubber stopper is open to the air to allow for volume changes during diffusion, and the lower capillary is fused to a small glass stopcock which can be opened prior to removal of the stopper. Although the stopcock is greased lightly, the grease does not readily contaminate the cell if care is exercised. As with the Northrup-Anson cell, the solution and solvent must be degassed and special precautions must be taken that no air is trapped in the pores of the glass disk during the filling operation. First the entire cell and diaphragm are filled with solution, then the solution in the upper compartment is removed by suction, and after several rinses solvent is added to this cell. Diffusion is allowed to occur with stirring for several hours. Again the upper compartment is emptied by suction; rinsing is performed and the liquid replaced by fresh solvent previously brought to the same temperature as the diffusion cell. Stirring is then resumed and the time recorded. Depending on the sensitivity of the assay procedure, one may have to allow a diffusion time of many hours. At an appropriate time the upper compartment is drained, the cell inverted, and finally the lower compartment is drained. The necessary determinations are made, and the diffusion coefficient is then calculated from equation 15. The cell is immediately refilled and the experiment repeated. If the newly determined diffusion coefficient agrees with the first one, then it can be assumed that a steady state had been reached and tl~e experimental values are valid. Thermostats are generally maintained at the desired temperature with variations no greater than _0.01 °. With the cell shown in Fig. 8, there appears to be little, if any, bulk flow of liquid through the diaphragm, and special precautions about leveling of the diaphragm are unnecessary. For calibration purposes it has been found that potassium chloride is an excellent standard. If the diffusion experiment is performed at 25 ° with 0.1 M potassium chloride diffusing into an equal volume of distilled water for a period such that the final concentrations are 0.075 M and 0.025 M for the solution and solvent, respectively, then the diffusion coefficient is 1.867 X 10-5 cm.2/sec. The volumes of the individual compartments of the cell are usually determined by weighing the cell empty and filled with water and then by converting the weight of the water into volume through use of the known density of water.

78

TECHNIQUES FOR CHARACTERIZATION OF PROTEII~S

[2]

This method gives satisfactory results with enzymes, although one must, of course, be certain that the enzyme molecules are not adsorbed on the surface of the diaphragm or that the pore size is not so great as to allow bulk flow. In view of the simplicity of the technique it is advisable to run experiments with different cells so that irregularities or deficiencies in an individual cell are readily detected. Free Diffusion Introduction. In the past ten years, major modifications have occurred in our techniques for the study of free diffusion with the result that today such diffusion studies can be performed routinely, and in commercially available apparatus, with an accuracy greater than that obtainable in the determination of sedimentation coefficients. This tremendous advance in experimental tech~fique was due largely to the rediscovery of interferometric optical methods and their adaptation to diffusion studies, As a consequence of these developments the older, more common procedures used in diffusion measurements of protein are now obsolete. We will therefore omit discussion of the older methods of handling the data obtained through the use of the familiar schlieren optical systems. This seemingly arbitrary decision is justifiable because the newer methods are neither more difficult nor more time consuming than the older methods, and further the accuracy of newer techniques is of the order of 10 times that of the latter. A thorough discussion of diffusion measurements involving schlieren optical systems is presented by Lundgren and Ward, 24 and the research worker who does not have available newer commercial apparatus equipped with interferometric optical systems should turn to that article for a detailed description of the different methods of handling the data. There are now available in this country three different instruments (and in Europe several more), each of which is completely equipped for precise diffusiometry by interferometric methods. Two of these, Model B, American Instrument Company, Inc., Silver Spring, Maryland, and Model H, Spinco Division, Beckman Instruments, Inc., Belmont, California, are versatile in that they possess two different interferometric optical systems in addition to the usual schlieren optical systems. The apparatus, Model 38A, made by the Perkin-Elmer Corporation, Norwalk, Connecticut, possesses one interferometric optical system along with the schlieren system. After many years of experimentation which led to a variety of ingenious designs for diffusion cells, there now seems to be widespread ~4 H. P. Lundgren and W. H. Ward, in "Amino Acids and Proteins" (Greenberg, ed.), Chapter 6. Charles C Thomas~ Springfield, Ill., 1951.

80


[2]

mercial models both the Rayleigh and the Gouy interferometric optical systems, and diffusion coefficients of comparable precision can be obtained by either method. A decision as to which is the method of choice in studying proteins is, at best, somewhat arbitrary; and the use in this laboratory of the Rayleigh method is based largely on convenience and ease of handling the data coupled with the ready availability of tables of probability functions necessary for the calculation of diffusion coefficients. For the Gouy method such detailed tables of the necessary mathematical functions are not yet generally available, and laborious, Enzyme Buffer

U

U

! 2

3

4

FIG. 9. Filling of Tiselius electrophoresis cell for a diffusion experiment. time-consuming interpolations within regions of the existing published tables are necessary. The Gouy method offers an important advantage, since it is very sensitive during the early stages of the diffusion experiment when the boundary is very sharp and the consequent interference fringe pattern is greatly spread. Early in a diffusion experiment the Rayleigh pattern is greatly compressed with a resulting loss in resolution; but the resolution of this method improves as the boundary broadens with time, whereas the resolution of the Gouy method decreases. This difference between the two methods can be decisive if one is dealing with a labile enzyme, since the length of a diffusion experiment using the Gouy method can be appreciably less than that with the Rayleigh method. It should be noted, however, that a long period for dialysis of the protein

[2]


81

solution against a buffer is required for either method, and the duration of the diffusion experiment itself may not be an overpowering factor. The Rayleigh method requires a reference optical limb with a refractive index close to that of the solvent, whereas the Gouy method requires no reference optical path. With present designs of diffusion cells, this constitutes an advantage for the Gouy method for solvents other than dilute salt solutions. In view of the foregoing brief, comparative comments about the two methods, the decision of the author to emphasize in this discussion the Rayleigh method should not be construed as an indication that it is a superior method but rather that it has been found in this laboratory more

F~G. I0. A representative schlieren and Rayleigh pattern from a diffusionexperiment on ribonuclease. convenient and simpler for work dealing with proteins. Where there is a desire for accuracy of the type desired by workers primarily interested in the theory of diffusion, the worker should examine the elegant studies of Gosting and his collaborators 28 employing the Gouy method and those of Longsworth 2~ with the Rayleigh method. The Rayleigh method can be used as well for electrophoresis, whereas the Gouy method cannot yield information about the position and number of boundaries. It is therefore important that enzymologists presently familiar with schlieren methods should acquaint themselves with the Rayleigh method because of its broad utility. In this regard most of the commercial equipment now gives simultaneous photographs of the schlieren and Rayleigh patterns as shown in Fig. 10 for a peptic digest of ribonuclease. E x p e r i m e n t a l Procedure. It is desirable to perform diffusion experiments at the isoelectric point of the protein, since charge effects there are 2~D. F. Akeley and L. J. Gosting, J. Am. Chem. Soc. 75, 5685 (1953). :7 L. G. Longsworth, J. Am. Chem. Soc. 74, 4155 (1952).

82


[2]

minimal. Frequently other factors such as insolubility or loss of activity preclude this practice, and experiments at other pH values can be performed satisfactorily if there is sufficient electrolyte to damp out charge effects which otherwise would lead to high values for the diffusion coefficient. Well-buffered solutions at an ionic strength of 0.1 suffice for this purpose. For the microcell in use in this laboratory, 3 ml. of protein solution at a concentration of 0.5 g. per 100 ml. and a volume of 300 ml. of solvent are ample. After the protein is dissolved in an aliquot of the buffer, it is dialyzed in the cold for a period of about 24 hours. The number of changes of buffer surrounding the dialysis bag and the length of the dialysis period depend largely on the purity and on other factors such as the stability of the enzyme. In certain cases, such as a study of the peptic digestion of ribonuclease, dialysis of the solution under investigation is precluded because of the permeability of the dialysis tubing to some of the products of the reaction, and diffusion experiments are conducted without dialysis as a means of equilibrating the protein solution against the buffer. For such situations special precautions are taken to subject the buffer to the same treatment as the protein solution so that the content of electrolyte in solution and solvent are as nearly the same as possible. Dialysis is conveniently performed by hanging the dialysis bag containing the solution in an Erlenmeyer flask in which is placed a glass-enclosed magnet and the appropriate solvent. The entire flask can be placed on a magnetic stirrer and stored in a cold room until equilibration is achieved. Dialysis tubing (obtainable from the Visking Corp., 6733 West 65th St., Chicago 38, Illinois) can be obtained in various diameters, and we find that 3~-inch tubing serves well for the small volumes used in this work, since ,it leads to a large surface relative to the volume of the protein solution, thereby ensuring a rapid equilibration. Before filling the diffusion cell it is advisable to centrifuge the solution and solvent for a few minutes at about 3000 r.p.m, to remove lint and any insoluble material such as denatured protein. It is important, before assembly of the cell to form a continuous U-tube as shown in Fig. 9, to lubricate each of the glass end plates of the three sections which slide relative to one another. For this purpose a grease made of two parts of petroleum jelly (Vaseline) and one part of mineral oil proves ideal for most work. The lubricant is readily mixed over a gently boiling water bath, and care should be taken to exclude lint from the grease after it is prepared. Some workers prefer as a lubricant a Dow-Corning silicone fluid, No. 200, of viscosity 7500 to 10,000 centistokes. This is satisfactory, but its removal from the cell at the completion of the experiment is difficult. In lubricating the glass plates, precautions should be observed to prevent the grease from entering the

84


[2]

due to thermal gradients is lower at temperatures near the maximum density of the solvent (ca. 1°). This is not nearly so serious as in electrophoresis, and if the material is stable at higher temperatures then diffusion experiments should be conducted at higher temperatures, since diffusion rates are much higher at 25 ° than at 1°. A 5-ml. syringe equipped with a 14-inch hollow stainless steel needle, No. 17 gage, with a blunt tip is used to transfer the protein solution to the cell. In filling the cell, the rack is tipped to the side so that the bottom section of the U-tube is at an angle and there is little tendency to trap air bubbles in the cell as the liquid is added. In this laboratory, observations of the diffusion process are generally conducted in the left-hand limb of the cell. Accordingly, the rack is tipped toward the right and the syringe needle is placed in the right-hand limb to avoid any contact of the needle with the windows of the diffusion channel. Sufficient solution is added so that the level of the solution in both limbs is about half to two-thirds the way up in the center section. The rack is righted and the needle withdrawn. At this point the cell looks like that illustrated by Fig. 9-1. By means of one of the knobs on the cell rack the bottom section is slid to the right, thereby disconnecting the left- and right-hand limbs of the cell as shown in Fig. 9-2. The syringe needle is again placed in the right-hand limb of the cell, and sufficient protein solution is added to raise the level in that limb just above the glass end plates of the center and top section. Less than 2 ml. of protein solution is involved in filling half of the left-hand section, the bottom section, and the entire right-hand section of the microcell. With the rack in place in the water bath, buffer is very carefully and slowly added down one of the side windows of the left-hand channel by means of a motor-driven syringe with a needle containing a very fine tip. This addition is continued until the buffer level in the lefthand limb corresponds roughly with that in the right-hand limb, as in Fig. 9-2. After a little practice this layering of solvent over solution can be done readily and reproducibly with the formation of rather sharp boundaries between the two liquids. In the microcell with a channel width of only 2 mm. there is little tendency for mixing if the operations are performed with care. The needle is then withdrawn and the center section is moved to the left, dragging with it the bottom section so that the cell sections are arranged as shown in Fig. 9-3. After rinsing of the upper, right-hand section free of protein, solvent is now added to both limbs slowly by means of a 25-ml. pipet whose tip rests against the wall of the extension tubes..This addition is repeated until the level of liquid is above the side arms of the extension tubes. Because of the connection between the two tubes, the level in the two vessels automatically reaches the same value; and there is consequently no danger of flow of liquid

[9.]


85

when the center section of the cell is moved to the right to form a continuous U-tube, as shown in Fig. 9-4. During the few minutes that have elapsed after the layering of solvent over the protein solution, some diffusion of the protein occurs. Furthermore, the layering itself and the subsequent handling of the cell produce distorted and relatively blurred boundaries so that we now create essentially a new boundary by the siphoning procedure of Kahn and Polson. ~5 This boundary sharpening technique can be performed in a variety of ways with equal success, and the method described here has its origin in the experiences of other workers. ~8 Owing to short supply of some of the diffusing substances, we have imposed the important additional restriction that the method require only a small amount of solution even if there is some sacrifice in the quality and sharpness of the initial boundary. A long, stainless steel needle with a special tip ground to an angle of 45 ° is filled with solvent and lowered into the left limb by means of a rack and pinion until the tip is at the level of the boundary. Care should be taken during this operation in order to prevent contact of the needle with either of the optical windows of the diffusion cell. The needle is 14 inches long, No. 17 gage, and onto its end is soldered a 2-inch length of tubing with inner diameter of 0.012 inch and wall thickness of 0.010 inch. The upper end of the needle is connected to a three-way stopcock either directly or by means of a short length of thick-walled, narrow-bore rubber tubing. If the needle and stopcock are rigidly coupled, extra caution is required in mounting the stopcock on the rack so that the mere turning of the stopcock doesn't impart any movement onto the needle. If there is a short length of flexible tubing between needle and stopcock, then a tubing should be selected which doesn't collapse when suction is applied; also the needle and stopcock must move in unison so that there is no bending of the tubing when the needle is raised or lowered. One end of the stopcock is connected by tubing to a motor-driven syringe of 5-ml. capacity. This tubing, like the other, should have a narrow bore and heavy wall thickness. A length of about 15 inches of Tygon tubing is connected to the third arm of the stopcock. This connection is for convenience in filling the syringe, the stopcock, and the sharpening needle without the risk of introducing air bubbles into the system. We have used a syringe of narrow bore so that linear movement of the plunger of the syringe involves only a small volume change. The motor-driven syringe should be capable of movement in either direction, and the speed of the motor must be adjustable. In connecting the piston of the syringe 28 Thanks are due Drs. H. Hoch, G. Kegeles, T. E. Thompson, and J. L. Oncley for their suggestions regarding different procedures for boundary sharpening in diffusion experiments.

86

TECHI~IQUES FOR CHARACTERIZATION OF PROTEINS

[2]

to the arm driven by the motor, care must be taken that the coupling is flexible so that the plunger won't bind with the barrel of the syringe in case of slight misalignment. This flexible coupling must allow for movement in all directions except that in the direction of movement of the plunger. Here there must be firm, continuous tension at all times with no elasticity as with most rubber connections. Otherwise the movement of the plunger and the liquid will occur in spurts and the boundary sharpening will be difficult to control. Many workers prefer to use a siphon and allow the liquid flow to occur through the sharpening needle under the influence of a fixed pressure head rather than trust a motor-driven syringe to produce smooth action. With the sections of the cell completely aligned to form a U, the needle, the stopcock, and only a small part of the syringe filled with solvent, and the needle in position such that its tip is near the center of the boundary, sharpening is begun. First, the connection between the side arms of the extension tubes is closed by means of a hemostat, and then withdrawal of liquid is started. The motor is adjusted so that the flow is only about 0.1 ml./min. This is continued for about 4 minutes during which time the boundary is considerably sharpened owing to the formation near the tip of the needle of an interface between solvent and solution. The motor is then turned off for a few minutes, and some spreading of the boundary occurs because of diffusion. Again the motor is turned on and another 0.3 ml. of liquid is withdrawn at the same rate of 0.1 ml./min. The withdrawal of liquid is then stopped for a few minutes, and the boundary broadens a little due to diffusion. For the last time the motor-driven syringe is started and liquid is withdrawn for about 3 minutes at 0.1 ml./min. The boundary should be examined during this time to see that the fringes are straight, right up to the position where the boundary is being sharpened. The motor is turned off, the stopcock to the sharpening needle is turned 45 ° to isolate the needle, and the bottom section of the cell is moved to the right in order to break the connection between the left- and right-hand limbs. It is important that these operations be done almost simultaneously, with the aid of another individual if necessary. The time of stopping the flow and moving the bottom section is then recorded as the starting time of diffusion. The needle is withdrawn by careful operation of the rack and pinion. When this is completed, it is advisable to slide the center section to the left, dragging with it the bottom section which will now be in its normal position. The center section of the diffusion cell is now isolated from both upper and lower sections. Thus any dust which falls into the open reservoir tubes or moisture which condenses on the cold extension tubes cannot cause a disturbance of the boundary. As soon as the center

88

TECHI~IQUES FOR CHARACTERIZATION OF PROTEINS

[9.1

symmetrically, and the b o u n d a r y width when sharpening is discontinued should be between 0.3 and 0.4 mm. Calculations. Figure 11 shows a series of typical Rayleigh interference patterns obtained in a diffusion experiment with a peptic digest of ribonuclease. I t should be borne in mind t h a t the observed p a t t e r n is essentially a section of a plot of refractive index versus distance in the cell. Since the refractive index of protein solutions can be considered as varying in a linear fashion with the concentration of solute, the Rayleigh m e t h o d gives a plot of the concentration versus distance in the cell, and if we were

Fzo. 11. Representative Rayleigh interference patterns from a diffusion experiment on a peptic digest of ribonuclease. Pictures from top to bottom were taken at 9, 38, 99, 337 and 1183 rain., respectively, after formation of the boundary. to plot the fringe n u m b e r as a function of fringe position the curve obtained would be the integral of the curves obtained b y schlieren methods. For this reason, the Rayleigh m e t h o d is frequently called the integral fringe method. Thus the total n u m b e r of fringes, J, is a measure of the change in concentration across the boundary, and it is equal to a hn/)~, where a is the cell thickness (along the optical path), An is the difference in refractive index between solvent and solution, and X is the wavelength of light. T o compute the diffusion coefficient from Rayleigh patterns we first must evaluate the total n u m b e r of fringes, J . This is done most readily b y using different photographs. An early picture, before the fringes are completely resolved as in the 9-minute photograph, is used to determine the fractional part of J ; and a later photograph, after the fringes are distinctly separated as in the 337-minute photograph, is used to measure

90


[2]

will be straight except for small regions at the top and bottom of the channel where cementing of the end plates onto the optical glass windows causes some distortion. If the fringes are not straight throughout the entire length of the diffusion cell, it is advisable to choose the best region and to form the boundary at that level. There are a variety of methods of handling the data for the calculation of diffusion coefficients. For example, the equivalent of the wellknown height-area method can be used, since J is a measure of the area and we can readily determine the maximum height (of the gradient curve) by finding the minimum separation of fringes. This method has been used by Svensson ~9 and others, but the method used by Longsworth ~7 is recommended both because of its accuracy and because it uses much more of the experimental data and is therefore capable of detecting deviations from ideal diffusion behavior. Computations by the method of Longsworth involve the assumption that the concentration distribution in the boundary is Gaussian, and therefore we can use the Tables of Probability Functions (Federal Work Agency, Works Project Administration, Superintendent of Documents, Washington, D. C., 1941). These tables are very complete and the calculations with them can be made without the need of interpolation. If the concentration and cell thickness are such that there are about 20 fringes in the boundary, then the position and number of all the fringes are tabulated. The number of the fringe with which the cross hairs coincide outside the boundary is termed the zero'th fringe. If there are as many as 50 fringes in the boundary it is advisable, in order to reduce labor, to record the position and number of every other fringe. The method of calculation is illustrated by Table IV showing data obtained on a peptic digest of ribonuclease. In column 1, headed fl, are listed the number of the different fringes up to a value less than J/2. The second column lists the computed value, jk* = (2jk - J)/J, for each value of jk. As long as j~ is less than J/2, the sign of the computed value, j~*, in column 2 can be ignored. Corresponding to each value of jk there should be a value of jr, where j~ is greater than J/2. The values of jl are listed in column 3, and the computed values, jz* = (2jl - J)/J, are tabulated in column 4. It should be stressed that the pairing of fringes can be done in a variety of ways. Thus the pairing of fringes 2 and 10, 3 and 11, 4 and 12, etc., could be changed to 2 and 11, 3 and 12, 4 and 13, etc., or 1 and .10, 2 and 11, 3 and 12, etc., or to a variety of other combinations. Pairing of the fringes in different ways can furnish valuable information about the homogeneity of the diffusing substance and the dependence of diffusion coefficient on concentration. We usually omit the first and last 29 H. Svensson, Acta Chem. Scand. 5~ 72 (1951).

[9-]

ULTRACENTRIFUGkTIO~ DIFFUSION, AND VISCOMETRY

i
,pH 4 below which the free acid is present as a zwitterion). The free acid will migrate to the positive side of the eleetrophoresis cell and will separate from the conjugate. With buffers of higher pH the protein can be shown to be free of traces of free sulfonamide a2 G. Weber, Discussions Faraday Soc. 13, 33 (1953).

212


[8]

(formed f r o m traces of a m m o n i a in the original sample) which do n o t m i g r a t e in an electric field. T o calculate the n u m b e r of conjugating groups bound to a unit weight (e.g., to 1 g. molecule) of the protein, the absorption s p e c t r u m in the region 300 to 450 m~ should be measured 19 for a solution of k n o w n protein content. D N S conjugates show an absorption m a x i m u m at 330 to 360 m~. T h e concentration of conjugating groups in the solution m a y be calculated f r o m the extinction coefficient of 4.3 X 10 e cm.2/g, mole for the absorption m a x i m u m . A b l a n k determination should be m a d e for u n c o n j u g a t e d protein so t h a t the observed absorption of the conjugate m a y be corrected for the absorption of the protein at the same wavelength. T h e correction will usually be small.

Coupling of ~-Anthrylisocyanate This is done as for the D N S conjugate except t h a t r a t h e r more organic solvent is used. ~2 F o r r e m o v a l of uncoupled fluorescent material, organic solvent precipitation would a p p e a r to be essential owing to the limited solubility of the hydrolysis products and their strong interactions with proteins. T h e molecular extinction coefficient is 3.04 × 10 e cm.2/mole. 22

[8] The Solubility Diagram as a Criterion of Protein Homogeneity By ROGER ~/[. HERRIOTT Introduction T h e solubility d i a g r a m is a powerful tool for detecting the presence of impurities in a n y t y p e of soluble material. T h e essential feature of this m e t h o d is simply t h a t in a s a t u r a t e d solution of a single solute the concentration of dissolved solute does not v a r y with the q u a n t i t y of s a t u r a t ing solid phase present.i This fact had been appreciated for a long time, b u t because of the complex nature of their protein p r e p a r a t i o n s the early workers 2-4 failed to produce convincing evidence of the value of the solu1The well-known effect of extreme differences in the size of the solid particles on the solubility is reported to be due to surface energy differences and is not considered in the present discussion, since it is probably a second-order difference. See the following references for a review of the effect of the size of solid particles: A. Hulett, Z. physik. Chem. 37, 385 (1901); 47, 357 (1904); M. L. Dundon and E. Mack, Jr., J. Am. Chem. Soc. 45, 2479 (1923); M. L. Dundon, ibid. 45, 2658 (1923). 2 G. Galeotti, Z. physiol. Chem. 40, 492 (1903); 42, 330 (1904); 44, 461 (1905); 48, 473 (1906). s W. B. Hardy, Brit. J. Physiol. 33, 251, 338 (1905).

212


[8]

(formed f r o m traces of a m m o n i a in the original sample) which do n o t m i g r a t e in an electric field. T o calculate the n u m b e r of conjugating groups bound to a unit weight (e.g., to 1 g. molecule) of the protein, the absorption s p e c t r u m in the region 300 to 450 m~ should be measured 19 for a solution of k n o w n protein content. D N S conjugates show an absorption m a x i m u m at 330 to 360 m~. T h e concentration of conjugating groups in the solution m a y be calculated f r o m the extinction coefficient of 4.3 X 10 e cm.2/g, mole for the absorption m a x i m u m . A b l a n k determination should be m a d e for u n c o n j u g a t e d protein so t h a t the observed absorption of the conjugate m a y be corrected for the absorption of the protein at the same wavelength. T h e correction will usually be small.

Coupling of ~-Anthrylisocyanate This is done as for the D N S conjugate except t h a t r a t h e r more organic solvent is used. ~2 F o r r e m o v a l of uncoupled fluorescent material, organic solvent precipitation would a p p e a r to be essential owing to the limited solubility of the hydrolysis products and their strong interactions with proteins. T h e molecular extinction coefficient is 3.04 × 10 e cm.2/mole. 22

[8] The Solubility Diagram as a Criterion of Protein Homogeneity By ROGER ~/[. HERRIOTT Introduction T h e solubility d i a g r a m is a powerful tool for detecting the presence of impurities in a n y t y p e of soluble material. T h e essential feature of this m e t h o d is simply t h a t in a s a t u r a t e d solution of a single solute the concentration of dissolved solute does not v a r y with the q u a n t i t y of s a t u r a t ing solid phase present.i This fact had been appreciated for a long time, b u t because of the complex nature of their protein p r e p a r a t i o n s the early workers 2-4 failed to produce convincing evidence of the value of the solu1The well-known effect of extreme differences in the size of the solid particles on the solubility is reported to be due to surface energy differences and is not considered in the present discussion, since it is probably a second-order difference. See the following references for a review of the effect of the size of solid particles: A. Hulett, Z. physik. Chem. 37, 385 (1901); 47, 357 (1904); M. L. Dundon and E. Mack, Jr., J. Am. Chem. Soc. 45, 2479 (1923); M. L. Dundon, ibid. 45, 2658 (1923). 2 G. Galeotti, Z. physiol. Chem. 40, 492 (1903); 42, 330 (1904); 44, 461 (1905); 48, 473 (1906). s W. B. Hardy, Brit. J. Physiol. 33, 251, 338 (1905).

[8]

SOLUBILITY DIAGRAMS

213

bility measurements as a criterion of purity. Sorenson and HSyrup ~ gave very careful consideration to phase rule aspects in their extensive studies on crystalline egg albumin. Landsteiner and Heidelbergeff made excellent use of solubility principles to differentiate hemoglobins of different species. The general subject received relatively little attention, however, until Northrop and Kunitz 6 re-examined and extended it to cover the various theoretically possible systems. In addition, these latter workers prepared proteins of such homogeneity that virtually ideal solubility diagrams were obtained experimentally which brought about an increased interest and confidence in the method. Perhaps even more important, this work demonstrated that proteins respond to thermodynamic principles and methods in a manner similar to that observed for small molecules, a concept not generally accepted in the early 1930's. It may be worth noting at this point that, although this solubility method originally was designed and applied almost exclusively to protein preparations, it is equally applicable to all types of molecules. General Procedure Briefly, the solubility method consists in (1) mixing a series of increasing quantities of the material under investigation with equal or known volumes of solvent until equilibrium is attained; (2) separation of the solid phases from the solutions; (3) determination of the concentration of the material dissolved in aliquots of the various solutions; and (4) plotting the concentration of dissolved material against the total (solid and dissolved) added per unit volume. A plot of the analytical results will take the form of one of the three general type curves shown in Fig. 1. Interpretation of Results

In those systems containing no solid phase at equilibrium, the dissolved concentration equals the total added, and therefore the points fall on the line O A in Fig. 1. This line has a slope of 1. As the total concentration is increased, a point will be reached where a small amount of solid will remain after equilibration. The key to any interpretation of the solubility diagram depends on the variation of the solubility (dissolved concentration) beyond the point where the first solid phase persists at equilibrium. As the total material increases beyond the point of first persistence of solid phase, the solubility may vary in any of three ways. 1. It may remain unchanged, as represented in Fig. 1 by curve O A E . 4S. P. L. SCrenson and M. H6yrup, Compt. rend. tray. lab. Carlsberg 12, 213 (1917) ; see also ibid. 18~ No. 5 (1930). s K. Landsteiner and M. tteidelberger, J. Gen. Physiol. 6, 131 (1923). 6j. H. Northrop and M. Kunitz, J. Gen. Physiol. 15, 781 (1930).

214

TECIIN'IQUES FOR CHARACTERIZATION OF PROTEIN'S

[8]

Such a curve represents a constant solubility and is obtained when the material is (a) a single component (i.e., pure); (b) a solid solution of two or more materials having identical solubilities; or (c) a mixture of two or more materials which are present in the preparation in direct proportion to their solubilities. Cases b and c are not often encountered, and they m a y be resolved b y performing the solubility determination in solvents of a different pH, temperature, kind of salt, or organic solvent composition. In general the ratio of the solubilities of materials would be expected to change under variations in conditions and the second component will be revealed. If the material under s t u d y has a constant solubility in several different solvents, it is probably a single component. However, a 1 : 1 racemic mixture of d and l isomers or perhaps a mixture of isotopic isomers would behave as a single component even in several solvents.

e 3P~ o~-

A

C

E

e~ex

~3

Total

concentration

FIG. 1. General types of solubility curves. See text for explanation of curves. 2. F r o m the point where the solid phase first persists the solubility m a y increase linearly at a different rate from before as the total increases and change a b r u p t l y to a still different linear positive slope or to one of zero. This t y p e of result is obtained when the components form simple mixtures in the solid phase and their solubilities are independent of one another. Each time there is an a b r u p t change in slope, a new solid phase appears. Curve O B C E represents such a system with pure solid phases appearing first at B and C. A mixture of d and 1 optical isomers in a n y proportion other than 1:1 would be expected to produce a curve of this type. F r o m certain algebraic considerations N o r t h r o p and Kunitz e,7 have shown t h a t the value of the intercept of the line B C with the ordinate is the solubility, S, of the pure component which first appears as a solid phase. The q u a n t i t y of this pure component, as a fractional part of the M. Kunitz and J. H. Northrop, Cold Spring Harbor Symposia Quant. Biol. 6~ 325 (1938).

[8]

SOLUBILITY DIXGRA~S

215

total, is given by 1 minus the slope of the line BC. The corresponding values of the second component are obtained by difference. Thus, the solubility of the second component equals the total less S, the value of the first component. The relative amount of the second component will be equal to the slope of line BC. Thorp 8 found that known mixtures of isomers of DDT respond quantitatively to this method of analysis, as Kunitz and Northrop 7 had shown for proteins. 3. Third, and finally, the solubility may vary continuously as the total is increased (see curve ODE), asymptotically approaching a constant value. Such a result is obtained with solid solutions. In such cases no quantitative predictions are possible. In general one may expect the first precipitate appearing along this curve to be richer than the starting material in one of the components. Similarly, the solution in equilibrium with a large excess of solid will be richer in the other components than is the starting material. It follows from what has been said above that in distinguishing between eases 2 and 3 it is only necessary to run a solubilitydiagramon the first or smallest solid phase to persist at equilibrium. In both eases the solubility of this fraction will be less than that of the original material. The key to the differentiation lies in the fact that a material behaving like a single component should be obtained from a mixture (ease 2), whereas another rounded curve similar in shape to ODE in Fig. 1 will be obtained if the material is derived from a solid solution (ease 3). Application of the Phase Rule. The phase rule is a useful thermodynamic generalization of heterogeneous equilibria relating the number of components, phases, and degrees of freedom. Use has been made of it in establishing the validity of the solubility method. It is not necessary, however, to apply the phase rule or even to understand it to make considerable use of the solubility method which generally is applied to decide whether a preparation consists of one or more components. The simple concept that in a saturated solution (equilibrium) the solubility of the solute is independent of the quantities of any of the phases will in most cases serve as a test of homogeneity, for, if the solubility value is independent of the quantity of solid phase, it is a constant solubility denoting a single component. For those interested in a proof of the various possible cases, the following is a brief description based on the phase rule. This rule in its abbreviated form is P + F = C + 2, where P equals the number of phases (physically separable portions which differ in structure), F equals the number of degrees of freedom (variables such as temperature, pressure, and concentration of components), and C equals the number of D. Thorp, J. Soc. Chem. Ind. 65, 414 (1946).

216


[8]

c o m p o n e n t s (the s m a l l e s t n u m b e r of i n d e p e n d e n t c o n s t i t u e n t s t h a t will define t h e s y s t e m ) 2 I n s o l u b i l i t y e x p e r i m e n t s t w o of t h e d e g r e e s of f r e e d o m , t e m p e r a t u r e a n d p r e s s u r e , a r e h e l d c o n s t a n t , so t h e e q u a t i o n simplifies t o P Jr F ' = C, w h e r e F J e q u a l s F - - 2. T h e m a t e r i a l in t h e t a b l e s h o w s h o w t h i s r e l a t i o n s h i p c a n be u s e d t o d i f f e r e n t i a t e t h e v a r i o u s k i n d s of s y s t e m s . PHASE RULE ANALYSES OF SOLUBILITY DIAGRAMS

Number of Region of curves of Fig. 1

Phases

Components Degrees of Solutes b freedom ~ Solvents

OB OAE

1

1 or more

1

1 or more

2

0

1

1

OBC

2

1

l

2

OBCE

3

0

1

2

ODE

2

1 or more

1 (liq.)

2 or more

Systems satisfylng these conditions A solution (no solid phase) Single pure solid in contact with a saturated solution Single pure solid in a solution of two components Mixture of two pure solids in a saturated solution of both Solid solution of two or more components in contact with liquid solution

a The degree of freedom observed is the variation in concentration of dissolved solute. Temperature and pressure are held constant. b Deduced from the phase rule. Such components as buffer, salts, etc., are considered as part of the solvent, since presumably they do not vary. I t s h o u l d be c l e a r t h a t t h e p h a s e rule is n o t c o n c e r n e d w i t h t h e t i m e r e q u i r e d to g e t t o e q u i l i b r i u m , n o r d o e s i t i n d i c a t e t h a t t h e s o l u t e in solut i o n is i d e n t i c a l w i t h t h a t in t h e solid p h a s e . I n s o m e i n s t a n c e s s o l u t e a n d solid a r e k n o w n t o differ. T h u s , t h e solid p h a s e m a y b e a c r y s t a l l i n e s a l t of a p r o t e i n a n d a p a r t i c u l a r ion, a n d in s o l u t i o n t h a t i o n m a y n o t be associated with the protein. A r e v e r s i b l y d i s s o c i a b l e s o l u t e d o e s n o t p r e s e n t a n y d i f f i c u l t y in t h e s o l u b i l i t y m e t h o d . I n s u c h a s y s t e m t h e r e is o n l y one m o r e c o m p o n e n t , e v e n if t h e s o l u t e d i s s o c i a t e s i n t o t w o p a r t s , for t h e c o n c e n t r a t i o n s of t h e u n d i s s o c i a t e d s o l u t e a n d one of t h e d i s s o c i a t e d p a r t s will define t h e c o n c e n t r a t i o n of t h e s e c o n d p a r t . I n t r o d u c t i o n of t h e a d d i t i o n a l c o m p o n e n t For more details regarding the phase rule and the definitions, see A. Findlay, "The Phase Rule and Its Applications." Longmans, Green, New York, 1938; J. A. V. Butler, J. Gen. Physiol. 24, 189 (1940).

[8]

SOLUBILITY DIAGRAMS

217

will be balanced by an additional degree of freedom; hence the number of phases will remain unchanged. The concentration of the dissociated components can also be considered as fixed by the concentration of undissociated material and the particular solvent used. Thus, reversible phenomena do not jeopardize the value of the solubility method by limiting it to nondissociating systems or requiring that the existence of such dissociation be known. If the dissociation is not reversible, then an equilibrium cannot be established and the method is not applicable.

Quantitative Limitations of the Method The resolving power of this method is dependent on the relationship of the components in the solid phase, i.e., whether mixtures or solid solutions, the difference in solubilities of the individual components, and the precision of the analytical method. If the solid phase is a solid solution, no quantitative prediction of the resolving power is possible. In general, however, the greater the difference in solubility of the individual components, the greater will be the resolution. No one analytical method can be followed with all materials, but the dry weight of solute has a very wide range of usefulness and can be a very precise measurement. It is open to a serious objection, however, if the solvent is a strong salt solution. For proteins the nitrogen content and the absorbancy at 280 mu are convenient measurements.

Conditions and Certain Details of Technique

Solvent. Although few definite rules apply to all proteins, it is obvious that certain general aspects of the problem must be kept in mind. Thus, it is important that the protein be stable under the conditions of the experiment. A solvent (usually a buffered aqueous salt solution) should be chosefi that will permit precise estimation of the concentration of dissolved protein and yet not so high that too large a proportion of the total protein is in solution in a concentrated suspension. Amorphous and Crystalline Forms. With some proteins the solubility experiment can be performed on either the crystalline or amorphous forms. Each has certain advantages, but other things being equal a solubility experiment on the crystals is preferred, for the exact nature of the amorphous state is not well understood. Preliminary Equilibration. Usually protein preparations contain additional ions sufficiently different from those of the solvent to be used that some preliminary adjustment is necessary before making the final distribution of suspension. In bringing the solid into equilibrium with the solvent much time can be saved by first washing the preparation in question on a Biichner funnel with small aliquots of the complete solvent

218


[8]

or by dialyzing the suspension in collodion or Visking tubing against an excess of solvent. To determine whether adjustment is complete a portion of the solid may be stirred or precipitated with successive aliquots of the solvent until the filtrates from several successive treatments contain the same amount of dissolved protein. If the amount dissolved is not constant after a number of washings, it may be the result of a change in the protein composition due to removal by dissolution of a small amount of a relatively soluble component and not because equilibrium of the salt is not reached. When several successive aliquots do contain equal amounts of dissolved protein, it may be presumed that equilibrium exists between the solid and solution. Criteria of Equilibrium. The equilibrium point may be approached from either the supersaturated or the undersaturated side. Both approaches should be made when the amount of material and other considerations permit. It is rather important that at least one or two points be obtained from both sides to establish that a true equilibrium value is being observed. Approach from the supersaturated side of the equilibrium can be conveniently carried out with amorphous preparations by using a split solvent. This is accomplished by dissolving the solid phase or suspension in water or buffer and adding the concentrated salt portion. It is difficult with crystalline preparations of proteins to get reproducible solubility figures when approaching the equilibrium solubility from the supersaturated side. This is particularly true for those systems in which the excess of solid phase is small. Equilibrium is usually approached from the undersaturated side for both amorphous and crystalline forms by stirring the solid phase with the complete solvent. The time required to attain equilibrium at any given temperature depends on a number of factors paramount among which are the quantity of solid, the size of the solid particles, and the rate of stirring. This time, therefore, must be determined experimentally in each case. To ensure against an unforeseen mistake the solubility value should be determined at two different time intervals. Distribution of Solute. The solvent and solid solute having been brought into salt equilibrium, the suspension is distributed in varying amounts in a number of glass or plastic test tubes and the solvent added. The amount of suspension in each tube is adjusted so that at equilibrium the series of tubes ranges from complete solution of the protein to a large excess of solid. It should be emphasized that the greatest interest is in those points which fall near the " b r e a k s " in the diagrams, in the regions where new solid phases appear. With a limited amount of material it is, therefore, important to adjust the concentrations of suspension with this in mind.

[8]

SOLUBILITY DIAGRAMS

219

It is possible to test for a one-component system with a minimum of effort and material by using only two points. From the preliminary equilibration data the solubility in the presence of an excess of solid is known. It is then only necessary to dilute such a suspension with solvent to the calculated point where there should be only a slight (10%) excess of solid present at equilibrium. In such a case, if the concentration of dissolved solute is the same as that observed when the excess of solid is large, the diagram is one of constant solubility and therefore indicates the presence of a single component. If, however, the solubility proves to be lower, the investigator will be better prepared to design the distribution of the remainder of the samples to get the maximum information. For reasons already discussed in the earlier sections of this paper, it is more important to have more experimental points in the region of small excess solid than of great excess solid. Final Equilibration. In stirring the protein solution and suspensions, the temperature should be kept constant and foaming should be avoided as it may represent considerable denaturation. It has been found that glass beads, marbles, or stainless-steel ball bearings in tubes that are completely filled, stoppered, and then rocked or rotated suffice to stir the suspensions. The time of stirring (necessary to attain equilibrium) must be determined experimentally and should be based on the time necessary to equilibrate the system with a minimum of excess solid phase. Separation of the Solid and Solution Phases. After equilibrium has been attained, the solid is separated from the solution by centrifugation or filtration. Care must be taken in solubility studies of amorphous material, when the approach is from the supersaturated side, that equilibrium is reached before the solid phase is removed. It has usually been assumed that a protein solution cannot be supersaturated with the amorphous protein, but certain experiments with pepsin seem to indicate that such supersaturation is possible. Filtrates from amorphous solids should remain clear for several hours unless crystallization occurs; in the event of cloudiness crystals usually may be detected by microscopic examination. Should the preparation crystallize in the complete solvent, one may depress this tendency by allowing the protein to stand in solution in the dissolving part of the split solvent for several hours before adding the precipitation part of the solvent. This treatment dissolves the last traces of crystals which would otherwise act as seeding crystals. The supernatant solution or filtrate must, therefore, be crystal clear. It has been found that filtration through Whatman No. 42 filter paper gives uniformly good results. ,If the amount of dissolved protein is under 0.5 mg./ml., the filter paper takes up a significant amount of the protein from the first portion of solution passing through so that the first few

220

TECHN'IQUES FOR CHARACTERIZATION OF PROTEIN'S

[8]

milliliters of filtrate may be low in dissolved protein and, therefore, are usually discarded. Estimation of Dissolved Protein. The amount of dissolved protein may be determined by any of a number of methods such as Kjeldahl nitrogen, turbidity, biological activity, color test for tyrosine and tryptophan, biuret, copper phenol value, 1° or any other easy and precise method. In the event that an unidentified substance having a specific property such as a biological activity is being examined, one can learn a great deal about the class of substances to which the unknown belongs by making as many different examinations on the test solutions as possible. For example, one may find that protein, carbohydrate, nucleic acid, specific absorbency, or another property parallels or separates from the specific biological property being studied, and this information should prove useful whether the solubility diagram is that of a preparation consisting of single or multiple components.

Separation of Protein Components by Methods Based on the Solubility Diagram From the discussion in the section on Interpretation of Results it follows that, if the system under examination is shown by the solubility diagram method to consist of two or more components, a procedure for a separation can be developed from this diagram. Thus, if the components occur as a simple mixture (case 2), the first solid phase appearing, represented in Fig. 1 by the region enclosed in the triangle BAC, will be a pure component having the solubility indicated by S. If the components form solid solutions (case 3), the first solid phase persisting at equilibrium will be richer in one of the components than was the starting material. The solution in equilibrium with an excess of solid phase will be enriched in respect to another component. Preparations of diptheria antitoxin H and swine pepsin ~2 yielded solubility curves showing a high degree of inhomogeneity. These materials were purified by procedures based on extraction under conditions which dissolved only a fraction of the total, precipitation of the dissolved protein from the supernatant solution, and re-extraction of this last precipitate. With both proteins the solubility curves of the dissolved fractions were improved (i.e., approached more nearly that of a single component) with each extraction step, being essentially constant after three extractions. For full details see refs. 11-13. 10 M. Heidelberger and C. MacPherson, Science 97, 405 (1943). H j . H. Northrop, J. Gen. Physiol. 25, 465 (1941). 18 R. M. Herriott, V. Desreux, and J. H. Northrop, J. Gen. Physiol. 24, 213 (1940). 13 R. M. Herriott, Chem. Revs. $0, 413 (1942).

[9]

DNP METHOD FOR AMINO ACID SEQUENCE IN PROTEINS

221

Summary The solubility method is a theoretically sound, highly selective procedure for detecting the presence of impurities associated with any kind of molecule. In general it can be made as accurate as the available methods of analysis permit and requires no special apparatus. When the results reveal additional components, it is possible to design a method of isolating one, and in some instances two, components as homogeneous fractions.

[9] Determination of Amino Acid Sequence in Proteins by the Fluorodinitrobenzene Method

By R. R. PORTER The complete amino acid sequence has been determined in insulin and in ACTH, and considerable progress has been made with ribonuclease. Extension to other proteins and polypeptides may be expected at an increasing rate. The procedure followed is: 1. Amino acid analysis. 2. Estimation of N terminal and C terminal amino acids. 3. Separation of individual peptide chains if more than one is present. 4. Determination of the sequence in the peptides produced by selective partial hydrolysis in order that the total sequence of one chain may be deduced. Only the application of the fluorodinitrobenzene technique to these procedures will be described here.

Principle1 If 1,2,4,-fluorodinitrobenzeue (FDNB) in solution in ethanol is shaken with a solution or suspension of a protein in aqueous NaHCO~ at room temperature it will react with the amino, phenolic, imidazole, and sulfhydril groups of the protein. Only the substitution of the amino groups gives rise to colored derivatives which are relatively stable to drastic acid hydrolysis of the proteins. The DNP amino acids obtained by hydrolysis may be fractionated, characterized, and estimated, and hence the N terminal amino acids and the lysine residues with a free NSNH2 group F. Sanger, Biochem J. 39, 507 (1945).

[9]


221

Summary The solubility method is a theoretically sound, highly selective procedure for detecting the presence of impurities associated with any kind of molecule. In general it can be made as accurate as the available methods of analysis permit and requires no special apparatus. When the results reveal additional components, it is possible to design a method of isolating one, and in some instances two, components as homogeneous fractions.

[9] Determination of Amino Acid Sequence in Proteins by the Fluorodinitrobenzene Method

By R. R. PORTER The complete amino acid sequence has been determined in insulin and in ACTH, and considerable progress has been made with ribonuclease. Extension to other proteins and polypeptides may be expected at an increasing rate. The procedure followed is: 1. Amino acid analysis. 2. Estimation of N terminal and C terminal amino acids. 3. Separation of individual peptide chains if more than one is present. 4. Determination of the sequence in the peptides produced by selective partial hydrolysis in order that the total sequence of one chain may be deduced. Only the application of the fluorodinitrobenzene technique to these procedures will be described here.

Principle1 If 1,2,4,-fluorodinitrobenzeue (FDNB) in solution in ethanol is shaken with a solution or suspension of a protein in aqueous NaHCO~ at room temperature it will react with the amino, phenolic, imidazole, and sulfhydril groups of the protein. Only the substitution of the amino groups gives rise to colored derivatives which are relatively stable to drastic acid hydrolysis of the proteins. The DNP amino acids obtained by hydrolysis may be fractionated, characterized, and estimated, and hence the N terminal amino acids and the lysine residues with a free NSNH2 group F. Sanger, Biochem J. 39, 507 (1945).

222


[9]

will be determined. This procedure may be expressed as follows: NO~ N O ~ ~ F

R

L

-[- NH2CH--CO--protein

?

N02

--~ N 0 , ~ N H C H - - C 0 - - p r o t e i n Ethanol

D N P protein ~- H F

NaHCO3

NO2 NO~~NHCHCO--protein DNP protein NO2

t~

RI

--* N O 2 ~ N H C H C O O H -t- NH2CHCOOH Boiling DNP amino acid -t- amino acids 5.7 N HC1 The error of the method arises chiefly from the breakdown of the D N P amino acids during hydrolysis. As this breakdown is in certain cases considerable and may vary from protein to protein, correction is difficult, but it is sufficiently exact to determine accurately the number of terminal residues per molecule if this is less than ten.

Technique 1. Preparation of 1,2,$-Fluorodinitrobenzene. 2 101 g. (0.5 mole) of 1,2,4-chlorodinitrobenzene is dissolved in 101 g. of dry nitrobenzene, and to the mixture is added 60 g. (1 mole) of finely powdered dry KF. The mixture is heated on an oil bath at 190 to 195 ° for 5 hours under reflux and with very vigorous stirring. The mixture is cooled and filtered through sintered glass, the inorganic salts remaining being washed with hot toluene. The combined filtrates are dried over anhydrous Na2SO4 overnight, filtered, and the toluene distilled over in vacuo. The residue is fractionally distilled at 1 ram. pressure, and the fraction boiling at 60 ° (nitrobenzene) discarded. The second fraction, boiling at about 130 °, is collected and if necessary purified by redistillation. The F D N B thus obtained is a pale-yellow liquid which crystallizes on cooling (melting point 24°). The yield is about 70 g. 2. Preparation of D N P Amino Acids. D N P amino acids are required as standards for colorimetric estimation and for chromatographic identi2 H. G. Cook and B. C. Saunders, Biochem. J. 41, 558 (1947).

[9]


223

fication. Most of the derivatives to be expected from the hydrolysis of a D N P protein have been prepared and examples of the methods used are as follows. (It has been reported from several laboratories 3,~ that quantitative or near quantitative yields of D N P amino acids are obtained if only a slight excess of bicarbonate and F D N B is used.) a. DNP-L-valine: This m a y be taken as typical of all the simple D N P amino acids. Three-tenths gram (0.3 mole) of L-valine and 1.1 g. of NaHCO3 are dissolved in 14 ml. of H20, and to this is added 1.1 g. (0.6 mole) of F D N B dissolved in 28 ml. of ethanol. The mixture is shaken for 2 hours at room temperature, concentrated to remove ethanol, dissolved in water, and the excess F D N B extracted with ether. The aqueous solution is acidified with N HC1, and the DNP-L-valine, which precipitates, is filtered off. I t can be crystallized from aqueous methanol and recrystallized from ether-ligroin. Yield, 0.45 g. b. NS-DNP-L-lysine: 5 To prepare NS-DNP-L-lysine and N4-DNI)-L ornithine, the NUamino group is blocked in a copper complex before reacting with F D N B . One-half gram of L-lysine is dissolved in 10 ml. of H20, and CuC03 is added slowly to the boiling solution until no more dissolves. The excess CuCO3 is filtered from the dark-blue solution and washed with 2 to 3 ml. of H20. To this solution excess NaHCO3 and 1.1 g. of F D N B dissolved in 20 ml. of ethanol are added. The mixture is shaken for 2 hours at room temperature, and the precipitate filtered off and washed with water, ethanol, and ether. I t is suspended in 5 ml. of H20, and just sufficient N HC1 is added to produce a clear solution. This is cooled in ice, H2S bubbled through for 2 minutes, a little charcoal added, and the mixture filtered immediately. The filtrate is rapidly taken to dryness in vacuo and the NS-DNP-L-lysine hydrochloride crystallized from water and recrystallized from 20% HC1. Yield, 0.45 g. c. N~-DNP-L-lysine: ~ 1.25 g. of NS-benzoyl-L-lysine is dissolved in 10 ml. of water containing 1.0 g. of NaHCO~, and to it is added 1.0 g. of D N C B which has been dissolved in 20 ml. of ethanol. The mixture is refluxed on a water bath for 4 hours. The ethanol is removed by evaporation i n vacuo, the residue dissolved in water, and after filtering to remove excess D N C B it is acidified with HC1. On standing, an oil separates which weighs 2.0 g. Of this N1-DNP-NS-benzoyl-L-lysine, 0.5 g. is boiled under reflux for 3 days with a mixture of 5 ml. of acetic acid and 5 ml. of concentrated HC1. The mixture is evaporated to dryness, taken up in water, and filtered 3 W. A. Sehroeder and J. LeGette, J. Am. Chem. Soc. 76, 4612 (1953). 4 A. L. Levy and D. Chung, J. Am. Chem. Soc. 77, 2899 (1955). R. R. Porter and F. Sanger, Biochem. J. 42, 287 (1948).

224


[9]

to remove unchanged substance. On neutralization with pyridine the N1-DNP-L-lysine crystallizes out. Yield, 0.2 g. d. O-DNP-L-tyrosine: 1 0.55 g. of N-acetyl-L-tyrosine 6 is heated with 2.0 g. of D N C B for 4 hours as in the p r e p a r a t i o n of N~-DNP-L-lysine. On acidification an oil forms which later crystallizes. I t could be recrystallized from aqueous ethanol as white needles. This is N - a c e t y l - O - D N P - L tyrosine. Yield, 0.1 g.; melting point 194 °. The p r o d u c t is boiled for 3 hours under reflux with 2 0 % HC1, cooled, and e v a p o r a t e d to small volume. The precipitate obtained is filtered off, dissolved in warm, dilute H N 0 3 , and neutralized with pyridine while hot. O-DNP-L-tyrosine crystallizes in white needles containing 1 mole of water. Crystallization of D N P Amino Acids Other D N P derivatives are prepared according to one of the a b o v e methods; convenient solvents for crystallization are listed in Table I. Thc melting points of crystalline D N P derivatives will be found in Table II. TABLE I SOLVENTS FROM WHICH D N P AMINO ACIDS HAVE BEEN CRYSTALLIZED

Amino acid derivative DNP-L-alanine DNP-L-arginine DNP-D~-aspartic Di-DNP-L-cystine DNP-D~-glutamic acid DNP-glycine Di-DNP-histidine DNP-D~-isoleucine Di-DNP-L-lysine DNP-DL-methionine N~-DNP-~ornithine N'-DNP-L-ornithine DNP-~-phenylalanine DNP-~-proline DNP-DL-serine DNP-D~-threonine DNP-L-tryptophan Di-DNP-DL-tyrosine

Solvents Ethanol and water Dilute and strong HCI Water 1. Ethylene glycol monoethyl ether and water 2. Acetic acid and water Water Acetic acid and water Methanol and water 1. Formic acid and water 2. Methanol and water Dry ether and ligroin Water Water and dilute HC1 Methanol and water 1. Ether and ligroin 2. Acetic acid and water Methanol Methanol and water Methanol and water Methanol and water

e V. du Vigneaud and C. E. Meyer, J. Biol. Chem. 98, 295 (1932).

[9]


225

TABLE II M E L T I N G POINTS OF D N P

Amino acid derivative DNP-DL-alanine DNP-L-arginine DNP-DL-aspartic Di-DNP-~cystine DNP-DL-glutamic DNP-glycine Di-DNP-L-histidine DNP-nL-isoleucine DNP-DL-leucine Di-DNP-L-lysine N~-DNP-L-lysine N1-DNP-b-lysine DNP-nL-methionine

Melting point 178 ° 252 ° 195 ° 109 ° 170 ° 205 ° 250 ° 168 ° 131 ° 173 ° 186 ° 260 ° 117 °

(d.) (d.)

(d.) (d.)

(d.)

DERIVATIVES OF AMINO ACIDS a

Amino acid derivative

Melting point

N4-DNP-L-ornithine N1-DNP-L-ornithine DNP-nL-phenylalanine DNP-L-phenylalanine DNP-L-proline DNP-DL-serine DNP-Db-~hreonine DNP-L-tryptophan Di-DNP-DL-tyrosine O-DNP-Tyrosine (1 H20) DNP-Db-valine DNP-L-valine

223 ° (d.) 227 ° (d.) 217 ° 188 ° 137 ° 198 ° 152 ° 180 ° 84 ° 202 ° (d.) 185 ° 130 °

There are some discrepancies in the literature; these figures are based on data generously supplied by Dr. H. Neurath, Dr. F. Sanger, and Dr. T. S. Work, as well as personal observation.

Preparation of DNP Proteins T h e p r o t e i n is d i s s o l v e d or s u s p e n d e d in t e n t i m e s i t s w e i g h t of N a H C O 3 s o l u t i o n ( 5 % w / v ) . T o t h i s is a d d e d t w i c e t h e v o l u m e of a n e t h a n o l i c s o l u t i o n ( 1 0 % w / v ) of F D N B a n d t h e m i x t u r e is s h a k e n for 2 h o u r s a t r o o m t e m p e r a t u r e . T h e D N P p r o t e i n , w h i c h is u s u a l l y i n s o l u b l e , is c e n t r i f u g e d a n d w a s h e d t h r e e t i m e s e a c h w i t h w a t e r , e t h a n o l , a n d e t h e r . I t is a i r - d r i e d for s e v e r a l d a y s . I f a s o l u b l e D N P p r o t e i n or D N P p e p t i d e is e n c o u n t e r e d , t h e d i f f i c u l t y of r e m o v i n g t h e s a l t m a y be a v o i d e d b y u s i n g a v o l a t i l e b a s e s u c h as t r i m e t h y l a m i n e i n s t e a d of N a H C O 3 t o k e e p t h e reaction mixture alkaline. 7 T o e s t i m a t e t h e c o n t e n t of t h e o r i g i n a l p r o t e i n in t h e a i r - d r i e d D N P p r o t e i n i t is n e c e s s a r y t o e s t i m a t e t h e c o n t e n t of a g r o u p or residue, which is u n a f f e c t e d b y t h e r e a c t i o n , in e a c h s a m p l e . E s t i m a t i o n of t h e a m i d e group has been found to be a simple and accurate method. ~ Amide estim a t i o n s a r e c a r r i e d o u t b y h y d r o l y s i s in b o i l i n g 2 N HC1 for 4 h o u r s , f o l l o w e d b y d i s t i l l a t i o n of t h e a m i d e a m m o n i a in a m i c r o - K j e l d a h l a p p a r a t u s a t 95 °, u s i n g a b o r a t e b u f f e r a t p H 9.5. A n a p p r o x i m a t e figure for t h e r a t i o s c a n be c a l c u l a t e d f r o m t h e m o i s t u r e c o n t e n t a n d t h e n u m b e r of D N P g r o u p s t o be e x p e c t e d f r o m t h e a m i n o a c i d c o n t e n t . I n m o s t cases t h e a i r - d r i e d D N P p r o t e i n c o n t a i n s a b o u t 75 % of t h e o r i g i n a l p r o t e i n . 7 F. Sanger and E. O. P. Thompson, Biochem. J. 53, 353 (1953).

226


[9]

Hydrolysis of D N P Proteins The weight of D N P protein required depends on the number of a-amino groups present, but for preliminary examination 50 mg., is normally taken; this should be sufficient to identify one end group per molecular unit of 100,000. Owing to the lability of some D N P amino acids, it is necessary to carry out the hydrolysis under three sets of conditions. 1. Hydrolysis in boiling 5.7 N HC1 for 24 hours will destroy all peptide bonds and leave sufficient of all the D N P amino acids, except the derivatives of glycine, proline, and hydroxyproline, for identification. Under these conditions, DNP-tryptophan is converted to a compound which no longer gives a color with Erlich's reagent but which behaves chromatographically in the same way as DNP-tryptophan. 2. Hydrolysis in 12 N HC1 (sealed tube) at 105 ° for 24 hours enables DNP-proline and DNP-hydroxyproline to be identified. The yellow substances remaining are not DNP-proline and DNP-hydroxyproline, however. As with DNP-tryptophan, they behave chromatographically as the original compounds, but have a different absorption spectra, s 3. Boiling in 5.8 N HC1 for 4 hours will leave sufficient DNP-glycine for identification. Under the last two conditions D N P peptides may be present, but these can readily be recognized by submitting the colored derivatives to further hydrolysis. It has been observed 9 that if DNP-glycylglycine is hydrolyzed in a mixture of 90% w / v formic acid (10 vol.), acetic anhydride (5.5 vol., i.e., enough to combine with the water in the formic acid), and 60% w / v HC104 (1.4 vol.) for 2 hours at 100 °, there is complete hydrolysis of the peptide with very little destruction of the DNP-glycine. Such a medium may be of value also for hydrolyzing D N P proteins. For the estimation of D N P amino acids in a hydrolyzate it is clearly desirable to use the least drastic conditions of hydrolysis which are sufficient to break the adjacent peptide bonds. The approximate recoveries after hydrolysis in HC1 are given in Table III, but as the presence of the protein influences the amounts lost it is essential to carry out a direct estimation in the presence of the protein being investigated and under the conditions being used. Fractionation of D N P Amino Acids The D N P method has been used extensively during the last ten years, and it is noteworthy that the many modifications suggested have been 8 H. Fraenkel-Conrat and B. Singer, J. Am. Chem. Soc. 76, 180 (1954). 9 C. S. Hanes, F. J. R. Hird, and F. A. Isherwood, Biochem. J. 51, 25 (1952).

[9]

DNP METHOD FOR AMINO ACID S E Q U E N C E IN P R O T E I N S

227

confined a l m o s t e n t i r e l y to the p r o c e d u r e for t h e f r a c t i o n a t i o n of the D N P a m i n o acids. 1°-1~ This i n d i c a t e s where the g r e a t e s t difficulty has been met, b u t in this article a d e s c r i p t i o n will be given only of the m e t h o d s with which t h e a u t h o r is familiar. P a p e r c h r o m a t o g r a p h y is now used in preference to columns, as smaller a m o u n t s can be h a n d l e d successfully, t h o u g h in certain circumstances columns m a y still be used w i t h a d v a n tage. The p r o c e d u r e w i t h b o t h m e t h o d s will be described. TABLE III APPROXIMATE BREAKDOWN OF DNP AMINO ACIDS ON ACID HYDROLYSIS Hydrolysis in boiling 5.7 N HC1 Time, Amino acid derivative hours DNP-alanine DNP-arginine DNP-aspartic acid Bis-DNP-cystine DNP-glutamic acid DNP-glycine DNP-hydroxyproline DNP-isoleucine DNP-leucine Di-DNP-lysine NS-DNP-lysine DNP methionine DNP-phenylalanine DNP-proline DNP-serine DNP-threonine DNP-tryptophan DNP-tyrosine DNP-valine

12 12 24 12 12 8 4 12 12 8 I2 12 12 2 12 24 12 12 12

Hydrolysis for 16 hours in 12 N HC1 at 105°

Amount Amount unchanged (minimum), unchanged, % % 80 90 60 25 75 40 40 80 80 95 95 75 70 10 90 90 90 75 80

75 75 75 0 75 50 50 75 75 75 75 75 50 50 75 75 0 50 75

Paper Chromatography A description of the complete s e p a r a t i o n of D N P amino acids b y twow a y p a p e r c h r o m a t o g r a p h y has been described b y Levy. 13 This was d e v e l o p e d as a m e t h o d of amino acid analysis, a n d t h e p r o b l e m of endgroup analysis where only one or two different D N P amino acids are to be s e p a r a t e d a n d identified is m u c h simpler I n L e v y ' s m e t h o d the 10 F. C. Green and L. M. Kay, Anal. Chem. 24, 726 (1952). 1~S. M. Partridge and T. Swain, Nature 166, 272 (1950). l~ S. Blackburn, Biochem. J. 45, 579 (1949). 19A. L. Levy, Nature 174, 216 (1954).

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[9]

toluene-chloroethanol-pyridine-0.8 N ammonia solvent (Biserte and Osteux ~4) is run first, and 1.5 M aqueous phosphate buffer second. We have had difficulty in getting satisfactory results with the solvent of Biserte and Osteux and prefer to use the tertiary amyl alcohol solvent described by Blackburn and Lowther, 16 followed by phosphate buffer if necessary. The procedure is as follows: The protein hydrolyzate is diluted several times with water and extracted three times with ether, the ethereal extracts being washed three times with small quantities of water. If large quantities of water are used for washing the extract, some loss of DNP-serine and DNP threonine may occur. This extract, which contains all the acidic DNP amino acids, is then taken to dryness i n vacuo, water added, and again taken to dryness. This is repeated until the HC1 has been removed. The DNP amino acids are now taken up in a few drops of ethanol for application to the paper for chromatography. The paper (Whatman No. 4) is washed, by irrigation, with 0.05 M phthalate, pH 6, before use. The tertiary amyl alcohol, which should be free of peroxide, is equilibrated with a small quantity of buffer. Known DNP amino acids are spotted onto the paper as markers (about 20 ~, gives a satisfactory color), together with the unknown. The paper is now hung in the chromatographic tank in which stands a beaker with the tertiary amyl alcohol saturated with buffer. It is allowed to equilibrate with the solvent vapor for several hours before addition of solvent to the trough. If this equilibration is omitted the spots may tail badly. In an overnight run satisfactory resolution should be obtained of most of the DNP amino acids except DNP-aspartic acid and DNP-glutamic acid. These may be resolved if the ehromatogram is continued for a longer period. After the paper is dried, the spots are cut out and eluted by standing with occasional shaking in 4 ml. of 1% NaHCO3 for 15 minutes at 60 °. The extract is read at 3600 A. in a spectrophotometer. In order to compensate for any losses during chromatography, comparison is made with the absorption of known aliquots of standard solution run on the same paper. If the identity of the unknown is in doubt, the solution is acidified and the DNP amino acid extracted into ether and again chromatographed with 1.5 M phosphate buffer (containing 1 M NaH2PO4 and 0.5 M Na~HP04). This procedure is considered to be more satisfactory than running a two-way chromatogram directly when only one or two unknowns are expected. 14 G. Biserte and R. Osteux, Bull. soc. chim. biol. 83, 50 (1951).

j6 S. Blackburn al~d A. G. Lowther, Biochem. J. 48, 126 (1951).

[9]


229

Fractionation with Silica Gel Columns

1. Preparation of Silica Gel. 16 One volume of commercial water glass. 1 vol. of H20, and 1 vol. of ice are well stirred in a large vessel; 12 N HCI is slowly poured in with continual stirring until the mixture of precipitated silica is acid to thymol blue. During the precipitation the mixture becomes very stiff, but it thins again as the correct pH is reached. It is allowed to stand for 3 hours, HC1 being added to maintain the acidity if necessary. The silica is filtered off under suction on a large funnel and washed well (approximately 2 1. of water per 250 g. of gel). This precipitate is suspended in 0.5 N HC1 for 2 days, filtered, and vcashed until free of chloride. It is then dried at about 100°. Only distilled water should be used throughout this preparation. There is some variation in the chromatographic behavior of different preparations, but in no case has an unusable batch been obtained. Generally the silica is prepared in large batches (say 5 kg.) in order that its characteristics may be fully understood and used to advantage. Samples are kept of any batch which proves to be particularly effective in certain fractionation steps. The variability of different batches of gel may be reduced if it is allowed to stand m HC1 solution of known concentration before washing and drying.17 The commercial water glass may be bought in 5-gallon drums from Joseph Crosfield Ltd., Warrington, England. 2. Preparation of Silica Gel Columns. The wet gel is prepared in several ways according to the solvent system in conjunction with which it is to be used. In most cases one-half its weight of water or buffer is well stirred into the dry gel. If intended for fractionation of basic DNP amino acids one-half its weight of N HC1 is used instead of the water, as N 5DNP-L-lysine is more stable in acid solution. If mixed organic solvents with a water-miscible component are used, two-thirds its weight of the aqueous phase is ground into the dry gel. To prepare the column, the wet gel (6 to 10 g.) is well mixed with wet organic solvent to give an even, freely running suspension. This is poured into a vertically held glass tube (30 X 1 cm. is convenient) whose lower end has been turned to support a perforated silver disk and filter paper. The column is rotated as it settles to remove air bubbles, and the supernatant solvent is allowed to drain through, care being taken that the bottom of the column is always immersed in the solvent in the receiving vessel. Unless this is done, the column will crack and have to be discarded. Despite this precaution frequent cracking of columns occurs if the gel has too high a water content. If a small positive pressure is used in the prepa,8 A. H. Gordon, A. J. P. Martin, and R. L. M. Synge, Biochem. J. 37, 79 (1943). ~7 p. Desnuelle, M. Rovery, and G. Bonjour, Biochim. et Biophys. Acta 5, 116 (1950).

230


[9]

ration of the column, more even packing and, therefore, sharper bands are obtained. The compositions of the solvent systems, and abbreviations used to denote them, are given in Table IV. All the solvents are kept in contact with a small a m o u n t of aqueous phase, and sufficient time for equilibration should, of course, be allowed. The only exception to this procedure is the glycol-benzene system, in which case the glycol replaces the H~O and the gel is prepared by grinding the d r y gel with an equal weight of glycol. TABLE IV COMPOSITION OF SOLVENT 1VIIXTURES

Abbreviation

Composition

Chloroform Chloroform saturated with H~O CB1 99 % chloroform, 1% n-butanol, saturated with H~O 97 % chloroform, 3 % n-butanol, saturated with H~O CB3 95% chloroform, 5% n-butanol, saturated with H~O CB5 85% chloroform, 15% n-butanol, saturated with H20 CB15 70% chloroform, 30% n-butanol, saturated with H~O CB30 1 vol. I-I~O, 2 vol. ethanol, 10 vol. petroleum ether (b.p. 90-100 °) AL 1 vol. ether, 2 vol. petroleum ether (b.p. 90-100°), saturated with H~O EL 1 vol. H20, 1 vol. acetone, 10 vol. cyclohexane AC 1 vol. propanol, 20 vol. cyclohexane, saturated with H20 PC 1 vol. H20, 1 vol. methanol, 15 vol. carbon tetrachloride MC 2 vol. methyl ethyl ketone, 1 vol. ether, saturated with H20 M66 Benzene saturated with glycol GB Chloroform should be well washed before use, as the 1% ethanol, frequently added by manufacturers as a stabilizer, greatly alters its behavior. 3. Fractionation of D N P Protein Hydrolyzate. The mixture of D N P amino acids obtained b y ether extraction as described previously is dissolved in 1 to 2 ml. of the desired solvent (wet chloroform in the first instance) and run onto the prepared column. A bent-tipped pipette is used to avoid disturbing the gel surface. Quantitative transfer is effected by washings of the same volume added after the previous extract has sunk into the gel. With certain solvents a considerable number of washings m a y be necessary, though taking to dryness after addition of a few drops of 12 N HC1 often assists subsequent solution of the D N P derivatives. After transfer has been completed, the head space of the tube is filled with solvent and an indication of the D N P amino acids present can be obtained b y measuring the R values. (The R value is the rate of m o v e m e n t of the front of the band relative to the rate of movement of the surface

[9]


231

of the solvent.) Synthetic D N P derivatives are run on parallel columns with the same solvent to give standard R values. In the hydrolyzate of a D N P protein it is usual to find a yellow band moving faster than any known D N P amino acid on a chloroform column. This is an artifact band of uncertain origin and should be ignored. If parallel columns of synthetic D N P amino acids are used, further fractionation according to the scheme in Fig. 1 should enable the components of an unknown mixture to be identified. To test the conclusions, the unknown is shown to be separable from other synthetic D N P derivatives, on an appropriate column, and inseparable from the identical derivative with any solvent system. It is most important in this work of fractionation and identification that only sufficient D N P derivatives are put on the column to make them clearly visible (50 to 100 ~ of D N P amino acid on a column 1 cm. in diameter). Greater amounts of color increase their R values and sometimes make separation impossible. Fractionation of the basic D N P amino acids, left in the aqueous residue after extraction with ether, follows the scheme shown in Fig. 2. In addition to the artifact band moving fast on a chloroform column, already referred to, other artifact bands may appear. Thus DNP-tyrosine and DNP-methionine on acid hydrolysis give rise to second colored bands stationary on a chloroform column, and DNP-tryptophan to a brown compound which is not soluble in ether. These artifacts are small relative to the DNP amino acids from which they arise and can be recognized by further fractionation. The possible presence of small amounts of D N P peptides must also be considered if uncharacterizable bands are found, as some of the D N P peptides are remarkably stable to acid hydrolysis. During work with D N P derivatives it is important that they be protected from direct sunlight, which causes discoloration and decomposition. If difficulty in fractionation is met, recourse to buffered columns with phosphate buffer in the range pH 3.5 to 6.5 may be helpful. 12 The rate of movement of the amino acids is greater with more acid buffers. Variation in the chromatographic behavior of different silica gel preparations still occurs with buffered columns, but perhaps less than when water is the stationary phase. 4. Estimation of DNP Amino Acids. In the quantitative assay of the end group of proteins it is desirable to use rather larger amounts of material (200 to 500 ~/ on a column 1 cm. in diameter) than are needed for identification because if only small quantities are taken the traces of D N P amino acids occasionally adsorbed onto the gel become a significant loss. The acidic D N P amino acids are collected as they run from the column, the organic solvent is removed in vacuo, and the acids are dissolved

232


~?

[9]

~. O

O

i

O CD ¢J

CD

O

__

.

[9]

DNP METHOD FOR AMINO ACID S E Q U E N C E IN P R O T E I N S

233

in 1 % N a H C O 3 solution for estimation in a photoelectric colorimeter at 3600 A. Calibration with s t a n d a r d solutions is, of course, necessary. The basic D N P amino acids are t r e a t e d similarly but are dissolved in N HC1 and estimated at 3900 A. Their m a x i m a l absorption is at 3600 A., as with the other derivatives, b u t c o n t a m i n a t i o n with colorless O D N P - t y r o s i n e , which also absorbs in t h a t wavelength, occurs. Original Mixture

t M66 I

1

t

1

I

2

i

3

4

[ N1-DNP-arginine N6-DNP-lysine NI-DNP-lyslne CB17

t

i

1.1 di-DNP-histidine

1

1.2 S-DNP-eysteine

FIG. 2. Scheme of fraetionation of water-soluble DNP amino acids. T a b l e V 18 gives the optical densities of some D N P - a m i n o acids and peptides, and Figs. 3 and 4 show their absorption curves. D N P - p r o l i n e is exceptional in t h a t its maximal absorption is at 3850 A. OPTICAL DENSITIES OF

TABLE V 20-~M. SOLUTIONS OF DNP AMINO ACIDS AND PEPTIDES Optical density (log Io/I)

DNP derivative DNP-glycine DNP-phenylalanine DNP-glyeyl-glycine DNP-phenylalanyl-valine NS-DNP-lysine O-DNP-tyrosine

Solvent

3500 A.

3900 A.

1% NaHCO3 1% NaHCO.~ 1% NaHCO~ 1% NaHC03 N HC1 N HC1

0. 309 0.313 0.316 0.310 0. 296 0. 058

0. 210 0. 214 0. 173 0. 178 0. 204 0.0

5. Determination of Amino Acid Sequences of Terminal Peptides of Proteins. I n a s t u d y of terminal peptides with the D N P technique it is preferable t h a t the work should be done with a single polypeptide chain; otherwise the confusion arising f r o m different sequences in different chains m a y p r e v e n t a d e q u a t e interpretation of the results obtained. ~sF. Sanger, Biochem. J. 45, 563 (1949).

234


[91

Insulin is the only protein where the different polypeptide chains have been separated from each other. In this case the disulfide bridges, apparently the only stable interchain bonds, were split by oxidation with performic acid. 19 The oxidation products could then be separated into two main fractions which corresponded to the two peptide chains. Other proteins made of dissimilar chains would no doubt present special problems.

0.4

Z

.t..,

0.2

o"

\

/ \

' ~,

/

%,

0.I 0 2500

"', I

f

3000

3500

4000

4500

5000

Wavelength (A) Fro. 3. Spectral absorption curves in 1 cm. cell of 23.7-~M. solution of D N P phenylalanine ( ) and DNP-phenylalanylvaline ( ....... ) in 1% NaHCO3.

If end-group analysis has shown the protein to contain only one open chain, or if the different chains can be fractionated, this material is treated with F D N B and subjected to partial hydrolysis. The terminal D N P amino acid and the terminal D N P peptides of increasing size may be separated from NLDNP-lysine and NS-DNP-lysyl peptides by extraction of the hydrolyzate with ethyl acetate and then separated from each other by chromatography in a similar way to the fractionation of D N P amino acids. Complete hydrolysis of these peptides should release the same terminal D N P amino acid and an increasing number of free amino acids to which it was bound. These amino acids may be identified by paper chromatography and hence the order of terminal sequence deduced. This method has been usefully applied to determine the structure of terminal penta- and hexapeptides. Larger D N P peptides are so little soluble that fractionation becomes impossible. 19 F. Sanger, Biochem. J. 44, 126 (1949).

[9]


235

PARTIAL HYDROLYSIS. The best conditions of hydrolysis to produce a series of D N P peptides of increasing size can only be found by trial with the particular protein or peptide to be investigated, as the stabilities of different peptide sequences vary greatly (cf. Sanger2°). Hydrolysis with HC1, with the strength, temperature, and time varied, may give the desired maximum yield of smaller peptides without too complete hydrolysis. FRACTIONATION. The partial hydrolyzate is diluted if necessary and treated as follows: (1) The hydrolyzate is extracted three times with ethyl acetate. (2) The ethyl acetate extracts are combined and extracted in countercurrent manner three times with 1% NaHC03 solution. (3) The

0.4

0.3

._~

0.2

o 0.1

0 .... 2500

I 3000

I ' 3500

~

1 4000

I 4500

5000

Wavelength(A) Fro. 4. Spectral absorption curves in 1 cm. cell of 22-~M. solutions of N L D N P lysine ( ) and O-DNP-tyrosine ( ....... ) in N HC1.

combined NaHCOa solution extracts are acidified with HC1 and extracted as above with ethyl acetate. This final extract should contain all the acidic D N P peptides and very few nonacidic D N P peptides. Some N L DNP-lysyl peptides may, however, persist in the ethyl acetate fraction, and, though none have been met, it is possible that terminal peptides containing a basic residue would remain in the aqueous fraction. For fractionation of the peptide derivatives oil silica gel columns, chloroform containing up to 30% by volume n-butanol has been found to give a most useful series of solvent mixtures. Thus, if the crude ethyl acetate extract is run on a CB15 column (see Table IV), all but the NLDNP-lysyl peptides, which have been extracted into the ethyl acetate, and perhaps very large terminal peptides, move fast. This fast~0 F. Sanger, Adxances in Protein Chem. 7~ 1 (1952).

236


.[9]

moving band is then put on a CB5 column (see Table IV), and the new fast-moving band again transferred to a chloroform column. The peptide bands which run slowly are developed, if necessary, with intermediate chloroform-n-butanol mixtures. Buffered gel systems have also been used to advantage on occasion, but some induce considerable tailing of the bands. In general, the more acid the buffer used, the more quickly will the DNP amino acids and peptides move down the column. Tailing is a difficulty met more frequently with DNP peptides than with DNF amino acids and varies with the batches of gel used. The running of the colored bands on different columns to establish homogeneity is particularly necessary when pronounced tailing occurs. In the partial hydrolysis products of both DNP-insulin and DNP rabbit ~,-globulin, small amounts of color, which are stationary on a chloroform column but which move on a CB1 column to split into a number of fractions, have occasionally been observed. They contain a complex mixture of amino acids which may arise from esterification occurring during the procedure. Other anomalous behavior, such as the splitting on a column of a homogeneous component to give identical fractions which subsequently behave similarly, rarely occurs with DNP amino acids but has at times been observed during work with DNP peptides. This fractionation procedure may have to be adapted considerably to meet the requirements of the great variety of peptide mixtures which can occur, but it has proved successful in the applications so far made. Estimation of the DNP peptides is carried out as for the DNP amino acids; the absorption is read at 3500 A., where it is uninfluenced by adjacent amino acids in peptide linkage. 6. Identification of D N P Peptides. The DNP peptides are collected as they run from the column, the solvent removed and the residue dissolved in 5 to 6 drops of 5.7 N HC1 by warming over a microburner. The solution is sucked into a capillary tube which is sealed and kept at 105° for 24 hours. The hydrolyzate is dropped into a small tube for extraction with ether and the DNP amino acid in the ethereal fraction identified as previously described. The aqueous residue is transferred by the capillary tube to a polythene strip and taken to dryness in a desiccator to remove excess HC1. A drop of water is added to dissolve the amino acids and the drop " p r i n t e d " on to a filter paper sheet so that the acids may be identified by partition chromatography. This method is based on that described by Consden et al. 21 for the identification of peptides. In any partial hydrolyzate a mixture of terminal peptides will result, and should the original material have only one polypeptide chain with, consequently, one terminal amino acid, the colored derivatives obtained ~ R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 41, 590 (1947).

[9]


237

under appropriate hydrolytic conditions will be constituted as follows: (1) D N P A, (2) D N P A + B, (3) D N P A -b B -[- C, (4) D N P A q- B ÷ C q- D, where A, B, C, and D correspond to different amino acids. If it is possible to account for the total amount of D N P A known to be originally present in terms of these peptides and to show that, say, peptide 4 will, on further partial hydrolysis, give only peptides 3 W 2 and D N P A, it is reasonable to conclude that the terminal sequence of amino acids in the chain is ABCD. Clearly the same method may be used to determine the terminal sequence of small or large peptides obtained by the hydrolysis of a protein. In this case fractionation of the hydrolyzate is the principal difficulty, and it is essential that the purity of the peptide to be investigated is established before partial hydrolysis of the D N P derivatives is undertaken. The presence of only one terminal amino acid would be good enough though not conclusive evidence of the purity of the peptide to be examined.

NS-DNP-Lysyl Peptides If only one or two lysyl residues are present in the peptide or protein being studied, it is possible to identify the adjacent amino acids in an analogous manner. These DNP-lysyl peptides will be in the aqueous residues from the ethyl acetate extraction together with the unsubstituted peptides. There is a possibility that the latter will not be separated from the DNP-lysyl peptides during chromatography. Sanger 17 overcame this by absorbing the D N P peptides onto talc from acid solution, other amino acids and peptides passing through. After the talc was washed with N HC1 the D N P peptides were eluted with 4 parts of ethanol and 1 part of N HCI. There is still some risk of the N~-DNP-lysyl peptides being contaminated by O-DNP-tyrosyl peptides which are colorless. Such contamination could be detected from the shape of the absorption curve of the lysyl peptides, as mentioned earlier. Fractionation of the NS-DNP-lysyl peptides may be effected with CB15, CB30, and M66 columns. It should perhaps again be stated that the methods of fractionating D N P peptides need to be varied according to the protein being investigated and the special problems the resulting partial hydrolysis mixture offers.

238


[10]

[10] The Pipsyl Method for Amino Acid Sequences in Proteins B y MILTON LEVY

Principle A reagent, p-iodobenzenesulfonyl chloride (pipsyl chloride), reacts with amino and phenolic hydroxyl groups to form stable derivatives. The amino derivatives have a molar absorption coefficient of approximately 16,000 at 250 m~. The reagent can be prepared with 1131 or S ~5 label. If the reaction between the reagent and the functional groups mentioned can be made stoichiometric, quantitative analyses of the derivative mixtures can be made on the isotopic dilution principle. Two forms of this method have been proposed. These are the carrier method,~ in which the diluent is the unlabeled compound of interest, and the indicator method, ~ in which the diluent is the compound of interest labeled with a different isotope than the unknown. These methods differ from the classical isotope dilution method by introducing a detectable label in the unknown rather than in the cartier. They can be used to demonstrate the absence of a particular derivative with a certainty not possible by any other method. The key to adequate quantitative work by this method is the initial derivative formation. Methods for quantitative derivative formation are available for most amino acids (see below) and for some peptides. The reactions of proteins require individual study2 In spite of the great resistance of the sulfonamide bond to hydrolysis and the use of internal control in the hydrolysis, the reagent is not yet firmly established as an end-group label for proteins. Qualitative but not quantitative agreement with the DNBF method has been demonstrated, s Independence of quantitative yield in the pipsylation reaction can be achieved by adding an isotopically labeled sample of the substance to be determined (i.e., C14-1abeled alanine) to the unknown and isolating the derivative from the mixture. 4 This method is closely related to classical isotopic dilution. Thus the absence of the substance is difficult to establish because the dilution of the C 14label is zero and the errors of counting and spectrophotometry obscure the result. 1A. S. Keston, S. Udenfriend,and R. K. Cannan, J. Am. Chem. Soc. 71, 249 (1949). A. S. Kestoa, S. Udenfriend, and M. Levy, J. Am. Chem. Soc. 72, 748 (1950). 3S. Udenfriend and S. Velick, J. Biol. Chem. 190~ 733 (1950). ' A. S. Keston and J. Lospalluto, Federation Proc. 11, 239 (1952).

[10]

PIPSYL METHOD FOR AMINO ACID SEQUENCES IN :PROTEINS

239

Preparation of Labeled Pipsyl Chloride Standard radiological safety precautions are necessary t h r o u g h o u t all procedures but will not be described here. The methods described have given radiochemical yields of 70 to 80 %.

Sulfanilic Acid (S 35) HOAC C H 3 C O - - N H C 6 H 5 + H2SO4 ' > CH3CONHC6H4SO~H (AcO)20 1 N HCI CHaCONHCsH4SO3H -t- H20 > CH3COOH -t- NH2C6H4SO~H The procedure is based on the method of SSll and Stutzer ~ as modified b y Lichtin 6 for high conversion of sulfur to the derivative. Isotopic sulfuric acid is obtained carrier-free from the Oak Ridge National Laboratories in lots of 100 to 200 mc. E v a p o r a t e the solution to 0.5 ml. in a 25-ml. flask under vacuum. (All glass apparatus referred to is standard-taper "Micro Organic. ") Add 80 rag. (0.8 millimole) of sulfuric acid, and heat the open flask until dense white fumes appear. Cool. Add 1.4 ml. of glacial acetic acid and 0.6 ml. of redistilled acetic anhydride. R e m o v e these solvents in vacuo at 55 ° (bath). Again add the same a m o u n t of acetic acid and the anhydride to the residue. Add 100 mg. (0.82 millimole) of acetanilid, and heat in a bath at 60 ° for 2 hours under a 100-cm. aircooled condenser. Let the mixture stand at room temperature for 16 hours. A copious white precipitate will be present. Add 15 ml. of 3 N HC1, and reflux for 2 hours. Remove all solvents b y distillation in sacuo to dryness. Add 10 ml. of water, and evaporate. The d r y residue contains a b o u t 0.8 millimole of SSS-labeled sulfanilic acid. Dissolve it in 1.5 ml. of 1 N N a O H for the next step.

Sodium Pipsyl (S ~5) HC1 NH2--CsH4SO3H + H N O 2 > + +N2C6H4SO~- -/- H20 +N2CsH4SO~ ~- I---~ IC6H4SO3H + N2 Add 55 mg. (0.8 millimole) of sodium nitrite. Cool on ice. Add 3.2 ml. of 3 N HC1. After 30 minutes add a solution of 34 rag. of sulfanilic acid (0.2 millimole) in a minimal a m o u n t of 1 N NaOH. Test for nitrous acid with starch iodide paper. If present, add sulfanilic acid. After 0.5 hour add a solution of 250 rag. of K I in water. Let stand overnight at room temperature, and warm to 60 °. Neutralize with solid NaHCO3, and J. SSll and A. Stutzer, Ber. 42, 4539 (1909). e R. A. Lichtin, M. S. thesis, New York University, 1945.

240


[10]

evaporate by letting the solution stand in an oven at 90 to 95 °. Sodium Pipsyl (I 131)

+N~C6H4SO~

-{- I---~ IC6H~SO3H

~ N~

Carrier-free I TM (I00 to 200 inc.) is obtained from the Oak Ridge National Laboratories in alkaline solution containing bisulfite. Transfer the main portion to a 25-ml. pear-shaped distilling flask. Rinse the remaining I TM to the flask using 10 mg. of K I in 1 ml. of water and t h e n 1 ml. of water. Evaporate the solution in vacuo to about 0.5 ml. Remove the still head, and wash the capillary inlet with 0.5 ml. of water. Dissolve 57 rag. (0.3 millimole) of sulfanilic acid in 0.5 ml. of 1 N NaOH. Add 21 rag. of NaNO~, cool on ice, and add 1 ml. of 3 N HC1. After 10 minutes a white precipitate appears. Add i ml. of 3 N HC1 to the cooled " i o d i d e " solution. Add 0.4 ml. at the diazo slurry. Shake until all has dissolved, and warm to 60 ° for 3 minutes. Cool. Add 10 rag. of KI, dissolve, and add 0.4 ml. of a diazo slurry. Mix and warm to 60 °. Cool, add 10 mg. of KI, dissolve, and add 0.1 ml. of diazo slurry. Warm to 60 °, let stand for an hour, and then add solid NaHCO3 cautiously until neutral. Evaporate overnight in an oven at 90 to 95 ° . Pipsyl Chloride (S ~5 or I TM) POCl3 4IC~H4SO3Na q- PC15

~ 4IC6H4SO~C1 q- NaC1 q- Na3PO4

Add 1 ml. of POC13 to the dried sodium pipsyl, and break up the solid with a glass rod. Add a pea-sized portion of PC16, and crush with the rod. Warm to 60 ° for a few minutes. Add the same amount of PC15 twice more and additional POC13 to keep the mixture liquid except for the NaC1 present. Fill a 250-ml. separatory funnel half full of crushed ice. Transfer the mixture in the flask to the separatory funnel with four 5-ml. portions of benzene. Shake to destroy the phosphorus chlorides, and allow the ice and benzene to melt. Add solid sodium bicarbonate cautiously until the aqueous layer is neutral to congo red paper. Discard the aqueous layer, and wash the benzene with 20 ml. of ice water. This washing should not be acid. Transfer the benzene layer to a 125-ml. Erlenmeyer flask, and dry it with a minimal amount of anhydrous Na~SO4. After an hour transfer the benzene solution to a distilling unit, and remove the benzene in vacuo. Wash the Na2SO4 residue with benzene containing 50 mg. of ordinary pipsyl chloride. Repeat this washing a second time. Evaporate all the benzene solutions to a volume of about 0.5 ml., and transfer to a cold-finger vacuum sublimation apparatus, using a little benzene. Remove the benzene in the sublimation apparatus at room temperature with no

[10]

PIPSYL METHOD FOR AMINO ACID SEQUENCES IN PROTEINS

241

water flow in the condenser. Sublime the pipsyl chloride in a bath at 150 ° and at a pressure of about 3 mm. The white crystalline deposit on the cold finger is washed with benzene into a 20-ml. vial containing 1 g. of ordinary pure pipsyl chloride. Bring all material into solution with as little benzene as possible, using gentle heat. About 2 ml. should be sufficient. After cooling add 8 ml. of petroleum ether to precipitate the pipsyl chloride. Evaporate the solvents with a stream of dry N2. Add another portion of petroleum ether, and again evaporate. Nonisotopic pipsyl chloride is prepared by the methods described by Weygand. 7 It should be sublimed at 150 ° and 3 mm. to ensure purity. It can be readily recrystallized from benzene. Nonisotopic pipsyl chloride does not discolor over long periods, but the radioactive samples do. The I131-1abeled material discolors more quickly than the S aS. The discoloration does not interfere with the utility of the reagents.

Carriers Dissolve about 30 millimoles of amino acid in 50 ml. of N sodium bicarbonate in a flask provided with an efficient stirrer. Add 6 g. (20 millimoles) of pipsyl chloride, and heat to about 90° to melt the chloride. With efficient stirring the pipsyl chloride goes into solution quite rapidly. Cool the solution, and add 1 g. of KC1. A small amount of impurity precipitates. Add activated charcoal, filter, and acidify the filtrate. Crystals of the derivative generally appear. If not, extract the aqueous layer with ether or ethyl acetate, extract the organic layer with a small volume of NaOH solution, heat to drive out solvents, and acidify. The materials are readily recrystallized from hot water or from water-acetone mixture.

Indicators These are used in much smaller amounts than the carriers. They are prepared from one of the isotopic pipsyl chlorides, usually S% Weigh 6 to 10 mg. of S 3s pipsyl chloride on a bit of aluminum foil, and transfer to a dry Folin-Wu blood sugar tube. Measure into the tube 0.1 m]. of amino acid (or other pure compound) solution containing 50 micromoles of amino group, and add 0.4 ml. of saturated borax solution. Clamp the tube on a ring stand to which is attached an electric massage unit of the motor-driven type. The object is to produce a vibration of 5- to 10-mm. amplitude at about 3600 cycles. Bring a hot water bath about the tube, and when the pipsyl chloride is molten start the vibration. At 90 ° the pipsyl chloride disappears in a minute or so. Stop, cool, and add a glass bead to the tube and 2 ml. of ether. Shake, remove the ether with a C. Weygand, "Organic Preparations," p. 112. Interscience, New York, 1945.

242


[10]

Pasteur pipet, and repeat the extraction twice more. Transfer the aqueous layer to a 3 g. column of Dowex I (2 % cross-linked in the chloride form, 200 to 400 mesh before cycling), and elute with a solvent made of 350 ml. of 95% alcohol, 10 ml. of 1 N HC1, and water to make 1 1. The rate of elution may be 3 to 6 ml. per hour, and hourly collections are made. The compound may be located by ultraviolet absorption (250 m~) or by radioactivity. The combined fractions containing the derivative may serve as indicator stock, or the solutions may be evaporated and the compound dissolved in a small volume of alkali.

Preparation of Radioactive Standards Pure DL-alanine obtained by hydrolysis of benzoyl alanine is pipsylated with the iodine or sulfur reagent and purified by the method described above. Evaporate the solution to dryness, and recrystallize the pipsyl alanine from 1 ml. of hot water by cooling on ice. After two recrystallizations, discard the supernatants and dissolve the residue in about 1 ml. of water with a few drops of dilute ammonia. Accurately dilute 25 ~l. of this solution to 25 ml. with 0.2 N HC1, and measure the absorption of the resultant solution at 250 m~ in a 1-cm. cell on the Beckman spectrophotometer. The absorption coefficient for pipsyl alanine at this wavelength is 15.8 per micromole per milliliter. Planchettes are prepared by evaporating suitable amounts of a dilution of the stock in water. The amounts of material on the planchettes are so small as to make self-absorption corrections unnecessary. The fresh reagents prepared as above have shown 1 to 2 X 10e (for S 35) and 8 × 10e (for 1131) counts per minute per micromole under mica window (1.7-mg./cm. 2) Geiger tubes. The counting apparatus must be rigorously standardized for geometry, dead-time losses, etc.

Preparation of Unknown The mlknown in 0.1 ml. of solution should contain about 1 micromole of total amino acompounds, but the amount may be increased simply by providing additional pipsyl chloride in about stoichiometric amounts. The water and amino compound in the mixture compete for the pipsyl chloride so that the hydrolysis at an absolute amount of pipsyl chloride (about 20 micromoles) is accompanied by the formation of pipsyl derivative from about 0.7 to 0.9 of the amino compound present. If attempts are made to drive the reaction further by addition of more pipsyl chloride, some of the product may be lost by "dipipsylation" or decomposition of amino acid. 8 To obtain quantitative conversion, the product is removed and the reaction is carried out in three steps. s E. Slobodian, in preparation.

[10]


243

Weigh 7 mg. of radioactive pipsyl chloride (generally the I TM reagent) into a Folin-Wu tube, add the sample in 0.1 ml. of water, and then 0.5 ml. of saturated borax (standardize by titrating with standard HC1 to methyl red). It is about 0.3 N in available alkali. Clamp to the vibrator and water bath unit, and carry out the reactions as indicated for preparation of indicators at a bath temperature of 90 to 95 °. Cool and acidify with 1 ml. of 3 N HC1. Add a glass bead. Extract three successive times with 3-ml. volumes of ether. Place the extracts in a 250-ml. Erlenmeyer flask. Connect a rubber-stoppered outlet tube to the top of the Folin tube, and evaporate while vibrating in the hot-water bath with an aspirator vacuum. Add 1 ml. of water, and evaporate again. To the residue in the tube add 10 mg. of the same pipsyl chloride preparation as previously, then 0.5 ml. of 0.3 N NaOH, and repeat the processes as described. A final reaction using 15 mg. of pipsyl chloride is carried out to ensure nearly quantitative conversion. To the combined ether or ethyl acetate extracts add a few drops of concentrated aqueous ammonia to react with any residue of pipsyl chloride. The process described above is suitable for amino acids. Peptides react more completely in Na2HP04 solutions. Instead of saturated borax add 28 mg. of Na2HPO4 and 0.5 ml. of water. Use 15 mg. (50 micromoles) of pipsyl chloride. The yield for tested peptides is 85 to 95 % in a singlestage reaction. After cooling add 1 ml. of 3 N HC1 and a glass bead. Extract with three portions of 3 ml. each of ethyl acetate. If desired, the aqueous residue may be evaporated and 0.5 ml. of 2 M NaOH added with additional pipsyl chloride (15 mg.). The process is repeated and the additional ethyl acetate extracts added to the others.

Analysis It will be assumed that the indicator method is to be used. The carrier method allows crystallization to be used in the purification, but this seems to be less powerful as a separation device than are the egraphic* (chromatographic) methods to be described. Add measured volumes of one or more of the indicator stock solutions to the organic layers; make sure that homogenization of the isotopic compounds is complete by adding sufficient NH3 and water to ensure solution. From this point on quantitative recovery of compounds is not necessary. The object is to obtain demonstrably pure samples for determination of isotope ratio. Separation techniques which have been used are countercurrent distribution between solvents (for distribution coefficients see Table I), ion exchange egraphy (chromatography) on Dowex 1 X 2 C1- with 35 % or 20 % alcoholic solvents * Egraph: a writing out; Latin e (ex) out, and graphus, written. The meaning is synonymous with chromatography b u t does not suggest color.

244


[10]

TABLE I DISTRIBUTION COEFFICIENTS OF PIPSYL DERIVATIVES (Systems: I, CC14-0.2 N HC1; II, CHC18-0.2 N HC1; III, ethyl ether-0.2 N HC1; IV, ethyl acetate-0.2 HCI; V, n-butanol-0.2 N HC1; VI, n-butanol-0.2 N NHs. Values entered are concentration in organic phase/concentration in aqueous phase) Pipsyl derivative of Alanine Aspartie acid Glutamic acid Glycine Hydroxyproline Isoleueiae Leucine Methionine Phenylalanine Proline Serine Threonine Valine Alanylglyeine Alanylalanine Glycylalanine Glycylglyeine Glyeylserine Glycylalanylglycine Alanylglyeylglycine Glycylalanylglycylalanine

I

II

0.04 0.00 0.00 0.01 0.01 1.60 1.24 0.26 0.75 0.67 0.00 -0.48 0.00 -0. 004 0.01 -----

2.9 O. 01 0.01 0.63 0.11 50 100 . . 47 0.05 0.16 22 0.06 0.23 0.15 0.05 ---0.20

III 100 15 17 43 4.7 100 100 . .

. . 56 0.42 7.9 100 2.4 4.8 4.2 1.5 0.34 0.34 0.20 0.41

IV

V

VI

100 90 100 100 30 100 I00 . . 100 22 45 100 31 39 37 30 6.9 6.9 3.5 4.5

83 54 74 78 28 100 -. . 33 8.8 43 -32 100 48 26 18.3 21 19 20

3.2 0.02 0.02 1.15 -6.5 9.5

T A B L E II RETARDATION OF PIPSYL COMPOUNDS ON DOWEX 1 X 2 CI- COLUMN (Eluant 350 ml., 95% ethanol, 10 ml. 1 N HC1, water q.s. 1 1.; 3 g. Dowex 1 X 2 CI-, 200 to 400 mesh) Pipsyl derivative of Alanine Aspartic acid Glutamic acid Gly(.ine Hydroxyproline Isoleucine Leucine Phenylalanine Proline Serine N-Tyrosine Glyeylalanylglyeylalanine

35% eluant, ml. 66 155 107 93 82 5t 57 128 56 78 210 53

-0.56 1.8 8.6 -1.13 ----0.09 --

[10]


245

containing 0.01 N HC1 as eluent, and paper egraphy using such solvents as butanol equilibrated with 1 N NH3 or tetrachloroethane-butanol mixtures equilibrated with 0.2 N HC1. Some data suggesting the possibilities are given in Tables II and III. TABLE III SOME RS VALUES FOR PIPSYL AMINO ACIDS ON WHATMAN NO. 1 PAPER (Eluants: I, butanol saturated with 0.1 N NH.~; II, 0.1 N HC1; III, 85 ml. propanol-15 ml. 1 N NH.~)

Pipsyl derivative of Alanine Aspartic acid Glutamic acid Glycine Hydroxyprolinc Isoleucine Leucine Methionine Methionine sulfone Phenylalanine Proline Serine Threonine Valine

I

II

III

0.5 0.03 0.03 0.40 0.30 0.75 0.75 0.65 0.40 0.65 0.40 0.40 0.50 0.70

0.7 .

0.7 0.13 0.13 0.60 0.53 0.90 0.86 0.82

0.60 0.60 0.60 0.35 0.70 0.60

0.60 0.50 0.63 0.90

Special Purification Technique, Washout This special technique is employed in the indicator method when separation in the countercurrent distribution on the limited basis used (10 plates) is not sufficient for the mixture of compounds. Thus combined plates of a couutercurrent distribution to obtain group I amino acids (glutamic and aspartic acids, serine, threonine, and hydroxyproline) contain a considerable a m o u n t of pipsylglycine. 2 To the mixture add nonradioactive pipsylglycine in q u a n t i t y (2 to 3 mg.) dissolved in alkali, acidify to precipitate it, and remove b y filtration or centrifugation. To the supernatant add additional pipsylglycine and repeat. The precipitated pipsylglycine removes the radioactive analog in proportion to the fraction precipitated. However, losses of other compounds b y adsorption m a y limit the extent to which this purification m a y be carried.

Paper Egraphy Any suitable arrangement for paper egraphy m a y be used. I t is well to spread the sample along a 20-mm. line about the origin. The spots

246

TECHNIQUES FOR CHAR,kCTERIZ~kTION OF PROTEINS

[10]

may be located by their radioactivity by a scanning method, by autography on X-ray film, or by observation under an ultraviolet lamp which shows the pipsyl compounds as darkened areas. Any suitable spot is cut perpendicular to the direction of development into three or more strips. Each strip is separately eluted with dilute NH3 and planchetted. A constant ratio of S 35 to I TM activity in the successive strips is an excellent assurance of purity. A trend indicates impurity. A second egraphic development with the same or a different solvent is often useful.

Ion Exchange Egraphy The ion exchange egraphy of the pipsyl compounds on Dowex 1 × 2 C1- is carried out on 0.9-cm.-diameter columns using various dilutions of 95% alcohol containing 0.01 N HCI. The resin is cycled from the commercial 200- to 400-mesh material through hydroxide and chloride forms. Three grams of air-dried resin are placed in the columns as a slurry in the 50% eluent (500 ml. of 95% alcohol, 10 ml. of 1 N HCI diluted to 1 1.). After settling, 50 to 100 ml. of the 50% eluent is forced through as rapidly as possible (5 to 6 seconds per drop). The sample is then added and allowed to flow into the resin by gravity, and elution is carried out with a suitable alcoholic eluent (35% for the amino acids, 20% for peptides) at a rate of about 3 ml. per hour (40 to 50 seconds per drop). The fractions are followed for radioactivity or ultraviolet absorption or both. Some retardation values are given in Table II. Pipsyl acid seems never to be eluted from the columns, but the higher the peptide molecular weight the earlier it comes out. With an 8 % cross-linked resin the bands are much wider and more retarded than with 2% cross-linked resin.

Counting Samples are mounted on metal planchettes from solutions in NH3. They are centered as accurately as possible. It is desirable to have sufficient counts on a planchette to reduce the time required but not so many as to produce dead-time losses. The samples will be obtained as successive fractions from columns or as elutions of the successive strips of paper egraphic separations. For final analysis it is necessary to count the planchettes without a filter (sin count, S) and with a filter (cum counts, C). A suitable filter is made from 0.003-inch thick aluminum foil mounted to be placed without disturbing the sample. The filter factors (f) are determined by measuring C and S for pure I TM pipsyl alanine (f~) and pure S 35 pipsyl alanine (f~). Our filters give about f, = 0.003 and f~ = 0.60. The calculations are based on the simultaneous equations S -- Ci - C,, where C~ and C, are the counts due to iodine and sulfur,

Ill]

METHODS FOR INVESTIGATING THE ESSENTIAL GROUPS

247

respectively, from the unfiltered sample, and C = f ~ C i - f8C8. Thus C , = (c -

f~S)/(f,

C8 =

-

(AS

C)/(f~

-

fs) -

f,)

The pipsyl alanines from the two reagents used are standardized by ultraviolet absorption, planchetted, and counted both with and without filters. Ci for the sample divided by the molar radioactivity of the standard gives the number of moles of I TM compound on the planchette. When this is multiplied by the ratio of total S 35 counts (added as indicator) to C,, the result is the number of moles of the unknown in the original derivative mixture and if derivative formation was complete is the desired analytical figure.

Sequences The determination of any peptide in a mixture derived from hydrolysis of a protein is pertinent data for sequence studies. The determination of the amino end group in pipsyl peptides follows the methods used for D N B peptides (see Vol. IV [9]) with the advantage that the sulfonamide bond is very stable (1% loss in I N HCI for 4 hours at 110 °) and can be internally controlled (in part) by adding the S35-1abeled pipsyl derivative of the end group to the I131-1abeledpipsyl peptide before hydrolysis. The isolated pipsyl amino acid will have both isotopes, and a quantitative estimate is made on the basis described above. W e have applied the hydrazinolysis method of Akabori et al2 directly to pipsyl peptides to find the C terminal acid. The value of nearly quantitative estimations of the amounts of particular peptides in determining sequences in proteins like silk where a few amino acids make up the greater part of the protein is illustrated in papers by Levy and Slobodian.1°,H 9 S. Akabori, K. Ohno, and K. Narita, Bull. Chem. Soc. Japan 25~ 214 (1952). ~0 M. Levy and E. Slobodian, J. Biol. Chem. 199, 563 (1952). H E. Slobodian and M. Levy, J. Biol. Chem. 201, 371 (1953).

[II] M e t h o d s

for Investigating the Essential G r o u p s for E n z y m e Activity By

H. FRAENKEL-CoNRAT

Introduction

The customary approach in an investigation of the essential groups of enzymes and other biologically active proteins consists in studying the effect of various chemical modifications on the specific biological activity. Thus the first step is the preparation of chemically modified enzymes.

Ill]


247

respectively, from the unfiltered sample, and C = f ~ C i - f8C8. Thus C , = (c -

f~S)/(f,

C8 =

-

(AS

C)/(f~

-

fs) -

f,)

The pipsyl alanines from the two reagents used are standardized by ultraviolet absorption, planchetted, and counted both with and without filters. Ci for the sample divided by the molar radioactivity of the standard gives the number of moles of I TM compound on the planchette. When this is multiplied by the ratio of total S 35 counts (added as indicator) to C,, the result is the number of moles of the unknown in the original derivative mixture and if derivative formation was complete is the desired analytical figure.

Sequences The determination of any peptide in a mixture derived from hydrolysis of a protein is pertinent data for sequence studies. The determination of the amino end group in pipsyl peptides follows the methods used for D N B peptides (see Vol. IV [9]) with the advantage that the sulfonamide bond is very stable (1% loss in I N HCI for 4 hours at 110 °) and can be internally controlled (in part) by adding the S35-1abeled pipsyl derivative of the end group to the I131-1abeledpipsyl peptide before hydrolysis. The isolated pipsyl amino acid will have both isotopes, and a quantitative estimate is made on the basis described above. W e have applied the hydrazinolysis method of Akabori et al2 directly to pipsyl peptides to find the C terminal acid. The value of nearly quantitative estimations of the amounts of particular peptides in determining sequences in proteins like silk where a few amino acids make up the greater part of the protein is illustrated in papers by Levy and Slobodian.1°,H 9 S. Akabori, K. Ohno, and K. Narita, Bull. Chem. Soc. Japan 25~ 214 (1952). ~0 M. Levy and E. Slobodian, J. Biol. Chem. 199, 563 (1952). H E. Slobodian and M. Levy, J. Biol. Chem. 201, 371 (1953).

[II] M e t h o d s

for Investigating the Essential G r o u p s for E n z y m e Activity By

H. FRAENKEL-CoNRAT

Introduction

The customary approach in an investigation of the essential groups of enzymes and other biologically active proteins consists in studying the effect of various chemical modifications on the specific biological activity. Thus the first step is the preparation of chemically modified enzymes.

248


[11]

This is in most instances very easy and requires no special techniques or apparatus. It is usually desirable to attack only one type of protein group at a time, however, and reagents or reaction conditions of high selectivity are rather scarce. Much research needs yet to be done to supply us with a specific reagent for each type of protein group. The fact that each of the various kinds of polar residues may show a wide range of reactivity within a given protein, as well as from one protein to another, makes it appear unlikely that absolutely selective reagents will ever become available. At present, certainly, thorough analytical characterization is necessary for most protein derivatives to ascertain to what extent the desired reaction has proceeded and side reactions involving other types of groups have been excluded. This analytical work is usually harder and more time-consuming than the preparation of the derived protein; however, the analyses are of great importance if any conclusions are to be drawn from this type of study. Actually, the drawing of valid conclusions concerning the chemical nature of the enzymatic site is generally more problematical than either the preparative or the analytical work. Only in the few instances where one single group per molecule is found essential for enzyme activity is there some justification in assuming that this group is located at or near the active site; and even then its direct implication in the mechanism of enzyme action cannot be regarded as definitely proved. ~ Inactivation produced only after extensive modification of presumably one type of group should be interpreted with the greatest caution because of the imperfect selectivity of all methods and the danger of nonspecific inactivation which may be due to such factors as denaturation, change in net charge, or solubility of the enzyme. In contrast, retention of enzymatic activity after chemical modification of all reactive groups of a certain type appears to be of much greater interpretive value in suggesting the noninvolvement of the modified group. One must not overlook the fact that one or a few groups per protein molecule are within the error of most protein analytical methods; but the assumption appears justified (until disproved) that the polar groups associated with the enzymatic site are at least as available to chemical reagents as the average group of its type. Thus the fact that various enzymes retain their activity after maximal acetylation of their amino groups ~-4 strongly suggests that amino groups do not occur in the active site of these enzymes. 1T. P. Singer, J. Biol. Chem. 174, 11 (1948). 2 R. M. tterriott, J. Gen. Physiol. 19, 283 (1935-1936). 3I. W. Sizer, J. Biol. Chem. 160, 547 (1942). 4 H . Fraenkel-Conrat, R. C. Bean, and H. Lineweaver,J. Biol. Chem. 177, 385 (1949).

[11]

METHODSFOR INVESTIGATING THE ESSENTIAL GROUPS

249

In the following brief description of some of the more useful methods for the preparation of chemically modified enzymes, analytical techniques available for the evaluation of each reaction will be indicated but no further discussion concerning the interpretation of the data will be included. For reviews of the literature and more detailed and critical evaluation of the methods of protein modification, the reader is referred to various publications. 5-8 Reactive Protein G r o u p s The chemical structure of apoenzymes, as of all proteins, is primarily determined by the order of amino acid residues along the peptide chains. I t appears probable that enzymatic specificity and, in the absence of coenzymes, enzymatic activity is largely a function of this arrangement of the R groups (formula I). However, the specific folding of chains, which R2

/

NH

\

/ CH

I

R1

CO

R4

I

\

/ NH

CH

\

/ CO

NH

\

/ CH

CO

I

\

/ NH

CH

\

/ CO

I

R3

can bring nonadjacent R groups into close spatial position, also probably plays an intrinsic role i n the shaping of the enzymatic site. The folded structure, in turn, is stabilized through disulfide bonds and through a neat pattern of hydrogen bonds between pairs of - - C O - - N H - - groups which also may play an important role in electron transport to or from the enzymatic site2 The side chains (R groups) of proteins fall into two main categories-i.e., the nonpolar hydrocarbon groups contributed by alanine, valine, leueine, isoleucine, phenylalanine, and proline, and the various groups carrying active hydrogen or reactive sulfur atoms. The nonpolar class may play a role in the active site of enzymes through their affinity for similarly hydrophobic groups on the substrate. The chemical inertness of the hydrocarbon chain renders this class unsuitable to chemical study, 5R. M. Herriott, Advances in Protein Chem. 3, 169 (1947). 6H. S. Oleott and H. Fraenkel-Conrat, Chem. Revs. 41, 151 (1947). 7H. Fraenkel-Conrat, in "Amino Acids and Proteins" (Greenberg, ed.), p. 532. Charles C Thomas, Springfield, Ill. 1951. a F. W. Putnam, in "The Proteins" (Neurath and Bailey, eds.), Vol. I, Part B, p. 893. Academic Press, New York, 1953. 9K. Wirtz, Z. Naturforsch. 2b, 94 (1947); W. Schmitt, ibid. 2b, 98 (1947); T. A. Geiasmann, Biol. Revs. 24, 309 (1949).

250

TECHNIQUESFOR CHARACTERIZATION OF PROTEINS

[11]

however, and probably justifies the belief t h a t their role is only secondary, their action radius being smaller than t h a t of the polar groups. The polar groups as listed below are in part dissociated under physiological conditions (the first four), but chemical modification is usually performed with the undissociated form. This probably accounts for the lack of good methods of a t t a c k on the completely dissociated guanidyl group. The various groups differ greatly in their chemical reactivity, decreasing in the approximate order listed (except for the low reactivity of the guanidyl and the high reactivity of free - - S H groups). Amino groups (lysine, N terminal amino acid) Imidazole groups (histidine) Guanidyl groups (arginine) Carboxyl groups (aspartic, glutamic acid, C terminal amino acid) Sulfhydryl-disulfide groups (cysteine, cystine) Phenol groups (tyrosine) Hydroxyl groups (serine, threonine) Indole groups (tryptophan) Amide groups (glutamine, asparagine, C terminal amide) Thio ether groups (methionine) (Phosphate ester groups in phosphoproteins; serine, threonine) The assumption t h a t the nature of the active site of any enzyme is a simple additive function of the properties of some of these polar and the nonpolar R groups m a y be an oversimplification. We now know of two specific instances in which reactions occur with proteins which cannot be duplicated with any simple mixture of amino acids or peptides, and in which a potentiating or synergistic effect of two or more of the typical R groups is believed to be operative. These are the inhibition of esterases b y D F P , to be discussed later, and the action of nitrogen trichloride on certain prolamines and no other proteins, causing the f o r m a t i o n - o f methionine sulfoximine presumably through cooperation of amide groups and methionine. 10 There exists also the definite possibility t h a t reactive groups of completely different nature from t h a t of any recognized protein building block m a y occur in certain enzymatic sites and m a y have escaped detection. Aldehyde or ketone groups have often been postulated, 11 and 10p. N. Campbell, T. S. Work, and E. Mellanby, Biochem. d. 48, 106 (1951); H. R. Bentley, E. E. McDermott, I. Moran, J. Pace, and J. K. Whitehead, Proc. Roy. Soc. B137, 402 (1950). 1~In one case the postulated occurrence of an aldehyde group in an enzyme [E. Werle and K. Heitzer, Biochem. Z. 299, 420 (1938)] has actually been confirmed later, though it was found to.be located in the coenzyme rather than in the protein (pyridoxal in histidine decarboxylase).

[11]

METHODS FOR I N V E S T I G A T I N G T H E ESSENTIAL GROUPS

251

hydroxylamino groups and others have been suggested. ~2 I t appears advisable to keep an open mind to this possibility, but also to shoulder the burden of proof before building extensive theories on such assumptions. The inactivation of an enzyme b y a so-called ketone reagent, for instance, is not sufficient proof for the ketonic nature of the enzyme until interaction of the reagent with all known types of protein groups has been thoroughly excluded.

Acylation of Amino Groups Discussion. M a n y different acyl residues have been introduced into proteins, b u t none appear to be preferable to the acetyl residue when water-soluble biologically active products are desired. Ketene has been often used for the acetylation of proteins1 ~ but acetic anhydride in neutral aqueous solution is easier to handle and appears as effective and less harmful to native proteins. 4,6-8 When it appears desirable to attach larger acyl groups to the amino groups, then phenylisocyanate is probably the reagent of choice. 13 In contrast to acetic anhydride, this reagent seems to attack also the masked - - S H groups of native proteins. TM Reaction with Acetic A n h y d r i d e . To the enzyme solution, preferably b u t not necessarily of high concentration (2 to 10%), is added an equal volume of saturated solution of sodium acetate; the solution (or suspension) is cooled in an ice bath and treated with a total a m o u n t of acetic anhydride approximately equal to the weight of enzyme used, but distributed over three to six additions in the course of 1 hour at 0 ° (e.g., 10 mg. of enzyme in 0.1 ml. of H:O in a small test tube, 0.1 ml. of sodium acetate, five times 2 ul. of acetic anhydride). The product is isolated b y dialysis. A n a l y t i c a l Characterization. This reaction is relatively specific for the amino groups. An easy approximation of the extent of acetylation of these groups is obtained by the ninhydrin test, 15 slightly simplified for use with proteins (see below). For critical evaluation the manometric Van Slyke m e t h o d is preferable. 1~ In such cases total acetyl introduced should also preferably be determined. 1~.17M a s k e d - - S H groups do not generally react,

1~S. Fiala and D. Burk, Arch. Biochem. 20, 172 (1949); H. Fraenkel-Conrat, ibid. 28, 452 (1950); H. Fraenkel-Conrat, N. S. Snell, and E. D. Ducay, Arch. Biochem. and Biophys. $9~ 97 (1952). la G. L. Miller and W. M. Stanley, J. Biol. Chem. 1411 905 (1941). 1~H. Fraenkel-Conrat, J..Biol. Chem. 1521 385 (1944). 1~S. Moore and W. H. Stein, J. Biol. Chem. 176, 367 (1948). 1~D. D. Van Slyke, J. Biol. Chem. 83~ 425 (1929). 17R. M. Herriott, J. Gen. Physiol. 19~ 283 (1935-1936).

252


[11]

but free - - S H groups become acetylated. In proteins containing - - S H groups, their involvement should be ascertained analytically (see pp. 257ff.). Phenolic groups are not generally acetylated, but this should be ascretained by means of the Folin test or by ultraviolet spectrophotometry (see below). No other protein groups seem to be attacked. Procedure for Ninhydrin Reaction for Amino Groups in Proteins 15 (see also Vol. III [76]). The reagent is prepared as follows: 0.8 g. of stannous chloride (SnC12.2H20) is dissolved in 500 ml. of citrate buffer, pH 5 (21.0 g, of citric acid, 1 H~O; 200 ml. of N NaOH and water to 500 ml.). To this is added a solution of 20 g. of ninhydrin in 500 ml. of methyl Cellosolve. The solution is saturated with N2 gas and kept in a glass-stoppered bottle in the refrigerator. To test tubes containing 0.2 to 1 mg. of protein in 0.1 ml. of water or salt solution is added 1 ml. of reagent. The tubes are heated in a vigorously boiling water bath for 20 minutes, allowed to cool, rapidly mixed with 5 ml. of 50% aqueous isopropanol, and read within 15 minutes after removal from the bath in a Klett colorimeter with the green filter No. 54 against a blank treated with reagent in the same manner. Alanine (0.05 to 0.2 ~M.) is the standard. Proteins generally give about 80 % of the color expected from their amino-N content under these conditions. Various acetylated proteins show chromogenic values ranging from 10 to 60% of the original. Procedure for Folin-Herriott Test for Phenolic Groups in Proteins 1~,18 (see also Vol. I I I [76, 87]). To 1 to 5 mg. of protein dissolved in 8.5 ml. of water, dilute salt, acid or alkali, are added simultaneously 2.5 ml. of buffer (60 ml. of 0.5 M Na~HPO4 ~ 40 ml. of N NaOH) and 1.5 ml. of the threefold-diluted Folin phenol reagent. The solution is held for 1 hour at 40 °, cooled, and read in a colorimeter with a blue filter against a blank sample. Tyrosine or tryptophan may be used as standard. To regenerate labile phenol esters (Herriott's pH 11 procedure) water is added to only 5.5 ml., followed by 1.5 ml. of 0.1 N NaOH. After 15 minutes at room temperature, 1.5 ml. of 0.1 N HC1 is added, followed by buffer and reagent as usual. The severe limitations of the Folin tes~ have been amply discussed. ~7 As a comparative method, however, particularly to show the intactness or the alkali-labile acylation of phenolic groups, it continues to serve a useful purpose. Spectrophotometric Determination of Phenolic Groups. In alkaline solution, tyrosine, as well as the tyrosine residues in proteins, shows a characteristic maximum at a higher wavelength than that due to tryptophan (295 versus 280 mg). A method of determination of the two amino acids has been developed on this basis.19 The protein is dissolved in 0.1 N NaOH is R. M. Herriott and J. H. Northrop, J. Gen. Physiol. 18, 35 (1934-1935). 19 G. H. Beaven and E. R. Holiday, Advances in Protein Chem. 7, 320 (1952).

[11]


253

and its absorption read at the point of intersection of the two curves (294.4 m~), as well as at 280 m~. From the formulas M(Ty,) = (0.592,2944 -- 0.263,~8oo) X 10-3 M(T~yp) ---- (0.263,~8oo -- 0.170,2944) X 10-3 where K2944 and K2800 represent the extinction coefficient of the protein at the two wavelengths, the gram moles of tyrosine and tryptophan per gram of protein, M(tyo and M(tryp), can be calculated. For more accurate results nonspecific absorption is determined at 320 to 360 m~, extrapolated to 280 m~, and subtracted from the observed optical density. With proteins rich in disulfide bonds, readings must be taken immediately after addition of the alkali, since progressive spectral changes have been observed. When dealing with, or searching for, alkali-labile tyrosine derivatives, such as O-acetyl tyrosine, this method cannot be applied. With proteins of a high tyrosine-tryptophan ratio, however, a good indication of the extent of blocking of the phenolic groups should be derived from a comparison of the absorption at 275 m~ of neutral solutions of the original, the modified protein, and the latter after exposing it to alkali (e.g., pH 11) and bringing it back to neutrality. An alternate technique is to determine spectrophotometrically the pH needed for dissociation of the phenolic groups of the protein under study. This varies greatly for different proteins, s° At this pH, then, the technique of Beaven and Holiday can be applied. If the absorption (at 295 m~) increases with time, indicative of hydrolysis of phenol esters, then extrapolation to zero time will be necessary to evaluate the extent of acylation of the phenolic groups. (See also under Iodination.) Reaction with Phenylisocyanate.1. ~ To the protein buffered at pH 7.5 to 8.0 and held at 0 ° is added with efficient stirring a similar amount (1 ml./g, of protein) of phenylisocyanate in small aliquots distributed over 2 to 3 hours. When the smell of the reagent has disappeared, the diphenylurea is centrifuged off and the protein solution dialyzed and analyzed by the same methods as the acetylated product. Esterification of Carboxyl Groups Discussion. Proteins are readily esterified if they are suspended in alcohols containing about 0.02 N to 0.1 N HC1. ~l This reaction is selective for carboxyl groups. Denaturation can be avoided or minimized with many proteins, including serum albumin, if the reaction is performed at 20 j. L. Crammer and A. Neuberger, Biochem. J. 37, 302 (1943). 21 H. Fraenkel-Conrat and H. S. Olcott, J. Biol. Chem. 161, 259 (1945).

254


[11]

lOW temperature, 6 but the danger of denaturation can never be completely excluded. T r e a t m e n t of proteins in aqueous solution with epoxides (e.g., propylene oxide) supplies a gentler means of esterifying carboxyl groups, but this reaction is not specific; amino, phenolic, and - - S H groups are also alkylated to a varying extent, depending on the p H of the medium. In acid solution, carboxyl groups react somewhat more readily than other groups, but the esterification never reaches completion. 22 M u s t a r d gas and related substances, including the nitrogen mustards, react in a m a n n e r similar to the epoxides, affecting primarily the - - S H and carboxyl groups below neutrality. 23,~4 Both diazoacetamide and diazoethylacetate seem to be specific reagents for the esterification of carboxyl groups. T h e y react under mild conditions, but a p p a r e n t l y with only a fraction of the carboxyl groups of serum albumin. 25 The esterification reactions are often reversible. M e t h y l esters are rather labile in dilute acid and alkali, and part of the original carboxyl groups can be regenerated by exposing esterified proteins to p H 2 or p H 10; 28 esters of higher alcohols form less readily and are more stable. T h e glycolic esters produced with the diazoacetyl reagents can also be split in dilute alkali. 25 Reaction with Methanol-HCl. ~I The d r y protein, preferably in the lyophilized state, is suspended in 100 parts of methanol cooled to - 5 to - 1 0 ° ; to this is added concentrated HC1 to a final concentration of 0.02 or 0.1 N, and the suspension is held at - 10 °, 0 o, or room t e m p e r a t u r e for one to several days, depending on the stability of the enzyme and the extent of esterification desired. M a n y proteins dissolve in the alcohol in the course of esterification. The reaction mixture is finally diluted with much ice water, dialyzed, and concentrated by pervaporation. A n a l y t i c a l Characterization. In view of the lability of m e t h y l esters, the only strictly q u a n t i t a t i v e m e t h o d of ascertaining the extent of methylation is methoxyl determination by the Zeisel method. ~7 Indications of the extent of esterification can be obtained through titration 22H. Fraenkel-Conrat, J. Biol. Chem. 154, 227 (1944). 2~R. M. Herriott, M. L. Anson, and J. H. Northrop, J. Gen. Physiol. 30, 185 (1946). ~4V. Desreux, E. Fredericq, and P. Fischer, Bull. soc. chim. biol. 28, 7 (1946). 2~We are indebted to Dr. P. E. Wilcox for letting us see a manuscript concerning this technique prior to publication; see also abstracts of papers, 12th Intern. Congr. Pure Appl. Chem., New York, pp. 60, 61 (1951). 36W. F. H. M. Mommaerts and H. Neurath, J. Biol. Chem. 185, 909 (1950). ~7E. P. Clark, J. Assoc. O~c. Agric. Chemists 15, 136 (1932).

[11]


255

of the protein and its derivative from pH 2.5 to 6.0, ~3and through electrophoretic examination. Reaction with Epoxides. 22 To the enzyme solution (about 2 %) adjusted to pH 3.5 to 4.0 with acetic acid is added 0.1 vol. of propylene oxide. The reaction mixture is held at room temperature for 1 to 6 days, then dialyzed and assayed. Analytical Characterization, Determination of Total Acid Groups. 28 The extent of involvement of amino, phenolic, and sulfhydryl groups can be ascertained by methods described elsewhere in this chapter. The esterification of the carboxyl groups can be determined by analysis for total acid groups of the enzyme and the esterified product based on the capacity of the protein to bind a basic dye in alkaline solution. This method is not applicable to the more alkali-labile methyl esters. To a series of test tubes containing 2- to 3-rag. samples of protein (0.2 ml. of a 1 to 2% solution) are added 1.0 ml. of a buffer of pH 11.5 (250 ml. of 0.2 M Na2.HPO4, 200 ml. of 0.1 N NaOH, and water to 1000 ml.) and 1.0, 2.0, and 3.0 ml. of a 0.2% solution of Safranine 0. 29 The tubes are shaken for 20 hours at room temperature to ensure equilibration. The protein-dye complex is then centrifuged off, and the concentration of dye remaining in the supernatant is determined photometrically. To this end 0.5- or 1.0-ml. aliquots are diluted to 100 or 250 ml., and the absorption at 520 m~ is read in a spectrophotometer2 ° The concentration of dye in the stock solution is similarly determined; insoluble impurites and moisture can be corrected for if the dye concentration is calculated from the optical density, as compared to that of a purified and dry sample (0.111 for a solution of I ~,/ml. and a l-cm. light path). The difference between the amount of dye added and that found in the solution is the dye bound by the protein. This amount per milligram of protein should be independent of the amount of excess dye, as indicated by agreement of the results obtained with increasing amounts of dye. For greater accuracy, repeat analyses are performed, avoiding too great an excess (only two levels of dye from 75 to 300 % in excess of the amount bound). Multiplication by 28.2 will convert the amount of dye bound per milligram to total acid groups per 104 g. of protein. 2s H. F r a e n k e l - C o n r a t a n d M. Cooper, J. Biol. Chem. 154, 239 (1944). 29 If the Safranine is impure a n d a b r o w n precipitate settles out, this m a y be separated. Purification can be achieved b y dissolving the dye in 95 % ethanol containing a few drops of 6 N HC1, a n d precipitating with m u c h ether. 30 A colorimeter, e.g. Klett, can also be used, with a combination of Corning filters No. 3060, 3389, a n d 5543. Safranine has a great t e n d e n c y to adsorb on glass, a n d Corex cells were found unsuitable with the B e c k m a n n Model D U spectrophotometer. Plastic cells were found more convenient for this purpose.

256


[llJ

Reactions o f - - S H Groups Discussion. Several classes of reagents are available for the - - S H group, and several of the reagents show a high degree of specificity for this group. On the other hand, part or all of the - - S H groups of most native enzymes are to a varying extent masked and unreactive toward one, most, or all of the reagents. Thus, lack of effect of one or even several - - S H reagents on the enzymatic activity proves neither the absence nor the nonessentiality of - - S H groups. On the other hand, inactivation by one of these reagents should not be regarded as absolute proof of the presence and essentiality of such groups. Analytical methods are available for the determination of the reactive and of the total - - S H groups of a protein, and analytical proof for the blocking of - - S H groups by the reagents under study ought to be sought whenever possible. Free, fully reactive - - S H groups occur only in a few proteins. Enzyme activities are rarely if ever directly dependent on these groups. The essential - - S H groups--i.e., those located at or near the enzymatic site and believed to be involved in reactions with the substrate--are usually more or less masked; they rarely give the nitroprusside test and generally react with only a few of the - - S H reagents. Often the most effective class of reagents for these groups are the mercaptide-forming mercurials and arsenicals, such as p-chloromercuribenzoate. These reagents fulfill most of the requirements of an ideal protein reagent: They react stoichiometrically under gentle conditions, they show high specificity, and they can easily be split off and the original - - S H group regenerated. Another reagent which often attacks masked - - S H groups is iodine. Iodine reacts also with tyrosine and histidine residue (see p. 260), however, and thus its effect on enzymatic activity can only be interpreted as due to oxidation of - - S H groups, if the iodine used up is quantitatively accounted for by a corresponding decrease of - - S H groups. This has actually been found to be the case with only one protein, egg albumin. 81 With two other - - S H proteins, serum albumin and tobacco mosaic virus, al,3~ about twice the theoretical amount of iodine was used up in oxidative reactions for the abolishment of the - - S H group. In the case of the virus, the transformation of the - - S H to a stable sulfenyl-iodide group has recently been demonstrated. 3~a Certain alkylating agents, notably iodoacetic acid or its amide, usually show lesser reactivity toward masked - - S H groups but are com31 M. L. Anson, J. Gen. Physiol. 24, 399 (1940); M. L. Anson and W. M. Stanley, ibid. 24, 679 (1941). 32 W. L. Hughes, Jr., and R. Straessle, J. Am. Chem. Soc. 72, 452 (1950). 3~ H. Fraenkel-Conrat, J. Biol. Chem. 217, 373 (1955).

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M E T H O D SFOR INVESTIGATING THE ESSENTIAL GROUPS

257

paratively specific and react rapidly with free - - S H groups at room temperature and neutrality. Another reagent, only recently introduced, ~3 is N-ethylmaleimide (NEM), which adds to the - - S H group to give a stable thio ether. It appears to be a specific - - S H reagent when used near neutrality but shows little affinity for masked - - S H groups. In view of the great variations in the reactivity of SH groups toward different reagents, no general procedure can be advocated, and many pilot experiments are needed to decide which reactions are most applicable to a given problem. In all cases, it should be attempted to confirm all conclusions by the use of more than one type of reagent. Also, isolation of the modified enzyme is strongly advocated. Much confusion has been caused by the tendency of enzymologists to add reagents directly to solutions to be assayed; they then study the effect of reagents on the enzymatic reaction, rather than on the enzyme. Thus inhibitions and activations have been observed and interpreted in terms of the enzyme which had actually no relation to the chemical nature of the enzymatic site but represented various types of interaction or competition of the reagent with the substrate or coenzyme. Removal of excess reagent by dialysis is a simple procedure and should be carried out whenever possible; this will greatly facilitate the interpretation of enzymatic assay data. A suggested sequence of tests concerning the sulfhydryl nature of an unknown enzyme available in amounts of 5 to 50 rag. is as follows: (1) Nitroprusside test, native, and (2) nitroprusside test after denaturation. With limited material one should be prepared for (3) an immediate titration of the same sample with N E M if the nitroprusside test is positive. Another sample of enzyme should be reacted with p-chloromercuribenzoate by the ultraviolet spectrophotometric method as an indication of interaction24 Other samples can be treated with small amounts of iodine, iodosobenzoate, or iodoacetamide, and the change in N E M titration subsequently determined. Amperometric titration methods can also be employed to advantage. All samples that have been treated with the various reagents in the native state can be isolated by dialysis or some other convenient procedure and subjected to enzymatic assay, and to various analyses. Titration o f - - S H Groups with N-Ethylmaleimide. 3~ The reagents are (a) guanidine hydroehloride, saturated solution in 0.1 M acetate, pH 4.8; 33 T. Tsao and K. Bailey, Biochim. el Biophys. Acta 11, 102 (1953); E. Friedmann, D. H. Marrian, and I. Simon-Reuss, Brit. J. Pharmacol. 3, 335 (1948). 34 p. D. Boyer and H. L. Segal, in " T h e Mechanism of Enzyme Action" (McElroy and Glass, eds.), p. 520. Johns Hopkins Press, Baltimore, 1953; P. D. Boyer, J. Am. Chem. Soc. 76, 4331 (1954).

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METHODS FOR INVESTIGATING TttE ESSENTIAL GROUPS

259

ins, and readings must be taken at intervals until constant before more of the reagent is added. The n u m b e r of reactive SH groups can be estimated from the increment over the calculated absorption at 255 m# with 6200 as the molar extinction increment for the mercaptide formed at t h a t wavelength27 The same reaction can be performed at pH 7 (phosphate buffer), although the - - S H groups of native proteins seem to react more sluggishly at t h a t pH. (Under these conditions the molar extinction increment at 250 mt~ is about 7600.) These solutions m a y be used for enzymatic assay, preferably after thorough dialysis. M e r c u r y analyses and spectrophotometric analysis after dialysis will supply further proof for the extent of interaction. Dialysis against cysteine-HC1 solution reverses the reaction and removes most of the mercuribenzoate. The same spectrophotometric technique performed in 1% sodium dodecylsulfate will permit determination of the total (free + masked) - - S H groups of proteins. Reaction with Iodoacetamide, etc. Iodoacetamide is easily prepared by refluxing equimolar amounts of chloroacetamide with a slight excess of sodium iodide in alcoholic solution for 6 hours, filtering off the inorganic salt, evaporating part of the alcohol, and adding some water. The crude product separates on cooling and must be recrystallized from water and alcohol, without heating above about 60 °, to obtain a pure white product (melting point, 95°). 28 If iodoacetamide is found to inactivate an enzyme at neutrality and room temperature, then - - S H groups m a y be involved. The total extent of interaction can be ascertained from the acid liberated according to the equation R - - S H + ICH2CONH2--~ R - - S - - C H ~ - - C O N H 2 + HI. For this purpose an a u t o t i t r a t o r 39 can conveniently be employed, but the alkali needed to maintain a constant p H (pH 8) can also be determined malmally. 4° Analyses for amide-N in the isolated product can also serve as a basis for determining the extent of reaction with proteins containing m a n y reactive - - S H g r o u p s ) ° The involvement of the - - S H groups is demonstrated by means of N E M titrations in guanidine (methods 2 and 3). Iodoacetic acid can be used in the same manner if a white (iodinefree) preparation is available. 37According to Boyer34the molar absorption increment varies somewhat for different mercaptides and proteins, so that it must be determined by the use of excess thiol, when exact absolute values are needed. ~s j. yon Braun, Bet. 41, 2144 (1908). ~ C. F. Jacobsen and J. Leonis, Compt. rend. tray. lab. Carlsberg, S~r. chim. 27, 333 (1951). 40H. Fraenkel-Conrat, A. Mohammad, E. D. Ducay, and D. K. Mecham, J. Am. Chem. Soc. 73, 625 (1951).

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Iodine, when applied as an - - S H reagent, is added in small amounts (no more than three times the amount needed for the oxidation of all - - S H groups) and preferably in acid solution (pH 3.2 to 6.8). The concentration of potassium iodide may play an important but variable role. 3~ The extent of oxidation of - - S H groups can be ascertained by N E M titrations in guanidine hydrochloride (methods 2 and 3). o-Iodosobenzene, when used near pH 7, 36,41 is probably the most specific of the oxidizing agents which, in general, have lost their formerly predominating role as - - S H reagents. One of the objections to these reagents, the need to find a pair of reactive - - S H groups for oxidation to the disulfide form, holds also for o-iodosobenzoate. 4~ Thus complete reaction can never be assumed. For details of this procedure, as well as of many other useful procedures for - - S H groups, the reader is referred to a recent review2 8 Iodination

Discussion. Most proteins rapidly decolorize iodine in neutral solution. When SH groups are absent, or when the consumption of iodine exceeds their content and iodine is actually incorporated into the protein, then iodination of phenolic groups is usually assumed. In most proteins this reaction is comparatively specific, but in some proteins the histidine residues react more readily than the phenolic groups. Thus, in the case of lysozyme, iodination caused loss of activity which was reversible in the early stages of the reaction and which was found to involve the single histidine residue of this protein. 48 Since oxidative side reactions can never be completely excluded and good analytical techniques for allocation of the iodine atoms are lacking, the effect of iodination on an enzyme can not readily be interpreted in terms of essential groups. Reaction. Varying amounts of an iodine solution (0.01 M Is in 0.5 M KI) are added to the enzyme solutions buffered with acetate (pH 5 to 6), ~4 phosphate (pH 7 to 8), 48 or carbonate-bicarbonate (pH 9 to 9.5) ~2 and held at 0 ° or room temperature. The rate of disappearance of the yellow color is an indication of the reactivity of the groups of a given protein; this varies greatly for different proteins. The most alkaline pH at which an enzyme is stable should be used to minimize oxidative reactions. ~ The 4~L. Hellermann, F. P. Chinard, and P. A. Ramsdell, J. Am. Chem. Soc. 63, 2551 (1941). 43 The possibility of oxidation of single masked - - S H groups directly to - - S - - O H groups appears not to have been considered, although some of the analytical data favor this concept (see ref. 32a). 4a H. Fraenkel-Conrat, Arch. Biochem. 27, 109 (1950). 44 R. M. Herriott, J. Gen. Physiol. 20, 335 (1936-37); 31, 19 (1947).

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METHODS FOR I N V E S T I G A T I N G T H E E S S E N T I A L GROUPS

261

extent of modification can be controlled b y the a m o u n t of iodine added (usually from 4 to 40 equivalents per mole of protein). Any residual iodine can be titrated back with thiosulfate after a suitable time interval. T h e enzyme is then dialyzed (or reisolated) and assayed. Analytical Characterization. Oxidation of - - S H groups, if any are present, must first be ascertained (see p. 258), or excluded b y blocking them with mercuribenzoate or iodoacetamide. Apart from the reaction with - - S H groups, which only in exceptional cases is one of substitution, TM most of the iodine used up rapidly in dilute solution generally acts in substitution reactions, further oxidation (of disulfide bonds, indole residues, etc.) occurring only under more extreme conditions, particularly with a great excess of iodine22 T h e total extent of substitution is established b y iodine analysis of the thoroughly dialyzed product; the extent of oxidation can be estimated by comparing the a m o u n t of iodine used up (added minus b a c k t i t r a t e d iodine) to t h a t found in the product; this ratio equals 2 for substitution, ~o for oxidation. The use of I TM is v e r y advantageous for analytical purposes, particularly if a well-type scintillation counter is available. Naturally, the dilution of the I TM used by the K I 1~7 serving as solvent must be taken into account. The main difficulty lies in ascertaining the relative extent of substitution on imidazole and mono- and disubstitution on phenol groups. Methods have been described for the determination of monoiodo- and diiodotyrosine, 45 b u t the lability of these and even more so of the iodinated histidines under conditions of hydrolysis prevents a definite quantitative allocation of the iodine atoms in the protein. The shift of the ultraviolet absorption maximum of both the dissociated and undissociated form of tyrosine on mono- and diiodination to higher wavelengths and absorptions 44 is observed also in proteins and can be used as an indication of the extent of involvement of these residues. ~2

Coupling with Diazo Compounds Discussion. The reaction of proteins with diazobenzene sulfonic acid or other diazobenzene derivatives occurs with great ease near neutrality. 4,4° The reaction, like iodination, involves phenolic and imidazole groups, b u t in this case the imidiazole groups of most proteins react at least as fast as do the phenolic groups. Thus this reaction, like iodination, is easy to perform, b u t generally difficult to interpret. Reaction with Diazobenzene Sulfonic Acid. Diazobenzene sulfonic acid 4~The Millon reaction serves to differentiate tyrosine from diiodotyrosin, which gives no color, and from monoiodotyrosine, which gives a color with a slightly different maximum [J. Roche, R. Michel, and M. Lafon, Compt. re~d. 224, 233 (1947)].

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this reagent can be bound through methylene groups to the amino groups of the protein and cross-linking of the methylol with other protein groups can be minimized. Similarly, addition of amines or amino acids to the reaction mixture directs most of the reaction of formaldehyde toward the amide and guanidyl groups of the protein. 4s Reaction and Analyses. The reactions are performed at room temperature and neutrality, usually with 1% formaldehyde and about 10 % concentration of the small-molecular additives for 24 to 72 hours. For analytical evaluation the following methods are employed: the ninhydrin or Van Slyke reactions for amino groups; NEM titrations for - - S H groups; amide N by autoclaving with 1.2 N sulfuric acid for 2 hours, distilling in a micro-Kjeldahl apparatus with an antifoam agent after addition of buffer (pH 7.8 to 9), and titrating the ammonia as in the Kjeldahl procedure. Formaldehyde is released and steam-distilled by distillation from 1 N H2SOt until the acid begins to fume. The distillate is collected in water, and aliquots analyzed by means of chromotropic acid. 4~ Reduction of Disulfide Bonds Some enzymes containing - - S H groups essential for their activity are autoxidizable when removed from their natural, presumably reducing, medium. They are characterized by being activated by reducing agents, and they are preferably maintained in a reducing medium throughout their isolation. 5° Suitable reducing media are 0.01 to 0.1 M cysteine, mercaptoethanol, thioglycolate, or cyanide at pH 5 to 7. The same reagents may be used to reduce the - - S - - S - - bonds originally occurring in most native proteins. These are usually, at least in part, masked and of a low order of reactivity so that quite high concentrations of the mercaptans are needed for extensive reduction (e.g., 0.1 to 1 M at pH 7.5), and complete reduction is possible in most proteins only in solution in denaturing and dispersion agents (e.g., saturated urea4°), if at all. Since the disulfide bonds play an important structural role, it is not surprising that their reduction usually causes inactivation. Because of the high reactivity of the resulting free - - S H groups, reduced proteins tend to undergo rearrangements and form aggregates leading to precipitation, gelation, etc., even if reoxidation is prevented. 5°~ It is, therefore, advisable to " f i x " the reduced product by adding a great 49D. A. MacFadyen, J. Biol. Chem. 168, 107 (1945). 5oj. R. Kimmel and E. L. Smith, J. Biol. Chem. 207, 515 (1954); A. A. Green and G. T. Cori, ibid. 151, 21 (1943). ~0~C. Huggins, D. F. Tapley, and E. V. Jensen, Nature 167, 592 (1951).

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excess of an SH reagent, such as iodoacetamide, to the reaction mixture, maintaining the pH near 8 until the reaction is complete and a stable derivative has been obtained. 4° No protein groups except the disulfide are known to be affected by mercaptans. Oxidation

Many reagents have been used for the oxidative modification of proteins, but most of them show a low degree of specificity. They generally attack sulfhydryl and disulfide groups, thio ether, indole, phenol, imidazole, and other groups. Hydrogen peroxide, under certain conditions, shows a specific affinity for the sulfur amino acid residues. A manometric technique of photooxidation with methylene blue as catalyst has been described ~1 which causes destruction of most of the histidine prior to any appreciable attack on the indole, and later on the phenol groups. This technique has supplied corroborative evidence concerning the essential histidine group of lysozyme and the mechanism of action of D F P on chymotrypsin. ~1 For the details of this technique the reader is referred to the original literature. A new technique for the selective oxidation of protein is by means of peroxidase and hydrogen peroxide. 5~ Tryptophan appears most susceptible under these conditions. Interesting results were again obtained with chymotrypsin. Sulfation

No gentle and specific reagents are known to attack aliphatic hydroxyl groups (serine, threonine). These groups can quantitatively and rapidly be transformed to sulfate esters by means of concentrated sulfuric acid at - 1 0 °. No peptide bonds are ~plit, and sulfonation of some of the phenolic groups is the only side reaction. 5~ Insulin proved active after sulfation, 53 and the derivative has been used in studies of its mode of fixation in the tissues. ~4 Yet this technique can definitely be denaturing and is not advocated for most enzymes. ~5 ~ L. Weil and A. R. Buchert, Arch. Biochem. and Biophys. 34, 1 (1951); L. Weil, A. R. Buchert, and J. Maker, ibid. 40, 245 (1952); L. Weil, S. James, and A. R. Buchert, ibid. 46, 266 (1953). 51~ H. N. Wood and A. K. Balls, J. Biol. Chem. 213, 297 (1955). 5~ H. C. Reitz, R. E. Ferrel, H. Fraenkel-Conrat, and H. S. Olcott, J. Am. Chem. Soc. 68, 1024 (1946). 53 M. B. Glendening, D. M. Greenberg, and H. Fraenkel-Conrat, J. Biol. Chem. 167, 125 (1947). ~ W. C. Stadie, N. Haugaard, and M. Vaughan, J. Biol. Chem. 199, 729 (1952). 65 Methods to produce the acyl shift have been studied in recent years [D. F. Elliott, Biochem. J. 50, 542 (1952)]. Extensive transformation of the peptide linkages

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265

Deamination Nitrous acid has long been in use as a protein reagent. For the analytical purpose of determining the amino-N it is still unsurpassed. 18 B u t no conditions have been found under which its action can be confined to the amino groups, and under gentle conditions (pH 4, 0 °) designed to minimize side reactions (diazotization of phenolic groups, oxidation of - - S H groups, etc.) the deamination is far from complete. ~8

Guanidination A technique has been described for the transformation of amino to guanidine groups 57 which m a y prove useful in characterizing enzymatic mechanisms. T h e protein is allowed to interact with 0.5 M O-methylisourea at p H 8.5 to 10.5 and 0 ° for several days. Depending on the pH, a variable fraction of the amino groups will be guanidinated, as indicated b y a drop in amino-N.

Enzymatic Attack on Enzymes D i s c u s s i o n . N a t i v e proteins are attacked b y endopeptidases at greatly varying rates and to different extents. T h e y are resistant to most other enzymes, with a few notable exceptions. Among these are carboxypeptidase which splits amino acids from the C terminal end, certain phosphatases which split phosphate from serine residues, ~s and tyrosinase which m a y attack the tyrosine residues in proteins. 59 A s t u d y of the effect of some of these enzymes, including limited proteolytic digestion on enzymatic activity, appears well worth attempting. Although the enzymatic attack is not v e r y likely to supply a direct indication of the nature of the active site, it m a y help to focus our attention on it b y trimming away unessential parts of the molecule. In view of the great variations in susceptibility to the proteolytic enzymes, no general techniques can be proposed. E n z y m a t i c activity (ATPase) has been retained after brief digestion with trypsin or chymo-

involving hydroxyamino acids to ester linkages with the --OH group is being achieved. This reaction is catalyzed by acids and occurs under prolonged attack of concentrated sulfuric acid. The peptide bond is re-formed above pH 7. It appears possible that such acyl-shifted linkages occur naturally in some proteins. 56j. S. L. Philpot and P. A. Small, Biochem. J. 32, 542 (1938). 5vW. L. Hughes, Jr., H. A. Saroff, and A. L. Carney, J. Am. Chem. Soc. 71, 2476 (1949). ~8D. L. Harris, J. Biol. Chem. 166, 541 (1946); G. E. Perlmann, Nature 166, 870 (1950) 59I. W. Sizer, J. Biol. Chem. 169, 303 (1947).

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[11]

trypsin near the pH optima of these enzymes.S° Pepsin may be used with advantage at a higher than optimal pH (e.g., pH 4). 6~ The extent of digestion after various time intervals can be ascertained by means of the ninhydrin reaction (see p. 252). The termination of the reaction represents a definite problem. Trypsin can apparently be specifically inhibited even in the presence of substrate by addition of the soybean trypsin inhibitor. 60 In other cases the substrate enzyme may be removable by fractionation methods (e.g., isoelectric precipitation, ultracentrifugation), or further digestion may be prevented by change in pH and/or temperature. Carboxypeptidase has been used extensively for the prime purpose of elucidating the C terminal and adjacent amino acids. In general, the biologically active proteins investigated have retained their activity after loss of their C terminal amino acid. 62,83 Digestion with Carboxypeptidase (see Vol. II [8]). Preliminary experiments may be performed with commercial crystalline carboxypeptidase, although it is recognized that traces of endopeptidases are often present in such preparations. The carboxypeptidase crystal suspension (1 mg. of enzyme) is dissolved with a minimum of alkali (about 0.015 ml. of 0.1 N NaOH), buffered with 0.04 ml. of 1% bicarbonate, and diluted to 0.35 ml. To the substrate, adjusted to pH 7 to 8, is added one-fiftieth of its weight of carboxypeptidase. Amino-N (ninhydrin) is determined prior to addition of the peptidase and again after 2 hours at room temperature; if no appreciable increase (of the order of 0.2 to 1 equivalents per mole of substrate) has occurred, the digest may be incubated at 40 ° and again analyzed at intervals. Aliquots are also taken at various stages for assay of the enzymatic activity of the substrate. Depending on the rate of digestion, more or less (1/~0 to 1/~o0) carboxypeptidase may be preferable. The nature and the approximate amount of amino acids liberated can often be very simply ascertained by chromatographying aliquots (corresponding to about 0.05 ~M. of amino-N increase) of the digest directly, usually first in butanol-acetic-water. If the particular protein causes streaking, then other techniques must be employed 63 the detailed description of which are outside of the scope of this chapter. Before reliance can be placed on any results obtained with carboxys0 J. Gergeley, J. Biol. Chem. 200, 543 (1953); 19th Intern. Physiol. Congr., Montreal, p. 389 (1953); E. Mih~flyi and A. G. Szent-Gy6rgyi, J. Biol. Chem. 201, 189, 197, 211 (1953). el M. L. Petermann and A. M. Pappenheimer, Jr., J. Phys. Chem. 45, 1 (1941). 62 j . I. Harris and C. H. Li, J. Am. Chem. Soc. 74, 2945 (1952); J. I. Harris and C. A. Knight, Nature 170, 613 (1952). 63 H. Fraenkcl-Conrat, J. I. Harris, and A. L. Levy, in " M e t h o d s of Biochemical Analysis" (Glick, ed.), Vol. II, p. 360. Interscience, New York (1955).

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267

peptidase, contaminating pancreatic endopeptidases (mainly chymotrypsin) must be inactivated, which is usually done by adding the very toxic inhibitor diisopropylfluorophosphate (DFP)64 (see next section) to the enzyme solution (molar ratio about 50:1, or 0.02 ml. of 2%, i.e., 0.1 M DFP per milligram of enzyme) at pH 8, and holding the mixture for at least i hour at room temperature prior to addition to the substrate. Action of Diisopropylfluorophosphate (DFP) on Esterases ~5

The protein reaction which pinpoints most clearly the enzymatically active site is the stoichiometric interaction of many esterases, including trypsin and chymotrypsin (but excluding papain, carboxypeptidase, and phosphate ester-splitting enzymes) with DFP and related polyalkyl phosphates. This reaction has not been completely elucidated, but it appears certain that a particular spatial arrangement of several amino acid residues, probably involving a histidine and a serine residue, is needed for the substitution of one H atom by the dialkyl phosphate group. ~6 It also appears very probable that this same site is functionally involved in the fixation of the substrate ester which, in contrast to the dialkyl phosphate ester, can be split and released. The high affinity and specificity of DFP for the reactive group which favors stoichiometric reaction with esterases in quite dilute solution, and yet prevents any interaction with all inert proteins including the zymogens (chymotrypsinogen) and the denatured enzymes, makes this the ideal reagent for the study of this particular enzymatic site. All enzymes should be investigated for their susceptibility to DFP, and an active search for new specific inhibitors of other enzymatic sites should be vigorously pursued. The technique of the reaction of esterases with DFP has been described in the preceding section. If inactivation is observed, then phosphorus and other analyses are required as evidence for the mode and extent of reaction. For these the reader is referred to the original literature. ~5 Conclusions

The specificities of each of the reactions which have been discussed are summarized briefly in the table. The danger of causing denaturation with the various reagents is also estimated in the table, although gen64All direct contact with DFP and its fumes should be avoided. 85E. F. Jansen, M. D. F. Nutting, R. Jang, and A. K. Balls, J. Biol. Chem. 179~ 189 (1949); E. F. Jansen and A. K. Balls, ibid. 194~ 721 (1952). ~6 T. Wagner-Jauregg and B. E. Hackley, Jr., J. Am. Chem. Soc. 75~ 2125 (I953); R. E. Plapinger and T. Wagner-Jauregg, ibid. 76, 5757 (1953); N. K. Schaffer, S. C. May, Jr., and W. H. Summerson, J. Biol. Chem. 202, 67 (1953); 206, 201 (1954); W. N. Aldridge and A. N. Davison, Biochem. J. 55, 763 (1953).

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eralizations in this regard are very hazardous, owing to the wide range in the stability of enzymes. A considerable number of reactions can be performed in aqueous solution near neutrality and at room temperature, and these are regarded as nondenaturing conditions for most enzymes. Yet, some of the more specific are not among the safest reactions. The investigator will have to choose those methods which are within the stability range of his enzyme and run suitable controls to ascertain the effect of the reaction conditions per se. Considerable information has been accumulated for many enzymes, concerning the groups which are termed essential and unessential for their activity. A review of these studies appears outside of the scope of this book, and the reader is referred to other sources. ~-7,67 At a recent symposium on the Mechanism of Enzyme Action the newer developments i~1 our understanding of the nature and the mode of action of certain enzymatic sites were described. I t now appears probable that primary bonds may be formed between certain protein groups and the substrate. Thus, in the case of glyceraldehyde-3-phosphate dehydrogenase an - - S H group is believed to react with the aldedydic substrate to yield a hemimercaptal which is oxidized to the thiol ester at the expense of the bound coenzyme, DPN. The free D P N enzyme is regenerated by another molecule of coenzyme and by an acceptor for the newly formed acyl group. 34 Although the complete structure of an enzymatic site has as yet not been unequivocally established, working hypotheses are under active study in many laboratories. Data of the type described in this chapter will be needed as a basis, and as a test, of any such hypotheses. ~7 R. R. Porter, in " T h e Proteins" (Neurath and Bailey, eds.), Vol. I, Part B, p. 973. Academic Press, New York, 1953.

[19.]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

273

[12] T e c h n i q u e s f o r t h e A s s a y of t h e R e s p i r a t o r y E n z y m e s

By BRITTON CHANCE Introduction The classic method for the identification and characterization of respiratory enzymes is based on the appearance of the sharp absorption bands of their reduced forms when the respiring cells become anaerobic owing to their own respiration. This oxidation-reduction reaction served as the basis for the historic studies of MacMunn ~ and of Keilin 2 on cytochromes. As more components of the respiratory mechanism have become identified, attention has been directed toward quantitative estimates of their concentration. For this reason there has been considerable development, pioneered by Keilin, 2,3 of specialized chemical reactions that reveal the characteristic absorption bands of only one component of the respiratory system. Such reactions have been especially useful in conjunction with spectrophotometric recording of the absorption bands of the pigments and have served as the basis of a better differentiation of the respiratory components and their quantitative estimation. 4-6 Various chemical reactions that we have found useful in our spectrophotometric studies of members of the respiratory chain are briefly outlined.

Specific Chemical Reactions Carbon Monoxide. Carbon monoxide is an example of the ideal chemical reactant, since it combines specifically with the iron atom of the mammalian terminal oxidase, cytochrome a3. m,9,~ In the visible region of the spectrum no very distinctive effects are obtained when the reduced form of cytochrome a3 is treated with carbon monoxide, but the differential recording method in the region of the Sorer band brings out very 1 C. A. MacMunn, J. Physiol. 6, 22 (1885). D. Keilin, Proc. Roy. Soc. B98, 312 (1925). 3 D. Keilin and E. C. Slater, Brit. Med. Bull. 9, 89 (1953). 4 B. Chance, in " T h e Mechanism of Enzyme Action" (McElroy and Glass, eds.), p. 399. Johns Hopkins Press, Baltimore, 1953. b B. Chance, Harvey Lectures 49, 145 (1954-55). B. Chance and G. R. Williams, J. Biol. Chem. 217, 395 (1955). 7 O. Warburg, Biochem. Z. 177, 471 (1926). s D. Keilin and E. F. Hartree, Proc. Roy. Soc. B127, 167 (1939). 9 B. Chance, J. Biol. Chem. 202, 383 (1953). 9, L. N. Castor and B. Chance, J. Biol. Chem. 217, 453 (1955).

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distinctly the absorption band due to the carbon monoxide compound of cytochrome a3. 9 In this method, two samples of the material containing the respiratory chain are used, and both are made anaerobic by their own respiration. The difference of absorbancy between the two samples is then recorded, with the split-beam method, l°,H over a convenient span of wavelengths, usually 400 to 480 m~. This trace, of course, should show no distinctive absorption band, since the concentration of pigment and the optical path lengths in the two samples have been adjusted to be identical. One of the suspensions is now bubbled with a fine stream of CO gas for about 1 minute. The spectrum representing the difference of absorbancy between the two samples is again, recorded, and if cytochrome a3 is present in sufficient concentrations ( ~ 10-7 M), the sharp absorption band having its peak at 429.5 m~ and a trough at 445 m~ will be clearly defined (Fig. 1). This is a completely satisfactory identification of cytochrome a3, and the quantitative amount present may be estimated by dividing the total optical density change (the increase at 430 m~ minus the decrease at 444 m~) by the change of extinction coefficient, 91 cm. -1 mM.-l. 12 The pigments of mammalian tissues that interfere with this assay are hemoglobin, myoglobin, and a pigment found in microsomes. 18Various protein hemochromogens will not interfere because they are not reduced by substrate under anaerobic conditions and require a chemical reducing agent such as dithionite. Several methods are available for reducing the contamination by blood pigments. First, erythrocytes are usually completely removed from mitochondria in the process of differential centrifugation necessary for preparation of mitochondria. In cases where hemoglobin or myoglobin contamination is unavoidable, oxidation of these pigments by nitrite, followed by dialysis to remove the nitrite, has often been used, for example, in the case of leucocytes 14 and of heart muscle preparation. 1~ The ferric forms of the blood pigments are not reduced to ferrous by anaerobiosis and hence do not contribute to the carbon monoxide spectrum. The ferrous forms of hemoglobin or myoglobin combine with carbon monoxide and give rise to an absorption peak at 419 m~ and a trough at 432 m~. The effect of hemoglobin upon the assay of cytochrome aa is illustrated by the traces of Fig. 2. It is seen that the trough at 444 m~ is very little affected while the peak at 430 m~ of the CO compound of cytochrome a3 is shifted to shorter wavelengths 10B. Chance, Science 121, 621 (1955). 11C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954). 12B. Chance, J . Biol. Chem. 202, 397 (1953). ~aM. Klingenberg,unpublished data. 14B. Chance and L. N. Castor, Science 116, 200 (1952). 15B. Chance, J. Biol. Chem. 197~557 (1952).

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?~%u) FIo. 1. Illustrating the measurement of the concentration of cytochrome a3 by the formation of its carbon monoxide compound; a reproduction of original experimental records of spectra representing the difference of absorption between the oxidized and reduced forms of the cytochromes of a Keilin and Hartree heart muscle preparation (trace a-b) and between the reduced forms and the carbon monoxide compound of cytochrome a3 (trace d-c). Trace a represents a base line with the two cuvettes of the split-beam apparatus filled with a suspension of Keilin and Hartree's heart muscle particles. On the addition of succinate to one of the cuvettes, and after anaerobiosis, trace b is recorded. Trace c is recorded after addition of succinate to the aerobic cuvette and after both cuvettes have become anaerobic. Trace d is the one referred to in the text and is obtained after bubbling carbon monoxide gas through one of the anaerobic samples. The peak at 429.5 m# and the trough at 445 m# identify the carbon monoxide compound of cytochrome a3. The optical density change due to this compound is read off the scale of the graph. Trace e is obtained after carbon monoxide treatment of both samples. The small displacement of trace d with respect to the others was accomplished electrically in order to avoid confusion with the remaining traces. This method of chemical-spectral analysis differs from that of Lundeg~rdh ~a who subtracts the spectrum of the known cytochromes from the total spectrum of all components. (Keilin and Hartree heart muscle preparation, pH 7.2, 0.15 M phosphate buffer, 13 raM. of succinate added. (Courtesy of The Journal of Biological Chemistry.) b y a low c o n c e n t r a t i o n of h e m o g l o b i n - C O c o m p o u n d a n d t h e 430 m # p e a k m a y be o b l i t e r a t e d b y t h e H b - C O peak a t h i g h e r c o n c e n t r a t i o n s . N i t r i t e t r e a t m e n t s h o u l d be used u n d e r these c o n d i t i o n s . 14,15 T h e m i c r o s o m a l p i g m e n t whose c a r b o n m o n o x i d e c o m p o u n d a b s o r b s a t 450 m # has n o t b e e n f o u n d to be a s s o c i a t e d w i t h c y t o c h r o m e a3 a n d i n t e r f e r e n c e of t h e two c o m p o n e n t s is r e n d e r e d u n l i k e l y b y the r a t h e r wide s e p a r a t i o n of t h e i r a b s o r p t i o n b a n d s . I n a d d i t i o n , t h e 450-m# CO c o m p o u n d f o r m s r a t h e r slowly 13 a n d m a y t h e r e b y be d i s t i n g u i s h e d f r o m t h a t of c y t o c h r o m e a3, w h i c h f o r m s rapidly.16 ~eB. Chance, J. Biol. Chem. 202, 407 (1953). 1~ H. Lundeg~rdh, Biochim. et Biophys. Acta 20, 469 (1956).

276


[12]

In bacteria, three other types of carbon monoxide compounds have been observed2 The first is cytochrome a~ the terminal oxidase of Acetobacter pasteurianum. This terminal oxidase is similar t o t h a t of the mammalian system in m a n y ways and, in fact, was mistaken for it by Warburg in his pioneer studies of the photochemical dissociation of their carbon monoxide compounds.~7 The peak of the absorption band of the carbon monoxide compound cytochrome a~ lies at 427 m~, and the corresponding trough is observed at 442 m~. ÷0.1~ I¢EJ v

e-, / s

'l I

i°

\

-o.i

I

•

¢.J

o

i

380

i

400

420

i

|

i

440

460

480

~(rnjj) Fro. 2. Illustrating the effects of hemoglobin contamination upon the estimation of cytochrome aa of a heart muscle preparation in terms of its carbon monoxide compound. A small contamination is apparent in the solid trace and a larger value in the dashed trace (cf. trace d of Fig. 1). T h e second cytochrome is called the " C O - b i n d i n g p i g m e n t " because of its characteristic reaction with carbon monoxide2 In this case, the peak of the absorption band of the CO compound in the Soret region lies at 416 m~, with a corresponding trough at 432 m~ (Fig. 3). This compound is readily observed in m a n y types of bacteria, particularly M icr ococcus pyogenes var. albus and A cetobacter suboxydans. 18 T h e third carbon monoxide compound is t h a t of cytochrome as. This one has an indistinct Soret band t h a t is difficult to distinguish in the living cell but has a readily observable 647-m~ band with a corresponding trough at 630 m~.18 In m a n y cases, the terminal oxidases of bacteria are mixed, and various combinations of the spectra due to the carbon monoxide compounds 1~O. Warburg, "Schwermetalle," Freiburg, 1949. is L. Smith, Bacteriol. Revs. 18, 106 (1954).

[12]

TECHNIQUES FOR &SSAY O ? REBPIR&TORY ENZYMES

277

of cytochromes al, a2, and the CO-binding p i g m e n t h a v e been observed. E x a m p l e s of the complicated form t h a t the difference s p e c t r u m result in are given elsewhere. 9 +0.04.

Reduced , . + 0.02.

E

u

Reduced + CO

C

E u

_c

O,

C

a O

~_ - 0 . 0 2 . (3. o 380

420 ),,(re,u)

460

Fie.-,. 3. Identification of a terminal respiratory enzyme, the "CO-binding pigment," by means of its carbon monoxide compound. The spectrum represents the difference between the carbon monoxide compound of the "CO-binding pigment" and the reduced forms of the cytochromes of Micrococcus pyogenes var. albus. In addition, the spectrum representing the difference between the steady-state oxidized and reduced forms of the respiratory enzymes of this bacteria is presented. (Courtesy of Journal of Biological Chemistry.) Carbon monoxide is the m o s t effective reagent for identification of terminal oxidases; it is highly specific, and the carbon monoxide compounds h a v e distinctive absorption b a n d s in the Sorer region. Other studies show t h a t these CO c o m p o u n d s are inhibitors of cellular respiration because of the correspondence of the photodissociation and photochemical action s p e c t r u m for the relief of carbon monoxide inhibition of respiration. 9~ Cyanide. Cyanide is not nearly so useful as carbon monoxide because of the lack of spectroscopically distinct c o m p o u n d s of the terminal o×idases. 8,18 I t s effects on the peroxidase of y e a s t cells and on the catalase of bacteria are m u c h more pronounced t h a n on the terminal oxidases.

278


[12]

In addition, its combination with oxidized hemoglobin and myoglobin will cause spectroscopic interference. Other r e a g e n t s - - a z i d e , ethyl hydrogen peroxide, and so f o r t h - - g i v e less distinctive spectroscopic effects 8 and do not compare with carbon monoxide as a reagent for q u a n t i t a t i v e analysis of the terminal oxidases. Antimycin A, Hydroxyquinoline-N-Oxirne. When the m a m m a l i a n respiratory chain is a p p r o p r i a t e l y t r e a t e d with one of these substances, the principal b a n d which a p p e a r s is t h a t of reduced c y t o c h r o m e b with

A c ~ ~cm) *.080"

J.

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+.060.

'+.015

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u

-

+.040'

•+.010

~

+.020.

•+.0O5 u~

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o

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~

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,_005 g~

-.0~0 °

o

._u -o040o =060

°

'E

,-.o10

8

o

4bo

4go

5bo

"A(m)J)

5io

660

-.ol5

FIG. 4. Identification of cytochrome b by means of antimycin A treatment of respiring cells; the spectrum represents the difference between antimyein A-treated yeast cells and aerobic yeast cells. The absorption peaks shown are those of yeast cytochrome b together with a small quantity of flavoprotein. A similar spectrum for liver mitochondria is given in Fig. 9. (Courtesy of Johns Hopkins Press.) its Soret b a n d at 430 mtL and its a - b a n d at 562 mt~. 8,19-2j I n order to obtain the m o s t distinctive results, two samples of the respiratory s y s t e m are oxygenated and t r e a t e d with an a p p r o p r i a t e s u b s t r a t e so t h a t electron transfer proceeds at a m o d e r a t e rate. A base line is then run with the s p e c t r o p h o t o m e t e r to indicate equality of a b s o r b a n c y of the two specimens. One is then treated with the a p p r o p r i a t e a m o u n t of the inhibitor, and the difference in a b s o r h a n c y between the two samples is again plotted. This will reveal a sharp p e a k at 430 m~ with a corresponding trough at a b o u t 412 mtL (Fig. 4). I n the region of the ~-band, the peak and trough lie at 562 and 575 mt~. Depending on the intensity of respiration, other spectroscopic changes will a c c o m p a n y t h a t of c y t o c h r o m e b; for example, flavoprotein and pyridine nucleotide will become reduced ~9B. Chance, Nature 169, 215 (1952). ~0E. C. Slater, Biochern. J. 45, 14 (1949). 31 V. R. Potter and A. E. Rief, Federation Proc. 10, 234 (1951).

[12]

TECHNIQUES F O R ASSAY OF RESPIRATORY ENZYMES

279

due to the action of the inhibitor. Cytochromes c, cl, and a will become more oxidized, an illustration of the "pseudo-reversibility" of the respiratory chain. The effect on cytochrome b, however, so dominates the spectroscopic results that no confusion will result. TM Antimycin A and hydroxyquinoline-NIoxime are the most useful inhibitors. They act rapidly and at a low concentration with the intact respiratory chain. Usual values for maximal spectroscopic change are of the same order as the amount of cytochrome b involved. In Keilin and Hartree's succinic oxidase preparations, the end point of the titration of cytochrome b with antimycin A has been found to be half that of the cytochrome b content.19 Intact mitochondria respond very sensitively to these inhibitors, as do yeast and ascites tumor cells, although the rate of penetration of the inhibitor may be quite slow. In intact muscle, for example, frog's sartorius muscle, rather high concentrations and long incubation periods have been found to be necessary in order to obtain proper penetration of the antimycin A. 22 Hydroxyquinoline-N-oxime also interrupts the respiratory chain of certain bacteria but causes oxidation of reduced cytochrome bl in E. coli and Pr. vulgaris. 23 Chin has reported that urethan acts on the respiratory chain of Acetobacter peroxydans, although his report has not been confirmed. 24 Taken all in all, these inhibitors of the oxidation of reduced cytochrome b give very distinctive spectroscopic effects which can easily be observed spectrophotometrically at both 562 and 430 m~. The molarity of cytochrome b may be estimated by the optical density change measured at 562 and 575 m~. This optical density change may be divided by 22 to give the millimolar concentration of cytochrome b (see discussion below and ref. 19) (optical path length, 1 cm.). The concentration of cytochrome b may also be estimated from the optical density change measured at 430 and 410 m~, the peak and trough of the changes at the region of the Soret band, but in this case there is some interference due to cytochrome c, which, as mentioned above, becomes oxidized when cytochrome b becomes reduced. If, however, cytochrome c is known independently already to be oxidized before the addition of the inhibitor, then estimation of cytochrome b in the region of the Soret band ~a A condition which would render the estimation of cytochrome b difficult b y this m e t h o d is the presence, in cells containing endogenous substrate, of a strong dehydrogenase a n d a weak oxidase system which would cause the cytochrome b to be already reduced before t h e addition of the inhibitor. This difficulty is readily avoided if the substrate is r e m o v e d from the system b y vigorous aeration for a reasonable interval (18 hours is sufficient for b a k e r ' s yeast). 22 A. M. Weber, u n p u b l i s h e d data. 23 j . W. Lightbown a n d F. L. Jackson, Biochem. J . 63, 130 (1956). 24 C. H. Chin, Dissertation, Cambridge University, 1952.

280


[12]

is satisfactory and an extinction coefficient of about 180 cm. -1 mM. -~ may be used for liver mitochondria (see below). Except for one recent report in which optical constants were not given, 25 cytochrome b, as distinguished from cytochrome bs, has not been highly purified, and the molecular extinction coefficients have been derived by analogy with other purified hemoproteins. We have first used the characteristics of cytochrome b obtained from bacteria by Pappenheimer et al. ~'6 In a personal communication he has indicated that the optical density difference between the oxidized and reduced forms of cytochrome b at 560 m~ is about the same as that obtained at 554 m~ for the pyridine hemochromogen, corresponding to a value of 22 cm. -1 raM. -~. This value is in good agreement with the change of molecular extinction coefficient that has been calculated from the original data of Bach et al. ~7 on cytochrome b2, which they state to be essentially the same as that of cytochrome c (19.1 cm. -~ mM.-1). Later studies of cytochrome b2 give a value of 23 cm. -~ mh/[.-1. 28-3° Recent studies of the cytochrome b5 system give a similar value.3° Strittmatter and Velick3~ purified cytochrome bs, and we read from their graphs changes of extinction coefficients of 22 cm. -~ mM. -~ for the a-band and 160 cm. -1 mM. -~ for the -~ band. We therefore take 22 cm. -1 mM. -~ to be correct for the a-band of cytochrome b, as measured at 562 and 575 m~. In difference spectra of cytoehrome b, as measured in various materials, the ratio of the optical density changes at 430 and 410 m~ to those at 562 and 575 m~ varies from 8:1 in liver mitochondria to 10:1 in heart muscle succinic oxidase preparations. A m y t a l . Amytal inhibits electron transport near the lowest member of the respiratory chain, 32,33and therefore the changes of the cytochromes are in the direction of oxidation; such changes are large enough to be noticeable only if adequate levels of substrate are already present. Thus, on addition of Amytal to a respiratory chain supplied with substrate and oxygen, the spectra shifts observed are oxidations of eytoehromes a, a3, 2~I. Sekuzu and K. Okunuki, J. Biochem. (Japan) 48, 107 (1956). 2s A. M. Pappenheimer, Jr., and E. D. ttendee, J. Biol. Chem. 171, 701 (1947). 27 S. J. Bach, M. Dixon, and L. G. Zerfas, Biochem. J. 49~ 229 (1946). 28 C. A. Appleby and R. K. Morton, Nature 175~ 749 (1954). 29 E. Boeri, E. Cutolo, M. Luzzati, and L. Tosi, Arch. Biochem. and Biophys. 56, 487

(1955). 30 B. Chance, M. Klingenberg, and E. Boeri, Federation Proc. 15, 231 (1956). 81 p. Strittmatter and S. Velick, J. Biol. Chem. 221, 258 (1956). a2L. Ernster, O. Jalling, H. LSw, and O. Lindberg, Exptl. Cell Research Suppl. 3, 124 (1955). 3~B. Chance, in "Enzymes: Units of Biological Structure and Function" (Gaebler, ed.), p. 447. Academic Press, New York, 1956.

[12]


281

b, c, c~, and, in the case of liver mitochondria, flavoprotein. The distinctive change that is observed is the reduction of pyridine nucleotides (Fig. 5). Thus, Amytal is a specific reagent for the presence of pyridine nucleotide in the respiratory chain. Such a specific reagent is necessary DPNH

/

* 0.03-

/

/

/'\

'\ ' \

/

\ '

\ \

÷0.01

I

'

\

/

tO02

\~

Amytol to State 5 \ (AOH as subs~rale

E

k C

0

\

¼_

/

E

,\

/

//

-OOI-

--0102

© -0,03

3~0

3~0

4bo

:k (m~u)

4~0

sbo

FIG. 5. Identification of reduced pyridine nucleotide in rat liver mitochondria by means of Amytal treatment; the spectrum represents tim difference between Amytaltreated mitochondria (2 mM. Amytal) and aerobic mitochondria in state 3 (supplied with phosphate acceptor, phosphate, substrate, and oxygen). The trough at 430 m# is due to the oxidation of reduced cytochrome b, and the peak at 340 m# is due to the reduction of pyridine nucleotide (experiment 522-A).

in cases where only small amounts of pyridine nucleotide are present: the transition from aerobiosis to anaerobiosis may be unsuitable for revealing pyridine nucleotide, since this change causes reduction of cytochromes that have their ~-bands in the region of 320 m#. An example of

282


[12]

this is afforded by the work of Holton, ~4 whose data show no distinctive band at 340 m~ in the aerobic-anaerobic difference spectra of the a-ketoglutarate oxidase system of the heart muscle sarcosomes. Amytal treatment, on the other hand, gives an optical density change attributable to reduced pyridine nucleotide (PNH), and this change is readily measurable with a sensitive spectrophotometer25 The concentration of PNH has been estimated by dividing the optical density change measured at 340 and 374 m~ by 6.38 Pyridine Nucleotide-Linked Substrates. Oxidizing and reducing substrates for PN-linked dehydrogenases are nearly ideal reagents for the assay of a single component of the respiratory chain. If, for example, a PN-linked substrate were added to material made anaerobic with a nonPN-linked substrate, one would record only the absorption due to PNH. Unfortunately, in mitochondria, all substrates, including succinate, cause reduction of pyridine nucleotide. ~3 On the other hand, the reversibility of the dehydrogenase system does provide an excellent opportunity to obtain a highly specific interaction of a substrate with a respiratory chain. Mitochondria are made anaerobic by the addition of phosphate acceptor plus a low concentration of the DPN-linked substrate, ~-hydroxybutyric acid. Addition in excess of the reaction product, acetoacetic acid, plus ADP, will cause oxidation of pyridine nucleotide without affecting any other component. No measurable amount of flavoprotein oxidation has been observed under these conditions. Similar reactions can be obtained by reduction of pyridine nucleotide with a small concentration of glutamate, followed by oxidation with ammonium chloride. Reactions such as these are applicable to intact yeast cells which contain an active alcohol dehydrogenase system. A low concentration of ethanol is used to reduce the pyridine nucleotide of the anaerobic cells. An excess of acetaldehyde causes oxidation of the pyridine nucleotide without affecting other components 5 (Fig. 6). Intracellular pyridine nucleotides can be oxidized by a-glycerol phosphate dehydrogenase activity. 4 DPN is reduced by addition of ethanol to anaerobic yeast. The cells are then heated with iodoacetate to inhibit glyceraldehyde-3phosphate dehydrogenase activity. The D P N H is reoxidized on addition of glucose from which dihydroxyacetone phosphate is formed and rapidly reacts with D P N H to give DPN and a-glycerol phosphate. Penetration of hydrogen ions into the yeast cells also causes an oxidation of reduced pyridine nucleotides because of the pH-sensitive equilib34 F. A. Holton, Biochem. J. 61~ 46 (1955). 35 B. Chance and M. Baltscheffsky, Biochem J. in press. 38 P a b s t Laboratories Circular OR-7 (1955).

[19.]


283

rium of the reduced and oxidized forms. Thus, ethanol-reduced DPN can be largely reoxidized by addition of organic acids or hydrazoic acid. ~ It is also possible to distinguish between D P N H and T P N H by these methods; for example, if glucose is added to the starved yeast cells in

I 500

520

I

I

I

+0.10

I

,~(m~ 540 560 I

I

380 I

400_

I

i

I -I- 0.05 E v ID

E '/I -

--

,~nuerobi~

~

~ e a s t

(ethano~

eect)

j,

~ ~-

0

I

\

a°

Addition of 15ran /

\~-'acetoIdehyde-y

\

I/

:-

~-'5

-o.o

\__J

0.10 FIG. 6. Identification of intracellular pyridine nueleotide in yeast cells by a shift of the oxidation-reduction equilibrium by means of acetaldehyde; the spectrum represents the difference between acetaldehyde-treated yeast cells and yeast ceils treated with alcohol alone. The broad trough indicates the oxidation of intracellular DPNH.

which the terminal oxidase is inhibited by addition of cyanide or by anaerobiosis, all the pyridine nucleotides become reduced. On the other hand, if the yeast cells are pretreated with iodoacetic acid in order to inactivate glyceraldehyde-3-phosphate dehydrogenase, the addition of glucose activates zwischenferment and causes reduction of T P N only.

284


[12]

With a particular yeast, only 10% of the total pyridine nucleotide was so reducible, a result that is in accord with chemical assays. 37 This result also indicates that the intracellular transhydrogenase activity of these yeast cells can be neglected; otherwise the reaction T P N H + D P N ~-* T P N -}- D P N H would have proceeded. 3s Apparently the reaction between D P N and flavoprotein is not sufficiently reversible to cause appreciable oxidation of flavoprotein on the addition of these DPN-linked substrates. This has the advantage in that there is no interference from flavoprotein, and the disadvantage that these methods cannot be used to assay independently for flavoprotein. In order to obtain a quantitative measure of the D P N H absorption, the absorbancy changes are measured at 340 and 374 mp for the following reasons: The choice of 340 m~ is an appropriate one for mitochondria and sarcosomes, since we find there that the pyridine nucleotide that is involved in the oxidative phosphorylation reaction is not bound in such a way as to shift its absorption maximum from 340 m~ (Fig. 7). It has been shown, however, in yeast and in certain bacteria that the absorption maximum occurs at a wavelength of about 330 m~, and this result has been verified by Lundeg~rdh. TM This shift has been attributed to binding of D P N H to alcohol or lactic dehydrogenase. 4,j9,~9,4° The appropriate molecular extinction coefficient in this case is 5.8 cm. -1 miV[.-1. 89 In metabolizing cells the possibility of pyruvate accumulation often exists and its absorption band may interfere with the assay of pyridine nucleotide. The difficulty is readily avoided by (a) reducing the oxygen concentration in the cell suspension to a low value before adding a substrate, such as glucose which leads to pyruvate formation, and then measuring the pyridine nucleotide as soon as anaerobiosis has occurred; (b) establishing anaerobiosis with ethanol, then adding glucose; or (c) oxidizing the reduced pyridine nucleotide by acetaldehyde, etc. The choice of 374 mu as the reference wavelength for the measurement of reduced pyridine nucleotide is based on the following considerations: First, a wavelength shorter than 340 m~ is undesirable because the opacity of the sample increases rapidly with decreasing wavelengths. Second, in cells which contain glycolytic as well as respiratory chains, a considerable amount of glyceraldehyde-3-phosphate-dehydrogenase-DPN compound tl may be present. This compound has an absorption peak at 365 m~ that 87H. yon Euler, F. Schlenk, H. Heiwinkel, and B. H6gberg, Z. physiol. Chem. 256, 208 (1938). 38S. P. Colowick, N. O. Kaplan, and M. M. Ciotti, Federation Proc. 10, 174 (1951). 39H. Theorell and R. K. Bonnichsen, Acta Chem. Scand. 5, 1105 (1951). 40B. Chance and J. B. Neilands, J. Biol. Chem. 199, 383 (1952). 41E. F. Racker and I. Krimsky, J. Biol. Chem. 198, 731 (1952).

[12]


285

disappears on the reduction of pyridine nucleotides or on addition of iodoacetate 4z (Fig. 8 4). On the other hand, this absorption band is so broad that it has about equal values of extinction at 340 and 374 m~. 4 Thus, measurements made differentially between these two wavelengths

-I- 0.01

I

-

E U v

C

E

o~

c

u~

-0.01

0

_~

-0.02

o

-0,03-

350

s6o (re,u)

FzG. 7. Identification of reduced pyridine nucleotide in mitochondria by treatment with adenosine diphosphate. The spectrum represents the difference between mitochondria treated with ADP and those to which only substrate and phosphate were added. The oxidation of reduced pyridine nucleotide is indicated by the trough at 340 m~. The oxidation of reduced cytochrome b is indicated by the trough at 430 m~ (experiment 522-A).

would not be affected by the glyceraldehyde-3-phosphate-dehydrogenaseDPN compound. Wavelengths longer than 374 m~ are undesirable, since the Soret bands of the cytochromes interfere, and, in addition, the difference between 340 m~ and the reference wavelengths should be kept at a minimum. As will be shown below, flavoprotein interference at 374 m~ is not significant. Judging from the published data on most purified flavoproteins, a major absorption band in the vicinity of 360 mu (for a recent summary see reference 42) should be observed in the intact cell and should interfere with the measurement of pyridine nucleotide when the reference wavelength is 374 m~. An exception is the flavoprotein component of purified 4~ H. R. Mahler, Advances in Enzymol. 17, 233 (1956).

286


[12]

succinic dehydrogenase which does not show a 360 m~ absorption band. 42, We have, however, obtained no evidence for such an absorption band in i n t a c t cells or in mitochondria. A r a t h e r crucial test for the presence of such an absorption band is afforded b y studies of guinea pig liver mitochondria in which the absorption of the oxidized form of flavoprotein at 465 m~ is nearly as great as the absorption of reduced cytochrome a~ at 445 m#. 6 Nevertheless, no definite evidence of an absorption band at 360 m# has been obtained in the oxidized minus reduced difference spectra IE ¢J v

>-,

ooo:t\

Qa

r~ 0

300

350

400

450

500

(mjJ)

Q.

0

FIO. 8. Identification of glyceraldehyde-3-dehydrogenase by means of the effect of iodoacetate on the spectrum of its DPN compound. The spectrum represents the difference between yeast cells treated with iodoacetate and aerobic yeast cells. The trough in the neighborhood of 360 m~ indicates the disappearance of the absorption band of the glyceraldehydephosphate dehydrogenase-DPN compound on the addition of iodoacetic acid. (Courtesy of Johns Hopkins Press.) or n the r a t h e r more critical test obtained b y adding antimycin A to mitochondria in the aerobic quiescent state (state 4). In this state the pyridine nucleotide is already over 99 % reduced and the flavoprotein is considerably oxidized. 43 T h e addition of the inhibitor causes reduction of the flavoprotein (and cytochrome b as well; see above) and would presumably reveal the second flavoprotein absorption band if it were of appreciable magnitude. Anaerobiosis. B y definition, those pigments whose oxidation state changes from oxidized to reduced under anaerobic conditions are respirat o r y pigments. Absorption bands due to cytochromes a3, a, c, cl, and b, and reduced pyridine nucleotide appear under these conditions, whereas the absorption band due to oxidized flavoprotein disappears (Fig. 9). Thus, m a n y absorption bands appear nearly simultaneously in anaerobiosis, but the several' components m a y be separately assayed b y measur42=T. P. Singer, E. B. Keaney, and V. Massey, "Enzymes: Units of Biological Structure and Function" (Gaebler, ed.), p. 417. Academic Press, New York, 1956. 43B. Chance and G. R. Williams, J. Biol. Chem. 217, 409 (1955).

[12]

287


ing their absorption at appropriate pairs of wavelengths. This procedure is necessary for four of the components that are not individually affected by the chemical treatment described. These components are cytochromes a, c, and c~, and flavoprotein.

I--

80

" o:

.

7O 6O 0

50 n."

40 U.

3O

UJ n U)

2O I0 0

MILLIGRAMS

Fro. 4. Replot of data from Fig. 3 as specific activity (c.p.m./mg.) versus milligrams of sample per planchet.

Because of the greater uncertainty attaching to the assay of very thin samples it is best not to use preparations of less than about 2 mg./cm. 2, especially for reference purposes, unless unavoidable. For the same reason, extrapolation to zero thickness may be deceptive and inapplicable to the experimental situation. As can be seen in Fig. 4, the scatter of experimental points becomes excessive at low sample weights. The empirical procedure described above will give a completely dependable basis for correction independent of theoretical considerations, provided the samples are uniformly and reproducibly plated and enough samples (15 to 25) are used to obtain a smooth curve covering the range of sample weights to be encountered. Samples must be dried to constant weight before counting. It has been found that the empirical self-absorption correction curve

[20]

THE MEASUREMENT OF RADIOISOTOPES

447

about 78%, and on lead to almost 90%. 71 Infinitely thin plates are sometimes useful when dealing with very high specific activity materials, but it is obvious that absolutely identical support material must be used to permit valid comparisons of samples. As the weight of sample increases, the contribution of backscattering from the support decreases. More and more of the downward-directed radiation is now scattered from the deeper layers of the sample (self-scattering) rather than from the planchet material. In the case of BaC1403 plated on aluminum, the percentage of backscattered radiation actually increases with increasing sample thickness, since the C ~4/3-particles reflect more strongly from BaCO3 than they do from aluminum. As the thickness of the sample counted is increased, it is observed that a larger fraction of the radiation is counted than would be calculated on the basis of known factors such as geometry, self-absorption, and simple backscattering. This anomalous effect is attributed to a "focusing" effect of the sample tending to direct a larger than theoretical fraction of its emitted radiation in the vertical direction. The magnitude of the selfscattering effect can be large, and it varies from one source material to another. For example, S 35 counted as a thick layer of PbSO4 can give a 25% higher counting rate than the same amount of S 3~ counted as a thick layer of benzidine sulfate. 72 Because of the potential magnitude of backscattering and selfscattering, it is important to be aware of these effects and to avoid directly comparing assays on different materials or materials plated differently. Because of the difficulty in evaluating these effects it is inadvisable in routine work to attempt to make explicit corrections for them. Experimentally determined self-absorption curves, discussed below, include implicitly any corrections for backscattering and selfscattering in the particular cotmting system used and for the particular materials being assayed.

D. Counting Efficiency Correction Only a fraction of the total radiation from a given sample is actually counted by the G-M tube. This fraction, designated efficiency, is a function of the geometrical relation of sample to counting tube, absorption in intervening air and in the end window, operating voltage, fluctuations in ~1D. Christian, W. W. Dunning, and D. S. Martin, Jr., Nucleonics 10, No. 5, 41 (1952). 73D. W. Engelkeimer, J. A. Seller, E. P. Steinberg, and L. Winsberg, Radiochemical Studies: The Fission Products, Vol. 9, Div. IV, p. 56. National Nuclear Energy Series, McGraw-Hill, New York, 1951.

448

TECHNIQUES FOR ISOTOPE STUDIES

[9.0]

sensitivity of the counting apparatus, and other factors. In general, the over-all efficiency of a thin end-window counter will be in the neighborhood of 20% for high-energy f~-particles like those of p3~ and in the neighborhood of 5 to 10% for weak ~-particles such as those of C 14 and S 3~. Considerably greater efficiency is possible with windowless flow counters (40 to 50%) or with liquid scintillation counters (50 to 90%). The efficiency of ordinary G-M tubes for -y-rays is generally less than 5 % of their efficiency for f~-particles and special counting devices are required (see Gamma R a y Counters). 1. Geometry. The spatial relationship between the sample, the sample supporting apparatus, the intervening absorbers, and the detecting device (geometry) determines the fraction of emitted radiation that can enter the detecting device. Optimal geometry is, of course, obtained by suspending the sample within the counting device so that all the emitted radiation can potentially be counted (4~- geometry). For solid samples plated on ordinary planchets the maximum theoretical efficiency if the sample is placed within the counting device is 50% (2~ geometry). The term geometry can be used in referring in a general way to the sample-counter arrangement, or it can be used to mean specifically the fraction of the total solid angle about the sample subtended by the counter. In a given end-window counting arrangement the efficiency falls off very sharply as the sample is moved down away from the counter. If the losses due to absorption and to scattering effects are ignored and only the geometric efficiency is considered, it can be shown, for example, that a sample placed 2 em. from the window will be counted with an efficiency less than one-half that obtainable with the sample placed 0.5 cm. from the window (window radius, 2.46 cm. ; sample radius, 1.91 cm.). J Exact centering of the sample is equally important. A point source of radioactivity if moved out so that it lies just under the edge of the tube will give only one-half the count it will give when coaxially centered under the tube. In addition to these purely geometric factors the losses due to absorption in the air layer between the sample surface and the window become very important when dealing with weak/~-emitters. Even a 1-mm. layer of air will absorb about 5% of C 14 radiation. Finally, it must be recognized that, owing to the complex nature of the interactions of f~-rays with matter, scattering from the sample supports, lead housing, etc., can lead to very different observed counting rates independent of the other factors discussed above. Beta-particles in a closed counting chamber are like bullets in a canyon, ricocheting crazily from surface to surface in an unpredictable manner. Because of their

450


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for example, to rely on a UX~ or R a D -~ E reference standard. The ideal reference standard is one made with the particular isotope to be assayed. BAC1403 STANDARD. An amount of BaC140~ that will give an observed counting rate of approximately 1000 is plated on a planchet identical with those to be used for assaying samples. To prevent mechanical losses and possible exchange reactions a thin coating should be layered over the standard, in which case the sample before sealing should have a somewhat higher count to allow for the absorption loss. Collodion or nail polish can be used, but a conducting material such as silver paint is less likely to give trouble due to electrostatic effects. Ordinarily C 14 is an adequate reference standard for work with C ~4, S 36 or Ca 4~. E. D e c a y C o r r e c t i o n

Radioactive decay is the prototype of a unimolecular reaction. Each unstable atom in a sample of a given radioactive material has an equal probability of disintegrating in a n y given time interval, and so the rate of disintegration, dN/dt, is directly proportional to the number of radioactive atoms, N, present at any moment:

dN

-- d--/ = k N

(7)

where k is the disintegration constant, the fraction of the atoms disintegrating per unit time. Rearranging and integrating we obtain: -(ln~ N2 - ln, N1) = h(t2 - t~)

(8)

If we let No = number of radioactive atoms at time zero, and N = number of radioactive atoms remaining at time t, we obtain the usual form of the decay equation: ln, - ~ = ),t

or

No 2.303 lOgl0 - ~ = ~t

(9)

When one-half the labeled atoms originally present (No) have disintegrated (N = N0/2), we have No lne N~-2 = lne 2 = Xt~

(10)

The time it takes for this decay to occur is called the half-life or t~ for the particular isotope. The natural logarithm of 2 is 0.693, so we have 0.693 t~ = ~, (11) The half-life (t~) and the disintegration constant (h) are simply alternative ways of expressing the decay rate of an isotope and can be

[@.0]


451

readily interconverted by the above relationship. The decay rate of radioisotopes is for all practical purposes an immutable property, having a fixed value for each isotope independent of its chemical form or the physical and chemical stresses to which it may be subjected. In the above equations N has been defined as the absolute number of radioactive atoms present. This can be expressed in terms of the absolute number of disintegrations per minute (d.p.m.) in the sample, since that, is directly proportional to N (equation 7). Most commonly, however, one actually uses the observed counts per minute (c.p.m.) in a particular detecting system which is in turn proportional to the absolute disintegrations per minute (d.p.m.). Provided the values in observed counts per minute for No and N in equation 9 are obtained under identical conditions and are appropriately corrected for other factors as discussed below, they will be directly proportional to the number of radioactive atoms at time zero and time t. Since only the ratio of the two count rates enters the equation, the absolute values are not needed. Half-lives and some other properties of the more frequently used radioisotopes are given in Table I. Comprehensive compilations of data for all known isotopes are available in a number of reference sources.3.~ If an experiment extends over a time period significantly long relative to the t~ of the isotope used, it is necessary before comparing samples to correct the observed activities of all samples to the activity they would have had if counted at the same time. A convenient rule of thumb to use in deciding if a decay correction is necessary is that in a time period equal to one-tenth the half-life the activity of a sample will decrease by 6.5%. Exact corrections can be made by substituting the appropriate disintegration constant in equation 9 and expressing the time in corresponding units. Much more convenient is the use of a graph of correction factors which can be simply prepared on semi-logarithmic paper. A straight line is drawn from the point 1 on the ordinate at zero time through the point 0.5 on the ordinate at a time corresponding to t~. The line can be extended to cover as many half-lives as may be needed. The counts for samples assayed at any time, t, from the beginning of the experiment are corrected back to the initial time simply by dividing by the fraction read off the ordinate. Although convenient, the accuracy of this method is limited by the scale of the graph paper used. More precise corrections can be made with log tables or, more quickly, with a log-log slide rule. The simplest way of dealing with decay corrections, however, is to set, aside an aliquot of the initial labeled material for use as an internal standard in a given experiment and count this along with each batch of samples. By expressing all sample activities relative to the activity of this standard counted at the same time, the decay correction is made auto-

452


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matically, thus eliminating tedious calculations. This direct comparison makes the decay correction independent of any errors in the published half-life value, and the repeated counting of the internal standard may, in addition, show up radioactive impurites of different half-life in the original material.

IV. Units of Radioactivity and the Reporting of Radioactivity Data The curie is that amount of any radioactive isotope in which 3.7 X 10 ~° atoms will disintegrate per second. More commonly used are the subunits, the millicurie (inc.), 3.7 X 107 disintegrations per second (d.p.s.) and the microcurie (~c.), 3.7 × 104 d.p.s. Since count rates are generally expressed in counts per minute (c.p.m.) it is useful to remember that 1 ~c. --- 2.22 × 10 s d.p.m. These units of radioactivity define a rate of disintegration and not an amount of material. However, for any particular isotope the rate of disintegration is, as discussed above, directly proportional to the number of radioactive atoms present and can be considered equivalent to an expression of the amount of labeled material. As can be seen from equation 7, the actual number of atoms of a radioactive isotope corresponding to 1 inc. of radioactivity will depend on the disintegration constant or the half-life (see equation 11). For example, 1 mc. of p3~ undiluted by stable phosphorus (carrier-free) would represent only about 3.5 × 10-6 mg. of phosphorus, whereas it would require 0.2 rag. of undiluted C ~4 to give the disintegration rate corresponding to 1 inc. Specific radioactivity is a measure of the amount of radioisotope per unit of sample, and there are many different ways of expressing it. When the absolute disintegration rate is known, it is most often expressed as millicuries per millimole (inc./raM.) or as millieuries per gram (mc./g.) or in subunits analogous to these. More commonly, when only the relative count rate is known in a given counting system, the millicurie is replaced by counts per minute (c.p.m.) or counts per second (c.p.s.) per unit weight of sample. In some cases, for example in studies of reaction pathways, it is more informative to express specific activity in terms of the atom being used as a tracer. Frequently C 14 results are expressed not per unit weight of compound but per unit weight of carbon in the compound. In some studies the specific activities of individual carbon atoms may need to be separately determined for comparative purposes. A host of expressions for specific activity have been proposed for application in particular studies, and a survey of some of these has been made by Schulman and Falkenheim. 74 74j . Schulman, Jr., and M. Falkenheim, Nucleonics 3, No. 4, 13 (1948).

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453

Unfortunately there is no accepted standard procedure for reporting radioactivity data in biological experiments. Because of the diversity both in the nature of experimental design and in the types of equipment used for radioassay, complete standardization is not possible. This being the case, every effort should be made to report such data in sufficient detail and in such a manner as to permit the reader, even though he may not be experienced in radioassay techniques, to evaluate the results properly. The following items are suggested as a minimum to be presented when reporting radioisotope experiments: i. Type of counting equipment. The kind of detecting device employed is particularly relevant in interpreting raw count data. 2. Background counting rate. 3. Observed counting rate--the actual range of counts per minute recorded for the samples as prepared for radioassay. 4. Statistical error of counting (see Section V). 5. Types of corrections made on the observed count, particularly the type of self-absorption correction made. Of course, there is no need to elaborate this basic information in detail along with the final tabulated data. It can easily be summarized in brief form along with the other data of the experimental section. Generally the radioactivity data will have to be presented in a derived form in the body of the paper. An attempt should be made, however, to avoid extrapolating too far from the observed counting data. For obvious reasons raw counting data obtained on microgram quantities should not, for example, be reported as counts per minute per milligram.

V. Statistics of Counting and Counting Artifacts A. Statistical Error of Counting Radioactive decay being a random process, the closeness with which the rate observed during a finite counting time approaches the " t r u e " rate increases with the total number of disintegrations observed. Since every determination involves a background correction, calculation of the uncertainty in the corrected count rate must take into consideration the uncertainty in this background rate. The standard deviation, S.D., of any measurement in which N random events are recorded is simply __+ ~ If a total of 400 counts is recorded, for example, S.D. = +_20 counts, and the chances are about 2 in 3 that the " t r u e " count lies between 380 and 420, and 19 in 20 that it lies between 360 and 440. Standard deviation expressed in counts per minute is +_ %/N/t, where t is the time in which N counts are recorded. What-

[20]


o

f h-

,8 1111

455

!! 2

I

5

I

~

B(Ickgfound (cprn}

20

I0

20

30

40

60

80

I00

150

-

Must be counted

-

250

RATE FOR SAMPLE + BACKGROUND[(c.pm)obs]

Fie. 5. Nomogram for estimating counting time needed to reduce standard deviation to +__5 %. The necessary time over which the sample must be counted is deter-

mined from the observed or gross count rate (sample + background) during the first minute or two and the measured background rate (counted for at least 30 minutes). counting time to obtain 5 % accuracy is shown by the curves in Fig. 5 for a series of different background counting rates.

B. Counting Artifacts The application of the statistical evaluation above is predicated on the assumption t h a t the detecting device is completely stable in response and t h a t the only variations are due to the statistical randomness of the radioactive decay process. However, as in all measuring devices, the introduction of artifacts is almost inevitable. The most serious problems arise in connection with transient artifacts, since t h e y can easily go undetected. For example, intermittent operation of high-voltage equipment, leak detectors, X - r a y apparatus, and the like m a y lead to false high count rates for isolated samples. Similarly, operating defects in the detecting or scaling device m a y go undetected if t h e y are intermittent. Errors of this type m a y be disclosed only if several independent counts on the same sample are made at different times. Since reference standards are usually made so as to give several thousand counts per minute an artifact t h a t introduces only 10 to 20 spurious c.p.m, will not be detected in the count rate of the standard. However, if samples with less than 100 c.p.m, are being assayed very serious errors will be made. More frequent counting

456


[20]

of background would help under such circumstances but again only if the spurious count source continued at the same intensity during counting of both samples and background. In other words, artifacts may be present and go undetected, particularly with samples of low activity, until specifically checked for. Memory effect, contamination with radioactivity from previously counted samples, can be an important source of error, particularly in immersion and gas-phase counting and occasionally in windowless flowgas counting. Such an effect will, of course, show up in the background if the latter is checked between samples. Spurious counts may be occasioned by electrostatic effects. Plastic and paper are particularly apt to cause this type of artifact. The count rate may be high during the first few minutes and then stabilize subsequently. As mentioned above, counter discharges can be initiated by photons, and so with transparent window counters light artifacts are a potential source of error. Failure of a scaling stage or, more frequently, of a mechanical register may be overlooked particularly if intermittent. Introduction of a known 60-cycle pulse source, provided with the newer scalers, offers a simple check. Visual inspection of the scaling indicator bulbs and the register during a slow count will suffice to pick up simple failures.

VI. Preparation of Samples The manner in which samples are prepared for counting will be determined by the character of the radiation, the amount of radioactivity, and the properties of the isolated compounds.

A. Solid Samples The most frequently encountered form of C 14 in radioisotope work is C1402, either directly formed or derived from organic compounds by oxidation or by specific decarboxylation reactions. In the former case, such as collections of expired C140s or C1402 formed during in vitro incubations, the gas can be trapped directly in a solution of COs-free NaOH. Many procedures for trapping expired COs by bubbling the respiratory gases through towers of NaOH have been described. ~ C~402 formed during in vitro reactions can be trapped in the center-well alkali of a Warburg vessel or in an alkali-soaked fluted filter paper appropriately supported in an ordinary stoppered Erlenmeyer flask. For quantitative collection, of course, the incubation medium must be acidified (pH 5) and sufficient time allowed for complete absorption of CO2 from the gas phase

[20]


457

at the end of an incubation. The alkali-soaked paper and washings from the center well are transferred to a 50-ml. glass-stoppered graduate cylinder which is made up to volume with water and shaken vigorously several times. Elution of carbonate from the paper is not instantaneous but is complete after 16 hours of standing at room temperature. When dealing with organic compounds, CO2 can be obtained by combustion procedures 75,7~ or by specific reactions such as ninhydrin decarboxylation. 7~ Frequently it will be necessary to modify published procedures for application to isotope experiments. For example, the net amount of carbon available in a tracer experiment may be less than the amounts for which a given method is designed. If only total radioactivity is to be determined, carrier can be added to ensure quantitative collection of CO2. Any conversion procedure should be checked with the specific material under investigation and with the quantities actually to be encountered. When small amounts of carbon must be handled and specific radioactivities are required it is obviously essential to avoid dilution with CO2 from room air during conversion procedures and during subsequent sample preparation. If the a m o u n t of sample is known or can be determined prior to radioassay (for example, manometric measurement of ninhydrin-labile CO2 or COs derived by wet combustion) subsequent dilution with unlabeled COs is no longer a problem. The specific radioactivity is calculated from the total counts in the sample and the amount determined before any unlabeled diluent is allowed access to the sample. In fact, it may be desirable, when the total radioactivity is not limiting, deliberately to add carbonate after measuring the amount of COs so that any losses during handling will represent a smaller percentage of the original amount and thus facilitate sample preparation. C1402 derived by any of the general methods discussed above is most commonly converted to BaC1403 for counting because the very low solubility of this compound permits quantitative recoveries and because its physical properties favor the preparation of uniform samples for counting. In some cases C~40~ can be directly trapped in Ba(OH)2 as, for example, in applying the ninhydrin amino acid decarboxylation method of Van Slyke. ~s Unless the COs is trapped in a centrifuge tube, however, such a procedure involves a transfer that is difficult to make 76D. D. Van Slyke and J. Folch, J. Biol. Chem. 136, 509 (1940). 76D. D. Van Slyke, J. Plazin, and J. R. Weisiger, J. Biol. Chem. 191, 299 (1951). 77D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J. Biol. Chem. 141, 627 (1941); see Vol. III [75]. 78D. D. Van Slyke, D. A. MacFadyen, and P. B. Hamilton, J. Biol. Chem. 141,671 (1941).

[20]


459

The disk should be removed from the filter-funnel assembly while still slightly moist and dried slowly at room temperature or in a vacuum oven at low temperature. After weighing, the sample can be placed directly on a planchet for counting. If it will not lie flat it can be clamped in place with a ring that slips over the edge of the circle of paper and fits snugly around a holder, 64 or it can be pasted down to the planchet surface. Flaking, especially at the edges, tends to be a problem, and the samples must be gently handled to avoid cracking. The method of Armstrong and Schubert 81 is advantageous in this respect. Instead of removing the filter paper the entire filter cup with the precipitate in place is removed and placed under the counter. 3. A technique for packing washed precipitates onto the planchet by centrifugal force has been described. 82 In this case the planchet is incorporated as a part of the base of a centrifuge cup. 4. When the size of available sample permits, BaCO3 can be ground in a mortar and pestle, transferred as a dry powder to the planchet, and tamped down firmly to obtain a solid, fiat plate. A tamper milled to fit snugly within the walls of the planchet is convenient. After the powder is distributed uniformly over the bottom, the tamper is gently lowered into the planchet. Several sharp taps with a hammer give a firmly packed cake. When the products to be examined are volatile or difficult to precipitate or crystallize, conversion to C02 and BaC03 is virtually a necessity. On the other hand, many compounds lend themselves very well to precipitation and direct counting. In such cases the time-consuming conversion to BaCO3 is not at all necessary. As long as the isolated material is reasonably pure and plates well, a self-absorption curve can be prepared as described above and the samples can be counted as isolated. Moreover, in studies involving a series of homologous or closely related compounds the self-absorption correction curves frequently will be the same provided the average atomic numbers of the materials are not too different. For example, the average atomic numbers of phenylalanine, tyrosine, dihydroxyphenylalanine, and epinephrine are practically identical, and it is found that the self-absorption correction curves for these compounds measured in a windowless flow-gas counter are so close that one curve accurately serves for all of them. sa Karnovsky et al. 66 have published selfabsorption data on a number of organic compounds showing again that the self-absorption curves are very nearly identical. It should be noted, 81W. D. Armstrong and J. Schubert, Anal. Chem. 20, 270 (1948). 8~F. C. Larson, A. R. Maass, C. V. Robinson, and E. S. Gordon, Anal. Chem. 21, 1206 (1949). 8sS. Udenfriend, unpublished data.

460


[9.0]

however, that the introduction of a heavy atom into a derivative (as in conversion to BaCOn) may significantly alter the shape of the selfabsorption curve. The degree of disparity depends to some extent on the counting system employed. 66 Studies with S ~ usually involve conversion to sulfate and then precipitation as either BaS04 or benzidine sulfate. The radiation produced by S 35 is similar to that of C 14, and the same problems arise with respect to self-absorption and sample preparation. Actually most of the techniques described above for C 14 are equally applicable to S 85 studies. A discussion of sample preparation techniques with S 35 has been presented by Tarver. 84 Because of its penetrating radiation, pa~ presents no serious problems with respect to self-absorption, and so the physical form of the sample is far less critical than with C 14 or S 35. In most cases the product can be plated and counted in the form isolated. 1131 is most conveniently measured in solution by using a well scintillation counter to detect its ~,-radiation. However, it also emits an 0.6-1V[ev. ~-ray (87.2% of the disintegrations) and can be counted in solid form with ordinary G-]VI counters. Tritium/~-particles are so feeble (0.018 Mev.) as practically to exclude counting solid samples.

B. Liquid Samples Labeled compounds in solution can be assayed either as thin layers in ordinary planehets or in immersion-type counters as discussed above. The outstanding virtue of both techniques is the high degree of sample uniformity obtainable. With the latter method no self-absorption correction is needed, since all samples are counted in layers of identical thickness, determined by the dimensions of the apparatus. With the former method no self-absorption correction will be necessary if "infinitely thick" layers of solution are counted. When thinner layers are counted the sample is always absolutely flat 86 and the thickness can be determined volumetrically from the density of the solution and the dimensions of the planchet dish used. Because of these virtues, liquid sample plating has been applied even in the demanding area of absolute standardization of isotopes. ~4 The vapor from volatile solvents and even from water may be incompatible with stable counting behavior in windowless gas-flow counters. However, m a n y of these counters can be 84 H. Tarver, Advances in Biol. and Med. Phys. 2, 281 (1951). s5 Some error can be introduced b y " m e n i s c u s effects" a t the edges a n d b y "creeping." These can be minimized b y the use of a n appropriate surface active agent.

[20]


401

fitted with an extraordinarily thin end window (0.2 to 0.8 mg./cm.2), which will prevent vapor effects and yet not decrease efficiency very much. Some solvents can be used directly in windowless counters because of their low vapor pressure (e.g., formamide), and when such solvents can be used the range of applications for liquid sample counting is greatly increased because of the gain in efficiency.88,87 Preparation of samples for liquid scintillation counting has been discussed above (Section II, G). Research in this area is increasing rapidly in intensity and in scope. Large strides can be safely predicted for the near future.

C. Gas Samples21 Materials to be counted in the gas phase must be converted to a gas that will not interfere with the operating characteristics of the counting tube or ionization chamber. The gases that have been successfully assayed by this technique include C1402,tS,76,s8 tritiomethane, 26-2m9 and C ~acetylene. 23.24 The preparation of the gaseous sample calls for a complex manifold system fitted with appropriate manometric equipment. The size of sample introduced is usually determined by measurement of its partial pressure in the system. In the case of many gases the amount of material that can be counted is limited, since at higher partial pressures they interfere with tube operation. For example, Robinson's system for counting tritium after conversion to methane permits the equivalent of 0.2 ml. of water to be introduced as methane. 2°,27 On the other hand, as much as 15 mg. of carbon in the form of C02 can be assayed by the procedure of Van Slyke et al. 22 The gas must be perfectly dry, since the introduction of even small amounts of water vapor will seriously alter the counting characteristics of the system. Although gas-phase counting offers several advantages, outstanding of which is the maximal efficiency obtained, it has not been widely used in biochemistry, no doubt because the procedures are complex and time-consuming.

VII. Detection of Radioactivity on Paper Chromatograms Radiochemical analysis utilizing paper chromatography permits detection and identification of minute amounts of material. The sensitivity and simplicity of the procedure make it most useful. 86A. Schwebel, H. S. Isbell, and J. V. Karabinos, Science 118, 465 (1951). 87D. L. Tabern and T. N. Lahr, Science 119, 739 (1954). 88K. Wilzbach and W. Y. Sykes, Science 120, 494 (1954). 8~K. E. Wilzbaeh, L. Kaplan, and W. G. Brown, Science 118, 522 (1953).

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463

components in a chromatographic strip may be close together and may in many cases smear into odd shapes, the use of a slit perpendicular to the strip will often give misleading results concerning separation. Improved resolution can be obtained only at the expense of reduced sensitivity. Furthermore, when isotopes with high-energy radiation are used (I TM, p~2, etc.) they may penetrate the masking piece so that an appreciable number of counts will be derived from portions of the chromatogram adjacent to that in front of the slit. 3. The autoradiographic procedure offers the maximum of sensitivity for visualization of radioactive chromatograms. The high resolving power of the chromatographic technique is excellently matched by photographic resolution. Exposure of a chromatogram to a photographic plate results in darkened areas exactly corresponding to the separated labeled components. It is then possible to trace these bands or spots from the photograph onto the original chromatogram and separate and elute each radioactive band no matter how close or misshapen the bands may be. 93 In addition, the photograph provides a permanent record of the chromatographic results. Although almost any type of film can be used for this purpose, it is best to select a fast, relatively coarse-grained emulsion, since the resolution even with such coarse-grained emulsions is adequate. Films which have been used successfully in many laboratories are Eastman No-Screen and Eastman Type K, Industrial. These can be obtained in many different sizes, making it possible to photograph iudividual strips or even large two-dimensional chromatograms. Before exposing the chromatogram to a photographic plate, all traces of solvents should be removed, since some solvents may damage the emulsion or produce an intolerably high background fogging. Guide marks may then be drawn on the chromatogram with "radioactive ink " ordinary ink to which some highly radioactive nonvolatile material has been added. These marks appear on the autoradiograph and later permit exact superposition on the chromatogram for definition of the radioactive areas. The next step is carried out in a dark room under the appropriate safelight for the film used. The marked chromatogram is placed in direct contact with the film and attached in such a manner that no movement of either will occur during exposure. This can best be achieved by use of commercially available lightproof cassettes. Weights placed on top of the cassette ensure the closest contact between film and chromatogram, which is essential for ~naximum resolution. The exposure time needed is determined by the type of film, the 93 S. Udenfriend and M. Gibbs, Science 110, 708 (1949).

464

TECH~IC~UES FOR ISOTOPE STUDIES

[20]

nature of isotope emission, the total number of counts, the thickness of the filter paper used for chromatography, and the area covered by the band. The times of exposure and amounts of radioactivity required for detection of a number of isotopes can be estimated from the data presented by Steinberg and Solomon24 For instance, with Type K Industrial film a spot with density 0.1 above background film density is obtained in 24 hours if the absolute B-flux is 900 d.p.m./cm. ~. With the average endwindow G-M counter this corresponds to an observed count rate of approximately 100 to 150 c.p.m, when the paper is placed 1 cm. below the tube for assay. In order to get this intensity of radiation emerging from Whatman No. 1 filter paper, three times this total amount of radioactivity must be contained within the paper. It should be emphasized that these values represent the minimum requirements and whenever possible more radioactivity should be used. When the amount of radioactivity is limiting, long exposure times can, of course, be used. The film density obtained is a simple function of the total number of f~-particles striking per square centimeter. Therefore, the density will, in the case of long-lived isotopes like C ~4, be approximately a linear function of exposure time. For S 35 and Ca 45 exposure requirements are very similar to those for C 14. In the case of pure H-emitters, the exposure time required when the emergent ~-flux is equal increases with increasing E ..... Losses by absorption in the filter paper will, of course, be less for the more energetic emitters. Gamma-ray emitters are extremely inefficient in producing photographic densities. Data for Ca 45, I TM, P~, and Zn ~5 have been published 94 and can serve as a rough guide in estimating required exposure times for a specific problem. After exposure the X-ray films are developed according to standard procedures. The dried film is placed on an X-ray viewer, and the chromatogram is again laid on top of it in exactly the same position as during exposure, with the "radioactive i n k " guide lines as coordinates. The dark bands can then be traced directly on the paper. Each one can then be cut out and eluted for further study. Historically, photographic methods were the earliest used to detect radioactivity, and this procedure is now widely employed to determine localization of labeled molecules in tissues. These histological techniques are adequately covered in a number of texts and will, therefore, not be discussed here25 ~4D. Steinberg and A. K. Solomon, Rev. Sci. Instr. 20, 655 (1949). 95G. A. Boyd, "Autoradiography in Biology and Medicine." Academic Press, New York, 1955.

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VIII. Multiple Labeling It is often desirable to determine the fate of more than one atomic species in a biological reaction. The two atoms may be independent, they may be part of the same compound, or they may combine to form a product. Instead of carrying out two separate experiments, each with a differently labeled material, double labeling makes it possible to make both observations in the same experiment, thereby eliminating biological variation. Studies have been carried out on the simultaneous incorporation of Clt-glycine and inorganic p32 into DNA and RNA 98 and on the simultaneous incorporation of Cl*-acetate and tritiated water into fatty acids2 7 Studies of this type yield information concerning relative rates and time sequences in a biosynthetic process which would be very difficult if not impossible to obtain any other way. Multiple labeling can also be used to determine time relations by introducing the same precursor labeled in different ways at different time intervals. Such a technique was applied by Green and Anker in determining the rates of antibody synthesis at different time intervals after the injection of antigen. 98 Double labeling used in conjunction with isotope dilution makes possible corrections for losses during isolation or chemical reaction. ,~ Another application of double labeling using S 35 and I TM has been made in the isotope derivative method, which is described below. This procedure has been applied to quantitative assay of amino acids and as a method for chemical identification at the microgram level. 100 The successful use of double labeling depends on methods for differential detection of the two isotopes. The methods for differential detection may depend on differences in rates of decay or in energy or quality of the radiation. Tait and Williams ~°~ have published an excellent paper concerning the assay of mixed radioisotopes in which each of the above methods for differential detection have been discussed. A simple technique for determining Na 24 and K 42 in a mixture by combining immersion counting and scintillation counting has been described by Robinson 9o p. M. Roll, tI. Weinfeld, E. Carroll, and G. B. Brown, J. Biol. Chem. 220, 439 (1956). 97j. H. Balmain, S. J. Folley, and R. F. Glascock, Nature 169, 447 (1952). 98 H. Green and H. S. Anker, Biochim. et Biophys. Acta 13, 365 (1954). 09 W. B. Stewart and H. H. Rossi, Nucleonics 11, No. 10, 66 (1953). 100 S.Udenfriend, J. Biol. Chem. 187, 65 (1950). 10~j . F. Tait and E. S. Williams, Nucleonics 10, No. 12, 47 (1952).

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467

per minute per mole (C~) gives the moles of amino acid in the original mixture: Moles of amino acid - Ts/S X I C~

IX. Isotope I~ffects The use of radioactive isotopes as tracers is predicated on the fundamental assumption that the labeled molecules will in all respects behave in a manner indistinguishable from the corresponding normal molecules in the system under investigation. In almost every biological application this assumption is fully justified, or at least the deviations are below the limit of experimental error in the study. However, it should be recognized that whereas the qualitative behavior of a radioisotope in chemical reactions will not differ from that of the stable isotope (the isotopes have identical electronic configurations), the quantitative aspects of their behavior (the isotopes have different masses) may be very different. Artifacts arising from such differences are termed isotope effects. Isotope effects are most likely when the percentage difference in mass between the isotopes is greatest, as in the case of hydrogen versus deuterium or, the extreme case, hydrogen versus tritium. When the labeled atom is a stably linked part of a large molecule and only the fate of that molecule as a whole is under investigation, then the effective mass difference is the difference in the formula weights of the unlabeled and labeled compound and becomes much less significant. For example, the mass difference between molecular hydrogen and molecular tritium would be 200 %, but the mass difference between stable valine and valine singly labeled with tritium would be less than 2 %. Even when the mass differences are very small, however, the possibility of isotope effects cannot be dismissed a priori, as will be seen in the examples below. Even though the differences in reaction rate for a single reaction may be all but undetectable, the amplification of such a small effect over a long series of sequential reactions can lead to highly significant isotope fractionation. After all, by patient protracted recycling U 23~ and U 238 can be separated on the basis of the minute differences in diffusion constant between their gaseous derivatives. Some examples of isotope effects that have been observed and their magnitudes may be useful. Weigl and Calvin ~°3 have shown that carbon dioxide uptake by algae from a C1202-C~40~ atmosphere results in considerable fractionation, the lighter form being taken up more rapidly. At the point at which 70% of the carbon dioxide had been fixed the relative specific activities of the carbon in the gas phase, in the liquid phase, and in the algae were 10.~j . W. Weigl and M. Calvin, J. Chem. Phys. 17, 210 (1949).

468


[20]

99, 106, and 81. Partition chromatography, as might be anticipated because of the repeated "adsorption-desorption" steps involved, has been found to effectively fractionate isotopically.labeled compounds. For example, Van Dyken TM has reported on the column fractionation of tritium-labeled formic acid from its stable analog. The original sample had a specific activity of 5.8 ~c./meq. but the specific activity of the eluted 1.5

GLYCINE i

"3"

ALANINE

,.o

,.~ 0.5

> . , ~ OI I3.0 V--z U ~JX 2.0 O. 0 ~ . 1

-

0.5 o:5. I J7s

r leo

I , las

t

t

I

I R f i oo

r

a

I t 19s

I

r

r

I ,, aoo

t

FRACTION NUMBER FIG. 6. Portion of the effluent curve from the chromatography of C14-]abeled amino acids on a 100 X 0.9-cm. column of Dowex 50: e, amino acid concentration as determined by ninhydrin; o, amino acid concentration as determined by C 14 count, 100% recovery assumed; and A, calculated specific activities.

material varied from 2.8 ~c./meq. at 10% elution up to 8.3 ~c./meq. at 90% elution. Similarly Piez and Eagle ~°5 have shown that the widely used procedure of Moore and Stein z°6 for separation of amino acids, using a 100-cm. column of Dowex 50, will separate CZ4-labeled amino acids from their normal analogs. Although the separation is not, of course, very great, it is enough to make the specific activity of the material in the first part of the eluted peak as little as one-fifth that of the last portion eluted (Fig. 6). On the basis of their studies Piez and Eagle suggest 104 A. R. Van Dyken, Abstracts of the 128th National Meeting of the American Chemical Society, Minneapolis, 1955, p. 280. z05 K. A. Piez and H. Eagle, Science 122, 968 (1955). ~08 S. Moore and W. H. Stein, J. Biol. Chem. 192, 663 (1951); 211, 893 (1954).

[9.0]


469

that the magnitude of the isotope effect may depend on just which positions are occupied by the C 1~ as well as the total amount of C 14 in the molecules. Unless effects of this kind are explicitly ruled out it is obviously advisable to pool all portions of a chromatographic band before taking an aliquot for determination of specific activity. Isotope effects of this kind could not be demonstrated in one-dimensional paper chromatographic separations of amino acids. 1°7 The strength of the chemical bond is a function of the masses of the atoms involved, and the differences in bond stability can be calculated on theoretical grounds. Yankwich and Calvin found that in the thermal decarboxylation of malonic acid a C12--C ~2 bond was split more readily than a C12--C 14 bond, the difference being about 12%. In the case of bromomalonic acid the difference was as much as 40%. ~°s Perhaps the most dramatic example of isotope fractionation in biological systems is found in the studies of Catch ~°9 on algae grown from seed culture with C~402 as the sole carbon source. On analysis it was found that the specific activities of individual amino acids in the proteins of these supposedly "uniformly labeled" organisms varied considerably. The carbon in glycine, for example, had a specific activity of 0.65 mc./mM., whereas that in phenylalanine had a specific activity of only 0.42 mc./mM. The possibility that isotope fractionation during the isolation procedures contributed to the observed differences must be considered. Artifacts may also arise in biological systems when the radiation from the tracer material is sufficiently intense or prolonged to alter the properties of the system. ~1°-H2 The sensitivity of detection devices is such that this source of artifacts should not be a serious problem but it must be kept in mind at all times.

X. IsotopicaUy Labeled Compounds A large number of labeled compounds are now commercially available including almost all the biologically important amino acids, fatty acids, vitamins, and steroids. Information concerning commercial suppliers and their products may be obtained through the Atomic Energy Com107 D. Steinberg, unpublished observations. 108 p. E. Yankwich and M. Calvin, J. Chem. Phys. 17, 109 (1949). 109j. R. Catch, in "Radioisotope Conference 1954/' Vol. I, p. 258. Academic Press, New York, 1954. 110 H. J. Curtis, Advances in Biol. and Med. Phys. 2, 1 (1951). HI Biochemical Aspects of Basic Mechanisms in Radiobiology, National Academy of Sciences, National Research Council, Publication 367, Washington, D.C., 1954. ~12A. H. Sparrow and B. A. Rubin, Effects of Radiations on Biological Systems, Brookhaven National Laboratories, Publication BNL 97 (T-22), Upton, New York, 1951.

[9.0]


471

and r a d i o a u t o g r a p h y is an excellent test of purity, particularly if several different solvent systems are used. However, even with these p r o c e d u r e , impurities can be overlooked if insufficient material is examined. If 20,000 c.p.m, of a c o m p o u n d were applied to W h a t m a n No. 1 paper, an i m p u r i t y appearing as a second spot representing over 1% of the activity would not be detectable after one d a y of exposure to an X - r a y film. Longer exposures and larger a m o u n t s of material would be needed to ensure detection of all impurities. Testing of purity should also be carried out periodically during the life of a labeled compound, even though the compound is originally pure and is k n o w n to be chemically stable. This frequent inspection m a y be needed to detect decomposition due to self-irradiation, ~3,H4 a problem which becomes more i m p o r t a n t as the specific activity of the compound increases. The problem of decomposition b y self-irradiation has been ably discussed b y L e m m o n . H5 Included in this article is a table listing rates of decomposition of several biologically i m p o r t a n t C ~4-1abeled compounds.

XI. Radiation Dosage and Protection Discussions of radiation hazards and methods of calculating radiation dosage are available in a n u m b e r of published texts. ~-3,H6 These and the m a n y excellent publications of the National Bureau of Standards H7 provide detailed information concerning the safe and proper handling of isotopes, radioactive waste disposal, monitoring techniques, and other aspects of radiation hazards. T h e isotopes of greatest interest to the biochemist (C ~4, S 35, H 3, and p32) emit only /3-radiation. T h e energy of the radiation from the first three is so low t h a t it is completely absorbed by the walls of the containers, and so the only hazard these present under ordinary circum~13B. M. Tolbert, P. T. Adams, E. L. Bennett, A. M. Hughes, M. R. Kirk, R. M. Lemmon, R. M. Noller, R. Ostwald, and M. Calvin, J. Am. Chem. Soc. 75, 1867 (1953). 1~4C. D. Wagner and V. P. Guinn, J. Am. Chem. Soc. 75, 4861 (1953). ~16R. M. Lemmon, Nucleonics 11, No. 10, 44 (1953). 116W. V. Mayneord and W. K. Sinclair, Advances in Biol. and Med. Phys. 3, 1 (1953). 117Handbooks of the National Bureau of Standards: No. 41, Safe Handling of Radioactive Isotopes; No. 47, Recommendations of the International Commission on Radiological Protection; No. 48, Control and Removal of Radioactive Contamination in Laboratories; No. 49, Recommendations for Waste Disposal of P~ and I T M for Medical Users; No. 51, Radiological Monitoring Methods and Instruments; No. 52, Maximum Permissible Amounts of Radioisotopes in the Human Body and Maximum Permissible Concentrations in Air and Water; No. 53, Recommendations for the Disposal of C 14 Wastes; No. 59, Permissible Dose from External Sources of Ionizing Radiation.

472


[20]

stances is through accidental ingestion or inhalation. Even radiation produced through ingestion of amounts up to 1 mc. of C14-1abeled compounds such as glycine is considered to be within permissible limits, l's Danger from mishandling of these isotopes is more frequently of concern because of possible contamination of the laboratory and counting equipment rather than because of threat to health. p32 is a f~-emitter of higher energy, and some radiation will penetrate the walls of the container. At preparative levels, with quantities of several millicuries, glass or plastic shielding should be used. In particular, safety goggles should be used to prevent radiation damage to the eyes. The ~-emitters with which the biochemist is mainly concerned are 1131, K 42, Na 22, Na 24, Cr 51, and Fe 59. Any large-scale studies with these v-emitters will require appropriate lead shielding and monitoring devices such as film badges and ionization meters. With these isotopes the problem of external radiation is a real one and should be given serious consideration. It should not be forgotten that ~/-emitters administered experimentally to animals or patients present potential hazards to the investigator also. The amounts of radiation emitted from experimental subjects given these isotopes are shown below (taken from the Radioisotope Guide of the N I H Radiation Safety Committee, February 23, 1954). Isotope 1in F e ~9 K .2 Na ~ N a 24

Approx. mr./hr, at 1 ft. from 1 mc. in subject 2.5 7.0 2.1 14.1 20.6

These quantites of radiation are appreciable. Thus, 2.5 hours of exposure at a distance of 1 foot to a subject who had received 1 mc. of Na ~4 would give 50 mr. which is the daily tolerance dose. Direct handling of millicurie amounts of ~/-emitting isotopes, as in a chemical experiment, should be avoided. Tongs and automatic pipets should be used.

Acknowledgment The authors are indebted to Dr. Charles V. Robinson for his generosityin reading the manuscript and making valuable suggestions and criticisms. 118A. M. Brues and D. L. Buchanan, Summary of Conference on the Toxicity of Carbon 14, Argonne National Laboratory Publication, ANL-4787, Chicago, 1925.

[21]

THE MEASUREMENT OF STABLE ISOTOPES

473

[21] T h e M e a s u r e m e n t of S t a b l e I s o t o p e s

By ANTHONY SAN PIETRO The use of stable isotopes for investigating biological problems, in which isotopes are employed as tracers for the constituent atoms of organic compounds, requires that a highly sensitive and accurate analytical procedure be available for determining the relative abundances of the isotopes. A number of methods which have been used for measuring isotope abundances in an element, such as the determination of atomic weights by chemical means or the analysis of optical spectra, are not applicable to biological investigations for one or more of the following reasons. First, the methods are not sensitive to small variations in the isotopic composition of the element under investigation. Second, large samples (of the order of 100 mg.) are required for analysis. In many biological experiments it is not feasible to obtain samples of this size. Third, the methods are too time-consuming to be useful for routine analysis. The only practical method for the precise determination of isotope abundance ratios of any stable element other than hydrogen involves the use of a mass spectrometer. For hydrogen the common procedures for determining isotope concentrations take advantage of the large difference in mass between hydrogen and its stable isotope, deuterium. In general, these methods are based on the variation of some physical property of water with its deuterium content. For example, the density of a purified water sample is a direct measure of its deuterium concentration. The more important of these methods have been described in detail in the excellent and authoritative book by Kirshenbaum 1 and will be discussed only briefly in a later section. The remainder of this article will, therefore, be devoted primarily to a discussion of the preparation of samples for mass spectrometric analysis of isotope abundance ratios.2 With the mass spectrometer it is possible to make accurate determinations of isotope abundance ratios on a routine basis and with a minimum of sample. In order to use this method, however, the element to be analyzed must first be converted into a convenient gaseous form. The gas should be such that the following requirements are fulfilled; (1) It should be easily preparable from the constituent atoms of organic com1I. Kirshenbaum, "Physical Properties and Analysis of Heavy Water." McGrawHill Book Company, New York, 1951. 2 See Vol. IV [37] for the preparation of gas samples of OTM for mass spectrometric analysis.

474


[21]

pounds. (2) Its structure should be as simple as possible from both the molecular and the isotopic viewpoint. (3) The gas should be readily p u m p e d out of the mass spectrometer; i.e., it should not be strongly adsorbed to the structural elements of the mass spectrometer.

The Mass Spectrometeff The mass spectrometer separates b y an appropriate combination of electrical and magnetic fields a beam of ions into a spectrum dependent on their masses. The relative abundances of particles of different masses is determined by collecting the ions on an insulated electrode (or electrodes). The ion current is a measure of the n u m b e r of positive ions falling on the collector electrode. A schematic diagram 4 of a mass spectrometer developed b y Nier is shown in Fig. 1. The gas sample is introduced into the gas-handling portion of the mass spectrometer and the pressure in this system adjusted b y means of the Toeppler pump. The sample is then continuously introduced into the mass spectrometer tube at such a rate t h a t the pressure in the ionizing region of the tube is maintained at approximately 10-4 mm. of mercury. The rate of flow is controlled b y means of a constriction between the gas-handling system and the mass spectrometer tube. In the ion source a heated tungsten filament emits electrons 5 which are accelerated and ionize the neutral gas molecules present in the mass spectrometer tube. Those gas molecules which are not ionized 6 are continuously removed from the mass spectrometer tube b y a high-speed diffusion pump. The positively charged ions are drawn out of the ionizing region and accelerated in the proper direction b y negative potentials on the electrodes indicated in the ion source. The ions are next subjected to the action of a uniform magnetic field. In this field t h e y travel in a eircu3 For a more detailed description of the mass spectrometer, see (a) A. O. C. Nier, in " A Symposium of the Use of Isotopes in Biology and Medicine," pp. 80-104. University of Wisconsin Press, 1948; (b) A. O. C. Nier, in "Preparation and Measurement of Isotopic Tracers" (D. W. Wilson, A. O. C. Nier, and S. P. Reimann, eds.), pp. 11-30. Edwards Bros., Ann Arbor, 1946. 4 Figure 1 appeared in an article by Nier ~ and is reproduced here through the kind courtesy and permission of the University of Wisconsin Press. 6The beam of electrons is collimated and held in a fixed position by the action of a weak magnetic field in the region of the ion source. This is not indicated in Fig. I. We are here concerned primarily with singly charged positive ions, that is, ions formed by the loss of one electron from the neutral gas molecule. It is not essential that the ionization of gas molecules be quantitative, since we are measuring the ratio of the relative abundances of ions of different masses rather than absolute amounts. The various isotopic molecules should, however, exhibit equal probability of ionization when bombarded by an electron beam of sufficient energy to cause ionization.

[9.1]


475

lar path. The radius of the orbit depends on the mass and energy of the ions. Heavier ions follow a path of greater radius than the lighter ions. In the usual situation where the isotope abundance ratio of a single element is being determined, the ion beam is thereby separated into two SAMPLE r~, CONSTRICTION GRIN[ TO ,"" PUMP

SOURCE

I

TOEPPLER PUMP

4i.,

M AG N ET--j~---~-* POLES MAGNET

LIGHTER

ION', HEAVIER IONS

NO.I

EXIT SLIT COLLECTOR NO.2

TO NO.I TO NO.2

VlERCURY DIFFUSION PUMP

PREAMPLIFIER

FIG. 1. Simplified schematic diagram of a mass spectrometer.

m a j o r ion b e a m s of different mass to charge ratio. These ion b e a m s are properly focused to fall upon the two collector plates, as indicated in Fig. 1, and the ion currents tO these plates measured. 7 T h e ratio of the intensities of the ion currents can be converted into a t o m per cent of the 7 I n Fig. 1, the mass spectrometer tube contains two collector electrodes for the simultaneous collection of ions of different masses. Alternatively, it is possible to employ a single collector a n d focus ions of different masses to fal! on the collector b y v a r y i n g either t h e accelerating voltage or the magnetic field, the other being held constant.

476


[21]

isotope under investigation b y appropriate formulas. These are presented in the later sections which deal with the individual elements. T h e p a t h of an ion in the mass spectrometer is given b y equation 1: B2r 2

me = 4.82 X 10-6- V where m e B r V

(1)

= = = = =

mass of the ion in atomic mass units. n u m b e r of electronic charges lost in ionization. magnetic field intensity in gauss. radius of curvature of the ion p a t h in centimeters. accelerating potential, t h a t is, the "potential through which the ion is accelerated, in volts. I t is evident from this equation that, if B and V are constant, the radius of curvature' of a given ion will be proportional to its mass to charge (m/e) ratio. 7 For reference, the stable isotopes of importance as tracers in biological systems are listed in the table together with their approximate natural abundances. NATURAL ABUNDANCES OF THE STABLE ISOTOPES OF BIOLOGICAL IMPORTANCEa

Element Hydrogen Carbon Nitrogen Oxygen

Isotope

Natural abundance, %

Hi H 2 (D) C1~ C 18 N 1~ N 15 Ole Ois

99.98 0.02

98.9 1.1 99.63 0.37 99.8 0.2

a Accurate values for the natural abundances of these stable isotopes have been obtained by Nier [Phys. Rev. 77, 789 (1950)]. Hydrogen 8 In order to use deuterium as a tracer for hydrogen in an organic compound, the isotope must be introduced into the compound in such a position t h a t it is nonexchangeable with the hydrogens of the medium. Thus, deuterium cannot be used to label the hydrogens of - - O H , - - N H ~ , - - S H , or COOH groups, since they undergo rapid exchange with the s H e a v y water can be purchased from the Stuart Oxygen Company, 211 Bay Street,

San Francisco, California.

[21]


477

hydrogens of water. 9 In general, any acidic proton will undergo exchange with the hydrogens of water; the extent of exchange will depend on the experimental conditions. For example, deuterium exchange under the influence of bases occurs on the a-carbon atom of ketones, esters, nitriles, etc. 10

Analysis by Mass Spectrometer ',~','2 Principle. Molecular hydrogen is the most suitable compound for the mass spectrometric analysis of deuterium. The procedure for determining the deuterium content of an organic compound involves the oxidation (or combustion) of the compound to water and the reduction of water to hydrogen gas. The oxidation is carried out in a standard combustion apparatus and involves no technical difficulties. The production of molecular hydrogen from water, however, must be accomplished in such a manner that no fractionation of the hydrogen isotopes occurs, that is, so that the isotope concentration of the hydrogen formed is the same as the water. Most of the reactions for liberating hydrogen from water cannot be employed, since they exhibit high fractionation factors (approximately 3 or greater) unless all the hydrogen of the water is quantitatively converted to molecular hydrogen. The method developed by Rittenberg T M and his co-workers, in which the reduction of water is carried out with zinc at about 400 °, has proved to be the method of preference. This procedure results in the quantitative production of molecular hydrogen which is equilibrated with respect to the reaction

H~ ~ D2 ~ 2HD

(2)

The equilibrium constant for this reaction has been calculated by Urey and Rittenberg 13 and is approximately 3.8 at 400 °. This calculated value has been verified experimentally. 14I t has further been demonstrated that the value of the equilibrium constant for this reaction does not vary greatly with temperature. '3.~4 The value of this constant is important in the derivation of equation 3. 9R. Schoenheimerand D. Rittenberg, J. Biol. Chem. 111, 163 (1935). 10For a detailed description of the structural requirements for an acidic proton, see L. P. Hammett, "Physical Organic Chemistry," pp. 243-245. McGraw-Hill Book Co., New York, 1940. 11j. Graft and D. Rittenberg, Anal. Chem. 24, 1 (1952). 1~D. B. Sprinson and D. Rittenberg, Supplement to the United States Naval Medical Bulletin, pp. 82-93, March-April, 1948. 13H. C. Urey and D. Rittenberg, J. Chem. Phys. 1, 137 (1933). 14D. Rittenberg, W. Bleakney, and H. C. Urey, J. Chem. Phys. 2, 48 (1934).

478


[21]

Apparatus and Procedure. The method developed and described by Graft and Rittenberg 11is the one most commonly used for the preparation of hydrogen samples for mass spectrometric analysis. A diagram of their apparatus is shown in Fig. 2.15 For the sake of simplicity of description, the apparatus m a y be considered to consist of two p o r t i o n s - - a combustion system and a system for the reduction of water and collection of hydrogen. TO Oil_ PUMP~,

Oz"-"~

IJ o 250CC( TO WATER

PUMP--"~

Fro. 2. Apparatus for the preparation of hydrogen samples for mass spectrometric analysis. 15 T h e combustion system consists essentially of a quartz or Vycor microcombustion tube, A, a tube filled with 5/[g(ClO~)2 for drying the oxygen, and a bubble counter for adjusting the rate of flow of oxygen during the combustion. A portion of the microcombustion tube is filled with cupric oxide or platinum wool which acts as the catalyst for the combustion. During the combustion process, the filled portion of the microcombustion tube is maintained at a t e m p e r a t u r e of 750 ° b y an electric furnace (which is not indicated in Fig. 2). The sample, which is introduced into the microcombustion tube in a platinum or porcelain boat, is cornbusted, and the resulting water is frozen out in trap C. The reduction of water takes place in the converter tube, B, which is filled with zinc granules. Approximately 200 samples of water can be reduced before the zinc filling has to be replaced. Reduction of water with a fresh zinc filling starts slightly above 360 °, although temperatures as high as 415 ° are required with progressive use. T h e time required for 15A detailed description of this apparatus has been published in Analytical Chemistry

by Graft and Rittenberg. ~1 Figure 2 is reprinted here from their original paper through the kind courtesy and permission of the authors and the journal.

[21]


479

the complete reduction of water to molecular hydrogen depends on the age of the converter tube but is usually about 5 to 8 minutes. The hydrogen formed is transferred to the sample bulb, 16 F, by means of the Toeppler pump, E. A detailed description of the operation of this apparatus has been published 11 and will not be repeated here. The advantages of this method are: (1) Small samples are required for analysis. In general, a sample of sufficient size to yield 3 to 5 rag. of water is used. In most biological problems, it is possible to obtain this amount of material. (2) The time required for analysis is short enough to be useful for routine analysis. Owing to the complexity of the apparatus, the time required for analysis will depend on the competence of the operator and will decrease with use. It is possible to carry out a complete analysis in duplicate, including the mass spectrometric measurements, in less than 1 hour. (3) A wide variety of compounds can be analyzed with this apparatus, for example, fatty acids, amino acids, sugars, and porphyrins. (4) Whereas the initial assembly of the apparatus may be time-consuming, the apparatus requires very little maintenance to keep it in operation once assembled. The major disadvantage of all apparatus of this type is that there is a large "memory effect" inherent in them. When, for example, an unlabeled compound is analyzed after one of high isotope content, the isotope concentration of the unlabeled sample will be greater than the expected value owing to holdup of water in the apparatus. The apparatus described previously suffers from this defect, as does the modified apparatus used by the author and all apparatus which Graft and Rittenberg 1~ investigated. Furthermore, the site of holdup does not appear to be restricted to any specific portion of the apparatus. It is interesting that the total amount of holdup of water, with both the original apparatus and the modification described below, is of the order of 50 to 100 ~ of water. With a sample sufficient to give 5 mg. of water, the holdup is only 1 to 2 %. It is evident that the effect of holdup can be neglected if the deuterium contents of successive samples are not too different. We have used a modification of the above method in which the reduction of water is carried out in individual conversion tubes which are discarded after use. T M The combustion apparatus is essentially that used le These sample bulbs m a y be o b t a i n e d from Eck a n d Krebs, Inc., 27-09 40th Avenue, Long Island City, New York. 1~ T h e a u t h o r wishes to express his gratitude to Drs. Theodore E n n s and Susanne yon Sehuching for their invaluable collaboration in the development of this modification a n d t h e assembly of t h e apparatus. 1~ F. P. C h i n a r d a n d T. Enns, Anal. Chem. 2§~ 1413 (1953).

480


[21]

by Graft and Rittenberg with the exception that the outlet end of the combustion train consists of a male ball joint. The receiver for the combustion water consists of a piece of 4-ram. tubing with a female ball joint (socket) at one end. A piece of 4-mm. tubing approximately 15 inches long will suffice for about three samples. These are held together with a clamp, and the ball joint is heated by shining an infrared lamp on it. This is done to prevent the combustion water from collecting in the area of the joint. Prior to burning a sample, the 4-mm. tubing is flamed, from the ball joint to the open end, with a microburner to remove any moisture. This is repeated several times. Then dry ice is placed around the 4-mm. tubing, about 3 inches from the open end, and the combustion is carried out. The combustion water collects in the 4-mm. tubing and is driven •

•

9"

2%

) ~\\

~'-ZINC DUST G L A 5 5 WOOL

-"

~

2"

:

Fro. 3. Sample tube for the reduction of water.

toward the dry ice by heating gently with a microburner. When all the combustion water has been collected and frozen, the 4-mm. tubing is sealed off with a hand torch. At this point, the combustion water is sealed in approximately a 3-inch length of 4-mm. tubing. (The sealed end of the 4-mm. tubing remaining on the combustion apparatus is cut off with a file. It is then flamed over its whole length as before to be sure that there is no water in it and used for the next sample.) The sealed 4-mm. tubing, which contains the combustion water, is scratched in the middle with a file to facilitate breaking it later and placed in a drying oven (80 °) until all the samples are combusted. The reduction of water is carried out in the individual reduction tubes of the type shown in Fig. 3. A piece of glass wool is placed next to the break seal in order to prevent contamination of the mass spectrometer with zinc dust. Approximately 2 g. of zinc dust is added to each reduction tube. The tubes are prepared prior to the combustions and left in the oven until needed. The zinc dust and the glass wool are kept in the oven at all times. The 4-mm. tubing containing the combustion water is placed in the zinc reduction tube which is then attached to a piece of pressure tubing and evacuated with a mechanical pump. A stopcock is present between the pressure tubing and the pump. During the evacuation (approximately 7 minutes), the reduction tube is cooled with dry ice to freeze the combustion water. After evacuation, the stopcock is closed and the reduction

[21]


481

tube tilted so that the 4-mm. tube slides into the pressure tubing. The 4-mm. tube is broken by bending the pressure tubing, and the two halves are allowed to fall back into the zinc reduction tube. The zinc reduction tube is cooled again with dry ice and the stopcock opened. (The 4-mm. tube can be broken quite rapidly so that the combustion water remains frozen.) The zinc reduction tube is then sealed off with a hand torch during evacuation. The reduction of water to molecular hydrogen is accomplished by heating the tubes for 4 hours at about 390 ° The hydrogen gas is released from the zinc reduction tube into the mass spectrometer by breaking the break-off seal with a piece of iron which is activated by an electromagnet. The method has been checked with samples of known deuterium content, and the expected values were obtained.~9 Since this is a modification rather than a new method, all the advantages of the original method described above apply here as well. There are two additional possible advantages of this modification: first, the simplicity of the apparatus required, and second, the fact that the sample for mass spectrometric analysis is in a sealed tube and can be stored indefinitely without fear of leakage. This latter fact is of considerable importance when a mass spectrometer is not immediately available--for example, when the samples have to be sent to another laboratory for mass spectrometric analysis. On the other hand, more time is required for the preparation of samples with this modified procedure. This is not a serious disadvantage unless the number of samples to be analyzed is very large. Calculation of Results. The deuterium content of a compound is expressed as atoms per cent excess of the isotope which is defined as the isotope concentration in excess of the normal abundance. This value is calculated from the mass spectrometric data by means of equation 3 in which R is the ratio of the intensities of the ion currents corresponding to the H-H and H-D molecules; i.e., R = (H-H)/(H-D). Atom per cent D -

100 2R+ 1

(3)

The term atom per cent D is defined as the ratio of the number of deuterium atoms in the sample to the total number of hydrogen and deuterium atoms times 100. The atom per cent excess D is obtained by subtracting the normal, abundance of deuterium, 0.02 atom %, from the value of atom per cent D calculated from equation 3. 19These samples were obtained through the kind generosityof Dr. David B. Sprinson.

482


[21]

Analysis by Density Methods A n u m b e r of density methods have been employed to determine the deuterium concentration of organic compounds. 1 These methods are based on the fact t h a t H~O and D20 differ in density b y approximately 105 parts per million. Since this difference is so large, the density of a sample of water is v e r y sensitive to its deuterium content. (A change of 1 part per million in the density corresponds to 0.001% deuterium.) The principal methods used for determining the density of water are the p y c n o m e t e r method, 1 the float method, 2° and the falling drop method. 21,22 Other methods such as the gradient tube method s3 have also been employed. T h e density can be determined conveniently to 2 to 4 parts per million (0.002 to 0.004 % deuterium) b y these techniques. T h e steps involved in these procedures are (1) oxidation of the compound to yield water, (2) purification of the water, and (3) the precise determination of the density of the purified water. T h e technical details of these procedures have been described elsewhere and will not be discussed here. I t should be noted, however, t h a t sufficient material to yield at least 100 mg. of water is required. Furthermore, the water must be extensively purified in order to obtain the precision indicated above; any slight i m p u r i t y will yield erroneous results. A satisfactory m e t h o d for the purification of small amounts of water has been described b y Keston et al. 2~ In addition, it is necessary to normalize the abundance of the oxygen isotopes if great precision is required, since the density of water depends not only on its deuterium content but also on the relative abundance of the oxygen isotopes. Finally, careful attention to details is required in the density determination in order to obtain reliable results.

Nitrogen 24 Principle. E l e m e n t a r y nitrogen is used for the mass spectrometric determination of the relative abundances of the isotopes of nitrogen. T h e procedure for determining the N 15 content of the nitrogen of an organic compound involves the conversion of the organic nitrogen to

20D. Rittenberg and R. Schoenheimer, J. Biol. Chem. 111, 169 (1935). 21A. S. Keston, D. Rittenberg, and R. Schoenheimer, J. Biol. Chem. 122, 227 (1937). ~ M. Cohn, in "Preparation and Measurement of Isotopic Tracers" (D. W. Wilson, A. O. C. Nier, and S. P. Reimann, eds.), pp. 51-59. Edwards Bros., Ann Arbor, 1946. 2s C. Anfinsen, in "Preparation and Measurement of Isotopic Tracers" (D. W. Wilson, A. O. C. Nier, and S. P. Reimann, eds.), pp. 61-65. Edwards Bros., Ann Arbor, 1946. 24N15 can be purchased from Distillation Products Industries, Rochester 3, New York. It is available as ammonium nitrate in which only the ammonium radical has been enriched, as potassium phthalimide, as nitric acid in approximately 2 M

[21]

THE MEASUREMENT

OF STABLE ISOTOPES

483

a m m o n i a and the oxidation of a m m o n i a to nitrogen gas. 12,25,26 The nitrogen is introduced into the mass spectrometer and the intensities of the ion currents corresponding to mass 28 (N14-N 14) and mass 29 (NI4-N la) are measured. The procedure employed for converting the organic nitrogen to a m m o n i a will depend on the structure of the compound being investigated and the nature of the information desired. The Kjeldahl procedure ~7,'-'8 (or one of its modifications) is the preferred method when the (~ompound contains only one nitrogen a t o m or where differentiation between constituent nitrogen atoms is unnecessary. It is often desirable to determine the isotope concentration of each nitrogen a t o m when the c o m p o u n d to be analyzed contains more than one nitrogen atom. I n such cases, a suitable method for the stepwise degradation of the compound must be devised. Unfortunately, there is no general degradative procedure which is applicable to all nitrogencontaining compounds. However, several reactions which have been used to liberate certain specifc nitrogen groupings as ammonia should be noted. The t r e a t m e n t of an a-amino acid with nit~hydrin results in the formation of ammonia, carbon dioxide, and an aldehyde having one less carbon atom. ~9 The a m m o n i a thus formed is suitable for the preparation of nitrogen for the mass spectrometer. The experimental procedure employed in this reaction has been described by ]V[acFayden.3° solution, and as potassium nitrate. All these are supplied in approximately 30 and 60 atoms % concentrations of N15; the ammonium nitrate is also available in 7 atom % concentration. Isotopic nitrogen enriched to more than 95% N Is is available from Isomet Corporation, P.O. Box 34, Palisades Park, New Jersey. It is available as nitric acid in approximately 5 to 9 M solution, as ammonium nitrate in which either the ammonium radical or the nitrate group or both are enriched, as potassium nitrate, as ammonium chloride, as potassium phthalimide, or as nitrogen gas. ~ D. B. Sprinson and D. Rittenberg, J. Biol. Chem. 180, 707 (1949). ~6D. Rittenberg, in "Preparation and Measurement of Isotopic Tracers" (D. W. Wilson, A. O. C. Nier, and S. P. Reimann, eds.), pp. 31-42. Edwards Bros., Ann Arbor, 1946. 27The Kjeldahl procedure has been described in detail by Ballentine; see Vol. I I I [145]. ~8If CuSO4 is used as the catalyst in the Kjeldahl procedure, a prolonged digestion (about 16 to 18 hours) is required for the complete conversion of the nitrogen of lysine, creatine, sarcosine, arginine, and ornithine. If a shorter digestion is used, the nitrogen gas, formed subsequently by the oxidation of ammonia, will be contaminated with an impurity which affects the measurement of the ratio of the intensities of mass 28 to mass 29 in the mass spectrometer, 1~ 29D. D. Van Slyke, D. A. MacFayden, and P. B. Hamilton, J. Biol. Chem. 150, 251 (1943). 30D. A. MacFadyen, J. Biol. Chem. 1§~, 507 (1944).

484


[21]

Ammonia can be liberated from amides and amidine derivatives by simple alkaline hydrolysis. For example, arginine is hydrolyzed to 2 moles of ammonia and 1 mole of ornithine by treatment with alkali. 3' The ammonia is derived from the two nitrogens of the amidine moiety of arginine. Enzymatic hydrolysis has also been used in certain instances. Thus, ammonia can be released from urea in the usual manner with urease. 32 TO AIR SUPPLY': (NH 3 FREE )

24/40

~ 19/58

l,~JELDAHL FLASK

125cc ERLENMEYER FLASK Fie. 4. Distillation apparatus for the preparation of ammonia samples for oxida-

tion with hypobromite. 3s Procedure. Enough material to yield 1 rag. of nitrogen is required for analysis. The organic nitrogen of the sample is converted to ammonia either by the Kjeldahl procedure or by a suitable degradative procedure. In the latter case, the ammonia formed is transferred by a stream of nitrogen into dilute HC1. The ammonia is liberated from the Kjeldahl digest, or the solution of the ammonia salt, with strong alkali and distilled in a stream of air (ammonia free) in the apparatus shown in Fig. 4 as follows: 33 The Kjeldahl digest is cooled, diluted with 25 ml. of water, and transferred to the standard taper Kjeldahl flask. Approximately 5 ml. of 40% NaOH is added through the addition tube, and the ammonia is distilled into

~, R. Schoenheimer,S. Ratner, and D. Rittenberg, J. Biol. Chem. 130, 703 ~1939). ~ P. B. Hawk and O. Bergheim, "Practical Physiological Chemistry," llth ed., p. 708. Blakiston Co., Philadelphia, 1944. 58Figures 4 and 5 appeared in an article by Sprinson and Rittenberg ~2and are reprinted here through the permission of the Department of the Navy.

[21]


485

5 ml. of 0.07 N H2S04. The distillation is continued until about 20 ml. of water has been collected in the receiver. Between successive distillations, the apparatus is disassembled and thoroughly washed to prevent cross-contamination of samples. ,o/3o The distillate is concentrated to a small volume (about 2 ml.) and transferred to one arm of the sample tube ~ shown in Fig. 5. 23 /40 Approximately 3 ml. of a strongly alkaline solution of sodium hypobromite 34 is placed in the other arm, and the vessel is evacuated to less than 0.01 ram. as indicated by a Pirani gage. A convenient vacuum system for evacuating four sample vessels simultaneously has been described by Sprinson and Rittenberg. s5 A diagram of their apparatus is shown in Fig. 6. 35 After evacuation, the vessels are closed by rotating the Y-tube and disconnected from the vacuum system. Immediately before analFIG. 5. Gas sample tube ysis, the sample is oxidized to nitrogen by mixing the hypobromite and the sample. When for oxidation of ammonium salts with hypobromite and the evolution of nitrogen has ceased, the liberation of carbon dioxide Y-tube portion of the sample vessel is im- from carbonates) s mersed in a dry ice-alcohol mixture to reduce the vapor pressure of water. The sample vessel is then attached to the gas-handling portion of the mass spectrometer by means of the 1°/~0 34 Alkaline hypobromite is prepared according to the procedure of Sprinson and Rittenberg. 26 "The hypobromite solution is prepared by the slow addition, with vigorous stirring, of 50 ml. of bromine to 150 ml. of 40 per cent (by weight) reagent quality N a O H held at 0 °. Since hypobromite decomposes less readily in strongly alkaline solutions, another 150 ml. of 40 per cent N a O H is added when the addition of bromine is completed. 1 ml. of this solution should oxidize from 10 to 12 rag. of NH~. W h e n stored in a refrigerator, it retains its activity for months. Freshly prepared hypobromite tends to liberate oxygen, but this effect generally disappears after the reagent has been allowed to stand for a few days. After this time the solution can be separated by decantation from the precipitated sodium salts. Occasionally a batch of hypobromite is obtained which continues to exhibit this decomposition. The cause is not known, but it can be demonstrated that the rate of evolution of oxygen is accelerated by cupric ions . . . . It is clear that contamination with copper of either the reagent or the ammonia sample must be avoided. For this reason HgS04 rather than CuS04 should be employed as a catalyst in the Kjeldahl procedure. Before use the hypobromite is diluted with an equal volume of water." 3s Figure 6 appeared in an article by Sprinson and Rittenberg ~5 and is reproduced here by permission of the authors.

486


[21]

joint and the sample introduced by rotating the Y-tube portion of the sample vessel. The sample vessels can be stored for several hours without leakage of air, provided the caps and the tubes are kept paired and not interchanged. If the mass spectrometric analysis cannot be carried out on the same day the samples are prepared, the vessels can be stored overnight in the refrigerator before mixing. They should be re-evacuated the next day prior to the liberation of nitrogen. This is necessary to prevent dilution of the

TO MERCURY _ 20 M M . ~ ~ PIRANI GAUGE .....I ~

© ]~40/'50 -15 MM. .40 MM. DRY ICE TRAP "30 MM.

F~. 6. Vacuum system for preparation of gas samples for mass spectrometric analysis, s5 nitrogen of the sample by the normal nitrogen of air which may have leaked into the sample vessels during storage. In spite of all these precautions, small amounts of air may contaminate the sample. The presence of air is indicated by the appearance of an oxygen peak at mass 32 and confirmed by a peak at mass 40 due to the argon content of air. The method of correcting for air leakage has been described by Sprinson and Rittenberg 12 and will not be repeated here. This correction should not be applied when the sample contains more than 3 % air. Calculation of Results. The atom per cent N ]~ is calculated from equation 4 in which R is the ratio of the intensities of the ion currents corresponding to masses 28 and 29; i.e., R = ( N 1 4 N I 4 ) / / ( N i 4 N ] 5 ) . 100 Atom per cent N 15 -- - 2R -t- 1

(4)

The atom per cent excess N ~6 is obtained by subtracting the normal

[21]


487

abundance of N 15, which is approximately 0.37 atom %, from the calculated value of atom per cent N 15. In the case of nitrogen the mass spectrometric analysis can be carried out with a precision of 0.003 atom % N 15 in the low concentration range. C a r b o n 3~

Within recent years, the radioactive isotope of carbon, C TM, has almost completely replaced the stable isotope, C 13, as a tracer for investigating the metabolic pathways of carbon. This is due to the increased availability and ease of analysis of the radioactive isotope. However, the stable isotope is still generally preferred in experiments with human subjects. Furthermore, it is often advantageous to employ multiple labeling of the compound under investigation. In such cases, the usual procedure is to label one carbon atom with the stable isotope and a different one with the radioactive isotope. Principle. The mass spectrometric determination of the C 13 concentration of an organic compound in general requires the preliminary conversion of the organic carbon to carbon dioxide. The carbon dioxide is introduced into the mass spectrometer, and the intensities of the ion currents corresponding to masses 44 and 45 are measured. 3~ A variety of methods have been employed for converting the carbon of an organic compound to carbon dioxide. The most common of these involves combustion of the compound in a microcombustion apparatus 26 or by a modification of the wet combustion method of Van Slyke and Folch. 38 The combustion procedure can be used only when all the carbon of the compound can be obtained in the form of carbon dioxide by combustion and when differentiation between constituent carbon atoms is unnecessary. The ninhydrin reaction can be used to liberate carbon dioxide from the carboxyl groups of amino acids. ~°,89 The reaction is carried out at pH 2.5 and the resulting carbon dioxide absorbed in Ba(OH)2 solution. at C13 can be purchased from Distillation Products Industries, Rochester 3, New York. I t is available as potassium cyanide a n d b a r i u m carbonate in approximately 60 atom % concentration of C 13. ~7 Although C 13 is most conveniently determined in the form of carbon dioxide, the analysis is complicated b y t h e contribution of C~201~O ~7 to mass 45. I n practice, however, where we are interested only in t h e excess above n o r m a l a n d not the absolute concentration of C la, t h e contribution of C1:0~"0 ~7 is eliminated when the normal a b u n d a n c e is s u b t r a c t e d from t h e absolute a b u n d a n c e (atom per cent value). as S. Weinhouse, in " P r e p a r a t i o n a n d M e a s u r e m e n t of Isotopic T r a c e r s " (D. W. Wilson, A. O. C. Nier, a n d S. P. R e i m a n n , eds.), pp. 43-49. Edwards Bros., Ann Arbor, 1946. a, This reaction c a n n o t be used for aspartic acid, since b o t h carboxyl groups are liberated as carbon dioxide.

488


[21]

Easterfield and Taylor 4° have described a useful reaction for the preparation of carbon dioxide from the carboxyl group of fatty acids. The decarboxylation is carried out in the presence of iron filings, and the carbon dioxide formed is absorbed in Ba(OH)~ solution. Procedure. The combustion system of the apparatus shown in Fig. 2 can be used to convert the carbon of an organic compound to carbon dioxide. 41 A 15-ml. centrifuge tube containing 5 ml. of a saturated solution of Ba(OH)2 is attached to stopcock 5. Stopcocks 1 and 2 are set so that the combustion gases pass through trap C, through stopcock 2, through stopcock 5 and into the tube containing the Ba(OH)2 solution. After the combustion has been completed and the system flushed, the centrifuge tube is removed and covered with a rubber cap. The precipitated BaCO3 is washed three times with water by centrifugation. The BaCO3 is suspended in about 1 ml. of water and transferred to one arm of the sample vessel shown in Fig. 5. A little dilute HC1 is placed in the other arm and the vessel evacuated. From this point, the procedure is identical to that described previously for the analysis of the nitrogen isotopes. Calculation of Results. The atom per cent C 13 is calculated from equation 5 in which R is the ratio of the ion intensities of mass 44 to that of mass 45; i.e., R = (44)/(45). 100 Atom per cent C 13 = - (5) R+I This formula ignores the contribution of the molecule C1~0~60~7 to mass 45. The atoms per cent excess C ~3 is obtained by subtracting the normal abundance of C ~, which is about 1.1 atom %, from the calculated value of atom per cent C ~a. 40 T. H. Easterfield a n d C. M. Taylor, J. Chem. Soc. 99~ 2298 (1911). 41 If the a p p a r a t u s shown in Fig. 2 is n o t available, a convenient combustion system has been described b y Rittenberg. ~6

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

489

[22] T h e S y n t h e s i s a n d D e g r a d a t i o n o f I s o t o p i c a l l y L a b e l e d Carbohydrates and Carbohydrate Intermediates B y S. ABRAHAM and W. Z. HASSlD

I. Synthesis of Labeled Carbohydrates A. Biological Methods

1. Photosynthetic a. Preparation of Uniformly Labeled Glucose-C 14, Fructose-C 14, and Sucrose-C 14. Principle. When green leaves are allowed to photosynthesize in the presence of C1402, randomly C14-1abeled glucose, fructose, and sucrose are produced. The sugars are extracted with aqueous alcohol, purified b y passing through ion exchange columns, and separated by the aid of paper chromatography. M ° Reagents

B a r i u m carbonate. 80 % lactic acid. Paraffin oil. 0.1 N sodium hydroxide solution, carbonate-free. 80% ethanol. Duolite ion exchange resins C-3 and A-3 (Chemical Process Co., Redwood City, California). Water-saturated phenol solution. Glucose, c.p. Fructose, c.p. Sucrose, c.p. n-Butanol (52.5%), ethanol (32.0%), water (15.5%) solution. 1E. W. Putman, W. Z. Hassid, G. Krotkov, and H. A. Barker, J. Biol. Chem. 175, 785 (1948). 2 E. W. Putman and W. Z. Hassid, J. Biol. Chem. 196, 749 (1952). 3 S. Udenfriend and M. Gibbs, Science 110, 708 (1949). 4 G. R. Noggle and R. A. Bolomey, Plant Physiol. 26, 174 (1951). s M. Gibbs, R. Dumrose, and F. Archer, Biochem. Preparations 2, 45 (1952). 6 A. P. Lamb and G. O. Burr, Federation Proc. 8, 217 (1949). 7 H. K. Porter and R. V. Martin, J. Exptl. Botany 3, 326 (1952). s A. N. Wick, M. E. Blackwell, and N. Hillyard, Arch. Biochem. and Biophys. 32, 274 (1951). 9 C. A. Dubbs, Science 109, 571 (1949). 10R. H. Burris, P. W. Wilson, and R. E. Stutz, Botan. Gaz. 111, 63 (1949).

490


[22]

Carboraffin (or any other decolorizing carbon with low sugar absorbency).

Apparatus. The photosynthesis chamber is made of a Pyrex glass tube (Fig. 1) 21 cm. in length and 5.5 cm. in inside diameter. The lower end consists of a drawn-down recurred entry tube fitted with a stopcock, a, and a 1°~ o tapered joint. The upper end bears a 6°/go joint, A1, to which a cover is fitted carrying an exit tube which is also fitted with a stopcock, b, and 10~o joint. The volume between stopcocks a and b is 500 ml.

b

B Fla. 1. Photosynthetic chamber, Erlenmeyer flask, and Florence flask.

When conducting an experiment, about 500 mg. of barium carbonate containing approximately 5.0 me. of C 14 is introduced into a 25-ml. Erlenmeyer flask, B, from which the neck has been removed. A cork disk, e, slightly smaller than the inside diameter of the chamber, A, is fitted to the top of the flask so that its position is fixed when it is placed in the chamber. The barium carbonate is then mixed with about 5 ml. of water, and a few drops of paraffin oil are added to prevent excessive foaming. One milliliter of 80% lactic acid is placed in a small test tube, f, cut off so that it rests at about a 45 ° angle when placed in flask B. This flask, containing the barium carbonate slurry and the test tube of lactic acid, is then placed in an upright position in the photosynthesis chamber, A, which was previously rinsed with water to assure a humid atmosphere. (Enough water should also be left in the recurring portion of the entry tube to serve as an indicator when the internal and external pressures are equalized.) A Florence flask, C, from which the neck has been removed is attached to a plywood disk, h, bored through with numerous holes. The flask is filled with water, and the petiole of the leaf, which has previously been kept in the dark for 24 hours, is inserted so that it reaches the bottom of the flask. The leaf and its container are placed in a vacuum desiccator

[22]

491


which is evacuated to about 20 cm. After the initial flow of gas bubbles from the petiole of the leaf ceases, the pressures are equalized and the water displaced in flask C is replaced. The leaf and container are then placed in the photosynthesis chamber, A, on top of the cork ring, e. A piece of moistened filter paper is placed over the plywood disk to prevent spattering of acid into the upper part of the vessel. The greased upperend ground taper, A 1, is fitted on, and with the entry tube, a, closed the chamber is partially evacuated through the exit tube which is then closed with stopcock b. With the chamber tilted about 30 ° the acid is dumped

=

[~..L.~z- --:

gz

:--',~:

I t]

"

line

i "-'~

gl

FXG. 2. Complete apparatus for photosynthesis.

into the barium carbonate slurry. (This should be done slowly so as to avoid excessive foaming.) When the reaction has subsided and all the barium carbonate has reacted, with liberation of the carbon dioxide, atmospheric pressure is restored by opening stopcock a. The chamber is then immersed in a cylindrical Pyrex water bath, 10 inches high and 18 inches in diameter, held in position by a condenser clamp on a heavy ring stand in the bath as shown in Fig. 2. Illumination is effected by two 100-watt bulbs in desk lamps gl and g2 placed opposite each other on the outside of the bath. A small fan (not shown in Fig. 2) is placed above the water bath so that a current of air passes over the surface of the bath. This maintains the bath temperature 3 to 4 ° above the prevailing room temperature. Illumination is continued for 18 to 24 hours. A relatively long period of illumination is used in order to ensure complete utilization of the available carbon dioxide and to increase the probability of obtaining uniformly labeled compounds. During the last hour of illumination a soda-lime tower, E, is attached to the entry tube of the photosynthesis chamber, and two carbon dioxide traps, F1 and F2, are attached in series to the exit tube. During this period about 5 1. of carbon dioxide-free air is pulled through the system by application of a vacuum at F2. Trap F1 consists of a 500-ml. jar fitted with

492


[22]

a sintered-glass aerator containing 335 ml. of 0.1 N sodium hydroxide; trap F~ consists of a 100-ml. test tube also equipped with an aerator and 65 ml. of 0.1 N sodium hydroxide. Titration of a 25-ml. aliquot of the alkali in traps F1 and F~ with 0.1 N hydrochloric acid after addition of 1 ml. of 10% barium chloride, with phenolphthalein as an indicator, usually gives the same value as the alkali control. Procedure. Two Canna leaves (Canna indica), each about 5.0 inches long and weighing approximately 5 g., are left in water for 24 hours in the dark to reduce the carbohydrate stores in the leaves. They are then allowed to photosynthesize for 22 hours in an atmosphere of radioactive carbon dioxide, obtained from 1.0 g. of barium carbonate containing C TM, in a chamber as shown in Fig. 1. Best results are obtained when photosynthesis is carried out at 17°, since it has been observed that the sucrose/ reducing sugar ratio is more favorable at that temperature than at 28 ° . (The ratio of sucrose to reducing sugars is 1.0 at 17 °, whereas it is 0.7 at 280.1 ) At the end of that period all the carbon dioxide is usually absorbed by the leaves. The leaves are removed from the chamber immediately, cut into small pieces, and immersed in boiling 80% ethanol. The ethanol used for killing the leaves is transferred to a small boiling flask of a Soxhlet extractor, the cut leaves placed in a Soxhlet extraction thimble, and the leaves extracted with 80 % ethanol during a period of 24 hours. The alcoholic extract containing the soluble sugars is concentrated under reduced pressure to a small volume to remove the alcohol, diluted with water, and partially purified by passing through two successive ion exchange columns containing Duolite C-3 and A-3. The combined neutral effluent and water washings are concentrated to a sirup under reduced pressure, transferred to a small beaker, and dried to constant weight in a vacuum oven at 40 °. The neutral residue, 217 mg., contains a small amount of pigmented chlorophyll decomposition products, is dispersed in 2.2 ml. of water, and usually amounts to 82% of the activity incorporated into the leaves. This solution is then placed in 0.01-ml. aliquots on eight 22]/~ X 181/~-inch Whatman No. 1 filter paper sheets. The aliquots are placed 1.5 cm. apart as a band 7 cm. from the long edge of the paper, leaving a margin on either edge of the paper. The beaker containing the sample is washed at least fourteen times with 0.2-ml. portions of water, and this water wash is also distributed on the eight papers, after the original spots have been dried in the manner previously described. After drying in air, the papers are placed in airtight cabinets, and the band chromatograms are developed for 24 hours by descending'chromatography with water-saturated phenol as the solvent. The chromatograms are then dried in a current of air at room temperature, placed in contact

[22]


493

with 14 X 17-inch Eastman Kodak medical "No-Screen" X-ray films, and the latter developed after about I hour of exposure. The dark bands on the resulting radioautographs coincide in position with those of fructose and glucose. Sucrose, which is also present, does not show up on the chromatogram, as its RI value in water-saturated phenol is approximately the same as that of glucose. These dark areas of the film radioautographs are used as templates to locate the exact position of the bands of radioactive sugars on the filter paper chromatograms. This is done by placing the film on an illuminated cabinet with a transparent upper side and tracing the position of the bands on the superimposed filter paper. The radioactive areas may be checked through the use of any type of C 14 assay counter. The sections of the paper containing the sugar bands are cut out, one margin end of each resulting paper strip is cut to a pointed tip, and the strips are eluted in a small chromatography cabinet by capillary descent with water as the solvent. ~ The eluates, about 2 ml. from each strip, are collected in separate beakers and concentrated i n vacuo to a sirup. Each of the sirups is dissolved in water and again placed on filter papers as described above. The band chromatograms are then developed for 72 hours with a mixture of 52.5% n-butanol, 32.0% ethanol (95%), and 15.5% water, by volume. Before placing the filter papers in the cabinets, their lower edges are serrated so as to allow uniform dripping of the solvent. These chromatograms are air-dried, and radioautographs are prepared again by using X-ray films. The radioautographs obtained from the eluates containing the fructose usually show only one distinct band after 1 hour of exposure, whereas the radioautographs from the sucrose-glucose mixture are resolved into two bands, corresponding in positions to the location of authentic samples of sucrose and glucose. With the autographs as a guide, the positions of the sugars on the paper chromatograms are located, excised, and eluted with water as before. After the eluates are concentrated i n vacuo, sirups of glucose, sucrose, and fructose are obtained. The sirups are then rechromatographed, first with water-saturated phenol and then with the butanol-ethanol-water mixture. These sugars are eluted from the papers and the solutions clarified with a little decolorizing charcoal (carboraffin is preferred, since it adsorbs little sugar). After the charcoal is filtered off, the solutions are concentrated i n vacuo, and the following yield of sugars is obtained: glucose 72.4 mg., sucrose 70.2 mg., fructose 61.2 mg. On the basis of the original C1402 used, approximately 70 % of the radioactivity is incorporated into these three sugars. Of this amount 21%, 29%, and 20% is represented by glucose, sucrose, and fructose, respectively. 1~ j. R. Hawthorne, Nature 160, 714 (1947).

494


[22]

In order to establish the radiochemical purity of these sugars, small amounts of each of them should be subjected to two-dimensional paper chromatography and the papers exposed to X-ray films for several days until visible spots due to radioactive impurities can be observed. The purity of each sugar can be determined by counting the activities of the sugar and the contaminant spots on the chromatogram. The following results have been obtained by this procedure: 2 glucose 99.8% pure, sucrose 99.5% pure, and fructose 97.8% pure. The degree of purity of these sugars can be further increased by once more repeating the paper chromatographic procedure. Chemical degradation of the C14-1abeled glucose 12-~4 and fructose 16,16 reveals that they are evenly labeled with C TM in each of their carbon atoms. Bacterial degradation with L. casei of the inverted sucrose also showed that this sugar is uniformly labeled with C14.5 b. Preparation of Randomly Labeled D-Galactose-C TM and Glycerol-C 14. Principle. The marine red alga Iridea laminarioides (Iridophycus flaccidum) is allowed to photosynthesize in an atmosphere of C14-1abeled carbon dioxide and the galactoside, a-D-galactopyranosylglycerol, isolated chromatographically. Enzymatic hydrolysis of this galactoside followed by paper chromatographic separation of the products leads to the isolation of D-galactose and glycerol.~7

Reagents C~4-barium carbonate. Lactic acid, c.p. 80 % ethanol. 95% ethanol. Duolite ion exchange resins A-3 and C-3. Water-saturated phenol. n-Butanol-ethanol-water mixture. Petroleum ether (b.p. 30 to 60°). Liquid nitrogen.

Procedure. Approximately 1 g. of Iridea thallus, devoid of reproductive structures or nodules, is moistened with water and placed in the 50-ml. 1~ S. Abraham, I. L. Chaikoff, and W. Z. Hassid, J. Biol. Chem. 195, 567 (1952). 13 S. Abraham, E. W. Putman, and W. Z. Hassid, Arch. Biochem. and Biophys. 41,

61 (1952). 14 p. V. Vittorio, G. Krotkov, and G. B. Reed, Science 115, 567 (1952). in S. Abraham, P h . D . thesis, University of California, 1953. 16 M. Gibbs, J. Biol. Chem. 179, 499 (1949). 17 R. C. Bean, E. W. Putman, R. E. Trucco, and W. Z. Hassid, J. Biol. Chem. 204, 169 (1953).

[~]


495

flask, A, of the photosynthesis apparatus (Fig. 3); 40 mg. of BaC1403 is introduced into the C02-generating tube, B, and about 0.4 ml. of concentrated lactic acid pipetted into the side arm, C. This flask is maintained at a temperature of 15 to 20 ° by means of a water jacket. The apparatus is evacuated to approximately 200 mm. through stopcock a, and the C140~ generated by turning tube B and allowing the lactic acid from side arm C to drop slowly on the carbonate. After cessation of gas production, t4/20~

,o

A

Fro. 3. Photosynthesis apparatus for Iridea laminarioides. tube B is gently heated with a small microburner and the internal pressure of the apparatus equilibrated to about atmospheric pressure by allowing air to bubble in through the water manometer, D. (A slight negative pressure should be maintained to avoid the loss of CO2.) The photosynthetic chamber, A, is immediately illuminated for a period of 10 hours with a 150-watt reflector spot lamp (about 10,000 foot-candles). At the end of this photosynthetic period the alkali trap, E, containing 2 ml. of 1 N NaOH (carbonate-free) is attached to the manometer stopcock and the apparatus slowly evacuated. The apparatus may then be flushed out by allowing CO2-free air to enter through the manometer and the apparatus re-evacuated through the alkali trap. The residual C1402 is thereby trapped, precipitated as barium carbonate, and saved. About 99.5% of the C1402 is usually absorbed by the thallus, as judged by the residual activity obtained in this manner. The thallus is then

496


[22]

removed from the chamber, dropped into a 40-ml. conical centrifuge tube containing liquid nitrogen, and the frozen material finely ground with a pestle shaped to fit the bottom of the tube. The plant material is extracted twice with 15-ml. portions of boiling 80% ethanol, centrifuged, and the extracts decanted. The residue is thoroughly mixed with 1 ml. of water, 10 ml. of 95% ethanol added, and the mixture boiled on a steam bath. The solution is removed by centrifugation and decantation and the extracts combined. This latter procedure is repeated twice to ensure complete extraction. The combined extract is concentrated on a steam bath and dried i n vacuo at 40 °. The resulting residue, weighing about 66 mg., contains approximately 70% of the original activity supplied to the thallus as C14-barium carbonate. The dried extract is then taken up in water and transferred to a 12-ml. conical centrifuge tube and partitioned several times with petroleum ether. The solvent phases are separated by centrifugation, and the ether phase containing pigments and fats removed with a capillary pipet. The inorganic material is removed from this defatted extract by passage through 3 ml. of Duolite A-3 and C-4 ion exchange resin columns. The combined neutral solution and washings are concentrated i n vacuo. The residue weighs about 18 rag. and now contains about 48% of the original C14-barium carbonate activity. This residue is dissolved in 0.25 ml. of water and applied in 0.01-ml. aliquots as a band on Whatman No. I filter paper and tbe band developed by descending chromatography with water-saturated phenol. ~ The paper chromatogram is allowed to dry at room temperature and is placed in contact with an X-ray film and exposed for about 1 hour; the film is developed and used as a template to locate the band of galactosylglycerol on the paper. This band is cut out, washed with ether to remove nonvolatile impurities, and eluted by capillary descent with water. When the water eluate is concentrated i n vacuo a sirup is obtained weighing approximately 17 mg. and possessing about 42% of the original C 14 activity incorporated into the thallus. The galactoside sirup is then hydrolyzed by incubating it for 20 hours with 0.5 ml. of a 1.0 % solution of yeast invertase containing a-galactosidase. The hydrolyzate is concentrated, dissolved in 0.25 ml. of water, and placed on paper, as described before. The galactose is separated from the glycerol by chromatographing in water-saturated phenol. After the bands are located by radioautography, they are eluted and separately rechromatographed with a mixture of 52.5 % n-butanol, 32.0 % ethanol, and ]5.5% water. These paper chr matograms are then exposed to X-ray film for 2 days. This procedure allows the detection of radioactive impuri-

[~]

C A R B O H Y D R A TSYNTHESIS E AND DEGRADATION

497

ties of the order of 0.1%. Elution with water and filtration of the eluates through sintered-glass filters serves to remove any solid impurities. The galactose eluate, when concentrated m vacuo, yields a sirup weighing about 8.6 mg. and possessing approximately 25 % of the original C140~ activity incorporated into the plant. The yield of glycerol is about 5.0 mg. with an activity of approximately 12% of the original C1402 activity. c. Photosynthetic Preparation of C t4-Labeled Starch. Principle. When green leaves, such as Turkish tobacco leaves, are placed in the dark to exhaust the available reserve starch supply and then exposed to an atmosphere containing C140~, radioactive starch is re.adily synthesized. ~.7.~-2~ Reagents

Perchloric acid, 72%, 11.2 to 11.7 N, reagent grade. Iodine solution (7.5 g. of Is and 7.5 g. of KI ground with 150 ml. of water, diluted to 250 ml., and filtered through a Whatman No. 3 filter paper with suction). Alcoholic sodium chloride solution (350 ml. of ethanol, 80 ml. of water, and 50 ml. of 20 % sodium chloride diluted to 500 ml. with water). Alcoholic sodium hydroxide solution, 0.25 N (350 ml. of ethanol, 100 ml. of water, and 25 ml. of 5 N sodium hydroxide diluted to 500 ml. with water and filtered as above). 60% ethanol. 80% ethanol. 95% ethanol. Absolute ethanol. Absolute ether. 2 N sulfuric acid. Barium carbonate. Duolite ion exchange resins A-3 and C-3. Procedure. The Turkish tobacco plants are grown in Hoagland's solution 22 or sand culture. The leaves, weighing from 3.0 to 3.5 g. and from 15 to 18 cm. in length, are taken from the middle portion of the stem of plants about 2.5 feet high. Leaves of plants grown in culture 18j. p. Nielsen, Ind. Eng. Chem. Anal. Ed. 15, 176 (1943). 19 G. W. Pucher, C. S. Leavenworth, and H. B. Vickery, Ind. Eng. Chem. Anal. Ed. 20, 850 (1948). 20L. G. Livingstone and G. Medes, J. Gen. Physiol. 31~ 75 (1947). ~1 D. W. Dutton, Biochem. J. §6~ xlviii (1954). 22 D. R. Hoagland and D. I. Arnon~ Calif. Agr. Expt. Sta. Circ. 347 (1938).

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[22]

solution are usually harvested in the morning and placed in water in the dark until the following morning. Plants grown in pots of sand are placed with the container in the dark for a similar period of time, and the leaves are harvested just before the experiment. On a fresh-weight basis the leaves starved in this manner usually contain about 0.25% reducing sugars (glucose and fructose) and 0.15% sucrose. The residue after the extraction of these reducing sugars does not give the characteristic blue starch-iodine color, indicating the absence of starch. After 24 hours of illumination in the chamber (described above) in an atmosphere of carbon dioxide derived from 500 mg. of barium carbonate, about 0.85% reducing sugars, 0.65% sucrose, and 2.5% starch are obtained. Radioactive starch can be prepared by the following technique: A Turkish tobacco leaf is harvested in the morning and allowed to remain in the dark for 24 hours to exhaust its starch content. The leaf is then placed in the photosynthetic chamber in an atmosphere of C1402, as previously described, and illuminated for 24 hours. At the conclusion of the photosynthetic period and after the chamber has been swept out with carbon dioxide-free air, the leaf is immersed in boiling 80% alcohol to inactivate the enzymes. It is then cut into small pieces and placed in a Soxhlet extraction thimble. The alcohol used in killing the leaf is transferred to the boiling flask of the extractor. The Soxhlet apparatus is assembled and the extraction continued for 6 to 8 hours, after which the alcoholic extract containing the soluble sugars (glucose, fructose, and sucrose) is set aside for subsequent isolation. At this time the leaf residue is completely devoid of plant pigment and is white in color. After the 80% alcohol extraction, the residue remaining in the Soxhlet thimble is dried at 50 ° in a vacuum oven for a period of 18 to 24 hours (0.3 to 0.4 g. of dry material is obtained). The dried leaf residue is quantitatively transferred to a small mortar, which is contained in an ice bath, 4.0 ml. of water and 3.0 ml. of 72% perchloric acid added, and the plant material ground with a pestle. The slurry is transferred to a centrifuge tube with a minimum of water and the residue spun down. The aqueous starch solution is decanted into a 250-ml. centrifuge bottle (previously marked at 100 ml. volume) and the residue re-extracted two more times in a similar manner. The combined extracts are then diluted to the mark with water and 6.6 g. of solid sodium chloride added. After the salt has dissolved, 10 ml. of the KI-I~ solution is added and the blueblack precipitated starch-iodine complex allowed to form for 30 minutes at room temperature and then 15 minutes at 2 ° . The bottle with its contents is centrifuged and the supernatant liquid tested for additional starch content by adding several drops of KI-I~ solution. If precipitation is complete, the supernatant liquid is decanted and the starch-iodine com-

[22]


499

plex is washed twice with the alcoholic sodium chloride solution which removes the excess iodine. The complex is decomposed and the starch precipitated by the addition of 2 to 4 ml. of 0.25 N alcoholic sodium hydroxide solution. The crude starch is then centrifuged down and washed two times with 60 % ethanol. The crude starch is dissolved in about 5 ml. of water, filtered through paper into a 50-ml. centrifuge tube, and reprecipitated with 3 vol. of 95% ethanol. When precipitation is complete, the starch is centrifuged and washed with 60% ethanol, twice with 95% alcohol, twice with absolute alcohol, and finally with ether. After the ether has evaporated and the starch is ground to a powder, it is placed in a v a c u u m oven and dried at 50 ° for 24 hours. The yields of starch with this m e t h o d are approximately 20 to 25% calculated on the d r y basis of the alcohol-extracted plant material. C14-Glucose from Hydrolysis of Radioactive Starch. Drying of the starch is unnecessary if it is to be used immediately for the preparation of radioactive glucose. The starch can be immediately hydrolyzed with sulfuric acid after washing with 60% alcohol. T h e radioactive starch is dissolved in sufficient water to make a 0.2% solution, and an equal volume of 2 N sulfuric acid is added. The mixture is refluxed for 30 minutes on a hot plate and, after refluxing, the theoretical a m o u n t of powdered barium carbonate needed to neutralize the acid cautiously added. The precipitate is centrifuged and washed well with water. The combined supernatant liquid and water washings are then passed through Duolite ion exchange columns C-3 and A-3, having a bed volume of 25 ml. Each column is washed with 100 ml. of water. The deionized solution, about 300 ml., is concentrated to a small volume in vacuo at 50 °, transferred to a 25-ml. beaker, and taken to a sirup in a v a c u u m oven at 50 °. The C14-1abeled glucose obtained in this m a n n e r has been shown to be uniformly labeled with C14.2~ 2. Intact Animal

a. Preparation of D-Olucose-3~4-C TM from Olycogen. Principle. On administration of various Cl4-1abeled compounds (NaHC1403,~4-27 succinate-l-C~4, 2s palmitate-l-C TM~) to animals, glycogen or glucose pro23 p. V. Vittorio, G. Krotkov, and G. B. Reed, Proc. Soc. Exptl. Biol. Med. 74~ 775 (1950).

24H. G. Wood, N. Lifson, and V. Lorber, J. Biol. Chem. 159, 475 (1945). 25 D. B. Zilversmit, I. L. Chaikoff, D. D. Feller, and E. J. Masoro, J. Biol. Chem. 176, 389 (1948). ~e H. R. V. Arnstein and R. Bentley, Biochem. J. 54, 493 (1953). ~ A. K. Solomon, B. Vennesland, F. K. Klemperer, J. M. Buchanan, and A. B. Hastings, J. Biol. Chem. 140, 171 (1941). 2s S. Abraham and I. L. Chaikoff, Arch. Biochem. and Biophys. 41, 143 (1952).

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[22]

duced by these animals has been shown to be labeled almost exclusively in the 3 and 4 positions.

Reagents 20 % glucose solution. 1 N sodium hydroxide. BaC140~. 0.5 M citric acid solution. Nembutal. 30 % potassium hydroxide solution. 95% ethanol. Ether, anhydrous. 2 N hydrochloric acid. 1 N sulfuric acid. Anion and cation exchange resins. Succinic acid-l-C 14. Physiological saline (0.9% sodium chloride solution). Saturated lead acetate solution. Saturated disodium phosphate solution. 97 % methanol. Acetic anhydride. Pyridine, anhydrous. 0.1 N sulfuric acid. Saturated barium hydroxide solution. Duolite cation exchange resin C-3 and anion exchange resin A-4. n-Butanol-acetic acid-water mixture 29 (500 ml. of n-butanol, 105 ml. of acetic acid, and 250 mh of water). Solution of phenol saturated with water.

Procedure. Isolation as glycogen. 26 Six rats (total body weight 1.5 kg.) are fasted for 24 hours, after which 6 ml. of a 20% glucose solution is administered to each animal by stomach tube. One hour later 2 ml. of an aqueous solution of NaHC1403 is injected into each animal. This solution is prepared by absorbing the C140~ liberated from 5 mc. of BaC1408 (200 mg.) in a slight excess of 1 N NaOH (1.5 ml.), diluting with water to 10 ml., and adjusting the pH to 7.5 with 0.5 M citric acid, making a final volume of 12 ml. After 2 hours the animals are anesthetized with Nembutal; the livers are extirpated and immediately placed in hot 30% K O H (80 ml.). After the solution has become homogeneous, 160 ml. of ethanol is added and the solution cooled at 0 ° overnight. The crude glycogen, amounting to 2 g., is centrifuged off, washed twice with ethanol, once with ether, and dried. The crude glycogen is then dissolved in 10 ml. of hot water and the :9 S. M. Partridge, Biochem. Soc. Symposium, Cambridge 3, 52 (1950).

[22]


501

insoluble residue removed by centrifugation. The supernatant solution is acidified to pH 4 with 2 N HC1, and 4 ml. of ethanol is added. The precipitate is centrifuged, and the pure glycogen is isolated from the supernatant by the addition of 4 ml. of ethanol. This procedure yields 1.1 g. of white product (12.7 gc.). The glycogen is then hydrolyzed for 3 hours at 100 ° with 1 N H~SO4 and the acid removed by passage of the solution through a column of Deacidite Ez ion exchange resin. Concentration of the neutral effluent to a thick sirup and crystallization with absolute ethanol yields D-glucose-3,4-C 14. b. Isolation of D-Glucose-3~4-C ~4 from Urine. 2s A dog weighing approximately 7 kg. is surgically depancreatized in the usual manner and, for a period of 3 months thereafter, injected twice daily with insulin and fed an adequate diet2 ° This period ensures complete healing of the wound and brings the animal into a good nutritional state. Insulin injections are discontinued, and about 30 hours later the dog is injected intravenously with a solution of 10.4 mg. of succinic acid labeled with succinic acid-l-C 14 which has been dissolved in 6.8 ml. of physiological saline. The urine is collected thereafter by catheterization. The dog is not fed after the injection of succinic acid-l-C TM but is allowed to have access to water. Isolation of Glucose. 12 The collected urine is clarified by the addition of 50 ml. of a saturated solution of neutral lead acetate, and the resulting precipitate is filtered through Celite. To remove excess lead, 30 ml. of saturated disodium phosphate is added and the precipitate once again filtered through Celite. Enough 95% ethanol is added to the filtrate to double its volume, and the resulting solution is concentrated in vacuo to a thick sirup at a bath temperature which should not exceed 60 °. This sirup is mixed with an equal volume of technical methanol (97%) and warmed on a steam bath until thoroughly mixed. The precipitate which forms is filtered and discarded. The clear filtrate is once again concentrated in vacuo to a thick sirup. The isolation of pure glucose from this sirup is accomplished by preparing the water-insoluble pentaacetyl-a-D-glucose and subsequent deacetylation as described below. Preparation of Pentaacetyl-a-D-glucose. L2 For every 5 g. of glucose in the sirup (as determined by reducing values), 23 ml. of acetic anhydride and 35 ml. of anhydrous pyridine are added. The reaction mixture is stored at 0 ° and shaken periodically for several days (usually 10 days) until the sirup is dissolved. The mixture is then poured into 125 ml. of ice water, with stirring. Within a short time crystals of pentaacetyl-a-D~0 M. L. Montgomery, C. Entenman, I. L. Chaikoff, and H. Feinberg, J. Biol. Chem. 185, 307 (1950).

502


[22]

glucose are formed. The crystals are filtered and washed with ice water. The yield of pentaaeetate usually ranges from 60 to 70%, based on the reducing value of the urine. Deacetylation of Pentaacetyl-a-D-glucose. 12 For every gram of the pentaacetate, 10 ml. of 0.1 N H2SO4 is used for hydrolysis. The reaction mixture is put into a three-necked flask and heated on a steam bath under a reflux. The reaction is allowed to continue, with constant stirring, for 4 to 5 hours, by which time all the pentaacetate goes into solution. An amount of Ba(OH)2 solution sufficient to neutralize the sulfuric acid is added, and the resulting BaSO4 precipitate is filtered through Celite. The combined filtrate and washings are then passed successively over a Duolite C-3 cation exchange column and a Duolite A-4 anion exchange column (3 X 25 cm.), which serves to remove the inorganic impurities. The effluent and washings are then concentrated in vaeuo to a thick sirup, and the glucose is crystallized by the addition of absolute ethanol to the sirup. The yield of crystalline glucose is about 97%, based on the pentaacetate. The glucose thus obtained, when subjected to two-dimensional chromatographic analysis with butanol-acetic acid-water and phenolwater, 29should give a single radioactive spot occupying an area identical with that of the color spot that appears after the filter paper chromatogram is sprayed with a solution of aniline oxalate, or p-anisidine hydrochloride. Chemical degradation I~,2s showed that glucose obtained in this manner is labeled exclusively in the 3 and 4 positions and that these two carbon atoms contain an equal amount of C ~a activity. 3. Enzymatic a. Preparation of Sucrose Labeled with C TMin the Glucose or Fructose Component. Principle. An enzyme (sucrose phosphorylase) isolated from Pseudomonas saccharophila is capable of catalyzing the reversible phos-

phorolysis and transglycosidation reactions of sucrose according to the following equations :31-83 _+phosphate a-D-Glucose-l-phosphate + enzyme ~_ Glucose-enzyme ~ ±fructose Glucose-l-fructoside ~- enzyme (sucrose) 81 W. Z. Hassid, M. Doudoroff, and H. A. Barker, J. Am. Chem. Soc. 66, 1416 (1944).

32M. Doudoroff,H. A. Barker, and W. Z. Hassid, J. Biol. Chem. 168, 725 (1947). aa M. Doudoroff,J. Biol. Chem. 151, 351 (1943).

[22]


503

Thus sucrose with C14-1abeled glucose can be prepared by allowing labeled a-D-glucose-l-phosphate to react with unlabeled fructose by the aid of P. saccharophila enzyme. Radioactive sucrose with " t a g g e d " fructose is prepared by allowing C14-1abeled fructose to exchange for fructose in the unlabeled sucrose molecule in the presence of the same enzyme. 34

Reagents Enzyme solution prepared from 4 g. of dried cells of P. saccharophila ~2,33 (see also Vol. I [28]). C14-1abeled glucose-l-PO4 35 prepared from C14-1abeled starch. 1 Citrate buffer, pH 6.64. Phosphate buffer, pH 6.64. Torula monosa, yeast. Duolite ion exchange resins C-3 and A-4. Absolute ethanol. Sucrose, c.p. C14-1abeled fructose2 Potato phosphorylase 35 (see also Vol. I [22]).

Procedure. Four grams of dry cells of Pseudomones saccharophila is extracted twice with M/30 SCrensen phosphate buffer at pH 6.64, ~2,33the extraction being made in 1-g. lots. The cells are well dispersed in about 10 ml. of buffer per 1 g. of bacteria, either by grinding in a mortar or by agitation in a Waring blendor, and centrifuged after 20 minutes at room temperature. The combined supernatant liquids are then treated with ammonium sulfate at 0.8 saturation and kept at +_5 ° for several hours. The precipitate is collected by centrifugation, resuspended in the original volume of buffer, and treated for 1 hour at 5° with 0.3 saturated ammonium sulfate. The precipitate is discarded, and the supernatant is brought to 0.63 saturation of ammonium sulfate and stored at 5° for 3 days or longer. During storage the interfering enzymes (invertase, etc.) are inactivated while the precipitate retains most of the phosphorylase activity. Enzyme solutions can be prepared by suspending the precipitate in phosphate, citrate, or bicarbonate-carbonate buffers at pH 6.4 to 7.0. If the preparations are not clear, the fractionation with ammonium sulfate can be repeated and the material that is insoluble in the buffer can be removed by centrifugation. If the presence of phosphate is not desired in the final preparation, the enzyme can be reprecipitated several 34 H. Wolochow, E. W. Putman, M. Doudoroff, W. Z. Hassid, and H. A. Barker, J. Biol. Chem. 180, 1237 (1949). 35 R. M. McCready and W. Z. Hassid, J. Am. Chem. Soc. 66, 560 (1944); see also Vol. I I I [16B].

504


[22]

times from M / 3 0 citrate adjusted to pH 6.64. Enzyme preparations can be dialyzed against tap water for as long as 18 hours with little loss of activity, but they are inactivated by dialysis against distilled water. C14-Labeled a-D-glucose-l-phosphate is prepared according to McCready and Hassid 3~from a sample of radioactive starch, with the use of potato phosphorylase. The potato phosphorylase solution can be prepared in the following manner. 36,~7 A sufficient number of potatoes to furnish i 1. of juice are peeled and sliced and kept under water until used, preferably within 1 hour. The slices are then drained quickly and pulped in a Waring blendor. The minced material is filtered through cheesecloth, rapidly heated to 50 °, and maintained at that temperature for 5 minutes. The heating serves to inactivate the ~-amylase present in the juice. While the coagulated suspension is still warm, solid ammonium sulfate is added to bring the specific gravity of the solution to 1.085. The precipitate is centrifuged within half an hour and discarded. The supernatant liquid is adjusted to a specific gravity of 1.152 with solid ammonium sulfate and then centrifuged. The precipitate, which is not entirely soluble in water, is suspended in 250 ml. of water, and fractionation is repeated in a specific gravity range of 1.095 to 1.145. The precipitated enzyme is dissolved in 100 ml. of water for two subsequent fractionations in the specific gravity ranges of 1.100 to 1.140 and 1.100 to 1.35, respectively. The pH is determined at frequent intervals through the preparation and is kept between 6.0 and 6.5 by the addition of dilute ammonium hydroxide. Rapid inactivation of the enzyme occurs below pH 5.8. The final enzyme precipitate is dissolved in 25 ml. of water for each liter of crude potato juice used and stored at I to 2 °. At that temperature the enzyme can be kept from 4 to 6 weeks with little change of activity. One of the important precautions in the concentration of the enzyme is a speedy separation of the freshly expressed juice from the pulp, as inactivation is particularly rapid at this stage. All the other steps in the procedure should be carried out as quickly as possible. Sucrose with C~4-Labeled Fructose. 34 An extract from 2 g. of dried cells from P. saccharophila is mixed with 2.5 g. of sucrose acid and 0.2 g. of C14-1abeled fructose in a total volume of 30 ml. of citrate buffer at pH 6.64 and incubated for 6 hours at 30 °. At the end of that time, the enzymatic digest is inactivated by heating on a steam bath and the remaining fructose fermented with a washed cell suspension of the yeast, Torula monosa. This enzyme ferments glucose, fructose, and mannose but does not attack sucrose or other disaccharides. The organism is grown 8, D. E. Green and P. K. Stumpf, J. Biol. Chem. 142, 355 (1942). ~7p. H. Hidy and H. G. Day, J. Biol. Chem. 160, 273 (1945).

[9.2]


505

aerobically for 24 hours at 28 ° on agar plates containing 0.5% yeast extract and 1% glucose. The ceils are washed twice by centrifugation and resuspended in 0.033 M phosphate buffer, pH 5. A yeast suspension prepared in this way will decompose approximately 15 rag. of glucose per hour per 100 rag. of dry yeast under anaerobic conditions at 37 °. The initial glucose concentration should not be above 3 %. The yeast is removed by centrifugation and the electrolytes by the use of ion exchange columns Duolite C-3 and A-3. The neutral solution is evaporated to 10 ml., 2.5 vol. of ethanol is added, and the alcoholic solution filtered. After evaporation of the solution and treatment of the sirup with absolute alcohol, the sucrose is obtained in crystalline form. The crude product should be recrystallized twice from ethyl alcohol. On the basis of the activity of the C14-1abeled fructose originally added, the radioactivity of the sucrose is usually found to be 76% of the possible theoretical activity of equilibrium. Sucrose with C~4-Labeled Glucose. 34 The reaction mixture consisting of 0.5 g. of a-D-glucose-l-phosphate containing C t4, 0.2 g. of inactive fructose, and 2.5 g. of inactive sucrose (carrier) is mixed with an extract from 2 g. of dry cells from P. saccharophila. This mixture is made up in a total volume of 30 ml. of citrate buffer at pH 6.64 and allowed to react for 6 hours at 30 °. The isolation of the sucrose is carried out in the same manner as previously described. The radioactivity of the sucrose is usually found to be about 72% of the theoretical activity at equilibrium. b. Preparation of Maltose Labeled with C ~4 in Either the Reducing or Nonreducing Glucose Component. Principle. A phosphorylase extract obtained from Neisseria meningitidis is capable of catalyzing the reversible reaction maltose phosphorylase Maltose + inorganic phosphate ÷ 13-D-Glucose-l-PO4 A- D-glucose When C14-1abeled ~-D-glucose-I-PO4 and nonlabeled D-glucose are employed in the presence of this enzyme, maltose is obtained with C l~labeled in the nonreducing unit. Nonlabeled f~-ester and C~4-1abeled D-glucose produce maltose labeled with C ~4 in the reducing unit. 3s

Reagents Uniformly labeled C14-maltose (from C14-1abeled starch) page 507). 3s

C. Fitting and E. W. Putman, J. Biol. Chem. 199, 573 (1952).

(see

[22]


507

starch) is incubated for 6 hours at 37 ° with the maltose phosphorylase extract in the presence of 20 tLN[. of sodium phosphate in a total of 1 ml. The reaction mixture is then chromatographed, as described above, with water-saturated phenol as a solvent. The radioactive bands corresponding to maltose, glucose, and hexose monophosphate are located by reference to X-ray film radioautography. The hexose monophosphate band is cut from the paper, eluted with water, and rechromatographed in a propanolammonia-water solvent, 4[ with inactive f3-D-glucose-l-phosphate as a side marker. After excision and elution of this band, a product is obtained which is presumably a mixture of sodium and ammonium ~-D-glucose-1phosphate. This hexose phosphate is then incubated with 30 ~M. of inactive glucose in the presence of the enzyme in a volume of 0.5 ml. for 4 hours at 37 °. The reaction mixture is chromatographed with water-saturated phenol, and the band corresponding to maltose is excised and eluted. A glucose impurity amounting to about 4% can be eliminated by a second chromatographic development in the butanol-acetic acid-water solvent.2 Following this procedure, a yield of 2.4 nlg. of maltose sirup is obtained which may be crystallized by the addition of inactive maltose, dissolved in water, concentrated, and ethanol added. c. Preparation of Randomly Labeled Maltose-C ~4. Principle. C 14labeled starch is hydrolyzed with a-amylase and the resulting C ~4maltose isolated by a paper chromatographic procedure. 3s Reagents C14-1abeled starch (see page 497). Human saliva, prepared by diluting with 1 vol. of distilled water and filtering through .Celite with the aid of suction. 4"~ Sodium chloride, c.p. 0.2 M phosphate buffer, pH 5.8. Water-saturated phenol. n-Butanol-acetic acid-water mixture. Procedure. A sample of C14-1abeled starch (10 rag.) is mixed with 5 rag. of solid sodium chloride and dissolved in 2.0 ml. of water. After the addition of 0.1 ml. of phosphate buffer and 1 ml. of diluted saliva, the mixture is incubated for 2 hours at 40 °. The salivary amylase is then inactivated by heating the mixture on the steam bath and the enzymatic digest concentrated in dryness in vacuo. The residue is dissolved in 0.25 ml. of water and chromatographed as a band on Whatman No. 1 filter paper 42 W. Z. Hassid, R. M. ]V[cCready, and R. S. Rosenfels, Ind. Eng. Chem. Anal. Ed. 12, 142 (1940).

508


[22]

with water-saturated phenol as solvent. The chromatogram is allowed to develop for 24 hours. T h e areas on the paper containing the separated sugars are located b y the radioautographic procedure, cut from the chromatogram sheet, eluted with water, and again chromatographed as a band with butanol-acetic acid-water as solvent. This chromatogram is developed for about 48 hours to ensure complete separation of the products of the enzymatic hydrolysis of the C14-1abeled starch. These products which are located b y the radioautographic technique are found to be maltose, isomaltose, and glucose. T h e maltose band is cut from the paper, eluted with water, and the solution concentrated i n vacuo. A yield of about 6 mg. of maltose h y d r a t e is obtained. T h e randomly labeled maltose contains approximately the same specific activity as the starting C~-labeled starch. B. Chemical Methods

1. Cyanohydrin

a. Preparation of Cyanide-C ~4. The cyanohydrin reaction has proved to be v e r y successful for the synthesis of m a n y carbon-labeled sugars and sugar derivatives and has been used extensively. Thus the preparation of cyanide-C 14 from barium carbonate-C ~4 is of considerable importance. I n order for such a conversion to be practical, it should meet the following standards. 48 T h e synthesis should (1) give high and reproducible yields, (2) utilize a minimum of steps, (3) require simple apparatus, and (4) not be time-consuming. P r i n c i p l e . Five general types of procedures have been used to prepare cyanide, involving the following reactions: 1. Carbon dioxide or carbonate with a metal and ammonia 44-~° 2. Carbonate with azido 51-55 48F. L. J. Sixma, H. Hendriks, K. Helle, U. Hollstein, and R. van Ling, Rec. tray. chim. 73, 161 (1954). 44R. D. Cramer and G. B. Kistiakowsky, J. Biol. Chem. 187, 549 (1941). 45R. B. Loftfield, Nucleonics 1, No. 1, 54 (1947). 46p. Olynyk, D. B. Camp, A. M. Griffith, S. Woislowski, and R. N. Helmkamp, J. Org. Chem. 13, 465 (1948). 4~R. Abrams, J. Am. Chem. Soc. 71, 3835 (1949). 4sj. A. McCarter, J. Am. Chem. Soc. 73, 483 (1951). 49j. A. Bos, Experientia 7, 258 (1951). 50j. K. Jeanes, Science 118, 719 (1953). 51A. W. Adamson, J. Am. Chem. Soc. 69, 2564 (1947). 52G. 0. Henneberry and B. E. Baker, Can. J. Research B 28, 345 (1950) 53B. G. van den Bos and A. H. W. Aten, Jr., Rec. tray. chim. 70, 495 (1951). 54V. I. Maimind, B. V. Tokarev, and M. M. Shemyakin, Doklady Akad. Nauk S.S.S.R. 81, 195 (1951). 55A. G. MacDiarmid and N. F. Hall, J. Am. Chem. Soc. 75, 4850 (1953).

[22]


509

3. Sodium amide with sodium formate 56 4. Carbon dioxide with triphenylmethylsodium 57 5. Barium carbonate with potassium in the presence of ammonium chloride 43 Only the first (1) and the last (5) appear to satisfy the above-mentioned conditions. Of the various procedures reported for the preparation of cyanide-C ~4, three methods have been described which give high yields. In the first method, C14-barium carbonate is heated in the presence of zinc dust and metallic sodium in a stream of anhydrous ammonia which has passed over hot iron. The C ~4 cyanide thus produced is distilled into potussium or sodium hydroxide. The yield of cyanide obtained by this one-step process is reported to be quantitative. 5° However, the results are not consistent. In the second procedure, reported by McCarter, 48 a mixture of potassium-carbonate-C 14 and zinc dust is heated in a stream of dry ammonia gas which has passed over hot iron before passing over the mixture of alkali carbonate and zinc. Consistent yields of 90% have been obtained by this method. However, in this method conversion of the barium carbonate-C ~4to potassium carbonate-C ~4is required. An excellent review of the methods of cyanide synthesis is presented by Sixma et al. 43 These workers also describe a third procedure which makes use of the reaction between barium carbonate, ammonium chloride, and potassium. With this method, consistent yields of 94 % were reported. This third procedure is likely to produce the best results. Reagents for Method 1 ~o

C14-barium carbonate. Zinc dust. Metallic sodium. Anhydrous ammonia gas. Iron wire or powdered iron. 2 N sulfuric acid solution. 1 N sodium or potassium hydroxide solution. Procedure for Method 1. An intimate mixture of 1.0 mM. of C14-barium carbonate, 1.0 g. of zinc dust, and 0.2 g. of metallic sodium cut in small pieces is placed in a porcelain combustion boat of suitable size. A 5.0-g. ball of iron wire or 5.0 g. of powdered iron distributed throughout two plugs of Pyrex glass wool is placed in the mid-portion of a Vycor or quartz combustion tube (600 mm. in length and 19 mm. inside diameter). ~e j . W. Spyker a n d A. C. Neish, Can. J. Chem. $0, 461 (1952). 5T B. Belleau a n d R. D. H. Heard, J. Am. Che~n. Soc. 72, 4268 (1950).

510


[22]

The boat is then placed in the tube and pushed until it touches or nearly touches the iron plug. The end of the tube nearest the iron is connected to a cylinder of anhydrous ammonia, and a stream of gas is allowed to flow through a gas bubbler at a rate of 3 bubbles per second in and through the tube containing the mixture. That portion of the tube containing the boat and the iron plug is brought to 650 ° and maintained at approximately that temperature with an electric muffle furnace for a period of 4 hours. At the end of that time, the tube is cooled to room temperature, the flow of ammonia being continued until the tube is completely cooled. The boat and the contents of the tube, except the iron ball, are then washed with water into a 250-ml. flask which can be attached to a distillation head for subsequent distillation of the hydrogen cyanide. The solution in the flask is acidified with 2 N sulfuric acid, and 20 to 30 ml. of distillate is collected in a receiver containing a 20% excess of 1 N sodium or potassium hydroxide. Analysis by the argentimetric method 58 for cyanide, together with the data obtained from analysis of C ~4 content, showed that the yield is quantitative and that the specific activity of the labeled cyanide is the same as that of the starting C~4-barium carbonate. Reagents for Method 2 48

Potassium carbonate-C ~4. Zinc dust (reagent grade). Iron wire (0.01 inch in diameter "for standardizing"). Anhydrous ammonia gas. 1 N sodium or potassium hydroxide solution. 2 N sulfuric acid solution. 4 N potassium hydroxide. Procedure for Method ~. C14-Labeled potassium carbonate is readily obtained by passing the C1402, produced from barium carbonate-C 1~, into a slight excess of 4 N potassium hydroxide and evaporating the solution to dryness. The powdered anhydrous potassium carbonate-C ~4 is thoroughly mixed with about 1 g. of zinc dust and the intimate mixture transferred to a porcelain combustion boat (Coors, Size 2). The l~oat is then placed in a Vycor combustion tube (750 ram. in length and 19 ram. in inside diameter) containing from 3 to 4 g. of iron wire in the form of a loose ball occupying the mid-portion of the Vyeor tube. The boat is pushed into the tube until it touches the iron wire. ~s I. M. Kolthoff and E. B. Sandell, "Textbook of Quantitative Inorganic Analysis," rev, ed.~ p, 574. The Macmillan Co., New York, 1943.

512

[22]


by vigorous shaking. The tube is then placed in an electric furnace which is kept at 640 ° for 1 hour. After cooling, the tube is carefully opened and the excess potassium is destroyed by the slow addition of ethanol. The resulting solution is then transferred to a 250-ml. round-bottomed flask which can be attached to a distillation head for subsequent distillation of the hydrogen cyanide. With boiling water, the last traces of radioactive cyanide can be removed from the Supremax reaction tube. The solution in the distillation flask is acidified with 2 N sulfuric acid, and 20 to 30 ml. of the distillate is collected in a receiver containing a 20% excess of 1 N sodium or potassium hydroxide. With this procedure the yields obtained from eleven experiments averaged about 99%. A blank value of approximately 5 % was observed when no barium carbonate was present, probably due to a slight absorption of carbon dioxide on the surface of the potassium, so that the net yield was therefore about 94%. 48 b. Preparation of D-Glucose-l-C TM and D-Mannose-l-C TM. Principle. The condensation of D-arabinose with C~4-1abeled cyanide leads to the production of D-glucose-l-C TM and D-mannose-l-C TM. In the presence of sodium bicarbonate and carbon dioxide, the ratio of the epimeric nitriles is about 3 parts mannonic nitrile to 1 part gluconic nitrile, but in the presence of sodium carbonate this proportion is approximately reversed. 59 This procedure is based on that of Isbell et al. ~9 Production of D-Mannose-l-C TM

D-Arabinose -~ NaC14N -~ NaHCO3 Jr COs-~ D-Mannono-a-lactone-l-C TM-~ barium-D-gluconate-l-C TM (20%) (67%)

$

D-Mannose-l-C i~ (50% over*all)

Production of D-Glucose-l-C 14

D-Arabinose -~ NaC~4N ~- Na~CO3-* Barium D-gluconate-l-C TM~ D-mannono-~-lactone-l-C TM (61%)

(30%)

(D_Glucono_~_lactone_l_C,4) --~ D_Glucose_l_C,4 (45% over-all) Reagents

Sodium cyanide-C '4. 59 H. S. Isbell, J. V. Karabinos, H. L. Frush, N. B. Holt, A. Schwebel, and T. T. Galkowski, J. Research Natl. Bur. Standards 48, 163 (1952).

[22]


513

Sodium bicarbonate. Sodium carbonate. Sodium hydroxide. Sodium oxalate. 5 % sodium amalgam pellets. (The 5 % amalgam is prepared in the form of small pellets by pouring it in molten condition through a heated alundum thimble, having a small hole in the bottom, into a 70-cm. "shot tower" of mineral oil. The pellets are stored under mineral oil. They are blotted dry just before use, weighed, and rinsed with benzene to ensure removal of any residual mineral oil.) Carbon dioxide, gas and solid. o-Arabinose. D-Glucose. D-~V[annose. D-Mannono-8-1actone. Barium gluconate trihydrate. D-Glucone-~-lactone. Acetone, c.p. Ether, anhydrous c.p. Ethanol, c.p. Methanol, c.p. Isopropanol, c.p. Chloroform, c.p. 5~[ethyl Cellosolve. Oxalic acid dihydrate. Benzoic acid. Barium hydroxide octahydrate. Phenolphthalein indicator. Celite (diatomaceous earth). Decolorizing carbon (carboraffin). Amberlite IR-100, analytical grade (cation exchange resin, regenerated, thoroughly washed with water; immediately before use the resin is washed successively with methanol and water, in order to inhibit microbiological action) (Resinous Products Division of Rohm and Haas Co., Philadelphia, Pennsylvania). Duolite anion exchange resin A-4.

Preparation Favoring the Synthesis of D-Mannose-l-C 14. A solution containing 2 mM. of sodium cyanide-C 14 and 2.24 mM. of sodium hydroxide in a total volume of 5 ml. is placed in a flask and the solution frozen in a mixture of dry ice (solid CO2) and acetone. To the frozen mixture are added 1 g. of solid carbon dioxide and 20 ml. of a solution

514


[22]

containing 2 raM. of D-arabinose. The mixture is allowed to thaw, and the flask is stoppered and stored in a refrigerator overnight. After standing at room temperature for 2 days the solution is hydrolyzed by heating for 4 hours at 50 ° under reduced pressure, and then for I hour on a steam bath. The cations are removed by passage of the solution through a column (1.4 X 24 cm.) containing Amberlite IR-100, analytical grade, and the combined solution and washings are lyophilized. The residue is moistened with methanol, seeded with D-mannono-~-lactone, and allowed to stand for 3 days. During this time a considerable amount of the lactone usually crystallizes out. The entire residue is then dissolved in 0.5 ml. of Methyl Cellosolve. The solution is treated with anhydrous ether to incipient turbidity and is again seeded with D-mannono-~-lactone. The D-mannono-~-lactone-l-C 14 that crystallizes out after several days is separated, washed with ethanol and ether, and dried. The yield is about 192 mg. (54%), m.p. 151 to 152 °, [a]~° = ~-53 °. The combined mother liquor and washings are then concentrated to a sirup, 180 rag. of D-mannono-~-lactone carrier is added, and the mixture is dissolved in 1 ml. of Methyl Cellosolve. Ether is then added to incipient turbidity, and the lactone is allowed to crystallize in the course of several days. This crystallization yields about 187 mg. containing about 13 % of the radiochemical yield. The total recovery of radioactivity as D-mannono-~-lactone-l-C 14 is thus about 67% .... In order to isolate the gluconic acid-l-C 14 which is also produced by the cyanohydrin synthesis, the mother liquor and washings from the crystallization of the mannonic lactone carrier are combined and evaporated to a sirup. After the addition of 1 mM. of barium hydroxide octahydrate (315 mg.) and 3 ml. of water, the mixture is digested on a steam bath for 1 hour. Carbon dioxide gas is then bubbled through the solution until it is acid to phenolphthalein, and the mixture filtered by suction on a funnel precoated vcith Celite and decolorizing carbon. The combined filtrate and washings are lyophilized and the residue dissolved in 0.5 ml. of warm water. Methanol is added until the solution is turbid and then seeded with barium gluconate trihydrate. After several days the crystalline barium gluconate-l-C 14 trihydrate is separated and dried. The yield usually amounts to about 42 mg. (7%). An additional yield of barium gluconate containing approximately 12% radioactivity can be obtained from the mother liquor and washings by the addition of carrier. Thus the radiochemical yield of barium gluconate-l-C ~4 amounts to about 19%, and the total radiochemical recovery from the sodium eyanide-C ~4 is approximately 86 %. Reduction of D-Mannose-~-lactone-l-C ~4. The D-mannono-~-lactone is reduced as follows: One millimole (178 mg.) of the material is dissolved

[22]


515

in 20 ml. of ice water and transferred into the apparatus shown in Fig. 4. Benzoic acid (1.5 g.) is added and stirred into the solution only long enough to effect dispersion; 4.6 g. of 5% sodium amalgam pellets is added, and the entire contents of the tube stirred for 1.5 hours at 0 °. At the end of this time, the mercury which forms is removed with a pipet, 1.2 g. of benzoic acid and 4.6 g. of sodium amalgam pellets are added, and the stirring is continued for an additional 2 hours or until all the amalgam

1/4" stainless steel stirrer ....

I|

li

tl

II II I I II

I I

T1 g

I

Stainless steel stopper g 24/40 fitted with greaseless bearings

I

i I i;i I ~, I.F-44

,,I I

,!,

Q,

i|

Heavy-walled glass tube

FIG. 4. Apparatus for reduction of sugar lactones by sodium amalgam.

is spent. The mercury is again removed as before, 1.26 g. of oxalic acid dihydrate is added, and the resulting benzoic acid is removed by extraction with chloroform. The aqueous solution is then mixed with 2 vol. of methanol and 2 vol. of ethanol which serve to precipitate out the sodium salts. The sodium salts are removed by filtration, washed with methanol, and set aside for recovery of any unreacted mannonic acid. The combined filtrate and washings are concentrated at reduced pressure to about 10 ml. and again diluted with 2 vol. each of methanol and ethanol. The salts are separated, ancl the solution is once again concentrated at reduced pressure to about 5 ml. and passed through a column (1.4 X 10 cm.) containing an equal mixture of Amberlite IR-100, analytical grade, and

[22]


517

dried. The yield of D-mannono-~-lactone-l-C 14 crystals amounts to about 0.11 g. Addition of carrier (0.2 g.) to the mother liquors and washings followed by crystallization yields another crop of about 0.18 g. of radioactive lactone. The total mannono-~-lactone-l-C 14 corresponds to a 30% radiochemical yield. The over-all radiochemical yield of the two hexonic acids by this procedure is about 94 %. Conversion of Barium Gluconate-l-C ~4 to o~Glucono-~-lactone. An aqueous solution containing 0.581 g. of barium gluconate (milliequivalents) is passed over a column (1 X 30 cm.) containing Amberlite IR-100, analytical grade, cation exchange resin, and the column is immediately washed with water. The effluent is lyophilized at once, and the residue taken up in a small amount of Cellosolve. (In the event that D-glucose1-C 14 is the desired final product, the Methyl Cellosolve solution of gluconic acid can be evaporated and lactonized in the reduction tube (see Fig. 4), thus avoiding transfer and possible loss of material.) The solution is seeded with D-glucono-~-lactone crystals, and the solvent is removed by evaporation in a stream of dry air. In the course of about 1 day, the solvent is almost removed, leaving a partially crystalline residue. This residue is then moistened with Methyl Cellosolve, and evaporation is continued until the material appears to be completely converted to the characteristic chunky crystals of the ~-lactone. This material, corresponding to about 90% of the theoretical yield, may be used directly for the preparation of D-glucose-l-C 14 by reduction with sodium amalgam. Reduction of D-Glucone-~-lactone-l-C ~4 with Sodium Amalgam and Separation of Crystalline a-D-Glucose-l-C ~. In order to reduce the D-glucono-~-lactone-l-C ~4, 0.7 g. of oxalic acid, 0.8 g. of sodium oxalate, and 20 ml. of ice-water are placed in the reduction tube containing 1 raM. (178 rag.) of the lactone. Immediately afterward, 2.3 g. of 5% sodium amalgam pellets is added, the mixture cooled in an ice bath, the tube closed, and the mixture vigorously stirred until the amalgam is spent. After removal of the mercury, 3 vol. of methanol is added, and the precipitated crystalline salts are removed by filtration, washed with methanol, and set aside for recovery of any unreacted gluconic acid. The alcoholic solution is then neutralized with aqueous sodium hydroxide and finally evaporated almost to dryness under reduced pressure. The residue is extracted with several small portions of methanol (total volume, about 10 ml.), and the extract filtered and diluted with water. The solution is then passed through a column (1.4 X 10 cm.) containing equal parts of cation and anion exchange resins (Amberlite and Duolite). The combined solution and washings are evaporated under reduced pressure to remove the methanol, and the concentrate is finally lyophilized to a sirup, weighing about 184 rag. The sirup is dissolved in a small

518


[22]

amount of methanol, transferred to a tared tube (total volume with washings, about 4 ml.), isopropyl alcohol added to incipient turbidity, and the solution seeded with crystals of D-glucose. Crystallization, which usually begins immediately, is allowed to take place over a period of several days. On decantation of the mother liquor and drying of the crystals a yield of 144 mg. of D-glucose is obtained. Specific rotation at equilibrium [a]~2 = +51o; m.p., 147 to 149 °. The yield of D-glucose-l-C TM from the reduction of the ~-lactone is thus about 80%. On addition of D-glucose carrier to the mother liquors and concentration of the solution the yield of radiochemical glucose is increased to about 45%. Frush and Isbell 6° have recently described two macroprocedures for the efficient reduction of aldonic acid lactones through the use of sodium amalgam in the presence of an oxalate buffer. The first employs a stainless steel flask equipped with a stainless steel stirring rod fitted with a curved vane that conforms in shape to the flask and sweeps the bottom. The second method makes use of a high-speed blendor. Either procedure is suitable for the preparation of rare sugars from their respective lactones in relatively large amounts. With these methods, L-glucose was prepared from L-glucono-8-1actone (10.0 g.) in 88% yields. c. Preparation of D-Glucose-6-C TM and D-Glucuronolactone-6-C TM. Principle. 5-Aldo-l,2-isopropylidene-D-xylofuranose prepared from the sodium periodate oxidation of 1,2-isopropylidene-D-glucofuranose is reacted with sodium cyanide-C TM. The product is hydrolyzed to the corresponding 1,2-isopropylidene-D-glucuronic acid-6-C 14, which is then lactonized 81 reduced with lithium aluminum hydride, and hydrolyzed to D-glucose-6-C14. 62 The procedure as outlined by Sowden 6~ gave yields of about 12%, and the method described by Shafizadeh and Wolfrom 63 gave yields of about 15%. A modification of these procedures has recently been reported by Schaffer and Isbell ~4 which increases the yield of D-glucose-6-C TM to 48% based on the starting sodium cyanide-C ~4.

Reagents Sodium metaperiodate. 1,2-Isopropylidene-D-glucofuranose. 6~.e so H. L. Frush and H. S. Isbell, J. Research Natl. Bur. Standards 54, 267 (1955). 61 j. C. Sowden, J. Am. Chem. Soc. 74, 4377 (1952). s~ S. Roseman, J. Am. Chem. Soc. 74, 4467 (1952). sa F. Shafizadeh and M. L. Wolfrom, J. Am. Chem. Soc. 77, 2568 (1955). 84 R. Schaffer and H. S. Isbell, U.S.A.E.C. Rept. No. NBS 4048 (1955); J. Research Natl. Bur. Standards in'press (1956). 86 D. J. Bell, J. Chem. Soc. 1935, 1874. ~6 C. L. Mehltretter, B. H. Alexander, R. L. Mellies, and C. E. Rist, J. Am. Chem. ~oc. 73, 2424 (1951).

[22]


519

Ethylene glycol. Chloroform, c.p. Acetone and dry ice. Benzene, c.p. Sodium cyanide-C 14 (see page 508). Sodium hydroxide. 1.0 N acetic acid. Sodium sulfite. Sodium carbonate. Amberlite IR-120 H. Barium hydroxide • 8H20. Carbon dioxide gas. Methanol, c.p. Toluene, c.p. Decolorizing carbon (Norit or Carboraffin). Anhydrous diethyl ether, c.p. (stored over sodium wire). Lithium aluminum hydride solution in ether. 67 Anhydrous ethanol. Glacial acetic acid. Sulfuric acid. Duolite ion exchange resin A-4. 2-Propanol. Methyl Cellosolve.

1,2-O-Isopropylidine-D-xylodialdopentofuranose. To a stirred solution of 50 g. of sodium metaperiodate and 400 ml. of water in a 1-1. flask surrounded by an ice bath was added during 30 minutes 50 g. of 1,2JOisopropylidine-D-glucofuranose.65,e6 The stirring was continued for an additional 20 minutes and the excess periodate then decomposed by the addition of ethylene glycol. The water was removed by freeze-drying, and the residue was extracted with four 100-ml. portions of chloroform. The combined chloroform extract was clarified by filtration through carbon and concentrated to a heavy sirup under reduced pressure. The sirup was dissolved in 100 ml. of water and once more concentrated under vacuum to remove all traces of chloroform. The concentrate was again dissolved in 50 ml. of water, filtered, and stored in the refrigerator for several weeks. The crystals that formed were separated, washed with water, and then recrystallized from water to give 29.9 g. of 1,2-O-isopropylidine-D-xylodialdopentofuranoside hydrate. The specific rotation of the hydrate is [~]~D° = 25.6 ° (in water). 67W. G. Brown, Org. Reactions 6, 484 (1951).

[9.~]


521

glucuronolactone-6-C ~4 mixed with some brown residue. The product was used without further purification for the preparation of D-glucose-6-C 14. D-Glucose-6-C 14. The reaction vessel consisted of a 300-ml. threenecked Grignard reaction flask into which were inserted a condenser and a pressure-equalized dropping funnel, both protected with drying tubes. Through the third neck, stirring with a magnetic stirrer, 30 ml. of a diethyl ether solution of 13.2 mM. of lithium aluminum hydride 67 was added. The crude C14-1abeled lactone was dissolved in 70 ml. of diethyl ether and then added at a dropwise rate from the funnel over a period of 30 minutes to the stirred lithium aluminum hydride solution. The residual labeled material was rinsed into the reduction flask with ether, and the reaction mixture was refluxed for 30 minutes in a water bath at 50 to 60 ° while the solution was stirred. The mixture was then allowed to cool to room temperature after which 25 ml. of anhydrous ethanol was added dropwise, with continued stirring, followed by 130 ml. of water containing 6 ml. of glacial acetic acid to destroy the excess lithium aluminum hydride. After the supernatant ether was evaporated by a current of air an aqueous solution remained, which was then passed through 100 ml. of cation exchange resin (Amberlite IR-120 H), and washed with water until 725 ml. of eluant was collected. The solution was concentrated at reduced pressure, and finally 10 mN[. of aqueous sulfuric acid was added to make 200 ml. of 0.1 N sulfuric acid solution. The solution was heated for 2 hours in a boiling-water bath (steam bath) and, after cooling to room temperature, was passed through 40 ml. of anion exchange resin (Duolite A-4). A total of 550 ml. of eluant was collected, concentrated i n vacuo to about 50 ml., filtered through a bed of decolorizing carbon, and lyophilized. The residue was dissolved in methanol and a small amount of insoluble material removed by filtration. On conoentration of the filtrate to about 5 ml., addition of 2-propanol to incipient turbidity, seeding with crystalline D-glucose and, as crystallization occurred, with periodic additions of 2-propanol, a crop of crystalline D-glucose-6-C 14 was obtained. The crystals were separated from the mother liquor and washed with a mixture of methanol and 2-propanol. Recrystallization yielded 485 mg. of D-glucose-6-C ~4 of a specific activity of 9.2 ~c./mg. Through the use of 2.0 g. of carrier D-glucose, an additional 993 ~c. of labeled glucose was obtained. The over-all radiochemical yield based on the sodium cyanideC 14 was 48%. S o d i u m D-Glucuronate-6-C ~4 Monohydrate. In a 100-ml. flask equipped with a magnetic stirrer, an electrical heater, and a reflux condenser, a mixture of 896 rag. of barium 1,2-O-isopropylidine-D-glucuronate-6-C~4 (590 t~c.), 10 ml. of Amberlite IR-120 H, and 40 ml. of water was heated to reflux temperature for 50 minutes. The cooled mixture was filtered

522


[22]

through a small bed of resin and washed until 175 ml. of filtrate was collected. The clear filtrate was neutralized with sodium hydroxide, concentrated under reduced pressure, treated with decolorizing carbon, and filtered. T h e concentrated filtrate was then treated with a few drops of ethanol and seeded with crystalline sodium D-glucuronate monohydrate. 6s The resulting crystals were separated and washed with aqueous ethanol. Recrystallization from aqueous ethanol yielded 595 rag. of the monoh y d r a t e with a total radioactivity of 509 ~c. B y using carrier, an additional 34 ~c. of sodium D-glucuronate-6-C 14 m o n o h y d r a t e was obtained from the mother liquor, giving a 92 % radiochemical yield. D-Glucurone-6-C 14. A solution of 927 rag. of barium 1,2-O-isopropylidine-D-glucuronate-6-C ~ (165 ~c.) in 50 m l . . o f water and 10 ml. of Amberlite IR-120 H resin was treated as described above for the preparation of sodium D-glucuronate-6-C 14. After removal of the resin, the solution was concentrated at reduced pressure, passed through a filter coated with decolorizing carbon, and then lyophilized. The residue was dissolved in 5/[ethyl Cellosolve, seeded with o-glucurone, and allowed to crystallize. Recrystallization produced 438 rag. of D-glucurone-6-C 14 having a total activity of 133.5 ~c. B y use of carrier an additional 19.3 #c. of labeled glucurone was obtained, making the total radiochemical yield 93 %.

2. Nitromethane a. Preparation of Glucose-1-C TM and M a n n o s e - l - C 14. Principle. As developed b y Sowden, 69,70the procedure consists in converting methanolC ~4to methyl iodide by the m e t h o d of Norris 7~and then to nitromethane. 7~ Condensation of the nitromethane-C TM with D-arabinose according to Sowden and Fischer 78 yields 1-nitro-l-deoxysorbitol and 1-nitro-l-deoxyD-mannitol and subsequent conversion of the two nitro alcohols to the respective hexoses b y the Nef reaction.74 Reagents

C14H~0H. Hydriodic acid. Silver nitrite. D r y sand. 88W. Hach and D. G. Benjamin, J. Am. Chem. Soc. 76, 917 (1954). B9j. C. Sowden, Science 109, 229 (1949). 70j. C. Sowden, J. Biol. Chem. 180, 55 (1949). 71j. F. Norris, J. Am. Chem. Soc. $8, 639 (1907). 7~V. Meyer, Ann. 171, 23 (1874). 7~j. C. Sowden and H. O. L. Fischer, J. Am. Chem. Soc. 68, 1312 (1944); 67, 1713 (1945); 68, 1511 (1946); 69, 1048, 1963 (1947). ~4j. V. Nef, Ann. 280, 263 (1894).

[9.~]


523

Solid C02-chloroform mixture. Calcium sulfate. D-Arabinose. Absolute methanol. Sodium methoxide solution. Ether, c.p. Petroleum ether (b.p. 30 to 60°). Amberlite IR-100, analytical grade, and I)uolite A-4 ioi~ exchange resins. Absolute ethanol. Anhydrous acetone. 2 N sodium hydroxide. Concentrated sulfuric acid. Sodium carbonate. Phenylhydrazine. Acetic acid. Benzaldehyde. Congo red indicator. Barium carbonate. D-Glucose. Procedure. C14-Methanol (16 g., containing 4 me. of C 14) is converted to methyl iodide by distillation with hydriodic acid. A yield of approximately 59 g., or 83%, is obtained. 69,7°,7~ To an intimate mixture of 120 g. of silver nitrite and 250 g. of dry sand the methyl iodide is added dropwise under a cold finger maintained at - 4 0 ° by means of dry ice and chloroform. After the addition of the methyl iodide, the mixture is heated for 2 hours on the steam bath. The condenser is warmed to - 5°, and the gaseous methyl nitrite is flushed out of the system with a slow current of dry air into a re('eiver at - 7 0 °. The yield of methyl nitrite is approximately 4 g. (l 6 %). The nitromethane is distilled from the reaction flask with a slow stream of dry air into 20 g. of silver nitrite and calcium sulfate. About 18 g. of nitromethane (71%) is obtained. A suspension of 44 g. of D-arabinose in 50 ml. of absolute methanol is added to the 17.9 g. of nitromethane, and the mixture is treated with a solution of sodium methoxide (8.8 g. of sodium in 350 ml. of absolute methanol). The mixture is stirred for 5 hours at room temperature in a closed system, and the precipitated sodium nitro alcohol filtered and washed in rapid succession with cold methanol (-20°), ether, and petroleum ether. The mixed nitro alcohols are dissolved in 350 ml. of 75 j . D. Cox, H. S. Turner, and R. J. Warne, J. Chem. Soc. p. 3167 (1950).

[22]


525

converted to methyl-D-glucopyranoside. The methyl derivative is oxidized to a dialdehyde which on hydrogenation produces the corresponding dialcohol. This dialcohol is hydrolyzed, and the resulting glycerol is separated from the glyceraldehyde by removal of the latter as the 2,4dinitrophenylhydrazone. The identity of the glycerol obtained by this method was established from its tribenzoate derivative and from paper chromatographic data. 7s Several methods for preparation of glycerol labeled in a single position are described in the literature. ~7-79 Reagents

Uniformly labeled glucose-C 14 (see page 489). 0.083 N hydrochloric acid in anhydrous methanol. Benedict's solution. 0.37 M sodium periodate solution. 0.1 N sodium hydroxide solution. ~[ethyl red indicator. Barium chloride. Raney nickel. Hydrogen gas. Concentrated sulfuric acid. 2,4-Dinitrophenylhydrazine solution. 35 to 38% formaldehyde solution. Barium carbonate. Anhydrous acetone, c.p. Water-saturated phenol mixture, s° Butanol-acetic acid-water mixture, s° Saturated solution of lead tetraacetate in benzene (anhydrous). Pyridine, c.p. Be~lzoyl chloride. Ether. Saturated sodium bicarbonate. Anhydrous ethanol. Procedure. Uniformly labeled C14-glucose with a high specific activity caa be prepared photosynthetically according to the method of Putman el al. ~ (see p. 489). The methyl glucoside is prepared by dissolving the

76S. Abraham, J. Am. Chem. Soc. 74, 6098 (1952). 77A. P. Doerschuk, J. Am. Chem. Soc. 73, 821 (1951). 78H. Schlenk and B. W. DeHaas, J. Am. Chem. Soc. 73, 2921 (1951). 7~M. L. Karnovsky and L. I. Gidez, Federation Proc. 10, 205 (1951). s0 S. M. Partridge, Biochem. Soc. Symposia, Cambridge 3, 52 (1950).

526


[22]

sugar (1.0 g.) in absolute methyl alcohol containing dry hydrogen chloride (0.083 N HC1) according to the method of Fischer sl as described in the section on the chemical degradation of glucose. The yield of the mixed isomers is approximately 84 %. A small sample of this material should give a negative reaction with Benedict's solution. The methyl glucoside is then dissolved in 35 ml. of water, treated with 35 ml. of 0.37 M sodium periodate solution, and the mixture allowed to remain at 2 ° for 24 hours. The formic acid formed is titrated with 0.1 N sodium hydroxide to the methyl red end point and is usually found to be 100% of theoretical. A small aliquot of the solution at this point gives a positive Benedict's test. Solid barium chloride (1.6 g.) is added to the neutralized solution, and the resulting precipitate is filtered and washed with water. The filtrate is concentrated under reduced pressure at a bath temperature of less than 60 ° (the formic acid is recovered in the distillate), and the additional precipitate formed during this time is filtered and washed with water. The dialdehyde in aqueous solution'is then hydrogenated at 2700 p.s.i. and 140 ° for 18 hours, with Raney nickel as the catalyst. An aliquot of this solution gives a negative Benedict's test. The filtered solution is acidified with 3.5 mh of concentrated H2SO4 and is hydrolyzed under reflux for 3 hours. At the end of that time the solution turns slightly yellow in color. Addition of 42 ml. of 2,4-dinitrophenylhydrazine solution (4 g. of 2,4-dinitrophenylhydrazine -{- 20 ml. of concentrated sulfuric acid -~ 30 ml. of water ~ 100 ml. of 95% ethanol) precipitates the glycolaldehyde as its hydrazone. This is filtered, washed, and dried with a resulting yield of 1.0 g. (approximately 90%). Addition of 0.7 ml. of 35 to 38% solution of formaldehyde removes most of the excess 2,4-dinitrophenolhydrazine as the hydrazone, which is filtered and discarded. The H2S04 is neutralized with a large excess of solid BaCO3, and the resulting precipitate filtered and washed with distilled water. The combined filtrate and washings are treated with charcoal and filtered. The resulting slightly yellow solution is concentrated in vacuo, at 40 °, to a thick viscous mass. Extraction with boiling, anhydrous acetone, and final evaporation of the acetone at room temperature yields the glycerol-C 14. The recovered weight is about 0.4 g. (64 % based on glucose corrected for 78% glycerol in water as judged by its refractive index). The refractive index of this glycerol is 1.4398 at 25 °. This represents 77.6% glycerol in water. 82 The specific activity of the resulting glycerol is the same as the original glucose. Two-dimensional ,paper chromatography with water-saturated phenol 81 E. Fischer, Bet. 28, 1145 (1895). s~ "International Critical Tables," Vol. 7, p. 68. McGraw-Hill Book Co., New York, 1930.

[22]


527

and butanol-acetic acid-water s° reveals only one radioactive spot, which is identical with the color spot obtained with inactive glycerol, with a solution of lead tetraacetate in benzene as the color spray. Experiments with inactive glucose by the same method yielded glycerol (65%) which was identified (1) by its refractive index, as given above, and (2) by its crystalline glycerol tribenzoate derivative. Preparation of Glycerol Tribenzoate. To 0.5 g. of the synthesized glycerol is added 5 ml. of pyridine, and the solution is cooled on an ice bath for 10 to 15 minutes. Two milliliters of benzoyl chloride are added to this acid mixture, and the resulting mass is allowed to stand at room temperature overnight. The solution is then poured into 150 ml. of ice water (acidified with 5 N H~SO4) and allowed to stand for 2 hours. A clear oil separates out and is extracted with ether. The ether solution is washed several times with a saturated solution of NaHCOa and finally with cold water. The ether is evaporated, and the resulting oil is dissolved in a minimum of absolute ethyl alcohol. After the alcoholic solution is allowed to stand at 0 °, the glycerol tribenzoate crystallizes. The product is recrystallized from ethanol, m.p. 74-75 ° (lit. 75°). A mixed melting point with the tribenzoate prepared from c.p. glycerol is not depressed. Degradation of Glyeerol-C ~4. A small aliquot of the glycerol-C ~ is dissolved in absolute ethanol, and inactive glycerol is added. The ethyl alcohol is removed in vacuo at room temperature, and the resulting diluted glycerol-C ~4may be degraded according to the procedure outlined by Kritchevsky and Abraham.83 b. Preparation of D-Xylose-l-C 14 from D-Glucose-l-C ~4 by Means of Periodate. Principle. D-Glucose-l-C ~4 is acetonated to 1,2,5,6-diisopropylidine-D-glucofuranose-l-C14. 84 Controlled hydrolysis of this derivative yields 1,2-isopropylidine-D-glucofuranose-l-C ~4, which on treatment with periodate leads to the formation of the substituted dialdehyde 5-aldo-l,2-isopropylidine-D-xylofuranose. When reduced with Raney nickel followed by mild acid hydrolysis this dialdehyde gives D-xylose1-C I4 in 55 to 60% yield. 85

Reagents D-Glucose-l-C 14. Sulfuric acid, c.p. Acetone, c.p. 83 D. Kritchevsky and S. Abraham, Arch. Biochem. and Biophys. 39, 305 (1952). s4 C. L. Mehltretter, R. L. Mellies, J. C. Rankin, and C. E. Rist, J. Am. Chem. Soc. 73, 2424 (1951). ~ J. C. Sowden, J. Am. Chem. Soc. 73, 5494 (1951).

528


[22]

50% NaOH solution. HC1, c.p. Benzene, c.p. Methyl red indicator. Sodium metaperiodate. Starch-iodide paper. Glycerol, c.p., or ethylene glycol, c.p. Absolute ethanol. Anhydrous sodium sulfate. Chloroform, c.p. Hydrogen gas. Raney nickel. Duolite ion exchange resin A-4.

Preparation. To a suspension of 5.0 g. of powdered D-glucose-l-C TM in 100 ml. of acetone at 0 °, 4 ml. of concentrated sulfuric acid is added dropwise. The mixture is stirred with a magnetic stirrer in a closed flask for 4 hours at room temperature. About 140 mg. of unreacted glucose is then separated from the solution by filtration. The clear filtrate is made just alkaline with a 50% sodium hydroxide solution at 15 to 20 °. The resulting sodium sulfate is filtered and washed thoroughly with fresh acetone. The combined filtrate and washings are concentrated at reduced pressure, water is added, and the concentration of the solution continued to a volume of 50 to 70 ml. The solution is adjusted to pH 2.0 with concentrated HC1 and stirred for 4 hours at 40 °. The solution is then adjusted to pH 8 to 8.5 with a sodium hydroxide solution and extracted with an equal volume (100 to 125 ml.) of benzene which serves to remove the unchanged diacetone glucose. Methyl red indicator is added, and the aqueous layer is stirred with portions of powdered sodium metaperiodate, keeping the pH as near as possible to the end point of the indicator by the dropwise addition of aqueous sodium hydroxide. After the addition of 7.3 g. of periodate, the presence of excess oxidant can be tested for by using starch iodide paper. The excess periodate is destroyed by the addition of a few drops of glycerol or ethylene glycol. The solution is then concentrated at reduced pressure to a thick sirup. Anhydrous sodium sulfate is added to the sirup, and the acetonate dialdehyde is extracted with five 40-ml. portions of chloroform. Concentration of the chloroform extract at reduced pressure yields about 5 g. of crude acetonated dialdehyde. The product is mechanically shaken in 65 ml. of 95 % ethanol with hydrogen in the presence of 5 g. of Raney nickel for 90 hours at room temperature and atmospheric pressure. The Raney nickel is then filtered and washed with 95% ethanol, and the combined filtrate and washings

530


[22]

After removal of the catalyst by filtration, the solution is evaporated to dryness i n vacuo, and the residue extracted with hot pyridine. On cooling of the pyridine solution, the sorbitol crystallizes as its pyridine complex. After two crops of the complex are thus isolated, 2 g. of sorbitol is added to the mother liquor as carrier, and two additional crops of this complex are isolated. The combined crops of the sorbitol-pyridine complex are recrystallized from pyridine two times and after drying i n vacuo over H2SO4 at room temperature. The product weighs approximately 9.2 g. The total yield of anhydrous sorbitol-C 14 is about 6.4 g. after quantitative removal of the pyridine by heating the complex at 80 ° and 1 ram. of pressure over H2SO, for 48 hours. Another method for the preparation of sorbitol by using sodium borohydride with nonisotopic glucose results in an approximate yield of 80%. 89 The method employed by Wick et al2 ° involves the use of Raney nickel 9~ as the catalytic reducing agent on glucose-C 14.

II. Degradation of Labeled Carbohydrates A . Biological Methods 1. Lactobacillus casei

See Vol. IV [23].

2. Baker's Yeast The method employed by Barnet and Wick 92 is based on the principle that when glucose is fermented by baker's yeast to ethyl alcohol and carbon dioxide, the ethyl alcohol represents carbons 1, 2, 5, and 6, and the COz represents carbons 3 and 4 of the fermented glucose. The ethyl alcohol can then be oxidized to acetic acid 9~ and further degraded by the Schmidt (azide) reaction described below. 3. Leuconostoc mesenteroides

See Vol. IV [23] for degradation to lactate and acetate. a. Degradation of C14-Labeled Lactate and Acetate (see also Vol. IV [23]). P r i n c i p l e . The following reactions are involved in the complete 89M. Abdel-Akher,J. K. Hamilton, and F. Smith, J. Am. Chem. Soc. 73, 4691 (1951). 90A. N. Wick, M. C. Almen, and L. Joseph, J. Am. Pharm. Assoc. Sci. Ed. 40, 542 (1951). 91S. D. Borisoglebskii, J. Appl. Chem. (U.S.S.R.) 13, 571 (1940). 92H. N. Barnet and A. N. Wick, J. Biol. Chem. 185, 657 (1950). 9sA. C. Neish, "Analytical Methods for Bacterial Metabolism," National Research Council of Canada Report No. 46-8-3 (rev.), pp. 21-22, Saskatoon, Sask., 1950.

[22]

CARBOHYDRATE SYNTHESIS

AND D E G R A D A T I O N

531

degradation and isotopic assay of the individual carbon atoms of lactate and acetate :94,95 KMnO4,H +

CH3CHOHCOOH * CH3COOH + C02 (3) (2) (1) (3) (2) (1) CH3COOH NaN3,H~SO~ CH3NH,. + CO~ (3) (2) (3) (2) KMnO4,0HCH~NH2 + CO.., (3) (3)

(a) (b) (c)

Although m a n y procedures for the degradation of these compounds have been reported 24,95-99 which involve elaborate equipment, the method described here permits the complete degradation to be performed in the combined combustion-diffusion vessel described below24,1°° A p p a r a t u s and Reagents

Reaction flask. Ordinary 50-ml. narrow-mouthed Erlenmeyer flasks provided with a center well for CO~ absorption are used. A center well similar to t h a t used in a conventional Warburg flask is sealed to the bottom of the vessel. The exact dimensions of the well are not critical; about 12 mm. in diameter and approximately 3 cm. high will be found convenient (Fig. 5A). Serum bottle stoppers, sleeve type. Catalog No. 16201, Braun, Knecht and Heimann Co., San Francisco; catalog No. 2319, A. H. Thomas Co., Philadelphia, 1950 (Fig. 5B). Siphon for carbonate sample transfer. A siphon which facilitates the quantitative transfer of the carbonate solution from the center well of the combustion flask is shown in Fig. 5C. 5 % potassium permanganate solution in 2 N sulfuric acid. Carbonate-free NaOH solution, approximately 1.0 N. Carbonate-free N a O H solution, approximately 0.1 N. Saturated barium chloride solution. Phenolphthalein indicator. Phenol red indicator. 100% sulfuric acid (3 parts of concentrated H~SO4 and 1 part of fuming H2SO4 9~). 94j. Katz, S. Abraham, and I. L. Chaikoff, Anal. Chem. 27, 155 (1955). 95E. F. Phares, Arch. Biochem. and Biophys. 33, 173 (1951). 56H. A. Barker and M. D. Kamen, Proc. Natl. Acad. Sci. U. S. 31, 219 (1945). 97S. Aronoff, H. A. Barker, and M. Calvin, J. Biol. Chem. 169, 459 (1947). 98S. Roseman, J. Am. Chem. ~oc. 75, 3854 (1953). 99L. Daus, M. Meinke, and M. Calvin, J. Biol. Chem. 196, 77 (1952). ~0oj. Katz, S. Abraham, and N. Baker, Anal. Chem. 26, 1503 (1954).

532


[22]

2 N sulfuric acid containing 1 to 2 % H20~ (freshly prepared). 30% sodium hydroxide solution. H~S04 solution, approximately 0.10 N. 5 % potassium permanganate solution. Sodium azide (twice recrystallized from water and alcohol and stored in a desiccator over calcium chloride). 2 N sodium hydroxide solution (carbonate-free). To Vocuum

To Vacuum

9 B

A

FIG. 5. Combined combustion--diffusion vessel and siphon. The Oxidation of Lactate (Equation a). Two hundred micromoles of lactate is introduced into the vessel, and 2 ml. of 5 % K M n 0 4 dissolved in 2 N H~SO4 is added. T h e total volume can be in excess of 10 ml. ; however, for subsequent ease in distillation of the resulting acetate a small volume is preferable. One milliliter of standardized carbonate-free base (1.0 N) is placed in the center well. The flask is immediately capped and evacuated by inserting through the rubber cap a 20- or 22-gage hypodermic needle which is connected to a v a c u u m line. T h e flask is heated for 30 minutes in an 80 ° water bath and allowed to cool. The carbonate solution in the center well is collected by means of the siphon, barium chloride is added, and the excess base is titrated to the phenolphthalein end point. The BaCO3 is mounted and assayed for C14.1°1 T h e acetate is recovered b y steam distillation of the permanganate solution in a small M a r k h a m still, I°~ 20 vol. of distillate is collected, and the acetic acid 101C. Entenman, S. R. Lerner, I. L. Chaikoff, and W. G. Dauben, Proc. Soc. Exptl. Biol. Med. 70~ 364 (1949). 102R. Markham~ Biochem. J. 36, 790 (1942).

[22]

CARBOHYDRATE S Y N T H E S I S AND DEGRADATION

533

titrated with standard base. Typical results of such experiments with variously labeled lactates are presented in Table I. 94 TABLE I a DEGRADATION OF LACTATE-C 14

Products of reaction Per cent recovery Compound Lactic Lactic Lactic Lactic Lactic Lactic

Reaction

acid-l-C 14 KMnO4oxidation acid-l-C 14 K2S208oxidation acid-2-C TM KMn04 oxidation acid-2-C TM K~S20~oxidation acid-3-C TM KMnO~oxidation acid-3-C '4 K2S~0~oxidation

CO.~ Acetate

COs

97 100 99 92 95 92

24.6 8.5 2.4 35.8 1.3 18.1

98 -96 -96 --

Per cent activity in carboxyl carbon of Acetate lactate

Specific activity~

0 ------

96.5 -2.3 -2.4 --

a j. Katz, S. Abraham, and I. L. Chaikoff, Anal. Chem. 27, 155 (1955). b Counts per minute of BaC14Os assayed with an end-window Geiger-Mueller tube [C. Entenman, S. R. Lerner, I. L. Chaikoff, and W. G. Dauben, Proc. Soc. Exptl. Biol. Med. 70, 364 (1949)].

The Degradation of Acetate (Equation b). The sodium acetate solution is evaporated to dryness on the steam b a t h and the residue dissolved in a small volume of water (1 or 2 ml.). Aliquots are taken for determination of total activity by persulfate combustion, described in this section, 1°° and for the azide (Schmidt) reaction. The combustion with persulfate yields the average specific activity of acetate, which represents the 2 and 3 carbons of lactate. The aliquot used for the Schmidt reaction is put into a shell vial which conveniently fits into the center well of the reaction flask, and the solution is evaporated to dryness in a v a c u u m oven. A b o u t 0.2 ml. of 100% H.,SO495 followed b y about 40 mg. of recrystallized sodium azide is added cautiously and the contents mixed with a small, fine stirring rod which is left in the vial. The vial is placed in the center well and the flask capped, evacuated as described before, and placed in a water bath at about 35 °. The temperature is brought to 70 to 80 ° within 15 to 20 minutes, and maintained for half an hour. The vessel is cooled, and about 2 ml. of C02-free alkali (1.0 N N a O H ) is carefully injected into the main c o m p a r t m e n t by means of a syringe and a 3-inch No. 20 hypodermic needle. After about half an hour for CO2 adsorption, the v a c u u m is released b y inserting a hypo-

[22]


535

standard, and CO2-free base is put in the center well. The flask is again capped, evacuated, and the COs liberated by injecting excess dilute sulfuric acid into the alkaline permanganate solution. The methylamine can also be combusted with a large excess of persulfate (page 549). As seen from Table II, the yields of both CO2 and methylamine range from 90 to 95 %. B. Chemical Method with Lead Tetraacetate I. Determination of C 14 Activity of Individual Carbon Atoms of the Glucose Chain (see Vol. IV [23] for Biological Procedures)

Principle. The activity of the individual carbon atoms in the glucose chain is obtained from the combined data of the degradation of glucose and several of its derivatives. Their oxidation with specific oxidizing agents yields C02 either from a single carbon atom or from groups of carbon atoms. The general scheme is outlined in Fig. 6. I~ Degradation of glucose-C ~4 from various biological sources has been performed by this method.12.1s.2s.~03 2. Preparation of Glucose Derivatives

Reagents for the Preparation of Glucose Derivatives Methyl-D-glucoside. D-Glucose. Methanol, anhydrous. Hydrochloric acid, dry gas. Liquid nitrogen. Ethanol, anhydrous. Decolorizing carbon. D-Glucose phenylosazone. Phenylhydrazine hydrochloride, recrystallized. Sodium acetate. Ether. D-Glucose phenylosotriazole. Copper sulfate pentahydrate. n-Butanol. 2-Phenyl-4-formylosatriazole. Sodium metaperiodate. Methyl red indicator. Sodium hydroxide solution, approximately 0.1 N. ~0s F. W. Minor, G. A. Greathouse, H. G. Shirk, A. M. Schwartz, and M. Harris, J. Am. Chem. Soc. 76, 1658 (1954).

536

[22]


I

CHO

COOH

I

I

CHOH

CH2

I

I

HOCH

CHs

I

HBr I

I

J

+ HCOOH ~(o) cos (C=I)

CHOH ----* CO CHOH

CH~

I

CHsOH CHO

II

CHO

r I

I CHOH [

CHO

I

CHO

[

I

Pb(OAc)~

\ /

CH20 III H OCH3

H

\/

t

I

HOCH

\/

OCH3

C

C CHOH

/

CHsO + 2 COs (C-1, C-2)

CHOH CHCH~

I

\

CHOH CHCH3

HOCH

,

J

CHO

I Pb(OAc)4--* O

0

+ COs (C-3)

CHO [ CH

l

CHOH

I

l

CHsOH

CH

I

IV H

CH2OH OCH~

H

\/

C

C

HOCH

I

OCH3

\/

O

Pb(OAc)(

)

CHO

CHOH

CHO

CHOH

CHs

J

O _ + COs

(c-4)

I

CH2-FIG. 6. General scheme for degradation of glucose.

[22]


V

HC~N

HC~N

1 b )NC6H5 C-=N HOCH

+ HCHO

I

(C-6)

(C-l, C-2, C-3)

+2C0~

1

CHOH

i

)NC6H.~ C---N CHO

Pb(OAc),

. . . . . . --~

(C-4, C-5)

CHOH

t

VI

CH20H CHO

I

CHOH

I

HOCH

f I

CItOH

NaI(h

5 HCOOH + HCHO

1(o)

CHOH

CO2 (C-6)

I

CH:OH Fro. 6. (Continued)

Calcium gluconate. Barium iodide. Iodine. 0.4 N barium hydroxide solution. Sulfuric acid, concentrated. Lead carbonate. Silver sulfate. Hydrogen sulfide. Duolite cation exchange resin C-3. Calcium carbonate. D-Arabinose. Barium acetate. Ferric sulfate. Ascarite tower (for CO2-free air). 0.5 N sodium hydroxide solution. 30% hydrogen peroxide (Superoxol). Duolite anion exchange resin A-4. Barium chloride monohydrate. D-Arabinose. Aniline oxalate spray reagent.29

537

538


[22]

Methyl-D-arabinopyranoside. Silver carbonate. Ethyl acetate, c.p. 4, 6-Ethylidine-D-glucose. Paraldehyde. Ammonium hydroxide.

Reagents for the Determination of the C 14Activity in the Glucose Carbons 10% hydrobromic acid. Phenolphthalein indicator. Mercuric acetate. Lead tetraacetate. Glacial acetic acid. Hydrochloric acid solution, standardized at 0.10 N. Pyridine, c.p. 1 N hydrochloric acid. Sodium arsenite solution (30 g. of As203 and 15 g. of NaOH in 500 ml. of solution, giving a solution of approximately 1.2 N Na2HAsO3 lo4). Potassium permanganate. a . Methyl-D-Glucopyranoside. Methyl glucoside is prepared in a sealed ignition tube according to the method of Fischer. 81 Ten milliliters of a 0.25% solution of dry HCI (0.086 N) in pure, anhydrous methyl alcohol is added to 1 g. of the dry, crystalline glucose in the tube. The mixture is refluxed with frequent shaking for about 30 minutes on a steam bath in which time all the glucose dissolves. The contents of the tube are frozen with liquid nitrogen, and the bomb tube is carefully sealed with a torch. The tube is then placed in a lead pipe as a precautionary measure. Glucoside formation is carried out at 100° for 50 hours in an oven. At the end of that time the sealed tube is immersed in liquid nitrogen. The tube is then cautiously opened, the contents removed from the tube with a minimum amount of anhydrous methyl alcohol, and the solution concentrated in a vacuum desiccator. When crystals of the a isomer separate out, the small amount of mother liquor is poured off and the crystals are washed with absolute ethyl alcohol. This first crop of methyl glucoside crystals is dissolved in water, treated with decolorizing carbon, and filtered. The solution is then concentrated to a thick sirup in a vacuum oven at 40 °, and crystallization is induced with anhydrous ethyl alcohol. The pure methyl-a-D-glucopyranoside melts at 166° and gives [a]~° = ~-158.9 ° (water, c = 10). Weight of dry crystals, 0.60 g.

~04W. C. Pierce and E. L. Haenisch, "Qualitative Analysis," 2nd ed., p. 203. John Wiley & Sons, New York, 1946.

[29.]


539

The mother liquors and alcohol washings are combined and concentrated. F u r t h e r t r e a t m e n t of the concentrate b y the method described above yields a second crop of crystals. T h e pure methyl-~-D-glycopyranoside melts at 105 ° and gives [~]eo = _ 3 4 . 2 ° (water, c = 10). Weight, 0.20 g. The two crops of crystals are dried and combined. Total yield, 0.80 g., or 75 % of theoretical. b. D-Glucose Phenylosazone. To 1 g. of glucose in a large test tube is added a solution of 5 g. of recrystallized phenylhydrazine hydrochloride and 8 g. of sodium acetate in 20 ml. of water. T h e mixture is heated on a steam bath for 1 hour. During this time, golden yellow crystals of the phenylosazone are formed. At the end of the hour, the tube is cooled to room t e m p e r a t u r e and then stored at 0 °. T h e crystals of the glucosazone are then separated b y filtration and dried. The crude crystals are dissolved in boiling ethanol (95%), and water is added until the osazone begins to crystallize. Crystallization is allowed to proceed at 0 °. The glucosazone is filtered and washed, first with small amounts of ice water and then with ether. T h e crystals are dried in a v a c u u m oven at 40 °. The yield of pure osazone is 1.2 g. (62% of theoretical). c. D-Glucose Phenylosotriazole. The osotriazole is prepared according to the m e t h o d of H a n n and Hudson. 1°5 An aqueous solution of 100 ml. of CuSO4 (7.0 g. of CuSO4'5H20 in 600 ml. of H~O) is added to 0.83 g. of finely divided, recrystallized osazone, and the resulting suspension, after the addition of about 2 ml. of n-butyl alcohol (wetting agent for the osazone), is refluxed for 2 hours. ]V[ost of the osazone dissolves after the first hour and, as the color of the solution changes from blue to green, a small a m o u n t of copper or copper oxide precipitates. At the end of the second hour, the solution, now dark in color, is filtered while hot. Crude osotriazole crystals appear when the solution is stored at 0 °. The solution is filtered, and the crystals are dissolved in a minimum of boiling water. Activated carbon is added, and the boiling solution is filtered in a steamheated Biichner funnel. T h e solution is allowed to cool to room tempera° ture and stored at 0 ° overnight. During t h a t time, crystallization of pure osotriazole is completed. Concentration of the filtrate from the crude osotriazole i n vacuo yields a second crop of the triazole; this is recrystallized in the aforementioned manner. T h e total yield of pure, asbestos-like crystals is 0.32 g. (50% of theoretical); m.p., 195 to 196 °. d. 2-Phenyl-4-formylosatriazole. A suspension of 0.3 g. of the osotriazole in 9.6 ml. of water is refrigerated for 1 hour. To this suspension is added 9.6 ml. of a saturated (0.37 M) aqueous solution of NaIO4, ~05 R. M. H a n n and C. S. Hudson, J. Am. Chem. Soc. 66, 735 (1944).

[@.2]


541

in the Erlenmeyer flask is warmed on the steam bath, and 2.4 ml. of 30% hydrogen peroxide is added from the dropping funnel. With the appearance of gas bubbles, the flask is removed from the steam bath and the reaction allowed to continue. The reaction is complete with the appearance of a purple color. A second 2.4-ml. portion of peroxide is added in the same manner, whereupon a similar reaction takes place. The dark reaction mixture is then filtered and passed over the large Duolite C-3 and A-4 ion exchange columns (3 X 25 cm.). The neutral effluent is concentrated to a thick sirup under reduced pressure, and crystallization is induced by the addition of absolute methyl alcohol. Subsequent recrystallization produces white crystals of D-arabinose. Approximate yield, 1.0 g. Fifty milliliters of a BaC12 solution (10 g. BaCI~'H20 in 100 ml. of H~O) is added to the NaOH in the trap. The resulting BaCO3 is filtered off, washed, dried, and weighed. The amount obtained is 1.8 g. (theoretical yield, 2.5 g.). This barium carbonate cannot be used as a valid measure of the activity of carbon 1.1~ The arabinose prepared in this manner is examined chromatographically 29 and gives a single C14-containing spot which occupies an area identical with that of the colored spot that appears after the filter paper is sprayed with a solution of aniline oxalate. g. Methyl-D-arabinopyranoside. The methyl arabinoside is prepared by a modification of the method of Hudson. 108One-half gram of arabinose is refluxed for 3 hours with 6.7 ml. of pure, anhydrous methyl alcohol containing 1.5% of dry HC1 (0.516 N). The acid is neutralized with about 0.6 g. of solid Ag2CO3; the solution is filtered, treated with charcoal, and refiltered. On reducing the volume of the filtrate in a vacuum desiccator, a gumlike material is obtained. This material consists of a mixture of the a and ~ isomers of methyl-D-arabinopyranoside. To separate these isomers, the mixture is extracted with hot ethyl acetate and the insoluble isomer is then crystallized from absolute ethyl alcohol. Re-extraction with hot ethyl acetate and recrystallization from ethyl alcohol yields pure methyl-~-D-arabinopyranoside with m.p. 169° and gives [a]~° = -245.5 ° (water, c = 7). The alcohol mother liquors are evaporated in a vacuum desiccator to a thin sirup which crystallizes slowly on standing. This crystalline mixture of a and ~ isomers is separated and fractionally recrystallized from hot ethyl acetate as previously described. Since the ~ isomer is less soluble in ethyl acetate, it crystallizes first, and the a isomer accumulates in the mother liquors. The total weight of the pure f~ isomer is 0.23 g., 43% of theoretical. lo8 C. S. Hudson, J. Am. Chem. Soc. 47~ 265 (1925).

542


[22]

h. 4,6-Ethylidine-D-glucose. One gram of crystalline glucose is converted to 4,6-ethylidine-D-glucose according to the method of Hockett et al. 109 A solution consisting of 1 ml. of paraldehyde and 2 drops of concentrated sulfuric acid is added to 1 g. of glucose in a 125-ml. glassstoppered flask, and the mixture is placed on a mechanical shaker. The contents of the flask are shaken for 24 to 30 hours at room temperature. At the end of that time the reaction mixture usually sets to a solid mass. On the addition of about 30 ml. of ethyl acetate, the flask is stored at 0 ° overnight. The crude crystals are then filtered and washed successively with ethyl acetate and ether. They are recrystaliized from 25 ml. of ethanol to which about 3 drops of aqueous ammonium hydroxide has been added. The solution should be neutral at this time. A small amount of insoluble salt is removed by filtration through a pad of Celite and the resulting clear solution concentrated to a small volume in vacuo at room temperature. The concentrated alcohol solution, about 4 to 5 ml., is then placed in the icebox to induce crystallization of the pure 4,6-ethylidine-Dglucose. Yield is approximately 310 mg. and has a melting point of 179 to 180 °. 3. The Quantitative Recovery of Carbon Dioxide in the Lead Tetraacetate Oxidation of Glucose Derivatives Principle. The action of 3 moles of lead tetraacetate on compounds which contain three adjacent hydroxyl groups leads to the formation of the corresponding dialdehyde with the middle carbon splitting out as COs. This reaction can be used in the oxidation of the various glucose derivatives for the isolation of some of the individual carbon atoms. 11° A schematic representation of this reaction is as follows:

R

R

I

I

I I

R'

HCOH CHO I 3Pb(C,H,O2)~ HCOH CHO + CO2 HCOH

I

R' It should be noted, however, that under these conditions the oxidation of vicinal primary hydroxyl groups yields formaldehyde which is not further oxidized. 109 R. C. Hockett, D. V. Collins, and A. Scattergood, J. Am. Chem. Soc. 73, 599 (1951). tl0 S. Abraham, J. Am. Chem. Soc. 72, 4050 (1950).

[22]


543

Apparatus and Reagents Lead tetraacetate (prepared according to McClenanhan and Hockettm). Glacial acetic acid. Phenolphthalein indicator. Methyl orange-methylene blue mixed indicator. 1 M barium chloride solution. 0.10 N hydrochloric acid solution. 0.10 N sodium hydroxide solution (C02-free). Ascarite, C02-absorbing column. The apparatus is shown in Fig. 7. A column (at G) is filled with ascarite and serves to obtain carbon dioxide-free air. The reaction tube (A) is 2 cm. in diameter and 17 cm. long. The inlet which is placed 1.5 ram. from the bottom of tube A has a diameter of 4 mm. (drawn down to 2 ram. at the bottom) and emerges from tube A about 8 cm. from the bottom. A 24/40 standard taper glass joint connects the reaction tube (A) with the gas washing tube (B). The buret inlet at H has a 5-ml. capacity and a stopcock (a) with a 2-ram. bore. The tip of this buret should extend below the glass joint 24/40. The bulb on tube B has a diameter of 3 cm. and is 15 cm. long. Connected to the gas washing tube (B) with a 10/30 standard taper glass joint are burets D and E ring-sealed on a 50/50 standard taper glass joint (F). Buret D has a 35-ml. capacity which is graduated into tenths, and buret E has a capacity of 5 ml. Both stopcocks b and c have a 2-ram. bore. The tips of both burets should go below the 50/50 joint. Buret E has a driptip with a 4-ram. diameter; D has a regular drawn-out buret tip made from standard 2-ram. capillary. The 50/50 glass joint (F) is now connected to the receiver (C). The top 7 cm. of C has a diameter of 4.5 cm. and is drawn down to 2 cm. for the bottom 9 cm. The gas bubbling tube (d) which has the 10/30 joint is made from standard l-ram, capillary and reaches through the ring seal (F) to within 1 cm. of the bottom of the receiver (C). The outlet tube which is 4 ram. in diameter is about 1 cm. down from the bottom of the 50/50 joint. The suction is applied at this point when the apparatus is all assembled. Procedure. The oxidizing agent, lead tetraacetate, is prepared according to McClenanhan and Hockett, TM and stored in a desiccator over phosphorus pentoxide. When needed, it is weighed out and put into 2 ml. of glacial acetic acid in the reaction tube (A). Two additional milliliters of the reagent is used to wash down the sides of the tube. The slight brown color, due to decomposition of the reagent, has no effect on the reaction as a large excess of the oxidizing agent is used in the reactions. 111 W. S. McClenanhan and R. C. Hockett, J. Am. Chem. Soc. 60, 2061 (1938).

[29.]


545

phthalein h a v e been added, is sucked into the receiver (C) through buret (c). A precipitate of b a r i u m carbonate is formed in the receiver (C). After increasing the r a t e of bubbling, the excess sodium hydroxide is titrated to the phenolphthalein end point, with care to avoid local concentration of acid. (In the case of C14-1abeled compounds, the b a r i u m carbonate m a y be assayed for a c t i v i t y at this point.) T h e yield of carbon TABLE III I~ECOVERY OF CARBON DIOXIDE a

Compound I)-Glucoseb D-Arabinoseb Methyl-I)-glucoside b Methyl-D-arabinoside b D-Glucose phenylosotriazoleb Glycerol ¢ 4,6-Ethylidine-D-glucose ~ Formaldehydeb

Amounts used, Time, g. hr. 0.012 0.025 0. 068 0. 0125 0. 044 0. 062 0. 0343 0. 060

8.0 6.0 4.0 3.0 5.0 4.0 4.0 4.0

CO2 calculated, %

Carbons of sugar as BaCO3

95.3 99 97.7 !)9.5 98.7 99.4 96.5 0.0

1,2,3,4,5 2,3,4,5 3 4 4,5 2 1,2 --

The temperature is maintained at 40 to 42° throughout these reactions. b S. Abraham, J. Am. Chem. Soc. 72, 4050 (1950). c D. Kritchevsky and S. Abraham, Arch. Biochem. and Biophys. 39~ 305 (1952). d S. Abraham, I. L. Chaikoff, and W. Z. Hassid, J. Biol. Chem. 195, 567 (1952). dioxide is then determined b y titration of the precipitated b a r i u m carbonate with 0.1 N hydrochloric acid to the m e t h y l orange end point. W h e n the sample was 0.0120 g. of glucose, the carbon dioxide yield as determined b y this m e t h o d was 95.3% of the theoretical. 110 I n the case of the D-glucose phenylosotriazole, the c o m p o u n d is dissolved in 1 ml. of pyridine and introduced into the reaction tube. W a t e r (0.5 ml.) is added to the reaction t u b e at the beginning of the oxidation, since water is needed to produce the carbon dioxide. Blanks are run before each reaction to determine t h e c o n t a m i n a t i n g carbon dioxide. An alkali absorption column (ascarite) is used to obtain carbon dioxide-free air which is sucked t h r o u g h the whole apparatus. T h e blanks thus obtained are usually negligible. 4. D e t e r m i n a t i o n of C 14 Activity of Individual Glucose Carbons Isolation of Carbon 1 (Fig. 6, Equation I). This m e t h o d is a slight modification of t h a t of Sowden. 1~ One g r a m of crystalline glucose is heated with 10 ml. of 10% h y d r o b r o m i c acid in a sealed t u b e at 130 ° for 112j. c. Sowden, J, Am. Chem. Soc. 71, 3568 (1949).

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[22]

24 hours. The black residue obtained is filtered off and washed with water. Sufficient solid silver sulfate is added to the filtrate to precipitate the unreacted hydrobromic acid and bromide ion. The precipitate is filtered and washed well with water. The filtrate is made acid with 1 N sulfuric acid and steam-distilled; 1500 to 2000 ml. is collected in a period of 3 to 4 hours. The major portion of the formic acid distills over in the first 1000 ml. The distillate is titrated with standard sodium hydroxide to the phenolphthalein end point. The yield of formic acid is approximately 83 %. The formic acid is oxidized to CO2 with mercuric acetate,11~ and the COs collected in sodium hydroxide solution. The carbonate is precipitated by addition of barium chloride, mounted on filter paper, and assayed for radioactivity. 1~° Isolation of Carbons 1 plus 2 (Fig. 6, Equation II). An aqueous solution of 4,6-ethylidine-D-glucose containing 34.3 mg./ml, is prepared and oxidized with lead tetraacetate ~° for 4 hours. The recovery of C02 is about 96 to 97%.

Isolation of Carbon 3, Carbon ~, and Carbons ~ plus 5 (Fig. 6, Equations III, IV, and V). The lead tetraacetate oxidations of methyl glucopyranoside, methyl arabinopyranoside, and D-glucose phenylosotriazole are carried out as previously described. The resulting BaCOa precipitates are mounted on filter paper, TM and C 1~ contents determined. Isolation of Carbon 6 (Fig. 6, Equation VI). A sample of glucose, when oxidized with periodate, yields 5 moles of formic acid and 1 mole of formaldehyde. T M This formaldehyde, which is derived from C6, is isolated and oxidized to COs with KMn04. To 0.18 g. of glucose dissolved in 15 ml. of water is added 15 ml. of 0.37 M NaI04. The solution is allowed to stand overnight at room temperature; by this time the oxidation is about 98% complete. The excess periodate is decomposed by the addition of 30 ml. of 1 N HC1 and enough sodium arsenite reagent to remove all the iodine color from the solution, usually about 40 ml. The solution is made basic to phenol red with NaOH, and distilled. The distillate is collected in an iced container, and the distillation continued until crystals begin to form in the distilling flask. Two grams of KMnO4 is added to the distillate, and the mixture is refluxed while a stream of CO2-free air is passed through the system. The CO2 produced from the reaction is collected in NaOH. After 1 hour of refluxing, the system is flushed out for 20 minutes, and the carbonate precipitated with 2 ml. of 1 M BaCl~ solution. The excess base is titrated lla H. D. Weihe a n d P. B. Jacobs, Ind. Eng. Chem. Anal. Ed. 8, 44 (1936) ; J. D. Reid a n d H. D. Weihe, Ind. Eng. Chem. Anal. Ed. 10, 271 (1938). H~ R. E. Reeves, J. Am. Chem. Soc. 63, 1476 (1941).

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547

to the phenolphthalein end point, and the BaC03 formed is mounted on filter paper and its C 14 content measured. TM Isolation of Carbons 1 through 6 and Carbons 2 through 6. Glucose is oxidized by the wet combustion method of Van Slyke and Folch H5 or by the persulfate method described on page 549, H6 converted to barium carbonate, mounted, and assayed for the activity of the complete hexose molecule. T M Arabinose representing carbons 2 through 6 of the starting glucose, obtained from the decarboxylation of calcium gluconate, is also converted to barium carbonate and assayed for C ~4 in a similar manner. The values for C J4 activity of four of the six glucose carbons, namely 1, 3, 4, and 6, are based on a procedure involving their individual isolation as BaCO3. The values for the other two carbons are determined indirectly, i.e., by difference. The isotopic content of carbon 2 can be obtained in two ways. Since carbons 1 and 3 are measured individually, the C ~4 content of carbon 2 can be obtained either by the lead tetraacetate oxidation of 4,6-ethylidine-D-glucose (a reaction yielding COs derived from carbons 1 and 2) or by the combustion of 2-formyl-4-phenylosotriazole (a reaction yielding COs derived from carbons 1, 2, and 3). This method yields activity measurements for four individual carbon atoms--l, 3, 4, and 6. In addition, values are also obtained for (1) the sum of carbons 1 and 2; (2) the sum of carbons 1, 2, and 3; (3) the sum of carbons 4 and 5; (4) the sum of carbons I through 6; and (5) the sum of carbons 2 through 6. Thus by this method it is possible to determine the activity of each carbon atom of the glucose molecule without assuming that the activity of carbon 3 is equal to that of carbon 4, that of 2 equal to 5, or that of 1 equal to 6.

C. Other Chemical Methods of Glucose Degradation Aronoff and Vernon 1~7 have employed a chemical method for the degradation of glucose based on the oxidation of glucosazone with periodate. However, certain difficulties with this procedure have been noted by Vittorio et al. TM The assumption that carbon 3 is equal to carbon 4, made in order to obtain the activity of all the glucose carbons, is a limitation of the procedure as outlined by Topper and Hastings. t~8 The scheme devised by Bishop H9 utilizes some of the reactions described in the method presented in the previous section. In this method periodate oxidation of glucose phenylosotriazole yields 1 mole of formaldehyde (C-6), 2 moles 11~ D. D. Van Slyke and J. Foleh, J. Biol. Chem. 156, 509 (1940). 1~ J. Katz, S. Abraham, and N. Baker, Anal. Chem. 26, 1503 (1954). 117 S. Aronoff and L. Vernon, Arch. Biochem. 28, 424 (1950). 118 y . Topper and A. B. Hastings, J. Biol. Chem. 179, 1255 (1949). ~19 C. T. Bishop, Science 117, 715 (1953).

548


[22]

of formic acid (C-4+5), and 1 mole of 2-phenyl-4-formylosotriazole ( C - 1 + 2 + 3 ) . On nitration of the latter compound with fuming nitric acid at room temperature, 1~0 it yields 2-(p-nitrophenyl)-4-formylosotriazole, m.p. 136 to 137 °. Alkaline permanganate oxidation T M produces the acid 2-(p-nitrophenyl)-4-carboxyosotriazole, m.p. 236 to 2370,12° which is converted to the silver salt with ammoniacal silver nitrate solution. The silver salt is decarboxylated to yield COs (C-3) and 2-(p-nitrophenyl)osotriazole, m.p. 183 to 184 °12° (C-1+2). Bishop finds that initial nitration is essential to this scheme as the corresponding 2-phenylosotriazole is an oil and is difficult to purify. A new chemical procedure for the determination of the C14-distribu tion in labeled glucose has been described by Boothroyd et al. 1~ By this method analysis may be performed on as little as 2 mM. of glucose. The method is based on the degradation of methyl-a-D-glucopyranoside with periodate. 12~ Oxidation of the glucoside with periodate yields carbon 3 as formic acid, and a dialdehyde which is oxidized with bromine and isolated as the strontium salt of D'-methoxyhydroxymethyldiglycolic acid. The purified salt is then hydrolyzed to a mixture of glyoxylic and glyceric acids. The two-carbon acid (C-142) is isolated from the mixture as the 2,4-dinitrophenylhydrazone and thermally decarboxylated to yield carbon dioxide from carbon 2. From the complete combustion of the hydrazone and the direct determination of carbon 2, the C 14 activity residing in carbon 1 may be determined by difference. Carbon 1 may also be assayed directly by the procedure outlined in the previous section (page 545). The glyceric acid ( C - 4 + 5 + 6 ) is then oxidized by periodate to yield carbon dioxide (C-4), formic acid (C-5), and formaldehyde (C-6). This method can also be applied to other hexoses with minor modifications. Thus, the complete degradation of labeled galactose can be accomplished by first converting it to the methyl-D-galactopyranoside and then oxidizing the glycoside with periodic acid. The subsequent steps wit1 be the same as those given in the procedure of Boothroyd et al., 122 since the removal of carbon 3 as formic acid from the galactose derivative will result in the production of a dialdehyde which is identical with the one resulting from the corresponding methyl glucopyranoside. Methods for Pentose Degradation. Using a similar method to that applied to glucose or galactose, Brown ~4 has assayed the individual i~o H. von Pechmann, Ann. 262, 265 (1891). 121 R. L. Shriner and R. C. Fuson, " T h e Systematic Identification of Organic Compounds," p. 164. John Wiley & Sons, New York, 1945. 122 B. Boothroyd, S. A. Brown, J. A. Thorn, and A. C. Neish, Can. J. Biochem. Physiol. 33, 62 (1955). l~S E. L. Jackson and C. S. Hudson, J. Am. Chem. Soc. 59, 994 (1937). 1~4S. A. Brown, Can. J. Biochem. Physiol. $3, 368 (1955).

550


[22]

persulfate. 128 The former method has been adopted for the oxidation of C14-1abeled compounds by Barker. 1.9 Many modifications of this procedure have appeared in the literature. 13°-13~ This method is limited to the oxidation of dry and nonvolatile compounds; it involves the handling of a highly corrosive fluid, constant attention during combustion, and the use of special apparatus. Although it has been modified for the combustion of certain volatile compounds, TM the procedure and apparatus are too complex for routine laboratory analysis. The persulfate oxidation as described by Osburn and Werkman l~s can be used only for water-soluble compounds; it has been adopted by Weinhouse 133 and Anthony and Long TM for C 14 assay. This method may be employed for the oxidation of volatile as well as nonvolatile compounds with the limitation that the compounds must be water-soluble. ~°° This reaction can be expressed by the following equation: H20 -}- K2S20s--* 2KHS04 -}- 1~O2

Apparatus and Reagents 50-ml. Erlenmeyer flask with a center well (see Fig. 5). Rubber serum cap (see Fig. 5). Siphon (see Fig. 5). Potassium persulfate, reagent-grade. 4 % silver nitrate solution. Standardized sodium hydroxide (C02-free) solution, approximately 1N. Standardized hydrochloric acid solution, approximately 0.2 N. Dilute sulfuric acid. Saturated barium chloride solution.

Procedure. About 500 to 600 mg. of solid potassium persulfate is placed into the main compartment of the flask so that none of the salt is introduced into the center well. This is conveniently performed with a porcelain spoon and a small large-mouthed funnel. The sample (which should yield from 10 to 80 mg. of BaC03) and water are added to the main comll80. L. Osburn and C. H. Werkman, Ind. Eng. Chem. Anal. Ed. 4, 421 (1932). 1,9 It. A. Barker, in "Isotopic Carbon" (Calvin et al., eds.), p. 93. John Wiley & Sons, New York, 1949. is0 A. Lindebaum, J. Schubert, and W. D. Armstrong, Anal. Chem. 20, 1120 (1948). 181j. A. Them and P. Shu, Can. J. Chem. 29, 558 (1951). 13~E. A. Evans and J. L. Huston, Anal. Chem. 24, 1482 (1952). lss S. Weinhouse, in "Isotopic Carbon" (Calvin et al., eds.), p. 94. John Wiley & Sons, New York, 1949. 18, D. S. Anthony and M. V. Long, Oak Ridge Natl. Lab. Rept. 1303 (1952).

[22]


551

p a r t m e n t t o g i v e a v o l u m e of 5 t o 15 ml. T h e flask is t h e n s w i r l e d in o r d e r to p a r t i a l l y d i s s o l v e t h e p e r s u l f a t e . T h i s m i x t u r e c a n b e acidified w i t h a few d r o p s of d i l u t e H2S04 in o r d e r t o l i b e r a t e a n y c a r b o n a t e p r e s e n t in t h e s a m p l e . O n e m i l l i l i t e r of 4 % s i l v e r n i t r a t e s o l u t i o n is a d d e d to t h i s TABLE IV CARBON RECOVERY ¥¢ITH PERSULFATE OXIDATIONa'b Compound Glucose Fructose Inositol Glycerol Sodium acetate Lithium lactate Succinic acid Citric acid Benzoic acid Potassium acid phthalate Ethyl acetoacetate Acetaldehyde Acetaldehyde + 20 mg. NaHSO3 Methanol Ethanol Histidine hydrochloride Thymine Sodium oxalate Zinc lactate Fumaric acid Acetone Adenine hydrochloride

Carbon taken, rag.

Recovery of C()2, %

2.40 2.40 2.40 4.40 2.40 3.60 2.40 2.40 2.82 2.40 1.72 2.80 2 80 2 34 2.52 2.40 2.10

100 97 100 98 99 98 98 100 95 99 98 100 100 94 102 97 96 101 c 97 ° 99 c 98c 80

2.05

All samples were prepared by weighing out analytical reagent-grade compounds and dissolving them in water. Aliquots of these solutions were used throughout The yields were determined by titration of the alkali (after the addition of barium chloride) to the phenolphthalein and the methyl orange-methylene blue end points. Each value is an average of at least two separate determinations. The duplicate titrations agreed within 2%. b j. Katz, S. Abraham, and N. Baker, Anal. Chem. 26, 1503 (1954). c D. S. Anthony and M. V. Long, Oak Ridge Natl. Lab. Rept. 1303 (1952). mixture, and enough standard C02-free sodium hydroxide to absorb the C 0 2 f o r m e d is d e l i v e r e d i n t o t h e c e n t e r well. T h e flask is s t o p p e r e d w i t h t h e r u b b e r cap. T h e vessels a r e e v a c u a t e d b y i n s e r t i n g a 20- or 2 2 - g a g e h y p o d e r m i c n e e d l e w h i c h is c o n n e c t e d t o a v a c u u m line. T h e v a c u u m s e r v e s t o r e m o v e a n y d i s s o l v e d CO2 a n d p r o v i d e s a t i g h t seal for t h e flask d u r i n g c o m b u s t i o n . S e v e r a l s u c h flasks m a y be p r e p a r e d in t h i s w a y .

552


[22]

T h e flasks a r e p l a c e d in a w a t e r b a t h a t 40 to 50 ° a n d b r o u g h t u p t o a b o u t 70 ° in t h e p e r i o d of 15 to 20 m i n u t e s . T h e c o n t e n t s of t h e m a i n well will d a r k e n a n d gas e v o l u t i o n c o m m e n c e . T h e r e a c t i o n p r o c e e d s s m o o t h l y a t 70 t o 75 ° a n d is u s u a l l y c o m p l e t e d w i t h i n 30 m i n u t e s . A t t h e e n d of t h i s p e r i o d t h e d a r k color d i s a p p e a r s a n d t h e s o l u t i o n b e c o m e s w a t e r - c l e a r . A l o n g e r o x i d a t i o n t i m e m a y be r e q u i r e d w i t h s o m e c o m p o u n d s , a n d in s u c h cases t h e flasks s h o u l d be h e a t e d u n t i l t h e m i x t u r e is clear. TABLE V COMPARISON OF VAN SLYKE AND 1)ERSULFATE COMBUSTION OF C 14 COMPOUNDS a,b

Persulfate oxidation

van Slyke oxidation

Barium carbonate, rag.

Specific activity, c.p.m./mg,

Barium carbonate, rag.

Specific activity, c.p.m./mg.

Glucose-C ~4 Glucose-C '4

35.1 37.3

9.1 9.4

37.5 36.8

9.2 9.3

Succinic acid-P.-C I' Succinie acid-2-C '4

18.2 16.4

21.1 20.5

18.3 18.0

21.2 20.7

Blank

0.8

0.8

The glucose was photosynthetically prepared ¢ and was shown to be evenly labeled, d The succinic acid used was labeled with C 14in the 2 position.' Identical aliquots were combusted in each case. The van Slyke combustion was carried out according to Barker,S and all samples after the addition of barium chloride and titration of the excess alkali were mounted on filter paper according to Entenman et al.g and assayed for radioactivity on a conventional end-window counter. b j . Katz, S. Abraham, and N. Baker, Anal. Chem. 26, 1503 (1954). E. W. Putman and W. Z. Hassid, J. Biol. Chem. 196, 749 (1952). S. Abraham, E. W. Putman, and W. Z. Hassid, Arch. Biochem. and Biophys. 41, 61 (1952). * E. C. Jorgenson, J. A. Bassham, M. Calvin, and B. M. Tolbert, J. Am. Chem. Soc. 74, 2418 (1952). s H. A. Barker, in "Isotopic Carbon" (Calvin et al., eds.), p. 93. John Wiley & Sons, New York, 1949. o C. Entenman, S. R. Lcrner, I. L. Chaikoff, and W. G. Dauben, Proc. Soc. Exptl. Biol. Med. 70~ 364 (1949). T h e flasks a r e left for a n a d d i t i o n a l 15 t o 20 m i n u t e s in t h e w a t e r b a t h t o e n s u r e c o m p l e t e CO2 a b s o r p t i o n . T h e flasks r e q u i r e l i t t l e a t t e n t i o n ; h o w e v e r , if t h e t e m p e r a t u r e is p e r m i t t e d t o i n c r e a s e t o o f a s t or exceed 75 ° t h e e v o l u t i o n of O3 m a y b e c o m e t o o v i g o r o u s a n d f o r c e t h e c a p out. I n l i e u of a w a t e r b a t h t h e c o m b u s t i o n m a y also b e c a r r i e d o u t in a n o v e n

[22]


553

initially set at 75 °. The reaction proceeds somewhat slower than in the water bath and requires about 1 ~ hours. After the flasks have been cooled to room temperature, the vacuum is released by the insertion through the rubber cap of a hypodermic needle connected to an ascarite tower. The flasks are opened and the contents of the center well are rapidly and quantitatively transferred, by means of the siphon, into a suitable vessel for titration of the alkaline carbonate solution. Addition of about 1 ml. of saturated barium chloride solution and titration of the excess base with standardized HCI solution affords a measure of completeness of oxidation. An analyst can easily combust and titrate the BaCO3 from over 20 samples in a few hours. The above procedure has been used extensively for the combustion of C14-1actate and C~4-acetate and to some extent for radioactive assay of C~4-ethyl acetoacetate and C14-glycerol with highly satisfactory results. ~00 This method is applicable to a large variety of water-soluble compounds including carbohydrates, organic acids, amino acids, alcohols, and some heterocyclic compounds, as demonstrated in Table IV. It gives satisfactory results with volatile compounds such as acetaldehyde, methanol, and the like. As can be seen from Table IV, the recovery of carbohydrate ranges from 94 to 100% in all compounds tested with the exception of adenine which gives a value of about 80%. The results obtained with C14-glucose and C~4-succinate in a comparative study using the Van Slyke and Folch combustion fluid ~1~and persulfate can be seen in Table V. The radioactive assay of these compounds by these two methods was found to be identical.~°°

III. Isolation of Labeled Carbohydrates from Biological Sources

A. Glucose-C TM 1. As the p-Nitrophenylhydrazone

Principle. This method of glucose isolation from urine is based on the preparation of its p-nitrophenylhydrazone, the removal of the p-nitrophenylhydrazine with benzaldehyde, and the subsequent crystallization of the glucose. ~35 Reagents 10% HgSO4 in 5% sulfuric acid. Barium carbonate. 95% ethanol. IV[ethanol. 135 S. Gurin, A. M. Delluva, and D. W. Wilson, J. Biol. Chem. 171, 101 (1947).

[22]


555

Saturated zinc sulfate solution. Duolite cation exchange resin C-3 anion exchange resin A-4. Phenylhydrazine. Glacial acetic acid. Ether. Ethanol. Procedure. Approximately 4 to 8 ml. of blood is withdrawn from the left ventricle of a rat into a heparinized syringe. Plasma, obtained by centrifugation, is deproteinized with Ba(OH)2 and ZnS04 by the method of Somogyi ~4~ and passed successfully through a Duolite C-3 cation and A-4 anion exchange column. The columns are then washed with 5 to 6 vol. of distilled water to remove all adhering glucose. The combined neutral effluent and washings are concentrated to about 4 to 5 ml., under reduced pressure, and transferred to a 15-ml. centrifuge tube. To this glucose solution, 100 rag. of phenylhydrazine and 186 rag. of glacial acetic acid are added and the mixture heated on the steam bath for 1 hour. Phenylglucosazone crystals usually appear within 15 minutes. The mixture is then cooled, first slowly to room temperature and then to about 4 ° . After centrifugation, the supernatant liquid is removed by decantation, and the crystals washed first with water (to remove electrolytes) and finally twice with ether (to remove aniline and excess phenylhydrazine). The glucosazone is dissolved in 2 to 3 ml. of hot ethanol, filtered, and recrystallized by the addition of water. The golden yellow needles thus obtained are dried in a vacuum oven set at 45 ° for several hours.

3. As the Pentaacetate This procedure as outlined on page 501 consists of forming glucose pentaacetate from the dried urine residue by treatment with pyridine and acetic anhydride. The pentaacetate is recovered in the crystalline form and hydrolyzed by refluxing with 0.1 N H2SO4. The contaminating ionic material is removed with ion exchange resins and the resulting glucose crystallized. 12,28,~43 C~4-Glucose may also be isolated from tissue slice experiments in this way through the use of unlabeled carrier glucose. 139

4. Chromatographically The method used by Katz and Chaikoff 144 for the isolation of glucose as well as a great number of other compounds from tissue slice experiments with C~4-1abeled substrates is a combination of paper chroma14~ M. Somogyi, J. Biol. Chem. 160, 69 (1945). 1~3 M. R. S t e t t e n a n d Y. Topper, J. Biol. Chem. 203, 653 (1953). 144 j . Katz a n d I. L. Chaikoff, J. Biol. Chem. 206, 887 (1953).

556


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t o g r a p h y in conjunction with r a d i o a u t o g r a p h y . T h e bulk of the inorganic salts in tissue extracts is easily r e m o v e d b y an electrolytic desalting procedure without loss of the labeled compounds. 145 This desalted solution is then subjected to two-dimensional c h r o m a t o g r a p h y first in waters a t u r a t e d phenol and then in butanol-acetic acid-water2 48 T h e chromat o g r a m s thus obtained are used to prepare r a d i o a u t o g r a p h s on X - r a y film. With the use of a S t a n d a r d X - r a y illuminator the radioactive areas seen on the negative m a y be located on the p a p e r c h r o m a t o g r a m and thus assayed for C ~4 a c t i v i t y as well as eluted and degraded. 147 B. Ribose-C 14

See Vol. I V [23]. C. Isolation of Conjugated Glucuronides f r o m Urine Principle. Glucuronic acid can be isolated f r o m the urine of a v a r i e t y of experimental animals, chiefly in the f o r m of conjugation products with phenolic substances and steroids. 148-167 T h u s the m e t h o d of isolation a n d purification of the glucuronide depends on the aglucone m o i e t y administered. I n the studies of the precursors of glucuronic acid, P a c k h a m and Butler 16~ h a v e administered Ci4-1abeled substrates to a - n a p h t h o l - t r e a t e d rats and h a v e isolated the uronic acid conjugate. Subsequent hydrolysis and t r e a t m e n t with b a r i u m hydroxide yielded b a r i u m glucuronate. Reagents

a-Naphthol. p-Toluidine hydrochloride. 6 N hydrochloric acid. 14~R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 41, 590 (1947). 14sA. A. Benson, J. A. Bassham, M. Calvin, T. C. Goodale, V. A. Haas, and W. Stepka, J. Am. Chem. Soc. 72, 1710 (1950). 147j. Katz, S. Abraham, R. Hill, and I. L. Chaikoff, J. Biol. Chem. 214, 853 (1955). 148E. H. Mosbaeh and C. G. King, J. Biol. Chem. 185, 491 (1950). 149W. Deiehmann and G. W. Thomas, J. Ind. Hyg. Toxicol. 25, 286 (1943). 150R. T. Williams, "Detoxieation Mechanisms." John Wiley & Sons, New York, 1947. 151H. Meyer and C. Neuberg, Z. physiol. Chem. 29, 256 (1900). 15~M. A. Packham and G. C. Butler, J. Biol. Chem. 194, 349 (1952). 153F. Eisenberg, Jr., and S. Gurin, J. Am. Chem. Soc. 73, 4440 (1951). 1~4E. Fromm and P. Clemens, Z. physiol. Chem. 84, 384 (1901). 15~M. Berenbom, Thesis, University of Toronto, 1947. x56N. E. Artz and E. M. Osman, "Biochemistry of Glucuronic Acid." Academic Press, New York, 1950. 1~ F. Eisenberg, Jr., and S. Gurin, J. Biol. Chem. 195, 317 (1952).

[22]


557

1 N sulfuric acid. Ethyl ether. Ethyl alcohol. Phenolphthalein indicator. Saturated barium hydroxide. Procedure. A suspension of 150 mg. of a-naphthol in water is administered by stomach tube to each of 4 well-fed 200-g. rats. One hour later the rats are given the C~4-1abeled compound, either by intraperitoneal injection or by stomach tube, and immediately placed in a cage fitted with a metal funnel to allow collection of the urine..The rats are supplied with water but are given no food during the following 8 hours during which time the pooled urine sample is collected. The urine is adjusted to pH 8 to 9 and stored overnight at 5 °. It is then filtered and the washed precipitate discarded. The combined urine and water washings are concentrated to about 5 ml. at 50 to 60° in vacuo, neutralized, and mixed with 600 mg. of p-toluidine hydrochloride. After chilling, the crude p-toluidine naphthol glucuronide is collected by filtration, washed with cold water, and dissolved in a minimum of boiling water. The resulting solution is treated with decolorizing carbon, filtered while hot, and then chilled. The white crystals which form are collected by filtration, washed, and dried. The crystals are then converted to naphthol glucuronide by the addition of 6 N hydrochloric acid (1 ml. per 100 rag. of p-toluidine naphthol glucuronide). The naphthol glucuronide is collected by filtration, washed with cold water, dried, and recrystallized, if necessary, from hot water (m.p. 197 to 198°). The glucuronide is hydrolyzed at 100 ° with 1.0 N sulfuric acid (2 ml. per 100 mg.) for 6 hours. After extraction of the a-naphthol with ether, the hydrolysis mixture is made alkaline to phenolphthalein with a saturated barium hydroxide solution. Carbon dioxide gas is then bubbled through the mixture for a few minutes, heated to 100 ° for 10 minutes, filtered, and the precipitate washed with boiling water. The combined filtrate and washings are concentrated to 2 ml. in vacuo and mixed with 100 ml. of ethanol. The barium glucuronate is collected by filtration and washed with alcohol. This barium glucuronate may be converted to the sodium salt by dissolving the precipitate in water and passing the resulting solution through a cation exchange column in the sodium form. Degradation of C ~4 Glucuronic Acid. In order to determine the location and specific activity of C 14 in biosynthetic glucuronic acid derived from administered radioactive precursors, a degradation scheme has been developed by Eisenberg and Gurin 1~3,157which yields a series of fragments sufficient to permit the measurement or calculation of C 1~ in all the

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carbon atoms of glucuronic acid. These workers have isolated the hexuronic acid as its menthol conjugate. Oxidative hydrolysis of methyl glucuronide with Br2 and HBr yields saccharic acid, which when treated with HI04 breaks into 2 moles of formic acid originating from C-3 and C-4. Glyoxylic acid is further degraded by known procedures to C02 and formic acid representing C-1~-6 and C-2-~5 respectively. Methyl methyl glucuronidate on oxidation with HI04 yields a substituted diglycolic aldehyde which on hydrolysis with semicarbizide hydrochloride yields the semicarbazones of glyoxal, representing C-1-~2 and mesoxaldehyde methyl ester, representing C-4-[-5~6. Decarboxylation of the substituted glucuronide yields C-6. T M D. Glycogen-C 14

Methods for the isolation of glycogen-C TM have been presented elsewhere (Vol. III [7]). One of the methods of isolation consists in digestion of the tissue in hot 30% potassium hydroxide solution and precipitation of the crude glycogen with ethanol in the presence of sodium sulfate. The polysaccharide is dissolved in water and dialyzed against distilled water to remove any contaminating amino acids and any other dialyzable substances. The dialyzed starch solution is then filtered into a volumetric flask and diluted to volume with distilled water. Aliquots of this solution may be plated directly on aluminum disks, the water evaporated, and the sample assayed for C14-activity, or they may be oxidized with potassium persulfate ~°° (see page 549) to barium carbonate and counted in any conventional manner.

IV. Miscellaneous C~4-Labeled Carbohydrates Table VI (pp. 558-559) summarizes procedures for the preparation of a number of C14-1abeled carbohydrates.

[23]

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

561

[23] Determination of Isotopic Carbon Patterns in Carbohydrate by Bacterial Fermentation B y I. A. BERNSTEIN 1 and HARLAND G. WOODla

The bacterial fermentation, when applicable, would appear to be the m e t h o d of choice for determining the pattern of tracer carbon in carbohydrates. Available chemical procedures, generally, are more involved, require greater time, and necessitate a larger sample. For example, the level of C ~4 in each carbon of glucose can be determined on as little as 0.5 millimole with Leuconostoc mesenteroides. Before a particular fermentation is used for degrading carbohydrate, the source of each carbon in the products should be ascertained. In addition, one must know the amounts and directions of "cross contamination," if any, which result from the organism's own metabolism. The following fermentations meet these criteria and make possible the determination of the level of isotope in each carbon of D-glucose and D-ribose. I t seems likely that the isotopic carbon patterns of D-galactose, D-xylose, and L-arbinose m a y also be determined in this manner. Other monosaccharides as well as other fermentations could possibly be included, but sufficient critical data do not seem to be available to ensure their validity for the intended purpose.

Fermentation of Glucose by LactobaciUus casei L. casei CH~OHCHOHCHOHCHOHCHOHCHO ----* 2CHaCHOHCOOH (C-l, 6)(C-2, 5)(C-3, 4)

This fermentation was first used to degrade hexose by Wood et al. TM when t h e y determined the distribution of C 13 in glycogen isolated from the livers of rats which had received NaHC13Oa. Lactobacillus casei, A T C C 7769, has since been extensively employed to determine the patterns of C 1~ and C TM in glucose from various sources. The carboxyl carbon of the lactic acid arises from carbons 3 and 4 of the glucose, the carbinol carbon from 2 and 5, and the methyl carbon from 1 and 6. 1Department of Dermatology, Medical School, University of Michigan, Ann Arbor, Michigan. 1, Department of Biochemistry, School of Medicine, Western Reserve University, Cleveland, Ohio. lb H. G. Wood, N. Lifson, and V. Lorber, J. Biol. Chem. 159, 475 (1945).

562


[23]

M a i n t e n a n c e of Cultures

Medium 1% glucose. 0.5% Difco yeast extract. 0.5 % Bacto-tryptone. 0.6% sodium acetate.3H~O. Distilled H20.

Procedure. Ten milliliters of medium is inoculated with several drops of the stock culture and incubated at 37 ° for 24 hours. Cultures are stored at about 5 ° and are transferred every two to three months. P r e p a r a t i o n of B a c t e r i a for F e r m e n t a t i o n of Glucose

Medium. The medium is the same as described above. Procedure. F i f t y milliliters of medium is inoculated with 1 ml. of stock culture and incubated at 37 ° for 24 hours. This entire culture is then added to 1.5 1. of medium in a 2-1. flask and incubated for 48 to 60 hours. The cells are harvested b y centrifugation and washed three times by suspending each time in 20 vol. of distilled H20. The wet weight of cells is determined, and the bacteria are dispersed 2 in 4 vol. (4 ml./g.) of H20 to make a 20% suspension. P r e p a r a t i o n of G l u c o s e for F e r m e n t a t i o n

The sample of glucose to be fermented is dissolved in distilled H20. If a glycogen hydrolyzate is to be used, the H2SO4 should be neutralized to p H 7 with N a O H and the molar concentration of S O 4 - - adjusted so as to be less t h a n 0.06 in the fermentation mixture.lb A nonalcoholic solution of b r o m o t h y m o l blue m a y be used as an internal indicator for the p H adjustment. Fermentation Procedure

Reagents 0.4 M NaHCOs. Glucose sample in H20. Distilled H20.

Procedure. The fermentation is carried out in a 125-m]. W a r b u r g In case it is necessary to prepare the cells the evening prior to use, the cells are not resuspended at this point but are stored as a paste at about 5 °. Before use, they are washed, centrifuged, weighed, and dispersed in H20 as a 20% suspension.

[9.3]


563

respirometer vessel with two side arms. 8 Four milliliters of the 20% cell suspension is divided between the two side arms. Six milliliters of 0.4 M NaHCOa and 1 millimole or less of glucose in 20 ml. of distilled H20 are put in the main compartment. The flask is " g a s s e d " with 100% C02 for 10 to 15 minutes 4 with frequent shaking to facilitate saturation with C02, is closed off from the atmosphere, and is immersed in the water bath at 37 ° . After equilibration, the fermentation is started b y transferring the bacteria to the main c o m p a r t m e n t and is followed manometrically b y the evolution of COs from the buffer resulting from the formation of lactic acid. 6 When the fermentation is finished (theory: 2 moles of COs per mole of glucose fermented), 2 ml. of 2 N H2S04 is added to acidify the medium. Manometric measurements generally indicate 1.8 to 1.9 millimoles of acid formed per millimole of glucose originally present.

Isolation and Purification of Lactic Acid After separation of the cells b y centrifugation, the lactic acid is removed from the supernatant solution (which should be acid to Congo red) b y continuous extraction with ether for 24 hours. About 25 ml. of H20 is initially p u t into the receiver, as this appears to decrease the destruction of the extracted lactic acid by impurities in the ether. The ether in the receiver is removed b y evaporation, and the aqueous solution is concentrated b y distillation to 20 ml. after which 200 ml. is distilled with steam at constant volume to remove any steam-volatile contaminants resulting from the fermentation or introduced with the ether. About 5 % of the lactic acid m a y be lost in the distillate during this procedure. The yield of lactic acid (usually 85% of the a m o u n t of glucose submitted to the bacteria) is determined b y titration with N a O H and with phenolphthalein as indicator. The lactic acid m a y be further purified by a number of procedures including preparation of the guanidine salt 8 and partition chromat o g r a p h y on Celite No. 535. 7 It has been found, however, that additional These vessels may be obtained from E. Machlett and Son, 220 East 23rd Street, New York 10, New York. The side arms have joints to facilitate introduction of materials and venting plugs for better "gassing." 4 If bromothymol blue is present internally, "gassing" may be terminated when the color of the solution changes from greenish blue to yellowish green. For a detailed description of the Warburg respirometer technique see W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques," Burgess Press, Minneapolis, 1949. 6 M. A. Packham and G. C. Butler, J. Biol. Chem. 194, 349 (1952). 7 H. E. Swim and L. O. Krampitz, J. Bacteriol. 67, 419 (1954); H. J. Saz and L. O. Krampitz, ibid. 67, 409 (1954).

564

[23]


purification of the lactic acid is unnecessary when purified glucose is fermented. Purification through Guanidinc Lactate. To prepare the guanidine salt of lactic acid, 2 molar equivalents of guanidine as the sulfate is added to the aqueous solution of sodium lactate and the H20 is removed by lyophilization. The guanidine lactate is extracted from the dried residue with hot absolute alcohol and crystallized. The salt is recrystallized from alcohol until the melting point (162 °) coincides with and is not depressed by mixture with an authentic sample. The free acid is then quantitatively recovered in the effluent when an aqueous solution of the salt is passed through a small column of Dowex 50 (hydrogen form). Purification by Partition Chromatography. Lactic acid may be purified by partition chromatography according to the procedure described in detail on page 563 for the purification of acetic acid. For elution of the lactic acid, however, 150 ml. each of 100% CHC13, 97.5% CHC13-2.5% n-butanol, 95% CHC13-5% n-butanol, and 92.5% CHC13-7.5% nbutanol (each equilibrated with 0.18 N H~SO4) in that order is used. Under the conditions described, the lactic acid first appears after about 75 ml. of the ]ast solvent has been put through the column.

Chemical Degradation of Lactic Acid (see also Vol. IV [22])

Principle. The isotopic level in each carbon of the lactic acid may be determined by a modification of the method of Wood et al. TM involving the following reactions: CHsCHOHCOOH MnO.~ CH3CHO ~- CO2 (C-l)

CH3CHO NaOI) CHI3 ~- HCOONa CHIa

Ag+

"iodic

HNOa

sulfate"

-~ CO - -

* CO2

HCOOH

Hg ++

--' CO2

Reagents Lactic acid sample in H20. MnSO4-HaPO4 reagent. Dissolve 100 g. of MnSO4.4H:O and 25 ml. of 85% H3PO4 in distilled H20 and make to 1 1. Aqueous NaHSO3, prepared fresh immediately prior to use. A 1% solution is used when 0.5 millimole of lactic acid is being degraded, 2% for 0.5 to 1.5 millimoles, and 2.5% for 1.5 to 2.5 millimoles. Acidified 0.25 saturated KMnO4. Add 30 ml. of distilled H20 and 1 ml. of 2 N H2SO4 to 10 ml. of saturated KMnO4. Approximately 2.5 N NaOH (carbonate-free).

[23]


565

0.02 M KS/[nO4 in carbonate-free H20. K2HPO4, solid. 1 N NaOH. Solution chilled to about 2 °. 0.1 N Is. Solution chilled to about 2 °. 2 N HsSO~. 2 M NaAsOs. 1 N acetic acid. 0.3 M mercuric acetate in dilute acetic acid. Dissolve 100 g. of reagent mercuric acetate and 30 ml. of glacial acetic acid in distilled H20 and make to 1 1. Boil gently for 30 minutes to remove any dissolved CO2. 0.2 M AgN03 containing 4 ml. of concentrated HNO3 per 100 ml. Absolute alcohol. Ascarite 8 or other CO2 absorbent. Anhydrous magnesium perchlorate. ZnCls, granular. Dry at 140° for several days prior to use. "Iodic sulfate." This is a mixture of IsO~ and concentrated HsSO~ suspended on silicic acid ~ and prepared as follows: 180 g. of IsOs, 45 g. of silicic acid, and 50 ml. of concentrated HsSO4 are mixed in a mortar and heated for 2.5 hours at 220 ° in a round-bottomed flask while being aerated with dry air brought into the flask with a vacuum pump. The material is adequate for use when it is evenly yellow throughout and must be protected from moisture. For a newer method of preparation see the abstract of Smiley. ~o

Decarboxylation of Lactic Acid. Lactic acid is converted to COs (carboxyl carbon) and acetaldehyde by a modification of the procedure of Friedemann and Kendall 11 in the apparatus 12 shown in Fig. 1. The sample in 75 to 100 ml. of H20, a boiling chip, and 10 ml. (per millimole of lactic acid) of the MnSO4-HsP04 reagent are put in the 200-ml. reaction flask (A) which is attached to the "degradation h e a d " (B). 1~ Bead tower F is charged with 30 ml. of NaHSO:~ (for concentration, 8 A COs absorbent m a r k e t e d b y A r t h u r H. T h o m a s Co., Philadelphia, Pennsylvania. 9 M. Sehultz, Bet. 77B, 484 (1944); Chem. Abstr. 40, 4858 (1947). 10 W. G. Smiley, Nuclear Sci. Abslr. 8, 391 (1949). IL T. E. F r i e d e m a n n a n d A. I. Kendall, J. Biol. Chem. 82, 23 (1929). ~2 The glassware shown m a y be purchased from the Corning Glass Works (Attention: Special A p p a r a t u s Section), Corning, New York. The numbers in Fig. 1 a n d 2 refer to the catalogue n u m b e r s of the pieces. is Instead of the degradation head (B) and r o u n d - b o t t o m e d flask (A), a round-bottomed flask with three necks (M) equipped with a condenser (bulb type) (O), a dropping funnel with stopcock (P), and an inlet tube (N) for aeration m a y be substituted.

566

TECHNIQUES

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ISOTOPE

[23]

STUDIES

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FIG. 1. Degradation train and accessories. Apparatus for decarboxylation of lactic acid: (A) reaction flask, round-bottomed, single-necked, 300 ml.; (B) "degradation head" with condenser (Allihn), dropping funnel, and inlet tube attached; (C) stopcock, double oblique bore; (C r) side view of stopcock (C); (D) drying tube with (a) anhydrous magnesium perchlorate (or CaCl2) and (c) CO2 absorbent (glass wool used at each end of tube and between a and c) ; (F) bead tower, with stopcock, containing glass beads (4 or 5 mm. in diameter); (G) same as (F) but without stopcock; (H) drying tube with anhydrous magnesium perchlorate (glass wool used at each end) and CO2 collector consisting of (I) "bubbler" containing 2.5 N NaOH (carbonate-free) and (J) drying tube with powdered anhydrous magnesium perchlorate (glass wool at each end). For a determination of yield, I and J with open ends covered by rubber "policemen" are weighed as a unit (wire hook shown on J used to mount collector in balance). CO2 collector (K) with coils partially immersed in liquid N2 in Dewar flask (L) may be substituted for I and J. Reaction flask (A) and "degradation head" (B) may be replaced by (M) flask, round-bottomed, three-necked, 300 ml.; (N) adapter for aeration; (0) condenser, Allihn; and (P) dropping funnel with stopcock. Apparatus for formic acid oxidation, same as above except that F and G are omitted. Apparatus for iodoform oxidation, as above except that F and G are replaced by tube (E) containing in order (a) CaC12, (b) ZnC12, (c) CO~ absorbent, (d) anhydrous magnesium perchlorate, (e) "iodic sulfate," and (f) anhydrous magnesium perchlorate (glass wool used at each end and between substances). All ground joints are standard taper 24/40. Designations in parentheses indicate catalog numbers of parts available commercially. 1~ The "degradation head," catalog number A-l, has been modified by replacing the condenser, West, with a condenser, Allihn.

[23]


567

see a b o v e s e c t i o n on r e a g e n t s ) , a n d b e a d t o w e r G w i t h 30 ml. of acidified 0.25 s a t u r a t e d K M n O 4 . D r y i n g t u b e D is filled w i t h A s c a r i t e or o t h e r CO2 absorbent, and tube H with anhydrous magnesium perchlorate. W i t h t h e CO2 c o l l e c t o r ( I or K ) r e m o v e d f r o m t h e t r a i n a n d t h e flask c o n t e n t s r e f l u x i n g g e n t l y , t h e s y s t e m is a e r a t e d ~4 (at a r a t e of a b o u t 3 b u b b l e s p e r s e c o n d ) w i t h c a r b o n a t e - f r e e a i r or 1 0 0 % N2 for 10 t o 15 m i n u t e s t o r e m o v e a n y CO2 p r e s e n t . T h e CO2 collectoV ~ is t h e n i n t r o d u c e d i n t o t h e t r a i n a n d t h e r e a c t i o n is c a r r i e d o u t b y a d d i n g t h e 0.02 M K M n O 4 d r o p w i s e f r o m t h e f u n n e l t o t h e b o i l i n g s o l u t i o n in t h e r e a c t i o n flask u n t i l t h e p u r p l i s h b r o w n color p e r s i s t s for s e v e r a l m i n u t e s . T h i s is f o l l o w e d b y 15 a d d i t i o n a l m i n u t e s of refluxing a n d a e r a t i o n . T h e a c e t a l d e h y d e is constantly ~e b e i n g a e r a t e d i n t o t h e N a H S O u s o l u t i o n w h e r e it is t r a p p e d as t h e b i s u l f i t e a d d i t i o n c o m p o u n d while t h e C 0 2 p a s s e s on t h r o u g h t h e K M n O 4 s c r u b b e r t o t h e CO2 collector. T h e K M n O 4 s c r u b b e r is i n c l u d e d t o p r e v e n t t h e t r a n s f e r of a n y SO2 f r o m t h e N a H S O a s o l u t i o n i n t o t h e CO2 collector. T h e r e c o v e r y of CO2 in t h i s r e a c t i o n is u s u a l l y b e t t e r t h a n 9 5 % , a n d m o r e t h a n 9 0 % of t h e a c e t a l d e h y d e is g e n e r a l l y r e c o v e r e d in t h e N a H S O 3 as d e t e r m i n e d b y t i t r a t i o n w i t h I2. TM Hypoiodite Oxidation of Acetaldehyde. T h e a c e t a l d e h y d e is r e c o v e r e d f r o m t h e N a H S Q s o l u t i o n b y d i s t i l l i n g t h r e e - f o u r t h s of t h e v o l u m e a f t e r t h e a d d i t i o n of 6 g. of solid K2HPO~. A b o u t 10 ml. of H 2 0 is i n i t i a l l y p u t i n t o t h e d i s t i l l a t i o n r e c e i v e r (a 300-ml. d i s t i l l a t i o n flask i m m e r s e d in a n ~4 Carbonate-free air can be passed through the system by applying a positive pressure of air at the start of the train (at tube D) or suction at the distal end of the CO~ collector. The authors prefer the former, since in this case a leak would result in some loss of yield but would not give a lowered specific activity determination as would be the case if a vacuum system were in use and nonisotopic COo. leaked in from the atmosphere. If a vacuum system is being used a 10 to 15-minute aeration should be carried out with the CO.~ collector in the system to check for a blank CO~ yield. If a pressure system is to be used, a simple mercury pressure stabilizer should be connected into the system, before the air enters the train, to "buffer" line pressure changes. If the COs collector is a "coil" immersed in liquid N2 and the CO~. yield is to be determined manometrically in a vacuum system, t00 % N,. rather than air should be used for aeration. Use of air results in a "blank" yield of unknown condensable gas. is The COo. collector may be a "bubbler" containing 2.5 M NaOH (carbonate-free) such as is shown in Fig. 1, or some other type of glass trap containing NaOH, or it may be a "coil" trap (see Fig. 1) partially (one-third to one-half) immersed in liquid N2 or air. Recovery of CO~, as such, with a "coil" is advantageous when the isotopic content is to be determined in the gaseous state, e.g., in the mass spectrometer or in a gas phase radioactivity counter. The COs yield can be determined as an increase in weight of the trap (in the case where NaOH is used) or manometrically. is If the degradation head (B) shown in Fig. 1 is used, the 0.02 M KMnO, should be introduced into flask A by quickly rotating stopcock C 360 °, thus adding a small amount of the reagent and interrupting the aeration for a minimum length of time.

568


[23]

ice bath), and the apparatus is so arranged that the distillate is delivered directly into this liquid to minimize loss of acetaldehyde. The iodoform reaction is carried out on the acetaldehyde (at a concentration no greater than 1 millimole per 100 ml.) in the receiver (still in the ice bath) by adding (per millimole of lactic acid degraded) 25 ml. of cold 1 N NaOH at once and, while the solution is being mechanically stirred, 70 ml. of cold 0.1 N I2 dropwise. The mixture is kept at about 2 ° for 2 to 14 hours after which 2 N H2S04 is added dropwise with stirring until the color of I2 appears. The I~ is destroyed with 2 M NaAsO~. The solution is then further acidified, if necessary, to Congo red, to make certain that all the I~ has been reacted. At this point, if the CHI3 is allowed to settle, the solution should be colorless or slightly yellow. The CHI3 may be separated from the supernatant solution (containing the HCOOH) by filtration through an asbestos pad on a Gooch crucible (and washed with H~O) or by removing the supernatant solution through a filter stick having an asbestos plug (and washing the remaining precipitate three times with 10 ml. of H20). The latter method generally gives a better yield when a small amount of CHI3 is involved, since the asbestos plug can be returned to the reaction flask and the subsequent oxidation of the CHI3 to CO~ can be carried out in the same flask without transfer. Any CHI~ on the filter stick is washed back into the flask with 10 ml. of absolute alcohol. Oxidation of Formic Acid (cf. volume III, p. 285). The supernatant solution of the CHI~ is neutralized with NaOH to phenolphthalein and evaporated to about 15 ml. The concentrated solution (with any salt which may have precipitated) is transferred to a steam distillation flask, acidified with H2SO4 to Congo red, and 10 to 15 vol. steam distilled. If the distillate shows traces of I~, several drops of 2 M NaAsO2 are added. The HCOOH (a-carbon) in the distillate is then oxidized to CO~ in a modification of the apparatus shown in Fig. 1 by the specific method 1~of Weike and Jacobs. TM The distillate, a boiling chip, and 5 ml. of 1 N acetic acid are placed in a 300-ml. reaction flask (A) which is attached to the "degradation head" (B). 13 The apparatus is the same as shown in Fig. 1 except that bead towers F and G are omitted and the top of the condenser on B is attached directly to drying tube H. With the collector absent from the train, the solution is refluxed and aerated with CO2-free air or N~ for 10 minutes to remove any CO~ present. The COs collector is then added to the system, and 40 ml. of the mercuric acetate reagent is added. Boiling and aeration are continued for 20 minutes, and the CO~ yield is 17 O. L. Osburn, H. G. Wood, a n d C. H. W e r k m a n , Ind. Eng. Chem. Anal. Ed. 5, 247 (1933). is H. D. Weihe a n d P. B. Jacobs, Ind. Eng. Chem. Anal. Ed. 8, 44 (1936).

[23]


569

determined. To determine whether the recovery has been complete, a further 10-minute collection period is made. T h e over-all yield of COs f r o m the a - c a r b o n of the lactic acid is a b o u t 50 %. Oxidation of Iodoform. T h e C H I 3 (E-carbon) is selectively oxidized to CO~ in the a p p a r a t u s shown in Fig. 1 as described b y Shreeve et al. 19 T h e reaction flask containing the sample in 50 ml. of 2 0 % alcohol is incorporated into the train shown in the figure. 13 T h e center t u b e of the " d e g r a d a t i o n h e a d " should extend u n d e r the solution in the flask. Bead towers F and G and t u b e H are replaced with t u b e E 2° containing, in order, CaC12 (a), ZnC12 (b), Asearite (c), a n h y d r o u s m a g n e s i u m perchlorate (d), "iodic s u l f a t e " (e), and m a g n e s i u m perchlorate (f). I n the reaction flask the CHI3 is converted to CO ~1 which is converted to COs on passage through the "iodic sulfate. ''22 With the CO~. collector out of the system, the train is aerated for 5 minutes to clear the COs from the a p p a r a t u s beyond the Ascarite (c). The collector is then put in and the mixture is refluxed gently. If the solution is boiled or aerated too vigorously some of the C H I 3 will pass b e y o n d the condenser and be lost. With the boiling and aerating continuing, 45 ml. of the acidified 0.2 M AgN03 (per millimole of lactic acid initially present) is added and the COs is collected over a 1-hour period or until there is no further recovery. The yield of CO2 for the E-carbon of lactic acid is a b o u t 50%. The specificity of this reaction procedure appears to eliminate the need for purification of the CHI3. ~9,2a Reliability of This Procedure. T h e validity of this procedure for the degradation of lactic acid is shown b y the d a t a in Table I. I n all reactions in which COs is collected, care m u s t be t a k e n to remove all extraneous COs present in the reaction mixture or in the train prior to starting the intended reaction. I t seems advisable to determine b l a n k COs values b o t h before and after the reaction to eliminate the possibility of u n k n o w n dilution of the COs sample. T h e a p p a r a t u s and reagents should be tested periodically b y determining the COs from a b l a n k run. 19W. W. Shreeve, F. W. Leaver, and I. Siegel, J. Am. Chem. Soc. 74, 2404 (1952). 2oGlass wool (not cotton) is used to separate the substances. A tube of 12-ram. inner diameter may be used to contain all the substances in the train--each occupying ~bout 10 cm. of the length. The CaC12 and ZnC12 are used to prevent any alcohol from passing on in the tube. If the alcohol is not adequately trapped, blank CO2 values will be obtained. 21 Any CO2 which may arise in the reaction flask is trapped by the Ascarite (c) prior to the conversion of the CO to CO2. ~ The part of the "iodic sulfate" which is used up changes from yellow to brown so that a tube containing the material may be used for many oxidations and need be replaced only when most of the reagent is brown. ~a I. A. Bernstein, K. Lentz, M. Maim, P. Schambye, and H. G. Wood, J. Biol. Chem. 215, 137 (1955).

570

[23]

TECHI~IQUES FOR ISOTOPE STUDIES

Other Methods for Degrading Lactic Acid (see Vol. IV [22]). Lactic acid m a y also be degraded by the procedure of Roseman. 2~ B y this method the acid is condensed with o-phenylenediamine to form the 2-(a-hydroxyethyl)benzimidazole which is purified by recrystallization. The derivative is treated with N a O I to give CHI~ representing the B-carbon of lactic acid. Another aliquot of the derivative is oxidized with K1V[nO4 to give 2-benzimidazolecarboxylic acid. This acid is decarboxylated at 190 ° to give CO~ (a-carbon of lactic acid) and benzimidazole (carboxyl carbon). T h e CHIs and benzimidazole, after suitable purification, are burned to C02 for isotopic analysis.

TABLE I ISOTOPIC DATA ON THE DEGRADATION OF SYNTHETIC LACTIC ACID-C 14 BY THE I~IETHOD OF WOOD e~ a l . a

Atoms, % excess or c.p.m./mfllimole of carbon Type of labeling

Isotope

CH~

CHOH

COOH

C14H3CISHOHCOOHb

C I~ C 14

0.00 28,600

1.34 25

0.00 0

CHsCHOHC14OOH c

Cl4

525

50

105,000

" H. G. Wood, N. Lifson, and V. Lorber, J. Biol. Chem. 159, 475 (1945). b V. Lorber, N. Lifson, H. G. Wood, W. Sakami, and W. W. Shreeve. J. Biol. Chem. 183, 517 (1950). c I. A. Bernstein, unpublished data. Using synthetic lactic acid-l-C ~4 and lactic acid-2-C 14 to check the validity of this method, Roseman found essentially po "cross contaminat i o n " of C 14 during the degradations. The yields reported for this m e t h o d are lower t h a n those generally obtainable b y the m e t h o d of Wood et al. Aronoff et al. 25 reported the oxidation of lactic acid b y CrOs to CO2 (carboxyl carbon) and acetic acid. The barium salt of this acid was then pyrolyzed to BaCOs (a-carbon) and acetone. T r e a t m e n t of the acetone with N a O I yielded CHI3 (B-carbon). Reliability of the Lactobacillus casei M e t h o d The data shown in Table I I allow an evaluation of the validity of this technique for determining the p a t t e r n of isotopic carbon in glucose. I t is 24S. Roseman, J. Am. Chem. Soc. 75, 3854 (1953). 35S. Aronoff, H. A. Barker, and M. Calvin, J. Biol. Chem. 169, 459 (1947) ; M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwick, "Isotopic Carbon," p. 250. John Wiley & Sons, New York, 1949.

[23]

571


seen t h a t , a l t h o u g h t h e m e t h o d m a y r e s u l t i n q u a n t i t a t i v e i n a c c u r a c i e s , it gives a r e a s o n a b l y t r u e p i c t u r e of t h e over-all p a t t e r n . TABLE II COMPARISON OF C 14 DISTRIBUTION IN GLUCOSE DETERMINED BY CHEMICAL AND L. casei DEGRADATION

Chemical degradation

L. casei fermentation

Type of labeling Glucose_l_C,, ~.d.~ Glucose-34-C ~4d

C-3,4 C-2,5 C-1,6 9.7 361

0 7.7

294 12.2

Glucose_3,4_C,4 I.g 2560 Glucose-C ~4 (differential labeling)1.g4140

57

49

Glucose_C,4 i h

28

6920 28

C-1

C-2~

70

1290

1670

1850 1970b 7280

5950

5550

428

71b

C-2,3,4,5 b C-3,4,5 ~ C-6

815i

20

20

33 ~ 21()0~* 34

By degradation of glucobenzimidazole. b By NaIO4 oxidation of gluconate. c Synthetic sample. d Isotopic data given in millimicrocuries per milligram of carbon. M. Gibbs, R. Dumrose, F. A. Bennett, and M. R. Bubek, J. Biol. Chem. 184, 545 (1950). i Isotopic data given in counts per minute per millimole of C02. g I. A. Bernstein, K. Lentz, M. Malta, P. Schambye, and H. G. Wood, J. Biol. Chem. 215, 137 (1955). h M. Cook and V. Lorber, J. Biol. Chem. 199, 1 (1952). i By HBr oxidation of glucose.

Applicability to Other Hexoses S c h a m b y e et al. 26 h a v e r e p o r t e d t h e d e g r a d a t i o n of g a l a c t o s e - C ~4 b y this p r o c e d u r e u s i n g cells a d a p t e d to f e r m e n t this hexose b y g r o w t h on all e q u a l m i x t u r e of D-galactose a n d D-glucose. F e r m e n t a t i o n of Glucose by Leuconostoc mesenteroides L. mesenteroides

CH2OHCHOHCHOHCHOHCHOHCHO CHaCHOHCOOH + HOCH2CH8 + CQ (C-6) (C-5)

(C-4)

(C-3)

(C-2)

(C-1)

T h i s d e g r a d a t i o n of glucose yields each c a r b o n s e p a r a t e l y - - a n a d v a n t a g e over t h e f e r m e n t a t i o n w i t h L. casei. D e M o s s et al. 27 r e p o r t e d 2s p. Schambye, It. G. Wood, and M. Kleiber, J. Biol. Chem. 226, in press (1957). 27 R. D. DeMoss, R. C. Bard, and I. C. Gunsalus, J. Bacteriol. 62, 499 (1951).

[9,3]


573

and are transferred at least every month. The cultures may also be maintained in 0.5% agar using the medium given below for growth of the culture. Preparation of Bacteria for Fermentation of Glucose Medium

1% glucose. The glucose is autoclaved separately as a 10 % solution. 1% tryptone. 1% Difco yeast extract. 0.5% K2HPO~. 10% (by volume) tomato juice. 2% (by volume) salt mixture (autoclaved separately), consisting of 0.96% MgSO4.7H20, 0.04% NaC1, 0.072% FeSO4.7H~O, 0.18% MnSO4-H20, and 0.002% ascorbic acid. Distilled H20. The broth (except the salt mixture and glucose) is autoclaved for 20 minutes, the denatured protein is removed by centrifugation and/or filtration, and the clear supernatant solution is sterilized by autoclaving for 20 minutes. The salts and glucose are added aseptically just prior to inoculation. Procedure. Ten milliliters of the complete medium is inoculated from a stab culture and incubated at 30 ° until "gassing" perfusely (about 24 hours). Successive subcultures are made into 10 ml. of broth using a drop of inoculum until "gassing" occurs at 10 to 12 hours. Cultures are then successively transferred into 100 ml. and 1 1. of medium, using 1 ml. and 10 ml. of inoculum, respectively. Each culture is incubated at 30 ° until the evolution of gas is obvious (8 to 12 hours). When the large culture has begun to " g a s " strongly, the cells are removed by centrifugation, washed twice with distilled H~O, resuspended in distilled H20 (4 ml./g, wet weight of cells), and used immediately. Occasionally inactive ceils have been obtained when harvesting has been delayed too long (cf. Altermatt et al. 31 for a discussion of this problem). Preparation of Glucose for Fermentation The sample of glucose to be degraded is prepared as described on page 562. Fermentation Procedure Reagents

1 M phosphate buffer, pH 6.0. 2.5 M NaOH (carbonate-free).

574


[23]

Glucose sample in HsO. Distilled HsO.

Procedure. The fermentation is carried out in a 125-ml. Warburg respirometer vessel 3 with two side arms into which is put a total of 4 ml. of the cell suspension. Fifteen milliliters of 1 M phosphate buffer and 1 millimole of glucose in 11 ml. of distilled HsO are put in the main compartment. Two milliliters of 2.5 M NaOH (carbonate-free) is placed in the center well to absorb the evolved COs. A filter paper " w i c k " is immersed in the NaOH solution to facilitate rapid absorption of COs. A small vessel containing one-tenth the quantities of each component except the NaOH is run simultaneously to observe the progress of the fermentation manometrically~ (by C02 evolution). The vessels are "gassed" with 100% Ns (COs-free) for 15 minutes, closed off from the atmosphere, and equilibrated in the bath at 30 ° . The bacteria are then transferred to the main compartment. When the evolution of CO2 has ceased in the control flask, 3s the NaOH-NaHC03 solution and the filter paper from the center well in the large flask are removed and put in a stoppered container and the cells are separated from the supernatant solution by centrifugation. The fermentation may also be carried out in a 100 ml. round-bottomed, two-necked flask equipped with two gas inlet tubes one of which is connected to a "bubbler" (as shown in Fig. 1) containing 2.5 M NaOH (carbonate-free). The equipment is set up on a shaker (e.g. the Warburg apparatus) with the flask immersed in a water bath at 30 °. The sugar and phosphate solutions are put into the flask and the flask is "gassed" as above with Ns ( C O j r e e ) . One of the inlet tubes is then removed and the bacterial suspension is quickly introduced. "Gassing" is continued while the fermentation is in progress in order to carry the C02 over into the "bubbler." The progress of the fermentation is followed by weighing the bubbler periodically. Isolation and Purification of the Fermentation Products

Carbon Dioxide. The CO2 (carbon 1 of the glucose) is recovered from the NaOH solution and filter paper " w i c k " by acidification with HC103 or lactic acid in a closed system or by the addition of BaCI~, depending on the method to be used for determination of radioactivity. The recovery is about 90 % based on the amount of glucose present at the start of the fermentation. Ethanol. The fermentation supernatant is neutralized to phenol red s~ T h e C02 yield as m e a s u r e d manometrieally in the control vessel is a p p a r e n t l y not a measure of the actual CO~ yield in the large flask.

[23]


575

(added internally as an aqueous solution) with N a 0 H and three-fourths the volume distilled to recover the ethanol (carbons 2 and 3 of the hexose). The ethanol in the distillate is oxidized to acetic acid in a glass-stoppered (held in place by rubber bands), round-bottomed flask by adding 15 ml. of a potassium dichromate solution 3~ and heating in a boiling H20 bath for 30 minutes. The acetic acid is recovered by concentration of the mixture to 15 to 20 ml. by distillation and steam distillation at constant volume of 10 to 15 vol. After neutralization of the acid in the distillate with NaOH, the solution is evaporated to dryness and the acetic acid is purified by partition chromatography on a column of Celite No. 53534 with mixtures of CHC13 and n-butanol.m5 A glass tube 20 mm. in diameter containing 15 g. of ether-washed, air-dried Celite No. 535 is used to purify not more than 1.0 millimole of the acid. The Celite is thoroughly mixed (but not ground) with 7.5 ml. of 0.18 N H2SO~. Enough CHC13 (equilibrated with 0.18 N H2SO,) 38 is added to make a slurry, and the material is put in the tube in small portions--each being packed well with a glass tamper. After all the Celite has been put into the tube, the column is further packed under positive air pressure, with care being taken not to allow the column to become dry. The dried sample of sodium acetate is dissolved in 1 ml. of H20, and, with 1 drop of thymol blue as an internal indicator, the solution is acidified with 2 N H2SO4 to a definite pink color. Two grams of Celite is added with mixing, and the damp mixture is put on top of the column and tamped. Glass wool is used to transfer any residual solid to the column and is put in the tube and pushed down on top of the column. Then 150 ml. of 100% CHC13 (equilibrated with 0.18 N H2SO,) 36 is passed through the column, being collected in 15-ml. portions. To each aliquot of eluate, 1 drop of 0.1% soluble phenol red in H20 and about 5 ml. of H20 (carbonate-free) are added. The tubes are vigorously shaken to facilitate extraction of the organic acid into the aqueous layer. The extracted acid is titrated with 0.01 N NaOH (carbonate-free). Acetic acid first appears in the tenth tube under these conditions. The eluting solvent is then changed to 97.5% CHC13-2.5% n-butanol (equilibrated with 0.18 N H2SO,), 36 and collection of 15-ml. aliquots is continued. The titrations are then carried out with 0.05 M NaOH (carbonate-free). Recoveries are 3~ 134 g. of K~Cr~O7 4- 675 ml. of 10 N H2SO4 diluted to 1 1. 34 A product of the Johns Manville Co. ~ C. S. Marvel and R. D. Rands, Jr., J. Am. Chem. Soc. 72, 2642 (1950). Some details of the procedure are modifications of E. Swim and K. Lentz. 3~ The organic phase is passed through W h a t m a n No. 1 filter paper on a gravity funnel to remove suspended droplets of H~O.

576


[23]

80 % or better, based on the a m o u n t of glucose present at the start of the fermentation. After separation of the aqueous phase and removal of residual CHC18 and butanol b y aeration, the solution is evaporated to 10 to 15 ml., acidified to Congo red with H2S04, and the acetic acid recovered by steam distillation (at constant volume) of 10 to 15 vol. Barium acetate is formed b y titration with 0.1 N Ba(OH)2 (carbonate-free) to bromothymol blue. 87 The solution is then evaporated to dryness in a porcelain boat or directly into tube C of Fig. 2.

C(C.20)

J

•IceI~ Fro. 2. Pyrolysis apparatus. (A) receiver, round-bottomed, 300 ml., containing 20 ml. of H~O and immersed in an ice bath; (B) adapter with vacuum take-off and stopcock; (C) pyrolysis tube; (D) porcelain boat containing dried barium acetate; (E) electrical combustion furnace with thermostatic control, and (F) thermocouple. Designations in parentheses indicate catalog numbers of parts available commercially.l~ All ground joints are standard taper 24/40.

Lactic Acid. T h e residue from the above 3~ vol. distillation is acidified with H2S04 to Congo red and the lactic acid (carbons 4, 5, and 6 of the glucose) contained therein is recovered by continuous ether extraction as described on page 563. Chemical Degradation of Acetic Acid (see also Vol. IV [22])

Principle. The isotopic content of each carbon of the acetic acid can be determined b y the following reactions: 3~This end point is difficult to see, but titration to about pH 7 is preferable to a more alkaline pH, since there is less likelihood that carbonate will be titrated. Any BaCOs formed at this stage from unlabeled carbonate will dilute the BaC1403 formed in the pyrolysis of the barium acetate.

578


[23]

thetically prepared acetic acid-l-C 14 containing 3.1 × 106 c.p.m./ millimole of compound was degraded by the above procedure, 0.17% of the activity appeared in the CHI3 (a-carbon). Similarly, 0.88% of the activity of the molecule appeared in the BaCO3 (carboxyl carbon) when acetic acid-2-C 14 containing 5.7 X 106 c.p.m./millimole of compound was analyzed. Nevertheless it should be noted that randomization of isotope has been observed in the pyrolysis of barium acetate by others 39possibly using somewhat different procedures from those described above. Other Procedures. Other procedures by which acetic acid may be degraded include (1) decarboxylation by the Schmidt reaction and oxidation of the resulting amine to CO24o and (2) formation of 2-methylbenzimidazole, its conversion to the 2-benzimidazolecarboxylic acid, and the decarboxylation of this acid. 2~ Both procedures are reported not to result in significant "cross contamination" of isotope. In the former procedure, the dried sample of sodium acetate, NAN3, and 100% H2SO4 are warmed together to give COs (from the carboxyl carbon) which is removed by aeration with air or N2 (COs-free) and methylamine (from the methyl carbon). The reaction mixture is then brought to pH 11 to 12 with NaOH and the amine recovered by aeration into dilute H2SO4. The methylamine sulfate is neutralized and oxidized to COs with alkaline KMn04. In the second method, acetic acid is condensed with o-phenylenediamine to form 2-methylbenzimidazole which is isolated. This compound is condensed with benzaldehyde, and the resulting derivative is oxidized with KMn04 to the 2-benzimidazolecarboxylic acid. This acid is heated at 190° to give COs (methyl carbon of the acetic acid) and benzimidazole (earboxyl carbon) which is burned to C02. Chemical Degradation of Lactic Acid Lactic acid is degraded as described above and also in Vol. IV [22]. Reliability of the Leuconostoc mesenteroides Procedure Tables III and IV present data which demonstrate the reliability of the method. The entire procedure appears to give results with an error no greater than 2 to 3%. Applicability to Other Hexoses Schambye et al. ~6 used this technique to degrade galactose-C ~4. The cells were adapted to ferment galactose by being grown on 10 ml. of 39 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," p. 248. John Wiley & Sons, New York, 1949. 40 E. F. Phares, Arch. Biochem. and Biophys. 85, 173 (1951).

[23]

ISOTOPIC

CARBON P A T T E R N S

IN CARBOHYDRATE

wz wz ;2

o0

[-.,.

¢q

(b

g~

~d O

L)

©

z

%

O

579

580


[23]

glucose m e d i u m until " g a s s i n g " occurred in 12 hours followed b y subculturing on 10-ml." aliquots of m e d i u m containing galactose instead of glucose until " g a s s i n g " was again observed in 12 hours. At this point the cells were p r e p a r e d as usual for f e r m e n t a t i o n in 100 and 1000 ml. of m e d i u m containing galactose as the carbohydrate. TABLE IV ANALYSIS OF GLUCOSE-1-C14 RY FERMENTATION WITH L. meser~teroidesa S. A.b of products formed S. A.b of glucose added

CO~ (C-l)

9.6 9.6

57 56

Ethanol (C-2,3) Lactate (C-4,5,6) 0.01 0

0 0.02

° I. C. Gunsalus and M. Gibbs, J. Biol. Chem. 194, 871 (1952). b S. A. -- millimierocuries per milligram of carbon (specific activity). Although known isotopic glucosamine has not as y e t been degraded, f e r m e n t a t i o n of unlabeled glucosamine b y cells a d a p t e d to this comp o u n d 41 yielded C02, ethanol, and lactic acid in the usual quantities. 42

Fermentation of Ribose by Lactobacillu$ S e n t o s u s CH2OHCHOHCHOHCHOHCHO

L. pentosus

* CH~CHOHCOOH ~ HOOCCH3 (C-5) (C-4) (C-3) (C-2) (C-1)

F r e d et al. 48 showed t h a t Lactobacillus pentosus 124-2 produced 1 mole each of acetic acid and lactic acid per mole of pentose utilized. L a m p e n et al. 44 found essentially all the isotope in the m e t h y l group of the acetic acid when xylose-l-C I4 was fermented. 45 Gest and L a m p e n 4e p o s t u l a t e d t h a t this organism cleaves pentose between carbons 2 and 3 so t h a t the source of the acids would be as shown above. Bernstein 4' c o m p a r e d the p a t t e r n s of C 14 found in samples of ribose b y chemical m e t h o d s with those obtained b y f e r m e n t a t i o n with this organism and concluded t h a t the 4x Glucosamine hydrochloride was substituted for glucose in the growth procedure. 4~I. A. Bernstein, unpublished data. 43 E. B. Fred, W. H. Peterson, and J. A. Andrews, J. Biol. Chem. 48, 385 (1921). 44j. O. Lampen, H. Gest, and J. C. Sowden, J. Bacteriol. 61, 97 (1951). ,5 D. A. Rappoport, H. A. Barker, and W. Z. Hassid [Arch. Biochem. and Biophys. $1, 326 (1951)] obtained similar results in the fermentation of L-arabinose-l-C TM with LactobaciUus pentoaceticus as did Altermatt et al. 31 with Leuconostoc mesenteroides fermenting D-xylose-l-C 14. 46H. Gest and J. O. Lampen, J. Biol. Chem. 194, 555 (1952). ~ I. A. Bernstein, J. Biol. Chem. 205, 309 (1953).

[23]


581

sources of the carbons in the products were as Gest and Lampen had postulated and that this fermentation could be used to determine the pattern of tracer carbon in ribose.

Maintenance of Cultures

Medium 1% xylose. The xylose is autoclaved separately as a 10% solution and is added aseptically before the medium hardens. 0.4% Difco yeast extract. 1% Difco nutrient broth. 1.6 % sodium acetate.3H20. 2% agar. 1% (by volume) salt mixture, consisting of 2% MgSO~-7H20, 0.1% NaC1, 0.1% FeSO4.7H~O, and 0.1% MnSO4.4H20. Distilled H~O.

Procedure. The agar medium is inoculated by a stab from a previous culture and incubated for 24 hours at 37 ° . Cultures are stored at about 2° and transferred at least once a month. Preparation of Bacteria for Fermentation of Ribose

Medium. The bacteria are grown for fermentation on the same medium (prepared in the same way) as is used for maintenance of the cultures except that no agar is used and the first transfer is made into a medium containing glucose instead of xylose. Procedure. Five milliliters of medium containing 1% glucose as carbon source is inoculated from a stock culture and incubated at 37 ° for 24 hours. Two drops of this culture is used to inoculate 10 ml. of medium containing 1% xylose. After 24 hours at 37 °, this entire culture is added to 100 ml. of "xylose" medium. The new culture is allowed to grow for 12 hours at which time it is added to 3 1. of medium containing xylose. The cells are harvested by centrifugation after 36 to 40 hours and are washed twice with 40 ml. of 1% KC1 and twice with the same amount of distilled H20. The wet cell weight is determined, and the cells are suspended in 0.1 M NaHCO3 (4 ml./g.) to make a 20% suspension. Preparation of Ribose for Fermentation The sample of ribose to be fermented is dissolved in distilled H20.

Fermentation Procedure

Reagents 0.1 M NaHC03.

582


[23]

Ribose sample in H20. Distilled HsO. Procedure. The fermentation is carried out in a 125-ml. Warburg respirometer vessel with two side arms.S Five milliliters of cell suspension in 0.1 M NaHCO~ is divided between the two side arms. Then 7.5 ml. of 0.1 M NaHC03 and 0.5 millimole of ribose in 17.5 ml. of HsO are put in the main compartment. A separate vessel is prepared in a similar manner without the ribose to measure the endogenous metabolism of the cells. 48 The vessels are "gassed" for 15 minutes with 5% CO2--90% Ns, closed, and equilibrated for temperature in the bath at 37 ° . The bacteria are then transferred to the main compartment, and the fermentation is followed manometrically by the evolution of COs from the buffer resulting from acid formation 5 (theory: 2 moles of COs evolved per mole of ribose utilized). When no more COs is being evolved, 2 ml. of 2 N H~SO, is added to acidify the medium. I s o l a t i o n and Purification of F e r m e n t a t i o n A c i d s

Acetic Acid. The bacteria are removed by centrifugation and the acetic acid is recovered by concentrating (by distillation) the supernatant solution (acid to Congo red) to about 15 ml. and then distilling with steam at constant volume until a total of 15 vol. of distillate is obtained. The distillate is titrated with NaOH to phenolphthalein and evaporated to about 15 ml. The acid is then purified as described on page 575. Lactic Acid. The lactic acid is recovered by continuous ether extraction from the combined steam distillation residues as described on page 563. C h e m i c a l D e g r a d a t i o n s of A c e t i c Acid and Lactic Acid

Acetic acid may be degraded as described on page 576, and lactic acid as on page 564. R e l i a b i l i t y of the

Lactobacillus pentosus P r o c e d u r e

The validity of the procedure is shown by the comparative data given in Table V. The C ~ patterns of labeled ribose samples were determined by this procedure and compared with data obtained by chemical methods only. When the values for carbons 1 and 2 by the fermentation were 48 The specific activity of the acetate formed from labeled ribose in the fermentation is corrected for the endogenous production of acetate as measured manometrically in this vessel. Gest and Lampen 4s report the endogenous metabolism of this organism to result almost entirely in the production of acetate.

584


[24]

The cells were grown on a medium containing 0.5 % glucose and 0.5% xylose. Although the fermentation was carried out and the products isolated and degraded by techniques different from those discussed in this article, it seems likely that the detailed procedures given here could be conveniently adapted to this fermentation if desired.

[24] Isotopic Experimentation with Intermediates of the Tricarboxylic Acid Cycle By H. E. SWIM and M. F. UTTER I. General Considerations The objective of this report is to discuss certain procedures applicable to isotopic tracer experiments with intermediates of the tricarboxylic acid cycle and the related compounds, lactic and acetic acids. Primary attention is directed toward procedures which appear to be particularly well suited for tracer experiments, and no attempt is made to review the voluminous literature on biochemical methodology of this group of compounds. We have attempted to select methods which seem generally useful in tracer experiments, but the choice is necessarily somewhat arbitrary and many methods of equal merit have undoubtedly been omitted. The rationale of isotopic tracer experiments is quite straightforward: the presentation of appropriately labeled compounds to a biological system followed by the extraction, separation, and degradation of suspected intermediates and end products. For reasons of convenience the discussion of methodology relating to tracer experiments with compounds of the tricarboxylic acid cycle has been divided into four sections: extraction and separation, determination, degradation, and synthesis. The coverage of these four topics is by no means uniform. Only the first of these, the extraction and separation of tricarboxylic acid cycle intermediates, is dealt with in the detail comparable to that usually achieved in these volumes. The presentation of other aspects of isotopic methodology departs somewhat from the usual format in that it has not been possible in most cases to provide complete details. The discussion is directed toward correlating diverse procedures with respect to their applicability and limitations with only an outline presentation of actual methods in most instances. Additional details can be obtained from the appropriate references which are cited.

584


[24]

The cells were grown on a medium containing 0.5 % glucose and 0.5% xylose. Although the fermentation was carried out and the products isolated and degraded by techniques different from those discussed in this article, it seems likely that the detailed procedures given here could be conveniently adapted to this fermentation if desired.

[24] Isotopic Experimentation with Intermediates of the Tricarboxylic Acid Cycle By H. E. SWIM and M. F. UTTER I. General Considerations The objective of this report is to discuss certain procedures applicable to isotopic tracer experiments with intermediates of the tricarboxylic acid cycle and the related compounds, lactic and acetic acids. Primary attention is directed toward procedures which appear to be particularly well suited for tracer experiments, and no attempt is made to review the voluminous literature on biochemical methodology of this group of compounds. We have attempted to select methods which seem generally useful in tracer experiments, but the choice is necessarily somewhat arbitrary and many methods of equal merit have undoubtedly been omitted. The rationale of isotopic tracer experiments is quite straightforward: the presentation of appropriately labeled compounds to a biological system followed by the extraction, separation, and degradation of suspected intermediates and end products. For reasons of convenience the discussion of methodology relating to tracer experiments with compounds of the tricarboxylic acid cycle has been divided into four sections: extraction and separation, determination, degradation, and synthesis. The coverage of these four topics is by no means uniform. Only the first of these, the extraction and separation of tricarboxylic acid cycle intermediates, is dealt with in the detail comparable to that usually achieved in these volumes. The presentation of other aspects of isotopic methodology departs somewhat from the usual format in that it has not been possible in most cases to provide complete details. The discussion is directed toward correlating diverse procedures with respect to their applicability and limitations with only an outline presentation of actual methods in most instances. Additional details can be obtained from the appropriate references which are cited.

[24]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

585

The planning and carrying out of isotopic tracer experiments depends oll a number of considerations some of which are mentioned briefly in the following paragraphs. In isotopic experiments the purity of the compounds involved is of even greater importance than in many other types of chemical studies, since the presence of even traces of a highly labeled contaminant can lead to erroneous conclusions. This requirement for high purity influences all stages of the methodology of isotopic experimentation. The problem of permissible dilution of the isotopic label faces every investigator in this field. Ultimately, the magnitude of dilution which can be sustained depends on the sensitivity of the counting methods. Dilution occurring during the biological stage and in the separation and degradation stages must be considered. In many cases it is expedient or necessary to add carrier compounds at various points during the experiment. Obviously, no set rules are possible and individual problems must be considered carefully prior to experimentation. Many isotopic experiments have been performed in a semiquantitative fashion and the data used to formulate qualitative conclusions. In such circumstances, the problems of dilution may be of lesser importance and also the specificity of the degradative procedures used need not be so rigid. II. Extraction and Separation of Acids Introduction

The procedures described in this section are applicable to the isolation and separation of intermediates of the tricarboxylic acid cycle and related compounds from a wide variety of sources. Acids are isolated from reaction mixtures by extraction with ether, acetone, or other suitable organic solvents. After removal of the solvent, a number of chromatographic procedures may be employed to resolve the mixture into its individual components. Separations are usually made on a two-phase chromatogram consisting of an immobile phase prepared by saturating a solid with water, the solid merely acting as a mechanical support, and a mobile phase which is some liquid immiscible with water. Separations on chromatograms of this type depend on differences in the partition of the compounds between two liquid phases. 1 Ion exchange techniques may also be used in the separation of organic acids. 2,8 This method, however, has not gained general acceptance primarily because of the difficulty in devising methods for determining small 1 A. J. P. Martin and R. L. M. Synge, Biochem. J. 3§~ 1358 (1951). 2 H. Busch, R. B. tturlbert, and V. R. Potter, J. Biol. Chem. 196, 717 (1952). 3 H. H. Schenker and W. Reiman, Anal. Chem. 25, 1637 (1953).

586


[24]

quantities of organic acids by titration in acidic or buffered column effluents. Water adsorbed on silicic acid, Celite, or paper has been used most frequently as the immobile phase in partition chromatography of organic acids. Excellent separations of acids of the tricarboxylic acid cycle are obtained by partition chromatography on paper, 4 but the method is of limited usefulness in isotopic experimentation because the quantity of acid that can be employed is too small to be readily determined by conventional methods. Procedures employing columns prepared with Celite or silicic acid are preferable because relatively large quantities of a mixture of acids can be resolved into its individual components. The selection of Celite over silicic acid is primarily one. of personal choice, since neither offers any outstanding advantages over the other and comparable separations are obtained when the same developing solutions are employed. The procedures described below in some detail have yielded satisfactory results in the extraction and separation of intermediates of the tricarboxylic acid from a variety of microorganisms, enzyme mixtures, and other sources. These techniques combine many features of a number of similar methods. 5-1° It is emphasized that additional procedures may offer certain advantages under specially circumscribed conditions.

Equipment and Reagents Chromatographic tube constructed from Pyrex tubing, 1.8-cm. inside diameter and 75 cm. in length, constricted at the bottom and sealed to a stopcock. A plug of glass wool at the constriction acts as a support for the column. Pressure device. A tank of nitrogen or air line fitted with a suitable pressure-regulating valve. Lyophile apparatus. Ethyl ether, free from ethyl peroxide and organic acids. The presence of peroxide can be detected by the liberation of iodine (brown coloration or blue coloration on the addition of a few drops of 1% starch solution) when a small sample is shaken with * R. J. Block, E. L. Durrum, and G. Zweig, " A Manual of Paper Chromatography and Paper Electrophoresis." Academic Press, New York, 1955. 5 F. A. Isherwood, Biochem. J. 40, 688 (1946). s C. S. Marvel and R. D. Rands, Jr., J. Am. Chem. Soc. 72, 2462 (1950). 7 C. E. Frohman, J. M. Orten, and A. H. Smith, J. Biol. Chem. 193, 277 (1951). 8 E. F. Phares, E. H. Mosbach, F. W. Denison, Jr., and S. F. Carson, Anal. Chem. 24, 660 (1952). 9 W. A. Bulen, J. E. Varner, and R. C. Burrell, Anal. Chem. 24, 187 (1952). 10F. E. Resnik, L. A. Lee, and W. A. Powell, Anal. Chem. 27, 929 (1955).

[24]


587

an equal volume of potassium iodide solution (2 g. of KI dissolved in 100 ml. of 0.05 N HC1). If the ether contains peroxide it can be removed by shaking 1 1. with 100 ml. of an acidic solution of ferrous sulfate (50 g. of FeSO4, 5 ml. of concentrated HC1, and 100 ml. of water) and distilled unless acids are to be removed also. The presence of organic acids can be determined by titrating a sample with 0.01 N NaOH (phenol red indicator). When free from peroxide, the ether is distilled over sodium hydroxide to remove organic acids. Celite 535, Johns Manville. Dry Celite is firmly packed into a Pyrex tube of suitable diameter and washed with ether (free from peroxide and organic acids) by allowil~g approximately 1 1. per 100 g. of Celite to flow through the column. The Celite is then spread in a thin layer on aluminum foil (hood) and allowed to dry at room temperature overnight. It is then heated in an oven at 100 ° for 4 to 6 hours and finally stored in a closed container. n-Butanol, reagent grade. Most lots are satisfactory, but some are contaminated with organic acids which can be removed by washing thoroughly with water or distilling over sodium hydroxide. Chloroform, reagent grade, extracted several times with water to remove ethanol. Chloroform-n-butanol mixtures equilibrated against 0.2 N H2SO4. Each mixture is thoroughly shaken in a separatory funnel with 0.5 to 1 vol. of 0.2 N H2SO4 and the two phases are allowed to separate. It is desirable to repeat this procedure several times. To assure the complete removal of water droplets from the chloroform-butanol solutions, each is finally filtered through a dry filter paper (12 cm. in diameter, any grade, in 15-cm. filter funnel, per liter of ether). For smaller volumes of solution the size of the filter paper should be reduced accordingly to avoid the possibility of removing acid from the solvent so it is no longer equilibrated. Phenol red indicator, 0.04%, prepared by mixing 100 rag. of solid (acid form) with 2.8 ml. of 0.1 N NaOH and diluting to 250 ml. with water. Extraction of Acids Procedure A. This method involves the adsorption of an acidified mixture containing intermediates of the tricarboxylic acid cycle on Celite followed by elution with ether. The procedure is applicable to tissue homogenates, to biochemical reaction mixtures, and to solutions in general.

588

TECHNIQUES FOR ISOTOPE STURIES

[24]

The following example is based on a 10-ml. sample. The pH of the mixture is adjusted to less than 2 (congo red paper) with 10 N H~SO~. Sulfuric acid is also used in experiments where it is necessary to terminate biochemical reactions prior to extracting the acids. The solution is then mixed thoroughly with 20 g. of dry Celite and transferred quantitatively to a chromatographic tube (18-ram. inside diameter) in 2-g. portions, each portion being uniformly packed with a stainless steel pestle. The organic acids are eluted from the Celite by allowing ether to run through the column, or it may be forced through under pressure at a rate not exceeding 24 ml./min. The volume of ether required to extract the acids quantitatively varies with the composition of the sample, although 300 to 500 ml. is usually sufficient. It is therefore advisable to determine the volume of ether required by collecting aliquots of the eluate at suitable intervals and titrating each with 0.01 N NaOH (phenol red indicator). The ether is distilled i n v a c u o (without application of heat) or evaporated in a well-ventilated hood with the aid of a gentle stream of air. The residue is chromatographed on Celite as described under Separation of Acids. This method is superior to the conventional liquid-liquid extraction in that all the acids of the tricarboxylic acid cycle are recovered quantitatively in a relatively short time. Furthermore, this method can be used at temperatures as low as 0 ° if necessary. In addition the method serves as a desalting procedure. This is particularly important when the original mixture contains high concentrations of phosphates and other inorganic salts which may interfere in the chromatographic separation of acids with peak effluent volumes (Table I) greater than 500 ml. The volume of material which can be extracted in the manner described is limited only by the amount of Celite which can be conveniently extracted with ether. The salient points to be considered in adapting the extraction method to larger or smaller volumes are as follows. (1) The ratio of sample (water) to Celite must not exceed 1:2. If too much water is present in the column, some will be eluted along with inorganic acids and other water-soluble materials which interfere with chromatographic separations. (2) The volume of ether required to elute the organic acids quantitatively should be determined by titrating aliquots of eluate, collected at intervals during the extraction with 0.01 N NaOH. (3) The diameter of the tubing should be such that a column of Celite 20 to 25 cm. is obtained. The volume of the sample may also be reduced i n v a c u o at temperatures usually not exceeding 30 ° prior to ether extraction on Celite. No other alteration in the procedure is required. The degree of concentration should not exceed 100-fold, since excess acid may contribute to the

[~4]


5~,0

decomposition of the more labile m e m b e r s of the tricarboxylic acid cycle series. A portion of the acetic acid is r e m o v e d b y distillation b u t can be readily recovered b y neutralizing the distillate and e v a p o r a t i n g to dryness on a s t e a m bath. T h e acetic acid is purified b y c h r o m a t o g r a p h y on Celite. I t will be noted t h a t at no point in the sequence is the mixture of acids neutralized. This is of considerable i m p o r t a n c e when q u a n t i t a t i v e TABLE I SUMMARY OF RESULTS IN SEPARA.TION OF ACIDS BY PARTITION CHROMATOGRAPHY ON CELITE ~

Peak number b

Acid

1 2 3 4 5 6

Propionic Acetic Pyruvic Fumaric Succinic a-Ketoglutaric

7

cis-Aconitic

8 9 10

Malic Citric d-Isocitric

Per cent (v/v) chloroform-butanol

Peak effluent volume/ ml.

Acid added, aM.

Acid recovered, aM.

100-0 95-5 90-10 90-10 85-15 80-20 70-30 70-30 60-40 50-50

70 150 210 260 360 420 630 690 860 950

40.0 20.0 30.0 35.0 35.0 25.0 10.0 20.0 20.0 10.0

39.8 20. l 29.1 34.8 35.1 23.7 9.6 19.5 19.6 9.2

" Compiled from H. E. Swim, Thesis, Western Reserve University, 1952. b Refers to chromatograph, Fig. 1. Average value obtained from several experiments. recovery of the keto acids is required. Oxalacetic acid is unstable at neutral or alkaline p H ' s . ll F u r t h e r m o r e , when mixtures are e v a p o r a t e d to dryness or lyophilized at neutral or alkaline p H ' s , only 60 to 80 ,~,~of the pyruvic and a-ketoglutaric acids is recovered on subsequent chromat o g r a p h y on Celite. T h e low recoveries are p r e s u m a b l y due to the formation of polymers of these keto acids under alkaline conditions which do not migrate on the column with the developing solutions employed. Procedure B. This m e t h o d is similar in principle to Procedure A and is well suited to the isolation of acids from d r y powders, tissues, and microbial cells. I t has been employed successfully ill the isolation of intermediates of the tricarboxylie acid cycle from microorganisms 12,~ in experiments employing 100 g. of cells (wet weight) (see ref. 14 for 1~p. Ostern, Z. physiol. Chem. 218, 160 (1933). 12j. A. DelVfoss and H. E. Swim, unpublished experiments. 13E. Fisher, Jr. and H. E. Swim, unpublished experiments. ~4H. E. Swim and L. O. Krampitz, J. Bacteriol. 67, 419 (1954); H. J. Saz and L. O. Krampitz, ibid. 67, 409 (1954).

590


[24]

examples of this type of experiment). Enzymatic reactions are terminated by adjusting the pH of the reaction mixture to 3.5 to 4 with HC1 and cooling the mixture to 0% The cells are recovered by centrifugation and washed twice with cold water. After washing, the packed cells are mixed with 150 ml. of distilled water to form a thick slurry, 4 ml. of 5 N HC1 is added, and the mixture is then lyophilized. The small quantity of HC1 vapor which is not condensed in the lyophile apparatus may be prevented from reaching the vacuum pump by employing a trap filled with sodium hydroxide pellets or one cooled in liquid nitrogen. The lyophilized material (approximately 20 g.) is mixed thoroughly with 40 g. of dry Celite. If the material contains C 14, this operation should be performed in a well-ventilated hood. The cell-Celite mixture is acidified (pH less than 2) by mixing uniformly with 15 ml. of 0.5 N H2S04. If the sample contains an unusually large quantity of various salts it may be necessary to use more concentrated H2S04 (volume not to exceed 20 ml.). The pH can be readily checked by mixing small pieces of congo red paper with the Celite. The acidified mixture is added quantitatively in 10-g. portions to a Pyrex tube (5 to 6 cm. in diameter), each portion being packed uniformly. The organic acids are eluted by allowing 3 1. of ether to flow through the column, or it may be forced through under pressure at a rate not exceeding 100 ml./min. The volume of ether required to elute the acids quantitatively may vary with the composition of the sample, and this can be determined as described in Procedure A. It is essential that the Celite remain acid (pH less than 2) throughout the extraction. Congo red paper can be used as an internal indicator by adding several small pieces to the cell-Celite mixture and by packing the column in a manner so that a number of these are visible. After evaporation of the ether (eluate), the residue is chromatographed on Celite as described under Separation of Acids. Since acetic acid is volatile, it is removed by lyophilization. This compound is readily recovered by neutralizing the distillate and evaporating to dryness on a steam bath. The acetate is purified by chromatography on Celite. A small portion of the pyruvic acid may also be removed by lyophilization, but the extent to which this occurs has not been established. The quantity of material to be extracted in this manner can be varied over a wide range. The important points to be considered in varying the quantities of sample are the same as described under Procedure A.

Separation of Acids Preparation of the Column. The preparation and characteristics of a 10-g. column are described. Ten grams of Celite 535 is thoroughly mixed

[24]

INTERMEDIATES

OF T H E TRICARBOXYLIC ACID CYCLE

591

with 5 ml. of 0.2 N H2SO4 in a 250-ml. beaker. The fine slurry prepared by flooding the mixture with chloroform (previously equilibrated with 0.2 N H2SO4) is added to the tube in 2-g. portions, each portion being packed uniformly with a stainless steel pestle. A more uniform column is obtained if 50 to 100 ml. of chloroform is forced through the column (after packing) under 10 to 15 pounds of pressure of nitrogen or air. Care must be exercised not to allow the level of the chloroform to fall below the top of the Celite, and the pressure must be reduced gradually to avoid cracking the column. Addition of Sample to Column. The mixture of acids obtained according to Procedure A or B, under Extraction of Acids is dissolved in 1 to 1.5 ml. of 0.2 N H2SO4 and thoroughly mixed with 2 to 3 g. of Celite. In the case of neutral or alkaline mixtures from other sources (when keto acids are not present) the pH is adjusted to less than 2 with H2SO4 prior to mixing with Celite. The Celite-acid mixture is transferred quantitatively to the top of the column and uniformly packed. This is conveniently accomplished by wiping the last traces of Celite from the container with a plug of glass wool which is then pushed down firmly on top Of the column. The glass wool plug serves to hold the Celite in place and should be sufficiently large to sweep down particles of Celite adhering to the upper portion of the tube. After packing, sufficient chloroform (equilibrated with 0.2 N H2SO4) is added to saturate the Celite. Separation Procedure. The developing solutions used in increasing polarity are as follows: chloroform, and chloroform containing 5, 10, 15, 20, 25, 30, 35, 40, and 50% (v/v) n-butanol. For a 10-g. column, 100 ml. of each solution is allowed to run through the column or may be forced through under pressure at a rate not exceeding 4 ml./min. The effluent is usually collected in 10-ml. fractions in 20 N 150-mm. tubes or by the use of an intermittent syphon constructed so as to deliver the correct volume. One drop of phenol red indicator and about 5 ml. of CQ-free water are added to each fraction which is then titrated with 0.01 N NaOH. During the titration, intimate contact between the two phases is obtained by bubbling CO:-free air through the mixture. It is necessary to apply a small blank correction to the titrations. These values increase gradually with increased concentrations of n-butanol (Fig. i). Heavy emulsions are encountered when the concentration of n-butanol in the eluant exceeds 40% which makes accurate titrations practically impossible. This difficulty is eliminated by addition of 5 ml. of water-washed chloroform to each tube prior to titrating. Recovery of Individual Acids from Column E~uent. After titrating with NaOH, the various acids, with the exception Of the keto acids, are recovered as their sodium salts by combining serial fractions of the effluent

592

TECHNIQUES

FOR

ISOTOPE

[24]

STUDIES

(aqueous phase plus nonaqueous phase) containing a single acid and evaporating the solvent on a steam bath (hood) with the aid of a gentle stream of air. Fractions containing keto acids are treated in a somewhat different manner to avoid possible decomposition and polymerization. The latter is of critical importance if enzymatic procedures are to be employed to degrade the keto acids. Immediately after titrating, the contents of a series of tubes containing one of the keto acids are combined. The aqueous phase containing the acid is separated from the chloroformbutanol mixture. The latter is extracted several times with small volumes of water. The combined extracts are added to the aqueous phase (keto •acid solution), the pH is adjusted to 1 with hydrochloric acid, and the solution is lyophilized.

z z

3.0" 4 ~j~ 2.0.~ ~ 1.0-

(5 .4

o-~

160

Ebo : ~

4bo e~o ~

MILLILITERS OFEFFLUENT ~

s~

9bo Ioo

FIG. l. Typical chromatograph of a mixture of known acids. The sodium salts or the free acids (in case of keto acids) are dissolved in water and transferred quantitatively to volumetric flasks. These solutions are subsequently examined for the presence of contaminants (see Discussion of Separation Procedures) and finally used for isotopic analyses. Discussion of Separation Procedures. A typical chromatograph of a mixture of known acids is illustrated in Fig. 1. The quantitative aspects of this experiment are summarized in Table I. Propionic acid is also included, since it is an intermediate in the degradation of succinic and a-ketoglutaric acids. The separation procedure is similar to that described by Marvel and Rands, e who employed silicic acid instead of Celite in the immobile phase, and the results obtained with the two types of columns are in excellent agreement. Peak effluent volume (Table I) is defined as the quantity of effluent collected while a given compound migrates to the bottom of the column and is measured at the point at which the greatest concentration of the compound is eluted. When variables such as dimensions of the column, solvents employed, strength of H2S04 used to prepare the Celite and developing solutions, and rate of change in polarity of eluant are

[9.4]


593

maintained constant, each compound has a characteristic peak effluent volume. This constant can be used in the tentative identification of an acid component from an unknown mixture. The quantitative addition of a sample to the top of a prepared column is facilitated by adsorbing in Celite prior to transfer. On the other hand, acids are usually dissolved in tert-amyl alcohol or other organic solvents before transfer to silicic acid columns. There are, however, certain technical difficulties associated with this procedure. The results obtained by Bulen et al2 indicate that a technique similar to that described above for Celite is applicable to silicic acid columns although this method has not been in general use. Zbinovsky and Burris 15 have described an additional method for adding aqueous solutions of organic acids to silicic acid columns. Simple devices for gradually and automatically increasing the nbutanol concentration and thereby the polarity of the solvent entering the column have been described. 16,1~These methods facilitate the use of a mechanical apparatus for the collection of effluent fractions and eliminate false chromatographic peaks which occasionally are produced when the polarity of the solvent is increased in discrete steps. Furthermore, the results obtained by Marshall et al. ~8 indicate that chromatographic resolution can be improved by rigid control of the rate at which the polarity of the eluant is increased. The quantity of a mixture of acids which can be separated on a 10-g. column varies with the acids involved. For example, the amount of two acids which can be separated depends on the difference between their peak effluent volumes and the sharpness with which they are eluted. For the mixture of acids shown in Table I, quantities up to 300 micromoles (total acid) can be separated. The same procedures can be used on a smaller or larger scale by simply applying proportionality factors to determine the materials required. The amount of Cetite used to adsorb the sample may also be varied according to the size of the column. It is essential that not more than 1 ml. of sample be added for each 2 g. of Celite. When unknown mixtures are chromatographed there is usually a possibility that fractions may be contaminated with organic acids with peak effluent volumes similar to those of the tricarboxylic acid cycle. The presence of a relatively large quantity of such a contaminant is indicated by irregularities in the contour or symmetry of the curves on a 15 V. is K. 17 R. is L.

Zbinovsky and R. H. Burris, Anal. Chem. 26~ 208 (1954). O. Donaldson, V. J. Tulane, and L. M. Marshall, Anal. Chem. 24, 185 (1952). R. Allen and D. N. Eggenberger, Anal. Chem. 27~ 476 (1955). M. Marshall, K. O. Donaldson, and F. Friedberg, Anal. Chem. 24, 773 (1952).

594


[9.4]

chromatograph (Fig. 1) and by an increase in the volume of eluant required to eluate a given quantity of acid (broader peak). In certain instances there may be no indication that a contaminant is present. The following procedures are useful in checking the purity of acids isolated by partition chromatography. 1. In instances where methods are available, the quantity of acid as determined by titration is compared with that obtained by a more specific method (see Section III, Determination of Acids). 2. Chromatographic procedures employing paper and a variety of developing solutions 19-~7 may be used to examine a portion of the sample. Paper chromatography is well suited for this purpose, since minute quantities of sample are employed and a variety of developing solutions can be tested with a minimum expenditure of time and materials. The phenol red indicator that is added during the titration of fractions of effluent from the column does not interfere in isotopic analyses because the quantity of carbon introduced in this manner is ordinarily insignificant in comparison with that of the organic acid per se. The ratio of phenol red to organic acid is reduced even further when samples are diluted with carrier (see Section IV). Phenol red may interfere, however, with the spectrophotometric determination of a number of compounds, particularly the keto acids. This difficulty can be circumvented by using a pH meter or a titrimeter instead of phenol red to determine the end point of the titration. Spectrophotometric methods can also be employed in place of titration, or phenolphthalein may be substituted for the phenol red indicator. If the aqueous phase is acidified after titration and the phases are thoroughly mixed prior to the separation of the two, as already described for the isolation of keto acids, the phenolphthalein is for the most part extracted into the chloroform-butanol, since in the acid form it is sparingly soluble in water. A small quantity of the organic acid is lost in the chloroformbutanol, and it is therefore necessary to determine the acid recovered in the aqueous phase by other means (see Section III). A d d i t i o n a l Separation Procedures. 1. Lactate and succinate are not lg j. W. H. Lugg and B. T. Overell, Australian J. Sci. Research A1, 98 (i948).

~oA. A. Benson, J. A. Bassham, M. Calvin, T. C. Goodale,V. A. Haas, and W. Stepka, J. Am. Chem. Soc. 72, 1710 (1950). ~1j. B. Stark, A. E. Goodban, and H. S. Owens, Anal. Chem. 23, 413 (1951). 2~F. W. Denison, Jr., and E. F. Phares, Anal. Chem. 24, 1629 (1952). ~3M. L. Buch, R. Montgomery, and W. L. Porter, Anal. Chem. 24, 489 (1952). ~4F. A. Isherwood and C. S. Hanes, Biochem. J. 55, 824 (1953). ~5F. Bryant and B. T. 'Overell, Biochim. et Biophys. Acta 10, 471 (1953). ~6A. R. Jones, E. J. Dowling, and W. J. Skraba, Anal. Chem. 25, 394 (1953). 2~R. W. Scott, Anal. Chem. 27, 367 (1955).

[24]


595

well separated by the procedure described, since the peak effluent volumes are 340 and 360 ml., respectively. Lactate can be removed from a mixture of succinate and lactate by treatment with permanganate, 2s and the residual succinate can be rechromatographed on Celite as already described. In instances where both lactate and succinate are required, the procedure described by Phares et al. 8 can be employed to resolve the mixture into its components. The procedure is similar to the one described except that Celite-0.5 N H2SO4 constitutes the immobile phase and the column is developed with ethyl ether which has been equilibrated with 0.5 N H2SO~. With the proper selection of eluants, silicic acid may also be employed for the separation of lactic and succinic acids. 7,0,~8,29 2. Mixtures of formic and pyruvic acids are not resolved on Celite with the chloroform-butanol solutions employed. A mixture of the two acids can be resolved by partition chromatography as described by Bulen et al. 9 The pyruvic acid can also be isolated and chromatographed as the 2,4dinitrophenylhydrazine derivative (see part 4, this section). 3. Oxalacetic acid is not sufficiently stable to be chromatographed in columns prepared with Celite-0.2 N H2SO4. Only 20% is recovered (peak effluent volume approximately 450 ml.), and the remainder appears as pyruvic acid and as an increased blank titration value between the pyruvic and oxalacetic acid peaks. This difficulty can be circumvented by using Celite containing 3 N H2SOt as described by Utter. a° Frohman et al. 7 used silica gel-4.7 N H~SO4 as the immobile phase, and the chromatogram was developed with mixtures of c h l o r o f o r m - t e r t - a m y l alcohol. The general application of procedures of this type is limited because large blank titration values are obtained with the more polar solutions which makes it difficult to determine small quantities of organic acid in the eluate by titration. This is of particular significance in the separation of c i s - a c o n i t i c , malic, citric, and isocitric acids. 4. Pyruvic, oxalacetic, and a-ketoglutaric acids may be conveniently separated by chromatography in the form of their 2,4-dinitrophenylhydrazones. 31-33 III. Determination of Acids Partition chromatography is useful not only in the isolation of pure intermediates of the tricarboxylic acid cycle from biological sources but 28H. G. Wood, C. H. Werkman, A. Hemingway, and A. O. Nier, J. Biol. Chem. 169, 377 (1941). 29A. C. Neish, Can. J. Research B27, 6 (1949). 30M. F. Utter, J. Biol. Chem. 188, 847 (1951). 3i G. A. LePage, Cancer Research 10, 393 (1950). 32D. Cavallini and N. Frontali, Biochim. et Biophys. Acta 13, 439 (1954). 33A. I. Virtanen, J. K. Meittinen, and H. Kunttu, Acta Chem. Stand. 7, 38 (1953).

[24]


597

also in the determination of these compounds. The values obtained in the determination of individual acids by titrating fractions of effluent from partition chromatograms are satisfactory for most purposes particularly when conditions are arranged in such a manner that the blank titration values are reduced to a low level. The method when properly applied is relatively specific and sufficiently sensitive that 5 to 10 microequivalents of acids can be determined quantitatively without sacrificing any of the sample. The latter is of particular importance in situations where all the experimental material is required for further isotopic analysis. The usefulness of partition chromatography in quantitating acids is evidenced by its extensive application in diverse fields and by the fact that few recent advances have been made in the development of additional methods. On the other hand, supplementary methods are frequently useful in detecting gross contamination of acids isolated by partition chromatography, in compensating for losses which may occur in the recovery of acids from column effluents prior to isotopic analysis, and in the routine analysis of large numbers of samples, e.g., during the course of preliminary experiments. It is apparent that such procedures should not only be simple and specific, but also applicable on a micro- or semimicro scale. 5~[ethods which have been employed frequently are listed in Table II. Few of these techniques satisfy all the requirements of ideal analytical procedures but are nevertheless suitable for a variety of investigations. The choice of a given method is largely dependent on factors peculiar to a given analytical problem. It is beyond the scope of this report to consider the applications and limitations of each method. For information of this type in addition to technical details, the references appended to Table II should be consulted.

IV. Degradation of IsotopicaUy Labeled Compounds Introduction The degradation of isotopically labeled compounds ordinarily represents the application of conventional techniques of organic chemistry, bt~t the small amounts of compounds involved and the special aims of isotopic experiments often necessitate modifications of the usual techniques. As noted earlier, the requirements for purity of the compounds under study are very stringent in many circumstances. In most tracer experiments it is necessary to localize the labeling within a compound by the use of specific procedures whereby the labeled carbon atoms representing a single position or group of positions within the molecule can be isolated and analyzed. The possibility of cross contamination with other

598


[24]

positions can be detected in many cases only by the degradation of compounds with known labeling. As will become apparent in the following discussion, information of this type is available for only a limited number of degradative procedures, although the limits of cross contamination in other methods can be inferred to some extent by examining data obtained from compounds whose labeling is not known with certainty. The practice of balancing the sum of the isotopic content of the individual carbons against the content of the entire compound provides a useful safeguard against many types of experimental error. Examples of this type of calculation are presented in the reports of Leaver et al. "~4 and Bernstein et al.3S The preparation of samples for degradation, the conversion of compounds to a form in which they can be counted, and degradative methods for individual acids are considered in this section.

Preparation of Samples for Degradation Before proceding with the degradation and isotopic analysis of the various compounds isolated by partition chromatography, it is advisable to establish the purity of each compound by independent means (see Separation of Acids under Section II). Once the criteria for purity are satisfied, it is usually necessary to dilute the labeled compound with a known amount of unlabeled material (carrier), since most of the following methods require from 0.25 to 5 millimoles of material with the apparatus usually available. It is apparent that the magnitude of such dilutions must be anticipated in planning experiments in order to avoid shortages of material containing sufficient isotope to be measured accurately. Such factors as the relative yields of products formed from a given substrate, dilution from endogenous sources, and the quantity of material required for a particular degradation procedure must be considered in the calculations. Much of this information can be obtained from preliminary experiments of a semiquantitative nature. On the other hand, situations arise where it becomes difficult to predict the yield or the specific activity of metabolic products obtained from a given substrate. It is therefore expedient to establish the specific activity of each compound prior to the addition of carrier to avoid excessive dilution of isotope. In the case of C 14, an aliquot of the sample may be evaporated in metal planchets and counted. This method is superior to converting the compound to CO~, s~F. W. Leaver, H. G. Wood, and R. Stjernholm, J. Bacteriol. 70, 521-530 (1955). 35I. A. Bernstein, K. Lentz, M. Malm, P. Schambye, and H. G. Wood, J. Biol. Chem. 215, 137-152 (1955).

[~4]

INTERMEDIATES

OF T H E TRICARBOXYLIC ACID CYCLE

599

since none of the material is sacrificed. The procedure is sufficiently accurate for the purpose, and the results m a y be expressed (approximately) in terms of BaCO3 by applying a correction factor for the difference (due to alterations in the geometric arrangement of the counting chamber) in counting efficiency between the two methods.

Combustion Methods Although compounds m a y be counted on occasion without conversion to C02 (see above), in most cases the compound or the fractions derived from it are converted to CO2 b y appropriate combustion techniques and

counted as CO2 or BaCO3. The two most commonly used combustion procedures employ either chromic acid ~e or persulfate. 37 The first of these is the method of choice, since it has been tested extensively and found to be effective for a wide variety of organic compounds. It has been found expedient by many investigators in these laboratories to employ double the concentration of chromic acid rather than that originally proposed. ~6 This method requires anhydrous samples, whereas aqueous solutions of pure compounds are conveniently combusted by the persulfate method. Ma~ny compounds, including all mentioned in this report, appear to be oxidized completely by persulfate on the basis of yield of CO2. On the other hand, Sakami ~7~ has found that a few compounds such as choline are not oxidized completely. In addition, Wood et al. 38 have observed that the values obtained with propionate-2-C 14 and propionate-3-C 14 by the persulfate method are lower than when chromic acid is employed, which suggests that carbons 2 and 3 are not converted quantitatively to CO2. This type of discrepancy may have been overlooked previously because it is within the error of CO2 measurements by the usual procedures. Special combustion procedures have been employed for several compounds which are intermediates in various degradative schemes; these include formic acid by mercuric ions; 39 CO by iodic sulfate; 4°,41 iodoform by hot silver nitrate; 42 and methylamine by alkaline KR'[nO4. 43 36D. D. Van Slyke and J. Folch, J. Biol. Chem. 136, 509 (1940). 37O. L. Osburn and C. H. Werkman, Ind. Eng. Chem. Anal. Ed. 4, 421 (1932). 37~W. Sakami, personal communication. 38 H. G. Wood, R. Stjernholm, and F. W. Leaver, J. Bacteriol. 70, 510 (1955). 39 N. Pirie, Biochem. J. 40, 100 (1946~. 40 M. Schutze, Ber. 77A, 484 (1944). 41 W. G. Smiley, Nuclear Sci. Abstr. 3, 391 (1949). 4~W. W. Shreeve, F. W. Leaver, and I. Siegel, J. Am. Chem. Soc. 74, 2404 (1952). 43 E. F. Phares, Arch. Biochem. and Biophys. 33, 173 (1951).

600

[24]


Degradation of Individual Acids

Succinic Acid. Succinic acid has been degraded by modifications of the Schmidt reaction by several investigators 44-4~ to yield C02 from the carboxyl carbons and ethylenediamine from the methylene carbons as indicated in equation 1. 4 3 2 1 NaN3~ 1,4 3 2 HOOC'CH2"CH2"COOH ........ 2C0~ -{- NH~CH2"CH2NHs oxidation 2,3 NHsCH2"CH2NH2 ............ * 2C02

(1) (2)

The method described by Phares and Long 44 appears to be the most satisfactory modification of this procedure in .terms of the methylene carbons. The yield of COs is approximately 70% of theoretical, and the yield of ethylenediamine obtained by vacuum distillation after treatment with azide is 40 to 45 %. The results obtained by the use of succinate-l-C ~4 and succinate-2-C 14 44 indicate that cross contamination between the carboxyl and methylene carbons is less than 1%. When succinic acid was converted to its anhydride prior to the Schmidt reaction, the yield of ethylenediamine was increased to 70% and the cross contamination virtually eliminated. The ethylenediamine was oxidized to CO~ by treatment with persulfate. 37 Succinic acid has also been degraded by a combination of biological and chemical techniques which are based on the observation of Johns 47 that Micrococcus lactilyticus (VeiUoneUa gazogenes) decarboxylates succinate quantitatively to propionate and COs. Swim 4s has utilized this observation for the degradation of succinate as follows. 43

2

1

M. lactilyticus

3

2

1,4

1,4

HOOCCH~CHsCOOH ................... * CH3CHsCOOH ~ COs

(3)

CH3CH2COOH ......... N a N ~ CH3CHsNH2 3 2 1,4 W CO2 alkaline KMn04 CH3CH~NHs ...................... ~ CH3COOH oxidation 2,3 CH3COOH ............ * 2COs

(4) (5) (6)

In order to avoid the possibility of endogenous dilution of isotope in reaction 3, it is desirable to use a minimum quantity of a highly active suspension of M. lactilyticus. To ensure highly active preparations, the culture should be grown from a young inoculum (12 hours) that has been 44 E. F. Phares and M. V. Long, J. Am. Chem. Soc. 77, 2556 (1955). 4~ M. Strassman and S. Weinhouse, J. Am. Chem. Soc. 75, 1680 (1953). 4~ H. G. Wood and F. W. Leaver, Biochim. et Biophys. Aeta 12, 207 (1953). 47 A. T. Johns, J. Gen. Microbiol. 5, 326 (1951). 4s H. E. Swim, Thesis, Western Reserve University, 1952.

[24]


601

TABLE III DEGRADATION OF LABELED SUCCINATEa

Quantity, ~M. Reaction Succinate ~M. lactilyticus

b

Propionate -{- C02 Propionate l HNz Acetate -t- COs Succinate I M. lactilytlcus

b

Propionate ~- CO2 Propionate l I~N~ Acetate T CO~

Specific activity c.p.m./~M.

Compound

Initial

Final

Initial

Final

Succinate_l,4_C~4 c Propionate CO~ Acetate

560 ----

90.6 455 470 22.6

258 ----

--254 0.03

Acetate CO2

800 ~ 800 d

726 750

---

0.5" 248"

Succinate-2,3-C '4 ! Propionate CO2 Acetate

560 ----

81.4 470 484 19.9

Acetate CO2

600 d 600 d

462 480

1453 ----

---

--11.8 0.12

1400' 11.6'

° H . E. Swim, Thesis, Western Reserve University, 1952. b Reaction volume, 25 ml. : 4.0 ml. of 10% suspension of Micrococcus lactilyticus, 0.1 M potassium phosphate buffer, p H 6.0, 0.004% sodium sulfide, 560 micromoles of isotopic sodium succinate. Two milliliters of 3 N N a O H in center well and 10 millimoles of H~SO~ in side arm. Gas phase, helium. Temperature, 37 °. Incubation period, 3 hours. c Synthesized from acetate-l-C 1~ by Escherichia coli under anaerobic conditions in the presence of fumarate [see H. E. Swim and L. O. Krampitz, J. Bacteriol. 67, 426 (1954), for discussion]. d Quantity of propionate degraded after dilution with unlabeled compound. ° Corrected for dilution of C14-propionate with unlabeled propionate. I Synthesized from acetate-2-C ~4 as described in (c); under the conditions employed, about 1% of the C 1~ appears in the 1,4 carbons. t r a n s f e r r e d d a i l y a t l e a s t t w i c e p r i o r t o use. F r e s h l y h a r v e s t e d cell s u s p e n s i o n s s h o u l d b e u s e d . P r e p a r a t i o n s ( 1 0 % s u s p e n s i o n in 0 . 0 1 % s o d i u m sulfide) can be stored at room temperature under helium for 4 to 6 hours w i t h o u t s e r i o u s loss of a c t i v i t y . T h e a c t u a l p r o t o c o l f o r a b a c t e r i a l d e g r a d a t i o n is s h o w n in t h e l e g e n d of T a b l e I I I w h i c h a l s o i l l u s t r a t e s t h e v a l i d i t y of t h i s s e r i e s of r e a c t i o n s a s t e s t e d o n s u c c i n a t e - 2 , 3 - C 14 a n d s u c c i n a t e - l , 4 - C ~4. T h e p r o p i o n a t e

602


[24]

produced from succinate by M . lactilyticus was chromatographed on Celite and degraded further (equations 4 to 6) by a modification of the procedure described by Phares. 43 The ethylamine produced by treating with azide was oxidized by permanganate and the resulting acetic acid was isolated by chromatography and converted to C02 by treatment with chromic acid. 38 It is apparent from the results shown in Table III that the yields of propionate and C02 from the bacterial decarboxylation are equivalent and that there is essentially no dilution of any fraction, and that cross contamination is insignificant. Suspensions of M . lactilyticus also yield a small amount of acetate of endogenous origin (unlabeled). The presence of acetate makes it necessary to separate the propionate by chromatography prior to degradation in order to avoid dilution of both the carboxyl and methylene carbons in later steps of the procedure. The Hunsdiecker procedure 49 for degrading carboxylic acids has been used successfully by Dische and Rittenberg, ~° who report that a sample of succinate-2,3-C 14 yielded 76% of the theoretical C02 from the carboxyls with a contamination from the methylene carbons of less than 1%. M a l i c Acid. Malic acid can be degraded by MnO2 oxidation, followed by degradation of the resulting acetaldehyde. 43

2

1

MnO2

1,4

3

2

H O O C C H s C H O H C O O H ......... 2COs + CHaCHO K2Cr,O7 CH3CHO ............. * CH3COOH

CHsCOOH

_.Na_N,_~ CH,,NH2 3 2 ~- CO2 CH,NHs _K__MnO~COs

(7) (8)

(9) (10)

This series of reactions (equations 7 to 10) is an adaptation of the oxidation of lactate by MnOs described by Friedemann et al., 51 coupled with the degradation of acetic acid as carried out by Phares. 43 The entire procedure has been used by Wood et al. 5s in a study of malate formation from COs in the propionic acid fermentation. The results obtained by these investigators when biologically formed malate was degraded by the above procedure and by an independent determination of the 1 position with HsSO4 as described below indicates that the contamination of CO2 (from positions 1 and 4) by the 2, 3 carbons is of the order of 3%. Cross contamination might be expected if some malonic acid is produced 49 H. Hunsdiecker and C. Hunsdiecker, Ber. 75A, 291 (1942). 60 R. Dische and D. Rittenberg, J. Biol. Chem. 211, 199 (1954). 61 T. E. Friedemann, M. Gotonio, and P. A. Shaffer, J. Biol. Chem. 73, 335 (1927). 52 H. G. Wood, R. Stjernholm, and F. W. Leaver, J. Bacteriol. 72, 142 (1956).

[2~]


603

during the initial oxidation which can then be decarboxylated to acetate in a random fashion. In this way, the 2 position may contribute to the C02. However, this difficulty is minimized by avoiding a large excess of KMnO4 and by using relatively mild conditions. 52 The 2 and 3 positions seem to be relatively free from contamination by the carboxyl carbons as might be expected, since the acetMdehyde is removed from the reaction mixture. The yields of COs and acetaldehyde are nearly theoretical and 70 to 90%, respectively. 52 The foregoing procedure has the disadvantage that it does not distinguish between the two carboxyl carbons of malic acid. These positions can be determined separately, however, by other methods. The 4 position (fl-COOH) can be obtained as CO2 by the use of a nmlate-adapted strain of L a c t o b a c i l l u s a r a b i n o s u s as reported by Korkes and Ochoa. 53 4 3

2

1

HOOCCH2CHOHCOOH

L. arabinosus 4

3

2

1

............. * CO2 + CH~CHOHCOOH

(1l)

The initial observations were made with whole cells, but Ochoa et al. 54 and Korkes et al. 55 describe the preparation of cell-free extracts and partially purified enzyme systems which can carry out the above reaction. Nossal ~6 has carried out extensive studies of the conditions of the decarboxylation reaction as carried out by whole cells. Freshly harvested cells contain fumarase in addition to malic enzyme, and, therefore, CO~ will be produced from the 1 position as well as from the 4. The fumarase can be reduced or eliminated by fractionation of the cell-free preparations or by storage of the whole cells in the cold. None of the above reports deals with this reaction as a tool in isotopic analysis. However, Utter a6 utilized intact cells (aged in the cold) of L. a r a b i n o s u s for this purpose. The 4 position of malate was found to randomize with the 1 position to the extent of about 10%, presumably because of the presence of residual fumarase activity. In addition, as with most preparations of whole cells, some dilution by endogenous materials undoubtedly occurs. The difficulties of endogenous dilution and randomization via fumarase could be obviated by the use of fractionated cell-free systems. As is frequently the case in degradations based on enzymatic procedures, the amounts of enzyme required to produce sufficient COs for counting purposes makes the method somewhat laborious. The lactate produced in the above reaction (equation l l) ~3 S. K o r k e s a n d S. Ochoa, J. Biol. Chem. 176, 463 (1948); see Vol. I I I [71]. ~4 S. Ochoa, J. B. V. Salles, a n d P. J. Ortiz, J. Biol. Chem. 187, 863 (1950) ; see Vol. I

[124]. 5~S. Korkes, A. del Carapillo, and S. Ochoa, J. Biol. Chem. 187, 892 (1950). 5s p. M. NossM, Biochem. J. 49, 407 (1951); 50, 349, 591 (1952).

604


[24]

could be isolated and degraded. T o the authors' knowledge this procedure has not been used, probably because of the serious obstacles introduced b y the endogenous production of lactate. The 1 position (a-COOH) of malate can be obtained as CO b y treatm e n t with concentrated H2S04'° and the CO converted to COs (equation 13) as described b y Schutze. 4° 43

2

1

H~SO4 1

4(?)

H O O C C H s C H O H C O O H .......... , CO + C02 iodi¢ 1 CO ........ * COs sulfate

(12) (13)

I t would appear from equation 12 t h a t the COs is derived from position 4, but in practice this fraction is contaminated b y other carbons. B y a combination of this method with the K M n 0 4 procedure described earlier (equations 7 to 10) it is possible to obtain an analysis of the individual carbons of malic acid, s~ although the 4 position must be calculated b y difference. Fumaric Acid. Fumaric acid can be converted to succinic acid b y reduction with Zn 5~ or b y hydrogenation. 5° The succinic acid can then be degraded b y one of the methods described in the section dealing with t h a t acid. Zn HOOCCH-----CHCOOH ............. ~ H O O C C H ~ C H s C 0 0 H (14) or Pd(H~) I t is significant t h a t Dische and Rittenberg s° report t h a t the Hunsdiecker m e t h o d 49 for the degradation of carboxylic acids is not effective with fumaric acid, since the yield of COs from fumaric acid-2,3-C 14 was only 25% and the COs was contaminated b y carbons 2 and 3 to the extent of about 4 %. Oxalacetic Acid. Krebs ~8 reported t h a t oxalacetic acid was decarboxylated rapidly in acid solution in the presence of a n u m b e r of metal ions of which A1+++ is one of the most effective. 43 2 1 Al+++ 4 3 2 1 H O O C C H s C O C O O H ......... * COs ~- CH~COCOOH

(15)

This method has been adapted 8° for the determination of isotopic content of the 4 position (f~-COOH) of oxalacetate. The p y r u v a t e can be decarboxylated b y ceric sulfate 59 to yield the 1 position of oxalacetate as COs. 3 2 1 Ce(S04)2 3 2 1 C H s C O C O O H ............. * CH3COOH ~- CO~ 5, H. A. Krebs, D. H. Smyth, and E. A. Evans, Biochem. J. 34, 1041 (1940). 5s H. A. Krebs, Biochem. J. $6, 303 (1942). 6~C. Fromageot and P. Desnuelle, Biochem. Z. 279, 174 (1935).

(16)

606


[24]

can be obtained as CO or C02 b y t r e a t m e n t with concentrated H2S04 according to Weinhouse et al. 65 54 3 2 1 cone. H2SO4 6 1,5 4 3 2 H O O C C H 2 C O H C H s C O O H ................ * CO -~ 2CO2 -{- CH3COCH3

(22)

I

COOH 6 The t e r t i a r y carboxyl is converted to CO which can be oxidized to CO~. 4° T h e p r i m a r y carboxyls are obtained as C02 and the remainder of the molecule as acetone, although the latter was not isolated from this particular procedure. T h e d a t a obtained b y Weinhouse et al., 6~ employing the quinidine salt of citric acid, indicate t h a t there is little contamination of the CO b y the p r i m a r y carboxyls. The C02 formed f r o m the p r i m a r y carboxyls m a y be diluted perhaps as m u c h as 6 % b y other portions of the molecule or b y impurities. Weinhouse et al. 65 obtained the noncarboxyl carbons of citrate as acetone b y t r e a t m e n t with dilute dichromate and isolation of the acetone as the Deniges complex in a b o u t 70% yield. 54 3 2 1 K2Cr207 4 3 2 H O O C C H 2 C O H C H ~ C O O H ............. * 3C02 ~ CH3COCH3

L

(23)

COOH 6

The acetone is free of c o n t a m i n a t i o n f r o m the p r i m a r y carboxyl carbons. Acetone can be further degraded b y the iodoform reaction. 66 4 3 2 NaOI 4,2 4,2 3 CH3COCH3 ......... * CH3I -~ C H 3 C O O H

(24)

T h e iodoform and acetate can be degraded as described in preceding sections. T h e a b o v e methods h a v e the d i s a d v a n t a g e t h a t t h e y do not distinguish between the 2,4 and the 1,5 positions of citrate which h a v e been shown to be stereospecific with respect to synthesis and breakdown in biological systems. Several investigators h a v e carried out partial 67,~8 or complete degradations 69 of citrate in which the stereospecificity of the a b o v e positions is maintained. All these methods necessarily require a biological component, usually pigeon breast muscle or pigeon liver s5 S. Weinhouse, G. Medes, and N. F. Floyd, J. Biol. Chem. 166, 691 (1946). 60L. F. Goodwin, J. Am. Chem. Soc. 42, 39 (1920). s7 V. R. Potter and C. Heidelberger, Nature 164, 180 (1949). 68V. Lorber, M. F. Utter, H. Rudney, and M. Cook, J. Biol. Chem. 18[i, 689 (1950). s9 E. H. Mosbach, E. F. Phares, and S. F. Carson, Arch. Biochem. $3, 179 (1951).

[9.4]


607

extracts, for the initial conversion of citric acid to a-ketoglutaric acid. 54 3 2 1 pigeon tissue H O O C C H ~ C O H C H 2 C O O H ...............

I

COOH 6

54 3 2 1 6 HOOCCH2CH2COCOOH + CO2

(25)

The resulting a-ketoglutaric acid can be degraded stepwise as shown in equations 26 to 30 below, thereby yielding a complete analysis of the citric acid molecule. Isocitric and cis-Aconitic Acids. Isocitric and cis-aconitic acids can be degraded by the stereospecific procedure described above for citric acid to yield CO2 and a-ketoglutaric acid and the latter compound degraded as shown below. (For preparation of isocitric dehydrogenases, see Vol. I [116].) a-Ketoglutaric Acid. Mosbach et al. ~9 have described a complete and apparently specific method for the degradation of a-ketoglutaric acid. 54 3 2 1 NH~ • 54 3 2 l HOOCCH2CH2COCOOH .......... H O O C C H 2 C H 2 C H N H 2 C O O H Pd(H~) chloramine T H O O C C H 2 C H 2 C H N H 2 C O O H ............... 1 H O O C C H 2 C H : C H O + CO2 NH2NH~ HOOCCH2CH2CHO ................... ~ HOOCCH2CH2CH~ (Wolf-Kishner) NaN3 5 HOOCCH2CH2CH3 ............ • CO2 + NH2CH~CH2CH3 (Schmidt) KMnO4 43 2 NH~CH2CH2CH3 ......... ~ HOOCCH~CH2

(26)

(27) (28) (29) (30)

The a-ketoglutarate is converted to glutamate (60 to 80% yield) by reductive amination. The glutamate is then converted to succinic semialdehyde by chloramine T, thereby yielding the 1 position of a-ketoglutaric acid as CO2. The succinic semialdehyde is not isolated but is reduced to butyric acid by a modified Wolf-Kishner reaction. The overall yield of these two steps (glutamate to butyrate) is 45 to 55%. The b u t y r a t e is treated by the Schmidt procedure yielding CO2 from the original 5 position of the a-ketoglutarate with propylamine as the other product. The latter is oxidized by KMnO4 to propionic acid which can be further degraded as described earlier (equations 4, 5, 9, 10). Mosbach et al. 69 tested this procedure on a-ketoglutaric acid-l,2-C :4 and found negligible dilution in any of the fractions and no cross contamination greater than 1 to 2 %.

608


[24]

a-Ketoglutaric acid can be oxidized with acid p e r m a n g a n a t e to C02 and succinic acid as shown below. 7° 54 3 2 1 acid KMn04 H O O C C H ~ C H ~ C O C O O H ................ 54 3 2 1 H O O C C H ~ C H ~ C O O H + CO~

(31)

The succinic acid can be degraded by one of the methods described in TABLE IV SYNTHESIS OF TRICARBOXYLIC ACID INTERMEDIATES

Compound

Reference

CH..CO-COOH

Anker •

CHa.CHOH-C00H

Cromer and Kistiakowsky ~

H00C.CHrC0.C00H

Heidelberger and Hurlbert ~

*

H00C.CHrCHOH'C00H

Jorgensen et al. ~

HOOC.CH=CH-COOH

San Pietro •

H00C'CH~'CHrC00H

Jorgensen et al. d

HOOC-CH2.CH2.COOH

Jorgensen et al. d

H00C-CH2-CHrC0.C00H

Kogl et a l /

H00C-CHrC(0H).CHrC00H

Rothchild and Fieldsg

~00H H00C.CHrC(0H)'CHrC00H

Wilcox et al. ~

~00H H. S. Anker, J. Biol. Chem. 176, 1333 (1948). b R. D. Cramer and G. B. Kistiakowsky, J. Biol. Chem. 137, 599 (1941). C. Heidelberger and R. B. Hurlbert, J. Am. Chem. Soc. 72, 4704 (1950). a j. A. Jorgenson, J. A. Bassham, M. Calvin, and B. M. Tolbert, J. Am. Chem. Sac. 74, 2418 (1952). • A. San Pietro, J. Biol. Chem. 198, 639 (1952). / F. Kogl, J. Halberstadt, and T. J. Barendregt, Rac. tray. ehim. 68, 387 (1949). g S. Rothchild and M. Fields, J. Am. Chem. Sac. 74, 2401 (1952). i p. E. Wilcox, C. Heidelberger, and V. R. Potter, J. Am. Chwn. ,goc. 72, 5019 (1950). the section dealing with t h a t compound. This m e t h o d has the obvious disadvantage t h a t it does not distinguish between the 5,2 and 4,3 carbons. The same method has also been used with the semicarbazide derivaT0H. G. Wood, C. H. Werkman, A. Hemingway, and A. O. Nier, J. Biol. Chem. 142, 31 (1942).

[9.5]

GLYCOLIC, GLYOXYLIC, A N D OXALIC ACIDS

609

rive of a-ketoglutaric acid by Buchanan et al. ~1 In the reaction, two moles of C02 are formed: one from position 1 of the a-ketoglutaric acid and one from the semicarbazide portion of the molecule. The 2,4-dinitrophenylhydrazone derivative of a-ketoglutarate has also been degraded by a modification of the above method. ~2

V. Synthesis of Labeled Substrates Chemical procedures for the preparation of most of the acids connected with the tricarboxylic acid cycle have been adapted for use with isotopes, and some of these compounds are available commercially in labeled form. For convenience, some of the procedures have been summarized in Table IV. Each compound can be labeled in several different positions or with combinations of C 14 and C is, but the compilation given merely lists procedures for one or two types of labeling with each compound although many of these procedures can be adapted to yield other types of labeling if so desired. Needless to say, the strictures concerning purity of compounds apply to substrates for isotopic experiments and appropriate procedures for establishing their purity must be used in the same way as in other phases of isotopic experimentation. ~1j. M. Buchanan, W. Sakami, S. Gurin, and D. W. Wilson, J. Biol. Chem. 159, 695

(1945). 7~H. J. Saz, Thesis, Western Reserve University, 1952.

[25] Synthesis and Degradation of IsotopicaUy Labeled Glycolic, Glyoxylic, and Oxalic Acids B y I~ATHARINE F. LEWIS and SIDNEY WEINHOUSE

Degradation Procedures Glycolic Acid Glycolic acid can be degraded to carbon dioxide and formic acid by several oxidizing agents, such as hydrogen peroxide, 1 cerie sulfate, ~ and the enzyme, glycolic acid oxidase 3 (equation 1). HOCH2COOH + 20--~ HCOOH + C02 + H20 1 H. A. Spoehr, Am. Chem. J. 43, 227 (1910). H. H. Willard and P. Young, J. Am. Chem. Soc. 52, 132 (1930). I. Zelitch and S. Ochoa, J. Biol. Chem. 201, 707 (1953).

(1)

[9.5]

GLYCOLIC, GLYOXYLIC, A N D OXALIC ACIDS

609

rive of a-ketoglutaric acid by Buchanan et al. ~1 In the reaction, two moles of C02 are formed: one from position 1 of the a-ketoglutaric acid and one from the semicarbazide portion of the molecule. The 2,4-dinitrophenylhydrazone derivative of a-ketoglutarate has also been degraded by a modification of the above method. ~2

V. Synthesis of Labeled Substrates Chemical procedures for the preparation of most of the acids connected with the tricarboxylic acid cycle have been adapted for use with isotopes, and some of these compounds are available commercially in labeled form. For convenience, some of the procedures have been summarized in Table IV. Each compound can be labeled in several different positions or with combinations of C 14 and C is, but the compilation given merely lists procedures for one or two types of labeling with each compound although many of these procedures can be adapted to yield other types of labeling if so desired. Needless to say, the strictures concerning purity of compounds apply to substrates for isotopic experiments and appropriate procedures for establishing their purity must be used in the same way as in other phases of isotopic experimentation. ~1j. M. Buchanan, W. Sakami, S. Gurin, and D. W. Wilson, J. Biol. Chem. 159, 695

(1945). 7~H. J. Saz, Thesis, Western Reserve University, 1952.

[25] Synthesis and Degradation of IsotopicaUy Labeled Glycolic, Glyoxylic, and Oxalic Acids B y I~ATHARINE F. LEWIS and SIDNEY WEINHOUSE

Degradation Procedures Glycolic Acid Glycolic acid can be degraded to carbon dioxide and formic acid by several oxidizing agents, such as hydrogen peroxide, 1 cerie sulfate, ~ and the enzyme, glycolic acid oxidase 3 (equation 1). HOCH2COOH + 20--~ HCOOH + C02 + H20 1 H. A. Spoehr, Am. Chem. J. 43, 227 (1910). H. H. Willard and P. Young, J. Am. Chem. Soc. 52, 132 (1930). I. Zelitch and S. Ochoa, J. Biol. Chem. 201, 707 (1953).

(1)

610


[25]

In the method of Spoehr, 1 which utilizes hydrogen peroxide in the presence of ferric ions, it is difficult to establish conditions in which stoichiometric amounts of products are produced. Consequently, the carbon dioxide, which should represent only the carboxyl carbon, is contaminated somewhat with methylene carbon. However, the formic acid arises solely from the methylene carbon. The oxidation of glycolic acid with ceric sulfate gives quantitative, isotopically pure yields of carbon dioxide and formic acid if the conditions of acidity, heating, temperature, time, and concentration of reactants established by Willard and Young ~ are employed. The following procedure was found to give stoichiometric amounts of carbon dioxide and formic acid. The apparatus consists of a 300-ml. three-necked roundbottomed flask through one neck of which there is inserted an inlet tube reaching to the bottom of the flask. This tube is attached to a drying tube filled with ascarite and a bubble counter containing 1 M sodium hydroxide. The center neck of the flask is attached to a reflux condenser to the top of which is connected an absorbing system. This is a 125-ml. Erlenmeyer flask closed with a two-holed rubber stopper; one hole carries a tube leading to the top of the condenser. The other carries a tower 25 cm. long and approximately 2 cm. in diameter filled through about 20 cm. of its length with solid glass beads of 3-ram. diameter. A 10-mm.-diameter extension of the tower leads to the bottom of the flask. The beads are held in place by a plug of glass wool. The third neck of the reaction flask is fitted with a dropping funnel equipped with a stopcock. Ten milliliters of COs-free 0.5 M sodium hydroxide solution is placed in the Erlenmeyer flask, and sufficient vacuum is applied at the top of the bead tower to draw one bubble of air per second through the apparatus. The sodium hydroxide is thereby drawn up into the bead tower where the effluent gases make contact at the surface of the beads. One millimole of glycolic acid in 65 mh of C02-free water, 32 ml. of 60% sulfuric acid (specific gravity 1.5), and 6.3 ml. of 0.1 N ceric sulfate are added successively to the reaction vessel through the dropping funnel. The reaction vessel is heated with a free flame until the contents boil, then are kept just below the boiling point (90 to 95 °) for 1 hour, and finally cooled to 50 °. The vacuum is broken,:allowing the absorbed COs to drain into the flask, the tower is washed down from the top with three successive 20-ml. portions of freshly boiled water, and the COs is precipitated as barium carbonate by the addition of 5 ml. of 1 M barium chloride solution. The contents of the reaction vessel are submitted to distillation with steam until no more volatile acid distills. The distillate is neutralized with standardized 0.1 N sodium hydroxide and evaporated to a volume of 20 ml. The solution is then quantitatively transferred with water to

[25]

GLYCOLIC, GLYOXYLIC, AND OXALIC ACIDS

(~l 1

another three-necked 125-ml. flask which is part of an apparatus identical with that described above. While CO2-free air is drawn through the flask by application of suction at the top of the bead tower, 2 ml. of 50% sulfuric acid is added to the flask and aeration is continued for 10 minutes to ensure removal of CO2. Ten milliliters of 0.5 M sodium hydroxide is then added to an Erlenmeyer flask, which is attached to the bead tower, and 10 ml. of a 10% mercuric sulfate solution is added to the formate solution through the dropping funnel. The mixture is heated to boiling, and air is drawn through the flask and bead tower for 30 minutes while the solution is refluxing. The bead tower is then washed down with CO2-free water, and the carbonate precipitated by addition of 5 ml. of 20% barium chloride solution. 4

Glyoxylic Acid Glyoxylic acid, in dilute aqueous solutions, can be oxidized quantitatively to formic acid and carbon dioxide by hydrogen peroxide. 5 However, since glyoxylic acid is most conveniently isolated from biological material by conversion to one of its carbonyl derivatives, a method utilizing one of these is preferable. The following procedure has been successfully employed 6 to determine the distribution of radioactivity in the carbons of glyoxylic acid isolated as the 2,4-dinitrophenylhydrazone from metabolism experiments. The glyoxylic acid portion of the hydrazone molecule is reduced to acetic acid, which is degraded to methylamine and carbon dioxide. The method of reduction is the Huang-Minlon micromodification of the Wolff-Kishner reaction. 7 The 2,4-dinitrophenylhydrazone (approximately 0.5 millimole) is transferred to a 200-ml. flask containing 1 g. of KOH pellets, 5 ml. of 85% hydrazine solution, and 25 ml. of diethylene glycol (redistilled). The solution is refluxed for 1 hour, then water is distilled off until the temperature of the vapors reach 180 to 190°. Refluxing is continued at this temperature for 1 hour. After cooling, the reaction mixture is acidified with dilute sulfuric acid and steam-distilled. Twenty-five-milliliter fractions are collected and titrated with base until no more volatile acids are recovered. About 90% of the hydrazone is recovered as acetic acid. The neutralized solution is evaporated to dryness on a steam bath. The recovered sodium acetate is degraded to methylamine and CO2 by the Phares s modification of the Schmidt reaction. The apparatus con4S. Weinhouse and B. Friedmann, J. Biol. Chem. 19"/, 733 (1952). 5 W. H. Hatcher and G. W. Holden, Trans. Roy. Soc. Can. Sec. I I I [3] 19, 11 (1925), H. I. Nakada, Thesis, Temple University, 1953. 7 Huang-Minlon (M. Huang), J. Ant. Chem. Soc. 68, 2487 (1946). s E. F. Phares, Arch. Biochem. and Biophys. 33, 173 (1951).

612


[25]

sists of a conical 50-ml. reaction flask equipped with an inlet tube leading to the bottom, and a condenser, the top of which is connected to two bead towers in series, the first containing 0.27 M KMn04 in 1 N H2S04 (to remove SO2 which is formed during the reaction by the reduction of H~S04 by hydrazoic acid) and the second to trap the evolved COs arising by decarboxylation of the acetic acid. The dry sodium acetate is placed in the flask, which is cooled in an ice bath, and 0.3 ml. of 100% H~S04 is carefully added. The salt is dissolved by warming and shaking, and 50 mg. of sodium azide is added to the cooled flask, which is then connected to the apparatus and slowly heated in a water bath to 60 to 70 °. After 1 hour at this temperature, the system is swept with a slow stream of air for 20 minutes. The contents of the bead tower containing the alkali is collected and the trapped COs precipitated as BaCO3. The methylamine is recovered from the residual solution by transferring the reaction flask together with the inlet tube to an aeration apparatus. This consists of an air condenser, bead tower, and 125-ml. Erlenmeyer flask of the same design as the oxidation apparatus described on page 610. Three milliliters of water is added to the reaction mixture, 5 ml. of 0.2 N H2S04 is added to the Erlenmeyer flask, and vacuum is applied to the top of the bead tower. The methylamine is liberated by the slow addition of 5 ml. of 4 N sodium hydroxide through the inlet tube. The flask is then heated in a boiling water bath for 30 minutes while air is drawn through the flask and into the sulfuric acid solution in the bead tower. The contents of the bead tower are washed into the Erlenmeyer flask, the pH is adjusted to 5.0 by addition of approximately 1.2 ml. of 1.0 N NaOH, and the methylamine is oxidized to COs by the persulfate wet combustion procedure) The yield of methylamine varies from 50 to 75%.

Detection of Glycolic~ Glyoxylic, and Oxalic Acids by Paper Chromatography The solvent formula developed by Jeanes et al. l° for the separation of mono- and disaccharides can be used for the detection of the three twocarbon acids by paper chromatography. The material is spotted on filter paper and irrigated overnight in a solvent mixture consisting of 3.0 vol. of butanol, 2.0 vol. of pyridine, and 1.5 vol. of water. After drying, the paper is sprayed with a 0.05% solution of bromophenol blue. Organic acids appear as yellow spots on a blue background. The Rj values are: 9 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," p. 94. John Wiley & Sons, New York, 1949. ~0A. Jeanes, C. S. Wise, and R. J. Dimler, Anal. Chem. 23, 415 (1951).

[25]

GLYCOLIC, GLYOXYLIC, AND OXALIC ACIDS

613

oxalic acid, 0.13; glycolic acid, 0.37; glyoxylic acid, 0.39. Though the RI values of glycolic and glyoxylic acids are quite close, this difficulty can be overcome by developing a second spot of the mixture on the paper and spraying this with a 0.2 % solution of o-phenylenediamine in ethyl alcohol containing 1% nitric acid. 11 On heating to 70 ° for 30 minutes, glyoxylic acid appears as a characteristic yellow spot on a steel-gray background. This color reaction is specific for carbonyl compounds.

Synthesis of Radioactive Compounds Oxalic Acid C14-Labeled sodium oxalate is conveniently prepared in essentially quantitative yield by heating dry sodium formate at 440 ° for 15 minutes. This is accomplished in the authors' laboratory by placing a test tube containing the dry salt in a vertically set-up tube furnace thermostated at the desired temperature.

Glycolic Acid Glycolic acid labeled in either carbon atom may be prepared from the correspondingly labeled glycine according to the method of N a k a d a ) Fifty microcuries of labeled glycine is washed into a beaker with sufficient carrier glycine to give a total of 6.64 mM. The calculated amount of barium nitrite (821 mg. of Ba(NO2)~.H~O) is added, and the volume is brought to 50 ml. With mechanical stirring, 10 ml. of 0.66 N H2SO4 is added over a period of 1 hour. The mixture is allowed to stand overnight, the barium sulfate is removed by centrifugation, and the clear supernatant solution is acidified with HC1 and extracted continuously with ether for 24 hours. Then 75 mg. of carrier glycolic acid is added to the aqueous layer, and extraction is continued for an additional 24 hours. The ether solution is evaporated by means of a jet of dry air until fumes of HC1 are no longer noticeable; the straw-colored sirup is transferred to a beaker with the aid of acetone and a wash solution of 125 mg. of glycolic acid in ether. The combined sirup and washings are evaporated to a small volume and allowed to crystallize. Three additional crystallizations from ether gives colorless crystals that melt sharply at 77 ° (recorded for glycolic acid, 78 to 79°). Approximately 30% of the initial radioactivity is recovered in pure glycolic acid. Singly labeled glycolates may also be prepared from the corresponding acetates according to the method of Hughes et al. TM The details of the chemical procedure are available in Document 3567 from the American 11 H. J. Koepsell, F. I-I. Stodola, and E. S. Sharpe, J. Am. Chem. Soc. 74, 5142 (1952). 12 D. M. Hughes, R. Ostwald, and B. M. Tolbert, J. Am. Chem. Soc. 74, 2434 (1952].

614


[26]

Document Institute (1719 N. Street, N.W., Washington, D.C.). The yield of calcium glycolate is 60 to 65%, starting with 20 millimoles of sodium acetate.

Glyoxylic Acid Doubly labeled glyoxylic acid can be prepared from labeled oxalic acid by the method previously described (Vol. III [49]). Sodium glyoxylate-2-C 14 may also be prepared from tartaric acid-2,3-C 14 according to the method of Weissbach and Sprinson. 13 In a typical run described by these authors 0.5 g. (3.3 millimoles) of tartaric acid in 10 ml. of water is treated with 7.8 ml. of 0.42 N sodium periodate for 10 minutes. The solution is extracted continuously with alcohol-free ether for 24 hours, and the ether solution (about 30 ml.) is extracted successively with a solution of 340 rag. (4 millimoles) of NaHCOa in 20 ml. of water and with 10 ml. of water. The combined aqueous extracts are neutralized carefully with NaHC03 to pH 7.0, concentrated i n vacuo to 1 to 2 ml., and treated with absolute ethanol until precipitation occurs. After storage overnight in the refrigerator, 442 rag. of sodium glyoxylate is obtained by centrifugation and washing with alcohol. An additional 158 mg. of sodium glyoxylate is obtained by concentration of the supernatant and washings; the total yield is 80 %.

Isolation by Column Chromatography Oxalic acid can be isolated in pure form by precipitation with calcium ions, after ether extraction to remove interfering inorganic anions. The first calcium oxalate precipitate may contain impurities, but these can usually be removed by subsequent reprecipitations. Oxalic and glycolic acids in amounts from 0.1 to 1.0 millimole can be separated from other earboxylie acids by the chromatographic procedure of Phares et al. ~4 These authors obtained satisfactory isolations and separations of a number of acids of biological importance by using Celite 545 as support for the immobile phase, and two solvent systems: butanol in chloroform-0.5 N sulfuric acid; and ethyl ether-0.5 N sulfuric acid. The choice of solvent system is dependent on the composition of the material to be separated, but, generally, pairs of acids which are not resolved by one system can be separated by rechromatographing with the other. Oxalic and glycolic acids are not separated by the chloroformbutanol system but are completely resolved in the ether system. la A. Weissbach and D. B. Sprinson, J. Biol. Chem. 205~ 1023 (1953). 14 E. F. Phares, E. tI. Mosbach, F. W. Denison, Jr., and S. F. Carson, Anal. Chem. 24, 660 (1952).

[2~]

PURINES AND PYRIMIDINES

[26] Purines and

615

Pyrimidines

B y EDWARD D. KORN

Synthesis For m a n y biochemical studies the use of uniformly labeled purine or pyrimidine compounds is satisfactory. These are most readily prepared, especially in the case of the nucleosides and nucleotides, by growing yeast on a medium containing labeled bicarbonate or ammonium salts. The R N A and D N A from this yeast m a y then be isolated and h y d r o l y z e d to the nucleotides or nucleosides, whichever m a y be desired. M a n y studies, however, require the use of purines and pyrimidines selectively labeled in specific positions. For these, it is usually best to resort to chemical syntheses. ~

Synthesis

of Adenine, Guanine, and 2,6-Diaminopurine (Fig. 1)

Adenine, guanine, and 2,6-diaminopurine m a y all be synthesized by the same general procedure. 2-5 This method is most applicable to the synthesis of 1,3-N 1~- and 8-C14-purines, although it may also be used to introduce labeled carbon into the 2 or into the 4 and 6 positions. An alternate procedure for the synthesis of 2-CI4-adenine is described later under Synthesis of Hypoxanthine. Formamidine Hydrochloride (I). Generate H C N from 10 g. of N a C N with acid, and bubble it into a flask containing 30 ml. of anhydrous ether and 10 ml. of absolute ethanol maintained at a temperature of - 1 0 to - 2 0 °. Then bubble a stream of anhydrou s HC1 through the solution until 10 minutes after the appearance of turbidity (about 30 minutes altogether). Maintain continual stirring an¢l anhydrous conditions throughout this reaction. Allow the mixture to warm gradually to 10 ° overnight, then cool it to - 2 0 °, remove the solvent, wash the residue with ether, and air-dry the crystalline product. The yield of ethyl formimino ether hydrochloride is 90 to 100%. Place 17 g. of this material in a bomb tube with 35 ml. of absolute ethanol, and cool the tube in a dry ice-ethanol bath. Then generate NH3 from 8.8 g. of NH4NO.~ by the addition of KOH, dry it over NaOH, and 1For the synthesis of nueleosides and nucleotides, see Vol. III [113]. 2L. F. Cavalieri, J. F. Tinker, and A. Bendich, J. Am. Chem. Soc. 71, 533 (1949). 3 V. M. Clark and H. M. Kalckar, J. Chem. Soc. 1950, 1029. 4M. F. Mallette, E. C. Taylor, Jr., and C. K. Cain, J. Am. Chem. Soc. 69, 1814 (1947). 5 A. Bendich, S. S. Furst, and G. B. Brown, J. Biol. Chem. 185, 423 (1950).

616

[26]


I

II 1

z--c~

II

Z

II

I

"7,

I N

T

=~-~=~ ~ II

~~o

I N

+

II

Z --r~ = Z

z

~z +

I

"

[26]


617

bubble it into the solution. Seal the tube, heat it to 100° with occasional shaking, cool, and aerate the contents at 50 to 60° for 30 minutes to remove any excess NH3 which might be present. If a precipitate of NH4C1 has formed, filter the hot mixture and then evaporate the filtrate to dryness in vacuo over P205 and H~SO4. To synthesize 2-C14-adenine, use NaC~4N in the above procedure; for 1,3-N ~5-adenine, use N ~SH4NO3. Guanidine Nitrate ( V I I ) . Slowly add a solution of 10.5 g. of KCN in 48 ml. of water to 6.4 ml. of bromine in 2 ml. of water, maintaining the temperature below 30 °. Distill the reaction mixture at 58 to 68 °, collect the distillate (cyanogen bromide), and dry it over P20~. The yield is about 50%. Generate NHs from 12.4 g. of NH4NO3 in 30 ml. of water, dry it over NaOH, and pass it into 65 ml. of ethanol cooled in a dry ice-ethanol bath. Add 9 g. of the cyanogen bromide dissolved in 50 ml. of ether, stopper the flask, and leave the mixture at room temperature overnight. Remove any excess NH3 by aeration and NH4Br b y filtration, and evaporate the filtrate to dryness in vacuo. The yield of cyanamide is approximately 85 %. Dissolve 1.65 g. of the cyanamide in 10 ml. of a solution containing 4.72 g. of NH4NO3, and heat the resulting mixture in a bomb tube at 155 ° for 3 hours. Chill the tube, and collect the guanidine nitrate. The yield is about 50%. As in the synthesis of formamidine, both C 14 and N ~5 may be introduced into the guanidine for the synthesis of 2-C 14- and 2-amino-l,3-N 1.~_ guanine, or 2,6-diaminopurine. N~5-Guanidine may also be readily synthesized by reacting N15H4NO3 with dicyandiamide. 6 Ethylcyanoacetate ( V I I I ) and Malononitrile (II). Add 740 mg. of NaCN to 5 ml. of 3.2 M sodium chloracetate, and boil the mixture gently for 5 minutes. After the mixture cools, add 2 ml. of concentrated HC1, and evaporate the resulting solution to dryness at 60 to 70 ° i~ vacuo. Repeatedly extract the sodium cyanoacetate from this residue with about twenty 5-ml. portions of ether. For the synthesis of ethylcyanoacetate/ add a slight excess of HC1 to the ethereal solution of sodium cyanoacetate, and remove the precipitated NaC1. Distill off the ether, wash the residue with ethanol, distill off the excess ethanol at 50 to 60°, and boil the residue under reflux for 3 hours in a mixture of 5 ml. of absolute ethanol and 1 ml. of concentrated H2SO4. Remove the ethanol and water by distillation in vacuo, 6 T. L. Davis, in "Organic Syntheses" (Gilman and Blatt, eds.), Coll. Vol. I, p. 302. John Wiley & Sons, New York, 1941. 7 j. K. H. Inglis, in "Organic Syntheses" (Gilman and Blatt, eds.), Coll. Vol. I, p. 254. John Wiley & Sons, New York, 1951.

618


[26]

and neutralize the H2SO4 with Na2CO~. Extract the ester with ether, and distill off the ether in vacuo. The ethyl cyanoacetate is in the distillate obtained at 94 to 99 ° (16 mm.). The yield is about 75%. Ethyl cyanoacetate is used for the synthesis of guanine. The ethereal solution of sodium cyanoacetate, the preparation of which was described above, may also be used for the synthesis of malononitrile. ~ Add the sodium cyanoacetate to an ethereal solution containing 2 g. of diazomethane. Slowly evaporate the solution at room temperature, treat the residual oil (methyl cyanoacetate) with 2 ml. of concentrated NH4OH, and warm the mixture gently. Add an additional 2 ml. of concentrated NH4OH, and leave the solution at room temperature overnight. Then evaporate it to dryness over P205. The product is cyanoacetamide. Place 400 rag. of the cyanoacctamide in a sublimation apparatus with 400 mg. of PC15, evacuate the apparatus, and place it in a boiling water bath. After the reaction has subsided, cool the cold finger to - 5 °, and heat the reaction mixture to 160 ° for 10 minutes. Collect the malononitrile in the cold finger. The yield is about 30 %. Malononitrile is used for the synthesis of adenine and 2,6-diaminopurine. To prepare 4,6-C14-adenine, 4,6-C~4-2,6-diaminopurine, and 4-C TMguanine, use NaC~4N in the preparation of sodium cyanoacetate. This is not a particularly efiicient synthesis, however, since the isotope is introduced at an early stage of the reaction sequence. ~,6-Diaminopyrimidine (III). Add 8 g. of malononitrile dissolved in 30 ml. of dry n-butanol to about 8 g. of formamidine hydrochloride. Then add 125 ml. of sodium butoxide (1 M sodium butoxide in anhydrous n-butanol) in several portions with continual stirring, and boil the mixture gently for 4 hours. Cool the mixture in ice, collect the precipitate, wash it with water and then with ethanol, and dry it at 110 °. The yield is 70 to 80 %. $,5,6-Triaminopyrimidine Bisulfile (IV). Prepare a homogeneous suspension of 5 g. of III in 15 ml. of water. Add glacial acetic acid until the mixture is neutral to litmus, and then an additional 3 ml. of glacial acetic acid to dissolve the material completely. Then add a solution of 3.4 g. of NAN02 in 8 ml. of water. Stir the solution vigorously during this addition. Quickly collect the bright red precipitate which forms, wash it with water, and suspend the solid in 30 ml. of water. Heat this mixture to 60 to 70 °, stirring continually, and add solid sodium hydrosulfite until the red color completely disappears. Then heat the mixture to boiling, filter it, cool the filtrate, and collect the precipitate. The yield is 50 to 60%. ~,6-Diamino-5-formamidopyrimidine Hydrochloride (V). Dissolve 400 mg. of sodium formate in 4.5 ml. of water, add 1.2 ml. of 5 N HC1, and

[26]


619

then add 1 g. of IV. Let the solution stand at room temperature for 2 hours, then cool it in ice and collect the crystals which form. The yield is about 70%. To synthesize 8-C14-adenine, use C14-formate in this step. Adenine Hydrochloride (VI). Add 117 mg. of V to a tube containing 0.75 ml. of freshly distilled 4-formylmorpholine, and heat the mixture at 200 ° for 80 minutes. Cool the tube, and leave it at 0 ° overnight. Collect the precipitate, wash it with ethanol, and recrystallize it from 2 N HC1. Dry the product at 70 °. The yield is about 60%. The formyl group of V exchanges with the formylmorpholine to a significant extent. This results in about a 20% reduction in the specific activity of the adenine when isotope is introduced into position 8. This can be avoided by using diethanolamine as the solvent instead of formylmorpholineJ~ Except for the differences in the initial reactants, as indicated in Fig. 1, guanine and 2,6-diaminopurine may be synthesized by this same procedure.

Synthesis of 4-Amino-5-imidazolecarboxamide (Fig. 2) The following procedure has been described by Shaw and Woolley. 8 It is most applicable to the synthesis of the 2-C~4-carboxamide. The original procedure has been modified slightly at the point of introduction of the formyl group in order to adapt the method to the synthesis of labeled material2 Imino Ether of Ethyl Cyanoacetate (I). ~° Place 11.3 g. of ethyl cyanoacetate, 7 ml. of absolute ethanol, and 7 ml. of anhydrous ether into a flask cooled in an ice-salt bath. Bubble dry HC1 through the solution until a weight increase of 4.1 g. is reached. Then stopper the flask, and leave it for 24 hours in the cooling mixture. Collect the product, and wash it with anhydrous ether. The yield is about 80%. Labeled ethyl eyanoacetate (see above) may be used at this point to prepare 4-C14-carboxamide. Malonamamidine Hydrochloride (II). Suspend 3 g. of I in 30 ml. of absolute ethanol, which has been previously saturated with NH3, and bubble NH~ through the mixture for 4 hours at room temperature with occasional shaking. Leave the flask at room temperature for about 5 days, and then collect the precipitate. The yield is 75 %. Phenyldiazomalonamamidine Hydrochloride (III). Dissolve 1 ml. of aniline in 6.5 ml. of 6 N HC1, and add 0.85 g. of NaNO2 in 5 ml. of water, maintaining the temperature below 5°. Pour this solution of phenyldiazochloride into a solution of 1.5 g. of II dissolved in 7.5 ml. of water, and 7~ R. A b r a h m s a n d L. Clark, J. Am. Chem. Soc. 73, 4609 (1951). 8 E. Shaw a n d D. W. Woolley, J. Biol. Chem. 181, 89 (1949). 9 I. Lin, personal communication. ~0 S. A. Glickman a n d A. C. Cope, J. Am. Chem. Soc. 67, 1017 (1945).

620


q~

•~ r ) ~

=r)~

I

;.-.

I N

t

Z

0

0

~,~ ~°,~ ~--0

~

----~

~ ~, .~

t

~

t

~

G

G

q G +

~5 z £ o °

~

[26]

[26]


621

adiust the mixture to pH 4 with concentrated sodium acetate. Leave this mixture at room temperature for several hours and then at 0 ° overnight. Collect the yellow needles, and dry them in vacuo. The yield is 90%. Formamidomalonamamidine Hydrochloride (IV). Add 300 mg. of III, 180 mg. of zinc dust, and 0.7 ml. of anhydrous formic acid to a small centrifuge tube, and heat the mixture at 60 to 65 ° for 15 minutes with continual stirring. Add an additional 30 mg. of zinc dust every 3 or 4 minutes and 0.1 to 0.2 ml. of formic acid if the mixture becomes dry. Chill the tube, centrifuge down the precipitate, and wash it well with water. Evaporate the combined supernatant fluids to dryness at 40 to 50°, and take the residue up in 20 ml. of water. Bubble H~S through the solution to remove any zinc ions which might be present, and reconcentrate the combined supernatant fluid and water washes of the zinc sulfide precipitate. Add 2 mh of absolute ethanol to the oily residue, and evaporate it to dryness. Repeat this procedure twice. Take the resulting yellow oil up into 10 ml. of absolute methanol, chill the solution, acidify it with methanolic HC1, and add 60 ml. of cold anhydrous ether to precipitate the product. Collect the precipitate, and dry it at room temperature. It may be recrystallized from about 10 ml. of 85% ethanol by the careful addition of ether. The yield is 65 %. 4-Amino-5-imidazolecarboxamide Hydrochloride (V). Slowly heat IV to 125 to 130°, and maintain it at that temperature for 1.5 hours, removing the water formed during the cyclization with a vacuum pump at 15minute intervals. Heat the residue with 4 ml. of saturated methanolic HC1 for 15 minutes at 80 to 90°, and then repeatedly evaporate the mixture to dryness at 40 to 50° to remove the HC1. Then dissolve the final residue in 1.5 ml. of water, and add 4 ml. of saturated sodium picrate. Chill the solution overnight at - 15°, and collect the carboxamide picrate which may be decolorized with charcoal and recrystallized from 85% ethanol. Dry this material in vacuo. The carboxamide hydrochloride, which is more useful in biological experiments, may readily be prepared from the picrate. Suspend the carboxamide picrate in 0.6 ml. of acetouic HC1, and collect the crystalline hydrochloride which precipitates. Repeat this procedure until the supernatant fluid is completely free of yellow color. Dry the hydroehloride in vacuo over P20~,. The yield is 60%. To synthesize 2-C14-4-amino-5-imidazolecarboxamide, use HC~OOII in the preparation of formamidomalonamamidine.

Synthesis of Hypoxanthine (Fig. 3) The following procedure is a modified version ~ of Shaw's. H If 2-C~*-4amino-5-iinidazolecarboxamide is used as the starting material, 8-C ~1~ E. Shaw, J. Biol. Chem. 185, 439 (1950).

622


[26]

hypoxanthine will be the product; if HC~4OONa is used in the first step, 2-C~4-hypoxanthine will be obtained. Note that the system of numbering is different for the carboxamide and hypoxanthine. 4-Formamido-5-imidazolecarboxamide (I). Add 100 mg. of 4-amino-5imidazolecarboxamide hydrochloride and 0.3 ml. of acetic anhydride to 50 mg. of sodium formate dissolved in 0.12 ml. of 98% formic acid. Heat this mixture at 70 ° for 1 hour with occasional stirring and then at 80 ° for 15 minutes. Evaporate the mixture to dryness at 50 °, wash the residue with three 3-ml. portions of water at 45 °, once with ethanol, and finally with acetone. Dry the product in vacuo. The yield is 80 %.

H2N--C--NH H2NC--

N

0 II HC--NH--C--NH H~N

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N

I

HN--C=O HCL ~ - N H --

N II

Fia. 3. Synthesis of hypoxanthine.

Hypoxanthine (II). Dissolve 75 mg. of I in 4.5 ml. of sodium ethoxide (0.1 M sodium ethoxide in absolute ethanol), and heat for 3 hours at 75 to 80 ° with occasional stirring. Chill the mixture in ice, and collect the precipitate. Take the residue up in a few milliliters of HC1, and heat the mixture at 80 ° for 15 minutes. Chill, collect the precipitate, and dissolve it in the minimal amount of warm 1 N KOH. Then adjust the solution to pH 4 with glacial acetic acid, chill overnight, collect the hypoxanthine, wash it with water, absolute ethanol, and ether, and then dry it in vacuo. The yield is 75 %. This procedure may also be used for the synthesis of adenine ~ from 4-amino-5-imidazolecarboxamidine dihydrochloride. This latter compound is synthesized by the method described for the preparation of 4-amino-5-imidazolecarboxamide with the substitution of malononitrile for ethyl cyanoacetate. This synthesis is especially useful for the preparation of 2-Ci4-adenine. S y n t h e s i s of Xanthine

Xanthine may be synthesized from urea and ethyl cyanoacetate by the procedure described for adenine. With appropriately labeled urea (the synthesis of which is described below) 2-C '4- and 1,3-N15-xanthine may be synthesized. Xanthine may also be synthesized from 4-amino-5imidazolecarboxamide hydrochloride. 1' Fuse equal amounts of the carboxamide and urea at 175° for 2 hours. Cool the melt, grind it with water, and collect the precipitate. Dissolve the xanthine in 1 N KOH, 12 A. R. P. Paterson and S. H. Zbarsky, J. Am. Chem. Soc. 75, 5753 (1953).

[26]


623

decolorize it with charcoal, and precipitate it with glacial acetic acid. Dry the product i n vacuo. The yield is about 75%. If 2-C~4-carboxamide is used, 8-C~4-xanthine is obtained; C~4-urea results in 2-C~4-xanthine. Note again the difference in numbering of the carboxamide and purine.

Synthesis of Isoguanine This compound may be synthesized from 4-amino-5-imidazolecarboxamidine dihydrochloride and urea by the procedure described above for the synthesis of xanthine from 4-amino-5-imidazoleearboxamide.

Synthesis of Uric Acid (Fig. 4) By the following procedure label may be introduced into the 2, the 8, or the 4 and 6 carbon and into the 1 and 3 or the 9 nitrogen positions of uric acid. Urea. Dissolve 300 mg. of cyanamide in 3 ml. of water and 0.5 ml. of concentrated HC1.13 Boil the solution under reflux for 10 minutes, cool, and neutralize it with Na2COa. Evaporate the neutralized solution to dryness i n vacuo, and extract the residue with small portions of boiling ethanol. Evaporate the combined ethanol extracts to dryness, and dry the residue over P~05 in vacuo. Extract this residue with acetone, evaporate the solution to dryness, and complete the drying over P~O~. The yield is 90%. Both C 14- and N15-1abeled urea may be synthesized from suitably labeled cyanamide, the preparation of which was described above. Diethylmalonate. Heat the residue of sodium cyanoacetate (prepared as described for the synthesis of ethyl cyanoacetate) in 5 ml. of a 1 : 1 mixture of ethanol and concentrated H2SO~ on a boiling water bath for 1 hour.I4 Cool the flask rapidly, add about 2 ml. of water, and remove the insoluble residue. Wash this undissolved salt with ether, and extract the supernatant fluid with ether. Distill off the ether i n vacuo from the combined extracts, and collect the product which distills at 92 to 94 ° (16 mm.). The yield is 50%. By using labeled cyanoacetate, diethyl malonate may be synthesized with C 14 in both carboxyl groups. Barbiluric A c i d ( I ) . The condensation of urea and diethylmalonate is carried out in the same manner as the condensation of formamidine and malononitrile described for the synthesis of adenine. If Cl*-urea is employed in this step, 2-C~4-uric acid will result; 4,6-C~4-uric acid is obtained from carboxyl-labeled diethyl malonate. 13S. H. Zbarsky and I. Fischer, Can. J. Research B27, 81 (1949). 14A. I. Vogel, "Practical Organic Chemistry," p. 469. Longmans, Green and Co., New York, 1951.

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Uramil (II). The amination of barbituric acid may be accomplished by the procedure described above (see p. 618) for the synthesis of 4,5,6-triaminopyrimidine. Uric Acid ( I V ) . Fuse 55 mg. of urea with 500 mg. of uramil at 150 to 170° for 45 minutes. 15 Extract the cooled melt with 20 ml. of boiling water, remove the insoluble material, decolorize the filtrate with charcoal, concentrate it to 6 ml., and collect the precipitated ammonium pseudourate (III). Dissolve this in aqueous NaOH, and precipitate the uric acid with HC1. The yield is about 35%. If C~4-urea is used in this step, 8-C14-uric acid is obtained; if N~B-urea is used, only position 9 of uric acid should be labeled. In practice, however, some N ~5 enters nitrogen 7.

Synthesis of Uracil~ Thymine, Isocytosine, and 5-Methyl Isocytosine (Fig. 5) These four compounds may be synthesized by the same general procedure. 16-1s It is most applicable to the synthesis of 2-C ~4- and 1,3-N ~6pyrimidines, although, through the use of appropriately labeled malic or methyl malic acid, isotope may be introduced into the 4, 5, or 6 positions. fl-Melhyl Malic Acid (ii).17.~9 Under anhydrous conditions, add 2.5 to 3.0 g. of ether-washed, powdered sodium to 7 ml. of ether. Add dropwise 175 ml. of absolute ethanol to this solution. When the reaction stops, immerse the flask in an ice bath, and slowly add a mixture of 12 g. of ethyl propionate and 17.5 g. of ethyl oxalate. Distill off the ether and ethanol, and solidify the residue by cooling. Dissolve the residue in 25 ml. of cold, 33 % acetic acid, and let the solution stand at room temperature for several hours. Extract this solution with four 20-ml. portions of ether, and then wash the combined ethereal extracts with water, 10% NaHC03, and water again. Remove the ether by distillation, and distill over the product between 114 and 116° (10 mm.). The yield of ethyl ethyloxalylpropionate is 70 %. Shake 15 g. of this material and 75 mg. of platinum oxide catalyst for 2 to 3 hours under a hydrogen pressure of 3 to 4 arm. Diethyl-~-methylmalate is obtained in 90% yield. To remove the ethyl groups, boil 10 g. of the ester in 40 ml. of 0.15 N HC1 under reflux for about 5 hours. Then 15L. F. Cavalieri, V. E. Blairi and G. B. Brown, J. Am. Chem. Soc. 70, 1240 (1948). 16D. Davidson and O. Baudisch, J. Am. Chem. ~oc. 48, 2379 (1926). 17H. W. Scherp, J. Am. Chem. Soc. 68, 912 (1946). 18W. T. Caldwell and H. B. Kime, J. Am. Chem. Soc. 62, 2365 (1940). 19R. F. B. Fox and S. M. McElvain, in "Organic Syntheses" (Gilmanand Blatt, eds.), Coll. Vol. II, p. 272. John Wiley & Sons, New York, 1951.

626


[26]

concentrate the solution to a sirup i n vacuo, and dry the product over NaOH again i n vacuo. The yield is about 95 %. f~-Methyl malic acid is used in the synthesis of thymine (III) and 5-methyl isocytosine (V). Uracil ( I ) . Chill 2 ml. of fuming sulfuric acid to 0 °, and gradually add 500 rag. of urea with stirring. Then add 500 mg. of malic acid, s° and heat the mixture on a steam bath for 1 hour. Cool, and pour the reaction mixture into 6 ml. of water. The uracil precipitates on further cooling. It may be decolorized with charcoal and recrystallized from water. The yield is about 50%. If C14-urea is used in this synthesis, 2-C14-uracil is the product; N 15urea results in 1,3-NlS-uracil. C ~4- and N~5-Guanidine may be used to synthesize similarly labeled isocytosine (IV) and 5-methyl isocytosine. This procedure has also been used for the synthesis of methyl-labeled thymine starting from sodium propionate-C14. ~°! Uracil and thymine may also be synthesized by the condensation of thiourea with ethyl formylacetate and ethyl formylpropionate, respectively, and the subsequent conversion of the 2-thio analogs to the desired pyrimidines. 2~

Synthesis of Cytosine and 2~4-Diaminopyrimidine (Fig. 6) Cytosine and 2,4-diaminopyrimidine may be synthesized by a procedure which differs only slightly from that described above for the other pyrimidines. 2~,~a By this method, the 2-C ~4 and 1,3-N ~5 compounds may be synthesized. Cyanoacetaldehyde Diethylacetal (1). 54 Cool a mixture of 11.5 g. of sodium, 30 ml. of absolute ethanol, and 200 ml. of absolute ether to - 17°. Then add 44 g. of ethyl formate (previously cooled to - 1 5 °) over a 30-minute period with continual stirring. Leave the solution for 3 hours at - 15° and 12 more hours at room temperature. Collect the solid, wash it twice with anhydrous ether, and dry it over H~SO4. This material contains the desired sodium ethyl formylacetate and the by-products sodium ethyl acetoacetate and sodium formate. The yield is about 50 %. Add 50 g. of the above product, in small portions, to 460 ml. of absolute ethanol containing 60 g. of dry HC1. Stir the mixture for 3 hours, neutralize it with NaHCO3, and remove the insoluble residue. Wash this residue with ethanol, evaporate the combined supernatant fluids to s0 For synthesis of labeled malic acid, see Vol. IV [24]. ~oi R. B. Henderson, R. M. Fink, and K. Fink, J. Am. Chem. Soe. 77, 6381 (1955). 21A. A. Plentl and R. Schoenheimer,J. Biol. Chem. 153, 203 (1944). ~ A. Bendieh, H. Getler, and G. B. Brown, J. Biol. Chem. 177, 565 (1949). 23A. Bendieh, W. D. Geren, and G. B. Brown, J. Biol. Chem. 185, 435 (1950). ~4S. M. MeElvain and R. L. Clarke, J. Am. Chem. Soe. 69, 2657 (1947).

[26]


627

dryness at 60° in vacuo, and distill over the residual oil. Ethyl/~fl-diethoxypropionate is obtained in 35 % yield. Stir 25 g. of this compound with 150 ml. of concentrated NH4OH at room temperature until the mixture becomes homogeneous. Then remove the water and excess NH3 at 50° in vacuo. Heat the residue at 60 ° (10 mm.) for 30 minutes, then dissolve it in 20 ml. of benzene and add 60 ml. of warm (50 °) ether. Cool this mixture to 0 ° , and collect the precipitated /3fl-diethoxypropionamide. The yield is about 80 %. NH2 CN NH~ CN N=-C--NH2 I I I L I I C---~O + CH~. --~ O = C CH2--~ O=-C CH I I L I I ]L NH2 CH(OC2Hs)2 N=CH HN--CH I

II

NH.2 CN N=C--NH2 I I I I C = N H ~ + CH2 --~ H2NC CH I I II tl NH2 CH(OC2H~)2 N--CH III

FIG. 6. Synthesis of cytosine and 2,4-diaminopyrimidine.

Under anhydrous conditions, slowly add 13.5 g. of P205 to a mixture of 11.5 g. of the above product, 25 ml. of benzene, and 14.5 g. of triethylamine. Stopper the flask, and stir the contents with a sealed stirrer. Heat the mixture at 80 to 90° until the exothermic reaction begins, and then remove the heat. When the reaction stops, boil the reaction mixture under reflux for 1 hour. Then distill off the benzene and triethylamine at 120° . When this is completed, gradually reduce the pressure and raise the temperature, and collect the resultant distillate in a dry ice-acetone bath. Redistill this material, and collect the portion which boils between 90 and 95 ° (12 mm.). Add this distillate to 34 ml. of absolute ethanol containing 5 mg. of sodium, and leave the mixture for 1 hour at room temperature. Neutralize the solution to phenolphthalein with glacial acetic acid, remove the ethanol, and redistill the product as above. The yield of cyanoacetaldehyde diethylacetal is approximately 80 %. Cytosine (II). Add 700 mg. of urea and 1.7 ml. of I to 9 ml. of n-butanol containing 270 mg. of sodium. Boil this mixture under reflux for 2 hours. Then chill the vessel, collect the sodium salt of the intermediate, and wash it with n-butanol and ether. (In the synthesis of 2,4-diaminopyrimidine, only sodium nitrate precipitates at this point. Acidify the supernatant with 6 N H2SO4, and proceed as below.) Dissolve the intermediate product in 15 ml. of hot 2 N H~SO,, decolorize the solution with charcoal, and add 2 vol. of hot ethanol. Cool

628


[26]

the mixture, and collect the precipitated cytosine sulfate. Dissolve it in the minimal amount of water, make the solution alkaline with NH3, decolorize with charcoal, and adjust the solution to pH 7 to 7.5 with glacial acetic acid. On cooling, cytosine crystallizes in approximately 50% yield. To synthesize 2,4-diaminopyrimidine (III), substitute guanidine for the urea. The use of C 14- or N15-1abcled urea and guanidine results in 2-C 14- or 1,3-N15-pyrimidines. In the case of 2,4-diaminopyrimidine, the 2-amino group will also be labeled. Synthesis of Orofic Acid (Fig. 7) The following procedure ~5 is applicable to the synthesis of 1-N ~5- and 4,5- or 6-C14-orotic acid from appropriately labeled aspartic acid ~6 and 3-N 1~- and 2-C14-orotic acid from labeled KCNO. Potassium Cyanate. Mix 0.875 g. of finely powdered dihydrous K2C08 and 1 g. of urea in a large porcelain crucible. ~7 Gently fuse this mixture over an open flame until the evolution of NH3 ceases and a clear melt is obtained. On cooling, the molten KCNO crystallizes. The yield is 98%. N 15- or C~4-Labeled KCNO may be synthesized from appropriately labeled urea. Ureidosuccinic Acid (I). Dissolve 1.64 g. of aspartic acid in about 10 ml. of 20% KOH, and adjust the solution to pH 5.5 with concentrated HC1. Chill the solution, and dissolve in it 1 g. of finely powdered KCNO. Allow the solution to stand for 45 hours at room temperature. Acidify the solution to pH 2 with concentrated HC1, cool it, and collect the crystalline product after 2 hours. The ureidosuccinic acid may be recrystallized from water. The yield is about 70 %. For the synthesis of orotic acid, DL-aspartic acid is used, since the final product is not optically active. If the ureidosuccinic acid, itself, is to be used in biological experiments, it is best to have the L-isomer, in which case L-aspartic acid must be used in the synthesis. L-Ureidosuccinic acid will not crystallize on acidification of the reaction mixture, but it may readily be isolated. 28 Adjust the solution to pH 4 to 5, and chromatograph it on a Dowex 2 resin column (chloride form, 200 to 500 mesh, 10 X 4 cm.). Wash the column with approximately 1500 ml. of 0.005 N HC1, and then elute the L-ureidosuccinic acid with 0.05 N HC1. The compound may be detected 25 j . F. Nyc and H. K. Mitchell, J. Am. Chem. Soc. 69, 2s For the synthesis of labeled aspartic acid see Vol. IV 37 A. Scattergood, in "Inorganic Syntheses" (Fernelius, Hill Book Company, New York, 1946. 28 p. Reiehard and U. Lagerkvist, Acta Chem. Scand. 7,

1382 (1947). [28]. ed.), Vol. II, p. 86. McGraw1207 (1953).

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631

Isolation Excretory Products of Purine and Pyrimidine Metabolism Uric Acid. Extract the excreta with 100 ml. of 97% ethanol on a boiling water bath for 10 minutes. 31,~2 Filter and grind the moist residue to a uniform powder. Repeat this extraction three times, and then boil off the remaining ethanol. Boil the dry powder in 0.15 N HC1 for 3 to 4 minutes, cool the solution, and add enough 0.25 N piperidine to make the contents alkaline to phenolphthalein. Heat the mixture on a steam bath for 45 minutes, cool, and filter it. Add 9 vol. of water to the filtrate and 16.5 g. of NH~C1 per 50 ml. of this solution. Heat the solution at 60 to 70 ° for 20 minutes, and allow it to cool slowly for 24 hours. Collect the crystals, wash them with 10% NH4SO4, then water, and finally dissolve them in sufficient HC1 to make the resulting solution acid to litmus. Concentrate this solution, and collect the crystalline uric acid. Allantoin. Add 35 ml. of 35% phosphotungstic acid (recrystallized from ether) to 50 ml. of urine, and remove the precipitate thus formed. 33 Add a little more phosphotungstic acid to ensure complete precipitation. Then add 35 ml. of 5 % basic lead acetate to the supernatant fluid, remove the precipitate, and again test for completeness of precipitation. Mix this supernatant fluid with 35 ml. of 5 % H2S04, and remove the PbSO4. Neutralize the filtrate to litmus with 5% NaOH, and precipitate the allantoin with 100 ml. of a solution containing 1 g. of mercuric acetate and 10 g. of sodium acetate. Leave the mixture overnight, and collect the mercury allantoin precipitate. Wash this with water, and dry it over P205 i n vacuo. Grind the dry powder up in water, and bubble H~S through the mixture. Mercuric sulfide precipitates, and the free allantoin dissolves. Remove the precipitate, aerate the solution to remove the H2S, and concentrate it to 2 to 5 ml. in vacuo. Leave the solution in the cold overnight, and collect the crystalline allantoin. This may be decolorized with charcoal and recrystallized from water. Urea. Add 2 ml. of urine to 24 ml. of water, and then add 50 ml. of glacial acetic acid followed by 5 ml. of a freshly prepared 5 % methanolic solution of xanthydrol. 34 A precipitate begins to form in a few minutes. After 30 minutes, add 10 ml. of water and leave the mixture overnight. Collect the dixanthydrol urea, and recrystallize it from glacial acetic acid.

n j. L. St. Johns and O. Johnson, J. Biol. Chem. 92, 41 (1931). 3~R. B. Fisher, Biochem. J. 29, 2192 (1935). 3s G. B. Brown, P. M. Roll, A. A. Plentl, and L. F. Cavalieri, J. Biol. Chem. 172, 469 (1948). 34O. Fosse and R. D. Bell, Ann. 6, 13 (1916).

632


[26]

Ammonia. Add Permutit to 5 ml. of filtered urine. '~5 Wash the Permutit well with water, and then release the NHa with NaOH and collect it in HC1.

Isolation of Nucleic Acid Purines and Pyrimidines Procedures for the isolation and hydrolysis of nucleic acids and for the separation of the resulting purine and pyrimidine derivatives have been discussed in some detail in Vol. III [100-103, 106]. Only a brief outline of such methods will be presented here, in which will be indicated some special precautions applicable to studies with labeled materials. The procedure of Schmidt and Thannhauser, 86 as modified by LePage and Heidelberger, 37 is very satisfactory for the separation of R N A and DNA from most animal tissues. Homogenize a weighed amount of tissue in cold 4% HC104, and centrifuge the mixture in the cold. Wash the residue with cold 4 % HC104 (to remove acid-soluble purine and pyrimidine derivatives), twice with ethanol (to remove the acid), and then remove the lipids with three extractions with ethanol :ether (3:1) at 40 °. The combined acid solutions may be retained for the further separation of the acid-soluble compounds. Suspend the dry, extracted tissue in 1 N NaOH (10 ml. per 2 g. of the fresh tissue), and incubate the mixture at 34 ° for 16 hours. Under these conditions, everything is dissolved and the R N A is hydrolyzed to its constituent nucleotides. Cool the solution in ice, and add 3.5 ml. of 65 % TCA for every 10 ml. of NaOH used in the hydrolysis. The protein-DNA complex precipitates, and the RNA-nucleotides remain in solution. Wash the precipitate with cold 5% TCA, neutralize the combined supernatant fluids to pH 8 with Ba(OH)2, and add 4 vol. of cold ethanol to precipitate the barium ribonucleotides. Wash the protein-DNA residue with 10 vol. of ethanol, and then suspend it in 10 ml. of a neutral 0.3 M barium perchlorate solution. Heat this suspension in a boiling water bath for 20 minutes, cool it to room temperature, add 70 % HC104 to a final concentration of 2 %, and remove the precipitated protein at room temperature. Neutralize the supernatant fluid to pH 8 with Ba(OH)~, and add 4 vol. of cold ethanol to precipitate the barium DNA. This procedure completely separates R N A and DNA, as can be shown by the fact that no thymine is detectable in the R N A fraction and no uracil in the D N A fraction. It does not work well for bacterial nucleic acids for which special methods are necessary. 35 O. Folin and R. D. Bell, J. Biol. Chem. 29, 329 (1917). 3e G. Schmidt and S. J. Thannhauser, J. Biol. Chem. 161, 83 (1945). 37 G. A. LePage and C. Heidelberger, J. Biol. Chem. 188, 593 (1951),

[26]


633

Hydrolyze 50 to 100 mg. of the barium ribonucleotides or barium DNA in 1.5 ml. of 1 N HC1 at 100° for 1 hour. The products of this hydrolysis are the free purine bases and the pyrimidine nucleotides. Add a slight excess of H2SO4 to remove the barium ions, and chromatograph the solution on a Dowex 50 resin column (hydrogen ion form, 200 to 400 mesh, 90 × 9 mm.). Elute the column with 2 N HC1. The pyrimidine nucleotides come off together in the first few milliliters, since they are not retained by the resin. The guanine and adenine are eluted separately, in that order. Guanine and adenine may be further purified by rechromatographing them on Dowex 50, eluting with less concentrated HC1 in order to bring them off more slowly. Paper chromatographic procedures may be used for further purification if necessary2 s The pyrimidine nucleotides, which are not retained on Dowex 50, are readily separable by chromatography on both Dowex 1 anion exchange resin and paper. 3s If desired, the pyrimidine nucleotides may be hydrolyzed to the free bases which may then be separated by chromatographic techniques. To perform this hydrolysis, add about 50 rag. of the mixed pyrimidine nucleotides to 1 ml. of 12 N HC104, and heat the solution in a glassstoppered tube at 100° for 40 to 160 minutes with occasional shaking. After the hydrolysis, most of the excess perchlorate can be removed as the relatively insoluble potassium salt by the addition of KOH. If this solution is passed through a Dowex 50 resin column, cytosine is retained and uracil and thymine are not. The cytosine may then be eluted with 0.5 N HC1. Uracil and thymine are separable on Dowex 1 resin.3S The DNA of T~, T4, and T6 bacteriophage contain hydroxymethylcytosine instead of cytosine. This compound is destroyed completely by HC104 hydrolysis. In order to preserve this base, 39 pyrimidine nueleotides from these sources should be hydrolyzed with 88 % HCOOH for 30 minutes at 175° . In all these chromatographic procedures the various compounds are identified and followed by means of their ultraviolet absorption spectra. The spectral purity of a given compound is best determined by the ratios of its optical density at various wavelengths. Concentrations of an impurity which are too low to alter the position of the maxima or minima of a given absorption curve may cause significant discrepancies in such ratios. Impurities might also be present which do not absorb ultraviolet light and are thus not dhtected. Although these will not interfere with the 38 For these techniques see Vol. I I I [106-108]. 39 G. R. W y a t t and S. S. Cohen, Nature 170, 1072 (1952).

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636


[26]

tion to 38 ° to decompose the hydroxyacetylene diureidocarboxylic acid (II) to allantoin (III) and COs, and aerate the solution for an additional 60 minutes, collecting the evolved COs in Ba(OH)~. This BaC03 represents carbon 6 of the original uric acid. Neutralize the solution to pH 7, make it 0.1 M with respect to Na2C03, and heat it on a boiling water bath for 20 minutes to convert the allantoin to allantoic acid (IV). Make this reaction mixture acid to thymol blue with 10 N HN03, and place it on a boiling water bath for an additional 4 minutes. Cool the solution, neutralize it to pH 7, and add 5 ml. of basic lead acetate to precipitate the glyoxylic acid (VI) as the lead salt. The supernatant fluid contains urea (V). Transfer the precipitate to a centrifuge tube in about 10 ml. of water, and add 1.5 ml. of 4 N H2SO4. Stir this well, and remove the PbSO4. Wash the precipitate, and combine the supernatant fluids. Add 250 to 300 mg. of semicarbazide hydrochloride to this solution, and leave it at 2 ° overnight. Collect the crystalline semicarbazone of glyoxylic acid (VII), wash and recrystallize it, and dry it in vacuo. Dissolve 20 rag. of the semicarbazone in 30 ml. of water, and add 1.5 ml. of 1.5 N KMn04 and 4 ml. of 1 N H2S04. Incubate the mixture at 38 ° in a C02-free atmosphere, and aerate over the evolved COs. The COp collected during the first 7 minutes of the reaction contains equal amounts of the carbazone carbon and the carboxyl carbon of the glyoxylic acid. The specific activity of the BaC08 derived from this is equal to onehalf the specific activity of carbon 5 of the uric acid. The C02 evolved during the next 7 minutes is not collected. The remaining C02 is derived from the aldehyde carbon of the glyoxylic acid and represents carbon 4 of uric acid. Evaporate to dryness the supernatant fluid obtained from the basic lead acetate precipitation, and extract the urea from the residue with absolute ethanol. Evaporate off the ethanol, dissolve the urea in water, acidify the solution, and aerate it with H2S to remove any lead ions which might be present. Neutralize the solution to pH 7 with 1 N NaOH, and add an equal volume of sodium acetate buffer (0.1 M, pH 5.0). Aerate the solution to remove any COs present, add 100 rag. of urease suspended in 3 ml. of acetate buffer, and incubate the mixture at 38 ° for 30 to 45 minutes. Aerate out and collect the COs which represents carbons 2 and 8 of the uric acid. The incubation mixture may then be made alkaline, aerated, and the NH8 collected in acid. This NH3 is derived equally from all four nitrogen atoms of the uric acid. Degradation B. This procedure 48 enables the separation of carbon 2 from carbon 8 and nitrogens 1 and 3 from nitrogens 7 and 9. 4s j . C. Sonne, J. M. Buchanan, and A. M. DeUuva, J. Biol. Chem. 178, 69 (1948).

[9.6]


637

Dissolve 60 mg. of uric acid in 0.45 ml. of 5.5 N HC1 in a conical 12-ml. centrifuge tube. Add 25 mg. of KClOa over a period of 20 to 30 minutes with continual stirring. Then add 0.45 ml. of water, and let the vessel stand at room temperature for 1 hour. Bubble H2S through the solution for 10 to 15 minutes, and let the vessel stand in the cold overnight. The oxidation converts the uric acid to urea and alloxan (VIII), and the subsequent reduction forms alloxantin (IX) from the alloxan. Collect the precipitate (a mixture of alloxantin and free sulfur) in the cold. Wash it with four 0.5-ml. portions of water, and adjust the combined supernatant fluids to neutrality with 1 N NaOH. Incubate this solution with urease as described above, and collect the CO~, derived from carbon 8 of the uric acid, and the NH3, which represents an equal mixture of nitrogens 7 and 9. Take the alloxantin precipitate up in 2 ml. of water, and heat it on a boiling water bath. Remove the insoluble sulfur, cool the filtrate, add 100 rag. of PbO2, and adjust the mixture to pH 2. Heat the mixture for 20 minutes in a water bath, remove the excess Pb02, and wash the precipitate once with hot water. Treat the combined supernatant fluids, which contain urea, with H2S to remove lead ions, and then degrade the urea as above. The resultant BaCO3 is derived from carbon 2 of uric acid, and the NH3 is derived equally from nitrogens 1 and 3. In practice, it is found that the BaC03 derived from carbon 8 of uric acid is contaminated to the extent of 10 to 15 % with carbonate derived from carbon 2. The reverse contamination does not occur. Degradation C. This procedure 44 gives carbons 4 and 5 and nitrogen 7 as glycine. Therefore the isotope content of nitrogen 7 is obtained directly and that of nitrogen 9 can be calculated by subtracting the value for nitrogen 7 from that obtained for nitrogens 7 and 9 by degradation B. Dissolve 12 rag. of uric acid in 1 ml. of concentrated HC1, and hydrolyze it in a bomb tube at 160° for 18 hours. Aerate off the NHa which is derived from nitrogens 1, 3, and 9 with a water pump at 50 °. Neutralize the solution, and concentrate it to 1 ml. Then add 50 rag. of citrate buffer (pH 2.5) and 50 rag. of ninhydrin, and heat the solution for 8 minutes to convert the glycine amino group to ammonia. 45 Treat the solution with H:S, dilute it to 10 ml., filter, make it alkaline, and aerate off the NH3. This NH3 is derived from nitrogen 7 of uric acid. The isotope content of nitrogen 9 can be independently calculated by subtracting the value for nitrogens 1W3 (degradation B) from the value for nitrogens 1~-3+9 obtained by this degradation. The glycine may also be isolated as hippuric acid for the determina44 C. Wulff, Physiol. Chem. 17, 468 (1892). 45 D. A. MacFadyen, J. Biol. Chem. 155, 507 (1944).

638


[26]

tion of the specific activity of the carbon atoms. This gives an independent value for carbons 4 and 5 of uric acid. Degradation D. This procedure 46,47 separates nitrogens 1 and 7 from nitrogens 3 and 9. Thus, by subtracting the value obtained for nitrogen 7 by degradation C from the value for nitrogens 1 +7, the isotope content of nitrogen 1 can be calculated. Similarly, the isotope content of nitrogen 3 can be calculated by subtracting the value for nitrogen 1 from the value for nitrogens 1~-3 (degradation B) or the value for nitrogen 9 (degradation C) from the value for nitrogens 3~-9 obtained in this degradation. Dissolve 20 mg. of uric acid in 2.5 ml. of water by the addition of 200 rag. of KOH. Then add 0.03 ml. of 30% H~02. Incubate this solution at room temperature for 24 hours with a continual slow aeration. The solution is aerated with acid-washed (NH~-free) air, and the NH3 which is evolved during the reaction is collected in 20 ml. of 2 % boric acid. This NH3 is derived equally from nitrogens 1 and 7. Treat the reaction mixture with a small amount of MnO~ to decompose the excess H~02 and then remove the MnO~ by centrifugation. Cool the supernatant solution, adjust it to pH 5 with 1 ml. of glacial acetic acid, and collect the potassium oxonate (X) which crystallizes overnight. Dissolve this material in the minimal volume of water, add 0.5 ml. of saturated BaCl~, cool, and collect the crystalline barium oxonate. This product can be digested by the Kjeldahl procedure for 6 hours to obtain nitrogens 3 and 9 as NHa.

Cytosine and Uracil (Fig. 10) Cytosine may be deaminated to uracil by the procedure described above for the deamination of the purines. With the aid of two methods, 48 the 4 carbons of uracil may each be obtained separately and the 2 nitrogens together. Degradation A. Dissolve about 20 rag. of uracil (I) in 3 ml. of water with the aid of 0.5 N NaOH and heat. Then add a 5% KMnO4 solution dropwise until the purple color persists. Cool the solution to room temperature, and add a few drops of concentrated H~S04 to precipitate the MnO. Destroy the excess KMn04 with H~O~, remove the precipitate, and wash it with 2 ml. of warm water. Add 1 ml. of 5 N NaOH to the combined supernatant fluids, and heat the solution at 100 ° for 15 minutes. Then acidify the solution to phenol red with 5 N H~SO4, warm it to 100 °, 4s H. Brandenberger, Helv. Chim. Acta 37, 641 (1954). 47 S. C. Hartman, S. B. and M. S. Thesis, Massachusetts Institute of Technology, 1954. ~s U. Lagerkvist~ Acta Chem. ~cand. 7t 114 (1953).

[26]


6~9

t o Izl o+c3

? oC.)-~O ~ t

=

z e3

+ I:I:I

.~

©

T t

z

0--~0-_ o

+

t

©

"~T

0

o--o--o--I~ t

©

I~ !zl I=1 Q--O~----O

0---0---0

II ~

©

©

0

[26]


641

in vacuo, and, after cooling the flask to 15°, add 0.3 ml. of 100% H2SO4. Warm and shake the flask until the material dissolves completely, recool the flask, add 50 mg. of sodium azide, and warm the flask again until all of this dissolves. Connect the flask to two traps, the first containing 5% KMnO4 in 1 N H2SO4 and the second 0.5 N NaOH, and slowly raise the temperature over a 30-minute period from 38 ° to 60 to 70°. After 30 minutes at the final temperature, aerate the solution with CO~-free air for 10 minutes. The evolved COs is collected in the alkali trap and precipitated as BaCO~ by adding an excess of BaC12. This carbonate is derived from the carboxyl group of the propionate and represents carbon 4 of the pyrimidine ring. Replace the KMnO4 trap with 5 ml. of 0.2 N H~S04, and adjust the original reaction mixture to pH 11 to 12 with 5 N NaOH. Aerate the solution for 15 minutes at 90 to 100°, and collect the ethylamine (VII) in the acid trap. Transfer the ethylamine solution to a small, roundbottomed flask containing 5 ml. of 5% KMnO4. Adjust the solution to pH 8, add an additional 0.5 ml. of 0.5 N NaOH, stopper the flask, and heat it at 90 to 100 ° for 5 minutes. Acidify the solution with H~S04, and steam-distill over the acetic acid. This acetic acid can then be degraded stepwise by the above procedure to obtain carbons 5 and 6 of the pyrimidine ring as BaC03.

Thymine Thymine may be degraded by means of two procedures 5° to obtain the methyl carbon and carbons 2 and 4 separately and carbons 5 and 6 together (Fig. 11). Degradalion A. Add 0.075 ml. of bromine to a suspension of 150 mg. of thymine (I) in 1.5 ml. of water. A rapid reaction ensues with the formation of a precipitate. When the layer of bromine has disappeared, heat the mixture on a steam bath until all the precipitate dissolves. Filter the hot solution. On cooling, 5-bromo-6-hydroxyhydrothymine (II) crystallizes. Dissolve this in a 5 % NaHCO3 solution, and then distill the solution. The distillate will contain acetol (III) and the residue urea. The urea may then be degraded as described above to yield carbon 2 of thymine as B a C Q and nitrogens 1 and 3 as NH3. Treat an aliquot of the distillate with an excess of acetic acid and phenylhydrazine and warm gently to precipitate acetol phenylhydrazone. This may be recrystallized from benzene. The acetol is derived from the methyl carbon and carbons 5 and 6 of thymine. Another aliquot of the acetol solution is reacted with an excess of NaOI at alkaline pH for 30 minutes at room temperature. The iodoform which precipitates is 50 O. Baudish and D. Davidson, J. Biol. Chem. 64, 233 (1925).

642


[26]

washed with water and recrystallized from methanol. It is derived from the methyl carbon of thymine. Degradation B. Add the bromohydroxyhydrothymine to a saturated solution of Ba(OH)~, and heat the mixture on a steam bath for 15 to 20 minutes. BaC03 precipitates. This is derived solely from carbon 4 of thymine. O H2NC--NH~-*

CO~ ~- NH3

+

CH.

I C~-O I

-~ CHI~

CH20H / HN--C:O l CH3 / O-~-C HN--CH

~Br

IMethyl CI

m

\

HN--CHOH \ II

~B

\ BaCO,

Fro. 11. Chemical degradation of thymine.

Orotic Acid Orotic acid may be degraded by the procedure B described for uracil degradation. 48 The product in this case is aspartic acid, instead of ~-alanine. The Dowex 50 column should then be developed with 0.5 N HC1 instead of 2 N HC1. Dissolve the residue obtained after chromatography on Dowex 50 in 1.5 ml. of water, neutralize the solution to pH 7 to 8 with NaOH, and add 0.4 ml. of saturated CuSO4. Adjust the solution to pH 5 to 6 with 0.2 N NaOH, and leave it in the cold for several days. Collect the crystalline copper aspartate. Dissolve this material in water, and aerate the solution with H~S to remove the copper. The aspartic acid may then be degraded by procedures described in Vol. IV [28]. In this way, the specific activity of each of the carbon atoms of orotic acid may be determined. The isotope content of nitrogen 1 of orotic acid may be obtained from the a~partic acid and nitrogen 3 by calculation from the isotope content of the original orotic acid.

[27]

BIOSYNTHESIS OF PROTOPORPHYRIN

643

[27] B i o s y n t h e s i s of P r o t o p o r p h y r i n

By DAVID SHEMIN The procedures described below are some of those which have been used in studies on the biosynthesis of protoporphyrin, the porphyrin moiety of hemoglobin. They include the isolation of hemin, the conversion of hemin to protoporphyrin, the reduction of the latter to mesoporphyrin, the oxidative cleavage of mesoporphyrin to crystalline fragments of the molecule representing particular structures of the porphyrin, and the degradation of the latter compounds to keto acids which on further degradation by standard procedures yield samples of carbon dioxide which arise from carbon atoms which had specific positions in the porphyrin compound. The procedures also include the synthesis of radioactive compounds which are intermediates in the biosynthetic chain and are not readily purchasable. Finally, references are given to the biological preparations used in which studies on the biosynthesis of protoporphyrin can be carried out. Preparation of Hemin ~ A 25-ml. sample of whole blood or washed red cells suspended in saline or cells hemolyzed with water is added dropwise, over a period of about 15 to 20 minutes, to a flask containing 75 ml. of glacial acetic acid and 0.5 ml. of a saturated sodium chloride solution, kept at 95 to 100 °. During this procedure the mixture is continuously and rather vigorously stirred. After addition of the blood sample, 2 to 5 drops of concentrated hydrochloric acid is added and the flask heated on a steam bath for about 1 hour. The formed hemin crystals gathered by centrifugation are washed twice successively with 50% acetic acid, water, alcohol, and ether, and then dried. The yield is approximately 70 to 100 mg. The hemin is purified by recrystallization. A 100-mg. sample of hemin in a 12-ml. centrifuge tube is dissolved in 0.5 ml. of pyridine by rubbing with a stirring rod. After solution, 4 ml. of chloroform is added, and the resultant mixture is filtered through a good grade of paper, by gravity, into a flask containing 15 ml. of a glacial acetic acid solution which has been previously saturated with solid sodium chloride. The filter paper is washed with small amounts of chloroform until it is almost colorless. The combined solution is heated carefully until the temperature reaches H. Fischer, Org. Syntheses 21, 53 (1941).

644


[27]

105 ° . At this time practically all the chloroform has been distilled off. Mter the addition of 2 to 3 drops of concentrated hydrochloric acid, the solution is kept at room temperature for 24 hours to complete crystallization. The crystals are recovered and washed as described above. Preparation of Mesoporphyrin

-Fe Hemin

[H] Protoporphyrin

~ Mesoporphyrin

A. Preparation of Protoporphyrin

To a three-necked flask, fitted with a reflux condenser and with an efficient stirrer, are added 1 g. of finely powdered hemin and 70 ml. of 98 % formic acid. The stirred mixture is heated to a gentle boil, and while boiling 2.3 g. of powdered iron is added in several portions during a 20- to 30-minute period. The solution becomes deep red in color. After the addition of the iron, the mixture is refluxed for another 15 minutes. The solution is then cooled and filtered through a sintered-glass funnel. The formic acid solution contains protoporphyrin and some mesoporphyrin, as ascertained by determining its absorption spectra3 B. Reduction of Protoporphyrin to Mesoporphyrin

The above formic acid solution is reduced with hydrogen, under atmospheric pressure, with the aid of about 100 to 200 mg. of palladium black catalyst (Baker and Company, Inc., Newark, New Jersey). 3 The uptake of hydrogen is quite rapid, usually complete in about 1 hour. The amount taken up, however, may be less than the calculated amount, since the solution contains varying amounts of mesoporphyrin formed from protoporphyrin during its preparation from hemin with iron and formic acid. After the uptake of hydrogen has ceased, the solution is filtered through a coarse sintered-glass funnel and poured into 5 vol. of a 30% ammonium acetate solution. The precipitate is collected by centrifugation and washed with water. The mesoporphyrin can be purified further by dissolving the material in about 100 ml. of 2% ammonium hydroxide and then causing its reprecipitation by the addition of acetic acid. The mesoporphyrin is washed with water and dried i n vacuo. The yield is about 80 to 90%. 4 H. Fischer and H. Orth, "Die Chemic des Pyrrols," Vol. II, p. 322. Leipzig, 1937. , S. Granick, or. Biol. Chem. 172, 717 (1948). 4 j. Wittenberg and D. Shemin, or. Biol. Chem. 186, 103 (1950).

[27]


645

Oxidation of Mesoporphyrin to Methylethylmaleimide and Hematinic Acid COOH

I I

CH 3

CH2

I

H~C Mesoporphyrin

CH2

o". ' ~ o H

tt~C

+ o' ~ ' ~ ~0

Methylethytmaleimidc

A.

CIT.~ iT

Hcrnatinic acid

Oxidation

One gram of mesoporphyrin is suspended in 80 ml. of 20% (w/w) of sulfuric acid and stirred with a magnetic stirring bar at room temperature. Over a period of about 30 minutes, 1.8 g. of Cr03 dissolved in a minimum of water is added, and the mixture is stirred for another 17 hours. After this period of time the greenish solution is extracted with ether in a continuous extractor for about 24 hours. The filtered ether solution contains the methylethylmaleimide and the hematinic acid. 5,6

B. Separation of Methylethylmaleimide and Hematinic Acid The above ether solution is extracted once with 40 ml. of a 5 % sodium bicarbonate solution. The separated bicarbonate solution is extracted five times with an equal volume of chloroform. The ether and chloroform solutions containing the methylethylmaleimide are combined. The bicarbonate solution containing the hematinic acid is acidified to about pH2 with 2 N sulfuric acid immediately after the last chloroform extraction. The hematinic acid solution, once acidified, can be stored.

C. Isolation of Methylethylmaleimide The combined ether and chloroform solution is taken to dryness under atmospheric pressure, and the residue is dried in a desiccator under atmospheric pressure. The crude methylethylmaleimide is purified by sublimation at 0.2 to 0.3 mm. pressure, with the bath temperature between 60 and 70° . The melting point of the purified material obtained on resublimation is 66 to 67°; yield, approximately 200 rag.

D. Isolation of Hematinic Acid The acidified aqueous solution containing the hematinic acid is extracted five times with an equal volume of ethyl acetate. The ethyl 5 W. Kiister, Ber. 45, 1935 (1912). 6 H. Fischer, A. Treibs, and G. Hummel, Z. Physiol. Chem. 181i, 33 (1929).

646


[27]

acetate solution is taken to dryness, and the residue, crude hematinic acid, is purified by sublimations at 0.2 to 0.3 ram. pressure, with the bath temperature maintained between 120 and 130°. The material can be further purified by recrystallization from ethyl acetate-petroleum ether or ethyl-ether-petroleum ether; yield, about 300 mg. ; m.p. 115 to 116°. 4,7

E. Decarboxylation of Hematinic Acid to Methylethylmaleimide and Carbon Dioxide The hematinic acid can be decarboxylated by heating the material at 300 ° for 30 minutes under a slow stream of nitrogen. 8 The formed carbon dioxide is collected in barium hydroxide; yield,. 90 to 95%. The black tarry residue is extracted with chloroform, and the filtered solution taken to dryness. The residue is purified by sublimations as described above and by recrystallization from 0.2 % ammoniacal water. The yield of the methylethylmaleimide is 40 to 50%; m.p. 65 to 66 °.

Cleavage of Methylethylmaleimide to Pyruvic and ~-Ketobutyric Acids

0

H

0

HO OH O""'N~"O H

" ~ CO I

+

C00H

I C----O I C00H

To 139 rag. (1 mM.) of methylethylmaleimide dissolved in 10 ml. of water is added 213 rag. (2 X mM.) of sodium chlorate followed by 60 mg. of osmic acid dissolved in 6 ml. of water2 (The precautionary measures, use of a hood and the wearing of goggles, in the handling of osmic acid should be taken.) The mixture, which turns brown, should be permitted to stand at room temperature for 20 hours. After this time the solution is extracted five times with an equal volume of benzene to remove the osmie acid. The conversion to the glycol is quantitative as determined with periodic acid. The tartarimide is then cleaved to pyruvic and a-ketobutyrie acids by means of periodate.10 An exact equivalent of solid sodium metaperiodate (214 mg.) is added. After standing for 20 minutes the solution is extracted with ether for 5 hours in a continuous extractor. The ether H. M. Muir and A. Neuberger, Biochem. J. 47, 97 (1950). 8D. Shemin and S. Kumin, J. Biol. Chem. 198, 827 (1952). 9N. A. Milas and E. M. Terry, J. Am. Chem. ~oc. 47, 1412 (1925). ~0p. Fleury and J. Lange, Compt. rend. 195, 1395 (1932).

[27]


647

solution is taken to dryness and the residue dissolved in 1 ml. of 0.5 N HC1 and heated for 1 hour at 80 °. The solution is then mixed with 1 g. of anhydrous silica gel and transferred, with the aid of a small volume of butanol-chloroform solution, to a chromatographic column for the separation of the acids. The pyruvic and ~-ketobutyric acids are separated by partition chromatography on silica gel. H A column 1.6 cm. in diameter and 21 cm. in length containing 10 g. of silica gel is used for 50- to 200-mg. quantities. The stationary phase is 0.5 N sulfuric acid, and the moving phase is a 7 ~ butanol-chloroform solution saturated with 0.5 N sulfuric acid. The f~te of flow through the column, 0.7 ml./min., is adjusted by a head of p~tsure. The effluent from the column is collected in 5-ml. fractions, and each fraction is slurried with water and titrated with 0.01 M sodium hydroxide to the end point of bromothymol blue. Usually the a-ketobutyric acid f~action (yield, 40 to 50%) is obtained between the 40- and 60-ml. fractioas, and the pyruvic acid (yield, 25 to 40%) is obtained in the 90to 120-ml. fractions. The respective keto acids are isolated as their 2,4dinitrophenylhydrazones. Each of the aqueous phases from the titrated effluent of the particular keto acid is combined, concentrated to a small volume, and acidified with HC1. The indicator is removed with DarcoG-60 and an equivalent amount of 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid is added. The pyruvic acid hydrazone is purified by dissolving it in sodium bicarbonate, filtering and reprecipitating with dilute hydrochloric acid, and then by recrystallization from ethanol; m.p. 217 to 218 °. The a-ketobutyric acid hydrazone is purified by dissolving it in half-saturated lithium carbonate, filtering and precipitating with dilute hydrochloric acidi and then by recrystallization by the addition of water to a hot glacial acetic acid solution; m.p. 198 to 200 °.12

Degradation of the Hydrazones of the Keto Acids The hydrazones of the a-ketobutyric and pyruvic acids can be convetted to propionic and acetic acids, respectively, by two convenient methods. In the method of Krebs ~3the hydrazones are oxidized with acid permanganate, whereas in the second method the hydrazones are treated with Ceric sulfate. TM The first method has the disadvantage that the carbo•yl group is diluted with 6 moles of carbon dioxide from the oxida11 F. 1~ D. 13 H. 14 R.

A, Isherwood, Biochem. J. 40, 688 (1946). Shemin and J. Wittenberg, J. Biol. Chem. 192, 315 (1951). A. Krebs, Biochem. J . 32, 108 (1938). H. Corzo and E. L. Tatum, Federation Proc. 12, 470 (1953).

648


[27]

tion of the phenyl group and further the oxidation of the phenyl group is not always quantitative. The second method has the disadvantage that about 10% of the formed carbon dioxide originates from the a-carbon atom, 15and thus the carbon dioxide from the carboxyl group is mixed with some originating from the a-carbon atom. The propionic and acetic acids are recovered by steam distillation. The propionic acid is converted to carbon dioxide and ethylamine by a modification le of the Schmidt reaction. It h a s b e e n demonstrated 17 that by this reaction each carbon atom of the acetic and propionic acids can be analyzed separately. The ethylamine is oxidized by alkaline permanganate to acetic acid. The samples of acetic acid, both from the decarboxylation of the propionic acid and from the pyruvic acid hydrazone, are similarly decarboxylated by the Schmidt reaction. The resulting methylamine is oxidized with alkaline permanganate. The carbon dioxide formed in each step can be collected as barium carbonate. • S y n t h e s i s of Labeled Substrates

Such compounds as glyeine, acetic, succinic, citric, and ~-ketoglutaric acids can be purchased with a particular carbon atom tagged with C 14 A. 5-Aminolevulinic Acid

The choice of the method of synthesis of this intermediate is in part dependent on the position one wishes to have labeled in the molecule. 1. 5-Aminolevulinic Acid-5-C14. TM Ethy! ~-keto-a-carbethoxyadipate was prepared according to the method of Riegel and Lilienfeld19 by the condensation of ethylmagnesiomalonate~° synthesized from 8 g. of diethyl malonate-2-C 14 with 9.3 g. of ~-carbethoxypropionyl chloride synthesized from succinie anhydride. ~1 Preparation of fl-Carbethoxypropionyl Chloride. Under anhydrous conditions 10 g. of succinic anhydride (see below) is refluxed with 10 ml. of absolute ethanol for 1 hour. After this period of time the solution is poured into an evaporating dish and after cooling is placed in a vacuum desiccator over CaCI~ for about 15 hours. The half ester is transferred to a flask fitted with a reflux condenser, 15 mt. of thionyl chloride is added, and the mixture, kept under anhydrous conditions, is kept for 3 hours 15j. C. Wriston, Jr., L. Lack, and D. Shemin, J. Biol. Chem. 215, 603 (1955). 16C. Schuerch, Jr. and E. H. Huntress, J. Am. Chem. Soc. 71, 2233 (1948). 17E. F. Phares, Arch. Biochem. and Biophys. 33, 173 (1951). 18D. Shemin, C. S. Russell, and T. Abramsky, J. Biol. Chem. 215, 613 (1955). 19B. Riegel and W. M. Lilienfeld,J. Am. Chem. Soc. 67, 1273 (1945). g0H. Lund and A. Voigt, Org. Syntheses, Coll. Vol. 2, 594 (1943). ~1U. Eisner, J. A. Elvidge, and R. P. Linstead, J. Chem. Soc. 1950, 2223.

[27]


649

at 40 °. The excess thionyl chloride is distilled off and the ~-carbethoxypropionyl chloride is obtained by distillation; b.p. 98°/15 ram. ;48°/1 ram. In a three-necked flask, fitted with a stirrer and reflux condenser and under anhydrous conditions, and containing ethylmagnesiomalonate synthesized from 8 g. of diethyl malonate-2-C 14, in 15 ml. of dry ether, are added dropwise, through an addition funnel, 10.5 g. of 2-carbethoxypropionyl chloride dissolved in 10 ml. of dry ether. The rate of addition should maintain a rather vigorous reflux. After the addition, the mixture (precipitate and solution) is refluxed for 2 hours and bumping avoided by judicious stirring. After this time the solution is cooled, and an ice-cold solution of sulfuric acid (1.5 nil. of concentrated H2SO~ in 30 ml. of water) is added slowly. The mixture is transferred to a separatory funnel and the ether layer washed once with water. The combined water layers are extracted once more with ether, and finally the ether layers are combined. After drying the ether solution over anhydrous sodium sulfate, the ether is removed by distillation and the residue distilled in vacuo. The boiling point of the triester is about 125 to 135 ° at 0.5 ram. pressure. The triester is then hydrolyzed and decarboxylated with concentrated hydrochloric acid. ~ For each gram of the triester 7 ml. of concentrated hydrochloric acid is added, and the lightly stoppered flask is allowed to remain at room temperature (about 25 to 28 °) for 18 to 20 hours. If the hydrolysis is permitted to proceed for a much longer time, the formed /~-keto acid may also decarboxylate. After this period of time the hydrochloric acid is removed by distillation in vacuo, while the outside bath temperature is not permitted to exceed 35 °. A flash evaporator is to be recommended. The crude ~-ketoadipic acid is suspended in about 50 ml. of glacial acetic acid. While the acetic acid suspension is kept at about 18 to 20 °, gaseous ethyl nitrite 22 prepared from sodium nitrite (10% excess) is passed into the suspension. The keto acid dissolves, and the solution is either slightly yellow or slightly brown. After standing at room temperature for about I hour, the acetic acid is removed by distillation in vacuo, and to the oily or semicrystalline residue a cold solution, made of 25 g. of concentrated hydrochloric acid and 25 g. of stannous chloride, is added. The resulting solution is placed in a refrigerator for about 48 hours. After this time the solution is diluted with 10 vol. of water and the tin removed with H~S. The filtered solution is taken to dryness in vacuo, and, after the residue has been dried in a vacuum desiccator, the crystalline product is obtained by crystallization from methanol-ethyl acetate. The material is recrystallized from these solvents; the yield, based on the malonate, is between 35 and 45 %.18 2~ W. L. Semon and V. R. Damerell, Ore. Syntheses Coll. Vol. 2, 204 (1943).

650


[27]

2. ~-Aminolevulinic Acid-l,4 or 2,3-C 14. This procedure is preferable if one wishes to synthesize $-aminolevulinic acid labeled in the succinyl moiety. 18.28 Preparation of Succinic Anhydride. 24 To 5 mM. of labeled succinic acid in a 12-ml. centrifuge tube with a standard taper joint, provided with a condenser and drying tube, 1.1 ml. of reagent-grade, freshly distilled acetyl chloride is added. The mixture is brought to a gentle boil in a water bath. In about an hour's time, solution occurs, after which heating is continued for another hour. After cooling, the stoppered vessel is placed in a refrigerator. The formed crystals are separated by centrifugation and washed three times with 0.5-ml. quantities of ether. The anhydride, dried in a desiccator in vacuo, is white; m.p. 118°; yield, 75 to 80%. 24 The anhydride is converted to ~-carbethoxypropionyl chloride as described above. To an ethereal solution of 1 g. of B-carboethoxypropionyl chloride is added an ethereal solution of diazomethane obtained from 2 g. of N-nitroso-N-methylurea, and the mixture is stirred for 2 hours at room temperature. After this time either dry HC1 gas is bubbled through the solution or an ethereal solution of HC1 is added dropwise until the ~volution of N2 ceases. The solvent is removed by passing a dry stream of nitrogen through the solution, and the residue is dried in vacuQ over NaOH. The oily chloromethyl ketone is treated with 4 ml. of dimethylformamide and 1.24 g. of potassium phthalimide (labeled with 34 .atom % excess N 15 ). T h e resulting orange suspension is stirred vigorously for 1 hour at room temperature and then mixed with 25 ml. of chloroform. The chloroform solution is washed with 50 ml. of water and the washings shaken with 2-ml. portions of chloroform. The combined chloroform solutions are then washed twice with 50-ml. portions of water, once with 25 ml. of 0.2 N sodium hydroxide, and finally four times with water. The chloroform solution is dried by filtration and taken to dryness in vacuo. The crude methyl 5-phthalimidolevulinate is purified by crystallization from methanol, m.p. 96 to 97 °. The phthalimido derivative is then hydrolyzed by boiling for 7 to 10 hours with 6 N hydrochloric acid. After removal of the phthalic acid by filtration, the crude ~-aminolevulinic acid hydrochloride is purified as above. The 5-aminolevulinic acid hydrochloride, m.p. 149 to 151 °, gives a positive ninhydrin test, reduces Benedict's solution in the cold, and on an ascending paper chromatogram has an R I of about 0.42 to 0.44 in a phenol-water mixture. The spot on paper, developed with ninhydrin, is

~3A. Neuberger and J. J. Scott, J. Chem. Soc. 1954, 1820. 24L. F. Fieser and E. L. Martin, Org. Syntheses Coll. Vol. 2, 560 (1943).

[27]


651

initially yellow and becomes purple after a few hours. The yield is about 30 % based on the succinic acid.

B. Preparation of Porphobilinogen T h e monopyrrole porphobilinogen m a y be prepared enzymatically from ~-aminolevulinic acid. A fraction from liver ~5 or duck erythrocytes 2~ is incubated with ~-aminolevulinic acid. The presence of porphobilinogen can be demonstrated b y treating a sample with Erlich's reagent, dimethyaminobenzaldehyde. The interference in the color development caused by glutathione, which is in the mixture, can be overcome b y the addition of a small a m o u n t of cuprous oxide. After the period of incubation, the protein is precipitated with trichloroacetic acid, and the porphobilinogen isolated. 27 To the filtrate, adjusted to p H 4 to 5 with a saturated solution of sodium acetate, a 15% solution of mercuric acetate is added until no further precipitation occurs. The precipitate is collected by filtration and washed with a solution of 1.5 % mercuric acetate. The precipitate is then suspended in water and the mercury removed with H~S. The filtrate, adjusted to p H 4 to 5 with 1 N NH4OH, is taken to dryness, and the residue is crystallized by dissolving the material in a minimum a m o u n t of 0.5 N N H 4 0 H and then adding 1 N acetic acid. The slightly pink crystals can be further purified b y repetition of the above crystallization procedure. The pink color is due to the spontaneous conversion of the monopyrrole to porphyrins. In an experiment in which 120 mg. of porphobilinogen was formed, as determined b y the Erlich reagent, 88 mg. were isolated.

Biosynthesis of Heine A s t u d y of the biosynthesis of heme can be carried out in vitro by using avian erythrocytes, 28 a hemolyzed preparation of the red cells, 8,29 or a cell-free extract of the avian cells. 18 Whereas glycine, acetate, succinate, a-ketoglutarate, and citrate are converted into heme in the hemolyzed preparation (permeability is a factor in the whole cell), one must, at present, start with ~-aminolevulinic acid when the cell-free extract is the preparation chosen. 18,3° 25A. Gibson, A. Neuberger, and J. J. Scott, Biochem. J. 68, xli (1954); 6J, 618 (1955). 26R. Schmid and D. Shemin, J. Am. Chem. Soc. 77, 506 (1955). 27G. H. Cookson and C. l~imington, Biochem. J. 57, 476 (1954). 28D. Shemin, I. M. London, and D. Rittenberg, J. Biol. Chem. 183, 757 (1950). 22I. M. London and M. Yamasaki, Federation Proc. 11, 250 (1952). 30D. Shemin, T. Abramsky, and C. S. Russell, J. Am. Chem. Soc. 76, 1204 (1954).

652


[28]

[28] Methods for Chemical Synthesis, Isolation, and Degradation of Labeled Compounds as Applied in Metabolic Studies of Amino Acids and Proteins

By

DAVID M. GREENBERG a n d ~¢~ORTON ROTHSTEIN

I. Methods for Chemical Synthesis of C14-Labeled Amino Acids 1 Introduction The use of isotopically labeled amino acids in biological research has become widespread with the advent of readily available isotopes of carbon, nitrogen, sulfur, iodine, and hydrogen. Almost all the known naturally occurring amino acids have been synthesized, incorporating one or more of these isotopes in the molecule. Because of the innumerable possibilities of multilabeling with combinations of isotopes, this chapter will confine itself to the synthesis and degradation of amino acids labeled solely with C TM. The basic starting material for synthesis of all C'4-containing molecules is BAC1403, available by permit from the Atomic Energy Commission at Oak Ridge, Tennessee. From this material, carbon dioxide-, cyanide-, formaldehyde-, methyl iodide-, and acetate-C TM, the primary building blocks of isotope synthesis, may be prepared. These compounds represent the starting materials for the synthesis of the vast majority of C'4-1abeled amino acids. In cases where their preparation is uneconomical or incon-' venient, they may be purchased from commercial sources. Synthesis of these intermediates will be discussed in detail only where they are an integral part of an experimental procedure for the preparation of a labeled amino acid. However, reference will be given to selected methods for their preparation, where necessary. There are often several different syntheses of a specifically labeled amino acid reported in the literature, not to mention numerous modifications of established procedures. Under these circumstances it becomes difficult to choose any single method as being the best available. This is especially true when one considers that, for many biochemists, the choice of method may depend more on convenience than on yield of product. In the final analysis, where two or more methods exist, the factors of time, cost, availability of elaborate equipment, and chemical yield must be balanced and decided on by the individual undertaking the synthesis. 2,3 Basically, Prepared by Morton Rothstein. 2 The book, "Technique of Organic Chemistry," Vol. VI (Micro and Semi-micro Methods), Interscience Publishers, New York, 1954, contains a great deal of useful

[9-8]

METABOLICSTUDIES OF AMINO ACIDS AND PROTEINS

~53

the author has attempted to select, for each amino acid, that procedure which is most efficient chemically. However, some consideration has been given to simplicity and ease of preparation, and, wherever possible, alternate methods have been outlined.

General Reactions for Synthesis of Amino Acids Containing C 14 The reactions used for synthesizing C14-containiag amino acids cannot be completely categorized. However, the majority of syntheses do fallinto certain general reaction patterns for the various positions of labeling. I. C 0 0 H - C ~4.Treatment of an aldehyde with cyanide-C 14and ( N H 4)~COa to form a hydantoin, which on hydrolysis yields the desired amino acid (Bucherer reaction), is a typical way of producing COOH-C~4-amino acids. (N H4) 2CO3

R C H O -~ KC*N - -

~. R C H

C*~O

I

t

NH

NH

\ /

hydrolysis

RCHC*OOH

I

NH2

C

H

0

3,4-Dihydroxyphenylalanine-l-C14, 4 lysine-l-C14, 5 and tyrosine-l-C1418 have been made by this general method. The reaction is very simple and requires no special equipment. The intermediates and products are all solids and thus can be manipulated with a minimum of trouble. In certain cases, the Strecker reaction can be applied, utilizing ammonia or NH4C1 instead of (NH4)2C03, as in the above, thus yielding the amino acid directly. Alanine-l-C~4, 7 phenylalanine-l-C14,8 leucine_l_C~4,9 and valine1_C~410 are among the labeled amino acids thus prepared. 2. 2-C14-Amino Acids. This type of labeling is generally achieved by condensing the appropriate halide with ethyl acetamidomalonate-2-C 14 information regarding small-scale, synthetic organic chemistry. 3 Both Metro Industries, Long Island 6, New York, and Scientific Glass Apparatus Co. Inc., Bloomfield, New Jersey, market a complete line of small, standard taper glassware which is excellent for carrying out small-scale organic reactions. 4 G. R. Clemo, F. K. Duxbury, and G. A. Swan, J. Chem. Soc. 1952, 3464. 5 Chem. Abstr. 48, 10588b (1954). s R. B. Loftfield, J. Am. Chem. Soc. 72, 2499 (1950). 7 R. B. Loftfield, Nucleonics 1 (3), 54 (1947). s G. O. Henneberry, W. F. Oliver, and B. E. Baker, Can. J. Research 29, 229 (1951). 9 H. Borsook, C. L. Deasy, A. J. Haagen-Smit, G. Keighley, and P. It. Lowy, J. Biol. Chem. 184, 529 (1950). 10 R. E. Selff and B. M. Tolbert, Univ. Calif. Radiation Lab. Rept. 1301 (1951).

654


[9.8]

(or a similar malonic acid derivative), followed by hydrolysis of the product to yield the desired amino acid. RX + CH--(COOR)~--~

I

NHAC

R* hydrolys~ C(COOR)2 RCHCOOH

I

f

NHAc

NH2

By this method, 2-Clt-labeled histidine, 11 lysine, 12 ornithine, 13 homoserine, ~4 and tyrosine 15 have been prepared. The yields from the condensation and hydrolysis steps are good, but unfortunately a rather arduous procedure is required to synthesize ethyl acetamidomalonate-2-C ~4. A good method is reported for the preparation of ethyl cyanoacetamidoacetate-2-C~4.16 Both compounds are available commercially, albeit they are somewhat expensive. 3. COOH or 2-C~4-Amino Acids. Labeling of these carbons can be secured by the a-bromination of a carboxylic acid followed by amination of the product with ammonia. This is, in general, a procedure giving reasonably good yields, since the acid is formed from an alkyl (or alkaryl) halide by carbonation of a Grignard reagent with C~402, or by cyanation with NaC14N or KC14N. •

Mg

RCOOH LihltI~) RCH2OH* --* RCH2Br - - - o o CO2 RCH2COOH --* R C H C O O H N_~ R C H C O O H

J

Br

J

NH2

A carbon 2-labeled amino acid results from reduction of a COOH-C 14acid to the alcohol, conversion to the bromide, and then treatment with Mg and CO2 or directly with KCN. A variation is the reaction of the radioactive bromide with acetamidomalonate to give a 3-C~4-amino acid. Among the C'4-containing amino acids prepared in this fashion are leucine-l-, 2-, and 3-C~4,le,17 alanine-l-, 2-, and 3-C~4,~8 and glycine-1- and 2_C14.19,20

11 G. Wolf, J. Biol. Chem. 200, 637 (1953). 1~H. Borsook, C. L. Deasy, A. J. Haagen-Smit, G. Keighley, and P. H. Lowy, J. Biol. Chem. 187, 839 ~1950). ~3D. B. Sprinson and D. Rittenberg, J. Biol. Chem. 198, 655 (1952). 1~E. P. Painter, J. Am. Chem. Soc. 69, 233 (1947). 16 M. Fields, D. E. Walz, and S. Rothchild, J. Am. Chem. Soc. 73, 1000 (1951). ~6H. Hauptmann, P. T. Adams, and B. M. Tolbert, J. Am. Chem. Soe. 74, 2423 (1952). ~TR. Ostwald, Univ. Calif. Radiation Lab. Repl. 2154 (1953). ~s R. Ostwald, P. T. Adams, and B. M. To!bert, J. Am. Chem. 8oc. 74, 2425 (1952). 19K. Bloch, J. Biol. Chem. 179, 1245 {1949). 20 R. Ostwald, J. Biol. Chem. 175, 207 (1948).

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

655

4. ~-C14H2NH~. This t y p e of labeling is customarily obtained b y the reaction of KC14N with an 00-halide compound followed b y reduction of the resulting nitrile:

Br(CH2)3C(COOEt)2 KCN-4 N C ( C H 2 ) 3 - - C ( C O O E t ) ~

I

[

NHAc

NHAc

H2 PtO= At20

hydrolysis H2NCH2(CH2) a C H C O O H

I NH2 Examples of labeled amino acids prepared in this way are lysine-6-C14, 21 f~-alanine-l-, 2-, and 3-C~4, 22 and ~-aminovaleric acid-~-C~4. 23 A variation of this procedure is found in the preparation of ~-HO-lysine-6-C 1424 where KC14N is added to the appropriate w-aldehyde, yielding a cyanohydrin which on reduction and hydrolysis yields the desired product. 5. Azlactone Reaction. This reaction makes use of the carbon chain of glycine to form the carboxyl and a-carbon of the amino acid being prepared. The reaction has been used to prepare thyroxine-l-C14, 2~ phenylalanine-l,2-C~4, 26 and benzene ring-labeled phenylalanine27 0 • o II • o ArCHO, Ac20 H2NCH~COOH --~ ~ C - - N H C H 2 C O O H AcONa ArCH~C--N

I?oo

P hydrolysis

• o ArCH~CHCOOH NH2

°C--O

II

0

A similar use of glycine to provide the earboxyl and ~-carbon atoms of an amino acid can be made by condensing an aryl aldehyde with h y d a n t o i n (prepared from glyeine). After reduction and hydrolysis of the produeL the desired amino acid is obtained. 2~M. Rothstein and C. J. Claus, J. Am. Chem. Soc. 75, 2981 (1953). 22p. Fritzson and L. Eldjarn, Scand. J. Clin. & Lab. Invest. 4, 385 (1952). ~s M. Rothstein, J. Am. Chem. Soc. 76, 3038 (1954). 34 S. Lindstedt, Acta Chim. Scand. 7, 340 (1953). 25S. C. Wang, J. P. Hummel, and T. Winnick, J. Am. Chem. Soc. 74, 2445 (1952). 26S. Gurin and A. M. Delluva, J. Biol. Chem. 170, 545 (1947) ; M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwieh, "Isotopic Carbon," p. 223. John Wiley & Sons, New York, 1949. 27 A. B. Lerner, J. Biol. Chem. 181, 281 (1949).

656

TECHNIQUES FOR ISOTOPE STUDIES *

o

#

*

-~=o I

ARCH0 -~ C H 2 - - C - ~ 0 --~ A r C H ~ - C

I

I

I

NH

NH

\/

NH

HI e

NH

\/

C

C

0

O

Ir

[28]

H

6----0 hydrolysi~ A r C# H 2 •C H Co O O H J I

ArCH,--CH

f

NH

\

/

NH

NH.o

C

J;

O

This procedure has been utilized for preparing tyrosine-fl-C TM28and 3,4-dihydroxyphenylalanine-fl-C 14.29 6. Acetamidomalonate-Type Condensations. Many labeled amino acids have been prepared by the synthesis of a C~4-containing molecule which will condense with ethyl acetamidomalonate, or a similar compound of this type. Hydrolysis of the product yields the amino acid. R X + CH(COOEt)2 •NaOEt * hydrolysis R* C H C O O H ~ RC(COOEt)2

NHAc

NHAc

NH2

Tryptophan-/~-C14, 3° serine-fl-C14, 31 phenylalanine-4-C14, a2 leucine-3- and 4-C14, 33 glutamic acid-l-C14, 34 aspartic acid-3- and 4-C14,85 and alanine3_C1418 have all been prepared in this way. A l a n i n e =C ~4

DL-Alanine-l-, ~-, and 3-C TM. The method of Ostwald et al.~S gives good yields and has the advantage of using the same procedure for alanine-l-, 2-, or 3-C TM. ~s j. C. Reid and H. B. Jones, J. Biol. Chem. 174, 427 (1948). 29 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," pp. 225-227. John Wiley & Sons, New York, 1949. 30 C. tteidelberger, J. Biol. Chem. 179, 139 (1949). 31 M. Levine and It. Tarver, Y. Biol. Chem. 184, 427 (1950). 32 R. Dische and D. Rittenberg, Y. Biol. Chem. 211, 199 (1954). 38 M. J. Coon and S. Gurin, Y. Biol. Chem. 180, 1159 (1949). 84 R. J. Speer, A. Roberts, IV[. Maloney, and H. R. Mahler, J. Am. Chem. Soc. 74, 2444 (1952). 85 R. M. Noller and B. M. Tolbert, Univ. Calif. Radiation Lab. Rept., 2041 (1952).

[28]


657

* o LiA1H, * o * o * o CO~ C H 3 C O O H - - - - - ~ CH3CH~OH ~ CH3CH2Br--* CH~CH2MgBr-----~ , o ° • o • NHa C H 3 C H 2 C O O H --~ C H 3 C H C O O H ----~ DL-Alanine-l-, 2-, or 3-C 14

I

Br The radioactive ethanol formed by the lithium aluminum hydride reduction of acetic acid was appreciably diluted with nonradioactive ethanol produced by splitting of the solvent (diethyl carbitol) first used for the reduction, thus lowering the specific activity of the product. Cox et al. 3~ reported that the use of t e t r a h y d r o f u r f u r o x y t e t r a h y d r o p y r a n ( T F T P ) as the solvent for lithium aluminum hydride reductions avoided the problem of contamination due to the splitting of solvent ethers. A modification of this procedure for the reduction of acetic acid by Pack and Tolbert 3t is incorporated below into the appropriate sections of the synthesis of alanine-C TM. On the basis of experience with T F T P as the solvent, it can be assumed t h a t the radiochemical yields from the reduction of acetic acid will be at least equal to, if not greater than, those reported originally with diethyl carbitol as solvent, but without concomitant dilution of radioactivity. Procedure: S o d i u m P r o p i o n a t e - l - C ~4. Propionic acid- 1-C 14was prepare d by carbonating 30 millimoles of ethyl magnesium iodide with 20 millimoles of C1402 (from 3.94 g. of BaC1403, 5.02 ~c./mg.)28 The yield was 1.87 g. of sodium propionate-l-C 14 (specific activity 10.4 ~c./mg.) (97.8%). a-Bromopropionic A c i d - l - C TM. D r y sodium propionate (937 mg., 9.87 mc.) from the above preparation was placed in a gas-solid reactor29 The bulb was evacuated to about 10 ~ of pressure and filled with an excess (one-half atmosphere) of dry, purified hydrogen chloride. The solid sodium propionate was then heated gently over a Bunsen burner. After the exchange reaction was complete, the mixture of propionic acid and excess hydrogen chloride was distilled i n vacuo into a large trap cooled with liquid nitrogen. The cooling bath was then changed to a dry ice-isopropyl alcohol mixture, and the excess hydrogen chloride pumped off. (Propionic acid may also be obtained from its salt by use of an apparatus similar to t h a t in Fig. 4--see Leucine-1- and 2-C14.) The propionic acid-l-C ~4, which contained 3 to 5 % water, was distilled 36j. D. Cox, H. S. Turner, and R. J. Warne, J. Chem. Soc. 1980, 3167. 37D. E. Pack and B. M. Tolbert, Univ. Calif. Radiation Lab. Rept. 1957 (1952). 38 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," pp. 178-79. John Wiley & Sons, New York, 1949. 39 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," p. 162. John Wiley & Sons, New York, 1949.

658


[9.8]

into the bromination vessel 4° which contained 0.04 g. of red phosphorus, 0.02 g. of iodine, and 0.2 ml. of propionyl chloride. The mixture was allowed to reflux on the steam bath for 1/~ hour to destroy the water, 1.5 ml. of bromine was added dropwise, and refluxing was continued for 3 hours. The low-temperature condenser was kept at dry ice-isopropyl alcohol temperature. After the excess bromine was swept out with a slow stream of air, the mixture of a-bromopropionic acid-l-C 14 and a-bromopropionyl bromide1-C 14 was cooled and hydrolyzed by slow addition of 2 ml. of water. Alanine-l-C ~4. The amination was performed in a three-necked flask by slow addition of a-bromopropionic acid-l-C ~4 to a mixture of 6 g. of (NH4) 2CO3 and 15 ml. of concentrated NH4OH. The reaction mixture was kept at 60 ° for 6 hours and then distilled to dryness in vacuo. The crude alanine-l-C ~4 was purified by (1) passage through an ion exchange resin column and (2) high vacuum sublimation. A glass column (30 × 2 cm. outside diameter) filled with 60 ml. of Dowex 50 resin (20 to 40 mesh) was treated by cycling to exhaustion three or four times with 2 N NaOH solution and 2 N HC1, respectively, ending with the acid. The excess acid was washed out thoroughly with water, and 600 to 900 mg. of crude alanine-l-C ~4, dissolved in a minimum amount of water, was added to the column, followed by 500 ml. of 1.5 N NH4OH, followed by 250 ml. of water. The resin was regenerated with 2 N HC1. The water effiuate, NH40H eluate, and HC1 regeneration solution were each evaporated to dryness and were found to contain 3, 88, and 5%, respectively, of the radioactivity put on the resin. In order to remove traces of ammonia, the dry residue of the NH40H eluate was made slightly alkaline with NaOH and evaporated to dryness. After it was redissolved and the pH adjusted to 6.8, the solution was transferred to the lower part of a sublimation apparatus with a 1-cm. sublimation gap, evaporated to dryness, and sublimed at 1 ~ of pressure and 160 to 200 ° for 2 hours with the cold finger at liquid nitrogen temperature. The sublimation residue was redissolved (pH 8.5), adjusted to pH 6.8, dried, and resublimed. The final residue contained 4% of the initial crude alanine activity. The yield of chromatographically and radioautographically pure alanine-l-C 14 was 802 mg. with a specific activity of 9.9 ~c./mg. This represents a radiochemical yield of 80.5% based on sodium propionate-l-C 14 used and a chemical yield of 76 %. Ethanol-l-C14; Preparation of the Hydride Solutions. T F T P (b.p. 124°/14 mm.) was prepared according to the method of Woods and 40 B. M. Tolbert, F. Christenson, F. N. It. Chang, and P. P. T. Sah, J. Org. Chem. 14, 528 (1949).

[9.8]


659

Kramer 41 from tetrahydrofurfuryl alcohol (TFA) and 2,3-dihydropyran. Lithium aluminum hydride solutions in T F T P were prepared by stirring the hydride (which had been ground in a mortar) with T F T P for 5 to (i hours at 50 to 60 °. The slurry was then filtered through a sintered-glass funnel, and the clear solution was stored in a flask sealed with a serum bottle stopper. The solutions, which were 0.7 to 0.8 M, were analyzed by decomposing an aliquot with butyl carbitol and measuring the volume of the evolved hydrogen. All manipulations were done in a nitrogen atmosphere.

DryN2 Inductionstirrer

~

J

k

TrapT1 FI(~. ]. Appa,rzttus for the reduction of acetic acid-]-C tl to ethanoI-t-C t4.

Sodium acetate-l-C 14 was converted to the free acid by the procedure given above for conversion of sodium propionate-l-C~4 to propionic acid1-C'4. The product was transferred to the dropping funnel of the apparatus shown in Fig. 1 with the aid of 10 to 15 ml. of T F T P . Dry nitrogen was then passed through the system, and trap T~ was immersed in liquid nitrogen. A bath of water at 60 to 70 ° was placed around the reaction flask, and the acid-TFTP mixture was added dropwise to the rapidly stirred hydride solution. The funnel was rinsed with several 1-ml. portions of T F T P . The mixture was then stirred for ]/~ hour longer. Excess hydride was decomposed with TFA, and the ethanol swept into the trap by placing 4, G. F. W o o d s a n d D. N. K r a m e r , J. Am. Chem. Sac. 69, 2246 (1947).

660


[28]

the flask in an oil bath at 150 ° and bubbling nitrogen through the mixture by means of a flitted-glass bubbler tube at a rate of 30 to 40 ml./min, for 5 to 6 hours. Exposed glass between the flask and the trap was wrapped with heating tape and heated to 200 °. The ethanol-l-C TM contained small amounts of T F A and T F T P , but this does not interfere with the subsequent bromination. 4~ Ethyl Bromide-l-C ~4. The ethanol-l-C TM was converted to the bromide by reaction with phosphorous tribromide according to Tolbert et al. 4° Sodium Propionate-2-C TM. The ethyl-l-C TM bromide thus prepared was converted to the Grignard reagent, and this compound was carbonated with inactive carbon dioxide. 43 Alanine-2-C ~4. Sodium propionate-2-C TM (0.935 g., 9.7 millimoles, specific activity 4.66 ~c./mg.) was converted to alanine-2-C ~4 as described for alanine-l-C TM. The yield was 0.689 g. or 67% chemically. The radiochemical yield was 63 %, specific activity 4.08 ~c./mg. Alanine-3-C TM. Acetic acid-2-C 1444 was converted to ethyl bromide2-C ~4 and thence to sodium propionate-3-C TM in the same manner as for propionate-2-C ~4. The propionate was then converted to alanine by bromination and amination. Other Syntheses. Alanine-l-C ~4has been prepared by a simple procedure in 35 % over-all yield (based on cyanide) by the action of labeled cyanide on acetaldehyde in the presence of ammonia :7 CHaCHO + HCN N~ CH3CH~OOH

I

NH2 ~-Alanine-C ~ fl-Alanine-l-, 2-, and 3-C TM. /~-Alanine has been prepared labeled with C TMin each of the three positions by the following sequence of reactions :22 o

C1CH~COOH ~- K C N -~ N C" C°H 2 C* O O H -~ H 2 N C H 2 C H , 5 0 0 H The over-all yield based on KC14N is 44.8%. The authors present only the synthesis of /~-alanine-3-C 14 but have also used chloroaeetic acid labeled in the 1 and 2 positions to obtain the correspondingly labeled 4~B. M. Tolbert, personal communication. 4s S. Gurin and D. W. Wilson, Federation Proc. 1, 114 (1942). 44Acetic acid-2-C ~4is best prepared by reducing C1402 to methanol-C TM in a fashion similar to the reduction of propionic acid reported above,~ and thence to methyl iodide-C 14.This is converted to the Grignard reagent and reacted with inactive carbon dioxide lB. M. Tolbert, J. Biol. Chem. 173, 205 (1948)]. The over-all yield for the conversion of C~40~ to sodium acetate-2-C 14is approximately 60%,

[9.8]


661

~-alanines. Labeled chloroacetic acid may be prepared in good yield from acetic acid by the procedure of Ostwald 2°,45 (also see Aspartie Acid-3and 4-C 14). Considerable care must be taken to neutralize the excess potassium hydroxide usually found in preparations of KCI*N. Outside of this, the synthesis is straightforward and offers no difficulties. The yield could probably be increased by fractionating the ~-alanine mother liquors by appropriate ion exchange techniques, thus avoiding the dilution with carrier required to obtain a second crop of product. P r o c e d u r e . To the vessel containing 22 mg. of KC14N (1 mc.) and 25 mg. of KOH was added 82.6 mg. of carrier KCN and 5 drops of water to dissolve the contents. One milliliter of partly neutralized monoehloroacetic acid was added dropwise with shaking. The solution of monochloroacetic acid was prepared by dissolving 3.75 g. of the acid in 10 ml. of water, adding 1.56 g. of dried Na2CO~ while heating moderately, and diluting to 25 ml. The excess of free acid was calculated to neutralize 92 % of the free alkali i~ the sample. After 40 minutes on the boiling water bath, the reaction vessel was cooled, 1.90 ml. of 1.124 N HC1 was added, and the solution was evaporated i n vacuo. A few milliliters of dry ether was added, and the mixture again evaporated. The residue was carefully extracted with four portions of dry ether, the extracts being transferred to a specially constructed microhydrogenation vessel where the ether was evaporated off with an air jet (Fig. 2). To the oily residue in the hydrogenation vessel was added 1 drop of water and 2 N KOH (0.9 ml.) to bring the pH to 10 to 11. Four milliliters of 35% aqueous ammonia and approximately 100 mg. of suspended Raney nickel were added, and the mixture was hydrogenated under a pressure of 2 atm. at 20 ° for 17 hours with continuous stirring. The amount of hydrogen absorbed was 99.6% of the theoretical. After hydrogenation, the catalyst was removed by centrifugation, and the supernatant fluid, together with the washings, was evaporated to a small volume i n vacuo. After addRion of 0.145 ml. glacial acetic acid and heating to 50 ° for a few minutes, the evaporation was continued until .~ thick oil remained. This was dissolved in 4 to 5 drops of water, 5 ml. of absolute ethanol was added, and the solution placed in the refrigerator overnight, after seeding with a few crystals of ~-alanine. The first crystallization gave a sticky precipitate. In subsequent crystallizations, however, silky needles of ~-alanine separated out, leaving part of the material as an oily residue. The crystals were separated from the residue by swirling the flask gently, whereupon the suspension of crystals could be decanted and a5 The reported yield of chloroacetic acid-C ~~ from acetic acid-C ~4 is 67% for a single radioactive run. A yield of 85 to 90%, however, is more typical of the procedure.

[28]

663


a-Arninoadipic Acid-C ~

DL-a-A minoadipic Acid-6-C 14. a-Aminoadipic acid-6-C 14 has been prepared as follows :2~ HO(CH2)aC(COOEt)2--~ Br(CH2)3C(COOEt)2 + K C N - - ,

I

I

NHAc

NHAc

NC(CH,~)~C(COOEt)2 hydrolysis HOOC(CH2) * 3CHCOOH

I

NHAc

I

NH2

I

The intermediate, I, yields lysine-6-C ~4 on reduction with hydrogen over Pt02 (see Lysine-6-C~4). The procedure for a-aminoadipic acid offers no difficulties. However, the time suggested for the refluxing of the KCI*N with the bromovalerate derivative should be adhered to strictly. Procedure: "y-Acetamido-%,y-dicarbethoxybutyraldehyde. This compound was prepared by the method of Moe and Warner with benzene as the solvent. 47 After neutralization with glacial acetic acid, the mixture was filtered and the benzene removed in vacuo without heating to more than 60 °. A nearly colorless, viscous product resulted. Ethyl a-Acetamido-a-carbethoxy-8-hydroxyvalerate. The aldehyde was reduced and isolated according to Moe and Warner, 47 except that six times the reported amount of catalyst was used. The yield of alcohol was 82% based on crude aldehyde. Ethyl a-Acetamido-o~-carbethoxy-5-bromovalerate. A vigorously stirred solution of 20 g. of the crude hydroxy ester in 90 ml. of dry benzene was cooled to 16° in a water bath, and a solution of 2.8 ml. of phosphorus tribromide in 20 ml. of dry benzene added dropwise over a period of 30 minutes. The mixture was then refluxed for 20 minutes, and the benzene evaporated on a steam bath. The yellow residue was repeatedly extracted with portions of low-boiling petroleum ether. The extracts, after being cooled in a refrigerator, yielded 16.8 g. (68%) of fine white needles. This material was used without purification for the next step. DL-o~-Aminoadipic Acid-6-C 14. To a solution of 2 g. of ethyl-a-acetamido-a-carbethoxy-~-bromovalerate in 20 ml. of ethanol was added 400 mg. of KC~4N in 5.4 ml. of water. An additional 2 ml. of water was used to rinse the last of the cyanide into the reaction flask. The mixture was ,70. A. Moe and D. T. Warner, J. Am. Chem. Soc. 70, 2763 (1948).

664

TECHNIQUES

FOR ISOTOPE

STUDIES

[28]

refluxed for 2.5 hours, distilled to dryness in vacuo, and the residue extracted four times with ether. 48 The ether extracts were evaporated and hydrolyzed for 5 hours with 30 ml. of concentrated HC1. The resulting solution was distilled to dryness under diminished pressure. Addition of water and distillation to dryness was repeated twice, yielding a crystalline residue which was dissolved in a small amount of water and filtered. The volume was made up to about 12 ml. with water, and 15 ml. of ethanol was added. The solution was treated with a slight excess of pyridine, stirred, and placed in the refrigerator overnight. The white, crystalline DL-a-aminoadipic acid-6-C TM was filtered, washed with 50% ethanol until halogen-free, and dried; yield 0.389 g. (41%, not allowing for recovered cyanide). A paper chromatogram (phenol-water) showed one spot only; R I = 0.33. Aspartic

Acid-C

TM

DL-A spartic A cid-3- and ~-C14. Several syntheses of C ~4-1abeled aspartie acid have been reported. They differ only in detail and are based on the condensation of bromo- or chloroacetic acid ester (methyl or ethyl) with acetamido- or formamidomalonate. The product is 3- or 4-C14-aspartate, depending on the labeling in the acetate. The following is the procedure of Noller and Tolbert. 35 The over-all yield is 66% based on acetate-C14: *

°

CH3COOH *

--

*

° OO

C h C1CH2C

H

(C2H5)2SO4

*

o

* C1CH2COOEt

o

C1CH~COOEt -b CH(COOEt)2 NaOEt o* --* EtOOCCH2C(COOEt)2

f

NHAc

P

NHAc hydrolysis o• HOOCCH2C--COOtt

I

NH2Ac One might consider esterifying the chloroacetate with triethyl phosphate rather than diethyl sulfate as directed below, since the former reagent is greatly superior for the esterifieation of sodium acetate. 49 Procedure: Ethyl Chloroacetate-2-C TM. Anhydrous acetic acid was prepared from dry sodium acetate-2-C TM(1.85 g., 22.5 millimole, 13.3 pc./mg.) by the method described for the preparation of propionic acid (see Alanine-C14). The acetic acid was distilled in vacuo into the chlorination apparatus (Fig. 3) which contained 30 rag. of red phosphorus, 15 mg. of iodine, and 0.2 ml. (2.8 millimole) of acetyl chloride. The reaction mixture 45In a similar run for the preparation of lysine, 36% of the starting cyanide-C14was recovered by trapping in dilute alkali the gases given off by the refluxing solution. Use of this trapping procedure would substantially increase the radiochemicalyield. 49G. A. Ropp, J. Am. Chem. Soc. 72, 2299 (1950).

[9.8]


6()5

was heated to approximately 40 ° to allow any water present to react with the acetyl chloride. Suitable precautions must be observed to accommodate any excess pressure which may develop. The bottom of the reaction tube was cooled in liquid nitrogen until all gaseous products were collected. The liquid nitrogen was then replaced by a dry ice-isopropyl alcohol bath and the excess hydrogen chloride removed i n vacuo.

Freezing mixture

C°ld conden! fingee~

/Reaction tube

I:H FIG. 3. Chlorination apparatus. The frozen mixture was placed on a steam bath where the condenser was filled with dry ice-isopropyl alcohol and opened to the atmosphere through a drying tube. Chlorine gas, passed through a phosphorus pentoxide tower, was bubbled through the reaction mixture at 100° for 2 hours. On cooling, the mixture solidified. The dry ice-isopropyl alcohol mixture was then poured out, most of the liquid chlorine was allowed to escape by evaporation, and the condenser stopcock closed. The reaction tube was immediately immersed in liquid nitrogen to prevent pressure from developing. The chloroacetic acid was distilled into the bottom of the reaction tube by filling the cold finger condenser with boiling water and by gently heating the other exposed parts of the apparatus with a small flame. The condenser and liquid nitrogen both were removed, and 1 ml. of water was added to the frozen mixture, which was then warmed and mixed, the chlorine being allowed to escape. The product was transferred to a 75-ml.

666


[28]

pear-shaped flask with the aid of about 5 ml. of water, immersed in an ice bath, and neutralized with about 20 to 25 ml. of 2 N KOH with phenolphthalein as indicator. The solution was frozen in a dry ice-isopropyl alcohol bath and then broken into small lumps with a spatula. The flask and frozen contents were then placed in a vacuum desiccator and freezedried (36 to 50 hours). Ethyl sulfate (15 ml.) was added to the dry solid, a reflux-distillation head was put in place, and the mixture was refluxed for 30 minutes by heating in an oil bath at 135 °. The temperature of the oil bath was then raised to 170 ° and the distillate collected for a period of about 2 hours. The reaction was judged to be complete when the black residue had acquired a granular appearance or when a blackish material began condensing in the reflux column. At this point, about 1.5 ml. of n-decane was added to the flask through the condenser, and the n-decane distilled out, carrying with it any residual ethyl chloroacetate. When the distillation was completed, the receiving vessel was removed and stoppered to prevent any access of moisture. If the reaction mixture is heated for too long beyond the stopping point described, it may suddenly decompose into a frothy carbonaceous mass. In addition to the desired product, ethyl chloroacetate, and n-decane, the distillate contains some ether, ethyl alcohol, and iodine. These impurities have been found not to interfere with any of the subsequent steps. The yield is 70 to 80 % based on sodium acetate. Aspartic Acid-3-C 14. Diethyl acetamidomalonate (7.32 g., 33.8 millimole, a 100% molar excess) was dissolved in 40 ml. of carefully dried ethanol in a 100-ml. round-bottomed flask fitted with a reflux condenser and protected from the air with a drying tube. To this solution was added 0.51 g. of metallic sodium (22.2 millimole, a 25% molar excess). When the sodium was dissolved, the ethyl chloroacetate was rinsed into the flask with about 5 to 10 ml. of dried ethanol, and the solution was allowed to reflux on the steam bath. After 4 hours, the ethanol was completely removed by distillation. Concentrated HC1 (50 ml.) was then added to the residue and the mixture refluxed for 72 hours. After evaporation to dryness on a steam bath and final drying in vacuo, the crude hydrolyzate was dissolved in a small quantity of water and transferred to a beaker. Copper chloride (7.67 g. of CuC12.2H20, a 33 % molar excess over theoretical total amino acid yield) was then added to the hydrolyzate solution and the total volume brought to 150 ml. This solution was adjusted to pH 5 with 1 N NaOH (approximately 97 ml.) and then placed in the refrigerator for at least 2 days. Approximately 4 ml. of Johns Manville Celite Analytical Filter Aid was then added to the cold mixture. The precipitate was filtered onto a 0.25-cm. bed of Celite and washed with ice water until the filtrate was chloride-free. The precipitate was redispersed in about 150 ml. of water,

668


[28]

The radioactive yield is 16% based on methanol-C 14 but is 47.5% based on the inactive thio compound. This indicates a conversion of methanol-C ~4 to formaldehyde-CTMof about 34%, although the method utilized claims a yield of 55% for this step. 53 The procedure of Murray et al. 54 gives formaldehyde-C~4from methanol-C ~4in 79 % yield, and that of Elwyn and Sprinson 5~ (see Serine-C 14) in 65% yield. Thus, by substituting one of these procedures, one might raise the over-all yield of cystine/~-CTM considerably. Methanol-C ~4may be readily prepared in nearly quantitative yield by the reduction of C~O2 with lithium aluminum hydride27 The procedure is the same as that for the preparation of ethanol-C 14 (see Alanine-C14). Procedure. C~4H~OH (approximately 150 mg., 0.9 me.) was converted to an aqueous solution of C~4H20 5~ (approximately 104 mg. per 5 ml.). This was added to a 10 % excess of ethyl N-benzylthiothiocarbonylaminomalonate (1.30 g.) in pyridine (10 ml.). 5~ The mixture was kept at room temperature overnight, 10 mg. of unlabeled formaldehyde (in dilute aqueous solution) was added, and after a further 7 hours the solvent was evaporated i n vacuo at 60 °. The residue was dried in vacuo overnight over concentrated H2SO4. Anhydrous ether (5 ml.) was added to the viscous oil, and the solution was cooled in an ice-salt bath. Purified thionyl chloride (10 ml.), 56 also previously cooled, was added dropwise to the ether solution at a slow rate. The mixture was allowed to stand in the cold for 1 hour after the addition of the thionyl chloride; as much of the thionyl chloride as possible was then removed by evaporation in vacuo at 20 °. 5 N NaOH in ethanol was then added to the residual oil until the solution was alkaline to litmus. After 20 minutes the alkaline solution was neutralized with 3 h r HC1 and the ethanol removed under reduced pressure. An excess of 3 N HCI was added, and the solution was refluxed for 2 hours. After removal of the HC1 under reduced pressure, water (25 ml.) followed by 1 N NaOH (25 ml.) was added to give a strongly alkaline solution which was allowed to stand for 30 minutes with occasional stirring. After additioa of concentrated HC1 (50 ml.), the solution was refluxed for 24 hours. The HC1 and some benzoyl mercaptan were removed by evaporation in vacuo, water being added to assist in the removal of the last traces. Water (50 ml.) was added, and the solution was twice extracted with ether, the ether layer being removed with a pipet. After evaporation of the aqueous layer, the residue was extracted with ethanol (10 ml.). The insoluble material was filtered off and washed with more ethanol. The filtrate (40 ml.) ~3 H. 54 A. 55 D. 58 E.

R. V. Arnstein, Biochem. J. 49, 439 (1951). Murray, III, C. W. Bells, and A. R. Ronzio, J. Am. Chem. Soc. 74, 2405 (1952}. Elwyn and D. B. Sprinson, J. Biol. Chem. 184, 465 (1950). L. Martin and L. F. Fieser, Org. Syntheses Coll. Vol. 2, 570 (1943).

[28]


669

was diluted with an equal volume of water, and the solution was oxidized with 0.2 N iodine (required: 10 ml., 52% yield), with starch as an external indicator. Excess iodine was removed with a drop of sodium thiosulfate solution, and the solution was adjusted to pH 5 by adding ammonia (specific gravity 0.880) until alkaline, followed by acetic acid. After 20 hours at 0 ° the cystine-~-C 14 was filtered off and dried (yield 0.2193 g., 47.5 % based on ethyl N-benzylthiothiocarbonylaminomalonate; specific radioactivity 0.63 ~c./mg.). The radioactivity of this material was unchanged after recrystallization from water. G l u t a m i c A c i d - C 14

DL-Glutamic Acid-5-C ~4. DL-Glutamic acid-5-C ~4 has been prepared by the following sequence of reactions in 36 to 42% over-all yield, based on KC14N.56~ O CH~CH2 ~- K C N --~ HOCH2CH2CN -~ C H 2 = C H C N C H 2 = C H C N + CH(COOEt)2 --+ NCCH2CH2C--(COOEt)2

]

NHAc

I

NHAc hydrolysis Glutamic acid-5-C 14

Procedure: Acrylonitrile-l-C 14. KC14N (325 mg., 5 millimoles) in 0.5 ml. of water was added to 1 ml. of a solution of 6.18 g. of 1VIgSO4.TH20 in 10 ml. of water. The solution was cooled to 0 °, and 0.5 g. of ethylene oxide at 0 ° was added. After standing at 0 ° for 2 hours with occasional shaking, the solution was allowed to stand at room temperature for 12 hours. Carbon dioxide was passed into the cooled solution for 30 minutes, the mixture transferred to a centrifuge tube, concentrated under vacuum at 30 °, and the residue extracted three times with 4 ml. and eight times with 2 ml. of ethyl acetate. The combined extracts were dried over calcium chloride. Evaporation of the solvent in vacuum left crude ethylene cyanohydrin. Thirty milligrams of diethylene glycol and 30 mg. of basic 1V[gC03 were added. The vessel was connected to a trap which was cooled in a dry icechloroform mixture and which contained about 80 rag. of finely powdered calcium chloride. A slow stream of nitrogen was passed through the apparatus. The tube connecting the trap and the reaction vessel was surrounded by a jacket, through which isopropyl alcohol vapor was passed. The reaction started shortly after the reaction vessel was placed in a bath at 190°, The yield of material boiling at 76 to 78 ° was ] 60 to 185 rag. (60 to 5~ H. Tiedemann, Biochem. Z. 326, 511 (1955).

670


[28]

70%). (For larger quantities, the ethylene cyanohydrin should be added dropwise to the magnesium carbonate-diethylene glycol mixture.) Glutamic Acid-5-C 14. Thirty-five milligrams of sodium ethylate in 2 ml. of dry ether was added to 1 g. of diethyl formamidomalonate (or 1.1 g. of diethyl acetamidomalonate). The vessel was connected to a high vacuum line, and 180 mg. of acrylonitrile was distilled into the reaction vessel under high vacuum through a tube containing a 2-cm. layer of finely powdered calcium chloride. During the evacuation, both vessels were cooled in liquid nitrogen, after which the liquid was removed from the vessel containing the acrylonitrile. The reaction vessel was shaken for 5 hours in a water bath at 50 ° and then allowed to stand for several hours at room temperature. After acidification with dilute H2SO4 the mixture was extracted with ether and the extracts dried. The ether was removed in vacuo and the residue hydrolyzed with 10 ml. of concentrated HC1 for 6 hours at 116 to 120 °. The solution was then evaporated to dryness under vacuum. Water was added, and the solution evaporated twice more. The residue was dissolved in a minimum of water, the pH adjusted to 3.0 with 0.1 N NaOH, and the crude glutamic acid precipitated with alcohol. In order to remove the glycine formed from unreacted formamidomalonate, the crude product was dissolved in 40 ml. of water and shaken for 30 minutes with 3 g. of Amberlite IR-45 (carbonate form). After centrifugation, the supernatant liquid was shaken twice more with 1 g. of the resin. The resin was washed twice with 10 ml. of water. The glutamic acid was eluted from the resin with 70 ml. of 0.2 N HC1. The eluate was concentrated under vacuum as before, the residue dissolved in 50 ml. of water, the pH adjusted to 3.0 as above, and the glutamic acid crystallized by concentration. The yield was 298 rag. (60%). Other Syntheses. A very similar reaction scheme was reported by Depocas and Bouthillier, 57 but few experimental details were given. Glutamic A c i d - l - C ~4. Carboxy-labeled glutamic acid has been prepared in 47% yield based on cyanide-C ~4 by Speer et al. ~4 The experimental details are available on microfilm; the reaction sequence is as follows: KCN + C1CH~CI-I~OH ------) HOCH2CH~SN--HB-~r BrCH2CH2COOH * 80 %

92 % phthalimido- hydrolysis

malonic ester

---* Glutamic acid-l-C 1~

70-75%

47 % over-all yield

Glutamic Acid-2-C 14. This compound has been prepared by a similrs procedure to that reported above for glutamic acid-5-C 14, but the label 5~F. Depocas and L. P. Bouthillier, Rev. can. biol. 10, 289 (1951).

[28]


671

originates from ethyl acetamidomalonate-2-C 14. The original procedure is t h a t of Albertson and Archer? s Glutamine-C ~4 DL-Glutamine-C ~4. Pure DL-glutamine-C ~4 m a y be prepared from glutamic acid-C ~4 in 34% yield. The position of the label corresponds to t h a t in the glutamic acid used as starting material. The procedure has been carried out in the authors' laboratory with OL-glutamic acid-2-C ~4 and is a small-scale adaptation of the method of King and K i d d ? 9 The procedure is not suitable for the preparation of L-glutamine-C ~4, as racemization of L-glutamic acid occurs in the course of the synthesis.

0

HOOCCH.~CH2~HCOOH

I

phth~lic A c 2 _ O / ~ N - - N J anhydrid--~ -0

l

~'"l 0

I

II

\ ~--~ 0

0 II • H~NNH~ [I • H2NCCH2CH2CHCOOH ~H~NCCH2CH~CHCOOH 0

I

N

I

NH2

O~-C C~O

N - - C H 3 ~ CH3NH2 --CO

Secondary Intermediates 1. Aldehydes and ketones are useful intermediates for the synthesis of straight-chain and branched-chain fatty acids, respectively. a. Acetaldehyde is obtained by hydration of acetylene in the presence of mercuric ion as catalyst: 1 H g ++

HC--~-~CtI + H20 ....

S04--

~ CH~--CHO

b. No general method for the synthesis of aldehydes from carboxylic acids is available. Among the many procedures ~ used, the controlled reduction with lithium aluminum hydride seems to be the method of 9 H. S. Anker, unpublished experiments. 10W. J. Arrol and R. Glascock, Nature 159, 810 (1947). 11R. D. It. Heard, J. H. Jamieson, and S. Solomon, J. Am. Chem. Soc. 73, 4985 (1951). 12j. D. Cox and R. J. Warne, J. Chem. Soc. 1951, 1896. ~3E. Mosettig, Org. Reactions 8, 218 (1954).

782

[32]


choice for the preparation of aliphatic aldehydes :18,

x,

i

e. Acetone is prepared by the pyrolysis of lithium acetate :~4 2CH3COOLi ~ CI-I3COC[-I3 nu Li2CO3 This method is also applicable for the preparation of other ketones. d. Several methods are available for the preparation of ketones using malonic esters and aeid chlorides by the following reaction scheme:

I

C/COOR''-

RCOC1 -{-

/ Na

i\

LR' C O O R " ,

-* RCOC

COOR" -~

i\

R' COOR" RCOCH2R / -{- 2CO2 -[- 2 R " O H

If R' is hydrogen, methyl ketones are obtained. Either the benzyl, 16 tetrahydropyran-2-ol, TM or tertiary butyl T M esters of malonic have been employed, since these can be decomposed without the use of acid or alkali, both of which frequently result in acid cleavage. The benzyl esters are hydrogenated followed by decarboxylation by heating. This method can only be used for nonreducible compounds. The tetrahydropyranol esters decompose with decarboxylation on heating in the presence of acetic acid as do the tertiary butyl esters with catalytic amounts of strong acids. 2. For both the cyanide and carbon dioxide addition the alkyl bromides seem the most generally useful starting materials, although in special cases the chlorides or iodides have been used. A large number of the alkyl halides are commercially available or can be prepared according to published methods. In some cases, however, it will be found that the required bromide is not available but the unlabeled analog of the acid F. Weygand, B. Eberhardt, H. Linden, F. SchRfer, and I. Eigen, Angew. Chem. 65, 525 (1953); G. Wittig and P. Hornberger, Ann. 577, 11 (1952). ~4I. Zabin and K. Bloch, .7. Biol. Chem. 185, 118 (1950). ~s R. E. Bowman, J. Chem. Soc. 1950, 322. 18 R. E. Bowman and W. D. Fordham, J. Chem. Soc. 195a., 3945. 1~G. S. Fonken and W. S. Johnson, J. Am. Chem. Soc. 74, 831 (1952). ~s W. H. Putherbaugh, F. W. Swayer, and C. R. Hauser, J. Am. Chem. ,%c. 74, 3438 (1952). 1as

[32]

CARBON-LABELED FATTY ACIDS AND RELATED COMPOUNDS

783

to be synthesized is. In such cases the bromide or iodide is readily prepared by decarboxylation of the silver salt of the acid with bromine or iodine respectively: RCOOAg ~ Br2 ~-~ RBr ~- AgBr ~- CO2 The preparation is carried out as described on p. 803. The halides prepared by this method have been tabulated. 19 Halides are furthermore available by reduction of acids with lithium aluminum hydride to the alcohols. ~°,~1 These on treatment with concentrated HI or concentrated HBr are converted to the iodides or bromides :22 RCOOH .LiA1H~ . RCH20H . .

HI

) RCH2I

Fatty Acids 1. The synthesis of carboxyl-labeled fatty acids can be effected with either cyanide or COs as starting materials :23-e5 a. RBr --~ RMgBr CO~ RCOOMgBr-~ RCOOH b. RBr -t- KCN --~ RCN -~ RCOOH The methods used differ mainly by the presence or absence of a vacuum line for the carbonation of the Grignard reagent. Carboxyl-labeled linoleic acid has been prepared by the following sequence of reactions starting from the natural product :26 CsH 11CH=CH--CH~--CH--CH--(CH2) 7--COOH

Br2

)

C ~H11CHBr--CHBr--CH2--CHBr--CHBr--(CH 2)v--COOH

Ag +

Br2

Silver salt - - - ~ C~HllCHBr--CHBr--CH2--CHBr--CHBr--(CH2)~Br Zn -~ C s H l l C H = C H - - C H 2 - - C H = C H ( C H ~ ) T B r Mg) Grignard reagent CO~ C~HllCH---CH--CH2--CH-~-CH--(CH2) TCOOH Carboxyl-labeled oleic and other unsaturated acids can probably be prepared by a similar set of reactions. 19 j . Kleinberg, Chem. Rev, 40, 381 (1947). 2o W. G. Brown, Org. Reactions 6, 469 (1951). 21 R. F. Nystrom, W. H. Yanko, and W. G. Brown, J. Am. Chem. Soc. 70, 441 (1948). 2~ It. N. Little and K. Bloch, J. Biol. Chem. 183, 33 (1950). ~3 H. S. Anker, J. Biol.'Chem. 176, 1333 (1948). ~4 S. Weinhouse, G. Medes, and N. F. Floyd, J. Biol. Chem. 155, 143 (1944). ~ H. S. Anker, J. Biol. Chem. 194, 177 (1952). ~6 D. R. ttowton, R. H. Davis, a n d J . C. Nevenzel, J. Am. Chem. Soc. 74, 1109 (1952).

784

TECHNIQUES FOR IOSTOPE STUDIES

[32]

c. Long-chain fatty acids can be esterified with glycerol to give the triglyeerides :27 RCOOH + SOC12 -~ RCOC1 + SO2 + HC1 3RCOC1 + CH~OH CH2--OCOR 1

CHOH :

pyridine

CH2OH

I

" CH--OCOR CH~--OCOR

An elegant synthesis of monoglycerides has been described :~s RCOCI -~- CH~OHCOOH --* RCOOCH:COOH ~ CH2N2

RCOOCH2COC1 He

RCOOCH2COCHN: --* RCOOCH~COCH~OH --; RCOOCH2 et l CHOH

I

CH2OH 2. For chain-labeled fatty acids a variety of syntheses are necessary, depending on the position of the labeled carbon atom. The following reactions have been used, yielding the label in the a-position :~m9-~1 a. RC*H2I Mg) RC*H2MgI CO., RC*H2COOMgI --~ RC*H2COOH OH~ RC*H~CN " RC*H2COOH c. RCOC1 + 2C*H.~N~ --~ RCOC*HN2 -t- C*H3C1 + N2 b. RC*H2OH + SO3 --~ RC*H2OSO3H

RCOC*HN2 -~- R'OH

KCN

~ * N~ + RC*H2COOR' ~ RC*H2COOH

base

In the Arndt-Eistert reaction a rearrangement occurs such that the carbon atom of the diazomethane becomes the a-carbon atom of the product. Diazoethane and propane can be used to synthesize branched chain acids2, s~ 27 W. G. Dauben, J. Am. Chem. Soc. 70, 1376 (1948). 23 H. Schlenk, B. G. Lamp, and B. W. DeHaas, J. Am. Chem. Soc. 74, 2550 (1952). ~9D. N. Hess, J. Am. Chem. Soc. 73, 4038 (1951). a0 F. A. Vandenbeuvel and P. Yates, Can. J. Research B28, 556 (1950). sl j. Links and M. S. DeGroot, Rec. tray. chim. 72, 57 (1953). a2 C. Huggett, 1%. T. Arnold, and T. I. Taylor, J. Am. Chem. Soc. 64, 3043 (1912).

[32]

C A R B O N - L A B E L E D FATTY ACIDS AND R E L A T E D COMPOUNDS

785

d. 5-Position :aa,a4

[

1

Na + --~ RC*H~--CH~--COOH

e. y-Position :,4 R'

COOEr

RC*H2--CO + CH~

m

\

CN

i

l

RC*H~--CH--CH--COOH

Pd

CN

R'

H +

" RC

H2CH--CH2COOH R'

f. ~-Position :as CH

CH

CH

CH--C*H2R

+ ICH2~C Ol ga --+CH~/)CO

RC*H2Br

[_

CH2

J

-~

CH2

RC*H~--CH2--CO--CH2--CH~--CH2--COOH

NH2-NH_~ ) RC*H--(CH2)~COOH

g. Any

position:2~,a6

2RC*H2MgBr CdCI2 (RC*H2)2Cd

COCI(CH~)~COOEt

.....

)

RC*H2CO(CH2)~--COOEt NH~-NH2 RC*H2(CH,2)n+ICOOH A similar synthesis has been described, which, even though it has not yet been applied to labeled fatty acids, seems suitable for this purpose :a~ EtOOC(CH2).COCI

4- Na[RC~--(COOCH2C6Hs)

EtOOC(CH2)

2] -+

~CO--CR-~(COOCH2C~H~)

2 Pd H2

EtOOC

A

(CH2) nCO--CR~(COOH)

EtOOC(CH2)nCO--CH2R

2 --+ H2N-NH~

"

" R(CH:)n+2COOH

aa A. W. Dox, J. Am. Chem. Soc. 46, 1708 (1942). ~4 W. Bleyberg and H. Werich, Ber. 64, 2504 (1931). a5 H. Stetter and W. Dierichs, Chem. Ber. 85, 61 and 1061 (1952). 36 j. Cason, Chem. Revs. 40, 15 (1947). a7 D. E. Ames, R. E. Bowman, and R. G. Mason, J. Chem. Soc. 1950, 174.

786


[32]

a,f~-Unsaturated acids are obtained from aldehydes or ketones :3s

/ R R ' C O ~ CH2

COOEt ~ RR'C~C

\

COOEt

/

RR'C~CH--COOH

\

CN

/ R C H O T CHs

CN

COOEt

/ ~ RCH~C

\

COOEt RCH:CH--COOH

\

COOEt

COOEt

h. A similar method has been used for the preparation of labeled dimethylacrylic acid :39-41 CH3

CH3

\

/

C*O ~ BrZnCH:COOEt--*

CH3

\ /

C*OH--CH2COOEt

--*

CH8 CI-I3

\

/

C*~CH--COOH

CH3 General methods for the synthesis of a chain-labeled fatty acid with double bonds at specified positions have not yet been reported. Several recent methods for total syntheses might be suitable.4~-46

Related Compounds 1. Pyruvic acid has been prepared by oxidation of butyl lactate.47 It is simpler to synthesize it from acetyl bromide and cuprous cyanide, s3 The position of the label depends on the starting material used: 3s A. C. Cope, C. M. Hofmann, C. Wyckoff, and E. Hardenbergh, J. Am. Chem. Soc. 68, 3452 (1941). 39 K. Bloch, L. C. Clark, and I. Harary, J. Biol. Chem. 211,687 (1954). 40L. Ruzicka, ~G. Dalma, B. G. Engel, and W. E. Scott, Helv. Chim. Acts 24, 1449 (1941). 41 G. A. R. Kon and K. S. Nargund, J. Chem. Soc. 1952~ 2461. 4~R. E. Bowman, D. E. Ames, and B. W. Boughton, J. Chem. Soc. 19[i0, 177; 19§1~ 1079, 2748; 19§2, 671, 677. 48K. Ahmad, F. M. Bumpus, and F. M. Strong, J. A~n. Chem. Soc. 70, 3391 (1948). 44 R. A. Raphael and F. Sondheimer, ]. Chem. Soc. 1950, 2100; Nature 165, 235 (1950). 4~H. M. Walborski, R. H. Davis, and D. R. Howton, J. Am. Chem. Soc. 73, 2590 (1951). 46 W. J. Gensler and G. R. Thomas, J. Am. Chem. Soc. 78, 4601 (1951). 47 W. Sakami, W. E. Evans, and S. Gurin, J. Am. Chem. Soc. 69, 1110 (1947).

[32]


787

CH~COOH --~ CH3COBr + CuCN --* CH3COCN --* CH3COCONH2 --+ CH~COCOOH 2. Lactic acid is obtained from pyruvic amide by catalytic hydrogenation and hydrolysis :9 CH~COCONH2 + H2(Pt) --+ CH~CHOHCONH2--* CH3CHOHCOOH Lactic acid can also be made from acetaldehyde and sodium cyanide ?7 CH3CHO W N a C N - * CH3CHOHCN--~ CHaCHOHCOOH 3. Methods for synthesis of labeled acetoacetic acids start from the correspondingly labeled acetic acids: 1- or 2-labeled acetoacetic acid is synthesized from 1- or 2-labeled ethyl bromoacetate, and 3- or 4-labeled acetoacetate from I- or 2-labeled ethyl acetate as follows:47 a. (CH3COOEt)Na + BrCH2COOEt Mg CH3COCH2COOEt -~ CH3COCH2COOH 3 or 4-labeled acetoacetic acid is more readily prepared from 1- or 2labeled acetyl chloride and e t h y l - t e r t - b u t y l m a l o n a t e ? 8

b. CH3COC1 + EtOMg

CH

-~ COOC(CH3)

/ CH~CO--CH

3

COOEt --* CH~COCH2COOEt

\

-~

COOC(CH~) CH3COCH2COOH Acetyl chloride is prepared as described for acetyl bromide from sodium acetate and benzoyl chloride (p. 800). This method is also applicable for the synthesis of substituted acetoacetic esters:49

2 RCOC1 +

CR'

Mg--~ COOC(CH~)3

2 COOEr

2

RCO

CR'

R'

\

H+ COOC(CH~)

~

RCOC--COOEt

3

4s W. G. Dauben and H. L. Bradlow, J. Am. Chem. Soc. 74, 5204 (1952). 49 D. S. Breslow, E. Baumgarten, and C. R. Hauser, J. Am. Chem. Soc. 66, 1288 (1944).

788

[32]

T E C H N I Q U E S FOR ISOTOPE STUDIES

B. Degradation Procedures

Stepwise Degradation of Fatty Acids 1. D e c a r b o x y l a t i o n of the silver salt: 25 R C H 2 C O O H --~ R C H 2 C O O A g --~ R C H 2 B r --* RCH2OAc --~ R C H 2 O H --~ R C O O H 2. Barbier-Wieland degradation :50

R C H 2 C O O H --* R C H 2 C O O E t --* R C H 2 C

\ OEt

2RC ~ H - - ~ C ~

2

-~

OH

3. Sehmidt reaction :51 R C H 2 C O O H I-IN~ R C H ~ N H 2 KMnO~ R C O O H 4. T h e r m a l cleavage:53 R C H 2 C H 2 C O O H ~ RCH2CH~COC1 ~ R C H 2 C H B r C O C I --~ R C H 2 C I - I B r C O O R t --~ R C H ~ C H - - C O O R ' --~ R C I - I m - C H - - C O O K --. R C O O K -~ C H 3 C O O K B y this m e t h o d two carbon a t o m s are removed simultaneously. 5. Imidazole degradation: 47

CH3COOH + I

I - - N H 2 -~ C H ~ - - C H

",/ KMnO, )

H---, /

--N%cH ~oE. R. Stadtman, private communication. ~ E. F. Phares, Arch. Biochem. and Biophys. 33, 173 (1951). 5~G. D. Hunter and G, Popj~k, Biochem. J. 50, 163 (1951).

[32]


789

Repetition of any of these procedures will permit the complete analysis of every carbon atom of the chain. 6. Chromic acid oxidation: ~5 C*H3CH~(CH2).COOH

CrO3) C*H3COOH + (n ~- 1)COo.

H2SO4

If branched-chain fatty acids are subjected to chromic acid oxidation every terminal methyl group yields 1 mole of acetic acid. However, only 1 mole of acetic acid is obtained from a terminal isopropyl group.

Degradation of Related Compounds 1. Pyruvic acid is oxidized by ceric sulfate to acetic acid and carbon dioxide. 58,54 Acetic acid can be degraded as previously described (p. 788) : CH3COCOOH

ceric sulfate

)CH3COOH + C Q

2. Several methods are available for the degradation of lactic acid. It has been oxidized with permanganate to acetaldehyde and carbon dioxide. 55 a. Imidazole method:5~ H ----N

NaOI < KMnO, ( / ~ - - - - N H '

N ~C--COOH H

(see p. 807) b. Oxidation by chromic acid:57 CH3CHOHCOOH CrO~ CHaCOOH -t- CO2

(see p. 808)

3. Two methods for the degradation of acetoacetic acid are commonly used: ~ M. F. Utter, F. Lipmann, and C. H. Werkman, J. Biol. Chem. 1§8, 521 (1945). 54 H. A. Krebs and W. A. Johnson, Biochem. J. 31, 645 (1937). 55 H. G. Wood, N. Lifson, and V. Lorber, J. Biol. Chem. 159, 475 (1945). 5~ S. Roseman, J. Am. Chem. Soc. 75, 3854 (1953).

5vM. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, in "Isotopic Carbon." John Wiley & Sons, New York, 1949.

790


[32]

a. Decarboxylation by acid:~.58 CH3COCH~COOH

Hg+ +

~ Acetone-mercuric sulfate complex + COs

Acetone is further degraded with hypoiodate to yield iodoform :24 CH3COCH3

NaOI

~ CH~COOH + CI~H

After removal of carbon dioxide and the acetone-mercuric sulfate complex the hydroxybutyric acid remaining in the reaction mixture can be analyzed by oxidation with chromic acid to yield more carbon dioxide and acetone-mercuric sulfate complex. b. Permanganate oxidation :sg CH3COCH2COOH KMnO¢ CH3COOH + HCOOH + C02 Formic acid is oxidized with mercuric sulfate: H C O O H + 2HgSO~--* COs + Hg~SO4 + H~SO,

II. Specific Procedures A. Synthetic Methods Preparation of NaCN (Method a)3 The apparatus consists of a length of glass tubing (A), approximately 2 ~ X ~ inch, sealed at one end, connected to one arm of a T-joint by rubber tubing. The stem of the T projects down into the first (B) of two vertical test tubes (about 1 X 6 inch, stoppered). The other arm leads through a right angle to the bottom of the second test tube (C) from the top of which glass tubing leads to a nitrogen inlet and a 50-ml. reservoir bulb (D). The test tubes are slipped snugly through 4-inch squares of asbestos. The BaCI~03 is placed on a small watch glass, and sufficient inert barium carbonate is added to it to give a total weight of approximately 0.1 g. This is then mixed into a paste with a little ethyl alcohol by using a small glass rod, and is allowed to dry in a desiccator. It is scraped from the watch glass onto black glazed paper, and any lumps are gently broken up by pressing with a spatula. The barium carbonate is then mixed well, while still on the glazed paper, with 1.5 g. of sodium azide which has been finely powdered, in small amounts in a mortar. ~8 D. D. Van Slyke, J. Biol. Chem. 32, 455 (1917). 6~ S. Weinhouse and R. H. Millington, J. Biol. Chem. 181, 645 (1949).

[$2]

CARBON-LABELED

F A T T Y ACIDS A N D

RELATED

COMPOUNDS

791

The apparatus is flushed out with dry nitrogen and a slow stream kept up throughout the experiment. The carbonate-azide mixture is introduced into A. Bunsen burners are then placed under the test tubes so that the inner blue cone of the flame just impinges on the bottom of the test tubes. The asbestos shields are placed approximately 1.5 inch above the bottoms of the test tubes, which should now glow with a dull red heat. Tube A is raised and is tapped gently so that its contents are slowly emptied into test tube B. This should take about 15 minutes. As the powder reaches the bottom of B, minute explosions occur and some of the undecomposed powder is blown over into C where it finally reacts. Metallic sodium collects on the sides of the tubes and remains there throughout the course of the experiment, since no air enters the tube after the miniature explosions because of the nitrogen reservoir, D. The asbestos shields are now removed, and the base and walls of both test tubes are heated to a dull red heat for 10 minutes by movement of the Bunsen burners. They are then allowed to cool, the nitrogen supply is turned off, and water is carefully added drop by drop to B and C to decompose the sodium. The contents of C are transferred with boiling water to B so that there is approximately 20 ml. of liquid in B. About 5 ml. of 0.1 N barium hydroxide is added to remove any carbonate formed; the solution is brought to boiling and filtered. The filtrate is placed in a distillation apparatus with boiling chips, acidified with 50% sulfuric acid, and the HC14N distilled into approximately 10 ml. of 0.1 N sodium hydroxide.

Preparation of NaCN (Method b) 6 A porcelain boat containing 140 mg. of Mg is placed in a Vicor tube inside a combustion furnace. A Pyrex tube containing 310 rag. of BaC03 well-mixed with 3 g. of lead chloride is attached to the Vicor combustion tube, and the entire apparatus is evacuated. The stopcock is then turned to seal the system from the pump. Slowly and carefully the lead chloride is heated (from the top down) with a microburner until it melts completely and until the side of the Pyrex tube collapses slightly. It is advisable to place a little wad of glass wool in the tube to prevent spattering. The furnace is now turned on and is allowed to produce "red heat." When the reaction appears to be complete, as evidenced by the fall of pressure in the manometer (about 1 to 2 hours), the furnace is turned off and the system is allowed to cool. The Pyrex tube is removed from the combustion tube and is replaced by an inlet tube for the dry NH3 gas. At the other end of the combustion tube is placed a safety trap followed by a trap containing 25 ml. of 0.7 N NaOH. Air is pulled through the system and through the alkali to trap any unreacted carbon dioxide. This alkali trap is then replaced by a gas wash bottle containing 4 ml. of 0.78 ~V

792


[32]

NaOH (slightly more than calculated on the basis of 100% conversion of the carbon dioxide to cyanide) and about 30 ml. of concentrated ammonium hydroxide. The ammonia valve is opened, and, when the correct flow rate is achieved, the furnace is turned on. After 4 to 6 hours of heating, the apparatus is disconnected, and the trap liquid is made up to 50 ml. An aliquot is titrated for CN ion by using standard silver nitrate solution (with a microburet) according to directions in Kolthoff and Sandall. The major portion is then concentrated to dryness in vacuo, water is added once or twice, and the material is concentrated to dryness and placed in the desiccator. The yield varies from 60 to 75 %. Preparation of CuCN 9

Commercial cuprous chloride is: dissolved in the minimum amount of concentrated hydrochloric acid, poured into20 vol. of water, washed with water by decantation until the supernatant is colorless, then with acetone on a Bfichner funnel, and dried in vacuo over phosphorus pentoxide. To 1 mole of solid cuprous chloride is added a solution of 1 mole of sodium cyanide in 100 ml. of water. The mixture is shaken for 12 hours, filtered, washed with water, and the precipitate dried at 125° for 12 hours. The yield is quantitative. Preparation of Barium Carbide 1°

By means of a vacuum system carbon dioxide is transferred to a stainless steel bomb containing a onefold excess of barium which has been scraped as clean as possible before being weighed. The bomb is closed, transferred to a furnace, and heated for 5 to 10 minutes. When it has cooled, the contents of the bomb are dissolved in water. The acetylene produced is purified by selective adsorption on charcoal; the mixture of gases is passed through a spiral dry ice-cooled trap to remove most of the water, and the ammonia, water vapor, acetylene, and traces of ethylene are adsorbed on active charcoal at dry ice temperatures. The charcoal trap is then warmed to 0 °, and the acetylene and ethylene are pumped into a liquid nitrogen trap. If not more than 10 minutes is taken for this last step, almost no water or ammonia will be desorbed and practically all the acetylene will be recovered. Synthesis of Labeled D i a z o m e t h a n e 11

Labeled hydrogen cyanide (4.2 millimole) in 9.4 millimole of sodium hydroxide solution is evaporated to dryness in vacuo. To the residue are added freshly reduced )~dam's catalyst prepared from 85 rag. of platinum oxide in 5 ml. of glacial acetic acid and 11.5 millimole of concentrated hydrochloric acid in 5 ml. of glacial acetic acid. The mixture is shaken

[32]


793

with hydrogen until the theoretical amount has been absorbed. The filtered reaction mixture is evaporated to dryness. The hydrogenation residue, in 1 ml. of water, is refluxed for 3 hours with 711 rag. of urea. Then 5.6 ml. of an aqueous solution of 790 mg. of carrierm ethyl urea and 1.3 g. of sodium nitrite is added, and, to this, 0.65 ml. of sulfuric acid in 7.1 g. of ice water is added dropwise and with stirring over 15 minutes. The precipitated and vacuum-dried nitroso-C~4 methylurea amounts to 1.04 g., m.p. 120 to 123 °. The yield is 55%, calculated from cyanide. The nitroso-C~4-methylurea is decomposed in the usual manner with 3 ml. of 50% potassium hydroxide solution and the liberated diazomethane-C TM distilled in 50 ml. of ether. The over-all yield of diazomethane is 319 mg. (41 to 42%, calculated from cyanide).

Preparation of Acetaldehyde ~ Acetylene is generated from about 0.6 millimole of barium carbide by the addition of water and collected in a trap. The trap containing acetylene is connected to a tube containing 10 ml. of a hydration catalyst prepared by diluting a mixture of 2 g. of mercuric sulfate and 6 g. of concentrated sulfuric acid to a volume of 100 ml. with water. After the acetylene has been distilled into the catalyst solution, the tube is sealed and the mixture is heated for 5 minutes at 100° to effect hydration. The yield is 75 %, based on acetylene. The acetaldehyde can be separated from water by distillation.

General Method for Aldehyde Synthesis TM To 0.1 mole of the acid chloride in 100 ml. of ether 0.1 mole of potassium carbazole is added in small portions with cooling to 0 °. The mixture is stirred for 1 hour at 0 °, then for 12 hours at room temperature, filtered, and the derivative precipitated by addition of petroleum ether. To 0.1 mole of the acid carbazole in 200 ml. of ether cooled to 0 ° is added slowly 25 ml. of a 1 M ethereal lithium aluminum hydride solution. After standing for 15 minutes at room temperature the product is decomposed by hydrochloric acid and the aldehyde isolated by an appropriate procedure.

Synthesis of Acetone 14 Labeled acetic acid is converted to its lithium salt and dried thoroughly i n vacuo at 100°. For pyrolysis, batches of about 2.5 g. of lithium acetate are placed in Pyrex boats and heated in a Pyrex tube at 350 ° for 30 minutes and subsequently at 400 ° for 1 ~ hours. Before the heating is begun, a slow stream of nitrogen is passed through the reaction tube, and this is continued throughout the pyrolysis. Acetone is condensed in a

794


[32]

trap which is cooled by dry ice and used immediately in the next step. Yields of acetone range from 80 to 95 %. General M e t h o d for Ketone Synthesis 1~

The requisite malonic acid (0.05 mole) is added in portions to a solution of dihydropyran (0.075 mole per carboxyl grouping in the malonic acid) in benzene (50 ml.) containing concentrated sulfuric acid (1 drop), with cooling to below 30 ° . Heat is evolved during the addition and for some time afterward. In all cases, the reaction is substantially complete when p clear solution is obtained, but in practice the mixture is left at room temperature for a further 0.5 hour. Traces of free acid are then removed by shaking or stirring the solution with solid potassium hydroxide (4 g.) for 1/~ hour, and the solution is decanted from inorganic material. Solvent and excess of dihydropyran are removed by distillation in vacuo, and the residual ester in benzene (50 ml.) is added to 0.05 g. atom of sodium powder in 100 ml. of benzene with cooling to below 35 °. When dissolution of the metal is complete, a solution of 0.05 mole of the acid chloride in 50 ml. of benzene is added. After ~ hour at room temperature, 5 ml. of acetic acid is added and the solution boiled under reflux until evolution of carbon dioxide ceases (ca. 11/~ hours). The collected mixture is washed with water or, if the final product is neutral, with dilute alkali. After removal of solvent, the resulting material is purified by distillation or crystallization. Reduction of Carbon Dioxide to M e t h a n o P 1

The carbon dioxide generated from 13.0 g. of barium carbonate by the slow addition of 30% perchloric acid, and diluted by a stream of nitrogen, is passed through Drierite into a solution of 3.8 g. of lithium aluminum hydride in 500 ml. of diethyl carbitol. Shortly thereafter, 120 g. of n-butyl carbitol is added, and, with continued flow of nitrogen gas, the mixture is heated. The product, collected in a trap cooled by a dry ice freezing mixture, is redistilled to separate a small residue of high boiling material. There is obtained as distillate 1.71 g. of methanol, n s0 D 1.3310, identified further as the N-(a-naphthyl)-carbamate, m.p. 124 °. The yield of methanol is thus 81% based on barium carbonate. A small fraction, 1.7%, of unreacted carbon dioxide is collected in an ascarite tube through which the effluent gas has passed. Methyl Iodide. Twenty millimoles of methyl alcohol is mixed with 15 ml. of hydriodic acid (specific gravity 1.7) and a trace of red phosphorus, and heated under a reflux for 1 hour while a slow stream of nitrogen is passed through the reaction flask. The methyl iodide is collected in a trap cooled by dry ice.

[32]


795

Preparation of 1-C14-Potassium Acetate 23

The apparatus consists of a carbon dioxide generator equipped with a dropping funnel, a gas wash bottle filled with concentrated sulfuric acid, a three-necked flask with a dropping funnel, and two traps. Twenty-five millimoles of barium carbonate-C 14 is introduced into the generator, 50 ml. of hydrochloric acid-water (1 : 10) into the dropping funnel, and a saturated barium hydroxide solution into the last trap. After a moderate stream of nitrogen has been started, 100 ml. of an ethereal 0.5 N methyl magnesium bromide solution is pipetted into the reaction vessel, which is cooled in an ice bath. After all the air has been displaced, 100 ml. of 0.1 N sodium hydroxide solution is added to the gas absorption bottle. The nitrogen stream is reduced and the hydrochloric acid added rapidly to the barium carbonate. The reaction vessel is continuously shaken. When the barium carbonate is decomposed, the nitrogen stream is increased again. The reaction product precipitates as the carbon dioxide enters the reaction vessel. After 20 to 30 minutes of shaking, 50 ml. of ice water is introduced into the reaction vessel. When the precipitate has dissolved, 30 ml. of 2 N sulfuric acid is added and the ice bath removed. All additions are made after pressure equalization and without interruption of the nitrogen stream, which is continued until the ether layer has evaporated completely. Sixty millimoles of silver sulfate and 50 ml. of concentrated sulfuric acid are added, and the mixture is distilled with steam in an all-glass distilling apparatus. The distillate is neutralized with 0.1 N potassium hydroxide and concentrated to dryness. The salt is dissolved in 100 ml. of hot methanol and filtered after addition of a small quantity of charcoal. The filtrate is again evaporated and dried. The yield is 65 to 85%, based on the barium carbonate used. Nearly all unchanged carbon dioxide can be recovered from the alkali trap. (Only a very small quantity of barium carbonate should have precipitated in the barium hydroxide trap.) Preparation of Carboxyl-Labeled n-Octanoic Acid ~4

Carbon dioxide is released from barium carbonate by means of sulfuric acid into a high vacuum system where it is purified and dried by repeated sublimation at - 8 0 ° and condensation at - 2 0 0 °. The CO2 is then condensed into a highly evacuated flask containing an excess of n-heptylmagnesium bromide in ether. After several hours are allowed for the reaction to go to completion, the reaction mixture is decomposed with dilute sulfuric acid and distilled with steam. The distillate is extracted with ether, the ether extract shaken with dilute sodium hydroxide, and the aqueous solution of sodium octanoate acidified and re-extracted with

[39.]


797

Preparation of 9.-C14-Acetic Acid 29 A 10.5-millimole portion of crystalline sulfur trioxide is introduced into a reaction flask equipped with a magnetic stirring bar. The flask is quickly attached to a vacuum line, and the 10-ml. bulb immersed in liquid nitrogen. A 10.06-millimole aliquot of labeled methanol vapor, measured manometrically, is added to the reaction flask. The liquid nitrogen bath is replaced by an ice bath, and the reaction mixture stirred. After the initial reaction has subsided, an additional 1/~ hour at room temperature is allowed for completion of the reaction. Complete reaction is demonstrated by the absence of methanol vapor pressure as determined with a McLeod gage. The flask is removed from the vacuum line, the bulb immersed in liquid nitrogen, and 10 ml. of 7.5 M potassium cyanide added dropwise. After the flask has been allowed to warm slowly to room temperature with stirring for 1/~ hour, the acetonitrile solution is distilled into a calibrated 40-ml. flask. Three successive 10-ml. portions of water are added to the reaction flask and distilled to ensure complete transfer of the acetonitrile. The acetonitrile is hydrolyzed by refluxing with 50 millimoles of potassium hydroxide for 24 hours. The alkaline solution of potassium acetate is acidified with 85 % phosphoric acid and titrated with a solution of potassium permanganate in order to destroy any formic acid. The solution is distilled to dryness after the addition of three successive small portions of water. The distillate is titrated with potassium hydroxide, and the water evaporated. The potassium acetate is dried in high vacuum. Preparation of 2-C14-Yatty Acids 3°,31 One gram of fatty acid is refluxed with 1 ml. of purified thionyl chloride for 2 hours. The excess thionyl chloride is removed i n vacuo. The residual acid chloride is taken up in ether and added slowly to a cold ethereal solution of 2.5 M excess of diazomethane. After standing for 12 hours, the ether is removed i n v a c u o and the residue dried over phosphorus pentoxide. One gram of the diazoketone is added with stirring to a mixture of 5 ml. of benzyl alcohol and 5 ml. of ~-collidine heated to 180°. The reaction mixture is kept at this temperature for about 10 minutes until nitrogen evolution subsides. 61 After saponification with methanolie potassium hydroxide, the free acid is purified by recrystallization from 90% ethanol and high vacuum distillation. Yield, about 80 to 85 %. 6~ h. L. Wilds and A. L. Meader, J, Or 9. Chem. 13, 763 (1948).

798


[32]

Synthesis of 3-C14-Octanoic Acid 83

Ethyl malonate (160 g., 1.0 mole) is added with stirring to a solution of 23 g. (1.0 g. atom) of sodium in 400 ml. of absolute ethanol. The mixture is warmed on a steam bath, and 165 g. (1.0 mole) of n-hexyl bromide is added gradually. The mixture is heated for 4 hours on the steam bath, after which it is neutral and most of the ethanol is then distilled off with stirring to prevent bumping. The residue is dissolved in water; the oily layer is separated, washed with water, and dried over calcium chloride. Fractional distillation of the oil yields 170 g. of ethyl n-hexylmalonate, b.p. 140 to 147°/15 ram. Redistillation gives a 73% yield boiling at 268 to 270°/749 mm. After hydrolysis with constant boiling hydrochloric acid, the product is isolated by extraction with petroleum ether and distilled. Preparation of 4-C14-Isovaleric Acid 14

Palladium black (0.12 g.), ammonium acetate (0.25 g.), and glacial acetic acid (0.36 ml.) are added to a small three-necked flask equipped with a dropping funnel, an inlet for hydrogen, and a stopper. Two grams of labeled acetone is transferred to this flask with the aid of 15 ml. of absolute alcohol. After the flask has been flushed with hydrogen, 1 ml. of ethyl cyanoacetate is added through the dropping funnel and the flask is shaken for hydrogenation. Four ml. of the ethyl cyanoacetate is added in 1-ml. portions at 2-hour intervals. Hydrogen uptake ceases after 9 ~ hours. The catalyst is removed by filtration, and 9 g. of sodium hydroxide in 25 ml. of water is added to the solution containing the ethyl a-cyanoisovalerate. The mixture is heated under reflux for 26 hours. After removal of alcohol by distillation, the solution is acidified with sulfuric acid and the isopropylmalonie acid obtained by continuous extraction with ether for 9 hours. The ether extract is dried over sodium sulfate, the solvent distilled, and the remaining liquid transferred to a small fractionating still. By heating at atmospheric pressure, the malonic acid derivative is decarboxylated and the resulting isovaleric acid distilled. The fraction distilling between 175 and 180 ° is collected. The yield of isovaleric acid based on acetone ranges from 65 to 76 %. Synthesis of 7-C~4-Yatty Acids 85

Eleven grams of dihydroresorcinol is dissolved in 22 ml. of 20 % KOH, 13 g. of allylbromide and 0.3 g. of copper powder added, and the reaction mixture stirred for 5 hours at room temperature. One hundred ml. of 5 % NaOH solution is added and the mixture extracted twice with ether. The residual aqueous solution is freed from ether by a current of air and

[32]


799

acidified to pH 4 with hydrochloric acid, and the precipitated 1-allyl-2,6cyclohexanedione is recrystallized from ethyl acetate. Ten grams of the product is added to 13 g. of sodium hydroxide dissolved in 100 ml. of warm diethylene glycol. To this solution are added 8.2 ml. of 85% hydrazine hydrate and sufficient absolute methanol (ca. 15 ml.) to bring the boiling temperature of the mixture to 110 °. It is refluxed at this temperature for 27 hours, then distilled until the inside temperature reaches 195 °, and refluxed for an additional 13 hours. After cooling 1 vol. of water is added and the mixture acidified with concentrated hydrochloric acid. The separating insoluble oil is extracted with ether and fractionated; b.p. 127 to 128°; yield, 6.5 g. (64% of theory). 1-Methyl-2,6-cyclohexanedione. 3~ Eleven grams of 1,3-cyclohexanedione is heated to refux with 3.9 g. of potassium dissolved in absolute methanol. After cooling 15.6 g. of methyl iodide is added and refluxed for 1 hour. The methanol is evaporated in vacuo, and unreacted materials are extracted with ether after addition of 100 ml. of 3 % sodium hydroxide solution. The aqueous phase is freed of ether by a current of air, acidified with HC1 to pH = 5. The product crystallizes from the solution. Yield, 6.5 g. (51% of theory); m.p. 204 °. The reaction can be carried out with other alkyl iodides in an analogous manner; however, in addition to the carbon-substituted cyclohexanedione oxygen-substituted products are formed. Preparation of 6-C14-n-Hexadecanoic Acid 27

The Grignard reagent is prepared from 0.15 g. (0.0062 mole) of magnesium turnings and 1.412 g. (0.006 mole) of n-hendecyl bromide, labeled at carbon atom 1, in 50 ml. of anhydrous ether. The resulting Grignard reagent is converted to the dialkylcadmium compound with 0.71 g. (0.00389 mole) of anhydrous cadmium chloride. After the addition of the cadmium chloride, the mixture is heated under reflux until a negative Gilman test for a Grignard reagent is obtained. This requires about 2 hours. The ether is replaced with benzene, and the resulting suspension is treated with 1.00 g. (0.0061 mole) of ~-carbomethoxybutyryl chloride. The reaction mixture, after heating under reflux for 1 hour, sets to a solid mass and then decomposes as usual. The crude reaction mixture is directly saponified with a solution of 0.4 g. of potassium hydroxide in 10 ml. of methanol. After dilution to 50 ml. with water, the mixture is extracted with ether to remove the neutral compounds. The alkaline layer is acidified and then extracted with ether. The crude keto acid is reduced by the modified Wolff-Kishner method using 6.4 ml. of diethylene glycol, 0.8 g. of sodium hydroxide, and 0.77 ml. of 100 % hydrazine hydrate. The crude acid is distilled onto a cold finger

800


[32]

type of condenser at a bath temperature of 110 ° and pressure of 1 ram. The distillate is recrystallized from 15 ml. of 10% aqueous acetone (Norit); m.p. 61 to 62°; yield, 700 mg.

Synthesis of Ethyl ~-Hydroxyisovalerate s9,4° Labeled acetone (1.5 ml.) is added to 2 g. of activated zinc ~" and 10 ml. of benzene and 10 ml. of toluene. After addition of 3 ml. of ethylbromoacetate and a crystal of iodine, the reaction starts. Heating is regulated to permit smooth progress of the reaction for about 1 hour. The reaction mixture is decomposed with sulfuric acid in the usual manner, the solvent removed, and the product distilled; b.p. 58 to 60°/9 mm.; yield, 1.0 g. The hydrazide gives m.p. 102 to 103 °. Dimethylacrylic Acid. ~9,4~ To ethyl /~-hydroxyvalerate is added an equal molar amount of fused and powdered potassium acid sulfate and the mixture is heated for 3 hours to 160 to 180 °. The cooled product is extracted with ether and an excess of alkali added. After standing at room temperature, the organic solvent is removed, and the aqueous phase is acidified with sulfuric acid and steam-distilled. The distillate yields 52 % of free acid. The product is characterized by Duclaux distillation. The free acid has m.p. 69 to 70 °, and the anilide m.p. 127 °.

Preparation of Acetyl Bromide 2~ and Pyruvic Acid Twenty millimoles of potassium acetate is pulverized, mixed with 16 millimoles of benzoic acid, and added to a distilling flask. Two portions of 2 millimoles each of benzoic acid are used to wash out the flask which contained the potassium acetate. Ten milliliters of benzoyl bromide is added to the mixture, a glass wool plug is inserted below the side arm, and the flask is heated carefully so that the acetyl bromide distills slowly into the tared receiving flask. The boiling range is 72 to 76 ° . Yield, 75 to 90 %. Acetyl Cyanide. Eight millimoles of acetyl bromide is added to 9 millimoles of dry cuprous cyanide contained in an ice-cooled ampoule. Sufficient dry cyclohexane to wet all the cuprous cyanide is added, and the ampoule is sealed off. After standing for 3 days at room temperature the contents are extracted with five portions of 2.5 ml. each of dry ether. The ether solution is used directly. Yield, 75 to 80%. Pyruvamide. The ether solution is introduced into a jacketed funnel with a fritted filter disk through which dry hydrogen chloride gas passes from the bottom. Ice water is circulated through the jacket, and a calcium chloride tube is attached at the top. After the solution is saturated with hydrogen chloride, 6.5 millimoles of water is added to the ether solution 6~ R. L. Shriner, Org. Reactions 1, 16 (1942).

802


[32]

Synthesis of Sodium Acetoacetate 47

Isotopic sodium acetate is converted into ethyl bromoacetate by the procedure of yon Auwers and Bernhardi. e3 Methyl acetate diluted with 15 ml. of anhydrous ether along with 3 g. of magnesium turnings is placed in a 50-ml. round-bottomed flask equipped with a side arm, dropping funnel, and reflux condenser. The stirrer extends through the bore of the condenser. Labeled ethyl bromoacetate (7.4 ml.) is added dropwise with gentle warming to start the reaction, which is maintained at a gentle boil by controlling the rate of addition of the ethyl bromoacetate. The product is isolated in the usual manner. The ethyl acetoacetate weighs 0.80 g. and on analysis is found to be 88 % pure. Preparation of Carbonyl-Labeled Ethyl Acetoacetate 4s

A mixture of 2.87 g. of magnesium, 100 ml. of absolute ethanol, 20 ml. of dry xylene, and 2 ml. of carbon tetrachloride is refluxed for 12 hours and then concentrated to dryness under reduced pressure on a steam bath. Benzene is added twice and removed in vacuo to ensure the absence of any alcohol. The residue is heated at 100 ° under reduced pressure for 3 hours, cooled, 40 ml. of anhydrous ether added, and the mixture stirred vigorously to break up the solid. Ethyl-t-butylmalonate (22.4 g.) then is added dropwise with stirring, and the mixture is refluxed until complete solution is obtained. Carboxyl-labeled acetyl chloride, prepared from 10.8 g. of sodium acetate, containing 9.9 mc. of C 14, by distillation from benzoyl chloride, is dissolved in 25 ml. of dry ether, and the solution is added dropwise with stirring to the magnesium derivative of the malonic ester. After refluxing for 30 minutes, the reaction mixture is cooled, diluted with water, extracted with ether, and combined with the above ether. The solvents are distilled, the residue dissolved in 100 ml. of benzene, a small amount of the solvent distilled, 0.75 g. of p-toluenesulfonic acid added, and the solution refluxed for 90 minutes. The cooled benzene solution is extracted with saturated sodium bicarbonate and saturated sodium chloride, and the benzene removed through an 18-inch fractionating column. The residue is distilled; b.p. 180°; yield, 12.2 g. (71.3% based on sodium acetate). B. Degradation Procedures

Degradation of Palmitic Acid 26

Palmitic acid (1.8 g.) is converted to the silver salt by dissolving it in methanol, adding an excess of concentrated silver nitrate solution, and 63 K. yon Auwers a n d R. Bernhardi, Bet. 24, 2209 (1891).

[~9.]

CARBON-LABELED

FATTY ACIDS AND RELATED COMPOUNDS

803

neutralizing with concentrated ammonia. The silver salt is centrifuged, washed, and dried at 110° (yield, 2.4 g.). It is suspended in 50 ml. of carbon tetrachloride which is refluxed under dry nitrogen. An excess of bromine (0.45 ml.) is added rapidly, and the liberated carbon dioxide is trapped by barium hydroxide. After 10 minutes, the reaction mixture is filtered, the precipitate washed with ether, and the solvents evaporated. The residue, dissolved in petroleum ether, is passed through a column of alumina. The residue of the petroleum ether eluate consists of pentadecyl bromide and weighs 1.8 g.; m.p. 18.0 to 18.4 °. The pentadecyl bromide is refluxed for 3 hours with an excess of silver acetate suspended in 30 ml. of glacial acetic acid and 1 ml. of acetic anhydride. Water is then added, and the solution is extracted with petroleum ether. The residue of the petroleum ether extract is hydrolyzed with methanolic potassium hydroxide. After extraction with petroleum ether the product (1.4 g.) is adsorbed on a column of alumina and fractionally eluted with petroleum ether-benzene (3:1). The middle eluates are combined, and the solvent evaporated. The residual pentadecanol amounts to 1.34 g.; m.p. 42 to 43 °. The pentadecanol is dissolved in 8 ml. of glacial acetic acid, 1 g. of chromium trioxide in 3 ml. of 66% acetic acid is added, and the mixture heated on a steam bath for 12 minutes. The reaction product is isolated by extraction with petroleum ether and amounts to 1.05 g. ; m.p. 49.5 to 50.0% The over-all yield from palmitic to pentadecanoic acid is 62 %.

Degradation of Methyl Caproate 5° To phenylmagnesium bromide prepared from 0.3 g. of Mg and 1.3 ml. of bromobenzene, 90 mg. of methyl caproate (0.1 ml.) and 1.5 ml. of anhydrous benzene are added. The mixture is refluxed for 2 hours on a steam bath. Then 2.5 ml. of benzene is added, and the solvent is distilled off over a period of 2 hours. The residue is then heated for an additional 2 hours, diluted with 3 ml. of benzene, and poured over crushed ice and ammonium chloride in a 25-ml. centrifuge tube. The flask is rinsed several times with benzene, and the rinsings also are poured over the crushed ice. A tight-fitting stopper is placed in the centrifuge tube, and the mixture is shaken thoroughly and then centrifuged. The clear benzene layer containing the tertiary carbinol is separated in a small separatory funnel and placed in a 50-ml. round-bottomed boiling flask with a groundglass neck. The aqueous portion is washed several times with benzene, and the washings are also placed in the 50-ml. boiling flask. The benzene is distilled off as completely as possible at a reduced pressure (4 cm.) using a small fractionating column 20 cm. tall and made from 10-mm. Pyrex tubing. The condenser is 12 cm. long and 6 ram. in diameter. Three

804


[32]

milliliters of 0.5 N alcoholic sodium hydroxide is added to the residue and refluxed for 30 minutes in order to saponify any unchanged ester. The alcohol is then distilled off under reduced pressure. The residue is dissolved in benzene, washed with water, and the benzene solution evaporated to dryness. To the residue containing the carbinol is added 2 ml. of acetic anhydride, and the mixture is refiuxed for 30 minutes. The excess acetic anhydride is distilled off under reduced pressure as above. The residue is an oil containing the hexene. Two milliliters of glacial acetic acid purified by distilling over potassium permanganate is added to the residue, and to this is added 0.7 ml. of the oxidizing mixture prepared by mixing 0.25 g. of Cr03, 0.2 ml. of water, and 0.5 ml. of glacial acetic acid purified as above. After 15 minutes on the steam bath, the mixture is cooled to - 1 0 ° and the excess Cr03 is decomposed with sodium bisulfite. The chromic acid digest is transferred to a 50-ml. separatory funnel and diluted with 5 ml. of water. This is extracted three times with about 10-ml. portions of ether. The ether fractions are consolidated and extracted with 5 ml. of 5 N sodium hydroxide. The aqueous portion is then acidified with a few drops of concentrated sulfuric acid and extracted with ether again. This ether fraction is placed in a 50-ml. round-bottomed boiling flask, and the ether is distilled off. Twenty-five milliliters of benzene is added to the acid residue from above, and the benzene is distilled off. Benzene forms an azeotropie mixture with acetic acid which boils at about 78 to 79 °. No valeric acid will be carried over by this procedure. If necessary more benzene is added and the distillation repeated until no more acetic acid comes over as determined by direct titration of the distillate. The residue from the azeotropic distillation is made to a volume of 10 ml. with water, a drop of sulfuric acid is added, and the mixture is steam-distilled. One hundred milliliters of distillate is collected. The identity of the volatile acid was determined by a Duelaux distillation. Degradation of Propionic Acid 6~ The apparatus used for the decarboxylation step consists of a reaction flask equipped with sweep tube, a scrubber containing 5% KMnO4 in 0.5 N H:SO4, and a trap containing 0.5 N NaOH. An inlet for CO2-free air is provided, and a vacuum source is connected through a mercury check valve. For the complete, stepwise degradation of propionic acid, about 0.5 mM. of the sodium salt is dried under vacuum in the reaction flask, and after the flask is cooled to about 15°, 0.3 ml. of 100 % H2SO4 is carefully added. The sodium propionate is completely dissolved by warming and shaking, and after recooling of the flask, 50 rag. (0.77 mM.) of sodium

806


[32]

slightly discolored. It is poured into a large excess of 20% H2S04 to remove the residual diethylaniline, and the resulting aqueous solution is extracted three times with an equal volume of ether. The ethereal extract is dried over anhydrous Na2SO4, and the ether then removed. The residue is distilled at 124 to 127°/19 ram., giving neopentyl octenoate (6.05 g., 62%) as a colorless, sweet-smelling oil. Neopentyl octenoate (0.48 g.) is dissolved in ethanol (3 ml.), and a solution of KOH (0.5 g.) in water (1.5 ml.) is added. The mixture is heated under reflux for 2 hours and evaporated to dryness on the steam bath. A further quantity of KOH (2.0 g.) is added, and fusion is then carried out at 300 to 350 ° until gas evolution practically ceases. The product is dissolved in water (30 ml.), and nonacidic impurities are removed by ether extraction. The aqueous solution is now neutralized with H2S04. The acidified aqueous extract is steam-distilled. The mixed acids in the steam distillate are neutralized, and the water then evaporated off. The free acids are liberated from the salts by treatment with a large excess of p-toluenesulfonic acid in dry benzene and fractionated by azeotropic distillation, first with benzene, and then with xylene, any octanoic acid remaining behind. Chromatographically pure acetic acid (0.131 g., 46%) and hexanoic acid (0.465 g., 85 %) are obtained. Thus the over-all conversion of octanoic to hexanoic acid is 51%. Degradation of Acetic Acid 56 (see also Vol. IV [22] and [23]) Glacial acetic acid (0.4 g.), 4 g. of o-phenylenediamine dihydrochloride, 10 ml. of water, and 1.5 ml. of 85% phosphoric acid solution are heated in a sealed glass tube for 2 hours at 135 °. The tube is cooled, opened, and placed in an oil bath at 135 ° for 2 hours. The resulting thick sirup is dissolved in water, neutralized with solid potassium carbonate, pH 3.5 to 4.0. The solution is then shaken with 10 ml. of benzaldehyde for approximately 30 seconds followed by three extractions with chloroform (70 ml. each). This treatment is repeated until the addition of benzaldehyde yields no further color. A final extraction of the colorless solution with petroleum ether is followed by neutralization with solid potassium carbonate. Thirty milliliters of concentrated ammonium hydroxide solution and a solution of 4 g. of silver nitrate, 4 g. of water, and 6 g. of concentrated ammonium hydroxide yield a white silver salt. The free benzimidazole is obtained by treating a suspension of the washed salt in an alcohol-water mixture with hydrogen sulfide. On concentration of the filtrate, 0.75 g. of white needles is obtained, m.p. 174 to 176 °. Recrystallized from dilute ammonium hydroxide, the 2-methylbenzimidazole n~elts at 175.5 to 176.5 °. 2-Methylbenzimidazole (0.2 g.) is heated at 190 to 200 ° with three

[32]

C A R B O N - L A B E L E D FATTY ACIDS AND RELATED COMPOUNDS

807

times its weight of benzaldehyde in a sealed tube for 2 hours. The resulting oil is dissolved in acetone-ether (1:1), and an excess of concentrated sulfuric acid is added with vigorous stirring. The white sulfate salt is washed, dried, and recrystallized from 4 vol. of 0.4 N sulfuric acid. The product is converted to the free base with ammonium hydroxide; m.p. 200 to 201°; yield, 0.3 g. To 60 mg. of this material, in 5 ml. of pyridine is added 10 rag. of potassium carbonate followed by a 10% excess of potassium permanganate solution. After 2 hours at 0 °, the pyridine is removed by steam distillation. A few drops of ethanol are added, and the mixture filtered hot. After the filtrate is adjusted to pH 6, about 30 mg. of 2-benzimidazolecarboxylic acid crystallizes in the cold. Additional quantities can be isolated from the mother liquors. It decomposes at 174°, yielding carbon dioxide and benzimidazole. The carbon dioxide originates from the methyl carbon atom of the acetic acid molecule; the benzimidazole nucleus contains the carboxyl carbon atom. Acetic Acid from the Methyl End of Fatty Acids 2~

One gram of fatty acid is added to a mixture of 10 g. of chromium trioxide, 80 ml. of water, and 40 ml. of concentrated sulfuric acid and refluxed under a stream of nitrogen for about 6 hours. The carbon dioxide liberated is recovered as barium carbonate, and the volatile acid steamdistilled from the reaction mixture.

Oxidation of Pyruvic Acid 53'54 An excess of ceric sulfate is added to a solution of pyruvic acid acidified with sulfuric acid. After 10 minutes at room temperature the excess ceric sulfate is destroyed by the addition of ferrous sulfate and the solution aerated with carbon dioxide-free air. The liberated carbon dioxide is trapped in saturated barium hydroxide solution. The acetic acid is recovered by steam distillation. Degradation of Lactic Acid (Method a) 5e (see also Vol. IV [22, 23 and 24])

The procedure employed for the preparation of 2-(a-hydroxyethyl)benzimidazole is identical with that described for acetic acid except that the 2-hour heating period in a sealed tube is omitted. The methyl carbon atom of lactic acid is converted to iodoform by treating 54 mg. of 2-(a-hydroxyethyl)benzimidazole with sodium hypoiodite at 60° for 30 migrates; yield, 22 mg. ; m.p. 117 to 119 °. The iodoform is purified before combustion. A solution of 2.90 g. of potassium permanganate in 100 ml. of boiling water is added all at once to a boiling solution of 810 rag. of 2-(a-hydroxy-

808


[32]

ethyl)benzimidazole and 200 mg. of sodium carbonate in 50 ml. of water. The mixture is boiled for 3 minutes and placed on the steam bath for 30 minutes. After the addition of small amounts of ethanol, filtration, and adjustment to pH 6, the colorless solution is placed in the refrigerator. After 2 days, 675 rag. of colorless needles of 2-benzimidazolecarboxylic acid is deposited, m.p. 174°.

Degradation of Lactic Acid (Method b) 57 To 10 to 30 mg. of lactic acid in 7 ml. of water is added 0.2 ml. of concentrated sulfuric acid and 2 ml. of chromic acid in water (l:1), and the mixture is refluxed under nitrogen for 40 minutes on the steam bath. The acetic acid formed is steam-distilled.

Degradation of Acetoacefic Acid (Method a) s4,58 For each milliliter of copper-lime filtrate are added 4 ml. of water, 0.4 ml. of 50% sulfuric acid, and 1.4 ml. of 10% mercuric sulfate solution. The mixture is refluxed under a stream of nitrogen and the carbon dioxide trapped in saturated barium hydroxide. Forty milligrams of acetonemercuric sulfate complex (Denig~s reagent) is dissolved in 2 ml. of 6 N hydrochloric acid. The acetone is distilled into a cooled mixture of 2 ml. of 15 N sodium hydroxide and 2 ml. of 0.1 N iodine solution. After warming to room temperature, the iodoform formed is filtered and washed.

Degradation of hcetoacetic Acid (Method

b) 59

A solution of 0.5 to 1.0 mM. of acetoacetate (copper-lime filtrates in many experiments) is washed into a 200-ml. three-necked flask carrying a dropping funnel, a lead-in tube extending to the bottom, and a condenser, the top of which has a tube leading to an absorption tower. The flask is immersed in an ice bath, and sufficient strong sulfuric acid and water added to give 100 ml. of a 1 M acid solution. COs-free air is drawn rapidly through the flask for about 5 minutes to remove any dissolved COs; then 10 ml. of an 0.5 M COs-free NaOH solution is placed in the bead tower and, with a moderate stream of air passing through the solution and into the bead tower, a sufficient quantity of 1.5 N KMnO4 solution is added to the acetoacetate to give a permanent pink color (8 to 10 ml. is required in these experiments). About 30 minutes is allowed for complete absorption of the COs. To recover the volatile acids the excess permanganate is destroyed by dropwise addition of 3 % hydrogen peroxide, any excess peroxide is decomposed by adding a few d?ops of very dilute permanganate to a permanent pink color, and the solution is steam-distilled until no more acid comes over, each portion of 50 ml. being titrated with standard alkali. The

[33]

LABELED

COMPOUNDS

AND

PHOSPHOLIPID M E T A B O L I S M

809

neutralized solution is evaporated to 50 ml., cooled, and acidified with 2 ml. of 18 N sulfuric acid. This is placed on the same setup used for the original oxidation, 10 ml. of 10% mercuric sulfate solution is added, and with a moderate stream of air passing through, the solution is refluxed for 1/~ hour. The CO2 resulting from the oxidation of the formate is again collected in a bead tower. The remaining acetate is recovered and determined by Duclaux distillation.

[33] L a b e l e d C o m p o u n d s i n t h e S t u d y of P h o s p h o l i p i d Metabolism B y CAMILLO ARTOM I. S y n t h e s i s

A. Building Blocks Ethanolamine

Principle. The procedure described below is based on the esterification of labeled glycine with ethanol and reduction of the ester with LiA1H4, according to Karrer et al.1 The method has been used by Weissbach and Sprinson 2 for the preparation of a triply labeled ethanolamine (1-C TM, l-D, 2-C 14) from 1-C 13, 2-C14-glycine and LiA1D4.

COOH

CO'O'C2H5 +C2H~'OH HCI

CH2NH2

CH20H +4H AILiH,

CH2'NH2.HC1

CH2-NH2"HC1

Reagents

Labeled glycine. 3 Saturated HC1 in absolute ethanol. 2 % NH3 in chloroform. Lithium aluminum hydride solution (420 rag. in 5 ml. of absolute ether). 2O% HC1. 1p. Karrer, P. Portmann, and M. Suter, Helv. Chim. Acta 31, 1617 (1948). 2 A. Weissbach and D. B. Sprinson, J. Biol. Chem. 203, 1031 (1953). 3 Glyeine_l_C14and glycine-2-CTM are available commercially. For these and other preparations of labeled glycine, see Vol. IV [28].

810


[33]

Procedure. Approximately 350 mg. of labeled glycine 3 is refluxed repeatedly with several portions of ethanolic HC1. Glycine ethyl ester HC1 (m.p. 142 to 143 °) is obtained in an almost quantitative yield. To the ester, suspended in 0.8 ml. of chloroform and cooled in an ice bath, 7 ml. of NH3 solution is added dropwise with stirring. The mixture is stirred for an additional 15 minutes and centrifuged in the cold. The precipitate is washed twice with cold chloroform, and the combined supernatant solutions are taken to dryness i n vacuo. The residual glycine ethyl ester is dissolved in a few milliliters of absolute ether and added dropwise to the lithium aluminum hydride (or deuteride) solution in a flask, equipped with a magnetic stirrer, reflux condenser, and dropping funnel. Fifteen minutes after the last addition of ester, 3 ml. of H20 (or D20) is added dropwise. The resulting mixture of water, ether, and Li and A1 hydroxides is transferred to a continuous extractor with the aid of 15 ml. of water and extracted for 48 hours with ether containing 3 ml. of 20% HC1. The ether extract is taken to dryness. Ethanolamine HC1 is crystallized and recrystallized from a small volume of hot absolute alcohol by the addition of dry ether. Yield, 200 mg. (50%) ; m.p. 74 to 76°; calculated for C2HTON'HCI: C1, 35.5 (found, 35.1 2). Comment. Labeled ethanolamine has been prepared also: (1) by synthesis of K phthalimide-N 15 with ethylene dibromide, and alkaline hydrolysis of the resulting bromoethylphthalimide (Bloch and Schoenheimer4); (2) by synthesis of ethylene-l,2-C ~4 oxide with NH3 and chromatographic fractionation of the mixture of mono- and polyethanolamines obtained in the reaction (Pilgeram et al.5).

Choline (Ethylene-Labeled) 6 Principle. The procedure described below (Walz et al. 7) is based on the conversion of ethylene-l,2-C ~4 into ethylene bromohydrin, and subsequent reaction of the latter compound with trimethylamine to yield choline bromide, as suggested by Renshaw.8

CI*H~

CI*H2.0H Br, OH (CH3.CONHBr)

I*H~

CI*H2OH (CH3)3N

CI*H2'Br

CH2"N(CH3) 3"Br

4K. Bloch and R. Schoenheimer,J. Biol. Chem. 138, 186 (1941). 5L. O. Pilgeram, E. M. Gal, E. N. Sassenrath, and D. M. Greenberg, J. Biol. Chem. 204, 367 (1953). 8For the preparation of methyl-labeledcholine, see Vol. IV [30]. 7D. E. Walz, M. Fields, and J. A. Gibbs, J. Am. Chem. Soc. 73, 2968 (1951). e R. R. Renshaw, J. Am. Chem. Soc. 32, 128 (1910).

[33]

L ABELED COMPOUNDS AND P H O S P H O L I P I D METABOLISM

811

Reagents

Ethylene-l,2-C142 N-Bromoacetamide 1° solution (276 rag. in 5 ml. of H20). 0.1 N H2SO4. Trimethylamine in anhydrous diethyl ether, saturated solution. Procedure. Two millimoles of the isotopic ethylene is distilled under high v a c u u m into a 50-ml. flask equipped with a break seal and containing the N-bromoacetamide solution and 0.2 ml. of 0.1 N H2SO4. The flask is sealed and shaken at room temperature for 18 hours. Unreacted ethylene is recovered by high v a c u u m distillation. The reaction mixture, saturated with NaBr, is extracted with several portions of diethyl ether. The combined ether extracts are dried over Na2SO4. Evaporation of the ether leaves the ethylene bromohydrin as an almost colorless liquid (yield, 90 %). T o this compound, without further purification, a few milliliters of the ether solution of trimethylamine is added. The mixture is refluxed at 55 ° for 5 hours, then maintained at room temperature for 15 hours. T h e ether and excess trimethylamine are distilled off, and the remaining choline bromide is crystallized from ethanol-ether; m.p. 287 to 289 ° with decomposition; yield, 83% of the ethylene bromohydrin. Comment. Renshaw's method s has been used also to prepare cholineN 15 from unlabeled ethylene chlorohydrin and (CH3)N15. ~1 A method of preparation of choline starting from acetate-l-C ~4 or acetate-2-C ~4, thus allowing labeling of either C of the ethylene chain, is described b y D a u b e n and Gee. ~ The synthesis proceeds through the stages of chloroacetate, ethyl chloroacetate, ethyl dimethylaminoacetate, and dimethylethanolamine which is finally m e t h y l a t e d to choline. Choline-N ~ has been obtained also by methylation of ethanolamine-N ~5 with dimethylsulfate. 4

Dimethylethanolamine (DME) (Methyl-Labeled) Principle. In the procedure of Artom and Crowder,~3 described below, methylethanolamine is methylated with HC~4HO in alkaline solution:

2HC14HO CH~'NH'CH2"CH20H + - - - ~ C14H3(CH3)'N'CH2CH~.OH + HC14OOH 9 Available commercially. The authors 7 describe briefly its preparation, starting from BaC1403, and progressing through acetylene-C ~4, which is finally reduced with CrC12. 10S. Winstein and R. B. Henderson, J. Am. Chem. Soc. 65, 2198 (1943). l~j. A. Muntz, J. Biol. Chem. 182, 489 (1950); S. Soloway and D. Stetten, Jr., ibid. 204, 207 (1953). ,2 W. G. Dauben and M. Gee, J. Am. Chem. Soc. 74, 1078 (1952). 13C. Artom and M. Crowder, J. Am. Chem. Soc. 74, 2412 (1952).

812

TECHNIQVSS FOR ISOTOPE STUDIES

[33]

Reagents HCI4HO.14 2.5 M methylethanolamine (2-methylaminoethanol).l~ Alkaline borate, pH 10 (6.2 g. of boric acid in 100 ml. of 1 N NaOH). 30% NaNO:, freshly prepared. Glacial acetic acid. Saturated solution of NaOH. 1% inactive HCHO. 5% NariS02. 1 N I2, 1 N NaOH, 1 N HC1. Procedure. Into a 25-ml. flask, fitted with a grouted-glass condenser, 2 ml. of the methylethanolamine solution (5 millimoles), 1.8 ml. of the HC14HO solution (containing 4.6 millimoles), and 0.7 ml. of the alkaline borate are introduced. The mixture is refluxed for 6 hours, cooled, and neutralized with 1 N HC1. The unreacted methylethanolamine is destroyed by adding 10 ml. of NAN02 solution and 5 ml. of acetic acid. After 10 minutes in ice, the mixture is aerated vigorously for 8/~ hour (to remove the oxides of nitrogen); it is then made alkaline with saturated NaOH and steam-distilled into 4 ml. of 1 N HC1. One milliliter of the inactive solution of HCHO, 1 ml. of 1 N I2, and a slight excess of 1 N NaOH are added to oxidize traces of active HCHO which may have been carried over in the distillate. The mixture is then acidified with 1 N HC1, and the excess I2 reduced with NaHSO~. The solution is concentrated to a few milliliters, made alkaline, and steam-distilled again into 2.5 ml. of 1 N HC1. The solution of D M E hydrochloride is of a sufficient isotopic purity to be used directly for most biological experiments. Yield, 40% based on the counts introduced as HC14HO. The picrolonic derivative prepared from the solution gave the expected values for the melting point (196 to 197 °) and per cent composition (calculated for C4HlINO" C10tI8N405: C, 47.59; H, 5.42; N, 19.82). Comment. D-Methyl-labeled D M E has been prepared by methylation of ethanolamine with D-formic acid and trioxymethylene (du Vigneaud et al.~). Ci4-Ethylene-labeled D M E is obtained as an intermediate in the procedure of Dauben and Gee ~2for the synthesis of choline. Soloway and Stetten 1~ prepared N15-1abeled D M E by pyrolysis of choline-N 15. Glycerol Glycerol-1-C~4 has been synthesized: (1) from N a C I 4 N and hydroxyacetaldehyde through ethyl-DL-glycerate, which is then acetylated and i~Availablecommercially. isThe commercial product should be fractionallyredistilledat 158 to 160°. 18V. du Vigneaud, J. P. Chandler, S. Simmonds, A. W. !V~oyer,and M. Cohn, J. Biol.

Chem. 164, 603 (1946).

[33]

LABELED COMPOUNDS AND PHOSPHOLIPID METABOLISM

813

reduced (DoerschukXT); (2) from NaC14N and bromoacetate, or from inactive NaCN and CH3CI40ONa (after conversion to the bromoacetate), through diethylmalonate and diethylacetoxymalonate (Gidez and KarnovskylS). By using C14H3"COONa (instead of CH3"C14OONa), glycerol2-C 14 can be prepared. Glycerol-l,3,C ~4 has also been synthesized from paraformaldehyde-C ~ and nitromethane through Na-2-nitro-l,3 propanediol, 2-amino-l,3-propanediol, and dicaproin (Schlenk and DeHass19). In view of the number of intermediates and the length of these procedures, their description is omitted, and the reader is referred to the original papers. Asymmetrically labeled glycerol-l-C 14 has been obtained by incubation of yeast with glucose-3,4-C 14 (Swick and Nakao~9~).

Serine and Fatty Acids See Vol. IV [30, 32].

B. Intermediates Glycerolphosphate (GP) DL-GP 3~. Principle. The procedure of Bailly 2° as applied by Kennedy 21 to the synthesis of DL-GP 8~ is described below. Glycerol is esterified directly with H3PO4 at high temperature. Pyrophosphate combinations are hydrolyzed with H2SO4, and GP 82 is isolated as the water-soluble, ethanol-insoluble Ba salt.

Reagents Na2Hp8204. Glycerol. 1 N H2SO~. Saturated Ba (OH)~.

Procedure. Na'H2PO4 is heated with an excess of glycerol for 6 hours at 150 ° under vacuum. The reaction mixture is taken up in several volumes of 1 N H2SO4, maintained at 100 ° for 2 hours, cooled, and brought to pH 8.4 with Ba(OH)2. The precipitate, consisting of Ba sulfate and phosphate, is centrifuged and washed repeatedly with H.20. From the combined supernatant and washings, GP is precipitated by the addition of an equal volume of ethanol. Comment. The preparation contains 90% of DL-o~-GP and approxi17 A. P. Doerschuk, J. Am. Chem. Soc. 73, 821 (1951). is L. I. Gidez and M. L. Karnovsky, J. Am. Chem. Soc. 74, 2413 (1952). 1~ H. Sehlenk and B. W. DeHaas, J. Am. Chem. Soc. 73, 3921 (1951). 19~ R. W. Swick and A. Nakao, J. Biol. Chem. 206, 883 (1954). 20 O. Bailly, Ann. chim. Paris [9]6, 96 (1916). ~1 E. P. Kennedy, J. Biol. Chem. 201, 399 (1953).

814


[33]

mately 10% of the/~ form. The latter can be eliminated by chromatography on a column of Dowex 1-acetate (Bublitz and Kennedy22). L-a-GP 32. Principle. The procedure of Kornberg and Pricer, 23 which is described below, is based on a study of the synthetic action of phosphatases by Meyerhof and Green. ~4 Human semen phosphatase is used to synthesize L-a-GP 3~ from glycerol and H~P~204. Reagents

H3P320~ solution. 1 M PO4 buffer (pH 5.8). Human semen. 0.08 M carrier L-a-GP. Magnesia mixture (0.5 M MgBr~, 2 M NH,Br, 2 M NH,OH). 1 M BaBr~, 5 N NaOH, 2 N ttC1. Dowex 50 (K+). Procedure. The solution of H~P3~O4 is neutralized and evaporated to near dryness under a stream of air at 40 °. Then 3.28 ml. of PO, buffer, 5.22 g. of glycerol, and 0.9 ml. of human semen are added, and the mixture is incubated for 40 hours at 40 °. About 20 ml. of water, 3.0 ml. of unlabeled L-a-GP, and 8.0 ml. of magnesia mixture are added. After 2 hours at 0 °, the precipitate is centrifuged and washed four times with 2.0-ml. portions of ten-fold-diluted magnesia mixture. The combined supernatant and washings are filtered, and the faintly turbid filtrate is reduced to about 8 ml. by vacuum distillation. The concentrated solution is transferred into a centrifuge tube and treated with 2.0 ml. of BaBr2, 0.5 ml. of 5 N NaOH (to bring the pH to about 9), and 3.5 ml. of ethanol. After 1 hour at 0 °, the small precipitate is centrifuged and discarded. Then 80 ml. of ethanol is added to the supernatant, and the mixture is left overnight at 0 °. The precipitate is collected by centrifugation, washed once with 10 ml. of ethanol, and dried in vacuo over K O H and CaC1,. To decompose the Ba salt, the dried sample (weighing 200 mg.) is dissolved in 6 ml. of water containing 4 drops of 2 N HC1 and passed through a Dowex 50 (K +) column (200 to 400 mesh) 2 cm. × 1 sq. cm. The column is washed with water, and the combined filtrates are neutralized. Comment. In experiments in which a solution of H~P3204 containing 0.05 mg. of P and 2 inc. of p32 was used, the final product contained 9.5% of the original radioactivity, no orthophosphate, and 550 micromoles of organic phosphate (corresponding to a 96% recovery of the a-GP added 2~ C. Bublitz and E. P. Kennedy, J. Biol. Chem. 211, 963 (1954). ~3 A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 204, 345 (1953). ~40. Meyerhof and H. Green, J. Biol. Chem. 178, 655 (1949); see also Vol. I I [79].

[33]


815

as carrier). The Ba salt was 80.9% pure (without correction for moisture content). Omission of carrier a-GP appeared to reduce the final yield. On the basis of periodate titration, some preparations contained free glycerol. 2~

Phosphorylethanolamine(PE) Principle. The procedure of Plimmer and Burch 2~ has been simplified by Ferrari and Ferrari. ~e An adaptation of this procedure to the synthesis of isotopic PE (C. Artom, unpublished data) is described below. Ethanolamine is reacted with H3PO4 in aqueous solution, and the resulting ethanolamine phosphate is dehydrated at high temperature in vacuo to give PE. This is isolated as the Ba salt, according to the original procedure of Plimmer and Burch. 25 Reagents

H3P32Oa solution. Ethanolamine, 27 24.4% solution. Ba(OH)~, saturated solution. 0.2 M H3PO4. Procedure. One milliliter of the H3P3204 solution (containing 196 mg. of H3PO4 and 2 mc. of p32) is placed in a graduated Thunberg tube, with about 1 g. of P20~ in the side arm. The tube is immersed in ice, and 0.5 ml. of the ethanolamine solution is added dropwise. The tube is transferred in an oil bath, the side-arm cap is placed on the tube, and the evacuation outlet connected with an oil pump. The mixture is then concentrated in vacuo (8 mm. Hg), while the temperature of the bath is slowly raised to 190° and maintained at that level for 3 hours. The melted mixture is dissolved in a few milliliters of hot water; then 1 ml. of inactive H3PO4 solution and enough water to bring the volume to at least 10 ml. are added. Saturated Ba(OH)2 solution is then added gradually until the mixture is alkaline to phenolphthalein. The precipitated Ba phosphate is washed twice with the Ba(OH)~ solution, diluted 1 : 10, and once with water. From the combined supernatant and washings, the Ba salt of PE is precipitated by adding slowly four volumes of ethanol. After centrifugation, the precipitate is redissolved with a few drops of HCI and reprecipitated with ethanol-water-Ba(OH)2, care being taken to maintain the reaction alkaline to phenolphthalein. Yield, 80 to 90%. The

25R. H. A. Plimmer and W. J. N. Burch, Biochem. J. 31, 398 (1937). ~6V. Ferrari and G. Ferrari, Arch. sci. biol. (Italy) 37, 1 (1953). 27The commercial preparation should be fractionally redistilled at 172°.

816


[33]

precipitate, dried at room temperature i n vacuo, analyzes correctly (calculated for C2H604NPBa.H20: N, 4.24; P, 9.39; Ba, 41.51 ~s). Comment. Chargaff and Keston 29 have prepared labeled PE from ethanolamine and Pa:OCl~. Phosphorylcholine (PC) P r i n c i p l e . The procedure described below is that of Plimmer and Burch, 2~ adapted to the synthesis of isotopic PC by Riley. 3° Choline is esterified at high temperature with H3P04, and the resulting PC is isolated as the Ca salt. By this or similar procedures, PC, labeled in the phosphoryl or in the choline moiety or both, has been prepared2 °-3a Reagents

H3PS~04 (or inactive H~PO4). Choline chloride (or bromide), labeled (C14-methyl, or C14-ethylene) or unlabeled. CaCI~. Ca(OH)s, saturated solution. Procedure. Labeled (or unlabeled) H3P04 (150 rag.) in 6 ml. of water is introduced into a pear-shaped flask of 35-ml. capacity. The flask is fitted with a standard taper No. 24 stopper and two outlets with glass stopcocks. The dilute acid is evaporated to 100% H3P04 by immersion in an oil bath, while the temperature is gradually raised to 180 ° and a stream of dry air is passed through the flask. When the H20 is completely removed, 214 mg. of dry choline chloride (or the equivalent amounts of isotopic choline bromide or chloride) is well mixed with the H3PO~. About 1 g. of P20~, mixed with asbestos, is placed in a Soxhlet thimble, suspended over the reactants. After cooling, the mixture is taken up in a few milliliters of It20, 100 mg. of CaC12 is added, and the solution is brought to the phenolphthalein end point with saturated Ca(OH)2. The precipitated Ca phosphate is centrifuged off, and the Ca salt of PC separated from the supernatant by adding an equal volume of ethanol. The compound is washed in succession by 60%, 80%, and absolute ethanol, and air-dried. Yield, 60 to 70%. The compound analyzes correctly (calculated for CsHlaO4NP'CaCI'4H~O: C, 18.2; H, 6.37; N, 4.25). It

280nly part of the water of crystallization is lost at 100°.~ ~9E. Chargaff and A. S. Keston, J. Biol. Chem. 134, 515 (1940). soR. F. Riley, J. Am. Chem. Soe. 66, 512 (1944). 8zj. Wittenberg and A. Kornberg, J. Biol. Chem. 202, 431 (1953). 8~A. :Kornberg and W. E. Pricer, Federation Proc. 11, 242 (1952). ss E. P. Kennedy, J. Am. Chem. Soc. 75, 249 (1953).

[33]

L ABELED COMPOUNDS AND P H O S P H O L I P I D METABOLISM

817

contains, as contaminating inorganic P, only 0.02% of p3~ (by isotope dilution) and less than 0.05 of p31 (by colorimetric determination). Comment. Kennedy 34 synthesizes isotopic PC by heating choline chloride with a mixture of P20~ and H3PO4, according to the original procedure of Plimmer and Burch. 25 After cooling, the mixture is diluted with H20 to a final concentration of 3 N H~PO4 and maintained for 20 minutes at 100° in order to hydrolyze pyrophosphates which otherwise contaminate the product. PC is finally isolated as the Ba salt, and recrystallyzed from acetone and H~O. Isotopic PC has been also synthesized enzymatically by the action of choline phosphokinase obtained from brewer's yeast. 3~ The method described by Baer a5 (in which choline is reacted with diphenylchlorophosphate in pyridine) is said to be the most satisfactory for the synthesis of PC. A study of its application to isotopic materials would be of interest. Other Intermediates Methods have been described for the synthesis of glycerylphosphorylcholine, 36 glycerylphosphorylethanolamine,a7 and phosphatidic acids. 38 Monoacyl phospholipids (" lysolecithins ") have been obtained by enzymatic hydrolysis of dipalmitoleyl- and dipalmityl-a-lecithins, isolated by chromatographic separation from natural phospholipid mixtures. ~' It seems likely that, with proper modifications, these procedures could be used for the chemical or biological preparation of the corresponding isotopically labeled compounds. The enzymatic synthesis of phosphatidic acids from Ci4-palmitic acid and a-GP 32has been reported. 23 For a detailed description of these methods, the reader is referred to the original papers. C. Phospholipids

The methods elaborated by Baer and his associates offer the possibility of synthesizing isotopic lecithins ~° and cephalins tl with a configuration identical to that of the naturally occurring phospholipids. The procedures are lengthy and rather complicated, however, and it is felt that the original sources should be consulted before an attempt is made to apply these 34E. P. Kennedy, personal communication. a~E. Baer, J. Am. Chem. Soc. 69, 1253 (1947); see also Biochem. Preparation 2, 96 (1952). as E. Baer and M. Kates, J. Am. Chem. Soc. 70, 1394 (1948). 37E. Baer and It. C. Stancer, J. Am. Chem. Soc. 76, 4510 (1953). a8E. Baer, J. Biol. Chem. 189, 235 (1951). 39D. J. Hanahan, IV[.Rodbell and L. E. Turner, J. Biol. Chem. 206, 431 (1954). 40E. Baer and M. Kates, J. Am. Chem. Soc. 72, 942 (1950). 4~E. Baer, J. Maurukas, and M. Russell, J. Am. Chem. Soc. 74, 152 (1952).

818


[33]

procedures to the conditions of isotopic synthesis. In a number of instances, mixtures of labeled phospholipids, obtained by biological synthesis, have been used. Two examples of preparation of such materials are given below.

Liver Phospholipids (Labeled with p82) Principle. The technique used by Artom and Swanson 4~ in a study of the intestinal adsorption of phospholipids is described. Labeled phospholipids were extracted from the livers of rats, previously injected with Na2Hp3204. The phospholipids were purified, emulsified in water, and administered to experimental animals.

Reagents NaH2P3~04 in Ringer's solution (prepared from a stock solution of H3p320~ containing 1 mc. and 25 ~, of p31). 0.1% Na glycocholate in Ringer's solution. All other reagents as described in the procedures for extraction, preparation, and purification of phospholipids.

Procedure. The solution of HaP320443 is adjusted to pH 7.4 and injected intraperitoneally into 4 to 6 rats. After 8 hours, the animals are killed by decapitation, and the livers are rapidly removed and pooled. The lipids are extracted with hot ethanol in a continuous extraction apparatus, the alcohol is evaporated under reduced pressure, and the residue taken up in chloroform. Phospholipids are separated by repeated precipitation with acetone ~- ~/~gCl2 44 and further purified. 45 The ether solution of purified phospholipids is poured over the solution of Na glycocholate in Ringer's solution. The flask is shaken mechanically, while the ~2 C. Artom and M. A. Swanson, J. Biol. Chem. 176, 871 (1948). 43 The amounts of HAP3204 to be injected depend on the desired level of specific activity in the phospholipids. In the experiments of Artom and Swanson 42 up to 25 ~c. per 100 g. of animals was injected. An average of 0.35% of the radioactivity was recovered in the purified liver phospholipids, with a specific activity in the range of 0.5 ~c./mM. of p31. Materials with a much higher specific activity may conceivably be obtained lay injecting larger amounts of p32. Indeed, the midlethal dose for a 21-day mortality ranges between 400 and 600 ~c. per 100 g. of rat or mouse [W. E. Cornatzcr, G. T. Harrell, Jr., D. Cayer, and C. Artom, Proc. Soc. Exptl, Biol. Med. 73, 492 (1950)]. 44 For greater details on the procedure for the extraction and isolation of phospholipids, see Vol. I I I [57], and below in this article. 46 To purify the phospholipids, Artom and Swanson 42 washed repeatedly the phospholipids in ether solution with an acetone-water solution of MgC12 and nonisotopic K2HPO4.

[33]

LABELED COMPOUNDS AND PHOSPHOLIPID

METABOLISM

819

ether is evaporated with a stream of nitrogen. The resulting emulsion of phospholipids was given b y stomach tube to rats.

Plasma Phospholipids (Doubly Labeled with p32 and C ~4) The technique used b y Weinman et al. 46 in a study of the turnover rate of the phosphate and f a t t y acid moieties of dog plasma phospholipids is described below. The plasma is obtained from a donor dog fed H3P3~O4 and C14-1abeled tripalmitin. T h e plasma containing the labeled phospholipids is utilized directly for biological experiments. Reagents

H3p3204 solution (approximately 4 me. of p32 and 0.1 mg. of p31). Tripalmitin, containing palmitic acid-C 1447(365 mg. and about 2 me., in 20 ml. of corn oil). Heparin. Procedure. The donor dog receives by stomach tube 6 ml. of the H3P3204 solution, then the C14-tripalmitin solution. After 24 hours, the dog is exsanguinated, and the blood is collected in a heparin-containing vessel. The plasma obtained by centrifugation is injected intravenously into recipient dogs. Comment. The procedure has the advantage t h a t the labeled phospholipids are obtained in the same state of dispersion as in the circulating plasma. On the other hand, the specific activities of the lipid C and lipid P in the preparation are rather low. Moreover, labeled compounds other than phospholipids are present, although in relatively small proportions. ~s

II. Extraction, Isolation, and Fractionation of Intact Phospholipids A . Extraction Principle. The direct extraction of blood or tissues with alcohol, alcohol-ether, or other organic solvents 4~ m a y be used in m a n y experiments with isotopic compounds. However, nonnegligible amounts of sub-

4~E. O. Weinman, I. L. Chaikoff, C. Entenman, and W. G. Dauben, J. Biol. Chem. 187, 643 (1950). 47Available commercially, or synthesized from palmitic acid-l-C 14 [W. G. Dauben, J. Am. Chem. Soc. 70, 1376 (1948)]. 48Twenty-four hours after the introduction of C14~tripalmitin, the specific activity of the C in the phospholipid fatty acids was ten times as great as in the nonphospholipid fatty acids. At ~he same time interval, the specific activities of the inorganic P and of the lipid P were of the same order of magnitude, but as much as threefourths of the total radioactivity was present as lipid P (Weinman et al.4s). 49See Vol. III [55].

820

TECHN'IQUES FOR ISOTOPE STUDIES

[33]

stances other than lipids which contain C, N and P are present in such extracts. Especially in experiments i n vitro, in which the tissue preparations are generally incubated with subs,rates of h i g h specific activity, contamination of the lipids b y these subs,rates a n d / o r by intermediary products becomes an i m p o r t a n t source of error. A satisfactory elimination of nonlipid contaminants can be obtained by precipitating the lipids with the proteins and washing the precipitate with aqueous solutions. The procedures described below, which are patterned after those of Schneider 5° and Folch and Van Slyke, ~ respectively, have been used in our laboratory in experiments on the incorporation of P32-phosphateS~ (method A), C ~4choline, 63 or C14-dimethylethanolamine s4 (methods B and B I) into the phospholipids of liver slices, homogenates, or mitochondria. M e t h o d A (TCA Precipitation) Reagents

T C A 10% with added MgC12 2.5%. 6~ Same solution diluted with H20 (1:1). 95% ethanol-diethyl ether (2:1). Procedure. The tissue, homogenized (or ground in a m o r t a r with icecold 10% TCA), is transferred to a 50-ml. graduated centrifuge tube, and T C A is added to bring the suspension to a total volume at least ten times the weight of the tissue with a final concentration of T C A not less than 7 %. After centrifugation, the supernatant is decanted (and filtered, if necessary). The precipitate is washed three times with the diluted TCA]V[gC12 solution and d e h y d r a t e d overnight with acetone. The acetone is filtered into a 250-ml. beaker. The residue in the centrifuge tube is then extracted three times each with boiling ethanol and with ethanol-ether, and the extracts are filtered through the same filter into the 250-ml.

50W. C. Schneider, J. Biol. Chem. 161, 293 (1945). TCA precipitation, as a preliminary stage in lipid analyses, had been used earlier by others [H. D. Kay, J. Biol. Chem. 93, 727 (1931); T. Cahn, J. Houget, and R. Jacquot, Ann. physiol, physicochim. 9, 205 (1932); B. Norberg and T. Teorell, Biochem. Z. 264, 310 (1933)]. 51j. Folch and D. D. Van Slyke, Proc. Soc. Exptl. Biol. Med. 41, 514 (1939). Precipitation with colloidal Fe203 was also used by E. Kahane and J. L6vy [Bull. soc. chim. biol. 21, 223 (1939)] to separate "hydrosoluble" and "lipid choline." 62C. Artom and M. A. Swanson, J. Biol. Chem. 193, 473 (1951). A very similar procedure IS. Chernick, P. A. Srere, and I. L. Chaikoff, ibid. 179, 113 (1949)] is described in Vol. III [55]. 58C. Artom, M. Crowder, and M. A. Swanson, Federation Proc. 9, 157 (1951). s4 M. Crowder and C. Artom, Federation Proc. 11, 199 (1952). 65The addition of MgC12 to the TCA solution has been suggested by R. M. Johnson and P. It. Dutch [Proc. Soe. Exptl. Biol. Med. 78, 662 (1951)].

[33]


82l

beaker. After addition of a few bumping stones, the combined acetone, ethanol, and ether extracts are evaporated almost completely on a steam bath. The beaker and the filter are than placed for 3 hours at 45 ° in a vacuum oven, containing CaC12. The dry residue in the beaker is then dissolved in warm chloroform, the solution is filtered, and the filtrate is brought to volume. A fraction is used for the determination of p3~. The remainder is shaken for 5 minutes with 1 g. of NaH~PO4 and a few drops of water, and then centrifuged. On a suitable aliquot of the supernatant, p32 is determined either by direct plating of the lipids, or by digesting the extract with H2SO-HNO~, isolating the P as strychnophosphomolybdate, 56 redissolving the precipitate in acetone, and evaporating the solution in an aluminum dish for counting.

Method B (Precipitation with Colloidal Fe20~)

Reagents 5 % colloidal Fe~O3.57 MgSO4"4H20, 1:2 solution in H20.

Procedure. The ground or homogenized tissue is placed in a 50-ml. centrifuge tube, and water is added to a volume of about 35 ml. In succession, with stirring, 2.5 m]. of colloidal Fe203 and 1.3 ml. of MgSO4 solution are added. The precipitate is centrifuged and washed four times by centrifugation with about 20 ml. of H20 plus 0.5 ml. of MgSO4solution. A fifth washing is made, with the 5/[gS04 omitted. The precipitate is suspended in ethanol, and the suspension is transferred into the thimble of a continuous-extraction apparatus of the Kumagowa-Suto or Wiley type. After a 6-hour extraction with boiling ethanol, the extract is transferred into a distillation flask, and the alcohol evaporated under reduced pressure at about 45 ° . When the residue is almost completely dry, the air inlet is closed, and the temperature of the water bath is raised to 80 ° for not more than 10 minutes. Chloroform is then introduced in the flask through the air inlet, and the suction discontinued. The chloroform solution is filtered through asbestos, and the filtrate brought to volume. Alternatively (method B~), the washed lipid-protein precipitate can be extracted with cold ethanol-ether-water, according to the original procedure of Folch and Van Slyke. 5~ The precipitate is suspended in 3 ml. of H20 and 3 ml. of 94 % ethanol and transferred to a 50-ml. glass-stoppered cylinder. To complete the transfer, one uses several portions of ethanol and ether (21 ml. each). This approximates the ratio of 1 vol. of 5e F. F. Tisdall, J. Biol. Chem. 50, 329 (1932). 57 Dialyzed iron, 5 % solution, Merck.

822


[33]

H~O to 5 vol. of alcohol-ether, which is optimal for complete extraction of the lipids. 5x The cylinder is inverted several times and left overnight. E t h e r is then added to the 50-ml. mark, and the extract is filtered. Isotopic and analytic determinations are made on appropriate aliquots of the filtrate. Comment. The results of comparative analyses carried out on separate portions of the same rat liver to which isotopic NaH2PO4 or choline had been added are illustrated in the accompanying table. I t appears t h a t the washing procedures by either method A or B effectively eliminate most of the radioactive substrates which m a y contaminate the lipids, obtained by direct extraction of the tissue. T h a t the lower N and P values in the extracts are not due to incomplete extraction, but rather to the removal of nonlipid materials, is indicated b y the minimal proportion of f a t t y acid in the extracted residues and b y the similarity in the choline and f a t t y acid contents of the lipids extracted by the various procedures. The values for the N : P ratios are higher in the lipids obtained b y direct extraction of the tissues, whereas in the lipids extracted b y procedure A or B such ratios approximate the values which m a y be expected on the basis of the lipid composition of rat liver. M e t h o d A is especially convenient for experiments with p3s, since it allows the simultaneous determination of the acid-soluble P and of its fractions in the TCA extract. However, the danger of a partial hydrolysis of the phospholipids is greater than in method B, as suggested b y the finding that, when colloidal Fe203 is used as a precipitant, choline and P values are higher in the lipid extract (see table) and lower in the hydroLIPID EXTRACTION FROM RAT LIVERS

Lipid extract

Extraction method

P, mg./g,

A B Direct

1.10 1.14 1.58

B BZ Direct

0.95 0.89 1.37

N, mg./g,

N P (atomic ratio)

Choline, mg./g,

Fatty acids, mg./g,

0.57 0.62 1.28

1.15 1.21 1.80

1.85 1.96 1.89

34 36 37

1.10 1.90

2.34 2.04 2.20

35 33 36

Isotopic Residual content, fatty c./see./g, acids, p82 ~ mg./g. 0.7 145

1.03 0.88 0.62

C 14b

0.44 1.18

0.9 2.1 1250

a H3P3~O4added to the samples = 16,200 c./sec./g. b C~4.(Methyl)_choline added to the sample -- 34,000 c./sec./g.

0.75 2.80 0.50

[33]


823

soluble fraction (Etienne-Petitfils and Kahane58). As compared with method B, method B ~yields slightly lower lipid values, but, in view of its greater rapidity, it may be preferred for routine analyses on a large number of samples.

B. Isolation This step (which may be omitted in many experiments with pz2 or labeled nitrogenous constituents) is obviously required when incorporation of the fatty acid or glycerol moieties into the phospholipids is being studied.

Method A (Precipitation with Acetone + MgCI.~)

Principles. In the presence of small amounts of salts of divalent materials, the precipitation of phospholipids with acetone is almost quantitative (96 to 98% of the chloroform-soluble P; see Artom59). However, the precipitate contains notable amounts of other lipids, especially neutral fat, which can be removed only by repeating the precipitation. 60 Reagent MgCl2, 5 % in ethanol 94 °.

Procedure. The lipid extract (preferably obtained by method B, see above) 62 is transferred to a centrifuge tube and the solvent evaporated to 2 to 3 ml. The tube is removed from the water bath, and about 25 ml. of acetone and 10 drops of MgC12 solution are added. After a few hours in the refrigerator, the tube is centrifuged and the acetone solution is decanted. Three milliliters of chloroform is added, and the tube is gently warmed until the precipitate is completely redissolved. The precipitation with acetone plus MgC12, followed by centrifugation and decantation, is repeated two more times, as described above. After the third precipitation, the phospholipids are washed with a small amount of cold acetone, 5s j. Etienne-Petitfils and E. Kahane, Bull. soc. ch, m. biol. 36, 354 (1954). ~9 C. Artom, Bull. soc. chim. biol. 14, 1386 (1932). 60 After two reprecipitations, the separated phospholipids are practlcaliy free of cholesterol. When relatively large amounts of iodized triglycerides (Artom~9), or free C14-palmitic acid (Borgstr6m61), were added to lipid extracts, less than 2% of the labeled materials was recovered in the precipitate. In experiments on tissue slices or homogenates, incubated with palmitate-l-C ~4, complete elimination of the substrate and labeled triglycerides was attained by the addition of 1 to 2 ml. of commercial oil before reprecipitation of the phospholipids [L. A. Jedeikin and S. Weinhouse, Arch. Biochem. and Biophys. 50, 134 (1954)]. 61B. BorgstrSm, Acta Physiol. Skand. 25, 101 (1952); see also D. L. Fillerup and J. F. Mead, Proc. Soc. Exptl. Biol. Med. 83, 574 (1953). ~ The nonlipid contaminants should be eliminated from the lipid extracts prior to the separation of the phospholipids, since such contaminants are precipitated to a large extent together with the phospholipids.

[33]

LABELEDCOMPOUNDS AND PHOSPHOLIPID METABOLISM

825

Reagents KOH 1 N. TCA, 10%. NaOH, 10%.

Procedure. Fifty milliliters of a methanol solution of phospholipids (containing not more than 0.06 mg. of P per milliliter) is placed in a centrifuge tube with 2.5 g. of MgO and a small amount of solid NaC1. e~ The tube is stoppered, shaken for 10 minutes, and centrifuged. The clear supernatant is siphoned off, and the residue is washed with methanol. The supernatant and the washing are combined and brought to volume. Aliquots of this solution are used for the determination of the p31 and p3~ content of CCP. The NCCP cannot be eluted from the adsorbent. However, 90% or more of their P can be determined as follows. After elution of the CCP, a portion of the MgO (1 to 2 g.) on which the NCCP has been adsorbed is suspended in 25 ml. of 1 N KOH and heated overnight on a water bath. The suspension is centrifuged, the supernatant is transferred to another vessel, and the MgO washed with 20 ml. of H20. On aliquots of the combined supernatant and washing, the p32 and p~l of the NCCP are determined. To separate sphingomyelin P from lecithin P, the solvent is evaporated from the methanol solution of the CCP. The residue is suspended in 5 ml. of 1 N KOH and incubated at 37 ° for 24 hours. Five milliliters of 10% TCA is added, and after 2 hours the mixture is centrifuged (or centrifuged and filtered). The precipitate is washed with a few milliliters of TCA. An aliquot of the combined filtrate and washing is used for the determination of lecithin p32 and p31. The precipitate is redissolved in 1 ml. of NaOH 10 % and a little water and brought to volume. Sphingomyelin p32 and p31 are determined on aliquots of the redissolved precipitate. HI. Hydrolysis and Degradation A. Preparation and Fractionation of Phospholipid Hydrolyzates Principle. Alkaline hydrolysis of the lipid extracts (or of the isolated phospholipids) will readily split phospholipids into nitrogenous constituents, fatty acids, and glycerolphosphate. This procedure may be safely e~Without the addition of NaC1, the methanol solution of CCP often remains quite turbid (Taurog et al.64).

826

[33]

TECHNIQUES FOR ISOTOPE STUDIES Acetone precipitate (phospholipids) Methanol-HCl Evaporation H20 + chloroform

/

I

Chloroform extract

Aqueous phase

Evaporation KOH Drying Absolute ether

i

1

Ether extract Picrolonie acid

Ether residue

1

Precipitate Fraction A (sphingosine)

Fraction B (fatty acids)

I

Aliquot I Alkaline borate Steam distillation

I

Aliquot II

I

Distillate [ Picrolonic J, acid Precipitate Fraction C (DME)

Residue I Reinecke J, acid Precipitate Fraction D (choline)

pH 7

Permutit

I

Effluent

lp

NaCl

H3 Amberlite IR-100-H Centrifugation

I

Supernatant

Eluate

I

Residue I NaOH

Eluate Ba*+ Ethanol

KIO~

Dimedon

Io._ I Hg *÷ Ba÷+

J,

~

$

Precipitate Precipitate Precipitate Fraction E Fraction F Fraction G (ethanolamine) (glycerolphosphate) (serine) FIG. 1. Hydrolysis and fractionation of phospholipid hydrolyzate.

[33]


827

used for the determination of the last two components. However, while choline seems to be unaffected by mild alkaline reagents (such as Ba(OH)2 solutions), a partial destruction of serine and ethanolamine occurs under such conditions. Accordingly, for the latter compounds, acid hydrolysis seems preferable. 68 A scheme for the fractionation of the hydrolytic products of phospholipids (Fig. 1) has been elaborated in a study of the incorporation of the a-carbon of glycine into the various components of phospholipids by liver slices, e9 With proper modifications, this scheme, or part of it, can be used in similar experiments with other isotopes or isotopic compounds in order to separate the hydrolytic products of phospholipids into crude fractions and obtain preliminary indications on the distribution of the isotope among these fractions. For more complete and definite information, isolation, purification, and degradation of the individual compounds in each fraction are of course required.

Reagents 6 N HC1 in methanol. 2 % KOH in methanol. Anhydrous ether (reagent-grade diethyl ether, freshly redistilled over Na). Permutit. Amberlite IR-100-H. Carriers: "crude sphingolipids, ''7° dimethylethanolamine (DME), ethanolamine-HC1, DL-serine, glycerolphosphate. Other reagents as in the procedures for the isolation and degradation of DME, choline, serine, ethanolamine, and glycerolphosphate.

Procedure. The phospholipids are isolated by repeated precipitation with acetone plus MgCl:. If the sphingomyelin content is low and isolation of fraction A is desired, 100 to 200 mg. of crude sphingolipids is added. The mixture is refluxed for 6 hours with 6 N methanolic HC1, which is then removed by suction on a water bath at 60 °. The residue is suspended in water, and the suspension is extracted with chloroform. The chloroform extract, after evaporation of most of the solvent, is transferred to a 15-ml. glass-stoppered centrifuge tube and dried on a water bath. It is then redissolved in warm methanol, made strongly 68 For a detailed description of some of these hydrolytic procedures, see Vol. I I I [59]. 69 Quoted by C. Artom, i;n "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 2, p. 203. The Johns Hopkins Press, Baltimore, 1952. 7o For this preparation, see H. E. Carter, W. J. Haines, W. E. Ledyard, and W. P. Norris, J. Biol. Chem. 169, 77 (1947).

828


[33]

alkaline to phenolphthalein, and refluxed for ~ hour (in order to saponify the methanol esters of the fatty acids). The methanol is evaporated, and the residue is thoroughly dried at 60 °. Then 10 to 15 ml. of anhydrous ether is added. The contents of the tube are shaken vigorously for 5 minutes and centrifuged. The extraction with anhydrous ether is repeated one more time. The combined ether extracts are concentrated, and about 20 ml. of an ether solution of picrolonic acid is added. The precipitate is separated by centrifugation, washed with ether, plated, and counted (fraction A, sphingosine). The residue from the ether extraction of sphingosine is dissolved in warm methanol and brought to volume. An aliquot is plated and counted (fraction B, fatty acids). If isotopic DME is expected to be present, the aqueous phase of the hydrolyzate, after extraction with chloroform, is divided into two aliquots, I and II. Carrier DME (8 to 10 rag.) is added to aliquot I, which is then steam-distilled in the presence of alkaline borate. In the distillate, D M E is precipitated with picrolonic acid, and the precipitate is plated and counted (fraction C, DME). The residue from the steam distillation is acidified with HC1 and concentrated to a small volume. Reinecke acid is added, and the radioactivity determined on the precipitate (fraction D, choline). To aliquot II of the aqueous phase of the hydrolyzate, 8 to 10 rag. of each of the ethanolamine-HC1, serine, and glycerolphosphate are added as carriers. The solution, brought to about pH 7, is passed through a Permutit column. The effluent is acidified to pH 3, shaken with 4 ml. of Amberlite IR-100-H for 10 minutes (Gidez and Karnovsky71), and centrifuged. The clear supernatant is brought to pH 10 with Ba(OH)2. An equal volume of ethanol is added. The precipitate is washed with 50 % ethanol, plated, and counted (fraction E, glycerolphosphate). The Amberlite is then resuspended in water, and NaOH is added to the pink color of phenolphthalein (pH 8.4). After centrifugation, the supernatant is treated in succession with NaIO4, I2, and HgCl~, as described for the degradation of serine. The C02 derived from the three carbons of serine is collected and counted as BaCO3 (fraction F, serine). From the Permutit column, ethanolamine and choline are eluted with a NaC1 solution. HIO4 is added to the eluate, and the solution is brought to pH 8.4. The HCHO, liberated from both carbons of ethanolamine, is precipitated as the dimedon derivative and counted (fraction G, ethanolamine). For more details on the determinations of fractions C through G, see the procedures described below. 71L. I. Gidez and M. L. Karnovsky, J. Biol. Chem. 206~ 229 (1954).

830


[33]

procedures, the CHa group of choline is also converted to T1V[A, but this compound is isolated as the reineckate, 79 or as tetramethylammonium iodide (SakamiS°). Reagents

Ag2SO4, saturated solution. BaC12, 10%. PtC14, saturated solution in ethanol. NaOH, 10%. KMn04, saturated solution. O.33 h r HC1. Procedure. The washed choline reineckate is dissolved in the minimum amount of acetone plus H20 (1 : 1), and the Ag~SO4 solution is added until the precipitation is complete. The insoluble Ag reineckate is separated by centrifugation and washed with water. The combined supernatant and washing are heated to boiling, and BaCl2 solution is added dropwise. The mixture is filtered. The filtrate is made slightly alkaline to litmus and concentrated in vacuo to remove NH3. It is then acidified with HC1 and taken to dryness. The residue is extracted repeatedly with absolute ethanol. The combined extracts are filtered, and the ethanolic solution of PtC14 is added. The precipitate is centrifuged, washed twice with absolute ethanol, and dried. It is then dissolved in the minimum amount of H20, and recrystallized. The P t (theory, 31.7 %) and isotope contents are determined. For the degradation to TMA, a weighed amount of choline chloroplatinate and 15 ml. of NaOH solution are placed in a three-necked flask, fitted with a dropping funnel containing the KMn04 solution. A slow stream of air is passed through the solution into two traps, immersed in ice, and each containing 3 ml. of 0.33 N HC1. The contents of the flask are heated to boiling, and the KMnO~ solution is added dropwise until a purple color persists. Heating and passage of the stream of air are continued for 15 minutes longer. The contents of the two traps are combined, evaporated to dryness under reduced pressure, redissolved in ethanol and filtered. T M A is precipitated with an excess of ethanolic PtC14, and the precipitate is washed with ethanol. The TMA-chloroplatinate is recrystallized from ethanol-H20 and analyzed for its Pt (theory, 37.0%) and isotope content. In the simplified procedure of Artom and Crowder, TM reinecke acid is added directly to the HC1 solution, into which T M A has been distilled. 79 C. Artom a n d M. Crowder, Arch. Biochem. 29, 227 (1950). so W. Sakami, J. Biol. Chem. 187, 370 (1950).

[33]


831

The precipitate is centrifuged and washed once with a few milliliters of water. It is then dehydrated by repeated additions of methanol and evaporation on a water bath. It is finally redissolved in an acetone solution of egg yolk lipids. ~s An aliquot is plated for counting, care being taken not to use temperatures above 55 ° in drying the sample.

Ethanolamine Isolation and Purification. Principle. In the procedure of Thierfelder and Schulze sl which is described below, ethanolamine is extracted with ether and converted into the ether-insoluble picrolonate (C~H7ON" C10HsOsN~). This derivative has been used as such in determinations of C TM (Artom, unpublished), or, after decomposition, in analysis of N ~5 (Stetten73). By a similar technique, NlS-ethanolamine can be isolated also as the 3,5-diiodosalycilate (C2HTON-C7H4OaI2: Stetten, s2 Chargaff, s3 Chargaff et al.S4). The latter derivative is obtained in a lower yield but has the advantage of containing no other N than that of the original compound.

Reagents CaO, freshly ignited. Picrolonic acid, 0.55 g. in 200 ml. of diethyl ether (U.S.P. ether for anesthesiaSS).

Procedure. The aqueous phase of the phospholipid hydrolyzate, after extraction of fatty acids and sphingosine with chloroform, can be used (see Fig. 1). If D M E is suspected to be present, it should also be removed (by steam distillation at pH 10.0). The water solution is then acidified and concentrated to about 0.5 ml. with repeated additions of ethanol. Enough CaO is added to obtain a dry and crumbly material, which is transferred into the extraction thimble of a Soxhlet apparatus. Then 200 ml. of the picrolonic acid solution is placed in the extraction flask, and the extraction is continued for 4 hours. The ether-insoluble ethanolamine picrolonate separates in the flask with a nearly quantitative yield. It is washed once with ethanol-ether (10:100), and repeatedly with ether, and can be recrystallyzed from hot ethanol (m.p. 126 to 128 °, with decomposition). The precipitate is suspended in methanol and plated, and its C TM content is determined. Or, it is decomposed by warming with 5 ml. of concentrated HC1, diluting with H20, and extracting the picrolonic s~ H. Thierfelder and O. Scbulze, Z. physiol. Chem. 96, 296 (1915-16). 82 D. Stetten, Jr., J. Biol. Chem. 188, 437 (1941). s3 E. Chargaff, J. Biol. Chem. 142, 491 (1942). s4 E. Chargaff, M. Ziff, and D. Rittenberg, J. Biol. Chem. 144, 343 (1942). 2~ Absolute ether is not satisfactory (MuntzLl).

832


[33]

acid with ethyl acetate (Warren and WeissSS). After two extractions, the colorless aqueous solution can be used for isotopic and analytical determinations of C or N. Comment. Sphingosine and D M E give ether-insoluble derivatives with picrolonic acid, and must therefore be removed from the hydrolyzate. On the other hand, when the procedure described above was applied to a mixture of nonisotopic ethanolamine and N15-choline, the isolated ethanolamine was free of N 15 (Stetten73). Alternate Procedure (Chargaff83). The material in the Soxhlet thimble is extracted for 40 hours with 60 ml. of a 1% solution of diiodosalycilic acid in diethyl ether. The ether is evaporated. The residue is washed six times with 2 ml. of ice-cold ether and recryst~llized twice from ligroinabsolute ethanol (4:1). Calculated: N, 3.1; I, 56.3: m.p. 198 to 199 °, with decomposition. Degradation. Principle. Ethanolamine is oxidized with alkaline periodate as follows (Artom 87) : H2N.CH2"CH:'OH -F NaI04 -* 2H.CHO ~ - N H 8 - k NaI03. Isotopic and analytical determinations are carried out on the dimedon derivative of H-CHO (Vorl~nder ~s) and/or on the NHa, separated by alkaline steam distillation.

Reagents 0.2 N HI04. 0.1 N HsP04. 1 N NaOH, 1 N HC1. 0.4% dimedon (5,5-dimethylcyclohexane-l,3-dione). Alkaline borate (6.1 g. of boric acid in 100 ml. of 1 N NaOH).

Procedure. The picrolonic acid derivative of" ethanolamine is decomposed (see above). To an aliquot of the colorless solution, an excess of the HI04 and 10 ml. of P04 buffer are added. The solution is made alkaline to phenolphthalein with 1 N NaOH, left at room temperature for 20 minutes, and then brought to about pH 5.6 with 1 N HC1 (orange color with methyl red). An excess of the dimedon solution is then added. After standing overnight, the precipitate is collected by filtration, or by centrifugation, and washed three times with water. It is then recrystallyzed from ethanol (or from water, by making the solution alkaline, and then acidifying again: Siekevitz and GreenbergSg). For determination of C 14, 88 W. H. Warren and R. S. Weiss, J. Biol. Chem. 8, 327 (1907). 8~ C. Artom, J. Biol. Chem. 157, 586 (1945). s8 D. Vorl~ader, Z. anal. Chem. 77, 241 (1929). s9 p. Siekevitz and D. $~I. Greenberg, J. Biol. Chem. 180, 845 (1949). These authors steam-distill the H.CHO, liberated by NaIO4, directly into a dimedon solution.

[33]


833

the precipitate (m.p. 189 °) is suspended in a methanol solution of egg lipids, plated, and counted. On another aliquot of the ethanolamine solution, periodate oxidation and steam distillation of the NH3 produced can be carried out simultaneously (Artom; 8~ see Vol. III [55, 59]) and N TM and N 15 determined in the distillate. Comment. In one experiment in which liver slices were incubated with glycine-2-C14 (Artom69), the following samples were prepared: (1) formaldimethone, after fractionation of the phospholipid hydrolyzate and periodate oxidation of fraction G (see Fig. 1); (2) ethanolamine picrolonate, isolated and plated directly from the phospholipid hydrolyzate; and (3) formaldimethone, by periodate degradation of the ethanolamine picrolonate. After the proper corrections had been applied for selfabsorption, and within the limits of error of the measurements, identical values were obtained for the specific activity of the ethanolamine C in each of these samples. Serine

Isolation. Principle. From the aqueous phase of the lipid hydrolyzate, ethanolamine is removed2 ° Serine is then isolated as the p-hydroxybenzene-p-sulfonate (C3HsO2N'C l~HllOsN2S), according to Stein et al.°l In the procedure outlined below, the indications given by Folch 9~ are followed. Reagents

p-Hydroxyazobenzene-p-sulfonic acid. Carrier DL-serine. Procedure. The aqueous solution of the phospholipid hydrolyzate is freed from fatty acids and sphingosine (by extraction with chloroform) and of ethanolamine (by adsorption on Permutit at pH 7). After addition of carrier serine, the solution is concentrated to 4 ml., and 800 rag. of the reagent is dissolved by heating on a boiling water bath. The tube is placed overnight in the refrigerator, then centrifuged in the cold. The separated crystals are washed twice with 3 ml. each of ice-cold water, and recrystallized twice from the same amount of water. The material, dried i n vacuo over CaCl:, contains 1 molecule of H20 (SakamiS°). Heated i n vacuo over

90p-Hydroxyazobenzene-p-sulfonic acid forms insoluble derivatives with both ethanolamine and serine. 91W. H. Stein, S. Moore, G. Stamm, C. Y. Chow, and M. Bergmann, J. Biol. Chem. 143, 121 (1942). 9, j. Foleh-Pi, J. Biol. Chem. 174, 439 (1948).

[33]


835

After the aeration, 5 ml. of the SrC12 solution is introduced, and the pH is raised to 6 by the addition of 2 ml. of 1.8 N NaOH. The mixture is kept at 6 ° for 5 hours. The precipitate, which consists of strontium iodate, periodate, phosphate, and p-hydroxyazobenzene-p-sulfonate, is removed by centrifugation, and washed twice with a few milliliters of cold water. The centrifugate and wash water are combined, adjusted to pH 3 with glacial acetic acid, boiled, and aerated to remove preformed CO2. Then 25 ml. of the HgCl2-acetate reagent is added, and boiling is continued for 1 hour to oxidize the formate to CO2. HCHO is not attacked. The CO2, formed from the a-carbon of serine is removed by aeration, trapped, and determined as above. The remaining solution is filtered from HgO and distilled to a volume of 10 ml. The distillate containing the H C H O is cooled to 6 °. Twenty milliliters of 1 N NaOH and 20 ml. of 0.1 N I2, both also at 6 °, are added. The mixture is then acidified to pH 4 with H2SO4, and the excess I~ is reduced with 0.1 N thiosulfate. The formate, derived from the E-carbon of serine, is oxidized to C02 which is displaced, trapped, and converted to BaCO3 for the analytical and isotopic determinations.

Dimethylethanolamine (DME) Principle. D M E has not been found as a component of natural phospholipids, but when it is added to preparations of isolated liver, it is readily incorporated in the phospholipids. In the procedure described below, 54,97the compound is separated from the aqueous phase of the phospholipid hydrolyzate by steam distillation and then isolated from the distillate as the picrolonic acid derivative28 Reagents Carrier DME. 0.2 N HI04. Alkaline borate, pH 10 (0.2 g. of boric acid in 100 ml. of 1 N NaOH). 0.1 N HC1. Picrolonic acid, 0.4 g. in 200 ml. of diethyl ether. CaO, freshly ignited.

Procedure. To an aliquot of the aqueous phase of the phospholipid hydrolyzate carrier D M E is added. The solution is transferred to the distillation flask of the Parnas-Wagner apparatus. Then 2 ml. of HIO4 97 C. Artom and M. Crowder, Federation Proc. 8, 180 (1949). 9s D M E can also be separated from alkaline aqueous solutions by extraction with ether in a continuous extractor for 6 hours (as in the experiments of Muntz11). This procedure is applicable to phospholipid hydrolyzates, provided that ethanolamine is first destroyed with either NaIO4 or HNO2 (C. Artom, unpublished data).

836


[33]

solution and 3.5 ml. of alkaline borate are introduced, and the mixture is steam-distilled for 20 minutes into an excess of 0.1 N HC1. After concentration of the distillate, D M E picrolonate is prepared and isolated by the same procedure, as described for the preparation of the ethanolamine derivative. Dh/[E picrolonate: (C4H110N'C10HsOsNt): m.p. 196 °, with decomposition. Calculated: C, 47.59; H, 5.42; N, 19.82. Comment. HI04 is added to destroy isotopic ethanolamine and monomethylethanolamine which may be also present. When mixtures of these compounds are steam-distilled in the absence of NaI04, traces of ethanolamine and a notable proportion of the monomethyl derivative are found in the distillate (see Vol. III [59]).

Sphingosine Isolation. Principle. The procedure outlined below is an adaptation of that used on large amounts of materials by Carter et al. 99 Sphingosine is isolated as the sulfate and converted into the triacetyl derivative. Similar procedures have been used by Zabin and Mead 1°° and by Sprinson and Coulon T M in isotopic experiments on the biosynthesis of sphingosine in brain and other tissues. Reagents "Crude sphingolipids, ''7° as carriers. 6 N HC1 in methanol. 1 N H2SO4 in ethanol. 1 : 1 pyridine-acetic anhydride, freshly prepared. Procedure. After addition of a suitable amount of carrier sphingolipids, the lipid extract is hydrolyzed for 5 hours with 6 N methanolic HC1 which is then removed by suction at 60 °. The residue is suspended in water, made alkaline with NaOH, and kept in a boiling water bath for 1/~ hour. Sphingosine is then extracted with several portions of ether, and the combined ether extracts are washed with H20. After evaporation of the ether, the residue is dissolved in the least volume of absolute ethanol, and 1 N ethanolic H~S04 is added to the first faint blue with congo red. From the solution, cooled in ice, crude sphingosine sulfate precipitates. The precipitate is separated by centrifugation, washed with a small volume of cold ethanol, and dried. Four milliliters of pyridine-acetic anhydride is added. The mixture is left at room temperature for 2 hours, in which time crystal99 H. E. Carter, W. P. Norris, F. J. Glick, G. E. Phillips, and R. J. Harris, J. Biol. Chem. 170, 269 (1947). 100 I. Zabin and J. F. Mead, J. Biol. Chem. 205, 271 (1953). ~ol D. B. Sprinson and A. Coulon, J. Biol. Chem. 207, 585 (1954).

[33]


837

line triacetylsphingosine separates. The tube is chilled and centrifuged in the cold. The precipitate is washed with cold acetone and air-dried. It is finally recrystallized from acetone to constant melting point (99 to 101 °) and specific activity. Comment. The crude " s p h i n g o s i n e sulfate," obtained after hydrolysis with methanolic HC1, is probably a mixture of the sulfates of sphingosine, O-methylsphingosine, and dihydrosphingosine. When a considerable proportion of dihydrosphingosine is present in the original material (as, for instance, in the cerebrosides of the spinal cord), pure triacetylsphingosine cannot be obtained easily by direct acetylation of the crude sulfate mixture, as in the procedure described above. In such cases, it may be preferable to prepare the N-acetyl derivative and hydrogenate it to N-acetyl dihydrosphingosine. For the preparation of this derivative (as well as of tribenzoyldihydrosphingosine), Zabin and Mead 1°° have followed the directions given by Carter et al. 99 Degradation (see also Vol. III [59]). Principle. Sphingosine is degraded by oxidation with periodate, as described by Carter et al. :~0~ CH2"OH

L

CH'NH2--

I

CH-OH

I

H'CH0

+

H.COOH + NH3

C02

+

CHO

I

CH

CH

CH

CH

L

(CH )I I

CH3

Hg ++

CH~

The procedure described below is patterned after that used by Sprinson and Coulon TM in experiments with C 14- and N15-1abeled compounds. In these experiments, C-1 of sphingosine is determined on the formaldimethone, and C-2 on the BaCO3, obtained after oxidation of H.COOH with Hg ++. The remaining carbons are calculated by difference, or counted on the 2,4-dinitrophenylhydrazone of the Cl~-aldehyde. N 1~ is determined on the NH3 separated by alkaline distillation. T M 102 H. E. Carter, F. J. Gliek, W. P. Norris, a n d G. E. Phillips, J. Biol. Chem. 170, 285

(1947). 103Zabin and Mead,1°°followingmore closelythe original procedure of Carter et al., 1°~ first convert sphingosine into dihydrosphingosine which is then degraded with NaIO4. The resulting palmitaldehydeis isolated as the semicarbazoneand oxidized with MkMineK2MnO4to palmitic acid. This is converted to the Ag salt, and decarboxylated with Br. The carboxyl C is counted as BaCOs.

838

TECh~NIQUES FOR ISOTOPE STUDIES

[33]

Reagents 0.5 N and 20% NaOH. 1% NaHC03. 10% NaIO~. Zn powder. 2,4-Dinitrophenylhydrazine (DNPH) (0.4 g. of D N P H is warmed with 2 ml. of concentrated H2SO4 and 3 ml. of H20. To the warm solution, 10 ml. of 95 % ethanol is added). Other reagents as for the degradation of serine.

Procedure. Sphingosine, isolated as the sulfate (see above), is suspended in a few milliliters of 0.5 N NaOH (enohgh to convert it to the free amine) and shaken in a separatory funnel with 30 ml. of ether. The aqueous phase is neutralized, and 30 ml. of NaIO4 solution is added. The mixture is shaken at frequent intervals during 4 to 5 hours. The ether layer is separated and shaken with 30 ml. of NaHC03 solution. The combined aqueous layers are treated with SrC12 at pH 6, and filtered. In the filtrate the H-COOH is oxidized to C02 with the HgCl~-acetate reagent, and the CO2 collected and converted to BaC03, as described for the degradation of serine (see pp. 834-835). The reaction mixture is then made strongly acid, and HCHO is distilled and isolated as the dimedon derivative. To the residue in the distillation flask, Zn powder and NaOH 10% are added, and NH3 is distilled off for N 15 analysis. The ether solution of the aldehydes is brought to dryness, taken up in 95 % ethanol, and 2,4dinitrophenylhydrazine is added. The resulting hydrazone is recrystallized from ethyl acetate to constant melting point and activity.~°4

Glycerolphosphate Isolation and Purification (see also Vol. III [58]). Principle. The method described below is an adaptation of the procedure used by Folch. 1°5 Glycerolphosphate is precipitated as the Pb X salt and converted into the water-soluble, alcohol-insoluble Ba salt.

Reagents 25 % Pb acetate (neutral). Ba(OH)2, saturated solution. lo4 The following data are reported by Sprinson and Coulon1°1 for the hydrazone obtained by periodate oxidation of a crude sphingosine sulfate fraction from rat brain and spinal cord: m.p. ffi 118°; C, 62.55; H, 8.45; N, 12.65; O, 16.4. These data correspond to the values expected for a mixture of palmitaldehyde, a,t3-dehydropalmitaldehyde, and 2-methoxy-3-heptadecenal, and thus indicate the presence of considerable proportions of dihydro- and O-methylsphingosine in the crude sphingosine fraction. 10aj. Folch, J. Biol. Chem. 146, 35 (1942).

840


[34]

The solution is filtered into a 15-ml. centrifuge tube and brought to a volume of about 8 ml. One milliliter each of the H~SO4 and HIO4 solutions are added. The tube is half immersed in boiling water for 1 hour, the volume being maintained by repeated additions of tt~O. After cooling, the solution is brought to 10 ml. One aliquot is treated with the HgCl~acetate reagent, and the/~-carbon of glycerolphosphate is obtained as CO2, and counted as BaCOn. After removal of COs, H.CHO is distilled off and precipitated in the distillate as the dimethone. On other aliquots, determinations of p31 and p32 (as ammonium phosphomolybdate, or as strychnophosphomolybdate) may be carried out. Comment. In natural phospholipids, the glycerolphosphate moiety is probably present only in the a-form, but acid as well as alkaline hydrolysis invariably yields a mixture of the two isomers.~'2.1~3 In the procedure outlined above, the a-glycerolphosphate, obtained by isomerization of the ~ form in hot acid, is removed by HIO4 as soon as it is formed. The equilibrium is thus shifted to the right, and, practically, a 100% conversion into the ~ form is reached. 11~O. Baflly and J. Gaum~, Bull. soc. chim. (Paris) [5] 2, 354 (1935). 11s E. Baer and M. Kates, J. Biol. Chem. 175, 79 (1948).

[34] Preparation and Analysis of Labeled Coenzymes

By SIDNEY P. COLOWICK and NATHAN O. KAPLAN I. Deuterium-Labeled Pyridine Nucleotides A. Preparation of Deuterium-Labeled Reduced DPN (Forms A and B)

I. Enzymatic Method Principle. This method is based on the discovery by Westheimer, Vennesland, and their collaborators that certain enzymes catalyze a direct transfer of hydrogen (or deuterium) from substrate to pyridine nucleotide. 1 Thus, in the presence of yeast alcohol dehydrogenase, the following reaction occurs: H D

V J ~,-~-CH~CDO ~

CH3CD2OH

I+ R

~ - ~

H+

I R

l H. F. Fisher, E. E. Conn, B. Vennesland, and F. Westheimer, J. Biol. Chem. 202, 687 (1953).

840


[34]

The solution is filtered into a 15-ml. centrifuge tube and brought to a volume of about 8 ml. One milliliter each of the H~SO4 and HIO4 solutions are added. The tube is half immersed in boiling water for 1 hour, the volume being maintained by repeated additions of tt~O. After cooling, the solution is brought to 10 ml. One aliquot is treated with the HgCl~acetate reagent, and the/~-carbon of glycerolphosphate is obtained as CO2, and counted as BaCOn. After removal of COs, H.CHO is distilled off and precipitated in the distillate as the dimethone. On other aliquots, determinations of p31 and p32 (as ammonium phosphomolybdate, or as strychnophosphomolybdate) may be carried out. Comment. In natural phospholipids, the glycerolphosphate moiety is probably present only in the a-form, but acid as well as alkaline hydrolysis invariably yields a mixture of the two isomers.~'2.1~3 In the procedure outlined above, the a-glycerolphosphate, obtained by isomerization of the ~ form in hot acid, is removed by HIO4 as soon as it is formed. The equilibrium is thus shifted to the right, and, practically, a 100% conversion into the ~ form is reached. 11~O. Baflly and J. Gaum~, Bull. soc. chim. (Paris) [5] 2, 354 (1935). 11s E. Baer and M. Kates, J. Biol. Chem. 175, 79 (1948).

[34] Preparation and Analysis of Labeled Coenzymes

By SIDNEY P. COLOWICK and NATHAN O. KAPLAN I. Deuterium-Labeled Pyridine Nucleotides A. Preparation of Deuterium-Labeled Reduced DPN (Forms A and B)

I. Enzymatic Method Principle. This method is based on the discovery by Westheimer, Vennesland, and their collaborators that certain enzymes catalyze a direct transfer of hydrogen (or deuterium) from substrate to pyridine nucleotide. 1 Thus, in the presence of yeast alcohol dehydrogenase, the following reaction occurs: H D

V J ~,-~-CH~CDO ~

CH3CD2OH

I+ R

~ - ~

H+

I R

l H. F. Fisher, E. E. Conn, B. Vennesland, and F. Westheimer, J. Biol. Chem. 202, 687 (1953).

[34]

L&BELED COENZYMES

84t

The same workers subsequently showed that two different stereoisomers of deuterium-labeled reduced D PN may be obtained, depending on the particular enzyme system used. Those systems which oxidize simple primary or secondary alcohol groups give rise to the same isomer (here termed form A) originally obtained with yeast alcohol dehydrogenase, whereas those systems which oxidize aldehydes and hydroxysteroids give rise to the other isomer (form B). 2,3 Pyridine nucleotide transhydrogenase belongs to the latter group of enzymes. 4 D

H

t

R Form A

H

D

I

R Form B

Procedure for Form A. The procedure for the preparation of DPND, form A, is exactly the same as that described in Vol. III [127] for the preparation of D P N H . The only difference is that 1,1-dideuterioethanol L is used instead of normal ethanol. The product should contain 1 atom of deuterium per molecule and should yield DP N devoid of deuterium when oxidized enzymatically with yeast alcohol dehydrogenase. Procedure for Form B. This procedure is also the same as that for D P N H in Vol. I I I [127]. In this case, however, one uses normal ethanol for the reduction of deuterium-labeled oxidized DPN. The preparation of the latter is described in Section B, Part 3, below. This product should retain all its deuterium when oxidized enzymatically with yeast alcohol dehydrogenase. 2. Chemical Method Principle. The reduction of D PN by dithionite in heavy water gives rise to a mixture of the two forms of DPN,1.5 about 70% of the product being in form A. Procedure. This method is exactly the same as that for the chemical preparation of D P N H (Vol. I I I [126]) except that the medium is D20 instead of H20 during the reduction stage.

2p. Talalay, F. A. Loewus, and B. Venneslaad, J. Biol. Chem. 212, 801 (1955). B. Vennesland, J. Cellular Comp. Physiol. 47, Suppl. I, 201 (1956). In form A, the side in which the deuterium is present is called either 1 or ~. The opposite position is called either 2 or 8. A. San Pietro, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 212, 941 (1955). M. E. Pullman, A. San Pietro, and S. P. Colowick, J. Biol. Chem. 206, 129 (1954).

842


[34]

The product should retain about 30 % of its deuterium when oxidized enzymatically with yeast alcohol dehydrogenase.

B. Preparation of Deuterium-Labeled Oxidized DPN 1. Enzymatic Oxidation of Labeled Reduced Compound 5 Principle. When chemically prepared D P N D (70 % form A) is oxidized with acetaldehyde and alcohol dehydrogenase, form A yields unlabeled D P N and form B yields D P N containing 1 atom of deuterium per molecule, so that the resulting mixture contains 0.3 atom of deuterium per molecule of DPN. Procedure. To a solution of 300 micromoles of chemically prepared D P N D (sodium salt) in 20 ml. of water, 10 ml. of 0.1 M Tris-HC1 buffer (pH 7.5), 2 ml. of 0.5 M acetaldehyde, and 0.05 ml. of crystalline yeast alcohol dehydrogenase (10 rag. of protein per milliliter) are added, and the reaction mixture incubated at room temperature. The reaction is almost complete in about 10 minutes, as determined by measuring the absorption at 340 mt~ of small aliquots diluted in 0.5 M Na2CO3. The oxidized D P N can be isolated in 50% yield by acidifying the reaction mixture with nitric acid until blue to Congo paper, precipitating with 6.5 vol. of cold acetone, washing with acetone, and drying in vacuo over P~Ob. The product is 75 to 80% pure by enzymatic assay. 2. Chemical Oxidation of Labeled Reduced Compound 5 Principle. On ferricyanide oxidation of the two isomers of D P N D , form A retains about 50% of its deuterium, whereas form B retains about 90% of its deuterium. 4 Ferricyanide oxidation of chemically prepared D P N D , containing 70 % form A, should therefore yield D P N containing 70 × 0.5 ~ 30 × 0.9, or about 60% of the original deuterium. Ferricyanide oxidation of reduced D P N does not proceed readily when both reactants are at the concentrations usually employed for direct spectrophotometric observations at 340 m# (10-4 M or less). In more concentrated solutions, however, the reaction goes to completion much more rapidly, as would be expected for a bimolecular reaction. Procedure. To a solution containing about 300 micromoles of D P N D (chemically or enzymatically prepared) are added 1 ml. of 0.5 Tris-HC1 buffer (pH 7.5) and 1 ml. of M potassium ferricyanide in a total volume of 20 ml., and the reaction mixture is incubated at 38 °. The D P N formed is measured with alcohol and alcohol dehydrogenase (Vol. III [128]) on 0.03-ml. aliquots. At 40 minutes, 90 % of the reduced D P N added can be accounted for as oxidized DPN. The oxidized D P N can be isolated as described in Section B, Part 1.

[~]

LABELED COENZYMES

843

3. Exchange of DPN-Cyanide Complex with D20 ~ Principle. When the DPN-cyanide complex is incubated with D20 at high pH values, an exchange occurs at the w-carbon of the pyridine ring: H CN D CN

\/

/

D20

)

I R

R

On neutralization, the cyanide complex dissociates, yielding DPN labeled with deuterium at the ~,-carbon: D

CN

N/

D -~

I

R

÷ CN-

I+

R

This appears to be the method of choice for preparing ~-labeled DPN. Procedure. To a solution of 420 micromoles of D P N (unneutralized) in 5 ml. of M KCN is added 0.13 ml. of 5 N KOH; the medium for all solutions is pure D20. The resulting orange solution (pH approximately 11.5) is incubated for about 2 hours at room temperature. Then 45 ml. of D~O containing 11 millimoles of KH2PO4 is added, and the H C N is removed by bubbling nitrogen through the solution; final pH 6.7. The recovery of DPN, as measured with alcohol and alcohol dehydrogenase (Vol. III [128]), is quantitative. It is necessary, however, to use more than the usual quantity of enzyme for the D P N assay, since a potent unidentified inhibitor of yeast alcohol dehydrogenase is present in the final mixture. Under the above conditions, the resulting D P N should contain over 0.9 atom of deuterium per molecule. The D P N may be isolated by acid acetone precipitation as described in Section B, Part 1. 4. Exchange of D P N with D20 in Alkali 7 Principle. When D P N is incubated with D20 in alkaline solution, deuterium exchange occurs in the a position (carbon 2) rather than at 6 A. San Pietro, J. Biol. Chem. 217, 579 (1955). 7 A. San Pietro, J. Biol. Chem. 217, 589 (1955).

844


[34]

the ~ position as is the case with the DPN-cyanide complex. The exchange reaction occurs more rapidly than the alkaline hydrolysis of the nicotinamide-ribose bond, thus permitting the accumulation of labeled D P N under appropriate conditions. It should be pointed out that, although this exchange reaction does not occur at pH 7 to 8 at room temperature, considerable exchange can occur at these pH values on boiling for 1 or 2 minutes) For this reason, it is recommended that enzymatic reactions involving deuterio-DPN should not be stopped by boiling, since boiling even briefly in D~O will introduce label at carbon 2, and boiling in normal water will remove deuterium from that position. Procedure. A solution of 420 micromoles of D P N in 5 ml. of pure D20 is adjusted to pH 11.0 by the addition of 1 N KOH (prepared in D20). After 2 hours at room temperature, when about 25% of the D P N has undergone hydrolysis, the remaining D P N is precipitated by acid acetone as described in Section B, Part 1. The product contains 0.9 to 1 atom of deuterium per molecule, and the label can be shown to be located exclusively at carbon 2 of the nicotinamide moiety. The product also contains the A D P R which was produced during alkaline hydrolysis of DPN. If necessary, this impurity can be removed by column chromatography (see Vol. III [124]). 5. Enzymatic Exchange of Deuterium-Labeled Nicotinamide with D P N Principle. Loewus et al. 9 have described the synthesis of monodeuterionicotinamide samples labeled with deuterium at carbon 2, 4 or 6. These compounds may be incorporated into D P N enzymatically by the use of mammalian DPNase according to the procedure of Zatman et al., 1° which is described below in Section II. One thus obtains 2-, 4- or 6-monodeuterio-DPN. This is the only known procedure for the preparation of the 6-derivative. For the preparation of the 2- and 4-derivatives, the nonenzymatic deuterium exchange reactions in alkali and cyanide, respectively, described above, are preferable.

C. Analysis of Deuterium-Labeled Pyridine Nucleotides 1. Determination of Deuterium Content in Intact Nucleotides Principle. One may analyze pyridine nucleotides directly by combustion, followed by conversion of the water to hydrogen gas and mass q S. Englard and S. P. Colowick, J. Biol. Chem. in press (1957). 9 F. A. Loewus, B. Vennesland, and D. L. Harris, J. Am. Chem. Soc. 77, 3391 (1955). t o L. J. Zatman, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 200, 197 (1953).

[34]

LABELED COENZYiVIES

845

spectrometry according to standard procedures (Vol. IV [21]). Because of the difficulty in obtaining the nucleotides in pure form, however, large errors may be introduced, the magnitude of which depends on the hydrogen and/or deuterium content of the impurities. If one assumes that the impurities contribute no deuterium to the sample, then one may apply the method of Fisher et al., ~ in which the solid sample is diluted with another pure solid (e.g., glycine) of known hydrogen content, prior to combustion. Procedure. Suppose, for example, that a 1.33-mg. sample of deuterioD P N is known by enzymatic analysis to be 50% pure and to contain therefore 1 micromole of pure DPN, corresponding to 27 microatoms of hydrogen (or deuterium). If one adds enough pure glycine (3.63 rag.) to dilute the hydrogen of D P N from 27 to 270 microatoms, and then finds on combustion of the mixture a value of 0.37 atom % excess deuterium, one may calculate ~ by correcting for dilution that the original D P N contained 3.7 atoms % excess deuterium (or 1 atom of deuterium per molecule). The error due to dilution by hydrogen-containing impurities is thereby minimized. 2. Determination of Deuterium in Nicotinamide Moiety 5 Principle. The labeled D P N is cleaved by a DPNase to yield adenosine diphosphate ribose (ADPR) and nicotinamide. The latter, which bears all the label, is isolated in crystalline form, usually after quantitative dilution with carrier nicotinamide. The pure product is combusted and analyzed for deuterium in the usual manner. This procedure is both more sensitive and more accurate than that described in the precedingsection. Procedure. To a solution of 300 micromoles of deuterio-DPN in 20 to 30 ml. of 0.1 M Tris-HC1 buffer (pH 7.5) is added 0.2 ml. (0.5 rag.) of a Neurospora crassa DPNase preparation, i.e., the fraction precipitating with 60% acetone at pH 2.7, containing about 15,000 units per milligram of protein, prepared according to Kaplan et al. ~1 (see Vol. II [114]). The reaction mixture is incubated for about 1 hour at 37 °, and the course of the reaction is followed by diluting 0.05-ml. aliquots to 3 ml. with M K C N and reading the absorption at 325 m~ (see Vol. III [128]). When the reaction is complete, the solution is passed through a Dowex 1 formate column (7 X 1 cm.) which removes the A D P R but permits the nicotinamide to go through. Nicotinamide is estimated in the effluent by the characteristic increase in ultraviolet absorption which takes place when neutral solutions of nicotinamide are acidified to pH 2 or less, as suggested by Rowen and Kornberg. 1~The extinction coefficients ~1 N. O. Kaplan, S. P. Colowick, and A. Nason, J. Biol. Chem. 191, 473 (1951). 12j. W. Rowcn and A. Kornberg, J. Biol. Chem. 195, 497 (1951).

846

TEC~QIUES FOR ISOTOPE STUDIES

[34]

for nicotinamide at 260 m~ are 2.85 and 5.02 X 10 e cm ~ per mole for pH 7 and 1, respectively. The column is washed with water (ca. 20 ml.) until most of the nicotinamide is recovered. The total recovery of nicotinamide should correspond to 95 % or more of the original DPN content. The nicotinamide content of the combined effluent and washings is determined carefully, and solid carrier nieotinamide (ca. 300 mg.) is added in an amount calculated to give a tenfold dilution of the deuteriumlabeled nicotinamide. The dilution factor is checked by spectrophotometric analysis of the resulting solution. The solution is taken to dryness in vacuo, and the las£ traces of water are removed by adding benzene and again evaporating to dryness. The dried residue is extracted with 25-mi. portions of hot benzene, and the benzene extracts are filtered through glass wool and chilled to 20° . Extraction is repeated until the extracts fail to yield nicotinamide on cooling. The crystalline nicotinamide is collected by centrifugation and dried in vacuo over P~O5 overnight. The yield is about 270 mg., or 75%. The resulting nicotinamide, after its purity is ascertained by determining the extinction at 260 m~ of a weighed sample, is combusted and the deuterium determined in the usual manner (Vol. IV [21]). The value is corrected for dilution by carrier, and the result expressed as atoms of deuterium per molecule of nicotinamide. For nicotinamide, a deuterium content of 1 atom per molecule corresponds to a value of 16.7 atoms % excess. The above procedure, as described, is on a much larger scale than the minimum required for deuterium determinations. The system can readily be scaled down for handling as little as 5 microatoms of deuterium (e.g., 5 micromoles of DPN containing 1 atom of deuterium per molecule, or 50 micromoles of DPN containing 0.1 atom of deuterium per molecule). Addition of 500 micromoles (ca. 60 mg.) of carrier nicotinamide will then result in a product containing about 0.01 atom of deuterium per molecule or 0.167 atom % excess. The latter value is still in the range for accurate determination by the mass spectrometer.

3. Determination of Site of Deuterium Labeling Principle. Any procedure which causes introduction of deuterium at a specific site in the DPN molecule can conversely be used specifically to displace deuterium from that site. Thus, if DPN contains deuterium at carbon 4 of the pyridine ring, this can be demonstrated in two ways: (1) by incubating with cyanide in normal water or (2) by reduction (either chemically in ~ormal water or enzymatioally with normal ethanol), followed by chemical oxidation with ferricyanide. 6 The resulting DPN in all these cases will show a diminished deuterium content if deuterium

[3~

LABELED COENZYMES

847

is present at carbon 4. If DPN contains deuterium at carbon 2, this should be lost on incubating with alkali in normal water. The above procedures, which depend on displacement of deuterium from the intact pyridine nucleotide, have all been described adequately in earlier sections. Another procedure, which depends on removal of deuterium after isolation of the nicotinamide moiety, will be described in detail here. In this procedure, which was first applied in ascertaining the structure of reduced DPN, 5 the nicotinamide resulting from enzymatic cleavage of oxidized deuterio-DPN is methylated with methyl iodide. The resulting N'-methyl nicotinamide is oxidized to yield a mixture of the 2- and 6-pyridone derivatives, which are isolated separately in pure form and analyzed for deuterium. With DPN labeled at carbon 4, the resulting pyridones show the same deuterium content per molecule as the nicotinamide from which they were derived. 5 With DPN labeled at carbon 2, the resulting 2-pyridone is unlabeled, whereas the 6-pyridone retains most of the labelY P r o c e d u r e ) A solution of 262 rag. of nicotinamide in 1.8 ml. of methyl alcohol is made up in a 15-ml. conical centrifuge tube. To this solution are added 0.2 ml. of methyl iodide and a boiling chip. A 3-foot air condenser is attached to the centrifuge tube, and the solution refluxed for 15 hours in a water bath at 58 to 62° in a cold room. The reaction appears complete at about 2 hours, the first crystals appearing at 30 minutes. After 15 hours the suspension is poured onto a small sintered-glass funnel and the precipitate is washed with cold methanol. The pale yellow crystals of N'-methyl nicotinamide iodide are dried in vacuo at 100 ° over P205 for 2 hours. Yield, 497 mg. (88% of theory). N'-!VIethyl nicotinamide iodide (453 rag., representing 1710 micromoles) is dissolved in 12 ml. of water. To this solution is added 8 ml. of alkaline ferricyanide solution to give a final concentration of 0.5 N NaOH and 0.3 M potassium ferricyanide. After incubation for 30 minutes at room temperature, the dark green alkaline reaction mixture is placed on a large Dowex monobed column consisting of 3 parts of Dowex 1, hydroxyl form, and 1 part of Dowex 50, hydrogen form (50 to 100 mesh, 5 cm. in diameter by 12 cm.). The effluent from the column should be nearly colorless and slightly acidic. The column is washed with seven 50-ml. portions of water, and the combination of effluent piLlS washings is taken to dryness in vacuo. The last traces of water are removed by adding benzene and repeating the drying procedure. The dried residue is then extracted with 4-ml. portions of hot chloroform until fluorimetric determinations indicate that all the 2-pyridone has been extracted. The 6-pyridone is then extracted from the residue with an excess of hot methanol.

848


[34]

For crystallization of the 2-pyridone, the combined chloroform extracts are taken to dryness; the residue is dissolved in a minimum of hot methanol and cooled in ice, yielding pale yellow needles. For crystallization of the 6-pyridone, the solution in methanol is concentrated and then chilled in ice, yielding white needles. Yields, 6-pyridone, 36 mg.; 2pyridone, 35 rag. The compounds are recrystallized from methanol and dried i n vacuo over P~05 at 100 ° prior to analysis.

II. C~4-Nicotinamide-Labeled Coenzymes A. Preparation of C~4-Nicotinamide-Labeled Exchange

DPN

by

Enzymatic

P r i n c i p l e . The hydrolysis of the nicotinamide ribose bond of D P N by mammalian DPNases is presumed to take place via the following mechanism. 10

ADP-ribosyl-nicotinamide -~ enzyme ~ ADP-ribosyl-enzyme -~ nicotinamide ADP-ribosyl-enzyme -{- HsO --~ ADP-ribose -{- enzyme This sequence explains the inhibition of D P N hydrolysis by added nicotinamide, as well as the rapid exchange of labeled nicotinamide into DPN. At high nicotinamide concentrations, there is a very slow hydrolytic breakdown of DPN, and the rate of exchange is almost equal to the rate at which the D P N would have been hydrolyzed in the absence of nicotinamide. Thus one can obtain good yields of D P N which has equilibrated completely with the added labeled nicotinamide. Both C14-1abeled 1° and deuterium-labeled 9 D P N have been prepared in this way. Procedure. 1° C14-Nicotinamide (CsH4N--CI4ONH2) is diluted with carrier nicotinamide and crystallized to constant specific activity from benzene. In a typical experiment, the specific activity was 7245 c.p.m. per micromole (thin-wall mica window counter). The incubation mixture contains 660 micromoles of D P N (65 % pure, neutralized with NaOH to pH 7), 3000 micromoles of C14-nicotinamide, 2240 units of spleen DPNase (see Vol. II [113]), and water to 50 ml. It is important that no buffer be added, and that the enzyme be prepared in water rather than bicarbonate, in order to avoid subsequent difficulty in chromatography of the DPN. After incubation for 23z~ hours at 37 °, analysis for D P N by cyanide reaction (see Vol. III [128]) indicates that 30% of the original D P N has undergone hydrolysis. (The enzyme added is sufficient to cause 100% cleavage in 1 hour in the absence of nicotinamide.) The reaction mixture is then heated to 70 ° for 10 minutes, a treatment which destroys the DPNase activity without significantly

[34]

LABELED COENZYMES

849

altering the DPN concentration. After cooling and centrifuging, the clear yellow supernatant fluid is transferred to a Dowex 1 anion exchange resin (formate form, 200 to 400 mesh, 4 X 2 cm. column). Distilled water is passed through the column under pressure, at the rate of about 45 ml./ hr., for about 16 hours, in order to remove all free nicotinamide. The latter is not detectable in the final effluent by 260 m~ absorption or radioactivity measurements. The DPN is then eluted with 0.1 M formic acid until the DPN content of the effluent, as assayed with alcohol dehydrogenase, falls to zero. The formic acid eluates containing the DPN are combined (134 ml., 232 micromoles of DPN), 1 ml. of 5 N HNO3 is added (blue to Congo Red paper), and then 1 1. of cold acetone. After standing in the cold (4° ) overnight, the precipitate is centrifuged, washed with acetone, and dried in vacuo. The yield is 142 mg. of an amorphous white powder, containing 180 micromoles of DPN by enzymatic assay, which corresponds to a purity of 84% (uncorrected for moisture). The ratio of absorption at 260 m~ before reduction to absorption at 340 mu after enzymatic reduction is 3.0. The specific radioactivity is 5430 c.p.m. per micromole, corresponding closely to the theoretical value for complete equilibration between the added radioactive nicotinamide and the unlabeled nicotinamide moiety of the starting DPN. The above procedure should be readily applicable to the preparation of labeled TPN, since the latter is acted on rapidly by the spleen DPNase. 1° C14-Labeled reduced nucleotides may be prepared, of course, by reduction of the labeled oxidized nucleotides according to the usual procedures.

B. Analysis of C~4-Nicotinamide-Labeled Pyridine Nucleotides Principle. Neurospora DPNase attacks only the oxidized forms of DPN and TPN and not the reduced compounds. When solutions containing both oxidized and reduced labeled nucleotides are incubated with the Neurospora enzyme, only that portion of the radioactive nicotinamide is liberated which is present in the oxidized nucleotides. This principle has been applied in studies on the pyridine nucleotide transhydrogenase mechanism. 13 Procedure. To 1 ml. of a reaction mixture containing about 10 micromoles of total pyridine nucleotides and about 20,000 total e.p.m. (thinwall mica window counter) is added 1000 units (20 ~,) of Neurospora DPNase (see Vol. II [114]), and the mixture is incubated for 20 minutes at 37 ° in the presence of 0.04 M Tris buffer, pH 7.5. The reaction mixture is then placed on a column of IRA-400 Amberlite (formate form, 50 La N. O. Kaplan, S. P. Colowick, L. J. Zatman, and M. M. Ciotti, J. Biol. Chem. 205, 31 (1953).

850


[34]

mesh, 1 X 4 cm.), which permits separation of nieotinamide from the other products of DPNase action as well as from the uncleared reduced nucleotides. The resin is prepared in the same way as Dowex 1 formate (see Vol. II [131]). Free nicotinamide is washed through the column with 1-ml. portions of water, and the effluents are collected separately and analyzed for nicotinamide by reading at 260 mu before and after acidification, as described above in Section C, Part 2. The samples containing nicotinamide are pooled, and suitable aliquots are dried on aluminum planchets under an infrared lamp for radioactivity determinations. The specific activity of the resulting nicotinamide represents directly the specific activity of the oxidized forms of the pyridine nucleotides in the original sample. III. Preparation of Ribose-Labeled D P N It has been found that when nicotinamide is administered to mice a tenfold rise in liver D P N takes place. 14 Through the use of C14-glucose or ribose and nicotinamide, it has been possible to isolate ribose-labeled D P N from mouse liver with relatively high specific activity. 15Since much higher specific activities were obtained with ribose than with glucose, it seems preferable to use labeled ribose as a source of the D P N ribose. Method. 15 Ten mice are injected intraperitoneally with 500 mg. of nicotinamide per kilogram. After an interval of l0 minutes, 5 /~c. of ribose-l-C14 is administered. After about 6 hours, during which the animals have access to water but not food, they are sacrificed by cervical fracture. The livers are removed, chilled on ice, weighed, and homogenized either in a glass homogenizer or in a Waring blendor with 5 vol. of cold 5 % trichloroacetic acid; the resulting protein precipitate is removed by centrifugation and washed with 2 vol. of 5% trichloroacetic acid. The wash is then combined with the first supernatant. The D P N is precipitated from the trichloroacetic acid extract by the addition of 5 vol. of cold acetone. After sitting overnight at - 1 8 °, the precipitate is removed by centrifugation, washed with acetone and then with ether, and then dried in a vacuum desiccator. The acetone precipitate is dissolved in water, and the D P N concentration determined by the cyanide method. This method gives values very close to the yeast alcohol dehydrogenase assay 16 or the fluorometric values after treatment with Neurospora DPNase. ~6 The yield from 10 mice is usually about 70 micromoles. 14 N. O. Kaplan, A. Goldin, S. R. Humphreys, M. M. Ciotti, and F. E. Stolzenbach, J. Biol. Chem. 219, 287 (1956). 15 L. Shuster and A. Goldin, in preparation. 1~ See Vol. I I I [128],

[~4]

LABELED COENZYMES

851

The crude DPN solution is adjusted to pH 7.5 with 1.0 M NH4OH and chromatographed on Dowex 1 resin, either in the formate or the chloride form. The flavin nucleotides are adsorbed to the top of the column. Glycogen is not adsorbed and is removed as an opalescent solution in the first few washes. After the resin is well washed with H~O and the eluate shows a negligible optical density at 260 m~, the DPN is removed from the column with 0.1 M formic acid in the case of the formate resin or with 0.003 N HC1 in the case of the chloride resin. The elution of the DPN can be followed by measuring optical density at 260 mu. Peak fractions are combined and lyophilized or evaporated under reduced pressure at room temperature. Approximately 50 micromoles of ribose-labeled DPN with specific activity of 30;000 c.p.m, per micromole are obtained. The radiopurity of the isolated DPN has been tested in a number of different ways. The specific activity was found to remain constant during elution from a Dowex 1 resin. A single ultraviolet quenching spot was obtained by paper chromatography in three different solvent systems. Paper electrophoresis in 0.02 M citrate (pH 3.5) also gave a single spot. The ratio of E2~0 to E325 in M KCN was the same as that of DPN, thus ruling out significant contamination by other nucleotides. Rechromatographing the material either on paper or on Dowex 1 did not significantly change the specific activity of the DPN. It is of interest to note that the ribose of the nicotinamide mononucleotide moiety of the DPN has a specific activity about three times as high as does the adenylic acid ribose part of the molecule. 1~ IV. P32-Labeled DPN from Yeast

Velick et al. 1~ have described a procedure for the preparation of P32-1abeled DPN from baker's yeast. This is accomplished by growing in a p32 medium. The culture medium is given in the accompanying table. To the whole of solution I plus 52 ml. of solution II are added 6.0 inc. of P3:-labeled phosphate and 55 g. of moist yeast cake (18 g. dry weight) as inoculum. The solution is vigorously aerated, and after 1~ hour the remainder of solution II is introduced at a rate so that addition is complete in 8 hours. The pH is adjusted to about 4.3 at l~-hour intervals by the addition of ammonium hydroxide. At the end of a 12-hour growth period, the cells are harvested by centrifugation and packed on a suction filter. The yield of moist yeast cake is 162 g. Velick et al. 1~ isolated the DPN by a modification of the procedure 17 S. F. Velick, J. E. Hayes, Jr., and J. Harting, J. Biol. Chem. 203, 527 (1953).

852


[34]

of Williamson and Green. 18 T h e yield of D P N f r o m the 162 g. of y e a s t was a b o u t 4 rag., and the specific a c t i v i t y (1 week after beginning the experiment) was 29,000 c.p.m, per micromole. T h e method of Horecker and K o r n b e r g (see Vol. I I I [124]) also appears to be a p p r o p r i a t e for the isolation of the P32-1abeled D P N from yeast. CULTURE MEDIUM FOR Pa2-LABELED YEAST

Solution I Corn steep water a KH~P04 Tap water to

Solution II 12 g. 3 g. 3600 ml.

Blackstrap molasses (beet) (NH4)2SO4 Tap water to

300 g. 8 g. 1200 ml.

a Commercial corn steep--water solids are dissolved in sufficient hot water and brought to pH 7.5 with NH4OH. The phytin fraction that separates is collected and discarded.

V. Preparation of P32-Labeled DPN from Nicotinamide-Injected Mice Phosphorus-labeled D P N of high specific a c t i v i t y can also be prepared f r o m mice injected with nicotinamide. 19 T h e D P N is isolated in the same m a n n e r as t h a t described a b o v e for ribose labeling. Usually 50 tLc. of o r t h o p h o s p h a t e (Na2HPa204) is administered per mouse, 10 minutes after the injection of nicotinamide. T h e animals are sacrified approxim a t e l y 3 to 4 hours after the administration of the labeled phosphate. Pa2-Labeled D P N with a specific a c t i v i t y as high as 100,000 c.p.m, per micromole of D P N has been obtained.

VI. P32-Labeled TPN I t is possible to prepare T P N labeled only in the monoester position through the use of A T P labeled in the p y r o p h o s p h a t e groupings, unlabeled D P N , and the D P N kinase. 2° T h e T P N can be isolated by column c h r o m a t o g r a p h y , and some preliminary experiments indicate t h a t this procedure is feasible. ~1 T h e labeling of T P N only in the 2' monoester position m a y be of value in preparing 2',5'-diphosphoadenosine, with only the 2' grouping labeled. T h e 2',5'-diphosphoadenosine can be obtained from T P N b y t r e a t m e n t with snake v e n o m p y r o p h o s p h a t a s e as described in a n o t h e r section (Vol. I I I [130]). 18S. Williamson and D. E. Green, J. Biol. Chem. 136, 345 (1940). 19T. Langan and L. Shuster, unpublished observations. 20See Vol. II [111]. 21 N. O. Kaplan, unpublished observations.

[~]

LABELED COENZYMES

853

VII. Preparation of P32-Labeled ATP and ADP A. Labeling with Mitochondrial Preparations Kielly ~2 has been able to prepare ATP or ADP with the labile phosphates labeled using the oxidative phosphorylation system from mitochondria. Well-prepared liver mitochondria from healthy animals will usually convert 90% of the inorganic phosphate to ATP. This makes it possible to obtain the polynucleotides with extremely high specific activity. Preparation of Mitochondria. The preparation described below is essentially that of Schneider ~8 but has been shortened by eliminating the steps for the quantitative recovery of the mitochondria. 1. Rats or mice arc killed by exsanguination or cervical dislocation, and the livers are rapidly removed and chilled in 0.25 M sucrose, cooled to about - 1 % The livers are then put through a tissue press and homogenized in a volume of cold 0.25 M sucrose to give a "10%" homogenate. From 5 to 10 g. of liver is used. 2. The homogenate is centrifuged in the horizontal head of the International refrigerated centrifuge at 2000 r.p.m, for 10 minutes. 3. The supernatant fluid is removed by syringe and recentrifuged in the multispeed attachment of the refrigerated centrifuge at a tachometer reading of 2000 r.p.m. (actually 9000 r.p.m, for the rotor) for 10 minutes. This step can also be done in the Servall SS-1 at a setting of 52 volts. 4. The supernatant fluid from step 3 is removed and discarded. The precipitate is resuspended in cold 0.25 M sucrose (15 to 20 ml. total volume) and recentrifuged in the multispeed head at a tachometer reading of 4000 (18,000 r.p.m.) for 10 minutes. 5. The supernatant fluid from step 4 is poured off, including the light pink layer on top of the sedimented mitochondria, and step 4 is repeated. 6. The supernatant fluid from step 5 is poured off and the mitochondria suspended in 0.25 M sucrose to a final volume equal to the weight of the original liver (i.e., 1 ml. of mitochondrial suspension per gram of fresh liver). The following system is used for the phosphorylation of adenylic acid: 0.90 0.20 0.15 0.60 0.15

ml. ml. ml. ml. ml.

0.2 M histidine, pH 6.8 2.5 X 10-4 M cytochrome c 0.1 M MgCl~ 0.04 M A M P 0.2 M a-ketoglutaratc

~2 We wish to thank Dr. Wayne Kielly for making this procedure available. ~3 W. C. Schneider, J. Biol. Chem. 176, 259 (1948).

854


[34]

0.05 ml. 1.0 M POt, pH 6.8 0.50 ml. mitochondrial suspension H20 or 0.25 M sucrose to a final volume of 3 ml. including the p82 solution (1 or 2 mc.). The reaction is carried out at 28 to 30 ° with shaking. Usually a pilot (" cold ") run is made prior to the "isotope" incubation to determine when the reaction is complete. Aliquots are removed at zero time, and at 10-minute intervals (up to 40 or 50 minutes), 5% perchloric acid is added and aliquots assayed for inorganic phosphate. Aliquots of 1 ml. are removed at zero time and at the termination of the "isotope" run for inorganic phosphate to determine actual conversion. The radioactive ATP is obtained after the reaction is stopped with 0.1 vol. of perchloric acid. The perchloric acid solution is neutralized with KOH and, after removal of the perchlorate, placed on a 2 % crosslinked Dowex 1 formate column. Except for washing the Dowex with strong formic acid, the details of the procedure are the same as given by Hurlbert et al. 24 and described in Vol. III [111]. After the ATP 3~ fractions are obtained from the column, they are lyophilized. Resolution and relyophilization will usually reduce the residual formate so that for most purposes it presents no interference. Preparation of Labeled ADP from A TP. ADP labeled in the terminal phosphate can be prepared from the labeled ATP by treatment with hexokinase and glucose. Usually 0.1 M glucose, 0.01 M Mg C12 and sufficient hexokinase (to transfer about 4 to 5 micromoles of P to glucose per minute) are used. The ADP can be purified by column chromatography. The labeled ADP can also be prepared with the adenosine triphosphatase of lobster muscle; the ADP can be isolated also as a barium salt (see Vol. III [118]).

B. Chemical Preparation of P82-Labeled Polyphosphates Lowenstein25 has recently described a procedure which can be used for the preparation of labeled adenosine polyphosphates. This procedure involves the reaction of orthophosphate with 5'-AMP, ADP, and ATP in the presence of N,N'-dicyclohexylcarbodiimide (DCC). The ADP so prepared is labeled exclusively in the terminal phosphate. The ATP and adenosine tetraphosphate prepared in this manner are labeled largely in the terminal phosphate. An important feature of this method is that 24 R. B. Hurlbert, H. Schmitz, A. F. Brumm, and V. R. Potter, J. Biol. Chem. 209, 23 (1954). 2~ j . M. Lowenstein, Biochem. J. 65, 197 (1957).

[34]

LA~ELED COENZYMES

855

carrier-free labeled orthophosphate can be used and hence nucleotides of high specific activities can be obtained. Lowenstein has purified the nucleotides by paper chromatography. There appears to be no reason why column chromatography could not be used for separation and purification of the polyphosphates on a somewhat larger scale than that used by Lowenstein. The following preparation of terminal-labeled ADP is given as an example of the method: A,3-ml. glass-stoppered flask was used as the reaction vessel. Thirty microcuries of P3~-orthophosphate was measured into this vessel and then evaporated to dryness. Then 10 rag. of AMP and 0.5 ml. of pyridine-water (10:1 v/v) were added; this was followed by the addition of 0.20 ml. of DCC. A glass bead was added, the flask was stoppered, and the stopper was secured by metal springs. The flask was shaken on a mechanical shaker. Complete solution occurred within 3 minutes and a precipitate (probably dicyclohexylurea) separated out after about an hour. The reaction was complete between 12 and 24 hours. Water (1 ml.) was added at the completion of the reaction, and the mixture was shaken rapidly for 1 minute. The flask was centrifuged slowly to separate the aqueous solution which was then transferred to a clean glass-stoppered tube by means of a drawn-out dropper. Free pyridine was removed by twice extracting the aqueous solution with 7-ml. portions of ether. The sample was then chromatographed. Approximately 50% of the total p32 was found in the terminal phosphate of ADP. The other adenine nucleotides showed little p32 incorporated. About 20 % of the p32 was still left as orthophosphate.

VIII. Coenzyme A No large-scale preparation of labeled coenzyme A has as yet been described. Sen and Leopold, however, have prepared p32_ or S35-1abeled coenzyme A from yeast grown either on radioactive sulfate or on phosphate. The labeled coenzyme was isolated by paper chromatography and elution. From the data reported by Sen and Leopold, 26 it would appear that the S 35 coenzyme A would be less subject to contamination with impurities than the p32 coenzyme prepared in the same manner. Preliminary experiments suggest that the dephospho-coenzyme A kinase system can be used as a method to introduce labeled p32 in the 3 ~position of the adenylic acid moiety of the coenzyme. 27 The dephosphocoenzyme A can be readily separated from the whole coenzyme by column chromatography. 28 26 S. P. Sen and A. C. Leopold, Biochim. et Biophys. Acta 18, 320 (1955). 2~ E. R. Stadtman, personal communication. 28 T. P. Wang and N. O. Kaplan, unpublished observations.

856


[35]

[35] Synthetic and Analytic P r o c e d u r e s Involving 1131Labeled Compounds

By ALVIN TAUROG and I. L. CHAIKOFF Introduction The I181-1abeled compounds described in this chapter are grouped into three categories: (1) iodinated amino acids and proteins related to thyroxine; (2) proteins which ordinarily do not contain iodine but which have been labeled by the introduction of I TM into the molecule (iodinated serum albumin, iodinated serum globulin, iodinated insulin); and (3) miscellaneous II~l-labeled compounds. The major portion of this chapter deals with the first group, i.e., with compounds of interest to thyroid biochemistry and physiology.

I. I~3~-Labeled Compounds Related to Thyroxine

A. Methods of Preparation Two general procedures are available for preparing I ~31_labeled, iodinecontaining compounds. In one method, I TM is included in the iodinating mixture used for introducing iodine into the molecule. In the second method, the iodine-containing compound is allowed to remain in contact with I TM under conditions that promote a high degree of exchange between the nonradioactive and radioactive atoms of iodine. Examples of both methods are described below.

1. I13~-Labeled Thyroxine a. Iodination of 3,5-Diiodothyronine--Method of Clayton et a[. 1 In this procedure labeled thyroxine is prepared by iodinating 3,5-diiodothyronine with I~ TM. T o 1 ml. of a s o l u t i o n of I TM as iodide (1 mc. or more) in buffered bisulfite, 2.3 mg. of NaI is added. The solution, in a centrifuge tube, is overlayered with 1 ml. of ether, and 1 drop of N HCI and excess 0.3 % H202 are added. The quantity of H202 used depends on the bisulfite concentration of the original I ~8~solution. The mixture is allowed to stand for 2 hours with occasional shaking, and then the lower iodine-free layer is removed with a pipet. 3,5-Diiodo-DL-thyronine (2 mg.) in 0.06 ml. of 33 % (w/v) aqueous ethylamine solution is added, and the mixture is shaken carefully to discharge the iodine color. The ether layer is removed 1 j . C. Clayton, A. A. Free, J. E. Page, G. F. Somers, and E. A. Woollett, Biochem. J. 46, 598 (1950).

[35]

I131-LABELED COMPOUNDS

857

by blowing a stream of air over the surface, and the aqueous layer is diluted with 1 ml. of water and adjusted to pH 4 to 5 with glacial acetic acid. The precipitated thyroxine is separated by centrifugation and washed with successive 1-ml. portions of water. About fourteen washings are required to reduce the nonthyroxine I TM to negligible proportions. The specific activity of the final product is about 100 ~c./mg. when 1 mc. of I TM is used in the starting mixture. b. Iodination of Triiodothyronine--Method of Critchlow and Goldfinch. 2 According to these workers, iodination of triiodothyronine with I2 TM yields labeled thyroxine which is less contaminated with IlaUtriiodo thyronine than the product obtained by iodinating diiodothyronine. The details of the method are as follows: Carrier-free iodide-I TM solution (70 mc., 3 ml.) is added to potassium iodide solution (2 mg. of I, 0.4 ml.) in a 10-ml. centrifuge tube, and the mixed solution is evaporated to 0.5 ml. on a water bath under a current of air. The cooled solution is acidified with 5 N sulfuric acid (0.5 ml.), and 10% w / v potassium nitrite solution (0.25 ml.) is added. The precipitated iodine is centrifuged down and washed with water (0.5 ml.). After removal of the washings, DL-triiodothyronine (4.7 mg.) in 1:1 (v/v) diethanolamine-water (0.4 ml.) is added to the iodine precipitate. The mixture is stirred to dissolve the iodine and allowed to stand for 18 hours. Then 2 N acetic acid is added to pH 4.5, and the precipitated thyroxine is centrifuged. It is redissolved in 33% (w/v) ethylamine solution (0.2 ml.) and water (0.2 ml.), any slight amount of insoluble material is spun down, and the supernatant liquor is transferred to a clean 10-ml. centrifuge tube. Thyroxine is reprecipitated as before, after addition of 2 to 3 drops of a fresh sulfurous acid solution. The dissolution and reprecipitation are repeated, and the precipitate is washed with 0.5-ml. quantities of water (the first two washings containing 2 drops of sulfurous acid) until the washings show a constant slight activity (about ten washes are usually required). The thyroxine is then dissolved in either N / 5 0 sodium carbonate or 1:1 (v/v) propylene glycol-water, the pH of which is adjusted to 8 to 9 with a drop of diethanolamine to give a final solution with activity about 2 mc./ml. The radiochemical yield is 20 mc. (29%). Both preparations deteriorate on keeping and should preferably be used within a few days. c. Microiodination of 3~5,3'-Triiodothyronine--Method of Roche and Michel2 This procedure was designed for the preparation of various I~3~-labeled iodothyronines of very high specific activity. The reaction is carried out in the bulb of a blood-diluting pipet (red cells), thus permit2 A. Critchlow and M. K. Goldfinch, Proc. 2nd Radioisotope Conf., Oxford, I. Med. and Physiol. Applications, pp. 271-280 (1954). 3j. Roche, R. Michel, P. Jouan, and W. Wolf, Bull. soc. chim. biol. 37, 819 (1955).

858


[35]

ting the use of small volumes of very concentrated solutions. The reaction products are separated first by paper electrophoresis to remove inorganic iodine, then by ordinary paper chromatography. Preparation of I2 T M Solution. The solution of tagged I2 is prepared by exchange between I~. and i TM in alcohol-water. A special bent micropipet is employed, consisting of a small bulb, with a capacity of about 50 ~l., connected to two outlet tubes, one with an internal diameter of 5 mm., and the other extremely thin and of small enough diameter to enter into the capillary of the blood-diluting pipet. A microdrop of ~3~ weighing 15 mg. and containing approximately 1 mc. is introduced into the bulb of the pipet by means of a fine capillary. 4 It is essential that no reducing agent be present in the I1~ solution. One milligram of 0.02 N HsSO4 is added to bring the pH to about 5. A solution of Is containing 52 mg. of Is in 20 ml. of ethanol is prepared separately. Ten microliters of this solution is introduced into the reaction bulb and mixed with the I T M solution. I t is advisable to keep the mixture at a temperature lower than 10° and to use it as soon as possible. Preparation of 3,5,3t-Triiodothyronine Solution. This solution is prepared by dissolving 3.0 mg. of 3,5,3'-triiodothyronine with 1.8 ml. of concentrated NH,OH. Iodination of 3,5,3'-Triiodothyronine. The iodination reaction is carried out in the bulb of the blood-diluting pipet. It is possible, after calibration, to use the graduated capillary portion of such a pipet to measure accurately the volumes of the solutions of Is TM and triiodothyronine which are used. These solutions are drawn into the bulb by suction, applied by means of a syringe attached to the other extremity of the pipet. Nine microliters of the triiodothyronine solution is drawn into the bulb through the capillary, and the capillary is then rinsed with distilled water to remove any ammonia which may be present. The rinse water should not be allowed to enter the bulb of the pipet. Nine microliters of the Is T M solution is then drawn into the bulb and mixed thoroughly with the triiodothyronine. The reaction mixture is allowed to stand for about 15 minutes. Separation of the Reaction Products. The products of the synthesis are transferred directly with the blood-diluting pipet to a sheet of Whatman No. 1 filter paper, 55 cm. in length, 30 cm. in width. These dimensions are chosen because both electrophoresis and chromatography are carried out subsequently on the same sheet. The solution is deposited on the If thyroxine of very high specific activity is desired, t h e n 150 mg. of 1 TM containing 10 mc. m a y be introduced into the bulb of the pipet. I n this case, however, the pipet should be placed in a v a c u u m desiccator over P20~ to reduce the volume of the 1181 solution to 15 to 20 ul.

[35]

Ilal-LABELED COMPOUNDS

859

paper along a line 10 cm. long in a narrow band. The line of deposition is situated 15 cm. below the center of the sheet, and midway between its edges (parallel to the 30-cm. dimension). Inorganic iodine is first separated by electrophoresis in 0.2 M ammonium carbonate buffer. The paper is soaked with the buffer, first on one side of the line, then on the other, avoiding possible diffusion of the reaction products in either direction. Electrophoresis is carried out for 150 minutes in the presence of 0.2 M (NH4)~C03 buffer (pH 9.5), using 120 volts and 10 ma. Under these conditions, only the iodide migrates appreciably. After electrophoresis, the paper is dried and the position of the iodide1131 located by radioautography. The section of the filter paper containing the iodide-I TM is cut off, and the remaining part of the sheet is employed for ascending chromatography with tert-amyl alcohol saturated with 2 N NH4OH. A microdrop containing a mixture of 3,5,3'-triiodothyronine and thyroxine is delivered alongside the radioactive band to serve as a reference standard. The Ii31-thyroxine is located by radioautography, and the corresponding zone of the chromatogram is cut out and eluted between two microscope slides by n-butanol saturated with water. Two to three milliliters is generally adequate to obtain a good elution. The n-butanol is eliminated by concentration in vacuo (temperature ~ 30°), and the residue is taken up in 50 ~l. of 0.1 N NaOH, followed by 0.2 ml. of water. The yield of thyroxine, based on the initial quantity of triiodothyronine, is about 50%. The I13~-thyroxine may best be preserved by allowing it to remain on the dried paper chromatogram, eluting each day only a portion of the radioactive compound as required. The microsynthetic procedure outlined above may also be employed for the synthesis of other I~3~-labeled iodothyronines, e.g., 3',3,5-triiodothyronine, 3,3'-diiodothyronine, and 3',5',3-triiodothyronine. For this purpose it is only necessary to change the nature of the starting iodothyronine compound, and to add the appropriate amount of I~ TM. The I~31-thyroxine prepared by methods a, b, and c above may be assumed to be labeled almost entirely in the 3',5' positions. The probability of multilabeling (two or more I TM atoms in the same thyroxine molecule) is extremely small. This is evident from the fact that it requires only one I TM atom for every eight hundred I ~27 atoms to produce labeled thyroxine with a specific activity of 100 uc./% d. I131-Thyroxine Labeled in 3,5 Positions Only--Method of Michel et al. 5 In this procedure, 3,5-diamino-4-(4~-methoxyphenoxy)-N-acetyl L-phenylalanine ethyl ester is used as starting material. This is converted by diazotization and subsequent treatment with a radioactive iodine soluR. Michel, J. Roche, and J. Tata, Bull. soc. chim. biol. 34, 466 (1952).

860


[35]

tion into Ila~-labeled 3,5-diiodo-4-(4'-methoxyphenoxy)-N-acetyl-L-phenylalanine ethyl ester (DIM). By treatment with concentrated HI, the DIM is converted into I131-1abeled 3,5-diiodo-L-thyronine. The latter is then iodinated with nonradioactive iodine to give L-thyroxine labeled only in the 3,5 positions, or with radioactive iodine to give thyroxine labeled in all four positions.

e. I131-Labeled Thyroxine Prepared by Exchange--Method of Taurog et al. 6 The possibility of preparing I131-1abeled thyroxine by exchange was first reported by Frieden et al. 7 These workers studied exchange between iodide-I TM and thyroxine in a butanol-water mixture (9:1) at pH 5. Exchange under these conditions is relatively slow, and usually only 10 to 15% of the I TM exchanges with the thyroxine during 12 hours of refluxing. We found that both the rate of exchange and the specific activity of the labeled thyroxine can be greatly increased by substituting elemental iodine (I2 ~8~) for iodide-I ~8~in the exchange reaction. The details of this procedure are described below. Preparation of Nonradioactive Thyroxine Solution. Approximately 1.5 mg. of L-thyroxine (Na salt pentahydrate, Glaxo) is dissolved in a 15-ml. centrifuge tube with 200 ~l. of 0.02 N NaOH. To this solution are added 0.5 ml. of water, 0.5 ml. of distilled alcohol, and 100 ~1. of 0.2 N acetate buffer, pH 4. I2 ~3' is then distilled into this solution as described in the following step. Distillation of I2 m. One milliliter of 1181solution containing 3 to 5 mc. of I m (or more, if desired) is placed in a small, round-bottomed flask (approximately 5-ml. capacity, 10/30 standard taper ]oint). To this are added 25 ~l. of iodide carrier (12.5 7), 50 #1. of 18 N H2S04, and 25 #l. of 30% H~O~ (Superoxol). The flask is connected to a small condenser (over-all length, about 15 cm.), and heated with a small Glas-Col mantle. A Carborundum chip facilitates smooth boiling. The receiver (containing the thyroxine solution) is set in position with the tip of the condenser dipping slightly below the level of the solution to avoid loss of radioactive Is. Complete distillation of the radioactivity requires about 5 minutes after the mixture has begun to boil. The entire distillation should be carried out in a hood behind a lead shield. Exchange Procedure. The receiver is corked and placed in a hot water bath at 60 ° for 1 hour. The thyroxine may be partially out of solution during this procedure because the water content of the solution is increased during the distillation. It is advisable, therefore, to shake the tube occasionally during the incubation. 6 A. Taurog, Brookhaven Symposia in Biol. No. 7, The Thyroid, 111 (1955). 7 E. Frieden, N. B. Lipsett, and R. J. Winzler, Science 107, 353 (1948).

[~]


861

Isolation of I131-Labeled Thyroxine. After the exchange reaction has been allowed to proceed for 1 hour, water is added to the 5- to 6-ml. mark to complete the precipitation of the thyroxine. The radioactive product is separated by centrifugation, washed twice with 0.001 N thiosulfate, then twice with water. About 50 to 75% of the starting thyroxine is recovered, and this contains almost 50% of the added I1% The specific activity of the product is 2 to 3 ~c./~ when 5 mc. of I TM is used in the exchange reaction. For animal experimentation, the radioactive thyroxine crystals are dissolved in 0.5 ml. of 0.02 N NaOH, and any insoluble residue is separated by centrifugation. This solution is then drawn off and diluted to the desired volume with isotonic sodium bicarbonate. Analysis by filter paper chromatography reveals that about 90 % of the 1131is in the thyroxine band. Most of the remainder of the I TM is present as I13Utriiodothy ronine, the latter arising most probably from triiodothyronine contained in the starting thyroxine. An exchange procedure for preparing I]~l-thyroxine is also described by Gleason. 8 In this method the exchange between I21~1 and thyroxine is allowed to take place at pH 7 to 8 in tert-butyl alcohol solution at room temperature. The distribution of the I ~3~in labeled thyroxine prepared by exchange has not been determined. It might be expected that the iodine atoms ortho to the hydroxyl group (3',5' positions) would be more labile, and hence would exchange more readily with I2 than would the iodine atoms in the 3,5 positions. Metabolic evidence supporting this view was presented by Roche and his co-workers, 9 who found that iodine in the 3',5' positions of thyroxine is excreted more readily into the urine than is iodine in the 3,5 positions. Further evidence was reported by Gleason, 8 who found that the rate of exchange between I21~1and 3,5-diiodothyronine is extremely slow. It is reasonable to assume, therefore, that I13~-labeled thyroxine prepared by exchange is labeled primarily in the 3',5' positions. The final answer to this problem, however, must await the development of suitable techniques which can distinguish between the iodine atoms in the 3,5 positions and those in the 3',5' positions of the thyroxine molecule. f. Biosynthesized Ira-Labeled Thyroxine--Method of Gross and Leblond. 1° Rats that have been fed an iodine-deficient diet are injected with approximately 50 pc. of I ~31. After 48 hours they are exsanguinated, and the heparinized plasma is first extracted with 2 vol. of n-butanol and 8 G. I. Gleason, J. Biol. Chem. 21a, 837 (1955). 9 j. Roche, R. Michel, and J. Tata, Compt. rend. soc. biol. 146, 1003 (1952). ~0j. Gross and C. P. Leblond, J. Biol, Chem. 184, 489 (1950).

862


[35]

then three more times with an equal volume of butanol. The combined butanol fractions are evaporated, i n vacuo, at room temperature, and the dry residue is taken up in distilled water. Because of the possibility of decomposition of thyroxine during the concentration of the butanol extract, the final solution should be tested by some procedure (isotope dilution, filter paper chromatography) for the presence of nonthyroxine I TM. The I~31-thyroxine prepared by this procedure is presumably evenly labeled and is of very high specific activity, but the purity of the product is difficult to control.

2. I131-Labeled 3,5,3'-Triiodothyronine a. Controlled Iodination of 3,5-Diiodothyronine. Roche and his co-workers 11 studied the iodination of diiodothyronine with radioactive iodine. They varied the molal ratio of iodine to diiodothyronine from 0.25 to 10 and found that the optimum formation of triiodothyronine occurred when 3.5 atoms of iodine per molecule of diiodothyronine was added. The following method is given by the French workers for preparing crystalline radioactive triiodothyronine: 100.5 mg. of 3,5-diiodothyronine is dissolved in approximately 20 ml. of concentrated Ntt4OH. To this is added an ether solution of radioactive iodine, prepared by the method of Clayton et al. ~ (see above), containing 3.5 atoms of iodine per molecule of diiodothyronine. The reaction products are separated by large-scale filter paper chromatography on a front of 50 cm., with isopentanol saturated with 6 N NH~OH as the solvent. The triiodothyronine bands (RI = 0.27) are cut out and eluted with 2 N NH4OH. After removal of the ammonia, in vacuo, the product is precipitated by the addition of acetic acid washed several times with water, and finally dried over P205. It is then dissolved in alcohol containing 5 % HC1, and an equal volume of water is added. After the solution has been allowed to stand at 1° for 24 hours, crystals are deposited whose elementary composition agrees with that of triiodothyronine. It may be assumed that triiodothyronine prepared by this procedure is labeled only in the 3' position. For the preparation of 1131-labeled 3,5,3'-triiodothyronine of very high specific activity, the microsynthetic procedure of Roche and Michel 3 may be used. The procedure is exactly the same as that described above for the preparation of I.l~Uthyroxine, except that 3,5-diiodothyronine (instead of 3,5,3'-triiodothyronine) is used as the starting material. The diiodothyronine solution should contain 2.4 mg. in 1.8 ml. of concentrated NH4OH. Wilkinson and Macl~gan TM report a procedure for preparing crystalline 11 j . Roche, S. Lissitzky, and R. Michel, Biochim. et Biophys. Acta 11, 215 (1953). a2 j. H. Wilkinson and N. F. Maclagan, Biochem. J. 58, 87 (1954).

[3~]


863

I131-triiodothyronine which does not involve chromatographic separation of the products. The details are as follows: A solution of KI (9.6 rag., 0.058 meq.) and NaI TM (5 mc.) in water (2 ml.) is acidified with 2 N HC1 (0.15 ml.) and covered with ether (5 ml.). The mixture is treated with 0.1 N H202 (1.3 ml., 0.13 meq.), added over 20 minutes, and shaken at intervals for 4 hours. The ether layer is then transferred slowly to a solution of 3,5-diiodo-L-thyronine (15.3 mg., 0.029 meq.) in 50% aqueous ethylamine (0.5 ml.). The iodination requires about 2 hours, toward the end of which water (2 ml.) is also added. The ether is removed in a current of air, and the mixture evaporated to approximately 0.5 ml. at 30°/1 mm. to remove excess ethylamine. The residue is transferred to a centrifuge tube, about 5 ml. of water being added, and the solution heated to 60 °. The pH is adjusted to 4 to 5 with acetic acid to precipitate the product. After several hours at 0 to 5 °, the solid is collected and washed three times with water (10 ml.). The crude product is dissolved in 2 N HC1 (5 ml.) at 70 to 80 °, the solution is filtered, and the filtrate, while still hot, is adjusted to pH 4 to 5 with NaOH and acetic acid. It is then stored at 0 to 5 ° for 2 hours, after which the crystalline deposit is separated in a centrifuge and washed three times with water (10 ml.). The product is again dissolved in 2 N HC1, and the crystallization procedure is repeated. The 3,5,3'-triiodo-L-thyronine is obtained in 80% yield and is free from thyroxine and iodide. According to these workers, solutions of I131-triiodothyronine (in 50% v / v aqueous propylene glycol adjusted to pH 8 to 9 with diethanolamine) are more stable than those of labeled thyroxine and can be stored for several weeks with no release of iodide. b. Exchange Method. I131-Labeled triiodo-L-thyronine has been prepared in the authors' laboratory (by Dr. Winton Tong) by the same exchange procedure described above for preparing It31-thyroxine. The two methods are similar in all respects except for the final method of purification. In the case of triiodothyronine, the washed precipitate obtained after the exchange reaction contains an appreciable amount of I13Ulabeled thyroxine, and for this reason the final purification is done chromatographically. The washed precipitate (1 to 2 mg.) is dissolved in 100 to 150 ~1. of 0.02 N NaOH and transferred to several filter paper strips (40 to 60 ul. on each). These are developed in collidine-water-NH3 or in butanol-ethanol-2 N NH4OH (see below), and the triiodothyronine bands are eluted with small volumes of 0.04 N NaOH. The eluates are combined, and the eluted product is reprecipitated with acetic acid, washed several times with water, and redissolved in dilute alkali. This solution, which is now contaminated with only a small percentage of I13~-thyroxine, contains approximately 50% of the added I~% The distribution of the I TM is not known, but most likely the 3 t position of the

864


[35]

triiodothyronine is more heavily labeled than are the 3,5 positions (see above for thyroxine).

3. II~-Labeled 3,5-Diiodo-L-Tyrosine and 3-Monoiodo-L-Tyrosine a. Exchange Method for Preparing Ii31-Labeled Diiodo-L-Tyrosine-Method of Tong et al. 18 The exchange reaction between diiodotyrosine and iodine was first studied by Miller et al. ~4 They found that diiodotyrosine exchanges very readily with I2 at pH 4 to 5, but slowly with iodide under the same conditions. In our laboratory, too, it has been found that diiodotyrosine exchanges readily with radioactive iodine at pH 4 to 5, and this is the basis for the method described here. The radioactive I~ is prepared and distilled exactly as described above for the preparation of I~31-thyroxine (by exchange). The receiving vessel in this case contains 0.25 to 1.0 mg. of diiodo-L-tyrosine in 1 ml. of solu-. tion containing 100 ~l. of 0.02 h r NaOH and 100 ~1. of 0.2 N acetate buffer, pH 4.0. The exchange mixture is kept at 60 ° for 1 hour, and at the end of that time 65 to 70% of the I TM has exchanged with the diiodotyrosine. Separation of the diiodotyrosine from the unreacted I ~3~ is accomplished by the use of Duolite A-4 anion exchange resin. One to two grams of resin in a 15-ml. centrifuge tube is first stirred with a solution of nonradioactive diiodotyrosine (40 mg. in 20 ml.) to saturate the resin with the iodinated amino acid. This treatment prevents appreciable loss of radioactive diiodotyrosine on the resin. The resin mixture is centrifuged, and the excess solution is drawn off. The exchange mixture, made strongly acid with 2 drops of 6 N HC1, is then added to the resin in the centrifuge tube. After the mixture has been stirred for a few minutes at room temperature, it is centrifuged once again. The supernatant is treated once more with a batch of resin prepared as above. The supernatant from the second treatment, which contains 113~ almost entirely in the form of diiodotyrosine, is neutralized with NaOH and diluted to the desired volume with NaHCO3. When the starting quantities consist of 3 mc. of I ~3~ and 1 rag. of diiodotyrosine, the specific activity of the final diiodotyrosine is approximately 1 ~c./~,. b. Iodination of Tyrosine with Radioactive Iodine--Method of Roche et al. 15 The radioactive, iodinating reagent is prepared by the method of Clayton et al.~ (see above), and the tyrosine is iodinated in concentrated NH~OH solution. The relative yields of mono- and diiodotyrosine can be 1, W. Tong, A. Taurog, and I. L. Chaikoff, J. Biol. Chem. 207, 59 (1954). u W. H. Miller, G. W. Anderson, R. K. Madison, and D. J. Salley, Science 100, 340 (1944). 15j. Roche, S. Lissitzky, O. Michel, and R. Michel, Biochim. et Biophys. Acta 7, 439 (1951).

[$5]

II3I-LABELED COMPOUNDS

865

varied according to the amount of iodine added to the tyrosine. When diiodotyrosine is the major product desired, 8 atoms of iodine per molecule of tyrosine is added. The iodinated amino acids are separated by unidimensional filter paper chromatography, using butanol-acetic acidwater (78:5:17). The compounds are located by radioautography and eluted from the paper with NH4OH. c. Iodination of Tyrosine with Radioactive Iodine--Method of Lemmon et al. 1~ This method was designed primarily for small-scale production of labeled diiodotyrosine of high specific activity. The radioactive iodinating reagent is prepared by the method of Horeau and Siie, 17 and the isolation of the desired product is accomplished by filter paper chromatography. 4. I131-Labeled Thyroglobulin Method of Roche et al. 18,~9 Roche and his co-workers prepared 1 l~lthyroglobulin from the thyroids of normal human beings, hyperthyroid human beings, ox, swine, and dog. Both i n vivo and i n vitro techniques were used to label the thyroglobulin. The method of isolation of labeled thyroglobulin varies somewhat, depending on the source, but in general the procedure is as follows: The thyroid tissue containing the labeled thyroglobulin is frozen and sliced, and the slices are immersed in isotonic saline at 0 ° for 24 hours, preferably with shaking. The mixture is filtered through gauze and centrifuged, and the extract (containing practically all the I TM) is treated with (NH4)2SO4 to 38% saturation at pH 6.5. The protein fraction which precipitates under these conditions is discarded. The (NH4)2SO, concentration is increased to 50% saturation, and the precipitate which then appears is collected by centrifugation, redissolved in water, and dialyzed. Studies carried out on such preparations showed that the precipitation of I TM with increasing concentrations of (NH4)2SO, followed closely the precipitation of total nitrogen. 5. I~31-Labeled Iodocasein Method of Courtier et al. 2° Eight milliliters of I TM solution (15 me.) containing 75 rag. of KI is treated dropwise with 4.5 ml. of concentrated ~6R. M. Lemmon, W. Tarpey, and K. G. Scott, J. Am. Chem. Soc. 72, 758 (1950). ~7A. Horeau and P. Stie, Bull. soc. chim. biol. 27, 483 (1945). 18j. Roche, O. Michel, G. H. Deltour, and R. Michel, Ann. endocrinol. (Paris) 13, 1 (1952). ~9j. Roche, R. Michel, O. Michel, G. H. Deltour, and S. Lissitzky, Biochim. el Biophys. Acta 6, 572 (1951). ~" R. Courrier, J. Roche, G. H. Deltour, M. Marois, R. Michel, and F. Morel, Bull. soc. chim. biol. 31, 1029 (1949).

866


[36]

HNO~. The precipitate of free Is (55 mg,), containing all the I ~81, is collected by centrifugation and washed with water to remove excess HN03. The radioactive iodine is powdered and added in small portions, with stirring, over a period of 2 hours, to a bicarbonate solution of purified casein at pH 7.4 and 37 °. Six atoms of iodine is added per molecule of tyrosine. Contrary to the findings of Reinecke, 2~ these workers report that thyroxine formation is not increased by elevating the temperature to 70 ° for 18 hours, after the iodine has been added. The product obtained after dialysis and isoelectric precipitation contains 9.5 % total I and 1.2 % thyroxine I. It has a specific activity of approximately 30 ~c./mg.

B. Methods for the Separation of D31-Labeled Amino Acids and Proteins For many years the standard procedure for separating the various iodine compounds of the thyroid gland was the butanol extraction procedure of Leland and Foster 22 or the modified butanol extraction procedure of Blau.33 These separation procedures were usually preceded by hydrolysis with 2 N NaOH or with 8 % Ba(OH)2.8H20--methods which were known to cause some decomposition of the iodinated amino acids. With the development of chromatographic techniques, improved methods for separating the hydrolytic products of the thyroid gland became available. Chromatographic methods are, in particular, suitable for the study of D3~-labeled compounds, because these labeled compounds are so easily located by their radioactivity. Enzymatic hydrolysis is also largely replacing the more drastic alkaline hydrolytic procedures. 1. Filter Paper Chromatography a. Sources of Error in the Hydrolysis and Chromatographic Separation of I~31-Labeled Compounds. Iodine compounds are relatively labile, and iodine itself is extremely reactive. It is important, therefore, in analyzing mixtures of Ii~l-labeled compounds to guard against the introduction of artifacts. The following possible sources of error should be considered: (1) Hydrolysis: There is good evidence that strong alkaline hydrolysis may introduce serious errors into the analysis of mixtures of I~31-compounds. 24,25 Enzymatic hydrolysis of thyroglobulin, although not usually so complete as alkaline hydrolysis, minimizes the breakdown 21E. P. Reinecke, Vitamins and Hormones 4, 207 (1946). ~ J. P. Leland and G. L. Foster, J. Biol. Chem. 95, 165 (1932). 23 N. F. Blau, J. Biol. Chem. 102, 269 (1933); 110, 351 (1935). ~4 p. G. Stanley, Nature 171, 933 (1953). ~5 j . Roche, R. Michel, and E. Volpert, Compt rend. soc. biol. 148, 21 (1954).

868


[35]

already have been formed. Sodium thiosulfate has been used for the same purpose by Dobyns and Barry. 3° b. Chromatographic Procedure of Tautog et al. 31 Twenty microliters of the solution or mixture containing I13Ulabeled compounds is delivered as a narrow band along a line 5 to 6 cm. long near one end of a sheet of Whatman No. 1 filter paper (10 X 36 cm.). Repeated deliveries may be made, if necessary, the quantity which can be successfully chromatographed being limited by the concentration of total material (protein, salt, fat, etc.). Several papers (up to six) are suspended, in a cylindrical Pyrex jar (18 inches tall, 8 ~ inches in diameter), and the chromatograms are developed for 15 to 16 hours by the ascending method at 23 to 25 ° . The solvent generally consists of a mixture of 100 ml. of collidine (redistilled, Koppers Co.) and 35 ml. of water, an ammonia atmosphere being provided by a small beaker of concentrated NH40H resting in the solvent. For many purposes a solvent consisting of butanol-ethanol-2 N Ntt4OH (5: 1:2) is employed (see below) in addition to, or instead of, the collidine solvent. We have employed two different systems for suspending the filter paper strips in the chromatography jars. The first procedure was to suspend the papers by means of paper clips from a stainless steel rack attached to the glass plate which forms the cover of the jar. More recently, a stainless steel rack has been built into the jar with the aid of a curved stainless steel band, tightened against the wall of the jar, close to its top, by means of a special screw arrangement. A flexible Teflon gasket between the steel band and wall protects the glass as the screw is tightened. Two notched cross pieces of stainless steel are set in place, supported at each end by metal supports silver-soldered to the steel band. Resting across the notches are six parallel stainless steel rods, each carrying two stainless steel spring photographic clips. The chromatograms are suspended by means of the spring clips, the lower end of the filter paper dipping 1 or 2 mm. below the surface of the solvent. After the chromatography is completed, the papers are dried in a hood at room temperature and placed in an exposure holder in contact with a sheet of X-ray film. The various components are located by the darkening of the film, which occurs only in areas where radioactivity is concentrated. The exposure time depends on the amount of radioactivity in the 1131labeled compounds. As little as 2 × 10-~ ~c. of I TM may be detected in a single component, but this requires an exposure of 2 to 3 weeks. The identity of a given band on the radioautograph is determined by reference to the positions taken by known carrier compounds which are added just before chromatography to the mixture of I~al-compounds a0 B. M. Dobyns and S. R. Barry, J. Biol. Chem. 204~ 517 (1953). 31 A. Taurog, W. Tong, and I. L. Chaikoff, J. Biol. Chem. 184, 83 (1950).

[3~]

II31-LABELED COMPOUNDS

869

being analyzed. T h e position of the added m a r k e r s m a y be determined b y spraying the filter p a p e r c h r o m a t o g r a m with diazotized sulfanilic acid2 ~ If carrier is not added, there is usually not enough iodinated amino acid on the c h r o m a t o g r a m , even f r o m a thyroid hydrolyzate, to give a color test with this reagent. For positive identification of a given radioactive component, its zone of darkening on the r a d i o a u t o g r a p h should coincide exactly, in location and in shape, with the colored area on the c h r o m a t o g r a m given b y an authentic sample of the carrier compound. Carrier compounds are not added routinely in our procedure, b u t only when there is some d o u b t a b o u t the identity of a given band on the radioautograph, or when analyses are carried out on tissues or mixtures with which we h a v e had only limited experience. The quantities of carrier compounds required to give a satisfactory color with the diazotized sulfanilic acid reagent are a p p r o x i m a t e l y 50 ~ for mono- and diiodotyrosine, and a b o u t 100 -~ for thyroxine and triiodothyronine. A m u c h more sensitive color test for the detection of the iodinated amino acids on p a p e r c h r o m a t o g r a m s is the ceric sulfate-arsenious acid test of Bowden et al. 3~ Lissitzky has suggested a means of using this test q u a n t i t a t i v e l y for the m i c r o d e t e r m i n a t i o n of the various iodinated amino acids in thyroglobulin24 The R I values for the various iodinated amino acids m a y v a r y considerably, depending on the nature of the solution which is delivered for c h r o m a t o g r a p h y . T h e values shown below are intended only to illustrate the general p a t t e r n of distribution on the c h r o m a t o g r a m .

Solvent Collidine-waterNH~ Butanol-ethanol2 N NH4OH

3',3,5MonoiodoTtriiodotyrosine Thyroxine Thyronine Iodide

Thyroglobulin

Diiodotyrosine

0

0.22

0.34

0.56

0.66

078

0

0.10

0.12

0.42

0.56

0.38

as This reagent is prepared as follows: 0.52 g. of recrystallized sulfanilic acid is dissolved with warming in 6 ml. 0f 2.5% Na2CO,. The solution is placed in an ice bath, and 0.19 g. of NaNO2 is added, foUowed by 7 ml. of 2 N HC1. On continued stirring a crystalline precipitate of the diazonium salt soon appears, and this is separated by filtration, washed with water at 0 °, and dissolved in 40 ml. of water at room temperature. For locating mono- and diiodotyrosine, the chromatogram is first sprayed with 2.5% Na2COa, then with the diazo reagent. For locating thyroxine, diiodothyronine, and 3',3,5-triiodothyronine, more stable colors are obtained if the chromatogram is sprayed with a single reagent prepared by diluting the diazonium salt solution 1:10 with 2.5% Na~CO,. 33 C. H. Bowden, N. F. Maclagan, and J. H. Wilkinson, Biachem. J. 59, 93 (1955), 3~S. Lissitzky, Bull. soc. chim. biol. 87, 89 (1955).

870


[35]

The butanol-ethanol-2 N NH~OH solvent is especially valuable for the separation of thyroxine and 3',3,5-triiodothyronine. Its usefulness is limited, however, since it does not separate mono- and diiodotyrosine, and since it gives only a poor separation of thyroxine and iodide. Satisfactory chromatograms can be obtained in the above solvents with whole-tissue homogenates, 29 provided not more than 20 ~1. of solution is delivered on the paper. Whole plasma or serum can also be chromatographed satisfactorily. 8~ Chromatogra]:hy of Plasma and Plasma Extracts. Filter paper chromatography is well suited for the identification of I TM compounds in the plasma of animals or patients that have received radioactive iodine. If the concentration of 1131 in the plasma is sufficiently high, then whole plasma may be chromatographed without further treatment. Best results are obtained if one or two deliveries of 20 ~l. each are made on the filter paper. If the concentration of I TM in the plasma is low, it is usually necessary to prepare a concentrated butanol extract of the plasma for chromatography. The procedure used in our laboratory is as follows: 1.5 ml. of plasma (containing 0.001 M thiouracil) is shaken vigorously with 3 ml. of distilled butyl alcohol in a tightly corked 15-ml. centrifuge tube. After centrifugation, the butanol layer is drawn off with suction into a second tube, and the aqueous layer is extracted again with 3 ml. of butanol. After centrifugation, the butanol layer is combined with the first butanol extract, and the aqueous residue is extracted a third time, with 2 ml. of butanol. The material is centrifuged again, and the third butanol extract combined with the other two. The protein residue is suspended in 0.2 ml. of water and extracted a fourth time, with 2 ml. of butanol. The combined butanol extracts, containing 80 to 90% of the total I TM, a r e transferred to a 10-ml. beaker, and approximately 0.3 rag. of solid thiouracil is added to minimize changes in I TM compounds which may occur during the concentration procedure. The butanol extract is concentrated just to dryness at 40 ° under reduced pressure in a stream of nitrogen. The residue is suspended in 300 ~l. of 1:1 ethanol-2 N NH4OH, and three or four deliveries of 20 ~l. each are made on filter paper for chromatography in collidine-water-NHa and in butanol-ethanol-2 N NH4OH. Chromatography of Thyroid Homogenates. Thyroid homogenates containing labeled I18~-compounds may be chromatographed either before or after hydrolysis. Before hydrolysis, practically all the I131is distributed between thyroglobulin and inorganic iodide, very little free, iodinated amino acids being present. 8e After hydrolysis, the 1 lal is distributed primarily among inorganic iodide, incompletely hydrolyzed thyroglobu3~A. Taurog, J. D. Wheat, and I. L. Chaikoff,Endocrinology 58, 121 (1956). **W. Tong, A. Taurog, and I. L. Chaikoff,J. Biol. Chem. 191, 665 (1951).

872


[35]

For enzymatic digestion, 0.5 ml. of the thyroid homogenate is added to 10 rag. of pancreatin (Merck), 1 drop of toluene is added, and the mixture is incubated at 37 ° for 24 hours. Under these conditions 85 to 90% of the I TM is released from peptide linkage. This percentage may sometimes be increased slightly by further digestion at 37 ° after the addition of more pancreatin (5 mg.), or of papain ~s (pH first adjusted to 5 to 6). Twenty microliters of the digest is delivered on filter paper for chromatography in collidine-water-NH3. A typical radioautograph obtained with a pancreatin digest of rat thyroids is shown in Fig. 1. ~61 Procedures for digesting I~31-thyroglobulin with trypsin prior to chromatographic separation of I131-amino acids are described by Roche et al. 37 and by Braasch et al. 8s c. Chromatographic Procedure of Gross and L e b l o n d 2 9,4° These workers employed a two-dimensional procedure for chromatographing Ilal-labeled compounds. The first solvent consists of the upper layer formed after shaking n-butanol with 2 N formic acid in a separatory funnel. (Gross and Pitt-Rivers later used n-butanol and 2 N acetic acid. 4~) For the second dimension, the upper layer of a mixture of 4 parts n-butanol and 1 part dioxane, shaken with 5 parts 2 N NH4OH, is used. This solvent is particularly useful for separating thyroxine and triiodothyronine. The chromatograms are developed by the ascending method of Williams and Kirby. 42 d. Chromatographic Procedure of R o c h e and Michel. 43,~4 These investigators have employed a variety of solvent systems for the separation of iodine compounds by filter paper chromatography. 43,44 Many of these solvents were introduced especially for the separation of the various iodothyronines. The use of newer solvents has led the French workers to report the presence in normal rat thyroids of both 3,3'-diiodothyronine and 3,3',5'-triiodothyronine (in addition to thyroxine and 3,5,3'-triiodothyronine). 44 3,3'-Diiodothyronine, which is reported to be 70 to 80% as active as thyroxine by the goiter prevention test in rats, has also been detected in the plasma of normal rats. 44 Listed below are nine different solvent systems employed by Roche 3s, Dr. W. Tong, in our laboratory, has recently found that the enzymatic release of thyroxine from thyroglobulin is improved by the addition of 0.005M Mn++. 3~j. Roche, R. Michel, S. Lissitzky, and Y. Yagi, Bull. soc. chim. biol. 36, 143 (1954). 8sj. W. Braasch, E. V. Flock, and A. Albert, Endocrinology 55, 768 (1954). 89j. Gross, C. P. Leblond, A. E. Franklin, and J. H. Quastel, Science 111, 605 (1950). 40j. Gross and C. P. Leblond, Endocrinology 48, 714 (1951). ~1j. Gross and R. Pitt-Rivers, Lancet 262, 439 (1952). ~sR. J. Williamsand H. Kirby, Science 107, 481 (1948). 43j. Roche, R. Michel, and J. Nunez, Bull. soc. chim. biol. 37, 809 (1955). ~4j. Roche, R. Michel, W. Wolf, and J. Nunez, Biochim. et Biophys. Acta 19, 308 (1956).

[35]

Ilal-LABELED COMPOUNDS

873

and Michel, together with information on the temperature and direction of the filter paper chromatography as used in their laboratory. Also shown is a table of their reported Rs values for various iodine compounds in each solvent. 1. 2. 3. 4. 5. 6. 7. 8. 9.

n-Butanol saturated with 3 N NH4OH (18 °, descending). n-Butanol-acetic acid-water (78:5 : 15) (18 °, descending). Aqueous collidine, NH3-saturated atmosphere (18 °, ascending). Isopentanol saturated with 2 N N H 4 0 H (12 °, descending). n-Butanol-dioxane (4:1) saturated with 2 N NH4OH (16 °, descending). Methanol-0.2 M ammonium acetate (1:2.5, pH 6.1) (3 °, ascending). 95% ethanol-0.2 M ammonium carbonate-0.2 M ammonium acetate (2: 1:1, pH 7.4) (16 to 17°, ascending). 95% ethanol-0.2 M ammonium carbonate (2: 1, pH 7.8) (16 to 17°, ascending). Tertiary pentanol saturated with 2 N NH4OH (18 °, ascending). R] values Solvent Substance

1

3-Monoiodotyrosine 3,5-Diiodotyrosine 3-Monoiodothyronine 3,5-Diiodoth~ronine 3,3'-Diiodothyronine 3,3~,5'-Triiodothyronine 3,5,3'-Triiodothyronine Thyroxine Iodide

0.13 0.08 0.60 0.66 0.51 0.43 0.64 0.48 0.31

2 0.43 0.59 0.70 0.65 0.70 0.68 0.70 0.75 0.20

3 0.32 0.35 0.65 0.68 0.58 0.63 0.70 0.59 0.85

4

0.70 0.75 0.50 0.40 0.60 0.50 0.20

5

6

7

8

9

0.51 0.34

0.45 0.30

0.01 0.01

0.64 0.64 0.48 0.40 0.60 0.46 0.38

0.45 0.35 0.00 0.00 0.23 0.00 0.00 0.00 0.68

0.70 0.63 0.71 0.60 0.70

0.70 0.60 0.74 0.60 0.71

0.50 0.35 0.20 0.40 0.25 0.18

2. Column Chromatography Column chromatography, although generally more tedious and timeconsuming than filter paper chromatography, allows much larger amounts of material to be used for analysis. This is of great advantage when the investigation requires chemical analyses of the various fractions or their isolation in pure form. a. Method of Dobyns and Barry2 ° A mixture of 14.3 g. of pure potato starch, 25.3 ml. of n-butanol, and 2.5 ml. of water is shaken and poured into a glass tube approximately 1 cm. in diameter. The starch settles on a sintered-glass disk, and air pressure of 7 cm. of mercury is applied. After approximately 1 hour, the starch is packed to a constant

874


[35]

height of 21 cm. The excess liquid is removed, the solvent reservoir is attached, and the same pressure is again applied for 36 hours to permit equilibration of the starch and solvent. The column is then freed of metallic ion impurities by passing 8-hydroxyquinoline (25 mg. in 2.5 ml. of solvent) through the starch. The sample to be analyzed is dissolved in a mixture of n-butanol-propanol-0.05 N Na2CO~ (1:2:1), and before it is applied to the column, a crystal of sodium thiosulfate is added to minimize exchange. A reservoir of this solvent is then applied with a pressure of 15 cm. of mercury and a flow rate of approximately 2 ml./hr. Fractions consisting of 0.5 ml. are collected in sequence in 300 tubes. The alkaline solvent elutes all the known iodine compounds, except diiodotyrosine, in convenient and well-separated fractions. For satisfactory elution of diiodotyrosine, the alkaline solvent is replaced, at about fraction 120, with an acid solvent composed of n-butanol-propanol-0.1 N HC1 (1:2:1). A thyroid hydrolyzate for use with the above procedure is prepared as follows: The tissue is heated at 100 ° in a sealed tube with 2 N NaOH for 17 hours in the presence of sodium thiosulfate. The 1181 compounds are extracted at pH 3 to 4 with butanol, and the butanol extract concentrated to dryness under reduced pressure at room temperature. The dried residue is extracted with 4 portions of ethyl alcohol, and the ethanol extract is similarly dried. The dried residue is then dissolved in the n-butanol-propanol-0.5 N Na2C03 solvent (with added thiosulfate) and applied to the column. The eluates from rabbit thyroid hydrolyzates, prepared as described above, contained thyroxine (fractions 5 to 10), iodide (fractions 40 to 60), monoiodotyrosine (fractions 90 to 130), diiodotyrosine (fractions 200 to 240), and two unknown 11~l-containing fractions. Triiodothyronine is not separated from thyroxine in this procedure. b. Method of Braasch et al. 3s This procedure is an extension of the kieselguhr column procedure of Gross and Pitt-Rivers. 45 The mixtures to be separated, dissolved in 0.5 N NaOH, in n-butanol saturated with 0.5 N NaOH, or in a butanol-ethanol-NaOH mixture, are applied to the kieselguhr column (Super-Cel Hyflo, Johns Manville). Elution is started by passing a 20% mixture of chloroform in n-butanol (v/v), saturated with 0.5 N NaOH, through the column. Thyroxine, triiodothyronine, and iodide are eluted in that order, and then the eluting solvent is changed to a mixture of n-butanol and n-propanol (9:1) saturated with 0.5 N NaOH. The latter solvent elutes diiodotyrosine followed by and well separated from monoiodotyrosine. Residual material on the column is eluted by water or propanol. ~5j . Gross and R. Pitt-Rivers, Biochem. J. 55j 645 (1953).

876


[36]

and actual electrophoresis are done in a cold room at 2 °. Carbon electrodes are introduced into separate vessels filled with buffer, the latter being connected to the main buffer vessels through slots filled with glass wool to prevent p H changes during the operation. Then 50 ~l. of serum is delivered on the paper through a small hole in the upper glass plate 9 cm. from the cathodal end. After 30 minutes, a potential of 300 volts direct current is placed across the electrodes, allowing approximately 1 ma. to pass through the paper strip. T w e n t y - f o u r hours is allowed for resolution at this current. The paper strip is then dried in an oven at 80 °, cut into 2-cm. segments, and counted. After the segments are counted, t h e y are subdivided into 0.5-cm. sections, and the protein bands are identified and quantitated b y the bromophenol blue elution method. More recently, Larson et a l . " have used starch electrophoresis to study the distribution of I TM in serum. The results are v e r y similar to those obtained with the above system. T h e I TM which was bound to the globulin fraction was extracted with butanol, rechromatographed on filter paper, and shown to consist entirely of thyroxine. T h e I ~3~ bound to albumin, on the other hand, was found to be inorganic. II. Preparation of Ira-Labeled Proteins Iodine m a y be introduced into certain protein molecules in small quantities without appreciably affecting the immunological or electrophoretic behavior of the protein. 57-~9 Advantage is taken of this fact in the labeling of protein molecules with I TM. The 1131 is bound primarily to the tyrosine residues of the protein e° and presumably is released only when the protein is degraded, el,e~ I~31-Labeled serum albumin is used for cardiovascular studies (blood volume, 68," cardiac output, 66 peripheral circulation"), for t u m o r localization, 67 and in the s t u d y of protein 56F. C. Larson, W. P. Deiss, and E. C. Albright, J. Clin. Invest. 33, 230 (1954). 57G. E. Francis, W. Mulligan, and A. Wormall, Nature 167, 748 (1951). 5~S. A. Berson, R. S. Yalow, S. S. Schreiber, and J. Post, J. Clin. Invest. 32, 746 (1953). 5gG. E. Francis, W. Mulligan, and A. Wormall, Biochem. J. 60, 363 (1955). 60 W. L. Hughes, Jr., and R. Straessle, J. Am. Chem. Soc. 72, 452 (1950). 61W. C. Knox and F. C. Endicott, J. Immunol. 65, 523 (1950). 6~L. R. Meleher and S. P. Masouredis, J. lmmunol. 67, 393 (1951). 68A. L. Schultz, J. F. Hammarsten, B. I. Heller, and R. V. Ebert, J. Clin. Invest. 32, 107 (1953). 64N. Freinkel, G. E. Schreiner, and J. W. Athens, J. Clin. Invest. 32, 138 (1953). s5 W. J. MacIntyre, J. P. Storaasli, I-I. Krieger, W. Pritchard, and H. L. Friedell, Radiology 59, 849 (1952)., " H. Krieger, J. P. Storaasli, W. J. MacIntyre, W. D. Holden, and H. L. Friedell, Ann. Surg. 186, 357 (1952). 67E. T. Yuhl, L. A. Stirret, and R. L. Libby, Ann. Surg. 137, 184 (1953).

[35]


877

metabolism. 58 I131-Labeled serum globulins are used primarily in immunological studies. A major difficulty in the interpretation of metabolic experiments with I13Ulabeled proteins is that one cannot be certain that the labeled protein is metabolized in the body in exactly the same manner as the native protein, even when the amount of iodine in the protein averages only 1 to 2 atoms per molecule. Another difficulty is that the I~31-labeled protein may not be uniformly iodinated, with the result that all the labeled protein molecule may not behave alike in metabolic experiments. A critical appraisal of the use of I~3~-labeled plasma proteins for metabolic studies has been made by Berson et al. b8 Several groups of workers have reported that the biological half-life of plasma albumin and of plasma v-globulin in human subjects is significantly greater when determined with plasma proteins biosynthetically labeled with S 35 than when measured with plasma proteins labeled in vitro with I~31.68-7° These observations emphasize the need for caution in interpreting the results of experiments with I~3~-labeled proteins. Recently, McFarlane 7' compared, in rabbits, the biological behavior of C'4-1abeled plasma proteins (biosynthetically prepared) with the behavior of plasma proteins labeled with I TM by a variety of iodination procedures. He observed marked differences when the I~3Uproteins were prepared by the usual techniques. However, he introduced a new technique (jetiodination) for preparing I~31-1abeled plasma proteins, whose rates of elimination from the plasma of rabbits followed very closely the pattern observed with C14-1abeled plasma proteins, vl,~2 A . I~31-Labeled S e r u m Globulins

1. M e t h o d of P r e s s m a n and Eisen 73

The iodinating mixture is made up as follows: 0.1 ml. of 0.01 M KI is added to 1.0 ml. of carrier-free I TM solution. One drop of 1 M NaNO2 is added, and the solution is acidified with 0.2 ml. of 2.5 N HC1. After 5 minutes, the pH is adjusted to 8 with 2.5 N NaOH. The radioactive iodinating mixture is added to 15.0 mg. of globulin in G8W. Volweiler, P. D. Goldsworthy, M. P. MacMartin, P. A. Wood, I. R. Mackay, and K. Fremont-Smith, J. Clin. Invest. 84, 1126 (1955). ~ S. It. Armstrong, Jr., J. Kukral, J. Hershman, K. McLeod,J. Wolter, and D. Bronsky, J. Lab. Clin. Med. 45, 51 (1955). 70S. Maxgenand H. Tarver, J. Clin. Invest. 35, 1161 (1956). 7, A. S. McFarlane, Biochem. J. 62, 135 (1956). 72 S. Cohen, R. C. Holloway, C. Matthews, and A. S. McFarlane, Biochem. J. 62, 143 (1956). 73 D. Pressman and H. N. Eisen, J. Immunol. 64, 273 (1950).

878


[35]

2 ml. of borate buffer of pH 8.0. After about 20 minutes, the mixture is dialyzed against 17 1. of saline for 2 hours at 5 °. A second dialysis against 17 1. of fresh saline is carried out overnight. Essentially all nontrichloroacetic acid-precipitable I ~3~ is removed. From 10 to 20% of the added I TM combines with the protein to yield a product containing 0.08 to 0.16 % iodine, corresponding to 1 to 2 atoms of iodine per molecule of antibody of molecular weight 160.000. 2. M e t h o d of M a s o u r e d i s el al. 7~

The iodinating mixture is made up by adding 1.0 ml. of carrier-free I TM to 0.2 ml. of 0.092 N Is in 0.1 M KI. The radioactive iodine solution is slowly added, with stirring, to 40.5 ml. of globulin solution (0.74 mg. N per milliliter) in 0.1 M phosphate buffer at pH 7.5. The iodine color disappears within a few minutes. After 15 minutes, the globulin preparation is dialyzed at 4 ° against 18 1. of 0.1 M phosphate buffer of pH 7.5. The globulin preparation recovered contains about 10 % of the added 1181. Only 1 to 2% of its 1131 is not precipitable with trichloroacetic acid. The average number of iodine atoms per molecule of globulin is 1.3. B . D31-Labeled S e r u m A l b u m i n

1. M e t h o d of Lutwak 76

This method substitutes treatment with ion exchange resins for the more time-consuming dialysis to remove inorganic 11~1from the iodinated protein. The iodinating mixture is prepared in a dropping funnel as follows: 1.2 ml. of 0.002 M NaI and 1.2 ml. of 0.02 M NAN02 are added to 1.0 ml. of I T M solution. The solution is acidified with 3 drops of 0.5 N HC1 to release radioactive iodine; it is then neutralized with 2 drops of 0.25 N NaOH, and diluted with 16.5 ml. of water. The iodinating mixture is added dropwise, with gentle stirring, over a period of 5 minutes, to a mixture of 20 ml. of salt-free human serum albumin (25% solution) and 5 ml. of 25% (w/v) Na2C03 solution. The funnel is rinsed with 5 ml. of water, and the stirring is continued for an additional 10 minutes. The reaction mixture is then poured onto a column of Amberlite IR-4B (analytical grade, backwashed with distilled water for 30 minutes). The column is prepared by pouring approximately 100 ml. of wet resin into a glass tube (20 mm. in diameter and 30 cm. long) fitted with a sintered-glass disk and stopcock. The effluent from this column is passed tl~rough a second column containing backwashed analytical grade Amberlite IR-100H. The first 20 ml. of effluent from the 74S. P. Masouredis, L. R. Melcher, and D. C. Koblic, J. Immunol. 66, 297 (1951). 7sL. Lutwak, Proc. Soc. Exptl. Biol. Med. 80, 741 (1952).

[35]


879

second column is made isotonic by the addition of 1.3 ml. of 15% NaC1 solution. The I TM contained in this solution is now completely precipitable with tungstic acid. It is filtered through a sterile Seitz-Manteufel filter into a sterile tube for human use. 2. M e t h o d of Gilmore et al. TM

In this procedure, I TM is added as iodide to the protein solution in phosphate buffer. Ammonium persulfate is added to oxidize the iodide-'3', and the iodination is allowed to proceed for 4 hours. The authors report that more than 90% of the added I TM is converted to I~3'-protein by this procedure. The details of the method are as follows: 150 mg. of bovine albumin is dissolved in 2 ml. of phosphate-saline-buffer (8.46 g. of NaC1, 7.24 g. of Na2HPO4-12H20, 0.53 g. of NaH2PO4"H~(), made up to 1 1.). To this are added 2 ml. of guanidine hydrochloride solution (2.5 % w/v), 1 ml. of KI solution (0.217 % w/v), and the desired amount of I TM (0.5 to 2 ml.). Ammonium persulfate (2 ml. of 10% w/v solution, made up in the phosphate-saline-buffer) is then added slowly (1 minute), with mixing, and the solution is allowed to stand for 4 hours. The reaction mixture is then dialyzed against tap water for 2 hours. This is followed by dialysis against 300 ml. of phosphate-saline-buffer for half hour. Francis el al. 59 use a slight modification of this procedure (omission of the guanidine hydrochloride) to label antibodies. Margen and Tarver use, instead of ammonium persulfate, a fresh, saturated solution of potassium persulfate. 7~ 3. M e t h o d of McYarlane 7~

The jet-iodination procedure of McFarlane is mentioned briefly above. The reader is referred to the original article for details of this method. C. I'~'-Labeled I n s u l i n

1. M e t h o d of Ferrebee e! al. 7s The mixture used for iodination contains 0.38 mg. of NaI, 0.16 mg. of Is, and 5 to 50 inc. of I TM. This mixture is added dropwise, wi'th mechanical stirring, over a period of an hour or more, to 10 rag. of hormone dissolved in a few milliliters of M/15 phosphate buffer at pH 7.4, in a cold bath. The solution is dialyzed to remove inorganic iodine, or the protein 76 R. C. Gilmore, Jr., M. C. Robbins, and A. F. Reid, N~cleonics 12, No. 2, 65 (1954). 77 S. Margen and H. Tarver, personal communication. 7s S. W. Ferrebee, B. B. Johnson, J. C. Mithoefer, and J. W. Gardella, Endocrinology 48, 277 (1951).

[~]


881

mixture of concentrated HN03 and H2SO,. The liberated iodine is extracted with chloroform, and a stream of chlorine is passed through the chloroform solution until the purple color just disappears. The labeled iodine chloride thus obtained is added to a chloroform solution containing 20 g. of olive oil. This mixture is allowed to stand for 24 hours, and then it is shaken repeatedly with a solution of Na2CO3, to remove free iodine. After the solution has been dried over anhydrous sodium sulfate, the chloroform is evaporated by means of an infrared lamp, leaving behind the I131-1abeled oil. The odor, taste, color, and consistency of the oil are unchanged after iodination. When petroleum ether solutions of the oil are repeatedly extracted with water, no radioactivity is detectable in the aqueous phase. Apparently, the I TM is firmly attached to the fatty acid chains at the double-bond positions. 2. M e t h o d of H o f f m a n 84

In this method, oleic acid is iodinated with I TM in ether solution by a procedure similar to that described above. B. I I~]-Labeled Diiodofluorescein

It was reported by Moore s5 that neoplastic tissue, especially of brain, has a much greater than normal capacity to concentrate injected fluorescein. Since diiodofluorescein behaves in the same manner, Moore suggested that I131-1abeled diiodofluorescein be used in the diagnosis and localization of brain tumors. The following method of preparation of 113L_diiodofluorescein is reported by Vigne and Fondarai.Se The reaction is carried out in a separatory funnel fixed rigidly to a mechanical shaker. The I TM solution is placed in the separatory funnel together with the theoretical quantity of iodine dissolved in chloroform (0.5 % solution). An equivalent quantity of recrystallized sodium fluorescein in M / 1 5 phosphate buffer at pH 7 (0.5% solution) is added, and the mixture is agitated for 20 minutes. The chloroform layer is removed, and the aqueous layer containing the 1131_diiodofluorescein is extracted several times with chloroform to remove traces of free iodine. The aqueous layer is acidified with 10 % KHSO4 until the diiodofluorescein precipitates, and the product is extracted with ethyl acetate. The ethyl acetate solution is washed with slightly acid water (saturated with ethyl acetate) to remove traces of iodide I TM. The solvent is then evaporated in vacuo, and the residue is dissolved in the smallest possible volume of 2% Na2CO3. To prepare crystalline Ira-sodium diiodofluorescein, the ethyl acetate solution is first dried by passing it through a column of Na~S04, and then 84M. C. I-Ioffman,J. Lab. Clin. Med. 41, 521 (1953). s~G. E. Moore, Science 106, 130 (1947); 107, 569 (1948). 86j. Vigne and J. Fondarai, Bull. soc. chim. France [5] $0, 331 (1953).

882


[36]

treated with the theoretical quantity of sodium ethylate in absolute alcohol. The product may be recrystallized by dissolving it in boiling absolute alcohol and then adding a quantity of xylene. C. I18'-Labeled Tetraiodophenolphthalein

This compound has been used for direct irradiation of carcinoma of the liver and biliary tract. Its method of preparation is described by Copher et al. 87 D. I13'-Labeled Pheniodol

This cholecystographic agent [a-phenyl-~(4-hydroxy-3,5-diiodophenyl)propionic acid] has been labeled with I TM by Free et al. 8s

IV. C~4-Labeled Thyroxine The labeling of the thyroxine molecule in its carbon atoms rather than in its iodine atoms has advantages for certain experiments, although higher specific activities are attainable with the use of I TM than with C ~4. The preparation of thyroxine-l-C 14 has been described by Wang et al. s9 s7 G. H. Copher, V. H. Wallingford, W. G. Scott, G. G. Zedler, B. Hayward, and S. Moore, Am. J. Roentgenol. Radium Therapy Nuclear Med. 67, 964 (1952). 8aA. A. Free, J. E. Page, and E. A. Woollett, Bioehem. J. 48, 490 (1951). s9S. C. Wang, J. P. Hummel, and T. Winnick, J. Am. Chem. Soc. "/4~2445 (1952).

[36]

Intermediates of Photosynthesis : Isolation and Degradation Methods 1 B y A. A. BENSON and M. CALVIN

In defining the path of carbon in photosynthesis a variety of new techniques were developed which have wide application in metabolism studies. The primary attribute of the tracer method, its ability to discriminate between the first few intermediates of a reaction sequence and the host of compounds in the organism, simplified the problem of isolation and identification of the intermediates of carbon dioxide fixation of the plant's numerous components; only a few become labeled as the label of carbon dioxide makes its way through the sequence of reactions leading to uniform distribution in the plant. When a plant engaged in steady-state photosynthesis at constant carbon dioxide pressure is given a negligible amount of high specific activity 1The work described in this paper was sponsored by the U.S. Atomic Energy Corn-

882


[36]

treated with the theoretical quantity of sodium ethylate in absolute alcohol. The product may be recrystallized by dissolving it in boiling absolute alcohol and then adding a quantity of xylene. C. I131-Labeled Tetraiodophenolphthalein

This compound has been used for direct irradiation of carcinoma of the liver and biliary tract. Its method of preparation is described by Copher et al. 87 D. I131Labele d Pheniodol

This cholecystographic agent [a-phenyl-~(4-hydroxy-3,5-diiodophenyl)propionic acid] has been labeled with I TM by Free et al. 88

IV. C~4-Labeled Thyroxine The labeling of the thyroxine molecule in its carbon atoms rather than in its iodine atoms has advantages for certain experiments, although higher specific activities are attainable with the use of I TM than with C ~4. The preparation of thyroxine-l-C 14 has been described by Wang e/al. 89 s7 G. H. Copher, V. H. Wallingford, W. G. Scott, G. G. Zedler, B. Hayward, and S. Moore, Am. J. Roentgenol. Radium Therapy Nuclear Med. 87, 964 (1952). s a A. A. Free, J. E. Page, and E. A. Woollett, Bioehem. J. 48, 490 (1951). 89S. C. Wang, J. P. Hummel, and T. Winnick, J. Am. Chem. 8oc. 74, 2445 (1952).

[36]

Intermediates of Photosynthesis : Isolation and Degradation Methods 1 B y A. A. BENSON and M. CALVIN

In defining the path of carbon in photosynthesis a variety of new techniques were developed which have wide application in metabolism studies. The primary attribute of the tracer method, its ability to discriminate between the first few intermediates of a reaction sequence and the host of compounds in the organism, simplified the problem of isolation and identification of the intermediates of carbon dioxide fixation of the plant's numerous components; only a few become labeled as the label of carbon dioxide makes its way through the sequence of reactions leading to uniform distribution in the plant. When a plant engaged in steady-state photosynthesis at constant carbon dioxide pressure is given a negligible amount of high specific activity 1 The work described in this paper was sponsored by the U.S. Atomic Energy Cornminion.

[36]

INTERMEDIATES OF PHOTOSYNTHESIS

883

C1402, the rate of photosynthesis can be measured by a linear accumulation of radioactivity. Such a C 14 fixation curve is a summation of a large number of radioactivity appearance curves for the intermediates and products. The curve for each compound, in turn, is made up of a summation of the rates of accumulation of label in each of its several carbon atoms. The observation of these rates has contributed to an understanding of the sequential relationships of carbon dioxide fixation and reduction. In the very shortest periods of steady-state C1402 fixation the major product was seen to be phosphoglyceric acid (PGA). 2 Such a primary carboxylation product has an "appearance curve" with an initial finite slope; all subsequent intermediates should have initial slopes of zero. Such curves have been obtained for photosynthesis by the green alga S c e n e d e s m u s 3 and are shown in Fig. 1. Phosphoglycerate and malate are direct carboxylation products; other compounds have negligible slopes at the origin. Each of these "radioactivity appearance curves" is made up of individual curves for label in each carbon atom of the compound. It is from these particular radioactive appearance rate data that one may deduce the path of carbon in photosynthesis. Securing such data requires several steps. One must be able to separate the labeled compounds and determine their total radioactivity. One must choose those which appear to be the major intermediates involved and identify them. This done, a method of chemical or biological degradation must be developed whereby the label of each carbon atom may be measured either as a one-carbon compound or its derivative like barium carbonate or formaldimedon or as the differences in label between that in known groups of carbon atoms. Some of the techniques applied in carrying out these steps with photosynthetic intermediates are described in the succeeding sections.

Separation of Labeled Compounds Many methods are available for fractionation of plant constituents. Since none is universally applicable, the method chosen is dependent on the type of compounds investigated and the standards of purity required. The classical methods for fractionation of amino acids or the sugar phosphates by their solubility properties are practical for large-scale separation when coprecipitated impurities can be tolerated. These methods are not applicable, however, when separating radioactive components of 2 M. Calvin and A. A. Benson, Science 109, 140 (1949). A. A. Benson, S. Kawaguchi, P. M. Hayes, and M. Calvin! J, Am. Chem. Soc. 74,

~477 (1952).

884

[36]

TECHNIQUES FOR ISOTOPE STUDIES UDPGURIO4NEOlPflOSPHATE GLUCOSE P PHOSPHOPYRUVICACID F-P FRUCTOSE MONOPHOSPHATE Ge GLUTAMIC ACID "A ASPARTIC ~ / ~ G GLYGINE SE SERINE S SUCROSE •AI ALANINE

O x

ACID

ACID

z

i at

ul lZ ::) 0 t.1 ). I:D I--

GLUCOSE MONOPHOSPHATE

_o

:ll BULOSE OIPHOSPHATE r MANNOSEAND ~EDOHEPTULOSE PHOSPHATES

_

o

~

m

~

•

f~

i

MINUTES

FIG. 1. Radioactivity appearance curves for photosynthetic intermediates in Scenedesmus2 Radioaetivities were measured by direct counting of the labeled com-

pounds on paper ehromatograms of extracts taken at 5-second intervals. Data up to 4 minutes may be considered "steady state." Thereafter, the supply of C140~ was insufficient and reservoirs were diluted by exchange with unlabeled plant constituents. widely varying radioactivities and specific activities. A negligible fraction of cocrystallized or adsorbed impurity of high specific radioactivity could easily lead to an erroneous identification. Methods must be chosen, therefore, which will be free of such limitation. Paper c h r o m a t o g r a p h y is such a method and has admirably complimented the tracer technique. Paper c h r o m a t o g r a p h y is a separation based largely on solvent par-

[36]


885

tition 4 and as such is remarkably free of those difficulties which plague the analyst--absorption, cocrystallization, and other interactions between substance and impurity. The partition coefficient of a substance is independent of concentration over a large range and not seriously influenced by moderate concentrations of impurities. By choosing suitable solvents a wide group of compounds may readily be separated. It has proved valuable to use chromatographing solvent systems which give optimum separation of the majority of soluble compounds becoming labeled during photosynthesis in C140~. Most plant extracts are near pH 6, and hence most of the acidic compounds are dissociated. Phenol-water has been chosen for the first dimension because it gives a remarkably good separation of amino acids, carboxylic acids, sugars, and phosphate esters and does not change the pH of the compounds applied to the paper. In order to achieve a different order of separation of these compounds, the second solvent contains acid capable of "swamping ''5 or acidifying the carbohydrate and phosphate salts, thereby radically changing their solubility characteristics. Bases, being largely in the form of their salts, will become more water-soluble, and their R / v a l u e s will, therefore, be reduced. Acids will become less soluble in the water phase, their R / v a l u e s increasing. The acidic compounds, therefore, move relatively farther in the acid solvent than do the neutral substances. Basic substances move much less at high pH than at pH 6. Position on the chromatogram, then, tells much of the acid or base character of a compound. A typical radiogram of such a paper chromatogram is shown in Fig. 2.

Sequence of Intermediates in Photosynthesis Photosynthetic incorporation of carbon dioxide has been found to follow the pattern outlined in Fig. 3. The kinetics of the appearance of C 14in the various carbons of this sequence is consistent with the available degradation data as well as with our knowledge of the enzymatic equilibria involved. The major synthetic pathway, leading to sucrose in most plants, follows the Embden-Meyerhof sequence to the condensation reaction by which sucrose phosphate is formed and from which free sucrose is liberated as the first free sugar. The cyclic system s serves for the regeneration of the carbon dioxide acceptor and involves equilibration of the ketose phosphates, fructose-6-phosphate, sedoheptulose-7-phosphate, and ribulose-5-phosphate by transketolase. This system was devised on the 4 R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 28, 224 (1944). 5j. W. H. Lugg and B. T. Overell, Nature 160, 87 (1947); Australian J. Sci. Research 1, 98 (1948). 6j. A. Bassham, A. A. Benson, L. D. Kay, A. Z. Harris, A. T. Wilson, and M. Calvin, J. Am. Chem. Soc. 76, 1760 (1954).

886


[36]

FT. 2. Radioactive products of 60 seconds of photosynthesis in C140~ by Scenedesmus. Developed in phenol-water (right to left) and butanol-propionie acid-water (bottom to top) on oxalic acid-washed Whatman No. 1 paper. (polysoccharides)

.,,/

/ ) /

akMolose

1.1/

(xylulose)

FIG. 3. Cyclic system for regeneration of CO2-aeceptor. basis of t h e isolation, identification, a n d d e g r a d a t i o n of t h e c o m p o u n d s which b e c o m e labeled w h e n p l a n t s are fed labeled COs. T h e relationships • of t h e labeled a t o m s t o a n u m b e r of externally controllable variables such as light a n d CO2 pressure v,8 m a d e possible a m o r e or less definitive 7 A. T. Wilson, Thesis, University of California, 1954. 8 M. Calvin and P. Massini~ Experientia 8 i 445 (1952).

[36]


887

specification of the path of carbon as shown in Fig. 3. Each of the individual enzyme reactions has now been demonstrated. Changes in Reservoir Sizes on Changing C02 Pressure

The carbon dioxide acceptor which accumulates when the carbon dioxide pressure is reduced appears to be ribulose diphosphate. Reservoir I

"-1% G O = - - - ~,~-

'..

'_

co,

'-

'-

.

C()z,,~.~aGA RDP ~TRIOSE-P

/f~

/

"~RMPJ

D

! ! RMP x IO

.J

w

I'

I

-IO0

I

O

I

I

I

I

IO0 200 TIME IN SECONDS AT 6 ° C.

I

500

TRANSIENTS IN THE REGENERATIVE CYCLE

FIG. 4. Dependence of reservoir sizes on changes in C().~ pressure in Scenedesmus. 7 Samples were t a k e n at 5-second intervals from a large vessel of Scenedesmus photosynthesizing in circulating 1% a n d 0.003 % C~402 in air. D a t a are obtained b y direct counting of radioactive areas on two-dimensional chromatograms.

sizes were determined by counting each compound on two-dimensional paper chromatograms prepared from extracts of samples of labeled algae taken at 5-second intervals while the carbon dioxide pressure was being reduced. The accumulation is demonstrated 7,8 in the curves of Fig. 4,

888


[36]

I m m e d i a t e decrease i n P G A c o n c e n t r a t i o n r e s u l t s f r o m t h e s u d d e n drop i n c a r b o n dioxide pressure. T h e first c o m p o u n d to increase in conc e n t r a t i o n is r i b u l o s e d i p h o s p h a t e . I t is seen t h a t p e n t o s e m o n o p h o s p h a t e s a n d triose p h o s p h a t e rise later. T h e s e r e s u l t s suggest t h a t ribulose d i p h o s p h a t e is t h e c a r b o n dioxide a c c e p t o r a n d are verified b y c a r b o x y l a t i o n of ribulose d i p h o s p h a t e w i t h CI~0: in t h e presence of a c r u d e cellfree p l a n t e n z y m e p r e p a r a t i o n 9 to give c a r b o x y l - l a b e l e d P G A . TABLE I EFFECT OF TIME, LIGHT INTENSITY, AND SOURCE IN DETERMINING C 14 DISTRIBUTION IN PRODUCTS OF PHOTOSYNTHESIS IN C1402

Time 27 0.4 2 5 5.4 15 15 60 60 1.5 3 4 4 5 10

hr. sec. sec. sec. sec. sec. sec. sec. sec. min. rain. rain. min. min. min.

Plant Sunflower Soybean Barley Soybean Scenedesmus Barley Soybean Barley Scenedesmus Sunflower Sunflower Sunflower Sunflower Soybean Sunflower

Light Phosphoglycerate I-Iexose intensity, Referfoot-candles COOH CHOH CH2OP 3,4 2,5 1,6 ence Dark 5,000 5,000 8,000 5,000 5,000 8,000 10,000 70 500 10,000 5,000 70

100a

0

0

70 85 81 49 44 44 56

15 5 5.3 25 21 30 19

15 6 5.6 26 35 25 25

41•

29

30

40 67~

35 16

38 17

87 98

6

7

48 26 25 88 3 9 41 29 30 37 33 30

b c d e f d e d g b h h b

43 52

3 25

3 23

37

34

32

70

h

14

16

.Manine degradation data. PGA labeling has been shown identical in many experiments. b M. Gibbs, Plant Physiol. 26, 549 (1951). c A. G. Zweifler, Thesis, University of California, 1953. a M. Calvin, J. A. Bassham, A. A. Benson, V. H. Lynch, C. Ouellet, L. Schou, W. Stepka, and N. E. Tolbert, Symposia Soc. Exptl. Biol. 5, 284 (1951). • S. Aronoff, Arch. Biochem. and Biophys. 82, 237 (1951). f J. A. Bassham, A. A. Benson, L. D. Kay~ A. Z. Harris, A. T. Wilson, and M. Calvin, J. Am. Chem. Soc. 76, 1760 (1954). S. Kawaguchi, this laboratory, unpublished data. h M. Gibbs, Arch. Biochem. and Biophys. 45, 156 (1953).

Distribution of C 14 in Photosynthetic Intermediates T h e earliest P G A is c a r b o x y l - l a b e l e d , a n d it a p p a r e n t l y is t h e p r o d u c t of t h e r e a c t i o n 6 9j. R. Quayle, R. C. Fuller, A. A. Benson, and M. Calvin, J. Am. Chem. Soc. 76, 3610 (1954).

890


[36]

thesis periods '° suggested that it may be related to the symmetrically labeled COs acceptor. The distribution of label in ribulose, sedoheptulose, and fructose phosphates is given in Table II. 6 These data are consistent with transketolase equilibration and led to the choice of the hexose as the only probable source of the C4 moiety required for the formation of sedoheptulose phosphate by an aldolase-type condensation. C C

C C ** * transketolase C

C** -{- C C** C C C C **

C **

C** -i- C C C C

C**

C** * C** + C C C C

atdolase

C )C C** C** C** C C

The rate of attainment of uniform labeling of these phosphates is dependent on the plant used and on the sizes of its reservoirs in intermediates. Five to ten minutes of photosynthesis is generally sufficient to attain C TM saturation of these compounds in steady-state light- and COs-saturated photosynthesis.

PGA-C 14 in Photosynthesis Photosynthetic Preparation of PC:rA-CTM. The PGA reservoir of most plants saturates with C TM during the first 2 minutes of steady-state photosynthesis. The green algae Scenedesmus and ChloreUa and the leaves of several higher plants are satisfactory sources. Heat-stable phosphatases 'I are more active in leaves; therefore more free glyceric acid also appears in their extracts. Procedure. Scenedesmus (ChloreUa is also satisfactory) culture: 12 One liter of inorganic culture medium contains the following salts: 5 ml. of 1 M KNO3; 0.5 ml. of 1 M K2HPO4; 0.5 ml. of 1 M KH2PO4; 2 ml. of 1 M MgSO4.7H20; 0.25 ml. of 0.1 M Ca(NO3)2; 1 ml. of trace element solution containing 1.43 mg. of HaBO3, 1.05 mg. of MnSO4"H20, 0.05 mg. of ZnCl~, 0.04 mg. of CuSO4"5H20, and 0.01 mg. of H2MoO4-H20; 1 ml. 10 L. Schou, A. A. Benson, J. A. Bassham, and M. Calvin, Physiol. Plantarum 3, 487

(195o). ~1 H. Boroughs, Arch. Biochem. and Biophys. 49, 30 (1954). 12 L. Norris, R. E. Norris, and M. Calvin, J. Exptl. Botany 6, 64 (1955).

[36]


89!

of iron a n d Versene (Versenes, Inc., formerly Bersworth Chemical Co., Framingham, Mass.) solution. The iron solution is prepared by addition of 24.9 g. of FeSO,.7H,O to a solution of 26.1 g. of Versene in 268 ml. of 1.0 N KOH (15 g. of KOH). After dilution to 1 1. the solution is aerated overnight and the pH is ~5.5. The medium should be pH 6.8. Algae are grown at 25 ° on 4% CO2 in air with sufficient agitation to prevent settling of the cells. Light intensity from a bank of daylight fluorescent tubes is 2000 foot-candles. The algae are harvested to give a yield of 3 cc. of packed cells per liter. Repetition of this culture with 18% inoculum and 82 % fresh nutrient gives a uniform daily harvest. A 1 to 2% suspension of algae in M/150 phosphate buffer, pH 6, is illuminated from both sides in a 1-cm.-thick vessel with two 300-watt reflector-spot incandescent lamps with suitable infrared filters. A bank of closely spaced white fluorescent lights (2000 to 5000 foot-candles) is also effective. After photosynthesis with 4% C02 in air long enough to overcome possible induction periods (up to 1 hour when algae have been stored in the cold or dark), air is flushed through the suspension for 1 to 5 minutes to remove excess COs. A solution of NaHCI*C03 (approximately 0.02 millimole per minute per gram of cells) is injected into the suspension, and the vessel is closed and agitated for 1 t o 5 minutes. A fixation rate of up to 1.5 X 108 dis./m./g, of wet cells may be anticipated using B a C Q with 25% C 14. The algae are then poured into 4-vol. of hot absolute ethanol. The extracted colorless cells are filtered through a filter aid (Hyflo Super-Cel) or centrifuged and the residue re-extracted with a smaller volume of hot 20% ethanol. The 20% alcohol extract contains most of the PGA and ribulose diphosphate as well as some polysaccharides. A similar experiment may be performed with young sugar beet or barley seedling leaves in an illumination chamber having a removable face (Fig. 5). The leaves should be cut from the plants in the light, placed in the chamber, and given C140~ without delay. The chamber air is partly removed quickly with a water aspirator and replaced with air passed through the U-tube or loop containing an excess of C1402. CI402 from 3 rag. of BaC~403 should be prepared for each gram of green tissue per minute of photosynthesis. After a chosen time the chamber is opened (in a well-ventilated hood) and the leaf(s) plunged into liquid nitrogen. It is ground while wet with liquid nitrogen, and the powder dumped into boiling 80 to 90% ethanol. Most of the PGA-C TM is obtained on re-extraction with hot 20% ethanol. The 20% ethanol extract is concentrated i n vacuo to about 1.0 ml./g. of cells and applied in a stripe for paper chromatography in phenol-water on oxalic acid-washed Whatman No. 4 paper. It is rerun, if necessary,

802


[36]

on a second paper in butanol-acetic acid-water. Purity of the labeled product may be determined by phosphatase hydrolysis and rechromatography of the resultant glyceric acid on Whatman No. 1 paper. Possible contaminants will be hexose monophosphates which would be detected as hexoses in the hydrolyzate radiogram.

FIG. 5. Photosynthesis chamber for leaves. C'402 is stored in the "loop" above the chamber which has a connection to a water aspirator. The rectangular water baths contain infrared absorbing filters. Hydrolysis and Degradation of PGA. Labeled phosphoglyceric acid eluates are hydrolyzed readily with phosphatase (Polidase-S, Schwarz Laboratories, Inc., Mt. Vernon, N.Y.) and chromatographed on Whatman No. 1 paper (R f: phenol, 0.28; butanol-propionic-water, 0.40). The glyceric acid is first oxidized with periodate at room temperature to give the ~-carbon activity in formaldehyde which is isolated as the dimedon derivative. The remaining glyoxylic acid oxidizes more slowly with excess periodate to give carbon dioxide from the carboxyl group and formic acid from the s-carbon atom. COOH

} HCOH { H2COH

COOH HIO,

I

, HC=O 1 mole

-~-

HCHO

CO2 HIO, -}~ HCOOH excess

[36]


893

Elution of Labeled Compounds from Paper Chromatograms: The radioactive spot, defined by the radiogram, is cut out in a rectangular area with one end pointed. The rectangular end is attached to a wet paper wick hanging from a small trough, surrounded by an inverted aquarium to maintain humidity (Fig. 6). The eluate is collected in a 2-ml. centrifuge tube as it flows from the pointed tip of the paper to the inner surface of the tube. The tube is briefly centrifuged to collect all the eluate in the bottom of the tube. Complete extraction is obtained with 50 ~l. for sugars and amino acids and 100 to 200 ~l. for phosphate esters. Paper

wick

/

Glasstrough

~

Water oc,se

I1

FI

O,a sco er -----'~ Eluate

Fro. 6. Elution apparatus for chromatographedcompounds. Procedure. 13 The glyceric acid is eluted from the paper and the eluate added to 100 to 200 mg. of calcium glycerate carrier. The mixture is dissolved in ca. 0.5 ml. of water and crystallizes on addition of several volumes of hot absolute ethanol. The labeled calcium glycerate is dried and its specific activity accurately determined. This is done by direct plating of samples with less than 1 mg./cm. 2 to avoid self-absorption corrections. Fifty milligrams of the labeled calcium glycerate (0.4 millimole) is placed in a flask with 0.80 ml. of 1.0 N periodic acid. After 2 hours at room temperature, the solution is made slightly alkaline and the volatile contents, including formaldehyde, are distilled in vacuo without heating into 13 ml. of a solution of dimethyldihydroresorcinol prepared from 1 g. dissolved in NaOH and diluted with 100 ml. of water, from which the dimendon compound of formaldehyde is precipitated by acidification to pH 6 to 7 and is isolated by centrifugation. It is recrystallized by dissolving in hot ethanol and adding water until incipient crystallization and then allowing the solution to cool. To the nonvolatile residue of sodium glyoxylate, 5 ml. of 0.1 N periodic 13j. A. Bassham, A. A. Benson, and M. Calvin, J. Biol. Chem. 18fi, 781 (1950).

894


[36]

acid is added, and after 24 hours at room temperature the volatile products are distilled in vacuo into 10 ml. of a carbonate-free 1 N sodium hydroxide solution. Excess barium chloride solution is then added dropwise, and the barium carbonate precipitate is centrifuged, washed with water and alcohol, dried, and its specific activity determined by direct plating and counting. Plates of less than 1 mg./cm. 2 may be counted without serf-absorption correction when specific activities are high ( ~ 50 c.p.m./ mg.). Otherwise thicker samples must be counted with adequate corrections. The supernatant solution is acidified and steam-distilled to collect the formic acid. The steam distillate is neutralized with barium hydroxide to the phenolphthalein end point and concentrated to dryness at reduced pressure. The barium formate is recrystallized from a small amount of water on addition of alcohol. The specific activities of the barium formate and dimedon compound are determined, and, with the theoretical yields, they give the radioactivities of the a- and f~-carbons (expressed as percentages of starting radioactivity). The measured weight of barium carbonate and its specific activity are used for calculation of the carboxyl activity. In general, even the measured barium carbonate yield will give a low result, since it is diluted by less active C02 derived from overoxidation, from CO2 from contact with the air, and from carbonate in the sodium hydroxide solution. Reasonably carbonate-free sodium hydroxide is obtained by filtering a saturated (50%, 21 N) solution which has stood a week or more while sodium carbonate separates. It is stored in a sealed vessel and diluted twentyfold for use. The method has been altered by Aronoff TM in which the oxidation of glyoxylic acid is done by a fresh solution of M / 2 perchlorato-cerate (G. F. Smith Chemical Co., Columbus, Ohio) whereby the oxidation time is reduced to 15 minutes at 40 ° . After distillation of the formaldehyde in vacuo, 5 ml. of the cerate solution is introduced with provision for trapping evolved carbon dioxide. The residual formate is then converted to carbon dioxide by excess mercuric oxide during 15 minutes of boiling.

Accumulation of Glycolic Acid during Photosynthesis When leaves or algae are illuminated aerobically in the absence of carbon dioxide (after C1402 photosynthesis), copious formation of glycolate is observed. Up to 30% of the C TM in the alcohol-soluble compounds of sugar beet leaf has been observed in glycolic acid. It appears that these conditions, which involve accumulation of ketose phosphates, lead to oxidative degradation of the transketolase-C~ complex to free glycolate. The active glycolic acid oxidase of most plant tissues rapidly oxidizes the glycolate when illumination ceases. 14 S. Aronoff, Arch. Biochem. and Biophys. 82, 237 (1951).

[36]


895

Degradation of Glycolic Acid-C 14 (see also Vol. IV [25]). TM Lead tetraacetate oxidation of glycolic acid yields formaldehyde and carbon dioxide. Since the reaction is best done in acetic acid solution, the 2,4-dinitrophenylhydrazone is chosen as the formaldehyde derivative. COOH

[ CH2OH

CO2 --* BaCO3

Pb(OAc)4

+

* HCHO -~ H ~ C ~ N - - N - - R

Procedure. A tracer quantity of C~4-1abeled glycolic acid, obtained by elution from a paper chromatogram, is added to 30.4 mg. of glycolic acid in 3-ml. of glacial acetic acid in a 50-ml. flask with a 15~0 joint. The solution is then frozen, about 0.5 g. of lead tetraacetate added, and the flask attached through a s t o p c o c k t o an inverted U-tube (14-mm.-diameter tubing) from which the air may be removed through a stopcock. The system is evacuated, the stopcock closed, and the reaction mixture heated on a water bath at 90 ° for 30 minutes. After cooling, the volatile contents of the flask are distilled (with due precaution to prevent bumping) through the U-tube into a second flask containing 80 mg. of 2,4-dinitrophenylhydrazine and immersed in liquid nitrogen. The stopeock is again closed, and the second flask warmed until a clear yellow solution of formaldehyde 2,4-dinitrophenylhydrazone is obtained. The first flask is replaced by a third flask containing 5.0 ml. of saturated, earbonate-free sodium hydroxide solution. Both flasks are immersed in liquid nitrogen for a few minutes, the stopcock is opened, and the system re-evacuated. The liquid nitrogen bath is then removed from the seeond flask, and the volatile contents are distilled into the third flask. The residue of formaldehyde-2,4-dinitrophenylhydrazone in the second flask may be crystallized from alcohol, purified chromatographically on a silicic acid column, and the specific activity determined. This specific activity, together with the theoretical yield based on the carrier taken, gives the total activity of the a-carbon atom. The third flask is warmed to room temperature, and the solution transferred to a centrifuge tube. It yields, on addition of excess barium chloride solution, a precipitate of barium carbonate which is washed, dried, weighed, and counted. The product of the specific activity of the barium carbonate and the total yield (slightly greater than theoretical owing to introduction of inactive carbon dioxide in reagents and manipulation) gives the total activity of the carboxyl carbon of glycolic acid.

Labeled Heptuloses in Photosynthesis The phosphate esters of sedoheptulose and mannoheptulose are intimately involved in phytosynthesis but do not accumulate to any great

[36]


897

Sedum spectabile. Tolbert and Zill used stalks of mature plants weighing 20 g. each; Nordal used young plants weighing 0.5 to 1.0 g. With a light intensity of 100 foot-candles, Tolbert and Zill found that 40 g. of plant assimilated 10 mc. of C140~ during 261/~ hours. The yield was satisfactory but not consistent with that expected from the known high concentration of sedoheptulose in these leaves. Nordal observed the expected sedoheptulose concentration only after 7 to 10 days of photosynthesis in C'402 using 20-minute periods of light and dark to maintain the health of the plant in the closed C1402-air atmosphere. In the cases examined, periods of a week or more were required to reach apparent saturation of the heptulose reservoir. At this point the amounts of sucrose, fructose, and glucose were relatively small. Preparation from Sedoheptulose-7-P of Soybean Leaves. The simplest photosynthesis of experimental amounts of sedoheptulose-C TM has been its recovery from sedoheptulose-7-phosphate formed during 1 to 5 minutes of photosynthesis by soybean leaves. These have a relatively high concentration of sedoheptulose-7-P in the monophosphate fraction. Soybean leaves freshly cut in the light are placed in a glass chamber (Fig. 5) of small volume having a detachable glass face. In direct sunlight or with suitable light source the chamber is rapidly evacuated and refilled with air admitted through a tube containing 100 to 200 ~c. of C'402 (5 to 10 mg. BaC~402) for each trifoliate leaf. After 1 or 2 minutes the leaf is taken from the chamber, plunged into ethanol, and extracted successively with 80% and 50% ethanol. The concentrated extracts are chromatographed two-dimensionally on paper (extract of 10 to 50 rag. of plant tissue per sheet), and a radiogram prepared. The total phosphate ester area or the "hexose monophosphate area" is eluted and hydrolyzed with 200 ~, of Polidase-S (Schwarz Laboratories, Inc., Mt. Vernon, N.Y.) in a volume of 100 to 500 ~l. The hydrolyzate is chromatographed on Whatman No. 1 paper with phenol-water and butanol-propionic acidwater to separate the resulting sugars. Sedoheptulose lies between glucose and fructose and can be separated from the former on Whatman No. 1 paper. In 1-minute photosynthesis the yield is approximately 10to 20% of the total fixed C TM. Longer photosynthesis gives more sedoheptulose-C TM but, of course, a lower fraction of the fixed activity in the heptulose. Five minutes of photosynthesis is required to attain essentially uniform labeling. Preparation from Sedum Leaves. The use of Sedum leaves for sedoheptulose-C TM preparation requires adequate temperature control and provisions for maintaining health of the leaf or plant for 24 hours or longer. The relatively large amount of free sedoheptulose in the plant serves to dilute the C '4 unless a great deal of C1402 is used over a long period.

898


[36]

When C1402 sufficient for 15 to 20 hours of photosynthesis is used (approximately 50 mg. of BaC1402 per gram of leaf tissue) the illumination chamber may be kept closed during alternating light and dark periods for a week or more. The C ~4is found largely in malic acid, sucrose, fructose, glucose and sedoheptulose, becoming greatest in sedoheptulose after a week. The plant may then be extracted with 80% ethanol for recovery of the product. The sedoheptulose may be isolated directly by paper chromatography ~° when the amounts involved are small With larger amounts the extract is passed through cation (Dowex 50) and anion (Duolite A-3) exchange resin columns. Fermentation of the neutral sugar fraction by added yeast can be repeated several times to obtain a solution free of hexoses. Sedoheptulose is converted to sedoheptulose anhydride which crystallizes readily. The anhydride is converted to a 20 % equilibrium solution of sedoheptulose by heating for 1 hour at 100 ° with a well-stirred suspension of 400-mesh Dowex 50-H +.

Ribulose-C ~4-Diphosphate Preparation. Inasmuch as the carbon dioxide acceptor of photosynthesis is required to accumulate in the absence of carbon dioxide at saturating light intensities, it is possible to obtain enhanced concentrations of the compound by killing the plant under such conditions. When the carbon dioxide pressure is suddenly dropped from 1% to 0.003 %, the concentrations of phosphate esters of Scenedesmus change according to the curves of Fig. 4. 7 There is a clear maximum in ribulose diphosphate concentration occurring 30 to 40 seconds after reduction of the carbon dioxide pressure. A similar transient occurs in soybean leaves and presumably all leaves, but the data available are not so complete as for algae. Procedure. A Scenedesmus suspension (1%) is allowed to photosynthesize at 5000 foot-candles with C~402 for 1 to 30 minutes, depending on the uniformity of labeling desired. A very rapid stream of nitrogen is introduced to flush out excess C1402. After 30 seconds, the algae are drained into 4 vol. of boiling absolute ethanol and re-extracted with 20% alcohol as described for the preparation of PGA-C ~4. Most of the ribulose diphosphate and phosphoglycerate is contained in the 20% ethanol extract. They are readily separated by paper chromatography in phenol (24- to 48-hour development) as a stripe on oxalic acid-washed Whatman No. 4 filter paper. One gram of wet cells contains up to 1 micromole of ribulose diphosphate. Larger amounts may be separated by anion resin column chromatography (cf. section on column chromatography of phosphate esters, Vol. III [15]). An application density of 50 cm. per extract of 4 g. of wet cells for a 5-mm.-wide stripe is satisfactory. The ribulose diphosphate is contaminated only by small amounts of labeled

[36]

INTERMEDIATES

OF P H O T O S Y N T H E S I S

899

polyglucose compounds and by a trace of fructose, glucose, and sedoheptulose diphosphates. The resulting stripe is excised and washed with absolute ethanol and ether. Elution is best performed on short sections of stripe or by suspending the stripe from a wick in a long trough of water and collecting the eluate from the serrated lower edge of the stripe ill a correspondingly long receiver. Degradation of Labeled Ribulose and Sedoheptulose G For lack of adequate enzymatic methods the chemical degradation of these sugars has been developed. The reactions used for the degradation H HC:N--N--(~ I Phenyl C=N--N--o

hydrazine

[

---~HC--OH

H

H HC=N--N--~ HIO~

NaHCOs

HC--OH

t

-, C=N--N--~ q- HCOOH + HCtIO !

H

CHO

CH20H CH+Ott--

]

C=O 1

Ce(C104)~- -

[

H+

HCOH

C02 + 4HC00H

HCOH [

CH2OH -CH~OH

E

HCOH H2

I

PtO~

I

---*HCOH HCOH I CH~OH

HIO4

* 2HCHO + 3HCOOH 11~51

12, a,

Fro. 7. Chemical degradation of ribulose. are shown on the accompanying flow sheets (Figs. 7 and 8). The free sugars obtained on phosphatase hydrolysis of chromatographed phosphate esters are purified by two-dimensional chromatography. Sedoheptulosan. The eluted heptulose is heated at 100° for 1 hour with a suspension acid-treated Dowex 50 (400 mesh) and separated in a small centrifuge tube. The resin is washed with water, and the solution is chromatographed to separate the 80 % yield of sedoheptulosan (Rj = 0.69 in phenol-water) from the equilibrium mixture.

900

[36]

TECHNIQUES FOR ISOTOPE STUDIES H H ttC~-~N--N--~ HC=N--N--¢ ~-~N--N--~b I I I O , ~-N--N--4~ q- 3H-=-COOH + I H NaHCOa ~HO It Phenyl HOC--H hydrazine,HC1 HC--OH CH~OH HC~--OH H~--OH

CH2OH--

4--0 ~o~

C]H~OH CH2OH

1

DO;o~, o~o

~oo. .~o.

~-~o~-c

NaI0,

I

HC00H q- 0 CH0 0 --CH I H2C---

l

~--OH 0

--~.

H ÷

* CO2 q- 6HCOOH

c~(cxo,),-- ~l CI-I2OH HO~H HO~H HC}OHRIO, H~OH ; 2HCHO + 5HCOOH

o~,o~-I HCOH H~OH H~OH ~H2OH

+

H~OR

~t

~ 1

~H20H

I suboxydans Acetobacter CH2OH I HCOH I HO6H H(~OH H(~OH

c~_-o

HCHO

~--~ Sedoheptulose q- Mannoheptulos,

H ÷

•

Ce(C1006-

) CO2 + 6HCOOH

-

~H20H Fro. 8. Degradation of sedoheptulose.

902


[36]

ethanol, and a drop of piperidine. After the mixture is warmed for 10 minutes oil the steam bath, 0.5 ml. of glacial acetic acid is added. Formaldimedon precipitates on standing. I t is recrystallized from an ethanol-water mixture and its specific activity measured by direct plating. From this, the activity in C-5 can be determined. The residue from the previous distillation contains sodium formate, sodium bicarbonate, and sodium iodate. This residue is dissolved in 5 ml. of water, and then 100 mg. of iodic acid is added. The solution is then distilled to dryness i n vacuo. The formic acid in the distillate is neutralized with barium hydroxide to a phenolphthalein end point, and after evaporation on the steam bath to ca. 1 ml. the barium formate is precipitated by the addition of absolute alcohol. The salt is recrystallized several times from a small volume of water by the addition of alcohol, and counted. From its specific activity the C TM percentage in carbon atom 4 of ribulose can be calculated. Cerate Oxidation of Ketoses. P r i n c i p l e . Carbonyl carbons in ketoses may be converted to C02 by cerate oxidation in perchloric acid solutions. The oxidation of the carbonyl carbon of a ketose to C02 by cerate ion was performed according to the method described by Smith. 24 Procedure. To a solution of an aliquot portion of radioactivity plus weighed carrier (sedoheptulosan or fructose) is added a slight excess of 0.5 M cerate ion in 6 N perchloric acid, the final concentration of acid being 4 N. The resultant C02 is swept with nitrogen into COs-free sodium hydroxide. The reaction is allowed to proceed for 1 hour at room temperature, and then the COs is precipitated and counted as barium carbonate. In all cases the theoretical amount of carbon dioxide was evolved. Catalytic Hydrogenation of Tracer Amounts of Sugars. Raney nickel is widely used for reduction of sugars but is not applicable for the small amounts obtained paper chromatographically. The alkaline nature of this catalyst results in almost complete and irreversible adsorption of the substrate. Platinic oxide (Adams' catalyst), on the other hand, is acidic by virtue of its method of preparation and adsorbs almost none of the sugar substrate. Procedure. The eluted sugar, usually with 50 to 100 -y of carrier sugar, is hydrogenated in 50 % alcohol solution with 5 to 10 mg. of platinic oxide. Although the reaction has been reported 25 with 2000 p.s.i, of hydrogen at temperatures of 80 to 100° for 6 hours, it may be possible to use milder conditions such as room temperature and 3 arm. of hydrogen pressure. 28 ~ G. F. Smith, "Cerate Oxidimetry." G. Frederick Smith Chemical Co., Columbus, Ohio, 1942. ~5A. A. Benson, J. A. Bassham, M. Calvin, A. G. Hall, H. Hirsch~ S. Kawaguchi, V. H. Lynch, and N. E. Tolbert, J. Biol. Chem. 196, 703 (1952). ~sj. X. Khym, D. G. Doherty~ and W. E. Cohn, J. Am. Chem. Soc. 76~ 5523 (1954).

[36]


903

Such an investigation with labeled fructose, from which mannitol is readily separable, can be readily performed. The catalyst is removed by filtration with Celite, and the polyol purified by two-dimensional chromatography. Since sugar alcohols often chromatograph closely to related sugars, it may be difficult to be certain of the yields in these reductions. Periodate Oxidation of Ribitol. Carrier ribitol (adonitol) or volemitol is added to an aliquot of the radioactive alcohol and treated at room temperature with a slight excess of paraperiodic acid. After 6 to 7 hours the formic acid and formaldehyde are distilled in vacuo. After titration of the formic acid with barium hydroxide, the formaldehyde is redistilled and precipitated as formaldimedon. Both the residual barium formate and formaldimedon may be recrystallized before plating and counting. Bacterial Oxidation of Heptitols from the Reduction of Sedoheptulose. The radioactive reduction products of sedoheptulose give only one spot on chromatography. After elution these are oxidized by Acetobacter suboxydans in a small-scale modification of the usual method. 27 Procedure. Two milligrams of volemitol and about 100 ~l. of solution of radioactive heptitols are placed in a 7-mm.-diameter vial, and an amount of yeast extract sufficient to make a 0.5% solution is added. The vial is sterilized, then inoculated from a 24-hour culture of Acetobacter and left for a week at room temperature in a humid atmosphere. The bacteria are centrifuged from the incubation mixture, and the supernatant solution chromatographed. Three radioactive spots were obtained. The two major spots were mannoheptulose and sedoheptulose, the oxidation products of volemitol. The third had R/values very similar to those of fructose and cochromatographed with authentic guloheptulose, the oxidation product of ~-sedoheptitol (R/in phenol = 0.47; R/in butanol-propionie acid-water -- 0.24). Both mannoheptulose and guloheptulose have carbon chains inverted from the original sedoheptulose and are suitable for oxidation to obtain sedoheptulose C-6 activity from their C-2. In the small-scale fermentations, however, the oxidation appeared to be incomplete. The original alcohol did not separate chromatographically from mannoheptulose. Therefore, the easily purified guloheptulose, despite its much poorer yield, was used for subsequent degradations with cerate ion to obtain C-6 activity of sedoheptulose. Degradation of Malic Acid (see also Vol. IV [24]) a-Carboxyl. Malic acid is readily isolated paper chromatographically. It has been degraded by permanganate oxidation, ~8 which gives 2 moles ~7L. C. Stewart, N. K. Richtmyer, and C. S. Hudson, J. Am. Chem. Soc. 74~ 2206 (1952). 2s It. G. Wood, C. H. Werkman, A. Hemingway, and A. O. C. Nier, J. Biol. Chem. 139~

377 (1941).

904


[36]

of carbon dioxide. The yon Pechmann Reaction ~9was adapted by Racusen and Aronoff 3° to the degradation of malic acid in order to differentiate between carbons 1 and 4. When malic acid is heated in sulfuric acid the product is coumalic acid, as shown in the equation. Coumalic acid is cleaved in dilute acid, and (4)

(3)

(2)

(1)

100% H~SO4

2HOOC--CH2--CH--COOH

[

100 °

OH

(2) CH

%(3) (1> CH -]- 4H20 ~- 2CO

/ HOOC--C(3)

li .o

Methods in Enzymology, Volume 4: Special Techniques for the Enzymologist

Methods in Enzymology, Volume 152: Guide to Molecular Cloning Techniques

Methods in Enzymology 492 - Biothermodynamics, Part 4

Nuclear Receptors, Volume 364 (Methods in Enzymology)

Methods in Enzymology. Liposomes

Methods in enzymology, Volume 167 - Cyanobacteria

DNA Replication, Volume 262 (Methods in Enzymology)

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Methods in Enzymology (Liposomes )