Advances in Protein Chemistry, Volume 32

ADVANCES IN PROTEIN CHEMISTRY Volume 32

CONTRIBUTORS T O THIS VOLUME Claude Balny Pierre Douzou Robert E. Feeney

Elvin A. K a b a t F. MacRitchie Yin Yeh

ADVANCES IN PROTEIN CHEMISTRY EDITED BY

C. B. ANFINSEN

JOHN T. EDSALL

Nationol Institute of Arthritis,

Biologicol Laboratories H a n a r d university

Metabolism and Digestive Diseases Bethesda, Maryland

Cambridge, Maswchusetts

FREDERIC M. RICHARDS Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut

VOLUME 32

1978

ACADEMIC PRESS

New Y o r k

S u n Francisco

A Subsidiary of Harcourt Brace Jovanovich, Publishers

London

COPYRIGHT @ 1978, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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LIBRARY O F CONGRESS CATALOG CARD NUMBER:44-8853 ISBN 0-12-034232-4 PRINTED IN THE UNITED STATES OF AMERlCA

CONTENTS

CONTRIBUTORS

TO VOLUME

.

32

vii

The Structural Basis of Antibody Complementarity

ELVIN A. KABAT I. Introduction . 11. Immunochemical Studies of the Size and Shape of . Antibody Combining Sites 111. Fragments, Chains, and Domains of Immunoglobulins and Antibodies IV. The Recognition and Role of Variable Regions . V. Hypervariable Regions or ComplementarityDetermining Segments (CDR) . VI. Correlation of Variability with Physiochemical Properties of Amino Acids in Immunoglobulin Chains . VII. Affinity Labeling Data and Hypervariable Segments . VIII. X-Ray Crystallographic Studies on the Combining Sites of Myeloma Proteins . IX. Predictive Attempts Based on Inserting CDR Sequences upon a Constant Framework . X. Predictive Attempts Based on Statistical Examinations of Sequences and Distribution of Amino Acids in CDR . XI, Other Directions . References .

1 6 15 18 21

26 30

32 37 45 61 69

Protein Fractionation at Subzero Temperatures PIERRE DOUZOU AND CLAUDE BALNY

I. Introduction . 11. The Properties of Aqueous-Organic Mixtures as a Function of Temperature: Physiochemical Data and Their Manipulation 111. Adaptation of Fractionation Techniques to Subzero Temperatures . IV. Applications . V

77 78

134 146

vi

CONTENTS

V. Conclusions Addendum References

.

184 185 185

. .

Antifreeze Protein from Fish Bloods

ROBERT E. FEENEY AND

YLN YEH

I. Introduction . 11. Freezing Resistances of Polar Fish Blood Sera . 111. Preparation and Properties of Antifreeze Glycoprotein . (AFGP) from Antarctic Fish Bloods IV. The Antilectin Activity of AFGP . V. Effects of AFGP on Freezing . VI. Activities of Chemical Derivatives of AFGP . VII. Other Proteins Reported To Have Antifreeze Activity . VIII. Theoretical Aspects of Actions of Substances Lowering Freezing Temperatures . IX. Discussions of Models for Mechanism of Action of Antifreeze Proteins . X. Summary and Conclusions . References . Note Added in Proof .

191 193 198 219 222 233 246 254 270 276 278 28 1

Proteins at Interfaces

F. MACRITCHIE I. Introduction . 11. Experimental Techniques . 111. Kinetics and Mechanism of Adsorption . IV. Conformation of Protein Molecules at Interfaces . . V. Equilibrium Aspects of Adsorption VI. Reactions at Interfaces . VII. Proteins at Interfaces in Biological Systems . References . AUTHOR INDEX

SUBJECT INDEX

. .

CONTENTS OF PREVIOUS

283 284 286

293 301 312 319 323 327 337

VOLUMES.

340

CONTRIBUTORS TO VOLUME 32

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

CLAUDEBALNY,INSERM, Unit6 128, B.P. 5051, 34033 Montpellier, and Znstitut de Biologie Physico-Chimique, Foundation Edmond de Rothschild, 75005 Paris, France (77) PIERREDOUZOU, ZNSERM, Unit6 128, B.P. 5051,34033 Montpellier, and Znstitut de Biologie Physico-Chimique, Foundation Edmond de Rothschild, 75005, Paris, France (77) ROBERT E. FEENEY,Department of Food Science and Technology, University of California, Davis, California 95616 (191)

ELVINA. KABAT, Departments of Microbiology, Human Genetics and Development, and Neurology, College of Physicians and Surgeons, Columbia University, New York, New York and the National Cancer Institute, National Znstitutes of Heulth, Bethesda, Maryland 20014 (1) F. MACRITCHIE,CSZRO Wheat Research Unit, North Ryde, New South Wales, 2113, Australia (283) YIN YEH, Department of Applied Science, University of California, Davis, California 95616 (191)

This Page Intentionally Left Blank

THE STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY' By ElVlN A. KABAT Departments of Microbiology, Human Genetics and Development, and Neurology College of Physicians and Surgeons, Columbia University, New York, and the National Cancer Institute, National Institutes of Health, Betherda, Maryland

I. Introduction

............................

Abbreviations and Definitions .................... 11. Immunochemical Studies of the Size and Shape of Antibody Combining Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Fragments, Chains, and Domains of Immunoglobulins and Antibodies . IV. The Recognition and Role of Variable Regions . . . . . . . . . . . . . V. Hypervariable Regions or Complementarity-Determining Segments (CDR) VI. Correlation of Variability with Physicochemical Properties of Amino Acids in Immunoglobulin Chains . . . . . . . . . . . . . . . . . . . . . . VII. Affinity Labeling Data and Hypervariable Segments . . . . . . . . . . VIII. X-Ray Crystallographic Studies on the Combining Sites of MyelomaProteins IX. Predictive Attempts Based on Inserting CDR Sequences upon a Constant Framework . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Predictive Attempts Based oh Statistical Examinations of Sequences and Distribution of Amino Acids in CDR . . . . . . . . . . . . . . . . . . . A. Analysis of Pairs of Residues in the CDR . . . . . . . . . . . . . . B. Identification of Residues Involved in Specificity by Examination of Sequences Showing Differences in Specificity but Limited Sequence Variation in the CDR . . . . . . . . . . . . . . . . . . . . . . . . . C. Recognition of Structural and Contacting Residues in CDR by Statistical Analysis of Frequencies of Occurrence of Different Amino Acids . . . XI. Other Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 5

6 15 18 21 26 30 32 37 45 47 49

53 61 69

I. INTRODUCTION Perhaps the most important problem in structural immunology and immunochemistry today is an understanding of antibody complementarity in terms of three-dimensional structure since this should provide new insights leading to the genetic basis for the generation of diversity and will open new perspectives for research in cellular immunology. More precisely one would like to know the mechanism by These studies were aided by grants from the National Science Foundation, BMS-72-02219 A04 and PCM76-81029. Work with the PROPHET computer system is sponsored by the National Cancer Institute, National Institute of Allergy and I n f e c tious Diseases, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institute of General Medical Sciences, Division of Research Resources (Contract No. NOl-RR-42147), National Institutes of Health. 1

2

ELVIN A. KABAT

which the immune system can produce an enormous repertoire of different receptor sites, estimates of the number of which range from at least lo4to as high as lo7,each distinct site showing a remarkable degree of specificity. Extraordinary progress has been made in the past 10-12 years both in sequencing and from X-ray diffraction studies on immunoglobulins and antibodies. I n a sense these have only opened to our view the magnitude of the problem and the need for a somewhat different approach if it is to be solved within a reasonable time. X-Ray diffraction studies on a particular class of protein, such as the cytochromes c , hemoglobins, snake venoms in a wide range of species, have shown that there is a remarkable preservation of secondary and tertiary structure of the molecule and indeed of sequence in the active-site region. Indeed, if one knows the three-dimensional structure of cytochrome c from one species one can approximate both the folding of the polypeptide chain and the shape of the active-site region of another cytochrome c; a stereo model of cytochrome c has been constructed using tuna cytochrome c for one chain and horse for the other (Takano et al., 1973). Th'is preservation of site structure over the evolutionary time span is the result of natural selection; individuals with mutations such that the polypeptide chain could not fold u p to form a functional molecule would not survive. Some mutations in proteins may not be lethal but may alter the molecule functionally, producing disease; many such hemoglobin mutants have been studied (for sequence differences, see Dayhoff,

1972, 1973,1976). Since mutations are occurring constantly, what one observes in examining the various species are those mutations which permit the proper three-dimensional structure to form. The resulting differences in amino acid sequence are recognized as species differences. Since the sequence for each species remains constant, there may well be some specific advantages of any given amino acid residue in one species, but from an overall point of view the differences are merely mutational noise compatible with satisfactory three-dimensional folding to give a functional molecule. Thus, if one has elucidated from X-ray diffraction studies at sufficiently high resolution the three-dimensional structure of one or two members of a class of proteins, such as the hemoglobins or the cytochromes c , one can predict, except for relatively minor differences, the threedimensional structures of all members of the class. One can also predict with substantial confidence the effects of mutations at a given position and validate the predictions crystallographically. This has been done for human hemoglobin mutants (Greer, 1971). One can

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

3

thus envisage acquiring a reasonably complete understanding of the three-dimensional structure of each of these classes of proteins within a finite time. Antibody complementarity is a far more complex phenomenon. Heterogeneity and diversity are the hallmarks of the antibody response (Kabat, 1961, 1966a,b, 1976a). A protein antigen generally contains many antigenic determinants, and the antibody response even to one determinant may result in a population of antibodies of different specificities, each recognizing different aspects of the same determinant. This has been shown most clearly with polysaccharide antigens containing a single sugar such as dextran, a dextran with 96% a1 + 6 linkages and 4% a1 + 3 like linkages giving rise to a population of antibody molecules with combining sites complementary to different lengths of the a 1+ 6 polysaccharide chain, e.g., to different numbers of al+ 6 glucose residues or, in other terms, having antibody combining sites of different sizes (Kabat, 1956, 1957, 1960, 1976a). Antibodies to polysaccharide antigens such as dextrans and levans produced in humans, while heterogeneous, often show restricted heterogeneity as shown by acrylamide gel electrophoresis (Yount et al., 1968), isoelectric focusing patterns (Cisar et al., 1975), starch gel electrophoresis of the separated polypeptide chains (Edelman and Kabat, 1964) and in possessing fewer genetic markers than found on the total immunoglobulin of the same individual (Allen et al., 1964; Yount et al., 1968). Antibodies to the streptococcal groups A and C carbohydrates and to the pneumococcal polysaccharides are also heterogeneous, but it has been possible in individual rabbits to obtain homogeneous antibodies or antibodies of substantially restricted heterogeneity (Haber, 1970; Krause, 1970) as demonstrated by the above criteria and by microzone electrophoresis (see Kochwa and Kunkel, 1971) in amounts suitable for sequencing; such studies have shown substantial differences in sequence among antibodies of relatively similar specificity (Jaton, 1974, 1975, 1976; Haber et al., 1975; Thunberg and Kindt, 1975; Braun et al., 1976a,b; for additional references, see Kabat et al., 1976b). It is doubtful whether studies on antibodies would have advanced as rapidly had not a substantial body of data on the specificity, sequence, and three-dimensional structure been accumulated on the immunoglobulins and related molecules associated with certain neoplastic diseases. A large proportion of patients with a disease of plasma cells called multiple myeloma had been known for over a century to excrete in their urine large amounts of a class of protein named

4

ELVIN A. KABAT

Bence Jones proteins. These proteins because of their unusual behavior of precipitating when heated to 60"-70"C at pH 4.5-5, again going into solution on boiling, and reprecipitating on cooling, have proved to be of great value in the diagnosis of multiple myeloma. Bence Jones proteins were shown by Edelman and Gally (1962) to be the light chains of immunoglobulins. It was recognized quite early that Bence Jones proteins from different individuals varied in chemical (Putnam and Miyake, 1954), physical (Gutman et al., 1941; Moore et al., 1943), and immunological properties (Bayne-Jones and Wilson, 1922; Moore et al., 1943; Korngold and Lipari, 1956; Migita and Putnam, 1963; Osserman et al., 1957); indeed among humans no two individuals have been shown to have identical Bence Jones proteins, and estimates of as many as 4000 different human Bence Jones proteins have been made (Quattrocchi et d., 1969). This diversity is intimately related to antibody specificity. The serum of patients with multiple myeloma often had high levels of serum globulin. When the Tiselius electrophoretic method was used and later with paper or gel electrophoresis, these sera were shown to contain high levels of proteins with a very narrow range of electrophoretic mobility unlike the broad heterogeneous immunoglobulin peaks. The mobilities of the sharp peaks in the sera of these patients varied from individual to individual. These serum immunoglobulins also differed in molecular weight. A macroglobulin now known as IgM was first isolated by Waldenstrom (1944,1948),and the neoplastic disease is termed Waldenstrom macroglobulinemia. Another crucial development was the finding that the BALB/c (Potter, 1972, 1977a) and later that the NZB strains of mice (Warner, 1975) when injected with paraffin oil develop a disease like multiple myeloma and also often excrete Bence Jones proteins. This not only provided an experimental model, but also permitted detailed comparison of mouse Bence Jones proteins and immunoglobulins with their human counterparts, an indispensable prerequisite for the study of antibody specificity. Relatively enormous quantities (kilograms in some instances) of Bence Jones proteins were obtainable from the urine of patients; plasmapheresis yielded substantial quantities of myeloma proteins. Large amounts of the corresponding mouse proteins were also obtainable; proteins from such neoplasms were in almost all instances monoclonal and homogeneous. About 12 years ago a number of myeloma immunoglobulins from both humans and mice were found to have specific receptor sites for various substances, and these are now considered to be monoclonal antibodies. These include human antibodies reacting with EDNP-

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

5

(see list of abbreviations below) or TNP-lysine, streptolysin 0 antiIgG, anti-blood group I and i, a hydroxy derivative of vitamin K, lipoproteins, etc., and mouse myeloma proteins with antibody sites reacting with DNP, phosphocholine, (Y 1 + 6 and (Y 1+ 3 linked dextrans, P2+ 1 and P2+ 6 linked fructosans, galactans, nonreducing terminal DG~cNAc,etc. (for a more complete listing, see Kabat, 1976a; Potter, 1977a,b). These, however, represent but a very small selection of the diversity of specific sites which the antibody-forming mechanism can generate. It is manifestly impossible to envisage, within a reasonable period of time, a solution to the problem of antibody complementarity by elucidation of the structure of all kinds of antibody combining sites. One must add at least another dimension to such attempts. This review will consider how one may gain more insight into the problem by attempting to make predictions as to the shape, structure, contacting and structural elements of complementarity regions of antibody combining sites which, if verified crystallographically, would provide insight into the structures of many kinds of antibody combining sites.

ABBREVIATIONS

AND DEFINITIONS

DNP-dinitrophenyl as in eDNP-Lys CDR-complementarity-determining regions or segments, the three portions of the light and of the heavy chain showing hypervariability and which were predicted (Wu and Kabat, 1970) and subsequently found to form the antibody combining site. DGlc-Dglucose DFru-Dfructose; in oligosaccharides the furanose or five-membered ring is indicated by f, as DFruf is Dfructofuranosyl. IM-the isomaltose or a1 + 6 linked series of oligosaccharides of D G ~followed C by a number which indicates the size of the oligosaccharide; thus IM3 is the trisaccharide ~ G l c a+ l 6DGlcal + 6DGlc. All glycosidic sugar units are in the six-membered pyranose form unless the furanose form is indicated. NMR-nuclear magnetic resonance ESR-electron spin resonance Idiotypic specificity-Antibodies and myeloma globulins of a given specificity show differences in antigenic specificity ascribable to differences in sequence in the variable regions of their light and heavy chains. Some of these idiotypic determinants are inhibitable by the antigenic determinant or ligand for which the antibody or myeloma protein is specific, whereas others are not. Those idiotypic determinants which are inhibitable are hypothesized to in-

6

ELVIN A. KABAT

volve residues of the CDR whose side chains project away from the interior of the combining site and whose three-dimensional structures are altered by conformational changes occurring when the combining site interacts with the specific ligand so that they no longer react with the antiidiotypic antibody (for a detailed discussion, see Kabat et al., 1976a). Noninhibitable idiotypes involve the V-region away from the site. Idiotypic specificity is thus another important parameter of antibody heterogeneity and diversity. No amino acid residues have as yet been implicated in the specificity of either inhibitable or noninhibitable idiotypes. Cross-reactions occur among idiotypes of a given specificity, and a given idiotype may be present in small amounts in the serum of normal animals. In an immunized animal, the antibody and nonantibody immunoglobulins may show substantial amounts of an idiotype which was not detectable in the serum prior to immunization (Oudin, 1974; Hopper and Nisonoff, 1971; Natvig and Kunkel, 1973; Childs and Feizi, 1975; Casenave et al., 1974). Immunoglobulin G (IgG) subclasses-There are four subclasses, called IgG1, IgG2, IgG3, and IgG4, which show sequence differences in the C-domains (see Fig. 4); they also show differences in their antigenic determinants.

11. IMMUNOCHEMICAL STUDIESOF THE SIZE AND SHAPE OF ANTIBODY COMBINING SITES Since Landsteiner showed that a low molecular weight hapten like arsanilic acid would compete for the antibody combining site with an antigen, prepared by coupling arsanilic acid to a protein, and inhibit precipitation, a tool was available to study the structure of antibody combining sites. Using synthetic dipeptides (Landsteiner and van der Scheer, 1932, 1934; see Landsteiner, 1945) and disaccharides (Goebel et al., 1934) coupled to proteins, it could be demonstrated that the C-terminal amino acid or the nonreducing end of the disaccharide in reacting in the antibody combining site contributed the major portion of the binding energy. In addition, with such azoproteins the antigenic determinant often involved not only the grouping introduced, but also a portion of the protein onto which it was attached (Hooker and Boyd, 1933). Thus arsanilic acid azotyrosine was a better inhibitor than arsanilic acid itself. Since the grouping diazotized to the protein could attach to any accessible tyrosine or histidine in the protein, and since the sequence around each tyrosine vaned, the antigen was very heterogeneous and the antibody response was

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

7

accordingly extremely complex and one could obtain no data on the size of antibody combining sites. A powerful approach to estimating the size of antibody combining sites came from the use of homopolymers as antigens. Dextrans were shown (Kabat and Berg, 1953) to be antigenic in humans, two injections of 0.5 mg a day apart giving rise to substantial amounts of precipitating antibodies and to the development of wheal and erythema skin sensitivity; these findings were confirmed by Maurer (1953). The antibodies were shown to be specific for chains of a1 + 6 linked glucoses, and when oligosaccharides of the isomaltose series became available (Jeanes et al., 1953; Jones et al., 1956; Turvey and Whelan, 1957) they could be used as a probe or molecular ruler to explore the sizes of human antidextran sites. It was observed that inhibiting power on a molar basis increased with increasing chain length but that the increment in inhibiting power per added glucose decreased and an upper limit was reached, generally with six sugars (Kabat, 1956, 1957, 1960). In its most extended form, the hexasaccharide (IM6), isomaltohexaose, measures 34 x 12 x 7 A. I t was also observed that each person did not produce a homogeneous population of antibody combining sites, since the ratio of inhibiting power of the tri- (IM3), tetra- (IM4), penta- (IM5), and hexasaccharides to one another differed for each individual. Moreover, absorption of the antidextran on Sephadex and successive elution with IM3 followed by IM6 fractionated the antibody into two populations (Schlossman and Kabat, 1962; Gelzer and Kabat, 1964) which varied in their relative inhibitability by small, as compared with larger, oligosaccharides, confirming that they were mixtures with combining sites of different sizes. Other studies (see Kabat, 1966a, 1976a; Goodman, 1969, 1975; Schlossman et al., 1968) showed that antibody combining sites for various kinds of antigenic determinants-carbohydrate, synthetic polypeptides, protein, nucleic acid, etc.-produced in various species varied from a lower limit of between one and two sugars, 4 to 6 A, to an upper limit of about 6 or 7 sugars, or about 34 A, in most extended length. From the standpoint of evaluating structural differences in antibody combining sites, these purified antidextrans gave relatively little insight because of their heterogeneity, and only when various myeloma antidextrans (Leon et al., 1970; Lundblad et al., 1972; Cisar et al., 1974,1975) were recognized could progress be made. Myeloma globulins have the disadvantage as compared with antibodies that one does not know precisely the antigen to which they are complementary. Nevertheless in a system such as the a1 +-6 dextran myeloma

8

ELVIN A. KABAT

antidextran, when their combining site sizes are at or close to the upper limit found for a1 +- 6 antidextrans and when they are tested with a variety of oligosaccharides of different structures and found to be a1 + 6 specific, the possibility is minimal that the myeloma globulin is more specific for other unrecognized determinants of an entirely different structure (Cisar et al., 1974,1975). Indeed the high specificity of antigen-antibody interactions makes it possible to obtain information about the size and shape of antibody combining sites as well as to elucidate the structures of antigenic determinants by identifying the structure which competes most effectively with antigen for the combining site (Kabat, 1961, 1976a). Figure 1 shows the findings with four IgA myeloma antidextrans. Three of these (W3434, W3129, QUPC 52) were specific for a1 +- 6 linked, and the fourth for a1 + 3 linked, glucose (UPC 102). The specificity of the inhibition and the reaching of an upper limit are seen. In both W3434 and W3129, IM5, IM6, and IM7 are equal as inhibitors on a molar basis and better then IM4 and IM3 (Fig. 1A and B), indicating a site complementary to a chain of five glucoses. With QUPC 52, however, IM6 and IM7 are clearly better than IM5, which in turn is better than IM4, etc., indicating a site complementary to six glucoses. Figure 1A and B also shows the a1 +- 6 glucosyl specificity, since disaccharides of glucose linked other than a1 +- 6 react much less strongly than IM2; ~ G l c a +l 6 DG~cNAcalso is not as good IM2, again indicating the need for a second glucose. The fourth myeloma antidextran is specific for a1 +- 3 linked glucose, nigerotriose, -tetraose, and -pentaose being equally potent and better than nigerose (Fig. lE), which in turn was better than diglucoses linked a1 +- 4, a1 +- 2 and a1 +-6. Two other myeloma antidextrans with a1 +- 3 specificity had been studied. One, an IgM myeloma, MOPC 104E, had a site complementary to the trisaccharide (Leon et al., 1970), whereas with the other, J558 belonging to the IgG3 subclass, nigeropentaose was the best inhibitor (Lundblad et al., 1972). The shapes of the inhibition curves in Fig. lA, B, and E are atypical since IgA myeloma proteins are mixtures of monomers and polymers. Using a lower molecular weight dextran N-l50N, rather than the higher molecular weight native B512, typical inhibition curves as shown in Fig. 1C were obtained. If the myeloma antidextran was separated into monomer and polymer portions, the usual inhibition curves could be obtained (Cisar et al., 1974). When two of these myeloma antidextrans were studied by equilibrium dialysis (Fig. 2A), a surprising result was obtained. QUPC 52, with the larger size site, had an association constant, K,, of 8.4 x lo3

b

0

GLUCOSE

A ISOMALTOSE A ISOMALTOTRIOSE

ME

0

4 NIGEROSE

0 GLUCOSIDE 4 GLUCOSIOE ME m D MANNOSIDE II ME 3-0-ME a Q GLUCOSIOE x a p Glc (1-61 Glc NAc V MALTOSE 0 KOJlBlOSE

A NIGEROTRIOSE

*

0 ME

0 ISOMALTOTETRAOSE b ISOMALTOPENTAOSE 0 ISOMALTOHEXAOSE 8 ISOMALTOHEPTOSE

NIGEROTETRAOSE NIGEROPENTAOSE NIGEROHEXA-HEPTAOSE 3’0 p GLUCOSYLISOMALTOTETRAOSE 34 a GLUCOSYLISOMALTOHEXAOSE

4

9

o

o 44,2

2 0 0 ~ 1W3434fdiluied 1/10) t 15pg B512

50fil W3129fdiluied l / I O ) t 15pg 651.2

GLUCOSYLISOMALTOHEXAOSE

TOTAL Volume:350pl

TOTAL Volume: ZOOp1

.!mld 50pl QUPC 52 (diluied 1/10)

loor

t

15pg B 512

TOTAL Volume: 2OOb1

r

20

I

0020.040060.080.10

0.14

0.18

0

2

4 0 ~ UPC 1 102t 2 0 p g B 1355-5-4

‘0

02

04

06

08

10 0 2 4 6 8 10

15

20

6

4

8

10

TOTAL Volume: ZOOpl

25

30

40

50

60

MICROMOLES INHIBITOR ADDED

FIG. 1. Inhibition by various oligosaccharides of precipitation of mouse,myeloma proteins by dextran. From Cisar et al. (1974), with permission.

10

ELVIN A. KABAT

FIG. 2. Scatchard plots of equilibrium dialysis data at 25°C with [3H]isomaltoheptitol. (A) Protein W3129 (0)at 10.5 mg/ml and protein QUPC 52 (0) at 20.6 mg/ml. (B) Purified human antidextrans, 1 Ds-w IM3 eluate 1 (0) at 6.68 mg/ml and 1 D,-,, lM6 eluates 1-6 (0)at 2.56 mg/ml. Calculations were done with a molecular weight of 150,000 for myeloma proteins and human antidextrans. From Cisaret al. (1975),with permission.

lo5M - l . This had not been observed for antibodies, those with larger size sites generally having higher K,. Figure 2B also shows findings with two portions of human antidextran which had been absorbed on Sephadex and eluted first with IM3 and then with IM6, the latter having the higher association constant. The heterogeneity of the antibody compared with the homogeneity of the myeloma antidextrans is clearly seen from the Scatchard plots (Cisar et al., 1975). An important insight into shapes of antibody combining sites emerged from attempts to resolve the dilemma of the W3129 and QUPC 52 K,'s. This was accomplished b y measuring, by competition in equilibrium dialysis, by fluorescence quenching, or by inhibition of precipitation, the relative contribution of each sugar in the oligosaccharides to the total binding energy. The two proteins differed strikingly. With W3129, methyl aDglucoside and IM2 contributed 55-60% and the trisaccharide over 90% of the total binding energy of the pentasaccharide. However, with QUPC 52 the first two compounds contributed less than 5 % but with the trisaccharide the value rose to 65-70% of the binding energy of the hexasaccharide (Table I) (Cisar et al., 1975). These data were interpreted as indicating that W3129 reacted with ends of chains and had a combining site in which the terminal nonreducing one or two sugars were firmly held in three M-' while W3129, with the smaller size, had a K , of 1 x

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

11

TABLEI Differences between Myeloma Antidextrans W3129 and QUPC 52" W3129 Oligosaccharide best-fitting site Association constant K, ( M - l )

IM5* 1 x 105

QUPC 52 IM6 8 x 103

Relative contribution to total binding of best oligosaccharide (%) Methyl aDglucoside IM2 IM3 IM4 IM5 IM6

56, 5CjC 61, 50 91, 94 91, 94 100,100 95,100

5 t 0 5 72, 64 88,BO 96,92 loo, 100

Data from Cisar et al. (1975); Kabat (197713). IM2, IM3, etc., denote numbers of sugar residues in these oligosaccharides of the isomaltose series ( ( ~ 1 - 6 linked). The first value was obtained by competition in equilibrium dialysis; the second by inhibition of precipitation.

dimensions while QUPC 52 could react at nonterminal locations along linear a1 -+ 6 linked dextran chains. Important supporting evidence came from the use of a synthetic linear a1 + 6 dextran (Ruckel and Schuerch, 1967) with about 200 glucoses but only a single nonreducing end. W3129 was not precipitated by this dextran, but it could inhibit precipitation, whereas QUPC 52 precipitated with the linear dextran. Concanavalin A, with specificity for terminal nonreducing CXDG~C, also does not precipitate with linear dextran (Goldstein et al., 1968). On this basis it was inferred that QUPC 52 had a combining site which was essentially a groove into which any six a1 + 6 linked gIucoses in the interior of a linear chain could fit. In this respect it resembled the lysozyme site (Phillips, 1966). On the other hand, the data on W3129 are best explained if the terminal nonreducing one or two glucoses were in a small pocket or cavity; whether the rest of the site is also a cavity or is a groove is not clear. Human antidextrans could be shown to consist of populations with both types of sites, another parameter of heterogeneity. Recently two NZB myeloma antidextrans (A. Wu et al., 1978) have been found to precipitate with linear dextran and thus have groovetype sites. Inhibition assays for site size were somewhat uncertain. IM3, IM4, and IM5 were equally potent on a molar basis, indicating

12

ELVIN A. KABAT

a site size complementary to a trisaccharide. However, I M 6 and IM7, which were also equal, were somewhat more active. There are two possible interpretations. One is that the site is complementary to IM3, with IM6 and IM7 providing a statistical advantage because of the additional ways in which they could compete with dextran for the site. Evidence for such statistical effects has been obtained (Kabat, 1966a). The alternative interpretation would consider the site as complementary to an internal chain of six glucoses, its contribution to binding of the fourth and fifth glucoses being minimal. It is evident that sequence and high-resolution crystallographic studies of antidextran myeloma proteins with specificity for terminal nonreducing a1 + 6 chains and for internal a1 + 6 chains would contribute immensely to our understanding of the structure of antibody combining sites. These two types of sites if thoroughly characterized might well serve for constructing models of many other kinds of anticarbohydrate sites. Knowing the shapes and sizes of these two kinds of sites and inserting sequences of complementaritydetermining regions or segments (CDR) of other anticarbohydrate antibodies should make it possible to arrange side chains so as to provide appropriate contacts for terminal and nonterminal carbohydrate chains. These models could then be evaluated by crystallographic studies. A few such predictions, if validated, would provide confidence in constructing models of all kinds of antibody sites specific for polysaccharides and probably of other sequential determinants. Myeloma proteins specific for fructosans also fall into two classes (Cisar et al., 1974), those that are p2 + 1 specific and precipitate with inulin and those that are p2 +-6 specific and precipitate with perennial rye grass levan, which is essentially entirely p2 + 6 linked. Those precipitating with inulin were inhibited by p2 1 linked fructosans, while those precipitating with perennial rye grass levan were not. One of the a 1 3 2 specific myeloma proteins, J606, had a site complementary to a chain of two p2 + 1 linked fructofuranosyl residues plus an additional p linkage either to carbon 6 of ~ G l or c to carbon 1 of DFru; since a tetrasaccharide with three p2+ 1 linked fructofuranosyl residues linked p2 + 6 to ~ G l cthis , tetrasaccharide, in which the D G ~ had C been reduced with borohydride, and a trisaccharide with two p2 + 1 fructofuranosyl residues linked /32 + 6 to D G ~ were C equally active on a molar basis and more active than the trisaccharide in which the DGIC ring had been reduced with borohydride. Two other anti p2 + 1 specific myeloma antifructosans, UPC 61 and W3082, had sites involving three p2 -+ 1 fructofuranosyl residues plus the p2 + 6 linkage to ~ G l c .The K , for both W3082 and --f

STRUCTURAL B A S I S OF ANTIBODY COMPLEMENTARITY

13

UPC 61 was 3.6 x 105 M-' by fluorescence quenching; precipitin inhibition as a function of chain length was typical for specificity for nonreducing chains of p2 -+ 1 linked residues. Yet both precipitated with the p2 + 1 linear polysaccharide, inulin, a criterion used with linear dextran as indicating specificity for internal chains. A model of the p2 -+ 1 linked oligosaccharide chain resolved this question, for it showed that most of each fructofuranosyl ring projects out so that a good portion could probably react as if it were a terminal nonreducing residue (Fig. 3) while the p2+ 6 chain is a truly linear molecule (Cisar et al., 1974, 1975). Streefkirk and Glaudemans (1977) have studied UPC 61 and three additional p2-+ 1 specific mouse myelomas ABE 47N, ABE 4, and EPC 109. All except EPC 109 precipitated with inulin in gel diffusion. K , values for proteins EPC 109 and UPC 61 increased up to four p2 + 1 linked fructofuranosyl residues linked 2 + 1 to D G ~ and C for ABE 47N and ABE 4 u p to three p2 -+ 1 linked fructofuranosyl residues linked p2 + 1 to D G ~ when C Fab' fragments were used. When they compared their K , values with those of Cisar et al, (1975)for UPC 61, it became evident that a /32 +- 6 linkage to D G ~ contributed C to binding whereas a p2 + 1 linkage to DGICdid not. Thus four p2 + 1 fructofuranosyl residues linked p2 + 1 to ~ G l gave c the same K , as three p2 +- 1 fmctofuranosyl residues linked p2 + 6 to DG~c.Three pZ -+ 1 ~ F r u flinked /I2 -+ 1 to DGIChad the same K , as two p2 + 1 ~ F m linked f p2 + 6 to ~ G l cetc. , Molecular models showed that the p2 + 6 D G ~structure C resembled p2 -+ 1 DFruf more than the p2 -+ 1 DG~c.It is not clear why EPC 109 does not precipitate with inulin, but this would be consistent with specificity for the nonreducing ends. In each instance it was assumed that only one molecule of inulin could react with an antibody combining site. EPC 109 was thought to be a monomeric IgA while the others were polymeric. Methyl CXDGIC was found to have K , values of 3.3, 1.5, and 1.4 x 102 for EPC 109, ABE 47N, and ABE 4; UPC 61 was not studied. Since the reducing fmctosyl residue in inulin is blocked by p2 la linked DG~c, inulin would conceivably have been capable of reacting by both ends and thus be divalent; however, methyl CXDG~C did not inhibit precipitation of the polymeric IgA myelomas by inulin and was considered to be bound to part of the molecule away from the combining site. Although the picture for p2 -+ 6 specific myeloma antifructosans is not complete, they would appear to be specific for internal chains of /32 + 6 Frufresidues since they precipitate with linear /32 -+ 6 linked perennial rye grass levan. While only relatively short p2 + 6 linked

*

14

ELVIN A. KABAT

FIG.3. Models of trisaccharides of fructose. Left: PDfructofuranosyl2 + 1 PDfruc6 tofuranosyl 2 + 1 Dfructose. Right: ~Dfructofuranosyl2 + 6 pDfructofuranosyl 2 Dfructose. The reducing end has a square of black tape on the hydrogen of the glycosidic hydroxyl. From Cisar et al. (1974),with permission.

oligosaccharides are available, their failure to show any inhibition at concentrations substantially higher than those at which sucrose gave good inhibition in the p2 + 1 system suggests that the 2 + 6 linked fructose disaccharide, like isomaltose with the antidextran myeloma QUPC 52, is not an effective inhibitor for sites reacting with internal chains and that larger oligosaccharides will be required. The approach outlined has also proved to be extremely useful in exploring combining sites of lectins (Kabat, 1978; Wood et al., 1978). In the case of concanavalin A the carbohydrate site originally proposed from X-ray data was incompatible with its capacity to react with

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

15

terminal nonreducing ends of branched polysaccharides, and it was shown that p-iodophenyl aDglucoside bound by the iodophenyl moiety to a hydrophobic region at which the two subunits were joined (Hardman and Ainsworth, 1973). Until X-ray crystallographic studies on these substances are carried out, the immunochemical approach will be indispensable to probing the structure of these and other receptor sites. A recent finding (Shankar Iyer et al., 1976) with the second lectin BSII of Bandeiraea simplicifolia showed it to be specific for terminal Nacetyl Dghcosamine and to react better with a linked than with /3 linked glycosides. However, some p linked disaccharides were better than a linked disaccharides. This anomalous finding was related to the shape of the molecule, flat disaccharides being more active than bent disaccharides (Wood et al., 1978). More important, it was found that the disaccharide chitobiose ~ G l c N A c p l +4~GlcNAcand the trisaccharide ~ G l c N A c a+ l 4 ~ G a l p l4j~ G l c N A cshowed equal inhibitory activity and that the trisaccharide could assume a conformation in which one side of the two DGIcNAc residues was almost superimposable on chitobiose and the molecule was flat. Thus the generally accepted concept that carbohydrate receptor sites in a linear oligosaccharide chain are sequential, with each sugar interacting in the site, no longer necessarily holds for lectins. It becomes possible that antibody sites also may not involve contact with each sugar in a linear sequence. AND DOMAINS OF AND ANTIBODIES

111. FRAGMENTS, CHAINS,

IMMUNOGLOBULINS

Over 40 years ago Parfentiev (1936, 1938) showed that horse sera containing diphtheria antitoxin could be digested with pepsin at pH 4.0-4.5 and yielded an active antitoxin. Subsequent studies by Pope (1939) and by Petermann and Pappenheimer (1941) established that digestion had split off one-third of the molecule, though the digested antitoxin gave the usual Ramon flocculation reaction with toxin. This procedure was applicable to other horse antitoxins, such as tetanus antitoxin, and to antivenoms. Such digested antitoxins came into worldwide prophylactic and therapeutic use. Papain was also shown to fragment horse antitoxins (Petermann, 1942). The nature of these phenomena remained obscure until Porter (1958, 1959) showed that papain split rabbit antibodies into three fragments each of molecular weight about 50,000, two of which retained their antibody combining sites, while the third, which crystallized spontane-

16

ELVIN A. KABAT

ously, was inactive. Fragments with antibody activity are designated Fab and the crystallizable fragment, Fc. The Fab fragments did not precipitate with antigen but could inhibit precipitation of undigested antibody by antigen, Nisonoff et al. (1959) subsequently demonstrated that pepsin-digested antibody precipitated with antigen but, on treatment with cysteine, lost its precipitating capacity while retaining antibody activity; such reduced peptic fragments are termed Fab’. Immunoglobulins were shown to be made up of two chains termed heavy (H) and light (L) by Edelman and Gally (1962),the light chains being Bence Jones proteins; the H and L chains were successfully separated at acid pH on DEAE cellulose (Fleischman et al.,

1962). From those studies, an overall picture of the immunoglobulin IgG molecule emerged (Fig. 4) as a bivalent molecule with a region susceptible to papain and to pepsin, the former splitting above, and the latter below, the interchain S-S bonds giving monovalent and bivalent fragments. Equilibrium dialysis studies showed the Fab fragments to have the same antibody activity as the intact molecule. The other classes of immunoglobulin, IgM, IgA, IgE, IgD, were all built on the same four-chain structure. All had the same light chains, but their heavy chains differed in size, sequence, carbohydrate content, number of disulfide bonds, etc.; some were oligomers of the monomeric structure; two, IgM and IgA, had a third chain termed the J chain (Koshland, 1975), and IgA in secretions was associated with another protein termed S or “secretory piece” (Tomasi and Bienenstock,

1968). In addition to their unique individual specificity, Bence Jones proteins from different individuals were shown by immunological methods to fall into two classes (Korngold and Lipari, 1956), now termed K and A; all five classes of immunoglobulins may contain either K or A light chains. Within K light chains, certain positions, 152 (Kern) and 190 (Inv), respectively, may have two amino acids, the genes for which are Mendelian alleles (Fig. 4). Another important principle in immunoglobulin assembly derives from the finding that the four-chain structure always consists of two identical light chains and two identical heavy chains. Thus in heterozygotes possessing the genes for two alleles, Inva and Invb, each cell secretes only one immunoglobulin, but not both. This is termed allelic exclusion and is another unique feature of immunoglobulin structure. A corollary of this necessitates that both antibody combining sites be identical. Efforts to show the natural occurrence of what was termed heteroligating antibody, a bivalent antibody molecule with

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

17

Fc FIG.4. Schematic view of 4-chain structure of human IgGK molecule. Numbers on right side: actual residue numbers in protein Eu (Edelman et al., 1969; Edelman, 1970); Numbers of Fab fragment on left side aligned for maximum homology; light chains numbered as in Wu and Kabat (1970)and Kabat and Wu (1971). Heavy chains of Eu have residue 52A, 3 residues 82A, B, C and lack residues termed 100 A, B, C, D, E, F, G, H and 35A, B. Thus residue 110 (end ofvariable region) is 114 in actual sequence. Hypervariable regions complementarity-determining segments of regions (CDR): header lines. VL and VH:light- and heavy-chain variable region; CH1,CH2,and CH3:domains of constant region of heavy chain; C,: constant region of light chain. Hinge region in which 2 heavy chains are linked by disulfide bonds is indicated approximately. Attachment of carbohydrate is at residue 297. Arrows at residues 107 and 110 denote transition from variable to constant regions. Sites of action of papain and pepsin and locations of a number of genetic factors are given. Modified from Kabat (1973).

each site having a different specificity (Lanni and Campbell, 1948), have been unsuccessful. Such antibodies may be produced artificially by preparing monovalent Fab’ fragments by peptic digestion from antibodies of two different specificities and reoxidizing them (Nisonoff and Rivers, 1961) to reform the S-S bond (Fig. 4). Sequence studies on the L and H chains established that each contained several disulfide loops of similar size (Fig. 4) and that each chain could be considered as made up of domains of about 110 amino acids; individual functions and properties of immunoglobulin mole-

18

ELVIN A. KABAT

cules tend to be domain associated. The Fab and Fc fragments are separated by an essentially open segment, a hinge region containing carbohydrate as well as the sites susceptible to pepsin and papain cleavage. Low-resolution X-ray crystallographic studies of an IgG myeloma protein showed it to be a T-shaped molecule with a flexible hinge (Sarma et al., 1971). The two-chain structure is appearing in other systems of immunological interest. The major histocompatibility complex (MHC) contains the genes for various transplantation antigens present on cell membranes HL-A in man and H-2 in the mouse, and these have been also shown to be made up of two chains. One chain, the pz microglobulin of molecular weight 12,000, first isolated from human urine (Berggird and Bearn, 1968) shows some homology to the CH3domain of immunoglobulin (Peterson et al., 1972), the other, of molecular weight 46,000, has the antigenic determinants of the transplantation antigens. The heavy chain of HL-A and H-2 antigens is split by papain to give a molecule of lower molecular weight. The Ia antigens, or immune response region-associated antigens, are also two chain structures, of molecular weights about 35,000 and 26,000 after papain treatment, and are present on the cell membranes of lymphocytes; the genes of the immune response region are also part of the MHC (Silver et al., 1976; Snary et al., 1976; Springer et al., 1976). Another set of antigenic markers associated with the same chromosome as the MHC in the mouse recognizes certain lymphocyte surface receptors (Ly antigens) and has been used to differentiate thymusderived lymphocytes (T lymphocytes) involved in helper and suppressor functions (Cantor and Boyse, 1975). IV. THE RECOGNITION AND ROLE

OF

VARIABLEREGIONS

Further insight into the problem of antibody specificity and structure came from sequencing chains of Bence Jones proteins. Hilschmann and Craig (1965) sequenced K Bence Jones proteins from two individuals and found that they differed strikingly in their aminoterminal halves while their carboxy terminal halves were identical. As additional sequences became available these findings were substantiated, and indeed the extent of variation from one Bence Jones protein to another in amino acid sequence of the N-terminal 107 residues was astonishing, and it became clear that antibody specificity could be accounted for by sequence variation in the variable domains. Four of 85 Bence Jones proteins gave a second peak on Sephadex G-100; on fingerprinting, this second peak was found in each case to

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

19

have a different set of peptides (Cioli and Baglioni, 1966). In comparing the fingerprints of the first peak of intact Bence Jones protein with the second peak, it was found that the former, if K , had, in addition to the unique peptides seen in the second peak, those characteristic of all K chains. If A, they had the set of peptides possessed by all A chains. This again showed the existence of distinct variable and constant domains. The existence of such variation in the N-terminal half of the light chains while the C-terminal half remained constant was a new phenomenon in protein chemistry. As the number of sequences increased and as such data on mouse Bence Jones proteins and later on heavy chains of immunoglobulins were accumulated, it became clear that understanding the nature of the structural and genetic basis for the variable domains of both chains was crucial to understanding antibody specificity. When the first two sequences of mouse Bence Jones proteins were compared with the two human sequences, it became evident that the constant half of the molecule was subject to the same evolutionary development as all other proteins, differences in amino acid sequences between the two species being generally similar in extent and distribution. The variable domains, however, were remarkably different. Indeed, each human sequence resembled a mouse sequence more than it did the other human sequence. Thus at many positions the same amino acid was present in a human and a mouse chain, while the other human or the other mouse chain had a different amino acid at that position. These differences, of course, are now recognized as variable-region subgroups (Hood et al., 1967; Milstein, 1967; Niall and Edman, 1967). When mouse and human variable and constant regions were compared, there was a paucity of such species differences in the variable regions (Kabat, 1967a). Since there are many species but only 20 amino acids, the term phylogenetically associated residue has been substituted for species speciJc residue (Kehoe and Capra, 1972). Another unique feature of the V-domain was the occurrence (Kabat, 1967b) of invariant glycines at certain positions. Most notable are the two Gly residues at positions 99 and 101 in the light chain and two corresponding GIy residues at positions 104 and 106 in the heavy chain; these Gly occur near the C terminus of the V-domains and were postulated to serve as a pivot, providing flexibility and permitting the walls of the site to move somewhat to ensure optimal binding of the antigenic determinant much as the walls of the lysozyme site had been shown to adjust to bind the hexasaccharide substrate. The role

20

ELVIN A. KABAT

of Gly as a residue in providing flexibility has been established by Bode et al. (1976) in trypsinogen by X-ray crystallography; Gly was present at five of seven positions at which the chain became flexible. In every light chain and in all but one heavy chain, Eu, the invariant Gly residues are separated by one residue; in Eu there are two adjacent Gly at positions 106 and 107. Saul et al. (1978) and Poljak et al. (1974) note the superimposability of the a-carbons of Phe-Gly-Gly-Gly residues 98-101 in VL and TrpGly-Gln-Gly in residues 103-106 in VH of Newm. They consider this as evidence against these glycines functioning as a pivot and attribute the invariance of the Gly residues to the need for VH-VLand intrasubunit contacts and the limited space available in this portion of the molecule. Whether these invariant Gly residues can serve as a pivot will ultimately be established by comparisons of the filled and empty sites when a ligand specific for the entire site is available. Invariant Gly, defined as occurring with a frequency of 0.95 or higher to allow for errors in sequencing and occasional acceptable mutations at a position, is present at positions 16, 57, 64 of the light chains and at positions 8,26, and 42 of the heavy chains of all species for which data are available. Gly at position 16 in V, and Pro-Gly at positions 41 and 42 in VH are important in making hairpin bends (Padlan, 1977b). Gly is the most frequently occurring amino acid in the heavy chains, followed by Thr, Leu, and Val, and is the third most frequent in the light chains after Ser and Thr (Kabat et al., 1977). Excluding immunoglobulins from Dayhoff s tabulation, Gly is the most frequently occurring amino acid, followed by Ala, Leu, and Lys. As additional sequences of Bence Jones proteins were elucidated, it became possible to classify light chains into subgroups; human K and A light chains into three and five subgroups, respectively designated as VKI,VJI, VJII, etc. (Hood et al., 1967; Milstein, 1967; Niall and Edman, 1967). From sequences, generally of the first 20 to 24 positions from the N terminus, 5 of 15 proteins in the VKIsubgroup and 3 of 9 VKIIIproteins had identical sequences. This provided the first evidence that antibody specificity could not be generated anywhere in the variable domain and that the first 23 amino acids would not be complementarity-determining. It was postulated, therefore, that there would be two kinds of residues in the V-domain, those making contact with the antigenic determinant and those involved only in three-dimensional folding, making up the framework. The first 23 amino acids were seen to be part of the framework (Kabat, 1968). Two regions of higher variability were noted, one consisting of residues 24-34, and the other of residues 89-97; each of these two segments began after an invariant Cys and was followed by an invariant

STRUCTURAL

BASIS OF ANTIBODY COMPLEMENTARITY

21

residue Trp 35 and Phe 98. These two hypervariable stretches would be brought together b y the disulfide bond 123-1188 and contained the positions with many amino acid substitutions noted b y Milstein (1967), Milstein and Pink (1970), and Fran&k (1970). It was postulated (Kabat, 1968, 1970) that these hypervariable segments would contain the complementarity-determining residues; episomal insertion was suggested as a mechanism (cf. also, Wu and Kabat, 1970; Capra and Kindt, 1975).

v. HYPERVARIABLE REGIONS OR

COMPLEMENTARITY-

DETERMINING SEGMENTS(CDR) To distinguish further between framework residues, which could show the usual mutational noise, and those positions which might be involved in antibody complementarity and thus show much greater variation, a parameter termed variability was defined by Wu and Kabat (1970): Variability =

number of different amino acids at any position frequency of the most common amino acid at that position

I n using this equation, sequences are first aligned for maximum homology. The data first used included complete and partial sequences on 77 Bence Jones proteins and human and mouse immunoglobulin light chains, which were considered as a single population and included human K , human A, and mouse K chains. In the original data at position 7, 63 proteins had been sequenced; Ser occurred 41 times, and four different amino acids were present, Pro, Ser, Thr, and Asp. The frequency of the most common was 41/63, or 0.65, and the variability was 4/0.65 = 6.15. In this equation an invariant residue would have a variability of 1, and the theoretical upper limit for 20 amino acids occurring with equal frequency would be 20/0.05 = 400. The variability plot showed three hypervariable segments (Fig. 5), the two seen previously (Kabat, 1968, 1970) and the third at residues 50-56 corresponding to that of Frankk (1970), who had noted a high incidence of nonhomologous replacements at four positions in this region as well as at positions in the other two regions. A number of the sequences examined differed in length; in the alignment for maximum homology such gaps or insertions, which varied up to six residues, occurred in or adjacent to the hypervariable segments. Variability is usually not calculated for the insertions. The variability equation was used to examine the sequences of the heavy chains as soon as a sufficient number became available (Kabat

22

ELVIN A. KABAT

Position

FIG.5. Variability at different positions for the variable region of light chains. GAP indicates positions at which differences in length have been found. From Wu and &bat (1970), with permission.

and Wu, 1971). Three segments of hypervariability were seen when sequences were aligned for maximum homology in positions corresponding to those in the light chains but displaced several residues from the corresponding Cys residues toward the C-terminus of the chain; these differed in length from those of the light chain and were also associated with differences in length. They comprise residues 31-35,5045, and 95-102 and include gaps or insertions tabulated as 35 A,B; 52 A,B,C, and 100 A,B,C,D,E,F,G,H. Another stretch at which differences in length were seen is allowed for as 85 A,B,C. The most recent computer plot of the heavy-chain data in the data bank (Kabat et al., 1976b) of all variable region sequences is seen in Fig. 6. High variability was noted also at positions 81, 83, 84, 85 when human heavy chains were examined, and this is considered another hypervariable region by Capra and Kehoe (1974). Unlike the other hypervariable regions it became less marked when several species of heavy chain were considered. It is not seen in mouse heavy chains (Vrana et al., 1977) and is not apparent when all heavy-chain data are combined (Fig. 6). Residues 84 and 85 of rabbit heavy chains, however, are involved in a allotypic specificity (Ansari et al., 1976; Mage,

23

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY ALL HEAVY CHAINS

120. 110.

100.

v

90.

A

R

BB. 70.

60. L

50.

T

40.

I

Y

30.

28. 10. 0.

J + - I

0.

10.

20.

30.

40.

60. RESIDLE 50.

70.

60.

98. 100

1 1 0

120

FIG. 6. Variability at different positions for the variable region of heavy chains. The plot was made by the PROPHET Computer System (Raub, 1974). From Kabat et al. (1976b).

1977), so that some variations in sequence would be expected. There are four subgroups of heavy chains, VHI, VHII, VHIII, and VHIV. The variability plot has become the most widely used reference standard for comparing variable domains. It has the advantage that it may be used to compare selected populations of light or of heavy chains, such as all human VHI chains, etc. It may be continuously kept up to date as more sequences accumulate. Most striking has been the retention of the three CDR as the numbers of chains sequenced have increased. Other parameters and correlations of other properties relate closely to findings by variability plots. The upper limit for variability depends upon n2 when n is the total number of sequences considered; since there are 20 amino acids the upper limit becomes 20 x 20 = 400 if more than 20 sequences are available. Especially noteworthy are the findings with mouse A chains. This class of chains had not been described when the variability parameter was introduced. Shortly thereafter, Weigert et al. (1970; Appella, 1971)found and sequenced the mouse Vx chains. Mouse A was found to be unusually highly selected, mutational changes in the framework being negligible. To date, twelve mouse A myeloma proteins have identical sequences throughout their entire variable regions; seven

24

ELVIN A. KABAT

instances have been found (Cohn et al., 1974; Weigert and Riblet, 1976) with sequence differences (Fig. 7), and these, with the exception of a substitution at position 48 of Leu for Ile, are confined exclusively to the hypervariable segments as originally defined (Wu and Kabat, 1970). Mouse A chains, however, have an insertion of three residues in the first hypervariable region, so that this position may also be in the second CDR. The original data base for defining the hypervariable regions in both light and heavy chains was made up of proteins generally not

1210 1A 1 1 A1 1A 1 1 h1

1 A1

lh2 1A4

25

50

75

lo0

POSITION

FIG. 7. Location and nature of amino acid substitutions in the mouse V, region superimposed on the variability plot of Fig. 6. The three CDR according to Wu and Kabat (1970) are shaded. All other residues in the sequence were identical. The number of instances of each sequence as well as the number of base changes to obtain it from A0 are given. Two NZB myeloma proteins, PC 2797 and PC 960, also have the A 0 sequence as far as studied, but the sequence of residues 11-24 is based on total composition and residues 70-87 have not been examined. Another NZB myeloma protein, PC 5060, has an Asn at position 93 as shown in the figure; the uncertainty at positions 1-24 is indicated in parentheses, and the absence of data at positions 70-87 by a gap. The 10 other sequences identical to A 0 are HOPCI, J698, H2061, W3159, Y5431, Y5485, Y5830, Y5669, xS104 and MOPC 511. IgA myeloma globulins S104E and J558 have specificity for a1 + 3 dextran, but their combining sites differ in size; Y5606 reacts with adenosine 5-monophosphate, purine, and trimethylamine. Myeloma globulins W3159, J698, and H2061 with identical A 0 light chains do not show anti-a1 + 3 dextran specificity.

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

25

known to possess antibody activity and essentially obtained without conscious selection, and it probably approximated more closely a random population of light chains. As such, it is desirable to retain the original numbering for these segments until sufficient evidence becomes available to justify changing a position. Thus, from the most recent data (Kabat et al., 1976b), position 24 in the light chain has a variability of 25 and is highly variable in human V,I chains, while positions 25 and 26 have variabilities of but 8.8 and 7.3 and are considered by some (Weigert and Riblet, 1976; Capra and Edmundson, 1977) not to be hypervariable. Nevertheless, the X-ray structures show them to be part of the loops forming the walls of the antibody combining site. It should be emphasized that, since almost all the more recently reported sequences are on antibodies to relatively few kinds of antigenic determinants or on myeloma proteins selected for antibody activity, the total data base is departing more and more from random. It thus becomes possible to include or exclude individual positions or indeed whole segments as being hypervariable. For instance, rabbit light chains from antibodies to the pneumococcal type-specific and streptococcal group-specific polysaccharides do not show the second hypervariable region (Haber et al., 1975); yet they hardly constitute a random population of antibodies and it is likely that these sites, highly restricted in specificity, might require similar sequences in this segment. They could well prove to be hypervariable if a wider spectrum of specificities were included. Another serious problem is the tendency to select for sequencing immunoglobulin chains in which the amino terminus is unblocked because of the ability to use the sequencer. This too is skewing the data base so that answers to many questions cannot be obtained. Pyroglutamic acid (pyrrolidonecarboxylic acid) was present at the amino terminus in 18/21 human VHI, 516 VHII, and 515 VHIVchains sequenced as well as in 10/10 human VAI,16/20 VAII, 3 / 3 VAV, 19/19 mouse VA chains and in the 1 pig VAchain sequenced (Kabat et al., 1976b). The variability plot for immunoglobulin light or heavy chains essentially revealed the converse of what is seen when a set of homologous proteins such as the cytochromes c are plotted in a similar manner. Figure 8 shows a variability plot for 67 cytochromes c. The site region is revealed by the stretch of invariant residues or those with very low variability, and no evidence of hypervariable regions is seen (compare Fig. 8 with Figs. 5 and 6). It should also be noted that the highest variability values are much lower than those found for im-

26

ELVIN A. KABAT CYTOCli?CtES

C

30.

28. 26. 24.

I

22.

v

20.

A

18.

1

16.

A

14.

I

12.

L

10.

R B

I

T 6. 4. 2. 0.

.20.

.

.

.

.

.

0.

10.

20.

30.

40.

RESIWE

T 50.

60.

L

70.

j.1 110.

60. 90.

FIG. 8. Variability plot for 67 cytochromes c. From &bat et nl. (197613).

munoglobulins. Since there are more than 20 sequences at every position, the theoretical upper limit for variability is 400 in each case. V I . CORRELATION OF VARIABILITYWITH PHYSICOCHEMICAL PROPERTIES OF AMINO ACIDS IN IMMUNOGLOBULIN CHAINS

Padlan ( 1977a) has compared two additional parameters, which depend on physicochemical properties of amino acids, with the variability plots. These were defined by Sneath (1966) and by Grantham (1974) as a way of evaluating properties which correlate most closely with the relative frequencies of substitution of one protein for another in evolution. The properties selected by Grantham (1974) were composition, polarity and molecular volume. The second, introduced earlier by Sneath (1966), involved a comparison of 134 properties including charge, aromaticity, bifurcation of side chain, optical rotation, number of bonds allowing rotation, Rf values in paper chromatography, ninhydrin color after paper chromatography in certain solvents, etc. For example, Grantham computed compositional differences involved in the atomic weight ratio of the noncarbon elements in end groups or in rings to the carbons in the side chains. Thus, for Ser for the COH group the value for OH/C would be 17/12 or 1.42, and for the Lys CCCCNH, moiety, the value for NH,/4C would b e 16/48 or 0.33; the compositional difference between Ser and Lys would be 1.42 - 0.33 = 1.09. The 190 comparisons of each

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

27

amino acid against the other 19 were made for each of the three properties, and estimates of the overall difference using the various properties between any two amino acids were made. Each investigator compiled a table comparing each of the 20 amino acids with the others by evaluating their dissimilarities relative to a value computed for randomly substituted amino acids. Thus at each position one may evaluate the average structural dissimilarity as a measure of the effects of various amino acid substitutions. Padlan (1977a) made these computations for the variable regions of the light and heavy chains using both methods. I n each instance the hypervariable segments of the molecule were excised and grouped according to length of the CDR. For the light chain, calculations were made for a first CDR of 11 residues, a second of 7 residues, and a third of 9 residues and for the heavy chain a first CDR of 5, a second of 17 residues in length and the region of 81 to 85 with 3 inserted residues; the third CDR of the heavy chain was not considered because of the limited number of sequences. For the non-CDR segments, one sequence was taken from each subgroup of various species. Variability was computed for 3 human, 3 mouse, 1 rabbit, and 1 guinea pig in the VHregion and for 9 human, 7 mouse, 1 rat, and 3 rabbit sequences in the V, region. Thus these non-CDR variabilities as well as the CDR variabilities are selected. Within each group a tabulation of variability and the average structural dissimilarity (ASD) by the two methods was made. There was with a few exceptions a good correlation between the values. Average dissimilarity indices were evaluated by examining the sequences for conservative replacements restricted to three groups (a) Ile, Leu, Val, Met; (b)Trp, Tyr, Phe; (c) Lys, Arg, His (Dayhoff, 1972); a fourth group, Pro, Ala, Ser, Thr, Gly, Asp, Asn, Glu, Gln, is generally quite heterogeneous and has been subdivided into three groups. Within each group, the average structural dissimilarity is relatively small whereas when members of different groups are compared it is larger. Thus the ASD-S ( h e a t h ) for the groups a, b, and c, respectively, were 37.4,35.8, and 46.9 (average 40.0, s.d 4.9) and the ASD-G (Grantham) 12.5, 21.6, and 21.5 (average 18.5, s.d. 4.3). Comparing one member of each group the average ASD-S and ASD-G are 66.9 (sad.6.1) and 47.3 (s.d. 4.7); a similar comparison for subgroups of the heterogeneous fourth group gives ASD-S of 67.6 (s.d. 7.0) and ASD-G of 51.3 (s.d. 4.7). Accepting these values plus one standard deviation provides a reference for comparisons of the VH and VL data. Values for ASD-S and ASD-G below 44.9 and 22.8 are considered conservative, while those above 66.9 and 47.3 are hypervariable. In Fig. 9, the Padlan data on ASD-S and ASD-G are plotted against

28

Lo

?i

I

$ 1

81

I I

I I I I

I

I I I

I

I

I

ELVIN A. M A T

I I

I I

100r

r

0

92

D

WO

OM

0

t

1

20

I

40

I

60 All Light Chains

I

'17

I'iO

530

I

I

1

I

I

80

20

40

60

80

All Heavy Chains

VARIABILITY FIG.9. Average structural similarity calculated according to Sneath (1966), ADS-S, and Grantham (1974), ADS-G, for light and heavy chains plotted against variability. The ADS-S and ADS-G values are from Padlan (1977a), and the variability values are from Kabat et al. (1976b); 0 , framework residues; 0, residues in CDR; X, residues 81-87 in framework considered to be hypervariable (Capra and Kehoe, 1974). For CDR residues and for residues 81-87 the number of the residue is given; values in parentheses are the number of different amino acids found at the position. The dashed lines represent the mean ADS-S and ADS-G (lower lines) and the mean plus one standard deviation (upper lines) (Padlan, 1977a).

30

ELVIN A. KABAT

variability for the entire set of VL and VH sequences (Kabat et al., 197613). Non-CDR residues are given as dots while those in the CDR are shown by open circles with the number ofthe residue alongside. A line has been drawn through the non-CDR points. The mean ASD-S and ASD-G and these values plus one standard deviation are shown by the lower and upper dashed lines. Many residues in the CDR show hypervariability by both parameters, but they are not strictly related. A group of residues in the CDR show low variability by both methods and are fairly close to the line for non-CDR while others show high variability by both. Values in parentheses in Fig. 9A and C give the number of different amino acids found at each position in the CDR; it is evident that those closest to the line for the non-CDR have fewer amino acids than those deviating from the line. The values with an X, residues 81-87 of the heavy chain, are those considered by Capra and Kehoe (1974) to be hypervariable but are not in the CDR; they generally fall very close to the non-CDR lines and are below the dashed lines considered to be the lower limit of hypervariability (Padlan, 1977a). VII. AFFINITY LABELINGDATAAND HYPERVARIABLE SEGMENTS Antibody combining sites have been studied extensively by affinity labeling in which a hapten covalently linked to a reactive group is added to antibody. The hapten will react in the combining site and a covalent link will form if the reactive group is so oriented that it can come into contact with a residue with which it can combine; as a control of specificity, in a second sample the combining site is usually blocked by a high concentration of the hapten not containing the affinity label. The residue to which the ligand is attached is located by obtaining and sequencing the peptide containing the covalently linked hapten; the control in which an excess of hapten was present should have blocked the sites so that they were not labeled. A variety of reagents have been used including several in which the haptenic group is at varying distances from the reactive group (for details and references, see Givol, 1974; Kabat, 1976a). A variety of studies have been carried out in which residues were labeled in light and heavy chains of antibodies and myeloma proteins with specificity for DNP, phosphocholine, and arsanilic acid azotyrosine. Table I1 lists residues identified in the heavy and light chains. All residues are in CDR. Tyr reported to be at residues 86 in the light chain and 90 in the heavy chain were stated to have been affmity labeled. There is some uncertainty as to whether these had been unequivocally identified and Goetzl and Metzger (1970) indicate that the labeled L-chain Tyr

TABLE I1 Affinity Labeling Studies on Myeloma Pmteins and Antihodies Myeloma protein or antibody specific for

Residue labeledfl Species

Light chain

Heavy chain

Referencc

Tyr 33, Tyr 59 (Tyr99-119)* Tyr 33, Tyr 93 Lys 58

Givol et al. (1971); Hairnovitch et (11. (1972) Hew et al. (1973) Cebra e t a!. (1974) Frangk (1971) Cehra et al. (1974) Chesehro et 01. (1973)

DNP hloiise 315

Arsanilic acid azo Phosphwholine

a

460 Guinea pig y2 Pig Guinea pig Mouse

‘ryr 32 Lys 50

Tyr 32

-

Tyr 32 Tyr 92

Lys 52

I

All residues are in sequences as aligned in Kabat et al. (1976b);see also Givol (1974).

’The exact position has not been determined.

32

ELVIN A. KABAT

probably was Tyr 32. The structures of the combining sites studied by X-ray crystallography also cast serious doubt on whether these two residues were correctly identified since they are part of the framework and could not come into contact with the reagents. It should be emphasized that the affinity-labeled residue is necessarily not a contacting residue for the hapten used since the hapten is fixed in the site although it could be contacting for a larger hapten. The affinity labeling technique rather identifies residues lining the combining site and the side chains of which can project into the site. Thus they could be involved in complementarity toward a determinant group of larger size than that used in the affinity label. Affinity labeling was most useful in helping to define combining sites before X-ray crystallographic data became available. It will probably b e used less in the future except for special reasons. It is unfortunate that it was used with only a few kinds of determinants. VIII. X-RAY CRYSTALLOGRAPHICSTUDIESON THE COMBINING SITES OF MYELOMA PROTEINS The X-ray crystallographic data at high resolution upon which sequence variability were related to site structure have been obtained on five proteins; one of these, Mcg (2.3 A resolution), is a V,V lightchain dimer (Edmundson et al., 1974); two, RE1 (2.0 A) (Epp et al., 1974) and Au (2.7 to 2.5 A), Fehlhammer et al., 1975) are V,I (Fv) dimers; in each the sequence of the two monomers is identical. The two others are Fab’ fragments, and thus each has a V, and a V, region and complete antibody combining sites: one, an CIK, McPC 603 (3.1A), binds phosphocholine (Segal et al., 1974a,b);the other, Newm (2.0 A) (Poljak et al., 1973, 1974), a y l h , binds an hydroxy derivative of vitamin K (Amzel et al., 1974). Mcg, the light chain dimer (Edmundson et al., 1974; Schiffer et al., 1973), binds a variety of substances, including DNP derivatives, caffeine, theophylline, 5 acetyluracil, menadione; no binding specificity has been established for REI’ or Au. The VL dimer Mcg and the Fab’ fragments McPC 603 and Newm show the domain structure (Fig. 10). Each chain is built of two distinct compact globular subunits which form a distorted tetrahedron. The binding site is at the tip of the two V, and of VH and VL domains which are arranged at an angle to one another and oriented to form a cleft, groove, or cavity at the surface accessible to solvent. The angles between the CL and CH domains and the VL and VH domains are not

* RE1 has recently been reported to bind one molecule of DNP (Huber, cited by Gavish et al., 1977).

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTAFUTY

33

(B)

FIG.10. Domain structure of a light-chain dimer: (A) The small bars indicate the S-S bonds. Monomer 1 is at the top and monomer 2 at the bottom, as in Fig. 10B. The angles relating the two V- and two C-regions are indicated; they intersect at an angle of about 120". In monomer 1, the 70" angle does not permit the extra S-S bond connecting the V- and C-domains between residues 82 and 174 of rabbit light chains to form while in monomer 2 with the 110" angle it is possible. Thus monomer 2 is in the light chain conformation, while monomer 1 assumes the role of the heavy chain in the dimer. (B) The orientation of the p-pleated sheets in the light-chain dimer. The interactions of C1 and C2 are through the four-chain layer (indicated by stripes), while in the V-regions these are rotated so that the V-regions interact by their three-chain ppleated sheets (rotational allomerism). From Edmundson et al. (1975). Reprinted with permission fromj. Am. Chem. SOC. Copyright by the American Chemical Society.

the same, and this difference in quaternary structure is characteristic and indeed is seen in the light-chain dimer (Schiffer et al., 1973). Even though the two monomers in the Bence Jones protein Mcg are identical in sequence and tertiary structure, they are not oriented in the same way, one monomer assuming the conformation like the heavy chain in the Fab, the V-domain making an angle of about 70" with the C-domain, while in monomer 2 the angle is 110" (Fig. 10). Monomer 1 had been identified as having the VH orientation since rabbit light chains are known (Strosberg et al., 1975) to have an extra S-S bond connecting the V, and C, domains; in Mcg the homo-

34

ELVIN A. KABAT

logized positions of this bond are in residues 82 and 174. No such bond would be possible for monomer 1 but could readily form in monomer 2, with the 110"angle between the VL and C, domains indicating that monomer 2 is oriented as the light chain with monomer 1 inferred to be functioning as the heavy chain. Even-in REI, which is an Fv dimer, this orientation of the two V-domains is maintained. The V- and C-domains of the light and heavy chains are each made up of two antiparallel twisted P sheets, one of three and the other of four &strands (Fig. 11);the two /3 sheets occur in different planes to form a sandwich-type structure; this has been termed the "immunoglobulin fold." The V-domains differ from the C-domains in having an additional loop between the two planes involving residues 43-61 and containing the second CDR of each chain. This loop is absent from the C-regions because the extended stretches are somewhat longer. An important property of immunoglobulin chains inferred from the light-chain dimer has been termed rotational allomerism (Edmundson et'ul., 1975). In Fig. 10B, the noncovalent interactions of the C-regions of monomer 1 and monomer 2 or of the CL and CH domains are by apposition of their four-stranded P-sheets intersecting at an angle close to 90" and stabilized by interactions between hydrophobic amino acids. The four-stranded sheets of the C-domain have a staggered arrangement of polar and apolar residues, the apolar residues being involved in CL-CL or CL-CH interactions while the hydrophilic groups are accessible to solvent. However, in the V-domains these two /3 sheets are rotated so that the three-stranded P-sheets of the two V-regions are adjacent and form the main binding cavity. In the V-domain the alternation of polar and apolar residues is not maintained and some polar residues are replaced by Tyr or Phe in the

3 FIG.11. Direction of the chain in monomer 2 of the Mcg dimer. Arrows indicate the chain direction; white arrows are in one plane, hatched arrows in the other. The black bars are the -S-Sbonds. The CDR are at the right. From Schiffer et al. (1973). Reprinted with permission from/. Am. Chem. SOC. Copyright by the American Chemical Society.

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

35

three-stranded P-sheet and contribute as contacting residues to binding the ligand in the site and to stabilize the site. If the a-carbon skeletons of the VL and V, of the McPC 603 are superimposed by aligning the disulfide bonds with the chains running in the same direction, and the average relative displacement of each a carbon of one chain from that of the corresponding one on the other chain is plotted against residue number, the result resembles a variability plot (Segal et al., 1974b; Padlan and Davies, 1975; Davies et al., 1975). The same result is obtained when VL-domainsfrom two different proteins, RE1 and McPC 603 and Mcg and McPC 603, were compared (Padlan, 197713). The framework proteins of the chains are thus conformationally homologous while the CDR show substantial differences. The most striking finding from the X-ray crystallographic studies is that the antibody combining site is formed by the CDR of the light and heavy chains. In the two Fab’ dimers the three CDR of the VH domain and the first and third CDR of the VL domain form the site. In McPC 603, an insertion of six residues in the first CDR of V, shields the second CDR from the site, while in Newm a deletion following the second CDR of VLpulls the chain away from the site. The McPC 603 site is a cavity while the Newm site is a shallow groove; in each instance the known ligand fills only a part of the site; the specificity of the remainder is completely unknown. In Mcg and in REI, all six CDR may form part of the site. Residues 81-85 of the heavy chain, which Capra and Kehoe (1974) considered hypervariable in human heavy chains, are not part of the antibody combining site. They lie in a region exposed to solvent. Residues 84 and 85 are involved in a allotypic specificity in the rabbit (Mage, 1977), and the variation in the human VHchains may be associated with some function other than site complementarity. Figure 12 shows stereodrawings of the a-carbon skeletons of VL and VH domains of McPC 603 (VHIII V,) and Newm (VHII VJ as they are oriented in the Fab’ fragments, the L-chain dimer Mcg, and the VL dimer REI; the three CDR of the four proteins are circled. The differences in the combining sites of the four proteins and the exclusion of the second CDR of the light chain in McPC 603 and Newm are evident. Figure 13 shows stereo views of the a-carbon skeletons of the three CDR of the four light chains, and Fig. 14 shows the three CDR of the two heavy chains. Even without the side chains the differences in orientation are striking. The differences in secondary structure clearly reveal the nature of immunological specificity in that no two backbones are alike in secondary structure; addition of different side

36

ELVIN A. KABAT

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

37

chains even if the secondary structures were the same could contribute additional degrees of variation, and thus of specificity. The problem of antibody complementarity thus resolves itself into the generatiqn of different-shaped receptor sites upon a constant framework by substitution of sequences of one set of CDR of each chain by another. This has led to several attempts to construct models of antibody sites of a different specificity from the known structures.

Ix. PREDICTIVEATTEMPTS BASEDON INSERTINGCDR SEQUENCES UPON A CONSTANT FRAMEWORK Using the framework of McPC 603, a model of a DNP binding site was attempted using the sequences of the CDR of MOPC 315 (Padlan et al., 1976a). A major first approximation in such model building is to select loops for the various CDR .which correspond, preferably exactly in length, to a CDR already studied crystallographically and to assume that both will have the same secondary structure (Padlan et al., 1976a), the specificity difference then presumably depending entirely upon the different side chains. This assumption is worth exploring although it is probable that it will prove not to be correct and that sequence differences will affect secondary structure. Indeed all methods developed to date for predicting the secondary structures of proteins from sequence have been based on the opposite assumption, namely that sequence differences among residues from n to n - 4 and n + 4 where n is any residue in a polypeptide chain are crucial to secondary structure (Anfinsen and Scheraga, 1975; Chou and Fasman, 1974; Schulzet al., 1974; Matthews, 1975; T. T. Wu et al., 1978). Unfortunately, since only four light chains and two heavy chains have been studied by X-ray crystallography, the number of CDR is very limited and, since differences in length have been noted in all except the second CDR of the light chain, predictive studies in which this assumption is possible are very limited until more X-ray structures become available. Variations in length may or may not provide additional contacting residues and also could influence whether or not other CDR residues are contacting. In the Newm structure the first CDR of the light chain contains an insertion of three residues after position 27; these form part of a helical loop consisting of a single turn which ~~~~

~~

~

~

FIG. 12. Stereodrawings of the a-carbon skeletons of the V-regions of the four proteins studied crystallographically. Each protein is in the same orientation. (Other views and coordinates used in Figs. 12, 13, and 14 may be found in Feldman, 1977). With a stereo viewer it is possible to see two adjacent models at the same time, so that a comparison may be made in three dimensions.

38 ELVIN A. KABAT

f

3

b

PP

c

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

H H W Y

tx>

FIG. 13. Sterodrawings of the a-carbon skeletons of the three CDR of the four VL regions studied crystallographically. Note in Newm that the second CDR is much farther from the other two CDR than it is in the other three chains. This is due to a deletion (see text).

39

40

ELVIN A. KABAT

T

P

4

FIG. 14. Stereodrawings of the a-carbon skeletons of the three CDR of the two heavy chains studied crystallographically.

could readily be deleted without disturbing the path of the polypeptide chain, but they are not considered to be contacting. It should also be emphasized that the insertion of these loops in attempts to predict structures of unknown sites applies only to the backbone structure of the CDR and tells nothing about the conformation of the side chains. Thus when one replaces one CDR by another of equal length, one is not changing the secondary structure whereas when one substitutes a CDR of different length, one is changing the gross form of the combining site. Nevertheless such efforts may be very informative and such models may serve as a point of departure for applying other techniques, such as nuclear magnetic resonance (NMR) (Dwek et al., 1977). For such attempts at model construction, the sequences of both the reference X-ray structures and the chains to be inserted must be absolutely correct or much time and effort may be wasted. In particular the sequence of the heavy chain of Newm has been revised (Poljak et al., 1977) several times, and it no longer has the same number of residues in the first V, CDR as MOPC 315 (Poljak et al., 1975). The earlier report of a Lys at homologized position 35 of the H chain of MOPC 315 has been corrected to Trp (Padlan et aZ., 1976a), but only after the incorrect assignment had led to serious difficulties which could have been avoided. Similarities in the gross orientation of the CDR of two Fab' structures, Newm and MOPC 315, were evident (Poljak et al., 1975), and a detailed effort to build the backbone of the site for MOPC 315 has re-

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

41

cently been made (Padlan et al., 1976a). This model has subsequently been refined using data obtained by N M R and with spinlabeled haptens (Dwek et al., 1977). The framework of the V-region of 315 was constructed based on the structure of the V-domains of mouse myeloma protein McPC 603. The CDR based on the sequence of 315 were then constructed, making as little change in the structure as possible. For the light chain, the first CDR of 315, Mcg, and Newm have the same length and, since all are A, the first CDR of 315 was constructed from the coordinates of Mcg. The second CDR was given the backbone conformation of McPC 603 since it, REI, and Mcg all are very similar. The third CDR was also built relatively like McPC 603, but to permit it to form the maximum number of H bonds it was built as two antiparallel strands. In the heavy chain the first CDR of MOPC 315 has one more residue than in McPC 603. The Trp at position 353 was assigned to the interior corresponding to Met 34, a residue in the domain interior shown by other considerations (see below) to be structural, and the extra residue was placed in the exposed loop at the beginning of the first CDR, the Gly at position 32 permitting a sharp turn at the position. The second CDR of 315 is three residues shorter than McPC 603, and the third CDR is two residues shorter. These extra residues were excised and fitted onto the framework to give a first approximation of the model. Since the second and third CDR of Newm and 315 each have the same length, the orientations were adjusted to resemble Newm more closely. At this point a reasonable binding site for the DNP was selected so that the NO2 groups would be hydrogen bonded to the protein and the stacking van der Waals interaction of the DNP ring and Trp 91 of the light chain would be maximal. The nitro groups were made coplanar with the phenyl ring, and in E DNP lysine the NH of the €-carbon was hydrogen bonded to one of the oxygens on the nitro group on carbon 2. The model was then modified to permit affinity labeling of Tyr 32 of the light chain and Lys 52 of the heavy chain as found experimentally (Givol et al., 1971; Haimovitch et al., 1972). This could be done with only relatively minor adjustments. The main feature of the site was its hydrophobic character. The structure of the model for the proposed anti-DNP site could be further improved by incorporation of information by N M R which prcvided definitive data on the orientation of the DNP moiety in the site, and the orientation of the aliphatic side chain could be assigned using All residue numbers are homologized according to Kabat et al. (1976b)throughout.

42

a

ELVIN A. KABAT

2-

'

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

43

spin-labeled haptens (Sutton et al., 1977; Dower et al., 1977). These techniques can provide additional experimental information which can substantially improve the characterization of antibody and of lectin (Brewer e t al., 1973) combining sites. Figures 15A and 15B show the orientation of the CDR in the predicted structure (Padlan et al., 1976a) and in the structure as modified by NMR and ESR (Dwek et al., 1977). The three CDR of each chain are labeled L1, L2, L3 and H1, H2, and H3. The revised model has a very different secondary structure in the third CDR of the H chain. In the other CDR, only the orientations of the side chains are altered. A recent study by Schechter et al. (1976) showed that a VL dimer as well as the intact light-chain dimer of the DNP binding protein MOPC 315 bound two molecules of E DNP lysine per mol of dimer with a K , of 2 x 107; this is about 1000 times lower than that of the Fv dimer (VL-VH)or of intact MOPC 315. The Mcg site also binds more than one DNP ligand while the RE1 site binds only one (Huber, cited by Gavish et al., 1977). In MOPC 315, a Scatchard plot shows that there is no cooperativity and that binding of each DNP is independent. However, in contrast to the 1000-fold difference with DNP lysine, the MOPC 315 V, dimer bound dinitrophenol much more effectively relative to the intact protein, for which K, was only 6.5 times greater. The light chain is thus interacting with the DNP moiety while the heavy chain is interacting with the side chains. From spectral shifts (Dwek e t al., 1977) and from circular dichroism analysis (Freed et al., 1976), it was clear that Trp 91 was involved in binding the DNP in accord with the predicted model; moreover, menadione and dinitrophenol also interact with Trp 91. Thus they involve at least a part of the same subsite as the DNP (Dwek et al., 1977). The NMR data assume explicitly that the aromatic residues are the only source of their observed shifts, and their model involves placing four aromatic residues in the vicinity of the DNP hapten (Fig. 15B). It will be important to verify this assumption, since there are many other causes for NMR shifts. It should be noted that the conformation of the E DNP lysine in the two models is not the same (Figs. 15A and 15B). This finding is of great importance in understanding antibody specificity. Richards and Konigsberg (1973) suggested that antibodies FIG. 15. Stereomodels of the predicted MOPC 315 site. (A) Constructed from its sequence and inserting the CDR sequences into the framework of McPC 603 (Padlan et al., 1976a). (B) Revised model with incorporation of data from nuclear magnetic resonence and spin-labeled haptens (Dwek et d.,1977).

44

ELVIN A. KABAT

were multispecific and concluded from rather weak evidence that menadione and DNP interacted at different substies. This was disputed by Michaelides and Eisen (1974) and by Johnston and Eisen (1976), who pointed out a structural similarity between the two compounds. The demonstration that they both react with Trp 91 leaves the existence of multispecificity possible but unproved (Gavish et d., 1977). The antibody combining site does not appear to have the same limitations in recognition as the investigator. Winkelhake and Voss (1970) showed that anti-TNP guanosyl antibodies prepared to a TNP-DNA complex with methylated bovine serum albumin bound guanosyl ligands directly. This shows that inferences of multispeci ficity from data of this type on antibodies whose specificity is unknown may be highly misleading. Cameron and Erlanger (1977) have described the cross-reaction of rabbit antisera to (AMP)z-gramicidinS. Three major anti-AMP globulin fractions separated by preparative isoelectric focusing in polyacrylamide behaved differently in their binding affinities relative to AMP toward a variety of seemingly structurally diverse ligands including hydralazine, caffeine, menadione, and DNP-lysine. Whether further study of these antibodies will provide convincing evidence for multispecificity or whether they will be another instance like the MOPC 315 case remains for the future. Since antibody combining sites are generally larger than the ligands used to study multispecificity, if a hapten filling the entire antibody combining site were compared with these haptens, so-called multispecificity would become another instance of cross-reactivity . A second predictive attempt (Davies and Padlan, 1977) was made by inserting sequences for the CDR for BS-5 and K25 (Jaton, 1975; Margolies et al., 1975; see Kabat et al., 1976b) into the framework of McPC 603 to construct a model of an antibody site specific for the type I11 capsular polysaccharide of pneumococcus which had been shown (Mage and Kabat, 1963) by immunochemical studies to be complementary to a hexasaccharide. The BS-5 model is of considerable interest because the substitution of its CDR sequences converted a deep cavity into a shallow groove capable of binding a hexasaccharide of the SIII structure and also of involving the second CDR of the light chain, which had'been shielded from the McPC 603 site by the insertion of residues 27A-27F (Segal et al., 1974a,b). The combining site became filled with residues capable of forming H-bonds with the SIII hexasaccharide. The positively charged Lys 50 in the L chain could interact with COO- of a glucuronic acid residue in SIII. All antipneumococcal type I11 and VIII antibodies have Lys or Arg at this position, as does an antibody to Micrococcus lysodeikticus, which has a

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

45

negatively charged lactyl group. However, rabbit antibodies to the group-specific carbohydrate of the group A variant (Braun et al., 1976a,b) and group C (Chenet al., 1976)streptococcus also have Lys or Arg although the antigen is uncharged; anti-p-azobenzoate despite the negatively charged hapten has Thr (Appella et al., 1973), and a homogeneous rabbit immunoglobulin produced by immunization with streptococci but showing no antistreptococcal group activity had Ala (Thunberg and Kindt, 1976) at this position (see Kabat et al., 197613). Table I11 lists the contacting residues in both chains McPC 603 and Newm and in the light chains of Mcg in actual contact with the various ligands as well as those predicted to be in contact in the models of the DNP site as modified by NMR and ESR data (Dwek et al., 1977) and in the SIII site (Davies and Padlan, 1977); many of the contacting residues occupy the same positions in both, but the data are too limited to provide any generalizations.

x. PREDICTIVE ATTEMPTS BASEDON STATISTICAL OF EXAMINATIONSOF SEQUENCES AND DISTFUBUTION AMINO ACIDS IN CDR The collected V-region sequences represent a vast body of information; indeed the numbers of V-region residues sequenced (over 23,000 in Kabat et al., 197613) comprise a substantial proportion of the total number of residues in all proteins sequenced (Dayhoff, 1972, 1973, 1976). Examination of these data by statistical and other methods has the potentiality of providing considerable insight into antibody complementarity. With so few X-ray structures available, and these being on myeloma proteins, the known determinants of which react occupying only a portion of the site (Segal et al., 1974a; Poljak et al., 1974), their development coupled with additional crystallographic data on purified antibodies of defined specificity could materially shorten the time necessary for solving the problem of antibody complementarity. Based on available data only limited inferences have been made, and many more sequences on myeloma globulins and antibodies of known specificity will be required. Indeed sequences of families of proteins of a given specificity, such as the mouse myelomas binding phosphocholine, a 1+ 6 dextrans, P l + 2 and P l - 6 fructosans, and the a 1j-6 galactans, are essential; it is of especial importance to have elucidated their site specificity so that a ligand filling the entire site is available. Sequences of many purified antibodies will also be needed, as will some method of classifying antibodies into groups with respect to the fine specificity of their combining sites. For instance, the diversities in sequences in the CDR of the rabbit anti-

TABLE I11 Contacting Residues in Predicted (P)Structures Compared with Those in McPC 603, Newm, and Mcg" Light chain CDR

MOPC315-Pb

First Tyr 32 Asn 34

BS-5P

McPC 603

Tyr 30 Ser 31 Asn 32

Heavy chain Newm

None

None

Tyr 32 Ser 34 Tyr 36 Tyr 49 Glu 50

Tyr 91

Ser 89 Tyr 91

Third Trp 91 Arg 93

Ser 91

MOPC315-Pb Tyr 33

Gly 30 Asn 31

Second Lys 50

Mcgd

Phe 50 Lys 52

BS-5P

McPC603

Asp 33

Tyr 33 Glu 35

Tyr 52 Ser 56 Tyr 58 Gln 95

Arg 52

Tyr 50

Glu 58 Asn 95 Leu 96 Ile 97 Ala 98

Thr 97 Thr 94

M e

95'

Ser 94 Leu 95

Asp 95 Phe 97 Phe 98

Newm

Val 100 His lOOA

Trp lOOA

All sequence numbers as in Kabat et al. (1976b). Predictions as modified by NMR and ESR (Dwek et al., 1977). Davies and Padlan (1977); Padlan (1977b). Edmundson et al. (1974); Asp 95 is considered structural in monomer 1 but could be contacting in monomer 2. Exact sequence not known; MOPC 21 has Tyr and Pro at positions 94 and 95.

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

47

bodies to SIII and SVIII and to the A, A variant, and group C streptococcal specific carbohydrates (see Kabat e t al., 1976b) are so great that one has not been able to see any unifying principles determining site specificity (see, for example, Lys 50 on page 44 in discussion of the SIII site). However, seven rabbit A variant light chains were identical in sequence for their first 40 residues, that is, through the first CDR (Braun et al., 1975). The myeloma proteins of a given specificity for some unaccountable reason show a much greater degree of similarity in sequence in the CDR than do the antibodies. Almost all the sequences available are on what are considered sequential rather than conformational determinants, and the problem of studying combining sites of protein determinants remains for the future (see Section XI). Several methods of examining the accumulated sequence data (Kabat et al., 1976b) statistically have been employed with a view of trying to locate contacting amino acids in antibody combining sites, as well as those which play a structural role.

A . Analysis of Pairs of Residues in the C D R (Kabat e t al., 1976a) The general principle involves the assumption that the data bank approximates a random collection of light and heavy chains of antibodies of diverse specificities and that any two amino acids in the CDR will be associated in a given chain largely by chance unless they are involved in specificity either as residues in contact with the antigenic determinant in the site or else exert a conformational influence on a contacting residue. Conversely, amino acids which occur in high frequency in CDR are likely to be playing a structural role in properly positioning the two kinds of residues determining specificity. For this method to be useful, data must be available on a sufficient number of sites of essentially the same specificity so that pairs deviating significantly from random can be selected (Kabat, 1977a,b). Thus far the data have provided one clear-cut demonstration of the potentialities of the method, since X-ray evidence was available to test the inferences. The original data for the frequencies of occurrence of each of the 20 amino acids as well as Asx and Glx at positions 32,33,34, and 35 of the heavy chains (Kabat et al., 1976a) are given (Table IV); values in parentheses include the more recent data (Kabat et al., 1976b). As originally computed assuming random association, Phe 32 and Tyr 33 should have occurred twice, Phe 32 and Glu 35 once, and Tyr 33 and Glu 35 twice; the combined probability of occurrence of Phe 32, Tyr 33, and Glu 35 would of course be much lower. These values are increased slightly but not importantly when the values in parentheses are used. Programming to select pairs occurring more frequently

48

ELVIN A. KABAT

TABLEIV Frequency of Amino Acid Residues at Positions 3 2 , 3 3 , 3 4 , and 35 of Heavy Chains" Position Residue TrP Ile TYr Phe Pro Leu Val LYS Met CYS Ala '4% Thr Ser GlY His ASP Asn Glu Gln Glx Asx

32

33

34

0 0 20 (29) 9 (12) 0 0 1

3 0 12 (18) 1 0 1 2 0 0 0 7 1 5 (6) 3

0 2 0 1 0 2 (6) 4 (5) 0 36 (46) 0

0

0 0 2 (3) 0

3 7 2 (3) 0 3 1 1 0 0

m.lL

49 (64)

35

0 1

0 1 0 0 0 0 0 0 0 0 47 (62)

a Data on completely and partially sequenced proteins at these positions are included. Data from Kabat et al. (1976a);values in parentheses from Kabat et al. (197613).

than expected shows cooccurrences in 9 instances of Phe 32 and Tyr 33,8 of Phe 32 and Glu 35 (including 1Glx), and 8 of Tyr 33 and Glu 35 (including 1 Glx). All nine proteins were phosphocholine-binding myeloma proteins, eight of mouse and one of human origin. One of these McPC 603 had been studied by X-ray crystallography, and Tyr 33 and Glu 35 had been shown (Segal et al., 1974a,b) to be residues in contact with phosphocholine in the site. Tyr 33 was also identified as a contacting residue in McPC 603 by the paired labeling technique (Grossberg et al., 1976). All nine sequences of the phosphocholine binding myelomas had Met at position 34 in the heavy chain. However, this amino acid was not selected as occurring more frequently than chance in that Met 34 is present in 37 other heavy chains; this defines Met 34 as a structural element whose function is to permit Tyr 33 and Glu 35 (or Asp 35 in the

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

49

human phosphocholine-binding myeloma) to assume the proper conformation for binding. The X-ray structure of McPC 603 is in accord with this and shows the role of Met 34 to be structural; its side chain points away from the phosphocholine. The variabilities at positions 32, 33, and 35 are 22,39, and 89 while that at 34 is only 9.4. These values are consistent with residues 32,33, and 35 being involved in specificity and 34 being structural. The sequence Phe-Tyr-Met-Glu would have a random frequency of occurrence of 1 in 160,000 assuming that all amino acids occurred in equal proportions. Since Met occurs much less frequently than most other amino acids, the frequency is even lower. It thus becomes highly significant that this sequence as well as Phe-Tyr-Met-Asp has been found only at positions 32-35 of the heavy chain and not in any other portion of the heavy or light chains nor in any other protein listed in the Atlas of Protein Sequence and Structure and its two supplements (Dayhoff, 1976). If this tetrapeptide is unique for phosphocholine binding, several interesting possibilities arise: (1) Would such a sequence occur in other proteins, such as enzymes, etc., which bind phosphocholine? When information on myeloma globulins with anticarbohydrate specificity and on lectins with similar specificities is available, will the contacting residues in the sites have the same amino acids? (2) Would the tetrapeptide sequence itself, or perhaps with the terminal NH2 and COOH groups blocked, bind phosphocholine? Five of the phosphocholine mouse myeloma H chains have Asx 31 while the remaining three and the human protein have Asp. Position 31 is seen to be a surface residue. If the remaining five proteins with Asx are found also to have Asp, it will probably not be in idiotypic4 determinant since the different phosphocholine-binding myeloma proteins have distinct idiotypic specificities although it could also exert a conformational role on other residues in the CDR. The accumulation of sequence data on the a 1 ---* 6 and a 1 --* 3 dextran, /32 + 1 and /32 + 6 fructosan, and /3l + 6 galactan specific myeloma proteins, for each of which substantial numbers are being studied, should soon provide further tests of this predictive procedure.

B . Identijication of Residues Involved in Specificity by Examination of Sequences Showing Differences in Specificity but Limited Sequence Variation in the CDR Sequence data in the CDR often show remarkable similarities and relatively restricted differences. Thus for example a VAII (Vil) and a VAV (Mcg) human light chain, despite 21 differences in framework, See abbreviations and definitions.

50

ELVIN A. KABAT

have an identical first CDR of 14 residues (Wu et al., 1975); only one, Mcg, is known to bind ligands, and it is relatively nonspecific. More recently Braun and Huser (1977) have found two different rabbits which produced completely identical V, chains specific for the group A variant carbohydrate; these were functionally identical in recombinants with their heavy chains. The light chains of two human IgM myeloma proteins of the same PO idiotype (Kunkel et al., 1973, 1974), LAY V,I and POM V,III (Klapper and Capra, 1976), the former being specific for and reacting equally well with human IgGl and IgG3 while the latter reacts only with IgG1, had identical third CDR of 9 residues despite 30 amino acid differences in their V-regions. Their second CDR differed at two positions, 55 and 56. The three CDR of the heavy chains of LAY and POM are identical except for one difference at position 31 in the first CDR, being Ala and Ser, respectively. Similarities in the first CDR of human V,II chains have been noted (Eulitz and Kley, 1977), but only one of these was associated with a myeloma protein of known specificity, the phosphocholinebinding myeloma protein FR. Light chains of myeloma proteins possessing the Wa cross-reacting idiotype (Kunkel et al., 1974) tend to fall into a separate subgroup called V,IIIb, but other V,IIIb chains lack this idiotype. Capra et al. (1977) have found the three CDR of the light chains of mouse anti-p-azo-phenylarsonate antibodies having a cross-reacting idiotype to be identical despite the presence of 16 substitutions in the framework. The framework substitutions were associated with at least two, and probably with more than three, distinct light chains; the inability to obtain overlap peptides did not permit determination of the exact number of light chains. At present since there is no information associating any amino acid residue at any position in V, or V, with idiotypic specificity, the only correlations which appear possible are those in which specificity differences in antibody combining sites may be ascribable to individual residues or to groups of residues. One instance in which some insight has been gained as to residues which could be involved in specificity differences in antibody combining site has been from an examination of the first CDR of certain human V,III light chains plus LAY, a human V,I (Kabat et al., 1976). LAY and POM show the most extensive sequence differences in the first CDR of their light chains; in the second CDR of the light chain they differ only at positions 55 and 56 and their heavy-chain CDR have identical sequences except for position 31. Thus these three positions and the first CDR of the light chain must be responsible for their differences in combining site specificity.

STRUCTURAL BASIS OF ANTIBODY

COMPLEMENTARITY

51

Table V shows the first CDR of seven other VKIIIlight chains and of LAY and POM (Kabat et al., 1976~).These comprise two additional IgM proteins, GLO and SIE (Capra and Kehoe, 1975), with specificity for human anti-IgG but of a different idiotypic cross specificity (Wa idiotype, Kunkel et al., 1973); two IgM cold agglutinins, DRE and TAK (Capra et al., 1972; Gergely et al., 1973), with anti-blood group I specificity; and three Bence Jones proteins with no known antibody activity. The Bence Jones proteins were probably not from IgM cold agglutinins with anti-I or from Waldenstrom IgM with anti-human IgG specificity, since such light chains almost never occur in urine as Bence Jones proteins. Anti-I specificity has clearly been established as anti-carbohydrate and involves Dgalactose and Nacetyl Dghcosamine residues (Marcus et al., 1963) and in one instance has been shown to be specific for DGalPl+ 4DGlcNAcPl + 6- (Feizi et al., 1971) while anti-human IgG specificity is directed against protein determinants. On the almost universally accepted premise that sequence differences are responsible for specificity differences in antibody combining sites, the remarkable similarities in sequence among these diverse proteins permit inferences implicating certain residues as involved in the differences in specificity. Since the differences in sequence in the CDR between LAY and POM involve predominantly the first CDR of the light chain, it is probable that some amino acids in this segment are associated with the specificity differences. On this basis the first CDR would be associated with antiprotein (anti-IgG) specificity, and one would thus consider it extremely unlikely that it could also contribute importantly to anticarbohydrate (anti-blood group I) specificity. DRE, GLO, and SIE have identical first CDR, and yet DRE is anti-I while the two others are anti-IgG; the second anti-I differs only at position 30 as does one Bence Jones protein, IKE, while two others, TI and WIL, differ at two positions, 30 and 32. P O M has the same residue at position 30 as the three Bence Jones proteins and differs from all others only in having Ile at position 28. Thus position 28 may be predicted to be involved in its specificity for IgG1; Asn at position 30 may also be of importance since POM differs from the two anti-I proteins and the two anti IgGs of the Wa idiotype at this position. Accepting these two residues in POM as possibly important to IgGl complementarity, LAY differs from POM at positions 24, 27a, 28, 29, 30, 34 in the first CDR and has a deletion, tabulated as residue 31. The first CDR of VKIprotein RE1 not known to have relevant antibody activity2 has the same number of residues as LAY, and both have

TABLEV Sequences of the First Hyperuariable Region of Human VJZZ Light Chains" Residue Protein DRE

24

25

Arg

Ala

26

27

Ser Gln

27a Ser

28

29

30

31

32

33

34

Val Ser

Ser

Ser

Tyr

Leu

Ala

GLO SIE TAK

Glxb

?'

IKE

Asn

TI

Asn

Phe

WIL

Asn

Met

POMd

Ile

Asn

VIJ LAY

Gln

Asn

Asn Ala

(

)"

RE1

Gln

Asp Ile

Ile

(

)

Lys

Antibody activity

Reference

Anti-blood group I; IgM cold agglutinin Anti-human IgG; IgM Wa idiotype Anti-human IgG; IgM Wa idiotype Anti-blood group I; IgM cold agglutinin Bence Jones protein; no known antibody activity Bence Jones protein; no known antibody activity Bence Jones protein; no known antibody activity Anti-IgG1; IgM Po idiotype

Capra e t al. (1972); Gergely et al. (1973) Capra and Kehoe (1975) Capra and Kehoe (1975) Gergely e t al. (1973) Capra and Kehoe (1975) Suter et al. (1969) Capra et al. (1971) Klapper and Capra (1976)

Anti-human IgGl and anti- Klapper and Capra (1976); Capra e t al. (1971) human IgG3; IgM Po idiotype Asn No known antibody activity' Epp e t al. (1974) Asn

All residues are the same as those in DRE except as indicated (from Kabat et al. 1976a, with additions). The sequences of 22 VJII proteins have been determined: 16 have Gln, 5 have Glx, and 1 has Ser. Capra et al. (1972) have Gln for residue 27; Gergely et al. (1973) determined it only as Glx. Gly or Trp are considered to be the only possibilities. The sequence originally reported (Capra et al., 1971) has been revised. Deletion. See footnote 2, page 32.

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

53

one less than the V,III proteins; residues 24-27 and 32-34 are identical in both. Moreover residues 25,26, 27, and 33 are the same in all proteins and 32 is Tyr in all but two Bence Jones proteins despite their different specificities. Thus these residues are unlikely candidates for determining specificity. The RE1 structure shows, moreover, that residues 24, 25, 26, and 33 cannot be contacting residues for antigen in the combining site; the side chain of 24 points in the opposite direction, 25 and 26 are shielded from the site by other residues, and 33 points toward the interior of the site. In the second CDR of the light chain of REI, residues 54 and 56 also cannot be contacting; the former points in the opposite direction while the latter is at some distance from the site. Exclusion of residues 24-26 33, 54, and 56 as noncontacting leaves residues 27,27a, 28,29,30,31,32,34, and 55 plus residue 31 of the heavy chain as the residues potentially involved in the difference in specificity between LAY and POM. The existence in the V,III proteins of the extra residue listed as 27a could have an effect on the overall conformation of the site, again leading to specificity differences; although residue 27 is Gln in all instances, it could play a different role in LAY and POM; Tyr 32 is perhaps excludable on the basis ofhigh frequency in antibodies of different specificity. It is not likely that all these residues will prove to be contacting residues in each protein. Rather one would suspect that the two sites differ sufficiently so that some would be contacting in LAY and others in POM. This analysis and the availability of the set of proteins offers a substantial possibility of attempting to verify these inferences. Recombination experiments using the heavy chains of LAY and POM with their reciprocal light chains as well as with the other light chains in Table V and evaluation of the specificity of the hybrids would shed substantial light on the residues involved in IgGl as compared with IgGl plus IgG3 specificity. Similarly, attempts to regenerate anti-I specificity by recombining anti-I heavy chains with the various light chains would test the hypothesis that the first CDR of the light chain is not important to anti-I specificity. It would be highly desirable to have complete sequences of the light chains in Table V.

C . Recognition of Structural and Contacting Residues in CDR b y Statistical Analysis of Frequencies of Occurrence of Different Amino Acids (Kabat et al., 1977) This procedure is based on the data bank of sequences (Kabat e t al., 197613). On examining the frequency of occurrence of each of the twenty amino acids at every position in the CDR, inferences may be

54

ELVIN A. KABAT

made (Kabat et al., 1977) as to individual amino acids playing a structural role at a given position or as being involved in specificity (see Section X,A). This procedure, when tested by comparison with the findings for that position by X-ray crystallography, in the few structures available shows remarkable agreement. The analysis materially improves our predictive abilities and may aid substantially in attempts to construct models of antibody combining sites of unknown specificity. If X-ray crystallography of an antibody combining site establishes a given amino acid residue in the CDR to be structural, not specificity-determining as a contacting residue, the information applies only to that residue in that particular protein. However, if an amino acid at a given position is inferred to be structural from this statistical analysis, this conclusion would hold for all antibody combining sites in which that amino acid occurred at that position. Moreover, the distribution of certain amino acids and their low frequency at each of many positions may suggest that they are playing a role in specificity either as contacting or as conformationally important residues as was inferred for Phe 32 of the phosphocholine binding myeloma; such an inference cannot be made by X-ray crystallography alone. All computations are based on the data bank of variable region sequences (Kabat et al., 1976b) aligned for maximum homology and for which variability was calculated. Several representative examples and a summary of the findings will be given, and the complete analysis can be found in Kabat et al. 1977). Table VI shows the distribution of the 20 amino acids and Asx and Glx at various positions in the CDR of the light and heavy chains, their frequency of occurrence in each CDR. Some unusual distributions are evident. These are indicated by boxes. Thus in the light chain Trp is found at 8 positions and occurred 184 times. Seven of the eight positions, or 88%, were in CDR. Of the 184 occurrences, however, 140 were at position 35,the invariant residue immediately following the first CDR. The remaining 44 Trp residues were distributed as follows: 9 at 2 positions in the first, 1at 1 position in the second, and 34 at 4 positions in the third CDR. All of these occurred at positions of high variability. Although 8 of the 9 occurrences in the first CDR are at position 32, variability 25, it nevertheless was present in only 6 of 27 human V,I, 2 of which had anti-human Ig activity; the remaining 2 occurrences were among the 9 rabbit type I11 antipneumococcal antibodies sequenced. In the third CDR, Trp occurred 17 times at position 91, variability 39: 42,15 times at position 96, variability 87, and once each at positions 92 and 97. At position

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

55

91, it occurred in 3/4 humanVhI,4/5VAIII, and 19/19 mouse VA. At position 96 it was seen in 1/22 human V,I, 5/9mouse V,, and 18/19 mouse VAchains. Since mouse A chains are very highly restricted, its absence from one mouse Vh at position 96 suggests a role in complementarity, but its invariance at position 91 makes its role at this position uncertain, and it could be structural. The distribution of all other Trp residues is in line with what one would expect from a residue involved in complementarity since for the 20 different amino acids to provide the enormous variety of antibody combining sites, one would have to b e distributed at low frequency among the various residues of the CDR. Met at position 34 in the heavy chain was shown by pair analysis to be a structural element in the first CDR in accord with the X-ray data (Segal et al., 1974a,b). Table VII summarizes the distribution of Met in the CDR of the heavy chain. This residue occurs at 20 positions in the entire V region, and 7 of these positions are in the CDR; it was found 121 times in the V-region with 57 occurrences, 47%, in the CDR. The distribution of some of these Met residues was quite striking. All 46 Met residues in the first CDR were at position 34 in the heavy chain. Met was present in a large proportion of the chains of human, mouse, and guinea pig V-regions. I t occurred in the three human and in the mouse V-region subgroups; it was present at this position in a number of antibodies of very different specificity, including human and mouse anti-phosphocholine, rabbit anti-SIII, and guinea pig anti-p-azobenzenetrimethylammonium antibodies as well as in many myeloma proteins and antibodies not shown to have antibody activity. Obviously it cannot play a role in specificity and is a structural element in the CDR. Table VII lists the six other amino acids occurring in low frequency at position 34 in the H chain. Some of these could play a role in specificity or some might be acceptable structural substitutions for Met. It is of interest that all are hydrophobic except for one Arg and one Ser. It should also be noted that, in the aligned sequences of MOPC 315 and in the predicted structures, Trp 35 occupies the same position as does Met 34 in McPC 603, e.g., two residues N-terminal to invariant Trp 36 but the first CDR of McPC 603 is one residue shorter with more MOPC 315 having Asn 35A. Table VIII gives an instance in the light chain in which Arg and Gln each play a structural role at position 24 and one in which Gln 27 is also structural. Arg was found at position 24 in 76, and 53 Gln + 10 Glx in 63 of the 190 sequences available. Their distributions are quite striking. Arg occurred in 11/11human VJI, 23/23 V,III and 19/19

TABLEVI Locations, Occurrences, and Proportions of lndividual Amino Acids i n CDR of Light and Heavy Chains".* Amino acid distribution i n all light chains ~

Trp

Ile

Tyr

8 2 1 4 7

44 9 1 5 15 34

29 9 3 7 19

Phe

Pro

31 4 1 7 12 39

30 2 2 5 9 30

Leu

Val

Lys

Met

~~

Cys

Ala

Arg

Thr

Ser

Gly

His Asp Asn

Glu

Gln

Asx Glx

u1 Q,

positions in 1st CDR in2ndCDR in 3rd CDR Total in CDR % totdl in CDR

# i j

44 7 4 4 15 32

37 4 4 6 14 38

occurrences in 1st CDR in 2nd CDR in 3rd CDR in all CDR % in 1st CDR % in 2nd CDR % in 3rd CDR % in all CDR

362 1123 471 1699 2306 1342 70 570 277 369 184 812 542 371 928 1139 11295 468 258 88 461 109 21 55 138 14 3 216 96 9 87 99 47 10 2 149 124 19 20 5 86 51 51 127 22 37 24 1 0 1 1 9 27 1 6 24 46 89 37 13 35 47 10 39 19 82 40 46 5 4 6 5 5 34 21 77 14 8 341 166 221 677 166 39 112 222 48 207 34 66 241 162 80 16 44 109 10 4 (23( 5 0.2 13 4 8 10 5 11 1 6 0.4 1 0.2 3 4 0.5 0.1 0 8 1 1 3 6 1 7 41131 7 1 3 4 5 4 3 p j 61171 3 4 1 2 3 1 1 4 1 4 4 4 2 13 121561 20 [801 13 13 17 6 m 1 3 1 3 8 / 9 7 21

1000 150 7 74 23 1

No. No. No. No.

No. No. No. No. No.

1651

57 9 3 9 21 37

65 10 3 9 22 34

41 9 3 4 16 39

23 3 1 3 7 30

7 1 0 3 4

[591

59 9 6 9 24 41

39 7 6 7 20

67 10 5 11 26 39

73 16 6 10 32 44

43 60 24 10 12 6 4 2 4 10 4 7 26 12 21 43 (501 49

40 11 4 9 2A

1601

p J

1181

13011351

1291

40 11 3 3 17 43

40 8 2 4 14 35

198 288 95 54 11 5 14 24 120 83 )151 48 18 0.7 6 2 7 7 8 60 28

1231

Amino acid distribution in all heavy chains No. positions No. in 1st CDR No. in 2nd CDR No. in 3rd CDR Total in CDR % total in CDR No. occurrences No. in 1st CDR No. in 2nd CDR No. in 3rd CDR No. i n all CDR % in 1st CDR % in 2nd CDR uI

% i n 3rd CDR % i n all CDR

15 2 4 5 11

35 2 5 7 14 40

149 4 18 8 30 3 12 5

147 3 31 11 45 2

1731

121( 7

32 3 9 13 25

60

39 5 8 12 25

33 0 5 7 12 36

51 2 7 9 18 35

34 20 2 1 9 2 1 0 3 4 24 14 7 40 41 35

295 261 52 16 76 13 55 31 183 60 (18(6

239 0 11 14 25 0 5 6 10

764 7 18

742 250 121 13 2 46 37 51 2 39 2 9 89 55 57 2 1(38( 5 m 2 5 1 7 12 (221

mm 12615

16211231

From Kabat et ul. (1977), with permission.

’Unusual distributions are indicated by boxes.

17 42 1 2 2 5

5 9

1471

6 1 0 3 4

1671 157 2 0 3 5

57 5 12 9 26 46

39 4 7 5 16 41

488 308 18 9 68 20 28 9 114 38

1 0 2

4

1141

3

m 1 2

6

3 6 3

60 4 7

74 6 13 9 1 2 31 20 33 42

63 5 11 1 27 43

479 23 41 20

983 27 203 112 380 21 4 12 7 9 62 12 37 31 15 39 9 35 6 1 122 25 84 44 25 2 ( 1 5 ( 6 6 2 6144111811281 4 0.2 41331117_1 5

84

884 38 102 34 174

4 5 9)121 4 4

18

14 39 2 4 4 10 1 6 5 12 19 49

1861

32 31 4 2 11 7 4 1 19 10 33

1591

[201121931(411(39(

7

24 0 4 4 8 33

35 23 5 2 10 5 8 1 2 3 8 66 35

333 0 8 4 12

87 65 16 3 2 5 9 21 1 62 13

0 2 1

18 29 24

5 14 1

4

71

20

58

ELVIN A. KABAT TABLEVII Distribution of Met in the V-Region of Heavy Chains and a Statistical Evaluation of the Role of Met 34 as Structural" Positions

Occurrences

Amino acid

Chain

Total

CDR

Total

CDR

Met

H

20

7 (35%)

121

57 (47%)

Distribution in CDR Positions

Occurrences

First

Second

Third

First

Second

Third

1

2

4

46

2

9

517 Ra VH 212 Mo VHI 313 GP VH 111 MOVHII 10110 MO VHIII (8 anti-P choline; (1 human anti-P choline) 2 no known Ab activity) Other amino acids at position 34: 2 Ile, 1 Phe, 6 Leu, 5 Val, 1 Arg, - 1 Ser Conclusion: Whenever Met occurs at position 34 in H-chain, it is a structural element Met 34

112 Hu VHI 314 HU VHII 21/27 Hu VHIII

Data from Kabat et al. (1976b).

mouse Vkchains while Gln was present in all 40 rabbit V, chains studied. In human V,I Arg makes up 14 and Gln 17 of the 36 chains; and in 30 mouse V, there were 15 Arg plus 4 Gln residues. Thus, this position is predominantly characterized by the two residues. Their role must be structural, and each will perhaps be associated with a particular type of conformation. Position 24 also has 3 Ile, 9 Lys, 3 Ala, 15 Thr, 15 Ser, 2 Gly, 2 Asp, and 2 Glu in V,I; their role cannot be specified precisely. The distribution of Gln at position 27 also readily establishes it as structural in V, chains (Table VIII). The 8 mouse V, chains in which Gln or Glx are absent include 2 Glu in chains lacking 6 residues 27A-F in the first CDR; 2 Thr in chains lacking 7 residues, 27A-27F and 28; 3 Lys, 2 in chains lacking residue 27F and 1 lacking 27E and F; 1 Ser in a chain lacking 27A-27F. Mouse V, chains with Gln or Glx at position 27 also have insertions of differing length after position 27 (Potter, 197713; Kabat et al., 1976b). In Vh chains there are many different amino acids at position 27 and the chains vary in the length of

59

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY TABLEVIII Euidence f o r the Structural Role of Arg and GZn pt Position 24 and Gln at Position 27 in the Light Chain" Positions

Occurrences

Amino acid

Chain

Total

CDR

Total

CDR

'4%. Gln

L L

39 37

20 (51%) 14 (38%)

471 1000

166 (35%) 231 (23%)

Distribution in CDR Positions Second 6 4

First 7 4

Gln

Occurrences Third 7 6

First 96 150

Second 51 7

Arg 24

761190 sequences

Gln 24

53

Conclusion: Arg

+ Gln are two structural elements at position 24

Gln 27

94 + 35 Gld185

+ 10 Glx/190

11/11 Hu V,II 23/23 Hu VJII 19/19 M o v, 40/40 Ra V,

33/34 Hu V,I 8/8 Hu V,II 21/22 Hu VJII 2/2 Hu VJV Conclusion: Gln 27 is a structural element in V, chains

Third 19 74

14/36 Hu V,I 1/2 Hu V,IV 15/30 Mo V, 1/6 Rat V, (5 Lys) 1/1 Pig V, 17/36 Hu V,I 1/10 HLIVhIII 112 HU VkIV 4/30 Mo V, 21/29 M o V, 5/6 Rat V, 1/1 Shark V,

Data from Kabat et al. (1976b3.

their first CDR so that residues at position 27 in these chains may play different roles. Table IX gives data on the distribution of His in the V, chains sequenced. Twenty-five of the 27 His residues were in CDR, and they were distributed at 12 positions in low frequency. Even when allowances were made for differences in length of the various CDR, the His residues did not appear in comparable positions. Thus, His in the CDR of the heavy chain is inferred in almost all instances to be complementarity-determining either as a contacting residue or in influencing the conformation of a contacting amino acid. Tables X and XI provide comparisons of the statistical inferences

60

ELVIN A. KABAT TABLE IX Distribution of His Residues in the Heavy Chain Favoring a Role for His as Complementarity-Determining' Positions

Occurrences

Amino acid

Chain

Total

CDR

Total

CDR

His

H

14

12 (86%)

27

25 (93%)

Distribution in CDR Positions

Occumences

First

Second

Third

First

Second

Third

2

4

6

4

12

9

31 1/3 Hu VHI 35 2/21 Hu VHI 1/1 Mo VHII

53 112 MO VHI 1/8 MO VHIII 54 1/5 HU VHII 58 7/12 HU VHIII (2 anti-IgG; 1 anti Bl.Cr.1) 1/2 MO VHI 61 1/1 MO VHII

95

1/1 MO VHII

1/5HU VHII 115 Ra VHanti-Pn 97 1/4 Ra VHanti-Pn 98 1/1 Mo VHI anti-DNP IOOA 115 Ra VHanti-Pn 102 3/11 HU VHIII (2 anti-IgG) 96

Second CDR: Distance in residues from 50-4,6,9,11,12 Third CDR: Distance in residues from 95-0,1,2,3,6,9,12 Conclusion: His in H chain is so distributed as to play a role in specificity, either contacting or conformationally influencing a contacting residue a

Data from Kabat et al. (1976b).

with the X-ray data in establishing various positions as structural together with the overall variability at that position (Kabat et al., 1977). The amino acid found at the position is indicated. Thirteen positions in the light chain and seven in the heavy chain were found to be structural, and these findings are in complete accord with the X-ray data. In some instances the X-ray data show the same amino acid residue at the same position as in the statistical study; in others the X-ray data show a different amino acid but nevertheless assign it a structural role. In two instances in which the X-ray data assigned a contacting role, position 29 in the light and 50 in the heavy chain, the amino acid was not the same as the one inferred to be structural from the statistical analysis. In the former instance, Ile was inferred to be structural in V, chains and Thr in chains lacking residues 27A-27F, while an

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

61

Ala was contacting in Newm a human VAI with three inserted residues 27D-27F. In the heavy chain, position 50 had Tyr in Newm a VHII chain while Ala was considered structural in VHI chains and was present at this position in McPC 603-but its role as a structural element was not certain. In McPC 603, Phe 32 in VH is assigned a structural role merely because it does not contact the phosphocholine, but statistical analysis strongly supports a conformational influence which cannot be established by X-ray crystallography. Zmplications of the Statistical Approach

This statistical analysis has potential applicability to building models of antibody combining sites. For example, one may fix the orientation of these structural elements to correspond to that in a known X-ray structure in which the amino acid has been found at the position and thus limit the conformations and positions in the site which the other amino acid residues may assume. When the ligand or antigenic determinant is known this could facilitate the identification or positioning of contacting residues. Similarly the inferences from Table VI (Kabat et al., 1977) that His, Tyr, Trp, etc., in the CDR are specificity-determining in almost all instances should influence the placing of such residues in model construction. The statistical analysis has further emphasized the important contribution of differences in chain length in the various CDR to the diversity of antibody combining sites. It thus makes imperative the accumulation of high-resolution X-ray data on sites with CDR of each chain length. This is a substantial limitation since X-ray crystalographers are reluctant to assign a role to a given amino acid in a CDR unless it is of the same length as one which has been studied. Obviously, when such data are also obtained on antibody sites of known specificity, our understanding and ability to construct sites will be greatly enhanced. XI. OTHERDIRECTIONS A major limitation of studies on antibody combining sites has been the unavailability of homogeneous combining sites to protein determinants. To date only one myeloma protein with specificity for a protein antigen, flagellin (Hood et d . , 1973), has been found. Thus the problem of studying combining sites for protein antigens will depend on obtaining purified homogeneous antibodies in adequate amounts. The first 92 residues have been sequenced on the light chain of fractionated rabbit antibody to the peptidoglycan of M . lysodeikticus (Margolies et al., 1975), which is made up of a pentapeptide linked to

TABLEX Comparison of Positions in CDR of Light Chain Considered To Be Structural f r o m Sequence Data w i t h Results of X-Ray Crystullographya Based on X-ray crystallography

a

M

Position

Variability

24 25 26 27

25 8.8 7.3 13: 19

29

33

36:39

9.9

Based on sequence data Arg in V,; Gln in rabbit V, Ala in V,; Gly and Ser in V, Ser in V, Gln in V,

RE1 Human VJ, no insertion 24 Gln 25 Ala 26 Ser

Ile in V,; Thr, in chains lacking 27A-27F Leu in V,

33 Leu

McPC 603 Mouse V, has 27A-27F

Mcg V,V has 27A-27C

Newni Human VAI has 27D-27F

24 Lys 25 Ser 26? Ser

24 Thr 25 Gly 26 T h r

24 Thr 25 Gly 26 Ser 27 Ser 27 D Ser; E Asn; F Ile

27 F Gly 28 Asx 29 Glx

29 Gly

29 Contacting Ala

33 Leu

33 Val

30? Gly 33 Val 34? Lys

51 Val involved in site shielded by insertion

50-56 not involved in site because of deletion

52 55

10 5.8 27:30

56

11

90

17:23

54

Ser in V, Leu or Arg Pro in human V,; (mouse V, at 56); Ala in V, except human V,I and mouse V, Ser (except perhaps in human VNI) Gln in human and mouse V,

54 Leu

52 Asn 54 Arg 55 Pro

56 Ala

56 Ser

90 Gln

w 92*

O,

w

95

31

Pro in human and mouse V,

95 Pro

97

29

Thr in human and mouse V,; Val in V,

97 T h r

a

Modified from Kabat et al. (1977).

* Not sequenced.

95 Asp (monomer I) 97b

89 Gln 90 Ser 92 Asp 93 Arg 95 Leu 97 Val

64

ELVIN A. KABAT

TABLEXI Comparison of Positions in C D R of Heavy Chain Considered to be Structural with Results of X-Ray Crystallography" Based on X-ray crystallography

Position

Variability

Based on sequence data

McPC 603 mouse VHIII has 52 A

32 Pheb 34 50 51

54

9.4 40 18 33

59

6

TYr

63

7.5

Ala?

101

3.6:6.5

Met in 1st CDR Ala in VHI Ile in VHI Ser

Asp or Asx

34 Met 50? Ala 51 Ser 54? Asp 55 Tyr; 57 Arg 59 Tyr 60 Ser; 61 Ala; 62 Ser 63 Val 64 Lys; 65 Gly 101 Asx

Newm Human VHII

31 Asn 32? Asp 33? Tyr 50 contacting Tyr 51 Val 54 His 55 Gly; 57 Thr 59 Asp 60 Thr; 61 Thr; 62 Pro 63 Leu 64 Arg 96 Leu; 99? Gly 100 Cys; lOOA Ile 101 Asp 102 Val

Modified from Kabat et al. (1977). From pair analysis, Table IV, Phe is considered to influence the conformation of Tyr in the tetrapeptide Phe-Tyr-Met-Glu while X-ray crystallography only establishes it as noncontacting. a

the muramic acid of the copolymer of DG~cNAcand Nacetylmuramic acid, but the specificity seems to be directed to the carbohydrate moiety. A most promising approach to obtaining homogeneous anti-protein antibodies was developed by Kohler and Milstein (1976; Milstein et al., 1976; Milstein and Kohler, 1977), who successfully fused antibody-secreting cells from the spleens of immunized mice to a mouse myeloma cell. Such hybrids usually continue to secrete antibody as well as the myeloma immunoglobulin, but variants have been cloned which have lost the ability to synthesize the myeloma protein and secrete only the specific antibody. These lines may be carried as tumors in mice, and large amounts of antibody obtained. Since each antibody-forming spleen cell secretes homogeneous antibody, antibody of any specificity is potentially obtainable. The study of the dif-

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

65

ferent kinds of combining sites which can be formed to individual protein antigens as well as to carbohydrate antigens of simple structure should contribute materially to evaluation of the diversity and heterogeneity of the antibody response and provide many monoclonal antibodies of known specificity for sequencing and for X-ray crystallographic studies. The enormous advances in nucleic acid sequencing (Maxam and Gilbert, 1977; Sanger et al., 1977, and earlier papers) and their relative simplicity and ease as compared with amino acid sequencing not only should provide confirmation of amino acid sequences of Vregions but may ultimately provide additional sequences of known antibodies, especially when carried out on antibodies of known specificity from hybridomas. Our ideas about the arrangement of the genome will have to be completely revised by the findings that mRNA is not transcribed from a continuous stretch of nucleotides in the DNA. Tonegawa et al. (1976, 1977a,b) showed that in the mouse embryo genome the V- and C-genes are separated but are brought closer together during differentiation to mature plasmacytoma cells; even in the myeloma DNA, the stretches coding for the V- and C-domains are not contiguous. It will therefore be essential to continue to do both amino acid sequencing as well as nucleic acid sequencing on heavy and light chains until the mechanism and function of this separation is understood. In the adenovirus (Berget et al., 1977) and SV40 (Aloni et al., 1977) genomes, the mRNA contained sequences transcribed from two or more separated regions of the DNA. Even more startling is the finding (Leder et al., 1977) that the structural gene for p-globin is interrupted by a stretch of 550 bases which is not transcribed in the mRNA for pglobin. These findings open new possibilities for the generation of antibody diversity. In very recent studies Kabat et al. (1978) have ordered the four framework segments FR1, FR2, FR3, and FR4 of the light and heavy chain variable regions from sequences compiled in “Variable Regions of Immunoglobulin Chains” plus additional published data. These framework segments comprised residues 1-23, 35-49, 57-88, and 98-107 of the light chain and 1-30,36-49,66-94, and 103-113 of the heavy chains, respectively. Sequences available for each framework segment were grouped into sets with identical sequences. Sets contained 1 to 18 members. When the V-region of each chain was traced from one FR to the next it was seen that members of the same set in FR1 could be associated with different sets in FR2, FR3, and FR4. Similarly, members of different sets in FR1 could be associated with the same set in FR2, etc. Data for the sets of rabbit V, sequences are given

TABLEXI1 Assembly of the Frameworks of Rabbit V, Chains from Minigene SetsaJ

FRl BS-1 BS-5 2348-3

I

I

FR2

-

FR4

BS-I

0

BS-5 K4820

A -

120

R2436-I

A

K4335d K29-213 K27-306 K9-106 K732-50 K6-14111 161 166 R4140FX4 R4140FX5

A A A A A A

BS-1 BS-5 K4820 K25 K30-267 3547 3368 120 K9-335 K27-489 K29-213 3374 3315 4135

4

I K29-2 13'

A A X

A A A 0 A

X X

+ +

3315 4 135 Key to symbols: Anti-type 111 pneumococcal polysaccharide 0 Anti-type VIII pneumococcal polysaccharide A Anti-streptococcal group A variant carbohydrate

A Anti-streptococcal group C carbohydrate + Anti-p-azophenylarsonate x Anti-Micrococcus lysodeikticus Mouse kappa light chain. Human kappa light chain subgroup IV. K9-335 has an identical sequence as K9-338. The sequence of position 36 (Kabat et al. 1976b) has been changed from Tyr to Phe for K29-213, K9-335 and K9-338 (Braun and Huser, 1977). From Kabat et al. (1978).

'

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

67

in Table 12. These findings suggest that the light and heavy chain V-region frameworks are assembled during embryonic development from sets of minigenes for each FR segment. FR4 from three sets of human V,I chains also contained members of V,II, VKIII,and/or VJV subgroups; one FR2 set contained eight rabbit V,, one human V,IV, and four mouse V, chains (Table 12) and FR4 set contained two human VHIII and one mouse VHIII, indicating substantial evolutionary perservation of these sequences and suggesting that the sets of minigenes are highly conserved in the germ line. Sequences corresponding to the sets of F R sequences, even those with representatives in several human subgroups and in several species, were not found among C-region sequences nor among other nonimmunoglobulin proteins in the Dayhoff data bank (Dayhoff, 1972, 1973, 1976), the highest degree of homology being a match at 6/11 positions for the FR4 set in which mouse and human VJII were identical; other homologies were less than 50%. Individual F R sets (Table 12) contained rabbit V, chains from animals with up to five different antibody specificites or with no known specificity. In the clone of Tonegawa et al. (1977b, 1978) from 12-day-old mouse embryo DNA in which nucleotide sequencing yielded a contiguous sequence for the first 96 amino acid residues of a mouse VA chain, there were nucleotide sequences corresponding to the amino acid sequence of MOPC315, a mouse VAII, with several replacements corresponding to MOPC104E, a mouse VAT; these occurred in the F R and CDR. In F R 1 and FR3 the nucleotide sequences were clearly VAII corresponding to the amino acid sequence of MOPC315; in FR2, however, the nucleotide sequence at position 36 was Val corresponding to VAI MOPC104E and this was ascribed to somatic mutation. In the light of the proposed germ line minigene sets, however, it would clearly be a recombinant in which FR1 and FR3 were from the MOPC315, VAII, and FR2 from MOPClWE, VAI. That FR4 was not joined to the rest of the V-region in the 12-day-old mouse embryo DNA is also in accord with the hypothesis and suggests that this piece had not yet been assembled in the cell of the 12-day-old embryo that donated the DNA sequence for the clone. Since Tonegawa et al. (197713) have shown that the extent of separation of the sequences coding for the V- and C-domains differs in the embryo and in the adult differentiated myeloma DNA, it will be essential to examine mouse and other species of sperm DNA to establish whether the sets of minigenes are separated by intervening sequences and whether the CDR are separated from the framework in the genome (Kabat et d.,1978). There will be need for continued characterization of antibody combining sites as to size and specificity differences by equilibrium dialy-

68

ELVIN A. M A T

sis, hapten inhibition, fluorescence quenching, NMR, spin-labeling studies (Hsia and Piette, 1969),especially on monoclonal antibodies to single antigens that are to be sequenced and studied crystallographically. Recently, Willan et al. (1977) have been able to demonstrate with spin-labeled haptens, differences in the sizes and shapes of the combining sites of three DNP-binding mouse myeloma proteins. The previously noted variation in sequence of rabbit antibodies to the pneumococcal type-specific and the streptococcal group-specific A, A variant, and C polysaccharides makes it imperative to define similarities and differences in the binding properties of antibodies to single determinants. In addition to antibody combining sites varying in size or extent of their complementary areas (Kabat, 1957, 1960, 1966a, 1976a) and with sites which may be grooves or cavities (Cisar et al., 1975), antibodies to type I11 pneumococcal polysaccharide have been shown to consist of two populations, one whose immunodominant group is directed toward the D G ~ C and another toward the Dglucuronic acid moiety, each consisting of subpopulations of antibody combining sites varying in size (Mage and Kabat, 1963). Moreover, recent studies by Zopf et al. (1978) have shown that some antibodies can be directed against one side of a tetrasaccharide while others can be specific for the opposite side, each of entirely different specificity; substitution of a sugar residue on one side can block reactivity with one population of antibodies without influencing reactivity with the other. These antibodies would be expected to have very different sequences even though they were each monoclonal and specific for the same antigenic determinant. If the findings on lectins (Wood et al., 1978) hold for antibody combining sites in that antibodies can also be directed against nonsequential residues of oligosaccharide determinants, another major dimension will be added to antibody diversity. Moreover, it is possible that antibodies may exist to different conformations of the individual sugars relative to one another in a carbohydrate chain (Kabat, 1957). It is not surprising therefore that the solution of the problem of antibody specificity will depend upon the precise understanding of antibody heterogeneity. ACKNOWLEDGMENTS The author is indebted to Mr. Richard J. Feldmann from the Division of Computer Research and Technology and to Dr. Eduardo A. Padlan and Manuel Navia, NIH, for preparing the stereo figures of the CDR of the light and heavy chains and of the four known antibody combining sites; to Drs. D. R. Davies and E. A. Padlan, NIH, and to Drs. Raymond Dwek and P. Gettins, Oxford University, for the coordinates of the two proposed models of the anti-DNP site from which the stereo figures were made; and to Dr. Jay L. Bock of the University of Pennsylvania for evaluation of the NMR data.

STRUCTURAL BASIS OF ANTIBODY COMPLEMENTARITY

69

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PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES By PIERRE D O U Z O U and CLAUDE BALNY INSERM, Unit6 128, BP 5051, Montpellier, a n d lnrtitut d e Biologie Physico-Chimique, Foundation Edmund d e Rothschild, Paris, France

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 11. The Properties of Aqueous-Organic Mixtures as a Function of Temperature: Physicochemical Data and Their Manipulation . . . . . . . . . . . . . 78 A. Density and Viscosity Changes . . . . . . . . . . . . . . . . . . . 82 B. Dielectric Constant Variations . . . . . . . . . . . . . . . . . . . 85 C. Acid-Base Equilibria . . . . . . . . . . . . . . . . . . . . . . . 100 D. Solubility of Salts and Solutes . . . . . . . . . . . . . . . . . . . 122 E. Association and Dissociation of Proteins at Subzero Temperatures . . 129 111. Adaptation of Fractionation Techniques to Subzero Temperatures . . . . 134 A. General Principles . . . . . . . . . . . . . . . . . . . . . . . . . 135 B. Fractionation Methods . . . . . . . . . . . . . . . . . . . . . . . 140 IV. Applications ............................. 146 A. Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . 146 B. Isoelectric Focusing . . . . . . . . . . . . . . . . . . . . . . . . 175 C. Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . 181 V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

I. INTRODUCTION The proliferation of powerful and diversified procedures for protein fractionation, generally based on relatively small differences in the physicochemical properties of the proteins in the mixture, has almost totally outdated the older procedures, particularly organic solvent fractionation based on the accentuation or induction of such differences. The reasons for such neglect are well known and have been emphasized by S. Kaufman (1971), who tried several years ago to revive organic solvent fractionation, which is frequently no more laborious or incomplete and of no lower yield than many other techniques claimed to be particularly suitable. There are two major reasons for using organic solvents as fractionation tools and low temperatures to delay or to minimize protein denaturation and as a further useful variable. 1. An increasing number of observations, including analysis of the contact regions between proteins in terms of surface area and of the volumes occupied by residues (Chothia and Janin, 1975), clearly indi77

78

PIERRE DOUZOU AND CLAUDE BALNY

cate that hydrophobicity is the major factor which stabilizes protein-protein association. This type of noncovalent binding is highly sensitive to “hydrophobic” solvents and to low temperatures. From a large number of earlier studies, the conclusion has emerged that interactions between hydrophobic residues will stabilize protein association at all temperatures, but considerably more at high than at reduced temperatures, as shown for instance by the rigorous thermodynamic treatment carried out by Brandts (1967). On the other hand, other weak bonds of an electrostatic nature and of a high degree of stereochemical specificity might show critical temperature dependence and therefore change classical patterns of protein association and dissociation leading to favorable or unwanted separation patterns. 2. Physicochemical data on a number of essential parameters in cooled aqueous -organic mixtures are now available; they allowed us to determine conditions necessary for enzyme studies in fluid mixtures at subzero temperatures (Douzou, 1975, 1976). They should open a new dimension in protein chemistry of homogeneous solutions at temperatures well below the freezing point of aqueous media, the methodology set u p to investigate enzyme systems at subzero temperatures being easily adaptable to most of the conventional procedures for protein fractionation. Such a task has been carried out over years in this laboratory. The results obtained up to now will be analyzed. Section I1 deals with knowledge and manipulation of quantitative physiocochemical data of a number of aqueous-organic mixtures and with the procedures designed to minimize the variations induced by cosolvent addition and/or temperature variations, which would permit improvement of current methods of protein fractionation. These methods, which include chromatography, electrophoresis, electrofocusing, and some of their applications for the separation of proteins and purification and isolation of “stabilized” enzyme substrate compounds, are described and discussed in Sections 111 and IV. Some applications to protein fractionation and purification described in Section IV are representative of the kinds of progress and problems raised by the investigation of protein systems in fluid media at subzero temperatures. 11. THE PROPERTIES OF AQUEOUS-ORGANIC MIXTURESAS A FUNCTION OF TEMPERATURE: PHYSICOCHEMICAL DATA AND THEIR MANIPULATION

When we started an extensive program of experimentation in the liquid phase at cryogenic temperatures, we were aware of the com-

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

79

plexity of the effects of the progressive addition of an organic solvent, and of the lowering of the temperature on water and on aqueous solutions. We therefore decided to measure the variations of physiochemical parameters such as density, viscosity, dielectric constant, and dissociation constants of the usual buffers under these different conditions, so as to pave the way for eventual experimentation with biomolecules at subzero temperatures. Methods emerged from these basic experiments for the preparation of supercooled solutions and for maintenance and change of any cryogenic temperature. Under such conditions we can observe biochemical reactions by various spectroscopic techniques of ordinary sensitivity and speed of response, as well as with fast kinetic techniques. Special attention has been given to the inspection, control, and significance of the factors that are modified by solvent addition and temperature change and are likely to influence the measurements of biochemical processes carried out under such unusual conditions. Although a great many isolated data existed, very little information was initially available on the effect of large concentrations of relatively innocuous organic solvents, such as alcohols and polyols, on biochemical reactions. The possibility of denaturation had certainly restricted the use of mixed solvents, and in the few cases where they have been used, lack of background data about their physicochemical properties led to caution in interpreting the results. Solvents such as glycerol, ethylene glycol, propylene glycol, dimethylformamide, and even methanol can behave as relatively inert reagents for many proteins although there are conflicting reports on this issue (Willsftter and Rohdewald, 1932; Farber and Wynne, 1935; Meyerhof and Ohlmeyer, 1952; Tanford, 1962; Castaneda-Agullo and Del Castillo, 1959; Hammond and Gutfreund, 1959; DOUZOU, 1977a). Even when there are data to suggest that an organic solvent is relatively inert for a given biomolecule, the possibility of special effects giving rise, for instance, to new reaction mechanisms remains and must be verified. The effects of the solvent on the dielectric constant, acidity and basicity, solvating ability, reaction rates and/or mechanisms should be measured. The values of many of these parameters are known or are easily measurable for various mixed solvents of different volume ratios in the normal temperature range. In the subzero temperature range a decrease in density (reflecting internal cohesion of the medium), an increase in viscosity and dielectric constant, and presumably a decrease in dissociation constant of electrolyte can be foreseen. Such changes might increase or decrease, according to circumstances, some of the effects originally due to the solvent and in some cases might

TABLE I Aqueous-Organic Mixtures Used in Low-Temperature Experimentation. The Freezing Points (“C) Are Indicated as a Function of Volume Ratioad ~

~~~

~

~

~~

Volume ratio, v/v 10

A. Binary Solvents

Ethylene glycol-water Methanol-water Propylene glycol-water Glycerol-water Dimethyl sulfoxide-water Dimethyl formamide-water Methylpentanediol-water

-4 -3.5 -3 -3 -2.5 - 1.5

20

- 10 -8 -8 - 12 -7

-5

30

40

50

60

70

- 17

-26 -40 -38 -29 -41 -25 -26

-44 -49

-69 -67

sc sc sc

sc sc sc

-40 -48

-62

sc sc sc -83 sc

- 17 -14.5 - 19 - 13 -10.5

80

90

100

-100

-83

-50

-85

-100

sc

-100

-90

-13 -97.68 -60 18.18 18.54 -60.43

sc

sc

sc

sc

sc sc

Volume ratio v/v

B. Ternary Solvents

11316 ~

Ethylene glycol-water-methanol

-71

-50

-69

From Travers and Douzou (1970, 1974); Travers et al. (1975). Reproduced with permission. SC, supercooling.

sc

sc sc sc

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

81

slow down the reactions af biomolecules at low temperatures. The measurements of these parameters at subzero temperatures were therefore carried out in this laboratory, in a selection of mixed solutions of various volume ratios. The aqueous-organic mixtures used are listed in Table I with their respective freezing points. Certain general requirements must be met by nonaqueous solvents if they are to be used: they must be chemically stable and chemically inert toward reactive groups of the biomolecules. It is also essential that the organic solvents used to prepare the “antifreeze” mixtures be highly purified, since traces of impurities can have considerable effects on the reactions. Since the ionic environment (ionic strength and acidity or basicity) is a critical parameter in biochemical reactions, knowledge of the variations caused by the addition of the organic solvent to water and the cooling of the mixtures is essential. Both the solubility of neutral salts and the pH might be influenced. Very little is known about the effects of solvent and temperature an acid-base phenomena in mixed solvents, and it is difficult to predict them in terms of changes in the dielectric constant (electrostatic effects) and basicity (nonelectrostatic effects). On the other hand, it is known that, because of the addition of solvent, one has to find practical scales of proton activity (pH = -log uHequivalent to pH when the medium is water) and to relate them to the conventional scale in aqueous solutions. These two important parameters were investigated as a preliminary to the experiments with biomolecules in mixed solvents at subzero temperatures and also to explain the effects observed under these conditions. Moreover, it is foreseeable that there will be dual and/or combined effects of the concentration of organic solvents and the lowering of temperature, according to the parameter considered. Because of the number and complexity of changing parameters when an organic solvent is added and temperature is lowered, any explanation based on a single phenomenon is probably an oversimplification, and detailed knowledge is needed about each parameter. In order to obtain any temperature between + 40°C and - 80°C, to induce heating f* cooling cycles with precision and sufficient flexibility, and to avoid temperature variation and unequal temperature distribution in the samples, special devices were built. These consist of three main parts: (1)a cryogenic temperature-production unit capable of delivering either cooled nitrogen gas or cooled organic liquid (methanol); (2) a temperature-regulation device; and (3)special thermostatted, double-walled measurement cells, as well as cell holders adapted to each particular type of determination. These various devices will be described in detail in Section 111.

82

PIERRE DOUZOU AND CLAUDE BALNY

A. Density and Viscosity Changes Although it is well known that increasing the percentage of organic solvent in aqueous solutions produces a volume contraction that is increased on lowering the temperature, no data were available for the subzero temperature range. This knowledge is essential to correct the spectroscopic recordings. Table I1 and Fig. 1 give density ( p ) values as a function of temperature for various binary or ternary mixtures. It can be seen from Fig. 1 that the variations of p are linear as a function of temperature, so that corrections are easy to make on spectroscopic recordings. In fact, the situation is somewhat more complex, since absorption bands of catalysts and reactants become sharpened and often undergo a displacement and splitting proportional to the variation. These marked changes in the absorption bands in the visible region of the spectrum at low temperatures were reported for several enzymes by Keilin and Hartree (1937, 1947, 1949). Thus the study of substances of biological interest would meet with serious difficulties in regard to the correction of data. However, in aqueous-organic mixtures supercooled and even frozen to transparent glasses, the absorption bands show sharpening with very little intensification, so that corrections for volume contraction are comparatively easy to perform. It should be mentioned that, when crystallization sets in, there is a rapid and very marked intensification in color and absorption bands; the latter may become more than 10 times as intense as the bands of original, uncooled material (DOUZOU, 1974), an observation corroborating earlier results from Keilin and Hartree (1949). Organic solvents added to water to depress its freezing point also change the network of hydrogen bonds, and therefore the viscosity. Increasing this network will produce very high viscosities, such as those obtained for glycerol-water mixtures. As the temperature is decreased, the number of hydrogen bonds increases with a corresponding increase in viscosity. Thus, although the viscosities of aqueous-organic solvents over the normal temperature range were known, data on the variations of viscosity as a function of temperature down to the freezing points of our mixtures were needed. We had to choose one of the earliest and least precise of the numerous methods for viscosity measurements that have been developed over the years, because the falling-sphere method was the only one that could be adapted to low-temperature measurements. The determination of viscosity was made by measuring the resistance to motion of a falling sphere in the fluid. Since the viscosities measured in our experiments on aqueous-organic mixtures at cryogenic tem-

TABLEI1 Densities of Some Water-Organic Solvent Mixtures as a Function of Temperature" Temperature ("C) Mixtures

+20

+10

0

-10

-20

-30

-40

-50

-60

20% Ethylene gylcol-80% water 30% Ethylene glycol-70% water 50% Ethylene glycol-50% water 50% Methanol-50% water 70% Methanol-30% water 40% Ethylene glycol-20% methanol-40% water 25% Ethylene glycol-25% methanol-50% water 10%Ethylene glycol-50% methanol-40% water 10% Ethylene glycol-60% methanol-30% water 50% Methylpentanediol-50% water 50% Propylene glycol-50% water 50% Glycerol-50% water 50% Dimethyl sulfide-50% water

1.026 1.041 1.067 0.928 0.888 1.025 0.994 0.934 0.916 0.990 1.039 1.136 1.077

1.030 1.044 1.073 0.935 0.897 1.032 1.003 0.942 0.924 0.996 1.045 1.142 1.084

1.034 1.048 1.079 0.943 0.906 1.038 1.010 0.950 0.933 1.003 1.051 1.147 1.091

1.037 1.052 1.085 0.951 0.915 1.044 1.017 0.958 0.942 1.009 1.057 1.153 1.097

1.092 0.959 0.923 1.051 1.023 0.967 0.951 1.016 1.062 1.158 1.104

1.098 0.967 0.932 1.057 1.030 0.975 0.960 1.022 1.068 1.163 1.110

1.104 0.974 0.941 1.064 1.037 0.983 0.969 1.029 1.074 1.168 1.117

0.950 1.070 1.043 0.992 0.977 1.079 1.174 1.124

0.959 1.077

a

From Travers et al. (1975).Reproduced with permission.

-

1.000 0.986

-

1.085 1.179 1.130

84

PIERRE DOUZOU AND CLAUDE BALNY

1.2

+20

0

-2 0

-40

-60

FIG. 1. Density ( p ) of some hydroorganic mixtures as a function of temperature. GIOH, glycerol; DMSO, dimethyl sulfoxide; EGOH, ethylene glycol; PrOH, propylene glycol; MPD, 2-methyl-2,4-pentanediol;MeOH, methanol. From Travers et al. (1975). Reprinted with permission of Biochirnie. Copyright by the Societk de Chimie Biologique.

peratures are high ( > 6 cP), the present method can be used without major risk of error, The device used to measure viscosity at low temperatures is shown schematically in Fig. 2. The viscosimeter was used only for relative measurements, and in each case numerous repetitive measurements under identical conditions were carried out. These showed the method to be both sensitive and relatively precise (the precision was approximately 8%). Results are given in Table I11 and Fig. 3. A semilogarithmic scale is used on account of the very large increase in r) as T is decreased. It can be seen that antifreeze mixtures can be classified into two groups: (1)Mixtures with a rather low alcohol content with viscosities, that never exceed 100 centipoise (cP) at low temperatures. (2) Mixtures with a higher alcohol content (>6Wo), which reach 300-500 CP at low temperatures.

PROTEIN FRACITONATION AT SUBZERO TEMPERATURES

I Regulation I

85

Transfer tube

Cryostat

FIG.2. Viscosimeter used between +4"C and the freezing point of various aqueous organic mixtures. The cryostat and the Dewar are not to the same scale. Courtesy of Debey (1968).

The viscosities of the ethylene glycol-water (in the volume ratio, 50:50, most often used) and glycerol-water mixtures are several times those of water at room temperature and attain levels of thousands of centipoise at subzero temperatures. It is known that the viscosity of the glycerol-water mixture (54:46, v/v) is 6.3 CP at +20"C and increases to 80 CP at -20°C. Slightly lower values are reported for the ethylene glycol-water mixture (50:50). In both mixtures, however, many rate constants of enzyme-catalyzed reactions might become diffusion controlled, and such high viscosities might therefore be responsible for changes in reaction kinetics. The density and viscosity changes both in mixed solvents and as a function of temperature are highly important parameters in protein fractionation methods. These values will be used and discussed in next sections. B . Dielectric Constant Variations

Water and a number of aqueous-organic alcohol or glycol mixtures are highly dipolar, and in most cases the effect of the individual dipoles is greatly intensified through their connections by hydrogen

86

PIERRE DOUZOU AND CLAUDE BALNY TABLE 111 Viscosity (Expressed in Centipoises) of Ethylene Glycol -Methanol -Water Mixtures as a Function of Temperature' Temperature ("C) Mixture

20% Ethylene glycol-80% 30% Ethylene glycol-70% 50%Ethylene glycol-50% 50%Methanol-50% water 70% Methanol-30% water 40% Ethylene glycol-20% water 25% Ethylene glycol-25% water 10% Ethylene glycol-50% water 10% Ethylene glycol-60% water

-10

water water water

-20

-30

-40

-50 -

-60

-

-

methanol-40%

1 1 1 3 - - 18 32 63 125 12 21 38 10 17 15 26 48 91

28 195

48 420

methanol-50%

10

19

34

66

145

-

methanol-40%

-

11

19

35

69

140

methanol30%

-

-

14

23

42

76

From Travers et al. (1975). Reproduced with permission.

bonds to form more extended oriented structures. The unique structure of water is reflected by its high dielectric constant, and the addition of any miscible polar solvent perturbs its oriented structure and lowers this value.

1 . Dielectric Constant of Mixed Solvents

a. As a Function of Solvent Concentration. Measurements carried out at a selected temperature (20°C) as a function of the concentration of the organic solvent from 10%to 100% are shown in Fig. 4. It can be seen that the dielectric constant decreases markedly as the concentration of organic solvent is increased, and that this effect is much more pronounced in the case of methanol and methylpentanediol than for dimethylformamide and polyols. At 50 volumes percent most of the organic solvents selected decrease the dielectric constant of water from 10 to 30 units depending on the solvent, except for dimethyl sulfoxide, which has a dielectric constant close to that of water (76.0 and 80, respectively at 20°C). b . At Subzero Temperatures. Akerlof (1932) published experimental values for dielectric constants of some aqueous -organic mixtures as a function of temperature between 0" and 80°C. Curves for the logarithm of D of pure solvents (water, methyl alcohol, ethyl-

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

87

I03 T

3.8

1

-10

1

1

-20

1

1

-30

I

I

I

-40

I

-50

I

- 60

Temp. 1°C )

FIG.3. Viscosity of water-ethylene glycol-methanol mixtures as a function of temperature. Numbers indicate the volume ratio for each mixture. From Travers et al. (1975). Reprinted with permission ofBiochimie. Copyright by the Societe de Chimie Biologique.

ene glycol, glycerol) were plotted against T and gave straight lines that closely fitted the following mathematical expression: log D

=u

-

bT

(1)

where a and b are empirical constants, and T is absolute temperature. This relation has great value for interpolation purposes and therefore is well worth testing over as large a temperature range as possible. It was tested using a number of previous measurements for ethyl bromide and chlorobenzene from -52 to +126"C, and for dimethylpentane from -120 to +80"C and was found valid within experimental error over a temperature range of at least 150°C.

88

PIERRE DOUZOU AND CLAUDE BALNY

80 70 60

a 50

40 30

0

10

20

30

40

SO

60

70

80

90

100

Solvent

FIG.4. Dielectric constant ( D )of hydroorganic solvents as a function of percentage in volume. DMSO, dimethyl sulfoxide; EGOH, ethylene glycol; MeOH, methanol; PrOH, propylene glycol; MPD, 2-methyl-2,4-pentanediol. Temperature, 20°C. From Douzou (197713). Reprinted with permission of Academic Press.

Every straight line obtained under these conditions seems to indicate that the above equation is valid. In this laboratory, Travers and Douzou (1970, 1974) measured D as a function of the volume ratio at constant temperature of various aqueous-organic mixtures, and then for selected volume ratios as a function of temperature between room temperature and the freezing point of mixtures. The results are reported in Tables IV-XI and in Fig. 5.

2. Cooled Aqueous -Organic Solutions Isodielectl-ic with Water It might be useful, in some cases, to raise the dielectric constants of mixed solvents by addition of suitable substances. It is known that dipolar molecules such as amino acids have this effect if added to pure water. Amino acids are virtually insoluble in nonpolar solvents, but, because of their highly polar structure, dissolve readily in aqueous salt solutions and in most mixed solvents. In such mixtures, the dielectric constant at a given temperature, T, is

D = D o + c x 6D

TABLEI V Dielectric Constant of Ethylene Glycol-Water Mixtures" ~

~

~~

~~

~~

Temperature ("C) Percent solvent

0

10 20 30

40 50 60 70 80 90 100

b +20

+10

0

80.4 77.7 75.1 72.0 68.1 64.5 61.1 56.9 53.0 47.5 41.9

84.2 81.4 78.4 75.7 72.1 68.4 64.6 60.0 55.6 50.5 44.7

88.1 85.3 82.5 79.5 76.3 72.4 67.9 63.4 58.8 53.5 47.6

-10

86.9 84.0 80.2 76.5 72.0 67.5 62.3 56.8 50.3

-20

84.4 80.7 76.3 71.3 66.2 60.2

-30

85 80.8 75.3 70.0 63.8

From Travers et al. (1975).Reproduced with permission.

-40

89.3 85.3 79.8 74.2 67.8

-50

90.1 84.5 78.5 72.0

-60

95.7 89.5 83.2

-70

94.6 88.1

-80

100.0 93.0

-90

106.1

-100

112.1

a

1.945 1.931 1.916 1.912 1.880 1.860 1.832 1.803 1.770 1.728 1.675

x 101

2.00 2.02 2.15 2.20 2.30 2.35 2.41 2.47 2.50 2.52 2.54

TABLEV Dielectric Constant of Methanol -Water Mixtures" Temperature ("C)

b

Percent solvent

+20

+10

0

0 40 50 60 70 80 100

80.4 63.8 60.3 55.1 46.3 43.7 33.6

84.2 67.7 64.0 58.7 49.4 46.4 35.4

88.1 71.9 67.8 62.5 53.0 49.5 37.9

-10

-20

-30

-40

-50 ~~

75.6 71.2 66.0 56.6 52.3 40.6

79.5 75.5 70.5 60.1 55.4 42.7

83.5 79.2 73.8 63.5 58.6 45.4

87.9 83.9 78.2 66.8 61.9 48.3

" From Travers et al. (1975).Reproduced with permission.

82.2 70.9 65.7 51.3

-60 ~

86.7 74.9 69.3 54.6

-70

-80

-90

-100

a

x 109

1.945 1.855 1.830 1.790 1.730 1.695 1.580

2.00 2.20 2.30 2.50 2.45 2.50 2.65

~

79.2 73.5 58.0

83.5 77.8 62.0

82.8 66.5

88.4

;j”

s TABLEVI Dielectric Constant of 2-Methyl-2,4-pentanediol -Water Mixtures“ Temperature (“C) Percent solvent

0

10 20 30 40 50 60 70 80 90 100 a

b +20

+10

0

80.4 75.4 70.8 65.7 59.6 53.7 47.7 41.3 35.6 30.6 26.3

84.2 79.2 74.7 69.7 63.7 57.0 50.6 43.8 37.8 32.6 28.0

88.1 83.2 78.8 73.5 67.3 60.5 53.5 46.8 40.3 34.7 29.9

-10

78.0 71.3 64.5 56.9 49.4 42.9 36.9 31.9

-20

75.0 68.3 60.5 52.6 45.3 39.1 31.0

-30

72.7 64.1 55.7 48.4 41.9 36.2

From Travers et al. (1975).Reproduced with permission.

-40

77.1 68.1 59.4 51.6 44.6 38.9

-50

72.0 62.6 54.9 47.3 41.0

-60

76.5 66.8 58.9 50.7 44.4

-70

81.2 70.6 62.1 54.3 46.9

-80

86.2 75.6 66.5 57.4 50.3

-90

91.7 79.6 70.9 60.9 53.5

-100

97.5 84.7 75.1 64.7 57.4

a

x 10s

1.945 1.920 1.895 1.866 1.829 1.783 1.728 1.668 1.605 1.540 1.476

2.00 2.13 2.33 2.48 2.50 2.60 2.59 2.60 2.71 2.72 2.80

80 5

3 0 Z

+ cl

‘

z

E

v)

TABLEVII Dielectric Constant of 12-Propanediol -Water Mixtures" Temperature ("C)

b

Percent solvent

+20

+10

0

0 10 20 30 40 50 60 70 80 90 100

80.4 76.2 72.0 68.0 63.6 58.3 53.2 47.6 41.6 36.2 29.4

84.2 79.8 76.0 71.7 67.4 61.6 56.4 50.4 44.2 38.5 31.3

88.1 83.6 79.8 76.6 71.3 65.4 59.7 53.5 46.9 40.9 33.4

-10

60.0 75.2 69.3 63.3 56.6 49.8 43.5 35.8

-20

79.4 73.3 67.1 60.1 52.8 46.2 38.0

-30

83.7 77.5 70.8 63.7 56.2 49.0 40.5

-40

81.8 74.7 67.6 59.5 52.3 43.3

From Travers et al. (1975).Reproduced with permission.

-50

86.7 79.3 71.6 63.1 55.6 46.0

-60

92.0 83.9 76.1 66.8 59.1 49.1

-70

97.6 89.0 80.6 71.1 62.8 52.3

-80

103.5 94.2 85.7 75.3 66.8 55.9

-90

108.1 99.6 90.6 79.6 70.6 59.4

-100

114.8 105.0 96.2 84.8 75.5 63.3

a 1.945 1.922 1.902 1.879 1.852 1.816 1.776 1.728 1.671 1.612 1.524

XlW

2.00 2.02 2.20 2.38 2.37 2.47 2.47 2.55 2.58 2.66 2.78

TABLEVIII Dielectric Constant of Glycerol-Water Mixtures" ~~

Temperature ("C) Percent solvent 0 10 20 30 40 50 60 70 80

90 100

b +20

+10

0

80.4 77.0 74.3 71.3 68.6 65.0 61.5 57.6 53.5 48.6 43.4

84.2 80.8 77.9 75.0 72.0 68.8 65.1 60.9 56.4 51.3 45.7

88.1 84.6 81.6 78.9 75.7 72.6 68.7 64.2 59.6 53.9 48.2

-10

83.0 80.0 76.6 72.8 67.9 63.0 57.1 50.5

-20

84.5 80.7 77.0 71.8 66.5 59.8

-30

85.1 81.3 75.9 70.1 63.3

-40

90.2 86.1 79.7 73:9 66.8

-50

95.3 91.0 84.7 78.2 70.2

-60

99.8 96.2 89.6 82.3 74.4

-70

106.0 102.5 94.7 87.2 78.2

-80

iii.5 108.5 100.1 91.8 82.4

-90

117.9 114.0 106.0 97.2 86.7

-100

123.0 121.1 112.1 103.0 91.7

a

x 10s

1.945 1.928 1.913 1.897 1.881 1.861 1.837 1.808 1.775 1.732 1.683

2.00 2.09 2.10 2.15 2.23 2.36 2.43 2.40 2.35 2.30 2.24

5

From Travers et al. (1975).Reproduwd with permission.

(D

w

TABLEIX Dielectric Constant of Dimethyl Sulfoxide -Water Mixturesa Temperature (“C) Percent solvent

+20

+10

0

0 10 20 30 40 50 60 70 80 90 100

80.4 79.4 78.8 78.6 77.2 76.0 73.6 70.4 65.4 58.1 45.0

84.2 82.9 82.7 82.3 80.8 79.6 77.4 73.4 68.0 60.3

88.1 87.0 86.5 86.0 85.1 83.9 81.0 76.8 71.0 62.4

-10

90.7 90.3 89.1 87.7 83.9 80.2 74.1

-20

94.7 93.0 91.9 88.7 83.9 77.1

-30

97.3 96.3 92.6 87.4 80.0

Travers et al. (1975).Reproduced with permission.

-40

101.2 100.5 96.4 91.2

-50

105.0 101.0 95.2

b -60

110.7 105.3 98.9

-70

115.1 110.9 103.9

-80

119.8 115.1 109.0

-90

125.8 122.0 113.7

-100

132.4 127.9 119.0

a

1.945 1.939 1.937 1.934 1.928 1.922 1.908 1.885 1.851 1.795 1.703

2.00 2.04 2.03 2.02 2.01 2.02 1.98 1.88 1.75 1.55 1.48

U 0

s

co

TABLEX Dielectric Constant of N,N-Dimethylformamide-WaterMixtures" ~~~

Temperature ("C)

b

Percent solvent

+20

+10

0

0 10 20 30 40 50 60 70 80 90 100

80.4 78.2 76.2 73.9 70.4 66.5 62.4 57.7 51.7 45.1 38.2

84.2 82.3 79.8 76.9 73.9 69.9 65.2 60.1 54.0 47.3 40.2

88.1 86.7 83.9 81.1 77.1 72.9 68.1 62.6 56.4 49.6 42.2

-10

~

84.6 80.8 76.2 71.3 65.3 58.9 51.8 44.6

-20

84.3 79.4 74.0 68.0 61.4 54.3 46.9

-30

83.1 77.2 70.8 64.0 56.9 49.5

-40

86.8 80.2 73.8 66.7 59.5 51.9

~~~~

From Travers et al. (1975). Reproduced with permission.

-50

84.0 76.8 69.7 62.5 55.0

-60

87.7 79.8 72.7 65.2 57.8

-70

83.4 76.1 68.7

-80

76.9 79.3 72.0

-90

82.6 75.6

-100

86.2

a

x 103

1.945 1.938 1.924 1.908 1.887 1.863 1.832 1.797 1.751 1.696 1.625

2.00 2.20 2.07 1.96 1.95 1.92 1.85 1.78 1.85 2.00 2.25

TABLEX l Dielectric Const,ant of Ethylene Glycol -Methanol-Water Mixtures" ~~~~~~~~~~~~~~

Temperature ("C)

b

Solvent

EGOH-MeOH-H,O 40%-20% -40% 25%-25% -50% 10%-50% -40% 10%-60% -30% a

+20

+10

0

-10

-20

-30

-40

-50

-60

a

x 109

59.6 62.1 57.3 52.2

63.3 65.8 60.5 55.4

67.0 69.5 64.3 58.7

70.8 73.6 68.1 62.4

74.8 77.7 72.1 66.1

79.1 82.2 76.2 70.2

83.6 87.1 80.9 74.7

88.3 91.9 85.5 79.3

93.8 97.5 90.8 84.0

1.825 1.842 1.808 1.769

2.45 2.45 2.50 2.60

From Travers et al. (1975).Reproduced with permission.

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES 1

I

I

I

97

I A

/

50% D M S O

120

110

100 90

n 80

70

60

50

/

P -20

I

0

I

-20

I

-40

I

I

-60

-80

I

-100

Tamp. ( " C )

FIG.5. Dielectric constant (D) of some hydroorganic solvents as a function of temperature. DMSO, dimethyl sulfoxide; EGOH, ethylene glycol; MeOH, methanol; PrOH, propylene glycol; MPD, 2-methyl-2,4-pentanediol. From Travers and Douzou (1970, 1974). Reprinted with permission oflournal of Physical Chemistry and Biochimie. Copyright by the American Chemical Society and Societe de Chimie Biologique.

where Do is the dielectric constant of the mixed solvent at T"C, C is the molar concentration of amino acid, and, 6D is the dielectric increment at T"C, which is characteristic of the amino acid. For example, in the case of glycine 6 varies from the value 22.58 in pure water to 20.4 in the presence of 60%of ethanol at 25°C in the concentration range in glycine from 0 to 0.133 M. Similar results are obtained for other mixed solvents used in cryobiochemical investigations, as indicated in Table XII. The addition of an amino acid to mixed solvents at selected temperatures can be a means of compensating partially for the decrease in

98

PIERRE DOUZOU AND CLAUDE BALNY TABLEXI1 Dielectric Increment 6D ( M )for Glycine in Aqueousarganic Mixtures as a Function of Temperature" 0°C

+20"c

-20°C

Solvent, %

Do

6D

Do

6D

Do

6D

Water, 100 Ethylene glycol, 50 1,2-Propanediol, 50 Glycerol, 50 2-Methyl-2,4-pentanediol, 50 Methanol, 50 Dimethyl sulfoxide, 50

80.4 64.5 58.3 65.0 53.7 60.3 76.0

22.5 22.6 23.4 23.0 17.0 21.4 21.0

88.1 72.4 65.4 72.6 60.5 67.8 83.9

23.0 23.8 25.2 24.6 18.8 22.4 22.4

80.7 73.3 80.7 68.3 75.5 91.9

_

Do is the dielectric constant of the solvent without glycine: D C is the glycine concentration ( M ) .

-

=

25.4 26.4 26.6 20.0 24.0 24.1 Do + C

-40°C Do

SD

-

-

89.3 27.4 81.8 28.2 90.2 28.0 77.0 87.9 25.5 100.5 25.6

. 6D, where

dielectric constant due to solvent addition. Limitations are imposed by the soIubility of the amino acid in such mixtures; for instance, there is a "salting out" effect in methanol-water 50:50 at 25°C when the limiting concentration of glycine is about 0.5 M (6 = 20). It can be seen that for solvent concentrations up to 80% the dielectric constant of any mixture reaches that of water (80) at 20°C at temperatures well above the freezing point. The lower the concentration of organic solvent, the higher the temperature at which the mixture reaches D = 80. For instance, in the case of ethylene glycol, for the fractional concentrations 10,20,30,40,50,60,70,and 80%,D reaches 80 at 13",So,0", - lo", -20", -30", -40", and -5O"C, respectively. Accordingly, one only needs to synchronize the addition of organic solvent and lowering of temperature to keep the dielectric constant of the mixed cooled solutions at the original value of the aqueous solution (80). This provides a convenient way to obtain cooled solutions of nucleic acids and proteins, starting from aqueous media at 4°C. Moreover, some problems of ''salting out," which are observed upon addition of organic solvent at 4"C, can be avoided by this synchronizing procedure, since the media with high dielectric constants retain their ionization properties. Finally, conformational changes and denaturation due to dielectric constant variations might be avoided by the procedure. In his pioneering experiments, Freed (1965) injected a solution of an enzyme in water cooled to O"C, as a fine spray into a previously cooled organic-water mixture by means of a micropipette. The spray froze at once to give floating particles of ice con-

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

99

taining the enzyme. The solution then became clear after periods varying from seconds to hours, depending on the temperature and the concentration of organic solvent. Such a procedure is hazardous and cannot be applied, generally, to a large number of enzyme systems or to the study of reaction kinetics. The synchronizing procedure described above appears to be definitely superior.

3. Bulk and Microscopic Dielectric Constants At atomic dimensions, the electric fields originating in neighboring molecules are strongly heterogeneous. In order to elucidate the interaction of the homogeneous macroscopic electric field with such superimposed molecular fields, the knowledge of the dipole moment of the molecules is not sufficient, but a rather more detailed description of the charge distribution in the molecules is needed. Unfortunately, the dipole moment is the only well-defined quantity that can be determined by direct experiment, and no more exact information about the detailed charge distributions is available. Thus the models depicting ions as charged spheres are far too simple to account for the real phenomena of electrostatics. The reason that chemical laws are not simply reduced to electrostatics is that the electrons behave under the influence of their own or applied electric fields, not according to classical mechanics, but according to quantum mechanics obeying the singular Pauli principle. In fact, electrostatics and dielectric constants are simpler applications of the electrical structure of molecules and use outside macroscopic homogeneous electric fields interacting with microscopic inhomogeneous fields. Dielectric constants cannot explain, quantitatively, most physicochemical properties and laws of solutions, and we shall soon see that they can become unimportant. The molecules of more polar solvents, which tend to cluster around the ions and dipole ions, produce a preferential or “selective” solvation that is reflected in measurements of such properties as solubility, acid-base equilibria, and reaction rates. Nonelectrostatic effects, such as the basicity of some solvents, their hydrogen-bonding, and the internal cohesion and the viscosity of mixtures, probably interfere with the electrostatic effects and thus reduce their actual influence. On the other hand, mixtures of water and nonaqueous solvents are enormously complicated systems, and their effective microscopic properties may be vastly different from their macroscopic properties, varying with the solute because of selective attraction of one of the solvents for the solute. For this reason, it may not be possible to explain the quantitative

100

PIERRE DOUZOU AND CLAUDE BALNY

behavior of proteins in mixed solvents in terms of molecular volume, viscosity, and dielectric constant. It can be assumed, therefore, that dielectric constant is not necessarily the “critical” value that explains the effects of organic solvents on protein structure, enzyme-catalyzed reactions, and, as we shall see below, acid-base equilibria. C. Acid -Base Equilibria The addition of organic solvents to water should modify acidbase phenomena, but assessment of such effects poses many problems, as only the measured pH of aqueous solutions can be interpreted in terms of hydrogen ion concentrations. The quantitative comparison of the acidities of partially aqueous solutions is therefore a problem of far greater complexity than the measurements of pH values in aqueous media. As mentioned earlier, a proton activity (paH)is defined in such a way that - log paHis equal to pH when the medium is water, and its value can be measured both by the electromotive force of a cell with liquid junction and by the spectrophotometry of colored indicators. The paH(which can also be termed the pa& or the p&)is a practical unit consistent both with the experimental methods for pH measurements and with the thermodynamic equations for acid-base equilibria in the solvents concerned, but it should be noted that pa, is a succession of scales rather than a single, universal scale of acidity. Consequently, two solutions in different solvent media may have the same paHbut behave in totally different ways in acid-base reactions. The paH value fails completely to indicate the increased acidity (proton availability) that accompanies the replacement of water by a solvent of lower dielectric constant and probably somewhat lower basicity. Nevertheless, the paH scale can be used to measure acidity in many partially aqueous media.

1. Spectrophotometric Determination of pa,. Use of Colored Indicators

A colored indicator is an ionizable molecule whose protonated (InH) and unprotonated (In) form have different spectra. The equilibrium InH -+In + H+ is dependent on the paHof the SOlution which gives

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

lol

and paH= pKf

+ log (yIn/yInH) + log ([Inl/[InH])

(4 1

where log y is given by the Debye-Hiickel equation: 10gyi = - (DT)3’2[l

AZ: (Id)’” + B (DT)-”2u

(5)

and where Z1 is the valence number of the i ion; a, the ionic radius in solution; I , the ionic strength; d, the density of the solvent; D, the dielectric constant of the solvent; T, the absolute temperature; and A and B are constants. In the usual conditions of ionic strength (-lo-’), considering the precision of the measurements, log yi is negligible. The ratio, T ([In]/[InHl), is spectrophotometrically determined. As an example, a series of spectra of 2,5-dinitrophenol, whose pK is 5.87 in water, is given in Fig. 6. Co is the total concentration of the indicator. At paH> pKT, i.e., pH = 9, [InH] = 0, and the absorbance is

(7)

AM= €In Co For an intermediary paH,near the pK, absorbance is A

= €InH

[InHl

+ €In

[In1

From Eqs. (6) and (7), we get A = (Am/Co) [InHl + (AM/CO)[In1 Since [In]

+ [InH] = Co we have r = [Inl/[InHl = (A - AM)/(AM - A )

and WH

2 .

pKT

+ log T = pKf + log (A - Am)/(A, - A)

(8)

From experimental values of r , this expression permits one either to determine paH,if the indicator pKT is known, or pKT if pa, is known. 2. Definition and Determination of a pa, Scale in Mixed Solvents It has been shown (Shedlovsky and Kay, 1956) that, in presence of an organic solvent whose concentration does not exceed 70%, lo-' M HCl is fully dissociated whatever the temperature. Under these conditions, the activity coefficient of the hydrogen ion, y,, paH = -log yH[H+] can be calculated by the Debye-Huckel formula, (Eq. 5), for which the parameters D and d have been measured in the mixed solvents used (Sections IIA and IIB). In this formula, the DT term is the only one in which temperature is involved. As it does not change with temperature, yH,and therefore paHof the HC1 solution, does not vary with temperature. The paHof a 0.01 M HCI solution, denoted as provides the starting point of the pa, scale to be established in the mixed solvent considered. A first indicator is selected whose pKf is slightly higher than paH,;r is determined as a function of T , and pKT can then be measured for each temperature using Eq. (8). In a second step, a buffer (I) is chosen, the P H ~of , which is slightly higher than pKT. Here again, r is determined as a function of temperature so that Eq. (8) allows pH(I, to be measured at any temperature. This procedure is then pursued step by step, alternating buffers and indicators of increasing basicity. Thus, with a judicious choice of buffers and indicators, the protonic activity scale (between paH2 and 11) can be established for mixed solvents at room and subzero temperatures. Currently used buffers in biochemistry have been selected. They

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

103

were prepared by appropriate dilution of an aqueous solution, contraction upon miking being taken into account. Their ionic strength was adjusted to lo-' or, in some exceptional cases, to 5 x to avoid precipitation on cooling. Routinely used colored indicators are given in Table XI11 as well as their pK in water at 20°C. Some of them possess more than one pK, so that care must be taken in order to avoid overlapping of the various zones. If the experimental results are valid, one should be able to measure the isobestic point as pa, is varied. Moreover, in order to obtain as high a precision as possible, r has to be kept between 0.1 and 9. This limitation requires a careful choice of indicators. With this spectrophotometric procedure for determinating pa,, the absolute error is 20.1.

3. Potentiometric Determination of paH a. Method. The potentiometric determinations were made using a glass-calomel electrode assembly (Larroque et al., 1976) as described in Fig. 7. The potential of a glass electrode, reversible with respect to H+ when immersed in a solution whose proton activity is a,, is given by Egms = E o g

+ ( R T / F ) In U H

E values are expressed in millivolts. Similarly, the calomel electrode, reversible with respect to C1-, has a potential

E,,, = Eo,

+ ( R T / F )In aci

TABLEXI11 Usual Colored Indicators with Their p K in Water Indicdtox Metanitroaniline Thymosulfone phthalein a-Naphthylamine 2,6-Dinitrophenol 2,5-Dinitrophenol Chloronitrophenol Paranitrophenol Metanitrophenol Parachlorophenol

pK, in water, t = 20°C 1.59 2.16

3.40 4.42 5.87 6.35 7.95 9.48 10.53

104

PIERRE DOUZOU AND CLAUDE BALNY pH meter

measuring cell

Junctions:

Ag*AgC1 H C I lo-*

1 I/ Glass

50E-0.

test solution

reference electrode

I

KCl 10" 50/50 calomel

Hg

FIG.7. General description of the electrodes and measuring cell: C.T., cooling thermostat; T.C., thermocouple; P.S., porous stone; R.T., room temperature thermostat; M.S., magnetic stirring. From Larroque et al. (1976). Reprinted with permission of Analytical Biochemistry. Copyright by Academic Press.

In these relations, Eog and Eoc are the standard potentials of the glass and calomel electrodes, respectively; F is the Faraday; ucl is the activity of solvated ion C1-. Let uH be the protonic activity of a solution to be tested and E the electromotive force (emf) measured between the electrodes E = E c a l - E g l a s s + Ej

where E , is the sum of both asymmetry and junction potentials. We have then W H =

-log

a H

=

(E" - Ej) F E x F - loguc1 + RT In 10 RT In 10

(9)

with E o = Eoc - EOg. In practice E" cannot be measured, and therefore absolute p a H determinations are not possible with this method. However, the term -log acl (E" - Ej)F / ( R T In 10) is constant for a selected tempera-

+

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

105

ture, so that relative paH measurements can be performed using a standard medium of reference termed (st) in these conditions.

In order to correlate emf measurements to paH,one has to know the response ( R e )of the glass electrode, which is defined as follows:

paH

Re From Eq.

=

AE/AwH

(lo), the ideal pHresponse is Re

=

(RT In 1O)/F

=

0.198 T

(11)

In this formula Re is a function of absolute temperature, only; in practice, however, no glass electrode has this theoretical response in all types of samples and over the whole paHscale. Nevertheless, provided too acidic or too basic paHare excluded, the departure from the ideal behavior is generally negligible. b. Electrodes and Measuring Cell (Fig. 7 ) i . The glass electrode. To perform paHmeasurements at subzero temperatures, the inner reference solution of the electrode must stay fluid in the whole temperature range investigated. For this purpose, we use a solution of 0.01 M HCl in a hydroorganic solvent mixture. The organic solvent must be the same as that present in the test solution. A volume fraction of 50% is chosen in order to allow a very low temperature to be reached without freezing. In such conditions, HCI has been shown to be fully dissociated (Shedlovsky and Kay, 1956) so that the inner solution reference p Hcould be calculated by the Debye- Huckel formula (Eq. 5). An empty glass electrode (Tacussel TB-HS) is filled with the HCI hydroorganic solution (one electrode for each organic solvent). When the inner reference electrode, an Ag, AgCl element (Tacussel) is immersed in this internal liquid the glass electrode is sealed with Araldite. When not in use, the electrode is stored in a 0.01 M HC1 hydroorganic solution of exactly the same nature and composition as that contained in the electrode. In order to obtain reasonably short equilibrium times, the glass electrode is equilibrated in the 0.01 M HC1 hydroorganic solution before measurements are made. ii. The calomel reference electrode. The calomel electrode is defined in Fig. 7. The potassium chloride is a 0.1 M solution in the hydroorganic solvent (50:SO) used in the investigation. The junction with the test solution is either with a capillary or a porous stone. When the capillary is used, a small hydrostatic pressure is maintained inside it in order to avoid any electrode contamination by the test SO-

106

PIERRE DOUZOU AND CLAUDE BALNY

lution. Most of the time we used a porous stone junction. The calomel electrode must be thermostatted at +20"C, as any temperature variations of this electrode cause appreciable emf variations that lead to errors in the paH determination of the order of 0.2-0.3 paH unit/10". iii. The measuring cell and temperature control. This cell, which is constructed of Pyrex glass, is described in Fig. 7 . The operational volume of the test solution is 10 ml. The cell is carefully stoppered, and a slow flow of dry N, is maintained inside it. Around this cell a thermostatic chamber controls the temperature of the sample. This chamber is isolated from the surrounding atmosphere by a vacuum jacket. The sample, which is continuously stirred magnetically, can be thermostatted from +50" to -45" r0.05"C, with a liquid thermostat. The temperature is directly measured in the sample with a chromel-alumel thermocouple. For all paH measurements at very low temperature (t < - l W ) , in order to avoid thermal shock on the glass electrode, solutions to be tested are previously cooled to the temperature of the electrode. In these conditions the electrical equilibrium is reached in about 5 minutes. Finally, the assembly glass-calomel electrodes and measuring cell were contained in a Faraday box. c . Practical Determinations of paH. With these conveniently modified electrodes, potentiometric determinations of paH can be performed in hydroorganic solvents at normal and subzero temperatures. Preliminary to any measurements, their paH responses have been tested using different buffers of paHvalues previously determined by the indicator method. The electromotive force ( E ) of the cell immersed in buffer solutions is thus plotted against paH(Fig. 8). It can be seen that, for the ethylene glycol glass electrode used, the practical response is in good agreement with the theoretical one between paH2 and 9 at +21", +lo, and - 19°C. The reproducibility of the determinations, estimated using two different electrode assemblies, is better than 21.0 mV, and the estimated error in the p a H determinations is kO.1 paH unit. The results obtained by the two methods are in good agreement. Once the electrodes have been prepared and calibrated for a given hydroorganic solvent, the paHdeterminations can be made at the required temperature, either graphically or by direct reading on commercial pH meters calibrated for paH measurement. In the graphical determination the pH meter is used as a millivoltmeter. A solution (A) (0.01 M HCl, in the hydroorganic solvent considered) is selected as the standard reference solution, and its paH is calculated for any temperature using the Debye-Huckel formula

PROTEIN FRACTIONATION

107

AT SUBZERO TEMPERATURES

10

10

9

9

0

0

7

7

r

0

x 6

6 ;

5

5

4

4

3

3 I

2

0

400

200 mV

0

I

I

I

I2

400

200 rnV

FIG. 8. Diagram of paHresponse of an ethylene glycol glass electrode at +21 and - 19°C. Abscissa: electromotive force determined between electrodes when immersed in ethylene glycol-water (50:50)buffer systems. Ordinate: paHof the same media determined by indicator spectrophotometry. The points are the experimental results; the straight lines represent the ideal behavior. (a) Chloroacetate, (b) acetate, (c) cacodylate, (d) Tris. From Larroque et al. (1976). Reprinted with permission ofAnaZytical Biochemistry. Copyright by Academic Press.

(Eq. 5), as described earlier. The electrodes are then immersed in this solution and the corresponding emf is determined. In the diagram OfpaH versus emf, shown in Fig. 8, this reference point is plotted, the theoretical straight line is drawn, and the slope is calculated using Eq. (11). The solution (B) to be investigated is then placed into the measuring cell, and the emf is determined. Using the diagram in Fig. 8, paH(B) can be determined graphically. Alternatively, the pH meter can be directly calibrated in paHvalues. The calibration is performed normally except that the two standard solutions are solution (A), 0.01 M HC1 in the hydroorganic solvent of calculated paH,and solution (B), the paHof which was determined graphically. Using this procedure, the pH meter is calibrated for paH measurements in the hydroorganic solvent considered. 4 . Results

Since weak electrolytes show a variation of their dissociation constants as a function of temperature, buffers containing such electro-

108

PIERRE DOUZOU AND CLAUDE BALNY

lytes should be similarly influenced in the same conditions, retaining their buffer capacity at each temperature. This was verified for buffers containing equimolar concentrations of a weak acid and its salt, in which the protonic activity is close to the pK, of the weak electrolyte component. The values of paHas a function of temperature for various buffers in different hydroorganic mixtures have been measured by the indicator method (Hui Bon Hoa and DOUZOU, 1973a; Maurel et al., 1975; Results are given in Tables XIV-XXII and Travers et al., 1975). Figs. 9-15. The graphs between 0 and -50°C (paHas a function of 1/T) are linear for each solvent, and the same behavior is found with nonequimolar buffers (dashed lines). We shall discuss later the thermodyTABLEXIV Values of paHas a Function of Temperaturea T ('C)

Acetate, 0.01 M

Chloroacetate, 0.01 M

~~

pH in water

+20 2.40 2.60 3.00 3.40

3.80 4.30 4.80 5.30

paH

+20 0 -10 -20 -30 -40

4.35 4.45 4.50 4.55 4.60 4.65

4.80 4.90 4.95 5.00 5.05 5.10

5.30 5.40 5.45 5.50 5.55 5.60

paHin 50%

+20 0 - 10 -20 - 30

4.25 4.45 4.55 4.70 4.80

4.70 4.90 5.05 5.15 5.30

5.15 5.35 5.45 5.60 5.75

5.60 5.80 5.90 6.05 6.20

5.20 5.20 5.25 5.25 5.30 5.35

5.65 5.65 5.70 5.70 5.75 5.75

6.10 6.10 6.15 6.15 6.20 6.25

5.05 5.09 5.15 5.16

5.56 5.60 5.66 5.67

6.04 6.08 6.14 6.15

in 50% ethylene glycol50% water

glycerol-50% water

in 50% propylene glycol50% water

paH

paHin 50%

3.30 3.35 3.35 3.40 3.45 3.45

3.70 3.75 3.75 3.80 3.85 3.85

3.10 3.25 3.35 3.45 3.55

3.50 3.65 3.75 3.85 3.95

3.90 4.05 4.15 4.25 4.35

+20 3.35 3.55 0 3.35 3.55 -10 3.40 3.55 -20 3.40 3.60 -30 3.40 3.60 -40 3.45 3.60

4.00 4.00 4.00 4.00 4.05 4.05

4.40 4.40 4.40 4.40 4.45 4.45

3.76 3.76 3.76 3.76

4.20 4.20 4.20 4.20

+20 0 methylpentanediol50% water -20 -25

2.95 3.00 3.00 3.05 3.10 3.10

2.75 2.75 2.75 2.75

3.24 3.24 3.24 3.24

4.55 4.59 4.65 4.66

5.60

6.30 6.35 6.35 6.40 6.40 6.45

a From Hui Bon Hoa and Douzou (1973a); Maurel et al. (1975). Reproduced with permission.

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

109

TABLEXV Values of paHas a Function of Temperature" T CC)

pH in water

+20

Chloroacetate, 0.01 ivf 2.40

paHin 50% dimethyl sulf- +20 oxide-50% water 0 - 10 -20 7-30 -40 paH in 50% dimethyl

formamide-50% watei

paHin 50% methanol-

50% water

paH in 70% methanol30% water

Acetate, 0.01 M

2.60

3.00 3.40

3.80 4.30 4.80

3.05 3.15 3.20 3.30 3.40 3.45

3.60 3.75 3.85 3.90 4.00 4.15

4.45 4.90 5.40 5.75 4.65 5.10 5.55 5.95 4.75 5.20 5.70 6.05 4.85 5.30 5.80 6.20 5.00 5.45 5.95 6.30 5.10 5.60 6.10 6.45

3.95 4.10 4.20 4.25 4.35 4.45

+20 0 -10 -20 -30 -40

4.25 4.35 4.40 4.45 4.50 4.55

4.45 4.75 4.55 4.85 4.60 4.90 4.65 4.95 4.70 5.00 4.75 5.05

6.05 6.45 6.95 6.20 6.60 7.10 6.30 6.70 7.20 6.40 6.80 7.30 6.50 6.90 7.40 6.65 7.05 7.55

+20 0 -10 -20 -30 -40

2.95 3.00 3.00 3.05 3.10 3.15

3.30 3.35 3.35 3.40 3.45 3.50

3.75 3.80 3.80 3.85 3.90 3.95

4.65 5.00 5.50 4.70 5.05 5.55 4.70 5.05 5.55 4.75 5.15 5.60 4.75 5.15 5.60 4.80 5.20 5.65

+20 0 -10 -20 -30 -40

3.25 3.25 3.25 3.25 3.25 3.25

3.75 3.75 3.75 3.75 3.75 3.75

4.20 4.20 4.20 4.20 4.20 4.20

5.15 5.45 5.25 5.55 5.25 5.55 5.30 5.60 5.35 5.65 5.40 5.70

5.30

6.00 6.10 6.10 6.15 6.20 6.25

From Hui Bon Hoa and Douzou (1973a); Maurel et al. (1975). Reproduced with

permission.

namic implications of these results. For the moment, it is sufficient to say that, from the slope of ApuHfor a given equimolar buffer, it is possible, starting from a different paHvalue at room temperature, to predict the paH value of a supercooled solution. This should allow any desired value to be adjusted from the data given in Tables XIV-XXII. Another interesting result concerns the variation of pa, itself. We can see that the protonic activities of buffers such as oxalate, acetate, cacodylate, and phosphate do not vary widely over small temperature ranges. On the other hand, with Tris buffer one is able to couple a

110

PIERRE DOUZOU AND CLAUDE BALNY

103 T

12

-

10

-

r 8 -

phosphate

P m

6.5

c8codyhte 5.5

6 -

4.8

acetate

4 -

21

I

I

20

0

I

I

- 20

-40

I

-6 0

Temp. ( " C )

FIG. 9. Proton activity of buffered solutions in 50% ethylene glycol-50% water and 50% glycerol-50% water (----) as a function of temperature. From Hui Bon Hoa and Douzou (1973a), and Maurel et al. (1975). Reprinted with permission of Journal of Biological Chemistry. Copyright by the American Society of Biological Chemists. (-)

change in temperature with a change of protonic activity in a paH range spanning 3-4 units. Working at subzero temperatures also introduces the important practical problem of the solubility of phosphate and Tris buffers as a function of temperature. At 0.01 M , phosphate buffer precipitates at -60" and -40°C when the solutions contain 70% and 50% methanol, respectively. In the same conditions, Tris buffer precipitates at -70" and -50°C in solutions containing 70% methanol or dimethylformamide and 50% methanol, respectively. In the presence of a high concentration of proteins or other solutes, the precipitation can occur even at higher temperatures. The same buffers do not precipitate at

111

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

concentrations of 1 mM, in 70%methanol or N,N-dimethylformamide. Moreover, phosphate buffer can eventually be replaced by cacodylate buffer, which is soluble up to 0.1 M over the whole temperature range in all the mixtures studied, even in the presence of solutes. Thus the results show that it is possible to choose a suitable buffer system at any required subzero temperature, in the presence of any amount of organic solvent; the properties of the buffer that have been described also allow their use to effect controlled simultaneous " jumps" of temperature and paH. With the development of the potentiometric technique as describe above, paHdeterminations can now be made very quickly and easily. This makes the screening of any buffer system in any mixed solvent much easier. Data concerning the paHof three buffer systems (cacodylate, phosphate, Tris) in three mixed solvents of water with ethylene glycol, methanol, glycerol at different volume ratios between +20° and their freezing points are summarized in Tables XXIIITABLEXVI Values of paH as a Function of Temperature"

T ("C)

Chloroacetate, 0.01 M

+20 2.20 2.60 3.00 3.40

pH in water

Acetate, 0.01 M 3.80 4.30 4.80 5.30

paH in 40% ethylene glycol- +20 2.80 20% methanol-40% water 0 2.80 -10 2.80 -20 2.80 -30 2.80 -40 2.80

3.40 3.40 3.40 3.40 3.40 3.40

3.90 3.90 3.90 3.90 3.90 3.90

4.25 4.25 4.25 4.25 4.25 4.25

4.55 4.60 4.65 4.70 4.70 4.75

5.10 5.15 5.20 5.25 5.25 5.30

5.55 5.60 5.65 5.70 5.70 5.75

6.10 6.15 6.20 6.25 6.25 6.30

paHin 25% ethylene glycol- +20 2.80 25% methanol-50% water 0 2.80

3.35 3.35 3.35 3.35 3.35 3.35

3.95 3.95 3.95 3.95 3.95 3.95

4.35 4.35 4.35 4.35 4.35 4.35

4.80 5.30 4.80 5.30 4.80 5.30 4.80 5.30 4.85 5.35 4.85 5.35

5.80 5.80 5.80 5.80 5.85 5.85

6.30 6.30 6.30 6.30 6.35 6.35

5.70 5.70 5.75 5.75 5.85 5.85

6.20 6.20 6.25 6.25 6.35 6.35

6.70 6.70 6.75 6.75 6.85 6.85

-10 -20 -30 -40

2.80 2.80 2.80 2.80

paHin 10% ethylene glycol- +20 3.15 3.80 4.35 4.70 60% methanol-30% water 0 3.15 3.80 4.35 4.70

-10 -20 -30

-40 ~

3.15 3.15 3.20 3.20

3.80 3.80 3.85 3.85

4.70 4.70 4.75 4.75

4.35 4.35 4.40 4.40 ~

From Travers et al. (1W5). Reproduced with permission.

5.15 5.15 5.20 5.20 5.30 5.30

112

PIERRE DOUZOU AND CLAUDE BALNY

12

10

8 I I P B

6

4

2

1

20

1

1

0

1

1

I

- 20

I

-4o\

I

1

- 60

Temp PCI

FIG. 10. Proton activity of buffered solutions in methanol-water as a function of 50%methanol; ---,70%methanol. From Hui Bon Hoa and Douzou temperature; -, (1973a). Reprinted with permission ofjournal of Biological Chemistry. Copyright by the American Society of Biological Chemists.

XXV. The composition of each buffer was chosen so as to give a pH close to that of the pK in water. The paH values obtained in 50% of organic solvents are in reasonable agreement with data obtained with the indicator method, and the absolute errors obtained are also similar (k 0.1 pH unit). The study of enzymic reactions in these conditions has shown that such precision is adequate for most of the biochemical experiments (Maurel e t al.,

1975). With this technique for measuring protonic activity at low temperatures, we were able to titrate proteins in mixed solvents at subzero temperatures. Such investigations using the potentiometric method, which allow us to study the solvent and temperature effects on the net

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

113

charge of the proteins as well as on their isoionic point, will be reported in Section II,C,6.

5. Validity of paHEvaluations Until procedures are available by which standard “universal” paH scales can be established, presumably with a very sophisticated procedure, it will be necessary to check whenever possible the hydrogen ion activity b y measuring its effect on a known pH-dependent reaction. Since acidity and basicity are chemical properties, their true measure would be their effects on reactions. A large number of enzyme-catalyzed reactions known to be markedly pH-dependent and

TABLEXVII Values of p,H as a Function of Temperature”

T (“C) pH in water

Cacodylate, 0.01 M

+20 5.50 6.00 6.50 7.00

paHin 50% ethylene glycol- +20

Phosphate, 0.01 M 6.50 7.00 7.50 8.00

0 -10 -20 -30 -40

6.00 6.10 6.15 6.20 6.25 6.35

6.50 6.60 6.65 6.70 6.75 -6.85

6.95 7.05 7.10 7.15 7.20 7.30

7.30 7.40 7.45 7.50 7.55 7.65

7.10 7.25 7.30 7.40 7.50 7.60

7.60 7.75 7.80 7.90 8.00 8.10

8.10 8.25 8.30 8.40 8.50 8.60

8.60 8.70 8.75 8.80 9.00 9.10

pa, in 50% glycerol-50% water

+20 0 -10 -20 -30

5.70 5.90 6.05 6.15 6.30

6.15 6.35 6.45 6.60 6.75

6.65 6.85 7.00 7.10 7.75

7.15 7.35 7.50 7.60 7.75

6.70 6.95 7.10 7.30 7.45

7.55 7.45 7.60 7.75 7.90

7.65 7.90 8.05 8.20 8.40

8.10 8.30 8.50 8.65 8.80

paHin 50% propylene glycol-50% water

+20 0 -10 -20 -30 -40

6.05 6.15 6.15 6.20 6.25 6.30

6.55 6.60 6.65 6.70 6.75 6.80

7.00 7.10 7.10 7.15 7.20 7.30

7.45 7.50 7.55 7.60 7.65 7.70

7.30 7.40 7.45 7.55 7.60 7.70

7.75 7.85 7.90 7.95 8.05 8.10

8.10 8.20 8.25 8.35 8.40 8.45

8.55 8.65 8.70 8.80 8.85 8.90

paHin 50% methylpentanediol-50% water

+20 6.08 0 6.09 -20 6.12 -25 6.12

6.77 6.78 6.81 6.81

7.34 7.35 7.38 7.38

7.80 7.81 7.84 7.84

7.36 7.47 7.78 7.87

8.05 8.16 8.47 8.56

8.57 8.68 8.99 9.08

8.90 9.01 9.32 9.41

50% water

a From Hui Bon Hoa and Douzou (1973a); Maurel et al. (1975). Reproduced with permission.

114

PIERRE DOUZOU AND CLAUDE BALNY

12

10

0

carbonate

10.0

Tris

8.0

phosphate

6.5

I

m

0

6

4

2

I

20

I

I

0

I

I

-20

I

I

-40

I

I

- 60

Temp. ("c)

FIG. 11. Proton activity of buffered solutions in 50% 2-methyl-2,4-pentanediol-50% water (-) and 50% propylene glycol-5Wo water (----) as a function of temperature. From Maurel et al. (1975). Reprinted with permission ofJuurna1 of Biological Chemistry. Copyright by the American Society of Biological Chemists.

showing characteristic pH-activity profiles could be used to check the values obtained by the indicator method. Several studies carried out in this laboratory using various biochemical systems have shown that our paHscales in mixed solvents at normal and subzero temperatures give satisfactory results (Maurel and Douzou, 1975; Maurel et al., 1975). For instance, the tryptic hydrolysis of benzoyl-i-arginine ethyl ester (BAEE) has been investigated: the similarity of the pH profiles obtained in various experimental conditions, and particularly the good fit to their theoretical curves of the experimental points gath-

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

115

ered in differexit buffers and for different temperatures, give reasonably convincing proof of the validity of the paH scales. Their precision appears to be adequate for biochemical experiments. However, it is obvious that if the adjustment of suitable pHvalues as well as the choice of the buffer are a prerequisite for enzyme activity in cooled mixed solvents, changes in pK of ionizing groups at the active site and corresponding shifts in the pH-activity curves must be first recorded to adjust new paHvalues to obtain an optimum enzyme activity. TABLEXVIII Values of paH as a Function of Temperature"

T ("C)

Cacodylate, 0.01 M

pH in water

+20

5.50 6.00 6.50 7.00

paHin 50% dimethyl sulf-

+20 0 - 10 -20 -30 -40

6.00 6.25 6.40 6.55 6.70 6.90

oxide-50% water

6.45 6.70 6.85 7.00 7.15 7.35

7.00 7.20 7.35 7.50 7.70 7.85

7.45 7.70 7.85 8.00 8.15 8.35

paHin 50% dimethyl form- +20

amide-50% water

0 - 10 -20 -30 -40

Phosphate, 1 mM 6.50 7.00 7.50 7.75 8.10 8.30 8.50 8.80 9.00

8.20 8.60 8.80 9.00 9.25 9.50

8.60 9.05 8.95 9.40 9.20 9.60 9.40 9.85 9.65 10.10 9.90 10.35

8.90 8.90 8.95 8.95 9.00 9.00

9.20 9.20 9.25 9.25 9.30 9.30

9.35 9.35 9.40 9.40 9.45 9.45

9.55 9.55 9.60 9.60 9.65 9.65

8.10 8.15 8.15 8.20

8.90 8.95 8.95 9.00

paHin 50% methanol-50% +20 6.40 6.80 water 0 6.40 6.80 - 10 6.45 6.85 -20 6.45 6.85 -30 6.50 6.90 -40 6.50 6.90

7.20 7.20 7.25 7.25 7.30 7.30

7.55 7.55 7.60 7.60 7.65 7.65

7.65 7.70 7.70 7.75 7.75 7.80

8.50 8.55 8.55 8.60 8.20 8.60 8.25 8.65

7.20 7.25 7.25 7.30 7.35 7.35

7.60 7.65 7.65 7.70 7.75 7.75

8.00 8.05 8.05 8.10 8.15 8.15

8.25 8.35 8.40 8.45 8.50 8.60

8.60 8.70 8.75 8.80 8.85 8.95

in 70% methanol-30% +20 0 water - 10 -20 -30 -40

paH

6.75 6.80 6.80 6.85 6.90 6.90

8.00

9.10 9.20 9.25 9.30 9.35 9.45

9.00

9.05 9.45 9.55 9.60 9.65 9.70 9.80

a From Hui Bon Hoa and Douzou (1973a); Maurel et al. (1975). Reproduced with permission.

116

PIERRE DOUZOU AND CLAUDE BALNY

I

4.2

3.8

3.4

I

I

I

4.6 I

I

I

12

Tris

8.0

10 phosphate

6.5 0

r

m

n

acetate 4.8 6

4

2 20

0

-2 0 Temp

-40

-60

("C)

FIG.12. Proton activity of buffered solutions in 50% dimethyl sulfoxide-50% water (--) and 50% dimethylformamide-50% water (----) as a function of temperature. From Maurel et al. (1975). Reprinted with permission ofJournal of Biological Chemistry. Copyright by the American Society of Biological Chemists.

6 . Titration of Proteins in Mixed Solvents as a Function of Temperature

As with buffers, ionizing groups on a protein surface are expected to b e affected by cosolvents and temperature variations. Examples of potentiometric titration will b e shown and discussed in Section IV. They were obtained with the following techniques adapted to mixed solvents and to subzero temperatures. a . Titration Curves. Potentiometric titrations can b e carried out with commercially available automatic titrators. paH values in mixed solvent (ethylene glycol-water, 1 : 1 volume ratio) and as a function of temperature can b e determined with a

PROTEIN FRACTIONATION

AT SUBZERO TEMPERATURES

117

glass-calomel electrode assembly modified as described in subsection 3,a above (Larroque et al., 1976). The KC1 bridge between the reference electrode and the measuring cell is 0.1 M, without hydrostatic pressure in order to minimize the effect of the slight leakage from the KCI bridge to the sample. The calibration and the control of the electrodes were performed at various temperatures before each determination with mixed solution (50% ethylene glycol) of 0.01 M HC1 and Tris buffer, paH = 8.00, 0.01 M . The Pyrex measuring cell, containing 2-4 ml of sample, is maintained at a constant temperature ( ? O.l°C) with a Colora thermostat or with a low-temperature cryostat (Hui Bon Hoa and Douzou, 1973b). The temperature is measured with a chromel-alumel thermocouple connected to a millivoltmeter. The sample is stirred magnetically and the cell is kept in an atmosphere of nitrogen which has been purified by bubbling through 0.1 M NaOH and then pure water (Butler et al., 1972). TABLEXIX Value of paHas a Function of Temperature" T ("C) pH in water

Cacodylate, 0.01 M

t 2 0 5.50 6.00 6.50 7.00

paH in 40% ethylene glycol- +20 0 20% methanol-40% water -10 -20 -30 -40

Phosphate, 0.01 M 6.50 7.00 7.50 8.00

7.60 7.60 7.65 7.65 7.70 7.70

7.45 7.45 7.45 7.45 7.50 7.50

8.00 8.00 8.00 8.00 8.05 8.05

8.45 8.45 8.45 8.45 8.50 8.50

8.80 8.80 8.80 8.80 8.85 8.85

paHin 25% ethylene glycol- +20 6.45 7.10 7.55 8.00 25% methanol-50% water 0 6.45 7.10 7.55 8.00

8.25 8.30 8.30 8.35 8.40 8.45

8.80 8.85 8.85 8.90 8.95 9.00

9.30 9.35 9.35 9.40 9.45 9.50

8.75 8.80 8.80 8.85 8.90 8.90

9.25 9.30 9.30 9.35 9.40 9.40

9.60 9.65 9.65 9.70 9.75 9.75

6.10 6.10 6.15 6.15 6.20 6.20

6.70 6.70 6.75 6.75 6.80 6.80

7.10 7.10 7.15 7.15 7.20 7.20

6.45 6.45 6.45 6.45

7.10 7.10 7.10 7.10

7.55 7.55 7.55 7.55

8.00 8.00 8.00 8.00

7.85 7.90 7.90 7.95 8.00 8.05

paHin 10% ethylene glycol- +20 6.65 60% methanol-30% water 0 6.65 -10 6.65 -20 6.65 -30 6.75 -40 6.75

7.15 7.15 7.15 7.15 7.25 7.25

7.60 7.60 7.60 7.60 7.70 7.70

8.10 8.10 8.10 8.10 8.20 8.20

8.25 8.30 8.30 8.35 8.40 8.40

-10 -20 -30 -40

From Travers et al. (1975). Reproduced with permission.

118

PIERRE DOUZOU AND CLAUDE BALNY 103 -

T

3.8

3.4 I

4.2

I

4.6

I

12

10

1

=

8

-*-*-*-*-t-

6

4

*t

,*-*--*--*-*-•

-*-*--*-*--*-•

chloroacetate 2.6

2 20

0

- 20

-40

-60

Temp. ("C

FIG. 13. Proton activity of buffered solutions in water (30%)-ethylene glycol (10%)-methanol (60?40)as a function of temperature. The number on each curve indicates pH value in pure water. From Travers et al. (1975). Reprinted with permission of Biochimie. Copyright by the Societe de Chimie Biologique.

The electrodes and measuring cell assembly are placed into a Faraday cage to minimize electrostatic effects on the electrodes, and the titrants are injected with a 1-ml automatic burette at a flow rate of 20 pI min-'. For low-temperature measurements, the titrant is precooled before it is introduced into the measuring cell. The titration curves are automatically calculated for each 0.1 pH or 0.1 paHunit with a Tektronix TK 31 calculator and two D M 501 multimeters placed in line with the titration assembly. b. lsoelectric Point Determination. Isoelectric points can be meas-

119

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

ured using the procedure of Sophianopoulos (1973). The conductimeter used in this laboratory is a CDM 3 Radiometer associated with a CM 03 Tacussel electrode. The signal is amplified with a DM 501 Tektronix multimeter. The method can only be used to evaluate the pZ in water and mixed solvent at +20°C of basic proteins. Relative positions of titration curves in water and in mixed solvent at various temperatures are determined as follows. The net proton charge of proteins in pure water at +20"C is calculated with ZH = 0 at the isoelectric point. In mixed solvent, at +20°C, the net proton charge is determined by the pH stat method relative to the 2, value in water at a given pH. The glass electrode is calibrated in pure water at a given pH, then recalibrated in mixed solvent at the TABLEXX Values of paH as a Function of Temperature"

T Tris, 0.01 M

("C)

Carbonate, 0.01 M

9.50

10.00 10.50 11.00

+20

7.95 8.45 8.85 0 8.70 9.20 9.55 - 10 9.10 9.60 9.95 9.50 10.00 10.35 -20 9.95 10.45 10.80 -30 -40 10.45 l0.95 11.30

10.50 10.85 11.15 11.25 11.45 11.70

11.10 11.45 11.65 11.85 12.05 12.30

11.85 12.20 12.40 12.60 12.80 13.05

+20 8.05 8.50 8.85 9.15 0 8.90 9.35 9.70 9.95 - 10 9.35 9.75 10.15 10.45 9.80 10.25 10.65 10.90 -20 -30 10.35 10.85 11.20 11.50

9.50 9.95 10.20 10.45 10.70

9.95 '10.40 10.60 10.90 11.20

10.25 10.70 10.95 11.20 11.50

10.45 10.90 11.10 11.40 11.70

7.80 8.20 8.60 8.90 8.40 8.80 9.25 9.55 8.80 9.20 9.60 9.90 9.20 9.55 10.00 10.30 9.65 10.00 10.45 10.75 10.10 10.45 10.90 11.15

9.70 10.00 10.15 10.35 10.55 10.75

10.25 10.55 10.70 10.90 11.10 11.30

10.60 10.90 11.10 11.25 11.45 11.65

10.75 11.05 11.20 11.35 11.60 11.80

8.10 8.54 8.90 9.20 8.64 9.08 9.44 9.74 9.39 9.83 10.19 10.49 9.60 10.04 10.40 10.70

10.67 10.97 11.47 11.62

11.16 11.46 11.96 12.11

11.61 11.91 12.41 12.56

pH in water

+20

paHin 50% ethylene glycol-50% water

paH in 50% glyceroI-50% water

paH in 50% propyl- +20 ene glycol-50% 0 water - 10

-20 -30

-40 paH in 50% methyl- +20 0 pentanediol-50% -20 water

-25

8.00

8.50

9.00

9.50

' From Hui Bon Hoa and Douzou (1973a); Maurel et al. (1975). Reproduced with permission.

120

PIERRE DOUZOU AND CLAUDE BALNY

3.8

3.4

I

=

I

t-

4.6

4.2 I

I

I

I

I

I

* t-

cacodylats

5.5

acetate

4.8

-+--tr-t-*

-*-

*--*--*-*-•

I

20

I

I

0

I

I

-20 Temp

I

I

-40

chloroacetats 2.6

I

I

- 60

('C)

FIG. 14. Proton activity of buffered solutions in water (50%)-ethylene glycol (25%)-methanol (25%)as a function of temperature. From Travers et al. (1975). Reprinted with permission of Biochimie. Copyright by the Societb de Chimie Biologique.

same proton activity paH. The calibration is performed at pH or pH 6, 7, and 8 with 0.01 M cacodylate and phosphate and Tris buffers, each at 0.01 M. The pH of the protein solution in 0.01 M KC1 (5-7 mg/ml) is adjusted to the desired value by addition of KOH or KC1 (0.01M). Ethylene glycol, at any concentration up to 50% by volume, containing 0.01 M KC1, is then added. With the electrode calibrated in mixed solvent, the paHvariation can be observed relative to the previous pH measurement in pure water. With the pH stat, a sufficient amount of similar titrant in mixed solvent is added to the sample to obtain a pH

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

121

to the pH obtained in pure water. The quantity of titrant introduced allows one to calculate the AZH between titration curves in water and in mixed solvent at a given proton activity. The pZ in mixed solvent, obtained at ZH = 0 using this method, is in good agreement with the results found by the differential conductimetric procedure. For proteins, the variations of pK,,, induced by cooling are very small in the acidic region (Tanford and Wagner, 1954). Moreover, the enthalpy of ionization is also practically zero for anionic buffers (Hui Bon Hoa and DOUZOU, 1973b). TABLEXXI Values of paHas a Function of Temperature' T ("C)

pH in water

+20 7.00

paHin 50% dimethyl

+20

sulfoxide-50% water

Carbonate, 0.01 M

Tris, 0.01 M 7.50

0 - 10 -20 -30 -40

9.50

9.50

7.20 7.55 7.95 8.25 7.90 8.25 8.70 9.00 8.35 8.70 9.10 9.40 8.75 9.15 9.55 9.85 9.25 9.65 10.05 10.35 9.80 10.15 10.55 10.85

10.75 11.30 11.60 11.90 12.30 12.65

8.00

8.50

9.00

paHin 50% dimethyl- +20 6.90 0 7.50 formamide-50% water -10 7.90 -20 8.20 -30 8.60 -40 9.05

7.35 7.95 8.35 8.65 9.05 9.50

paH in 50% methanol- +20 7.25

7.70 7.95 8.40 8.85 8.25 8.50 8.95 9.40 8.60 8.85 9.30 9.75 9.00 9.25 9.70 10.15 9.35 9.60 10.05 10.50 9.80 10.05 10.50 10.95

50% water

0 7.80 -10

-20 -30 -40

8.15 8.55 8.90 9.35

7.60 8.20 8.50 8.20 8.80 9.10 8.60 9.20 9.50 8.90 9.50 9.80 9.30 9.90 10.20 9.75 10.35 10.65

7.70 8.00 8.55 9.10 paHin 70% methanol- +20 7.30 30% water 0 7.95 8.35 8.65 9.10 9.65 -10 8.40 8.80 9.10 9.65 10.20 9.20 9.50 10.05 10.60 -20 8.80 -30 9.25 9.65 9.95 10.50 11.05 -40 9.80 10.20 10.50 11.05 11.60 From Hui Bon Hoa and Douzou (1973a);Maurel et al. (1975).Reproduced with permission.

122

PIERRE DOUZOU AND CLAUDE BALNY 103 T

3.8

3.4 I

1

4.6

4.2 I

I

I

I

I

12

10

0 I 6 a

cacodylate 5 . 5

6

-A-A-A-A-A-A

4 chloroacetate 2 . 6

-A-Ad-A-A-A

2

1

20

1

1

0

1

I

-20

I

I

-40

1

1

-60

Temp . ( " C 1

FIG. 15. Proton activity of buffered solutions in water (40%)-ethylene glycol (40%)-methanol (20%)as a function of temperature. From Travers et al. (1975). Reprinted with permission of Biochimie. Copyright by the Societe de Chimie Biologique.

D. Solubility of Salts and Solutes

1 . Ionic Strength The activity of most biomolecules is greatly influenced by the nature and concentration of the ions present. Salt effects can activate, stabilize, or inhibit enzyme activity, depending on the enzyme and the nature of the salt and its concentration. Addition of significant quantities of organic solvent to aqueous solutions, followed by a lowering of temperature, might influence the dissociation of neutral salts and their solubility, thus changing the ionic strength of the cooled mixtures.

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

123

It has been found that sodium chloride, which is frequently used in biochemical experiments, is completely dissociated at any temperature and has a solubility of 0.1 M in mixed solvents of 50% volumic ratio. Other neutral salts, such as sodium and potassium iodides, are even more soluble in these conditions. Thus, it is easy to preserve the ionic strength of many samples in cooled aqueous-organic mixtures. It must be realized, however, that in aqueous -organic mixtures at subzero temperatures we are dealing not with the familiar preparations of classical biology, but with abnormal solutions involving major changes in the properties of water, and background data are needed to overcome the problems caused by such unusual conditions. Therefore a systematic investigation of the behavior of neutral salts in mixed solutions at subzero temperatures should be carried out, as for buffers, before use of these solutions as antifreezes or cryoprotective agents as they are often used in cryobiology.

TABLEXXII Values of paHas a Function of Temperature" T ("C)

Tris, 0.01 M

Glycine, 0.01 M 9.50 10.00 10.50 11.00

pH in water

+20

9.00

9.50

paHin 40% ethylene glycol-20% methanol-40% water

+20 8.05 8.65 8.95 0 8.65 9.25 9.55 - 10 9.00 9.60 9.90 9.30 9.90 10.20 -20 9.70 10.30 10.60 -30 -40 10.15 10.75 11.05

9.20 9.80 10.15 10.45 10.85 11.30

paH in 25% ethyl-

8.40 8.80 9.25 9.50 9.05 9.45 9.90 10.15 - 10 9.35 9.75 10.20 10.45 9.70 10.10 10.55 10.80 -20 -30 10.05 10.45 10.90 11.15 -40 10.55 10.95 11.40 11.65

10.55 10.85 11.10 11.50

11.05 11.35 11.60 12.00

11.40 11.70 11.95 12.35

11.75 12.05 12.30 12.70

7.90 8.35 8.85 +20 0 8.50 8.95 9.45 8.80 9.25 9.75 - 10 -20 9.20 9.65 10.15 -30 9.60 10.05 10.55 -40 10.05 10.50 11.00

9.45 10.00 10.30 10.75 11.25 12.05

10.15 10.70 11.00 11.45 11.95 12.75

10.55 11.10 11.40 11.85 12.35 13.15

11.05 11.60 11.90 12.35 12.85 13.65

ene glycol-25% methanol -50% water

paHin 10% ethyl-

ene glycol-60% methanol-30% water

a

8.00

8.50

9.50 10.00 10.30 10.65 10.95 11.25

9.95 10.45 10.75 11.10 11.40 11.70

10.35 10.85 11.15 11.50 11.80 12.10

10.60 11.10 11.40 11.75 12.05 12.35

+20

9.80 10.30 10.65 11.00

0

10.30 10.80 11.15 11.50

9.30 9.90 10.20 10.60 11.oo 11.45

From Travers et al. (1975).Reproduced with permission

124

PIERRE DOUZOU AND CLAUDE BALNY

TABLEXXIII paHValues of 0.01 M Cacodylate Buffer System in Mired Solvents at Various Volumetric Ratios and Temperatures” Percent ethylene glycol

T (“(2)

+20 0 - 7 - 15 -22 -35

0

10

20

30

40

50

6.00

5.90 5.85

6.00 6.00 6.00

6.05 6.10 6.05 6.15

6.20 6.20 6.20 6.25 6.25

6.30 6.40 6.40 6.40 6.40 6.40

Percent glycerol

+20

0

10

20

30

40

50

6.05

6.05 6.05

6.10 6.05 6.05

6.15 6.10 6.10 6.05

6.25 6.15 6.15 6.10

6.40 6.25 6.20 6.15

+ 3 - 4 - 12

Percent methanol

+20 0 - 15 -24 -32

0

10

20

30

40

50

6.05

6.00 6.05

6.15 6.15 6.10

6.35 6.30 6.30 6.30

6.45 6.45 6.40 6.40 6.45

6.65 6.60 6.70 6.60

From Larroque et al. (1976). Reproduced with permission.

2 . Proteins a. Solubility and Structural Integrity. As stated by Cohn et al. (1946) in their classic paper on the separation of the protein components of biological fluids and tissues, “by balancing precisely the solvent action of the electrolyte with the precipitating action of the organic liquid widely different conditions can be defined, such that the solubility of the proteins under consideration remains constant.” Owing to the relatively low concentration of enzyme solutions, the solubility in the presence of both the suitable ionic content and the cosolvent is not a problem when enzymes are single-chain proteins. Some oligomeric proteins do precipitate under such conditions at

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

125

room temperature but remain in solution when the samples are prepared using the synchronizing procedure (solvent addition-cooling) described in Section I11 (Balny et al., 1971). As the freezing points of a number of mixtures are very low and since some enzyme-catalyzed reactions are carried out at rather low temperatures, temperature can become a very powerful variable markedly decreasing protein solubility. To keep protein solubility at a constant level, a concentration must be employed that is low enough to avoid precipitation at low temperature, yet as high as possible to permit dependable study. Last but not least, the soluble proteins must keep their native conformation. This must be checked by suitable enzyme activity tests described elsewhere (Douzou, 1974). TABLEXXIV paHValues of 0.01 M Phosphate Buffer System in Mixed Solvents at Various Volumetric Ratios and Temperatures" Percent ethylene glycol

T ("C) +20 0 - 7 - 15 -22 -35

0

10

20

30

40

50

7.10

7.00 7.10

7.15 7.20 7.30

7.25 7.30 7.40 7.50

7.40 7.50 7.50 76 0 7.60

7.60 7.60 7.65 7.70 7.65 7.65

Percent glycerol

+20 + 3 - 4 - 12

0

10

20

30

40

50

7.10

7.15

7.15 7.15 7.15

7.20 7.20 7.15 7.15

7.25 7.25 7.20 7.20

7.25 7.30 7.25 7.25

Percent methanol

+20 0 - 15 -24 -32

0

10

20

30

40

50

7.05

7.25 7.30

7.40 7.50 7.60

7.65 7.70 7.80 7.80

7.90 7.90 8.00 8.00 7.95

8.10 8.10 8.20

~~

From Larroque et al. (1976). Reproduced with permission.

126

PIERRE DOUZOU AND CLAUDE BALNY

TABLEXXV paHValues of 0.01 M Tris Buffer System in Mixed Solvents at Various Volumetric Ratios and Temperatures” Percent ethylene glycol

T 0

(“0

10

~~~~~

+20 0 - 7 - 15 -22 -35

20 ~

8.00

7.80 8.40

~

30

40

50

7.80 8.45 8.60 9.00

7.80 8.40 8.60 9.05 9.20

7.80 8.40 8.65 9.00 9.20 9.70

~

7.80 8.45 8.65

Percent glycerol

0

10

20

30

40

50

8.05

8.00 8.40

8.00 8.40 8.55

8.05 8.35 8.50 8.80

8.05 8.45 8.65 8.90

8.10 8.50 8.70 8.95

~~

+20 + 3 - 4 - 12

Percent methanol

+20 0 - 15 -24 -32 a

0

10

20

30

40

50

8.00

7.75 8.45

7.75 8.40 8.95

7.75 8.40 8.95 9.30

7.75 8.35 8.95 9.30 9.50

7.70 8.30 8.90 9.25 9.50

From Larroque et al. (1976). Reproduced with permission.

i. Native proteins (membrane-bound enzymes after solubilization).

The transition from the native to the denatured state of proteins in aqueous-organic mixtures is usually an endothermic process. The equilibrium involves an enthalpy change, and any decrease in temperature would favor the native state. Denaturation, being a rather slow process if the temperature is low enough, would be delayed almost indefinitely. One may therefore be able to maintain solutions of proteins in their native forms, a consideration that was fundamental to the development of the old methods for fractionation of proteins and might be useful for the isolation and stabilization of a number of solubilized membrane-bound enzymes that are thermodynamically unstable under normal conditions. As described in Section IV, this pro-

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

127

cedure has been applied successfully to an enzyme bound to microsomes and could be extended to many other systems. i i . Enxyme-substrate complexes. A large number of enzyme systems dissolved into aqueous -organic mixtures have been studied at subzero temperatures; cooling -+heating cycles often made it possible to obtain a temporal resolution of reactions, step by step, direct spectroscopic investigation giving details of reaction mechanisms through the detection and analysis of stabilized enzyme-substrate intermediates ( DOUZOU, 197713). Combined with fast kinetic techniques, such as rapid mixing of reactants, the above procedure can be used to study fast reactions and to determine mechanisms in terms of molecular structure. Kinetic determinations can be carried out upon selected, individual steps connecting one intermediate and the next (Douzou, 1975). With enzyme concentrations usually lower than 1 mM it is possible to maintain protein solubility at a satisfactory level and then to prepare soluble enzyme-substrate complexes. These are almost indefinitely stabilized at subzero temperatures and are readily decomposed on warming. Some of these intermediates have been isolated, purified by column chromatography, and then used as “pure” reactants in experiments designed to study their further change (see Section IV). In summary, the solubility of a large number of enzyme systems can be preserved in mixed solvents at subzero temperatures, allowing their investigation and isolation by classical fractionation techniques. Knowledge and manipulation of physical-chemical parameters considered in this section should’help one to avoid failures due to absence of control of these parameters rather than to a diiect cosolvent effect. b. Precipitation. As stated by Kaufman (1971) one of the reasons for the neglect of organic solvent fractionation is the fact that exposure to these solvents can denature proteins. It should be possible to minimize such denaturation if some of the data gathered on mixed solvents as a function of temperature were taken into consideration. Since the solubility of most proteins in these mixtures decreases as temperature is decreased, lower temperatures should become a useful variable, together with the proton activities of suitably selected buffers. Proteins do have a reasonable solubility in most polyolwater mixtures, thus allowing a significant starting concentration, particularly with careful selection of pa, values. The precipitation of proteins involves five essentially independent variables: protein concentration, salt concentration, organic solvent concentration, pH, and temperature. In such a complex system, there is therefore a

128

PIERRE DOUZOU AND CLAUDE BALNY

wide range of possible conditions, which can be varied, individually and simultaneously. Large-scale fractionation of plasma proteins has been carried out at low temperature and at low ionic strength in the presence of water-ethanol precipitants (Cohn et d.,1946). This organic solvent, as well as acetone, was used instead of high salt concentrations for “salting out” in aqueous solutions. Since ethanol and acetone are denaturing agents, irreversible changes in labile protein molecules can generally be minimized if the temperature is maintained sufficiently low-below 0°C and down to -10°C. If three of the five independent variables are maintained constant and the other two are chosen so that one will increase and the other decrease solubility, conditions will be found and curves constructed that define the constant solubility of any protein. Some of the theoretical aspects of this subject have been reviewed (Edsall, 1947; Singer, 1962), and we shall only summarize some technical aspects. Certain protein separations can be carried out in solutions containing an organic solvent and dipolar ions, such as glycine, which increase the dielectric constant of the solution. It is known that small variations of the dielectric constant of the medium can induce large changes in solubility because of the dipole, quadrupole, and higher electric moments arising from the distribution of positive and negative charges within the protein molecule and over its surface; therefore, the addition of amino acid will introduce new solubility conditions. Unlike the dielectric constant effect in aqueous -organic mixtures, the salting-out effect in highly concentrated salt solutions is relatively insensitive to the specific chemical characteristics of the protein and depends mostly on unspecific factors, such as size and shape. Low-temperature fractionation in low salt-ethanol mixtures has been applied to the systematic fractionation of plasma since 1940. There has been a gradual evolution and decisive advance both in respect to the number and specificity of the fractions separated and to the preservation of components in undenatured form. In order to minimize protein-protein interactions the concentration of protein is kept as low as possible, but high enough to permit practical large-scale processing. Concentration of the organic solvent (mostly ethanol) and the temperature are maintained as low as possible in order to minimize protein denaturation. The mole fractions of ethanol in the mixture do not generally exceed 0.163, and the temperature is maintained between 0°C and the freezing point. Concen-

PROTEIN FRACTIONATION AT SUBZERO TEMPERATURES

129

tration of salt is kept as low as possible to take advantage of the diversity of protein-electrolyte interactions and to avoid dialysis. The salt concentration is generally 10 mg/ml) AFGP was much less efficient and the plot curved into a plateau. 2 . The Hysteresis between Freezing and Melting One of the most significant characteristics of AFGP is that it lowers the freezing temperature without affecting the melting point (DeVries, 1971; Hargens, 1972; Feeney and Hofmann, 1973). This hysteresis not only shows the discontinuity of the overall process (Feeney et al., 1972), but also is in agreement with many of the other characteristics of AFGP and its activity. A detailed study of the differences in freezing temperatures and melting points of AFGP solutions has proved an “absolute” hysteresis and different sharp freezing temperatures and melting temperatures (Table XIV) (Feeney and Hofmann, 1973). (These experiments were performed with the specially constructed apparatus described in Section 11,C). In one series of trials, solutions of chicken egg-white ovomucoid were used in the control well of the microscope slide. The AFGP and the ovomucoid were both tested as 1% solutions in water. In all these trials, both the melting and freezing points of the ovomucoid solution were - 0.02 0.01”C. The melting temperature of an AFGP solution was -O.Ol”C, and the freezing temperature of the so-

*

224

ROBERT E. FEENEY AND YIN YEH

TABLEXIV Freezing and Melting of Water and Solution of Antifreeze Glycoproteins (AFGP)in Waterad Observed changes Temperature adjustments ("C phase)

In water containing ice crystals

0.0 Holding -0.1 Lowering -0.7 Lowering -0.8 Holding

Melt and freeze Frozen Frozen Frozen

-0.7 Raising -0.1 Raising 0.1 Holding

Frozen Frozen Melt and freeze

In water solution of 1% AFGP containing ice crystals Crystals melt Crystals do not melt, liquid does not freeze Crystals grow, new crystals form until all solution frozen All frozen, no melting All frozen, no melting Melt ~

~

~

From Feeney and Hofmann (1973), reproduced with permission. *Water and AFGP solution was initially frozen at -3°C and then allowed to melt at +O.l"C until 5-10% of solution remained as ice crystals. The temperature was then adjusted to 0.0"C and periodically lowered and then raised as indicated. The times at each temperature intermediate between freezing and melting were 5-10 minutes. All observations were made microscopically. a

lution containing ice crystals was - 0.80"C. In another typical series of trials, a solution of AFGP containing a few crystals of ice froze at - 0.78"Cand melted at 0.00"C. After freezing, the sample was melted at + 1°C until only a small bundle of crystals remained (only approximately 0.1% of the solution had crystals). The sample was then adjusted to -0.60"C and held at this temperature for 300 minutes. N o growth or melting of the crystals occurred. Similar observations were made when the AFGP solutions containing ice crystals were maintained at a temperature slightly lower (- O.lO°C)than the melting temperature or slightly higher (- 0.70"C) than the freezing temperature. From these experiments it was concluded that (1) ice formed in a solution of AFGP seems to be normal ice-that is, it melts at 0°C; (2) freezing and melting of AFGP solutions occur at rates approximately similar to those at which water freezes and melts when equivalent amounts of heat are applied or removed at the respective melting or freezing temperatures. Thus, there was no evidence indicating a comparatively rapid development of crystals in AFGP solutions as described by Hargens (1972).

ANTIFREEZE PROTEINS FROM FISH BLOODS

225

3. Possible Functions as an Inhibitor of Nucleation Solutions may supercool (i.e., not crystallize when the temperature is lowered below the melting point) in the absence of nuclei. Although the prevention of freezing by AFGP in the presence of ice crystals would indicate that AFGP must also function at a postnucleation stage, whether or not AFGP functions to prevent nucleation has not been proved. One set of attempts to prove that inhibition of nucleation was caused by AFGP was unsuccessful, however. These experiments were done under the supervision of Dr. Hansa Ahrend at the Department of Solid State Physics, the Swiss Federal Technical University, Zurich (Feeney and Hofmann, 1973). Differential thermal analytical experiments were carried out in aluminum vessels in a Mettler vacuum thermal analyzer. Controls of water or solutions of 1% chicken ovomucoid in water and the solutions containing 1% AFGP froze at the same temperatures and had similar freezing and melting curves. Freezing of small volumes (10 p1) of such solutions usually occurred between - 15 and -2O"C, depending on the purity of the water and the experimental rates of lowering the temperature. The freezing temperatures in these conditions are usually considered to be related to the rates of nucleation, not to their freezing temperatures in equilibrium with ice. These experiments did not, therefore, indicate that the AFGP functions by inhibiting nucleation, that is, by inhibiting the initial formation of points of crystallization. Under these conditions, however, extensive supercooling occurs and freezing initiates at a much lower temperature than that at which the AFGP is known to function. B . Effects of Freezing Solutions of AFGP on the Characteristics of the Ice and AFGP In comparison to most studies on the mechanisms of action of a protein, similar studies on AFGP must necessarily be done under conditions that present special problems, namely, the phase change of water to ice. These require the design of experiments to make observations on the ice, the surface of the ice, the surface of growing ice crystals, and solutions in the presence of ice crystals. 1 . Distribution of AFGP between Ice and Liquid Phase

Duman and DeVries (1972) found that the concentration of active antifreeze glycoproteins in the liquid phase was unchanged upon partial freezing of the solution and concluded that the active glycoprotein

226

ROBERT E. FEENEY AND YIN YEH

is incorporated into the ice phase. They also reported that the inactive component 8 was partially retained in the ice phase and that it functioned as a weak antifreeze agent. The incorporation of the active AFGP into the ice phase has been confirmed, but the inactive component 8 has not been found to be incorporated into the ice phase or to have weak antifreeze activity (Tomimatsu et al., 1976). The studies of Tomimatsu and co-workers (1976) used Raman spectroscopy with a specially constructed capillary cell (Fig. 18). The procedure allows a single crystal to be grown at determined rates and with or without sweeping the surface of the growing crystal with the solution. By this technique it was found that the active AFGP component 4 was distributed approximately equally in the ice and in the solution whether the crystal was grown slowly with sweeping or fast without sweeping (Table XV). In contrast, inactive AFGP component 8 was completely excluded when the crystal was swept even though the freezing was fast. When the crystal was not swept, then appreciable quantities of component 8 were found in the ice phase. Even sodium chloride was incorporated when growth of the crystal was rapid without sweeping of the growing edge of the crystal.

4

I

E

z -b

-9xii --e

C

t

Py

20

G

F

FIG. 18. Schematic diagram of the optical arrangement for single-crystal work. A, tungsten lamp; B1 and B2, Polaroids; C, focusing lens, D, light-collection lens; E, capillary cell showing lens (l),solid phase (ice, s), liquid phase (solution, w), interface (i), and laser beam (b);F, filler tube; G, monochromator slits; and H, analyzer. Coordinate system is as indicated. Cold air flow is along the X direction. From Tomimatsu et al. (1976), reproduced with permission.

227

ANTIFREEZE PROTEINS FROM FISH BLOODS

TABLEXV Relative Intensities of Methyl Symmetric CH Stretching Mode of Glycoproteins 4 and 8 in 1 % Solutions and Frozen 1 % Solutions. Fraction of Glycoprotein in the Ice Phase” ~

TemperatureC CH, band (“C) (cm-’)

Glycoprotein sampleb

4, 1% Solution 8, 1% Solution 4a, Ice (swept, slow, -4.5%) 4b, Ice (very slow, -0.7”C) 8a, Ice (swept, fast, -4.5”C) 8b, Ice (very slow, -0.5”C) 8c, Ice (fast, -3.5”C) ~

~~

~

-0.3 -0.3 -5.0 -5.0 -5.0 -5.0 -5.0

2948 2948 2945 294s 2945 2945 2945

Intensityd (CP4

4500 4200 4200 4200 0 1050 4200

Fraction of glycoprotein in icee

0.93 0.93 0 0.25 1.00

~

” From Tomimatsu et al. (1976), reproduced with permission. * For ice samples, continuous sweeping with fresh glycoprotein solution is indicated by “swept”; for these samples the filler tube remains in the capillary during crystal growth. For all other ice samples the filler tube is withdrawn during crystal growth. Crystal growth was at the temperature indicated. Temperature of Raman measurement. Normalized to 200,000 cps for the OH stretch peak for water at 3240 cm-’ for solution data. For ice data, normalized to 200,000 cps for the OH stretch peak for ice at 3150 cm-’ and adjusted for the ratio of OH stretch intensity of water to that of ice. Ratio of intensity in ice to intensity in water.

2 . Characteristics of the Ice Frozen from Solutions of Active Antifreeze Since the melting points of ice frozen from solutions of active AFGP are normal (i.e., approximately WC), the crystal structure of the ice would most likely be similar to the crystal structure of ice from water. The similarity of the AFGP ice to normal ice has been reported by Raymond and DeVries (1972), who cited that from X-ray studies the ice from AFGP solutions was ordinary hexagonal ice. The similarity of the ice from AFGP solutions to ordinary ice has also been confirmed by the Raman studies (Tomimatsu et al., 1976). Raman spectra (2600 to 3800 cm-I) of water and 1% solutions of AFGP components 4 and 8 at -0.3”C were used to determine the distribution of these components in water or ice (see Table XV). The 2948 cm-I and 2945 cm-I bands of the difference spectra were used to determine the amount of glycoprotein (Figs. 19 and 20). All spectra comparisons indicated that the ice from AFGP solutions was ordinary ice. The Raman spectrum in the OH stretch region shows no dif-

228

ROBERT E. FEENEY AND YIN YEH

3140

RAMAN SHIFT a c m - l

3000

2800

2600

RAMAN SHIFT Acm-1 FIG. 19. (A) Raman spectra, 2600 to 3800 cm-', parallel polarization experiments, at -0.3"C of water (curve W) and 1%solutions of glycoprotein 4 (curve 4) and of glycoprotein 8 (curve 8). (B) Difference spectra, 1%glycoprotein 4-water (curve 4) and 1%glycoprotein 8-water (curve 8). Experimental conditions: 300 mW of power, 6 cm-I slit width, and 0.5 second count time per cm-'. From Tomimatsu et al. (1976),reproduced with permission.

ANTIFREEZE PROTEINS FROM FISH BLOODS

3800

3600

3200

3400

3000

2800

229

2600

RAMAN SHIFT Ocrn-'

-

v)

W Z O W Z +

;z

W W

LL>

LLF O 4

w U

3100

3000

2900

2800

2700

2600

RAMAN SHIFT Acrn-' FIG. 20. (A) Raman spectra, 2600 to 3800 cm-I, parallel polarization experiments, at - 5.0"Cof ice from pure water (curve 1)and ice frozen from 1%solutions of glycoprotein 4 (curve 4a) and of glycoprotein 8 (curves 8a and 8b). (B)Difference spectra: ice sample 4b-ice (curve 4b), ice sample 8a-ice (curve 8a), ice sample 8b-ice (curve 8b), and ice sample 8c-ice (curve 812). Experimental conditions: 300 mW of power, 2 seconds count time per cm-I, and 6 cm-' slit width except 4 cm-' for curve 1. From Tomimatsu et al. (1976), reproduced with permission.

230

ROBERT E. FEENEY AND YIN YEH

ference from the spectrum of pure ice (Fig. 20). Similarly, the spectra in Fig, 19 shows that the water spectrum of 1%solutions of AFGP component 4 is like that of pure water. Although the ice formed from active AFGP solutions appeared to be ordinary ice, disorientation of the crystal and poorly defined icesolution interfaces were observed (Tomimatsu et al., 1976). When a 1% solution of glycoprotein 4 was used to grow an oriented single crystal of ice, it was impossible to obtain a reasonably uniform crystal of ice, even with very slow growth and continuous sweeping of the ice-solution interface with fresh glycoprotein solution. The ice formed showed poor polarization properties with numerous regions of uneven extinction between crossed polaroids (sample 4a, Fig. 20). Slow growth at -0.7”C with the filler tube withdrawn resulted in an even more nonuniform ice (sample 4b, Fig. 20). Both samples showed appreciable Rayleigh scattering. The salt and 1% glycoprotein 8 solution systems showed a sharp growing interface between ice and solution. In contrast to this, the ice-liquid interface for 1%glycoprotein 4 solutions was always “fuzzy” in the growing region.

3. Characteristics of the AFGP in Solution and in the Ice Phases Raman spectroscopic studies of active AFGP frozen into the ice phase or remaining in the liquid phase showed no structural differences that could be attributed to a mechanistic interpretation (Figs. 21-23, Table XVI) (Tomimatsu et al., 1976). However, Raman spectra showed that vibrational modes, which are structure-sensitive, differed in relative intensity or band location between active and inactive glycoproteins. The indicated differences in local conformation could alter the affinity of the carbohydrate hydroxyl for ice and could be reflected by a difference in Raman bands to which the COH vibrational mode makes a significant contribution. Bands near 1400,1303, and 1260 cm-’ appeared as though they might have use in studying differences in hydrogen bonding of the carbohydrate hydroxyls between active and inactive glycoproteins. A more detailed interpretation of the differences and changes in the Raman spectra of active and inactive glycoproteins, which requires deuteration studies of the glycoproteins and of suitable model structures, might provide a clearer picture of the mechanism by which the active glycoprotein anomalously lowers the freezing temperature of water. Direct observation of the AFGP in solution by quasi-elastic light scattering also did not show any conformational changes that could be related to function (Ahmed et al., 1975). Comparisons of measurements of diffusion coefficients, at different temperatures and at

23 1

ANTIFREEZE PROTEINS FROM FISH BLOODS

A

* t m

z

W I-

z

Y

2

I-

4 Y

a

Vb9

1800

1600

1400

1200

1000

RAMAN SHIFT

8 00

600

400

Om-'

c >

-

v)

E

z

LU

5 M

3800

1600

1400

3700

RAMAN SHIFT

1000

7800

lb00

am-'

FIG.21. Raman spectra at 25"C, perpendicular polarization experiments, of solid samples of glycoprotein 4 (curve 4) and glycoprotein 8 (curve 8). (A) 300 to 1800 cm-I; (B) 2600 to 3800 cm-'. Experimental conditions: 450 mW of power, 4 cm-' slit width, and 1 second count time per cm-'. From Tomimatsu et al. (1976),reproduced with permission.

TABLEXVI Tentative Assignments of Selected Bands from the Raman Spectra of Antifreeze Clycoproteins 4 and 8 as Solids, in 5% Aqueous Solutions, and in Rapidly Frozen 5% Aqueous Solutions" Glycoprotein 4

Solid (cm-')

2941 1690 1677 1657 I458 1380 1332 1315

-

1270 1250 1217 980 969 894

a

Frozen 5% s o h tion (em-')

5% Solution (ern-') ~~

~

~~

1662 1675 1641 1459 1403 1379 1333 1304

1277 1258

-

977 963 891

Glycoprotein 8

Solid (cm-')

5% Solution (cm-')

Frozen 5% solution (cm-')

-

-

-

2941

1673 1655 1458

1668

-

1653 1680 1641 1460 1397 1380 1332 1316

1381 1330

1459 1380 1323

1303

-

-

1670 1656 1459 1398 1384 1332 1317 1303

1275 1258

1270 1253

1274 1260

1279 1260

979 962 884

976 961 886

977 961 893

980 963 886

-

-

-

Tentative assignmenp

~

~

-

-

-

-

-

-

P: CHI symmetric stretch P: amide I P: amide I P: amide I P: CH3 asymmetric deformation C: CCH, OCH, and COH be nd C: CCH, OCH, and COH bend P: CC and CN stretch HCC and NH bend P: HCCH3 and HCN bend P: amide 111 C: CCH, OCH, and COH bend P: amide 111 P: amide 111 C: CC, CCH, OCH, COH coordinates P: amide 111 P: CO bend, CC stretch P: CH3 symmetric deformation P: CCH3 stretch C: CCH coordinate

From Tomimatsu et al. (1976),reproduced with permission. Tentative assignment for P (polypeptide) and C (carbohydrate) moieties of glycoproteins.

ANTIFREEZE PROTEINS FROM FISH BLOODS

I

1800

'

233

l4,bO

1600

1400

1200

1000

800

600

400

RAMAN SHIFT Om-' FIG.22. Raman spectra, 300 to 1800 cm-', perpendicular polarization experiments, of 5% aqueous solution of glycoprotein 4 at -4.3"C (curve 4) and of glycoprotein 8 at - 10.9"C (curve 8). Experimental conditions: 6 cm-' slit width, 420 and 300 mW of power, and 10.5 and 10.0 seconds count time per cm-', respectively, for curves 4 and 8. From Tomimatsu et al. (1976), reproduced with permission.

-0.2"C in the presence of ice crystals, did not show that any conformational changes in the proteins occurred under conditions where the protein was functioning, i.e., preventing ice crystal growth at - O.2"C (Table XII).

VI. ACTIVITIES OF CHEMICALDERIVATIVES OF AFGP Although the chemical modification of proteins can be a powerful tool for relating the structure of proteins to their functions (Means and Feeney, 1971),there can be formidable difficulties in applying and interpreting the modifications (Feeney and Osuga, 1975). The AFGP present particular difficulties because they are glycoproteins for which the development of specific methods has been largely neglected. In addition, almost all chemical methods for proteins have been developed for the reactive side-chain groups, i.e., amino, carboxyl, imidazole, sulfhydryl, guanidyl, indole, or thioether. AFGP

234

1800

ROBERT E. FEENEY AND YIN YEH

1600

1400

7

.

1200

1000

800

600

400

RAMAN SHIFT Ocrn-' FIG.23. Raman spectra, 300 to 1800 cm-', perpendicular polarization experiments, of rapidly frozen 5%aqueous solutions of glycoprotein 4 at - 9.0"C(curve 4) and of glycoprotein 8 at - 1O.O"C (curve 8). Experimental conditions: 240 mW of power, 6 cm-' slit width, and 20 seconds count time per cm-'. From Tomimatsu et al. (1976),reproduced with permission.

contains no such side-chain groups, but only the amino and carboxyls as terminal amino acids and, of course, the carbohydrate side chains. Consequently, the methods employed in studying AFGP have been primarily those directed at the carbohydrate side chains. These have included both the formation of complexes and covalent derivatives. Two main conclusions resulted from the modifications: (1) With the exception of the removal of a few residues from the amino-terminal end and the oxidation of the C-6 carbinol of the galactoses to the aldehyde, a11 other treatments caused inactivation. (2) The losses of antifreeze and antilectin activities largely paralleled one another (Table XVII). A. Shortening of the Peptide Chain

1 . The Activities of Small Glycopeptide Fragments Small glycopeptides obtained by extensive subtilisin hydrolysis of active AFGP were all reported to have no detectible antifreeze or antilectin activities (Vandenheede, 1972; Chuba et al., 1973). The largest

TABLEXVII Comparative Effects of Different Treatments of Antifreeze Glycoproteins on Antifreeze and Antilectin Activitiesa Activity remaining Treatment Reagent

Modification

Carbohydrate modificatilon Alkaline Iz oxidation None Neutral Brz oxidation General oxidation Alkaline periodate Oxidative scission of galactose Glucose oxidase Oxidation of C-6 carbinols to aldehydes Glucose oxidase + Oxidation of C-6 carbinols alkaline Iz oxidation to carboxyls Glucose oxidase + Formation of aldehydeexcess bisulfite bisulfite complex Sodium tetraborate Formation of carbanol borate complexes Acetic anhydride in Acetylation of carbinols pyridine High pH P-Elimination of disaccharide Shortening of peptide chain Edman degradation Sequential removal of amino acids Short-time proteolysis Hydrolysis into large (elastase) glycopeptides Extensive proteolysis Hydrolysis into small (elastase) gl ycopeptides a

Group affected

(%)

General carbohydrate Galactose

0

Reaction

Galactose N-Acetyl galactosamine Galactose N-Acetyl galactosamine C-6 positions Cis hydroxyls

? 90 70 15 70 15 Excess bisulfite >90

-

Antifreeze (%o)

Antilectin (%)

790 < 10 16

>90

-

-

89 15

-

80

Formation of peptide of -30 amino acids Formation of glycopeptides of 5-7 amino acids

0

0

0

0

Data from Ahmed et a1. (1973) and Chuba et al. (1973).

236

ROBERT E. FEENEY AND YIN YEH

pure peptide tested was a pentapeptide consisting of Ala-Ala-ThrAla-Ala, but mixtures of hexa- and heptapeptides containing two of the carbohydrate side chains were also inactive. 2 . The Effects of Edman Degradations Progressive degradations of AFGP component 5 by the Edman procedure caused no significant losses of antifreeze activity for the first three or four degradations (Vandenheede, 1972). At this point > 95% of the activity was retained, but with further degradations losses begin to rapidly occur. After six or seven degradations, approximately 85% activity was retained. Further degradation caused a precipitous drop in activity. It was thus possible to remove the first glycotripeptide with no apparent loss of activity. Removal of more than the second glycotripeptide caused extensive losses, but it was not known whether these losses were due to shortening of the peptide chain, or side reactions, such as accumulated degradations of the carbohydrate side chains.

3. Losses of Activity on Proteol ysis Treatment of AFGP with subtilisin or fungal protease resulted in loss of the antifreeze activity (Komatsu et al., 1970a). When this loss was plotted against the number of amino groups present and a line was extrapolated to zero activity (Fig. 24A,B), it was found that the antifreeze activity was lost when an average of 1.5-1.8 peptide bonds per 10,000 g of AFGP had been cleaved. Therefore, complete loss of activity occurred when only a few of the bonds in the polypeptide chain were split. No cleavage of glycosidic bonds by contaminating glycosidases occurred during the hydrolysis. Acrylamide gel electrophoresis of the inactivated glycoprotein indicated that none of the original glycoprotein remained. It was stated that examination of the products in the analytical ultracentrifuge indicated that no large glycopeptide remained after proteolysis, but no values for molecular weight were given. Incubation of AFGP with trypsin, a-chymotrypsin, leucine aminopeptidase, or carboxypeptidase A did not result in loss of activity or in the appearance of new amino groups. No loss of activity or cleavage of glycosidic linkages resulted when AFGP was incubated with pgalactosidase from bovine liver or in almond emulsin (containing both p-glucosidase and P-galactosidase activities). 4 . Activity of Larger Peptides Produced b y Proteolysis

A single attempt to estimate the minimum size of the polypeptide chain needed for antifreeze activity indicated that chains shorter than

ANTIFREEZE PROTEINS FROM FISH BLOODS

0

1 2 3 4 5 AMINO GROUPW10,WOg AFGP

237

6

AMINO GROUPS/10,000 g AFGP

FIG. 24. Loss of antifreeze activity as a function of the appearance of free amino groups when a mixture of antifreeze glycoproteins (AFGP) 3 and 4 was hydrolyzed with subtilisin (A) or Pronase (B) at 37°C. From Komatsu et al. (1970a), reproduced with permission. Antilreeze Glycoproleins:

-1 I

I

I

origin

I

I

I

+

FIG.25. Polyacrylamide gel electrophoresis of a 1-hour elastase digest of antifreeze glycoproteins 1-5. The gel was stained for carbohydrate, using a-naphthol and concentrated sulfuric acid. Two standard antifreeze glycoproteins 1-8 were included for localization of component 5. From Vandenheede (1972).

238

ROBERT E. FEENEY AND YIN YEH

two-thirds of the smaller active AFGP component 5 were inactive (Vandenheede, 1972). Thus, a minimum size with approximately 10-1 1glycotripeptides or approximately 30-33 amino acids appeared necessary. This estimation was obtained by determining the activities of peptides formed on short-time proteolysis with elastase (Fig. 25). Mixtures of components with apparent molecular weights less than approximately 7000 were inactive. B . Effects of Covalent Modifications of the Carbohydrate Side Chains

1 . Oxidative Scission Oxidation of AFGP components with 0.01 M periodate at 4°C for 5 hours resulted in complete loss of antifreeze activity (Komatsu et al., 1970a). Upon reduction of the oxidized products with sodium borohydride and cleavage of the acetal bonds with 0.05 M HzS04 at 80°C for 1 hour, only 18%(3residues) of the galactose remained intact. (A higher value of 40% for galactose before cleavage of the oxidized products was considered to have been due to a nonspecific reaction of these products with phenol-sulhric acid, the reagent used in these experiments for determinations of the galactose.) No loss of galactosamine, threonine, or alanine was detected. Most (84%) of the antifreeze activity and 89% of the antilectin activi-

’

10

20

30

ID

50

60

70

M

98

0

I6-ElIMlNAllON

FIG.26. Inactivation of antifreeze and antilectin activities of antifreeze glycoprotein by 8-elimination of carbohydrate. Antilectin activity was determined by inhibition of Osage-orange lectin. Antifreeze activities were determined by measurements of freezing temperatures. 8-Elimination was done with glycoprotein concentrations of 0.1 mg/ml in 0.1 N NaOH at 20°C. Extent of 8-elimination was determined according to data of Table VIII. From Ahmed et 01. (1973), reproduced with permission.

ANTIFREEZE PROTEINS FROM FISH BLOODS

239

ties were destroyed when 90% of the galactose was destroyed by a similar periodate oxidation (Ahmed et al., 1973). 2. @-Elimination /3-Elimination (Figs. 9 and 26) of the disaccharide destroyed both the antifreeze and antilectin activities (Ahmed et al., 1973). When 80% of the carbohydrate was @-eliminated,approximately 10 and 20% of the antifreeze and antilectin activities, respectively, remained.

3. Acetylation All the antifreeze activity was lost when approximately 35% of the hydroxyl groups (and the one NH2-terminal group) were acetylated (Komatsu et al., 1970a). Recovery of >95% of the activity was obtained on O-deacetylation with hydroxylamine. When 32% of the hydroxyl groups were acetylated, 18% of the antifreeze activity and 40% of the antilectin activity were retained (Ahmed et al., 1973). 4 . Enzymic Oxidation of the C-6 Carbinols with Galactose Oxidase Oxidation of the C-6 hydroxyls of the galactoses with galactose oxidase does not destroy activity (Shier et al., 1972; Vandenheede et al., 1972). When the C-6 hydroxyls were oxidized to aldehydes with galactose oxidase in a preparative experiment, 79% of the terminal Dgalactose and 19% of the D-N-acetylgalactosamine were oxidized with 90% of the antifreeze activity still remaining (Vandenheede et al., 1972) (Fig. 27). However, when this active product was further modified so as to place a negatively charged group at these C-6 positions, the activity was lost. Only approximately 20% of the activity was found after further oxidation of the C-6 aldehydes to the carboxylic acids with iodine. Examinations of the oxidized preparations by gel electrophoresis did not indicate any fragmentation of the products. Both the enzyme-oxidized preparations and an iodine-treated control had patterns indistinguishable from those of the original glycoprotein. In the gel electrophoretic pattern of the two-stage oxidized glycoproteins (i.e., the carboxylic derivative), there was a shift of the original bands toward the anode. This indicated that a higher negative charge was developed on the molecules in each band due to the formation of a carboxyl group on the C-6 positions. A possible explanation for these losses of activity could be that the negative charges cause undesirable ionic effects, possibly repulsions. This hypothesis appears to be at least partially substantiated by measuring the antifreeze activities under conditions where the negative charges would be decreased. When the antifreeze activities were

240

ROBERT E. FEENEY AND YIN YEH

79 ’A

I

c-0.

A

BlSULFlTE 19%

nc-on

79 96

.

I

C--0

IODINE 19%

7

L O-

I

79 96

I

NH

NH

I

I

G=O I

I

I

CH3

FIG. 27. Reaction schemes for oxidizing and formation of adduct products of antifreeze glycoproteins. From Vandenheede et al. (1972),reproduced with permission.

measured at neutrality and pH 3, the activities of the oxidized samples were 20 and 44%,respectively, of the original glycoprotein, which is fully active between pH 3 and 11. Since the carboxyl groups of the oxidized sample might be largely (- 80%)protonated at pH 3, a much higher activity might have been expected at pH 3. In addition to possible unrecognized side chemical reactions, the low recovery may possibly be explained on the basis of the structural characteristics of the nonionized carboxyl group (i.e., an intrinsically lower activity) or

ANTIFREEZE PROTEINS FROM FISH BLOODS

24 1

of residual unfavorable (nonlinear) ionic effects. No attempts were made to examine the activities at a lower pH because it was found that the native glycoproteins lost activity when kept below pH 2. Direct oxidation with bromine water, a stronger oxidizing agent, resulted in the loss of all the activity. The addition of bisulfite to the galactose oxidase-oxidized preparation caused extensive decreases in the activity (Fig. 27, Table XVIII). Lowering the pH of the bisulfite-adduct complex to obtain partial protonation was not possible as this would have required a lower pH, which would have resulted in destruction of the complex. These effects of oxidation and addition of sulfite were confirmed (Ahmed et al., 1975) and found to be very similar for the antilectin activities. Upon oxidation with &galactose oxidase, over 85% of both activities were still present. The extent of oxidation was 15 and 70% for Nacetylgalactosamine and galactose, respectively. However, when the aldehyde groups of this active product were either oxidized to the carboxyl groups or complexed with bisulfite, both the a.ntilectin and antifreeze activities were lost. Only approximately 15%of both activities remained after oxidation to carboxyl groups or on the addition of bisulfite to the aldehyde derivative.

C . Znactivations by Formation of Complex with Borate The addition of borate (Vandenheede et al., 1972; Ahmed et al., 1975) to solutions of AFGP causes reversible losses of antifreeze activity, TABLEXVIII Effect of Bisuljite on Actiuity of Galactose Oridase-Treated Antifreeze Glycoprotein' Activity of antifreeze glycoprotein Sodium sulfite added

(mM)

Native (96)

0 2.5 5.0 10.0 15.0

100 86 79 75 73

Galactose oxidase-treated (%)

lo@ 74 53 29 28

From Vandenheede et al. (1972), reproduced with permission.

* The Dgalactose-oxidized treated preparation had 91% of the activity of the native glycoprotein.

242

ROBERT E. FEENEY AND YIN YEH

1 . Effect of p H on Inhibition by Borate Both antifreeze and antilectin activities are lost when 2 mol of borate are bound per disaccharide side chain, and these losses and bindings are pH dependent (Ahmed et al., 1976). When borate was added to AFGP components 1-4 there was a gradual decrease in antifreeze activity as the pH was raised from 7.0 to approximately 8.4, at which point about one-half of the activity had been lost. This was followed by a sharp decrease from pH 8.4 to pH 8.8 (Fig. 28). Similar effects were obtained when AFGP polyaldehyde (i.e., the product from oxidation by galactose oxidase) was used. Although borate was also found to inhibit the antilectin activity of AFGP, the results were more difficult to interpret because borate inhibited the activity of the lectin itself in the hemagglutination assay.

7.0

8.0

PH

FIG.28. Effect of pH on antifreeze activity of antifreeze glycoprotein in the presence of borate. Antifreeze glycoproteins 1-4 (5mg/ml) were measured in 0.1 M phosphate and 0.1 M borate and adjusted to the pH values indicated. The freezing temperatures of the solutions were measured by the sensing of the heat of fusion. The values for the freezing temperatures of control solutions were subtracted from the values for samples containing the antifreeze glycoprotein. From Ahmed et al. (1976), reproduced with permission.

ANTIFREEZE PROTEINS FROM FISH BLOODS

243

There are many sites of interaction between carbohydrate and borate in the hemagglutination test and many more in the inhibition of hemagglutination. Antilectin activity of AFGP decreased as the pH of the borate buffer increased, but since there was inhibition by both the borate and the AFGP, only an approximate value could be estimated for the effect of borate on the AFGP. Borate appeared to decrease the inhibition caused by the AFGP to approximately one-sixth of the decrease in the absence of borate. The effect of pH and borate on the antilectin activity of the glycoprotein polyaldehyde was approximately the same as on native glycoprotein, similar to the results when tested for antifreeze activity. 2. Amount of Borate Bound The amount of borate bound to the antifreeze glycoprotein was strongly influenced b y the pH of the solution between pH 7.0 and 9.0. The number of moles of borate bound per disaccharide unit was only about 0.5 at pH 8.0 but approached 2 at pH 9.0 (Table XIX). Comparison of these data with those of Fig. 28 shows that approximately 20% of the antifreeze activity would be lost when 0.5 mol of borate were bound per mole of disaccharide, and almost 100% of the activity would be lost when 2.0 mol of borate were bound.

3. Effect of Borate Binding on Conformation Since both the antifreeze activity in the presence of borate and the amount of borate bound to the glycoprotein were shown to be funcTABLEXIX Effect of pH on Binding of Borate to Antifreeze Glycoprotein"

Moles of free borate ( x 105)

Moles of bound borate ( x 105)

Moles of borate bound per mole of disaccharide in glycoprotein

PH

A*

B C

A

B

A

B

8.0 9.0

14.03 10.96

13.91 10.66

0.97 4.04

1.09 4.34

0.42 1.75

0.47 1.88

From Ahmed et al. (1976), reproduced with permission. mol of borate and 2.31 x mol of antiA: experiment started with 15.0 x freeze glycoproteins 1 - 4 (calculated as a trimer of repeating glycopeptide) in 1 ml of solution inside the dialysis bag, which was dialyzed against 2 ml of water. B: experiment started with 2.31 x mol of antifreeze glycoproteins 1 - 4 in 1 ml of water inside the dialysis bag which was dialyzed against 15.0 x mol of borate in 2 ml of water. a

244

ROBERT E. FEENEY AND YIN YEH

tions of the pH, the effects of pH and borate on the conformation of the AFGP were studied in an effort to see whether or not the inactivation was a result of a conformational change on the binding of borate (Ahmed et al., 1976). No significant changes were observed in the diffusion coefficients as determined by quasi-elastic light scattering or equilibrium centrifugation or in the s2,, values as determined by sedimentation velocity ultracentrifugation (Table XX). When borate forms complexes with organic polyhydroxyl compounds having the required spatial relationships, the product in acidic solutions is described as a borate anion complexed with two hydroxyl groups (Fig. 29A), whereas in more basic solutions one borate can complex with four hydroxyl groups (Fig. 29B) and thus form crosslinkages (Acree, 1973; Aronoff et al., 1975). Consequently, in addition to the possible isomers formed by complexing with different hydroxyl groups, several crosslinked structures according to the model of Fig. 29B were considered (Ahmed et al., 1976). These included: (a) cross-linkages between two sets of hydroxyl groups on different glycoprotein molecules, (b) cross-linkages between hydroxyl groups on different disaccharide side chains of the same molecules, or (c) cross-linkages between hydroxyl groups of the galactose and Nacetylgalactosamines of individual side chains (intradisaccharide). Any such crosslinked structures were judged improbable, however, because of the near identity of the diffusion coefficients and molecular weights of the glycoprotein and its borate complex (Table XX). Although it was not proved unequivocally which specific hydroxyl groups were involved in the formation of the diborate inactive complex, it was concluded that the 3,4 hydroxyls of the galactose residues and the 4,6 hydroxyls of the N'-acetylgalactosamine residues were the ones involved (Ahmed et al., 1976). Inspection of ball-and-stick A

r--

FIG.29. Possible ligands between borate and hydroxyl groups: (A) in acidic solutions; (B) in alkaline solutions. These are two possible alternative structures. The crosslinked structure (B) seems less likely with the antifreeze glycoprotein. From Ahmed et al. (1976), reproduced with permission.

TABLEXX Effect of Borate on Sedimentation and Diffusion Coefficients of Antifreeze G2ycoproteinoSb

U1tracentrifugation szo x 1013 Antifreeze gl ycoprotein

D~ x 107 Antifreeze glycoprotein

Quasi-elastic light scattering D~~ x 107 Antifreeze glycoprotein

Buffer

4 (sec)

7 (sec)

4 (em2 sec-')

7 (cm' sec-I)

4 (cm' sec-I)

7 (cmZsec-I)

H*O Borate, 0.1 M, pH 7.0 Borate, 0.1 M , pH 8.6

1.48 1.43 1.44

0.645 0.662

5.64 5.09 4.97

11.60 10.74

5.04 4.80 4.68

9.30 9.10

a

From Ahmed et al. (1976), reproduced with permission. The molecular weights are approximately 19,000 and 4400 for antifreeze glycoproteins 4 and 7, respectively.

246

ROBERT E. FEENEY AND YIN YEH

models (Dreiding) of the carbohydrate side chain proved that the borate could bind to the 3,4 or 4,6 hydroxyl groups of the terminal galactose residues and to the 4,6 hydroxyl groups of the N acetylgalactosamine residues. The C-6 hydroxyl groups of the galactose residues, however, were shown to be unessential for activity. This was shown by the retention of antifreeze activity by the polyaldehyde AFGP in which oxidation had occurred at approximately 80% of the C-6 hydroxyl groups of the galactose residues and only 20%of the C-6 hydroxyl groups of the N-acetylgalactosamine residues.

VII. OTHER PROTEINSREPORTEDTo HAVE ANTIFREEZE ACTIVITY The original observation that an antifreezelike substance existed in northern polar fish has been more recently confirmed by Scholander and Maggert (1971), Hargens (1972), and Raymond and co-workers (1973, who found it in the saffron cod (Eleginus gracilis), Raymond and co-workers (1975), who found it in the sculpin (Myoxocephalus verrucosus), and Ananthanarayanan and Hew (1977a),who reported it in the winter flounder (Pseudopleuronectes americanus), and Osuga and Feeney (1978), who found it in the polar cod (Boreogadus saida). Antifreeze proteins are thus not restricted to southern polar regions but are common in northern regions. Of great significance has been the finding of AFGP very similar to those from Antarctic species as well as antifreeze proteins with no carbohydrates but with nearly similar high contents of alanines (Table XXI). A . AFGP from Northern Cod Although the families of the Antarctic fish and the northern cod (Nototheniidae and Gadidae, respectively) are widely separated, their AFGP have been found to be very similar. 1 . Saffron Cod (Eleginus gracilis)

The AFGP from the saffron cod (from Alaskan waters) was found to contain alanine, threonine, galactosamine, galactose, and the acetyl group in nearly the same proportions as found in the antifreeze from the Antarctic fish, T. borchgreuinki (Raymond et al., 1975). The northern glycoprotein was also prepared by only a slight modification of the procedure used in the Antarctic glycoprotein. Although a 40% lower antifreeze activity and slightly different amino acid composition of the two indicated that they were not identical (Raymond et al., 1975), further studies have not been reported to settle the question of identity.

TABLEXXI Comparative Compositions of Different Antifreeze Proteins ~~

~

~

~

~~

Protein (residues/10,000 g)

Residue

Antifreeze glycoprotein 5 Trematornus borchgrevinki (Antarctica)”

Antifreeze glycoprotein 8, T. borchgrevinki (Antarctica)a

Antifreeze glycoprotein “5”, Boreogadus saida (Arctic)b

Antifreeze glycoprotein “8”, B . saida (Arctic)b

Antifreeze protein, Pseudopleuronectes americanusc

Alanine Threonine Proline Galactose Galactosamine Aspartic acid -asparagine Leu ci n e Serine Lysine Glutamic acid-glutamine Arginine

32 16 0 16 16 0 0 0 0 0 0

30 15 7.5 15 15 0 0 0 0 0 0

32 16 0 16 16 0 0 0 0 0 0

30 15 7 15 15 0 0 0 0 0 0

63 10.1 0 0 0 11.6 5.0 3.4 3.4 1.7 1.7

a

Data from DeVries et al. (1970). Data from Osuga and Feeney (1978). Data from Hew and Yip (1976). Calculations based on a total of 100 residues/10,000 g.

248

ROBERT E. FEENEY AND YIN YEH

2 . Polar Cod (Boreogadus saidu) The polar cod frequents the far north Atlantic Ocean, the Barents Sea, and the Arctic Ocean, and, as such, and as its name implies, it is truly a polar fish. It is not surprising then that, when four species of fish were caught in the Barents Sea with temperatures varying from - 1.2"Cto - 1.3"Cat 79"N and 10-40 miles south of the southern end of a loose ice pack (frequent, but separated, small icebergs), only the polar cod was found to contain an antifreeze protein (Osuga and Feeney, 1978). This area would represent the near northern latitudes where the other three fish would go at that time and ocean temperature, while the polar cod is also found in more northern waters. Initial studies on the polar cod blood serum have shown that its antifreeze protein is a glycoprotein with most properties similar to those from the Antarctic species T. borchgrevinki. Both active and inactive components were obtained. The active components had the same ratios of alanine to threonine (approximately 2:l) as found in the Antarctic glycoproteins and the same contents of galactosamine and galactose. No other amino acids were found. In contrast to the comparatively low antifreeze activity of the saffron cod reported by Raymond and co-workers, the AFGP from the polar cod had an antifreeze activity similar to that from the Antarctic species. Numerous other similarities exist between the active antifreeze from both fishes. All the threonines appear to b e attached to galactosamine with a p 1 + 3 linkage to the galactose moiety. The N- and C-terminal residues were alanine. Since elastase digestion of the glycoproteins caused a marked increase in the freezing temperature, size was reported to be an important factor. Antifreeze from both species caused the inhibition of hemagglutination reaction of Osage-orange lectin. In addition, the inhibitory activity was blocked by the presence of borate. The only notable distinguishing feature was the electrophoretic patterns of the active antifreeze. The polar cod active components were not present in the same distinctive multiple molecular forms of 1 to 5 as were present in the T. borchgrevinki, but they were distributed into fewer than 5 forms with a preponderance of forms similar in size to those numbered 2,3, and 4 in the T. borchgre-

vinki. The active and inactive components were also found to be in approximately the same relative amounts as the Antarctic ones. The smaller inactive component number 8 had the same relative amino acid contents, which included, of course, the presence of proline in the approximate ratio for a1anine:threonine:proline of 4:2:1. This similarity in composition explains the identical electrophoretic migra-

ANTIFREEZE PROTEINS FROM FISH BLOODS

249

1. b o r c h g r e v i n k i :

NH2-Ala-Ala-Thr-Ala-Ala-Thr- Pro 75) Ala-Thr- Pro(25 1

2

3

4

5

6

f!7-8

9

-B. -saida: NH2-Ala-Ala-Thr1

2

3

Pro -Ala-Thru

5

6

Ala

7

-Ala-Thr8

9

a

-

Ala-Thr1

1

ri Pro

1

-Ala-Thr11

12

Pro -Ala-COOH 2

1

14

Ala -Ala-COOH 13

14

FIG. 30. Differences in positions of prolines in small antifreeze glycoproteins (AFGPS) from Antarctic Trernutomus borchgrevinki and the northern cod (Boreogodus soidu). The prolines are enclosed in squares, and their positional heterogeneity in T. borchgreoinki is expressed numerically as approximate percentages. A small amount (less than 10%) of a still further positional heterogeneous material may exist in T . borchgreuinki.

tion of the inactive components. Circular dichroism spectra of the two were also indistinguishable (Osuga and Feeney, 1978; Raymond and DeVries, 1977). However, three important differences were found in the sequence positions of the prolines (Osuga and Feeney, 1978). Proline was absent from position 13 of the polar cod material and present in position 4, and there was no heterogeneity (Fig. 30).

B . Other Antifreeze Proteins (AFP)from Northern Fish

1 . The Sculpin (Myoxocephalus v e m c o s u s ) A northern species, tentatively identified as the sculpin ( M . verrucosus), &om off the coast of St. Lawrence Island at a water temperature of -1.7"C, was found to contain an antifreeze substance with amino acids other than alanine and threonine and with no carbohydrate (Raymond et al., 1975). But it was similar to the antifreeze glycoprotein in having alanine comprising approximately two-thirds of its total amino acids. In addition to alanine, appreciable amounts of aspartic acid or asparagine, glutamic acid or glutamine, leucine, lysine, threonine, as well as other amino acids were reported. The total amount of alanine was only slightly less than ten times the combined amount of threonine and serine, much more than the relative amounts in the antifreeze glycoproteins, 2. The Northern Flounder (Pseudopleuronectes americanus) Two different laboratories (Duman and DeVries, 1975; Ananthanarayanan and Hew, 1977a; C. L. Hew, personal communication) have reported another antifreeze protein from the northern flounder (P.

250

ROBERT E. FEENEY AND YIN YEH

americanus) which resides in the northern Atlantic coastal waters of America and Canada. Both confirm that it is not a glycoprotein and has a high alanine content approximately similar to the antifreeze protein from M . verrucosus. Three active components with molecular weights estimated at 6000, 8000, and 12,000 by sodium dodecyl sulfate (SDS) gel electrophoresis were reported by Duman and DeVries (1975). The antifreeze activity was apparently somewhat different from the activities of the AFGP, being slightly less active at lower concentrations and more active at higher (nonphysiological) concentrations. Duman and DeVries (1975) reported an alanine content of 64.6 residues from a total of 105.4 residues per 10,000 g, giving an alanine frequency of approximately 60% of the total residues. The amino acid composition in residues per 10,000 g was: Ala, 64.6; Arg, 2.0; Asp, 14.0; Glu, 1.7; Leu, 5.6; Lys, 2.9; Ser, 3.4; Thr, 11.2. These authors suggested that the aspartyl and glutamyl groups might substitute for the carbohydrate groups present in the AFGP. The laboratory of C. L. Hew has more recently described the composition and structure (C. L. Hew, personal communication), the physical properties (Ananthanarayanan and Hew, 1977a), and the biosynthesis (Hew and Yip, 1976) of the antifreeze protein from P . arnericanus. Molecular exclusion through Sephadex G-75 was used to identify the numbers and sizes of the antifreeze proteins in the

FRACTION NO.

FIG. 31. The identification of flounder antifreeze glycoproteins on Sephadex G-75 chromatography. Freshly prepared concentrated sera (2.5 ml) were applied on the column (1.5 x 86 cm), and 7 0 fractions (2.5 ml each) were collected. The antifreeze activities of each fraction were monitored with an advanced osmometer. From Hew and Yip (1976), reproduced with permission.

ANTIFREEZE PROTEINS FROM FISH BLOODS

25 1

flounder’s serum. In this, most of the serum proteins eluted in the void volume and had little antifreeze activity. Two distinct antifreeze activities were detected (Fig. 31). However, the slow-running peak corresponded to the inorganic salts present in the concentrated, nondialyzed serum. Only one major antifreeze protein was found with a molecular weight of approximately 10,000 as compared to the cytochrome c marker. When the protein was prepared from serum by precipitation of the other proteins in 10% CC1,COOH and by dialysis, the soluble and nondialyzable fractions chromatographed identically to that present in whole serum as in Fig. 31. The amino acid composition was approximately similar to that reported by Duman and DeVries (1975) with the possible exception of a slightly higher content of alanine. The protein contains only eight amino acids (C. L. Hew, personal communication). Alanine accounted for 60% of its total residues. The first N-terminal sequence of positions 1-28 was determined on a Beckman sequencer and confirmed by the isolation and analysis of overlapping thermolysin- and subtilisin-digested peptides. This sequence is: Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Leu Thr Ala AlaAsn Asx Lys Ala Ala Ala Glu Leu Thr Ala Asp Asn Lys

There are clusters of 6 alanine residues in positions 6-11. Preliminary experiments with carboxypeptidase A digestion indicate the presence also of a cluster of alanine at the C terminus. In contrast to the antifreeze glycoprotein, an internal repeating sequence was not found, except possibly for the sequence of Leu Thr Ala Ala Asn Asx Lys (positions 12-18) and Leu Thr Ala Asp Asn Lys (positions 23-28). The flounder protein has been shown from circular dichroism measurements to possess a large proportion (- 85%) of the a-helical conformation in aqueous solution (pH 8.0) at - 1°C (Ananthanarayanan and Hew, 1977a). The helical content decreases as the temperature is raised (Fig. 32). Viscosity data at - 1°C indicate an asymmetric shape for the protein molecule compatible with its high helical content. Thus, the secondary and tertiary structure of this protein, as well as its primary structure, are found to be different from its counterpart glycoproteins isolated from other fish. No data are available concerning the assumption that this highly helical structure at - 1°C is related to its function. The levels of the antifreeze protein in the serum follow seasonal variations. The highest concentration is in January and February, when the water is the coldest, and the protein is absent from the serum in the summer, when the water temperature may exceed

252

ROBERT E. FEENEY AND YIN YEH

WAVELENGTH, nm

FIG.32. Circular dichroism spectra of flounder antifreeze proteins in 0.05 M NH4HC03 (pH 8.0) at the indicated temperatures ("C). From Ananthanarayanan and Hew (1977a), reproduced with permission.

+7"C (C. L. Hew, personal communication). DeVries (1974) reported the disappearance of the proteins when the fish are kept warm for a few weeks. Chuba and co-workers (1973) did not find any such change in the AFGP when T . borchgrevinki were kept as warm as considered feasible to maintain life at 3"C, for a month. TABLEXXII Incorporation of [3H]AZanineinto 10% CCl&OOH-Soluble, Nondialyzable Proteins from RNA-Znjected and Control OocyteP Experiment No. 1

2

Oocytes

Cpm/2O M I

Control RNA-injected Control RNA-injected

338 14 14 379 1909

From Hew and Yip (1976), reproduced with permission.

ANTIFREEZE PROTEINS FROM FISH BLOODS

-0.4

-0.3

-

253

FLOUNDER'S PROTEIN-

-

0

5 W

a SYNTHETIC POLYPEPTIDE

U 0

5

W

0

a ALBUMIN

10

20

30

40

50

CONCENTRATION (mglml)

FIG. 33. Lowering of the freezing temperature by the antifreeze protein of the flounder and a synthetic polypeptide composed of alanine and aspartic acid. The synthetic polypeptide is a random polymer of alanine and threonine in the molar ratio of 2:l. From Ananthanarayanan and Hew (1977b), reproduced with permission.

Hew and Yip (1976) have shown that a 6-10 S, poly(A)-rich RNA from the flounder contained mRNA for the synthesis of the antifreeze protein. When injected into Xenopus oocyte, the RNA preparation from the fish liver polysomes stimulated a 4-fold incorporation of rH]alanine into the antifreeze protein fraction (Table XXII). Ananthanarayanan and Hew (197713) have recently described the synthesis of a polypeptide with structural similarities to the antifreeze protein from the flounder. The product was a random polymer of TABLEXXIII Properties of the Synthetic Polypeptidea

Composition (mol Sample No. SAF-Ib SAF-11' ~~

Alanine

Aspartic acid

Molecular weighte

a-Helical conten6

65.5

34.5 35.2

45,000-70,000 50,000-70,000

15 20

64.8 ~

~

%)d

~

~~

~

~~

~~

~

From Ananthanarayanan and Hew (1977b). * Synthesized using an HF-pyridine debenzylation procedure. Synthesized using an HBr debenzylation method. Determined on a Beckman Model 121M amino acid analyzer after 24-48 hours of hydrolysis in 12 N HCI at 110°C. The compositions of the polypeptides before deblocking the side-chain benzyl group were almost identical to those ofthe final products. Estimated using a Sephadex G-75 column calibrated with protein markers. Estimated on the basis of circular dichroism data. a

'

254

ROBERT E. FEENEY AND YIN YEH

alanine and aspartic acid in a molar ratio of 2 :1 (Table XXIII) and had weak but definite antifreeze activity (Fig. 33). As in the study of the structure -function relationships of other proteins, the syntheses of homologous structures of the antifreeze proteins should provide excellent tools for understanding the mechanisms of action.

VIII. THEORETICAL ASPECTS OF ACTIONS OF SUBSTANCES TEMPERATURES LOWERING FREEZING

A . Theory of Antifreezing Mechanisms An explanation of antifreezing mechanisms can first be done invoking only generally accepted principles of solvent-solute interaction. Our discussion initiates with a review of freezing temperature depression in ideal solutions. Here, the basic assumptions are as follows: (1)When the two types of molecules are in solution, they are of sufficiently similar geometric shape and size that they essentially occupy the same volume of space. (2) The energy of interaction between the solvent-solvent pair, the solute -solute pair and the solvent-solute pair are similar in strength and are strictly painvise additive. We can then consider the potential of interaction to be a constant for any pair. Thus the partition function, Z, may be written as:

where there are N A solvent molecules and NB solute molecules. The total number N, is the sum of N A and NB;AA and AB are the thermal de Broglie wavelengths associated with the respective species. Note here that the configurational integral becomes that of an ideal gas system. The entropy of mixing, ASmix,may be evaluated by calculating the difference of the entropies of the mixture and those of the pure solvent and solute. A s m i x = Smixture - ( S A

+S J

(2)

In this case, the contribution of ASmixcomes only from the placement of the two different species in the lattice. Here, Asmix =

- N k [ x ~ l m+~XBlUB]

(31

where X A = NA/(NA + NB) and X B = NB/(NA + N B ) . Since the energy of mixing is a constant, as per our definition of the ideal system, it can be arbitrarily set to zero. Accordingly, AG,,,ix = -TASmix = NJCT[XAIUA+ X B I ~ X B ]

(4)

ANTIFREEZE PROTEINS FROM FISH BLOODS

255

In order to evaluate the chemical potential of the solvent, one calculates / - h ~- /-hi = (aGmjx/d2v~)= k T h A

(5) Here p i is the molar free energy of the species A in its pure state. Utilizing the thermodynamic relationship (Eggers et al., 1964) that

where ship

AH is the specific enthalpy of the solvent system, the relation(7)

In X A = -(AH/kT2)AT

where AT = To - T is the freezing point lowering is obtained, T o being the pure ice-melting temperature. As is typically the case, X A is a quantity near unity, and it is then possible to expand I w A about X A = 1.

Thus the leading term gives AT

(kT,2/h,)XB

(9)

where hl = AfT is the molar latent heat of fusion. We note here that freezing-point lowering for colligative systems as defined above has a linear concentration dependence on the solute, but no dependence on the structure of the solute. This appears to be well satisfied for a number of aqueous solutions (Table XXIV). TABLEXXIV Molar Concentration of Salts, Hydrocarbons, and S m a l l Sugars When Their Freezing Point-Depression Temperature is -0.8"C ~~

Molarity Species

(M)

Ethanol Ethylene glycol Glucose Glycerol Sucrose Methanol Antifreeze glycoprotein

0.23 0.23 0.43 0.43 0.40 0.42 0.39 0.43 1.0 x 10-3

NaCl KCI

256

ROBERT E. FEENEY AND YIN YEH

In order to improve upon the theory discussed above, one can first envision that the requirement of equal energy of placement or mixing be relaxed. In this case, we consider that there will be a difference between the placement of a solute molecule in a lattice of solvent, e.g., configuration energies for the nearest neighbors A-A, B-B, and A-B are now considered different (Henderson and Leonard, 1971). Econf = N A A E A A

+ NABCAB + NBBEBB

(10)

Here eij is the specific energy of a particular type of nearest neighbors. It is useful to define E =

EAB

-

+

(EAA

+ EBB)

(11)

as the difference between the unlike pair interaction energy and the like pair interaction energy. Using the above equation with expressions governing the number of nearest neighbors: N A

=

N B

= 2NBB

2NAA

+ +

NAB

(12b)

NAB

where z is the coordination number, we get B

Now the configuration integral can be reduced to a sum over all allowed N A B values:

where Eaa =- +ZN,E, and g(NA,NB,NAB) = N ! / ( N A W B ! ) . Since the sum over all sets of N A B pairs subjected to N = N A NB can be replaced by its maximum term, the entropy of mixing for this system is the same as that of the ideal mixture. Here, however, the free energy includes the new interaction energy term. In this random mixing approximation, N A B is related to xA and xB by (cf. Pathria, 1972)

+

The free energy of mixing is then given by

Correspondingly,

ANTIFREEZE PROTEINS FROM FISH BLOODS

257

Using this expression in Eq. (6),and again assuming that xA is nearly unity, we then write:

( A ~ / / C T $ ) A T= X B +(J

-

x)xB~

(18)

where x = z ~ / k T , . For solutes which statisfy this concept of regular solution, it is most generally true that x is a small quantity. Accordingly,

AT

=

(kT;/hi)

[XB

+ (*

-

X)XB'I

(19)

and this provides the first enthalpic correction to the expression for the colligative freezing temperature depression. Should the solute be a polymeric unit, the assumptions leading to the above expression become increasingly difficult to justify. For example, how can one justify the fact that a globular protein of molecular weight 50,000 would not occupy more space than a solvent molecule. Such disruptions in a solvent lattice obviously cause other entropies of arrangements to increase in addition to the greatly differing energy of placement. This problem, in general, is rather difficult to deal with in an analytical fashion. For a random-chain polymer molecule, however, the theories of Flory and Huggins provide us with an elegant means of examining freezing-temperature depression (Flory, 1970). Within this model the NB polymers each has m subunits. The assumption is that each of the subunits is still of the same size as the solvent molecule, and therefore volume change in mixing is still unaltered. Since the links between these subunits are assumed not to occupy any volume, one treats them as massless, volumeless, and interactionless links alone. Defining N = NA + mNB, X B = mNB/N, x A = NA/N, it was shown that there is indeed an additional entropic term (Tanford, 1961). However, this entropic term, due only to the arrangement or configurations in the polymer, is a feature of the pure polymer even in the absence of the solvents. Accordingly, it belongs only to SB,the total entropy of the polymeric solute. The resultant entropy of mixing, that term responsible for the freezing temperature depression, is not very much affected by the polymer configurational entropy. A s m i x polymer =

- N ~ [ x A ~ ~x A B1mB1

(20)

The free-energy and chemical potentials of the solvent is slightly altered from the case of small solute molecules (Flory, 1970): /.&A

= kT[lmA

-

+ (1 - l/m)XB + XxB2]

(21)

It then follows that

AT

=

(kT;/h,) [ ( x B / ~ )- (X

-

*bB21

(22)

258

ROBERT E. FEENEY AND YIN YEH

FIG.34. Freezing point lowering AT (or raising, - A T ) as a function of polymer concentration according to Eq. (26)and Eq. (27). (a) Normal polymer with no surface interaction contribution. KILT 5 l/m. Note “saturation” possible for x > $. (b) Polymer with strong surface interaction contribution. K / k T >> l / m . Note large initial depression slope and “saturation” possibility for x > 1. (c) ( K / k T + l/m) +.

W e note that the linear term is now proportional to the total number of polymer solute molecules, xB/m = N , / N , which for polymers is typically very low. Thus, the colligative effect from polymeric solutions is typically very small. Furthermore, if x < f, then Eq. (22) and Eq. (19) are completely similar. However, if x > 3, the AT vs xB plot would exhibit a downward curvature (Fig. 34). This could lead to a decrease of freezing point-lowering effect. It is well known (Tanford, 1961) that the interpretation that x is related to the energy of polymer placement in a polymer solution is incomplete. This is because the assumption of segments of a polymer having the same volume as that of the solvent is very much in error. Consequently, x has become an empirical factor to be experimentally determined. Flory has shown that, for a number of systems, the contribution of x is 2-fold, enthalpic and entropic.

Whereas xE is the energy or enthalpy of placement of nearest neighbors and is typically small, xs, the entropic term, can be very large. In fact x 2 f is commonly encountered in polymers if the entropy of mixing is significantly different from that of the assumptions for a regular solution. In extending the theory of Flory-Huggins to the situations encountered in the antifreezing systems of interest here, we suggest that a plausible mechanism must involve surface disruption in some general way. Accordingly, we shall introduce an additional term to the vol-

ANTIFREEZE PROTEINS FROM FISH BLOODS

259

ume chemical potential of the solvent, h. At the phase equilibrium of the solid-liquid interface, the chemical potential for volume must now be decreased by a surface interaction term (Solms and Rijke, 1971). PT

= ~v

ui (aAi/aNd

i

(a)

where pT is the total chemical potential,Ai is the ithfacet of the surface area, and ui is the interaction energy between that facet and the solute. If we assume that each facet of the nucleated embryo has the same ui = 8 and the total area of facets interacting with the solute is AxB, A being the area of an interacting facet, then

where p is a geometric factor, MA and pAare the molecular weight and density of the solvent, respectively, and Z is the characteristic length of the embryo. Defining K = %(MAP/pAZ)e, and using Eq. (21) for pv,we find that

For K/kT >> l / m , i.e., where the contribution of the surface term is large, we see that the freezing temperature lowering may be given by

A comparison of freezing temperature lowering via the mechanisms for the disruption of surface and for the Flory-Huggins model is shown in Fig. 34. Our result, Eq. (27), is similar to that obtained by Solms and Rijke (1971). In their case a polymer system where x < 3 is used to test the results of the theory. Raymond and DeVries (1977) have recently postulated that the functional saturation can be realized even with the simpler theory previously developed by Kuhn (1956). Reasoning that Z-’m(aA/dV), the differential surface-to-volume ratio, an irregularly shaped surfacewill lead to larger surface area for the same spherical volume, these investigators showed that I = [2p(r~]-”~, where p is related to polymer size, (Y is an entrapment coefficient of the polymer in ice, and c is the concentration of polymers in solution. If this value of 2 is used for the evaluation of K in Eq. (27),then even when the Flory-Huggins factor is not incorporated (x = +), ATLoweringw1’2 can be seen. Qualitative

260

ROBERT E. FEENEY AND YIN YEH

agreement with experimental results was demonstrated by these investigators. The present theory brings into focus the additional interactive parameter, namely x. As seen in Fig. 34,it is possible to achieve the opposite effect, heterogeneous nucleation, by simply allowing the quadratic term with x > 4 to be strongly dominant over that of any linear terms. It is further evident that should a,the average surface energy, be negative, AT will also be negative, leading to heterogeneous nucleation. This development thus suggests that, in a general sense, processes for nucleation and antifreezing are in fact just two different manifestations of solvent-solute interaction. We next want to examine how nucleation initiates. In particular, how does heterogeneous nucleation involve these same interactive parameters?

B . Nucleation and Propagation 1 . Homogeneous Nucleation There are two approaches in developing the conditions that govern favorable nucleation of a crystal embryo out of the liquid phase of a fluid without dissolved substances. From a structural point of view, nucleation implies that under certain favorable conditions the bond formation rendering a crystalline lattice may be competitive with the thermal motion trying to randomize and destroy bonds. A detailed molecular theory of freezing is necessarily a many-bodied problem. There exists, however, a classic thermodynamic theory of freezing that provides much insight into the process of ice nucleation (Hobbs, 1974). It is first necessary to establish the criteria for nucleation in the absence of any nucleation centers except for the molecules to be frozen. We may simplify this problem by considering an isotropic system. That is, let each direction of growth be equally favorable. Furthermore, we deal with a scaler surface tension (idealized, but simpler to consider) at the liquid-solid boundary. Here the surface interaction energy is denoted by mLs. Let us first ask the question how can a species exist in clusters of molecules at equilibrium with its own monomers? We necessarily must consider the fluctuation of any system about its equilibrium. Under normally liquid conditions, the density fluctuations are small in extent (localized) and persist for short times. A microscopic description of the phenomenon may be that of temporary formation of weak bonds, which are then quickly dissipated. If, however, these fluctuations lead to clusters that have a nontransient existence, then

ANTIFREEZE PROTEINS FROM FISH BLOODS

26 1

such a cluster might be considered to behave as a unit in thermodynamic equilibrium with its surroundings. In the classical theory no attempt is made to explain the actual formation of the cluster. Instead, we search for conditions that will lead to cluster stabilization. The thermodynamic theory of homogeneous nucleation is based on four basic assumptions. First of all, it is assumed that the embryo cluster is large enough so that thermodynamic parameters can be defined. Second, cluster density, ps, is uniform, as is the liquid density, pL. Third, the interface is sharp; thus, a surface tension between the phases is definable, and, correspondingly, a surface free energy uLs exists. If a further assumption is made that the volume change in the liquid is negligible as the cluster forms, then the equivalence of Helmholtz and Gibbs free energy can be made (Walton, 1969). Under these conditions, let us assume a cluster of N, molecules existing in liquid ofNLmolecules. Ifp, and pLare the respective chemical potentials for the two phases, then G for the system can be written as

G

= NSps

+ NLpL + 4daLS

(28)

where r is the radius of the spherical embryo cluster. The expression can be put in the form: G

=

NPL

+N

s

-~ YL)

+4 ~ ~ ~ s f l

(29)

where N = N L + N, is the total number of molecules of the system. At equilibrium, the change in free energy with respect to a change in the number of particles in the embryo is zero and is given by dG/dNs = 0

= (ps- p L )

+ ~ T U , ,(dfl/dN,)

(30)

Since uniform density, ps, is assumed

N , = (477T3/3) Ps then 8 N s

=

4?rpSr28r,and Ps -

P L

+ (2ULslPsr) = 0

(32)

Note that if r + 30, then the macroscopic equilibrium condition of p L = ps is obtained. In the present situation, the surface interaction energy, mh, helps to define a parameter r*, the critical radius,

(33) r* = 2CLs/[PS(PL - PJl Since the total change in Gibbs free energy in this process of embryo creation is AG

=G -

N/..LL= (/L, - pL)NS+ 4~1-20-b

(34)

262

ROBERT E. FEENEY AND YIN YEH

FIG.35. Free energy barrier for homogeneous nucleation process. For sizes of nuclei greater than r*, spontaneous growth will be favorable.

we can substitute Eq. (33) into Eq. (34) and obtain

AG

=

47raL,2[1- (2r/3r*)1

(35)

It is interesting to note that AG (Fig. 35) will be positive for small values of r, reaches a maximum when r = r*, and becomes negative for r > 6 r*. The AG at critical radius is denoted by

Thus far, the nucleation equations have been derived with the assumption that the homogeneous interfacial energy, uIs, is independent of the critical cluster size. There is obviously some relationship between surface tension and droplet size, but thus far the theoretical predictions of this relationship and the experimental nucleation data do not seem to be in accord (Walton, 1969). One classical way to examine the interfacial problem is to treat this interfacial energetics in terms of bond breaking. In such a picture, the incorporation of a surface first causes certain liquid phase bonds to be broken. The atoms then readjust from their initial positions to new equilibrium positions via relaxation mechanisms. For a general interface, the interfacial energies may be given by: ah =

(+L

+ us- (U,/2A)

(37)

where aLand a,are specific liquid and solid surface energies. ULs is the total interaction between solid and liquid, and A is the interfacial area. UL, may result from a composite of ionic, polar, dispersive, or hydrogen-bonding forces. It can be seen that the larger UIs is, the lower is the interfacial energy, and correspondingly AG is lowered. We note here that the relaxation term, which leads to dynamic changes of the structure in the interfacial region, has not been ac-

ANTIFREEZE PROTEINS FROM FISH BLOODS

263

counted for in this expression, Eq. (37), even though its importance is evident. The growth of an embryo may be considered as an equilibrium reaction between a metastable cluster of size x and the monomers (x = 1). The growth of a cluster by such a unit addition may be written

A1 + A, =A,+, (38) The number of clusters per unit volume of size x is then given by n,, where

n, = nexp[ - (AG,/kT)I

(39) and n is the density of monomers, or the liquid phase. We note from Eq. (37)that as long as r is less than r*, AG, is positive and increasing so that the probability of n, formation is low. For r > r*, the critical size has been reached, and it becomes increasingly more favorable to find these x-sized clusters. The rate of nucleation of particles or clusters of size x can be written as the product of the number of clusters of size x and the probability that another molecule gets to the interface by overcoming kinetic barriers which provide an activation energy barrier, A g . This latter t e r n includes viscosity and diffusion effects of the bulk liquid medium as well as solvent association reactions that deplete monomers. If x is the critical size then the nucleation rate, J x is Jx = J ( r * )=

( k T / h )exp [ - (AG/kT)ln(r*)

(40)

Using Eqs. (36) and (39),J(r*) may be written

A most interesting feature of this equation is its dependence on ub. Owing to the cubic dependence on uLswithin the exponential, variation of the nucleation rate can be extremely rapid. Mason (1957) has shown that, should a 5% change occur in uu,there will be a 5-fold change inJ(r*) at T = - 40°C, a 250-fold change inJ(r*) at T = - 30"C, and a l@-fold change in J ( r * )at - 20°C. Thus, small surface-energy variation does indeed lead to large changes in the nucleation rate, especially at higher supercooling temperatures. In summary then, the rate of homogeneous nucleation can be regulated via two different mechanisms: (1) kinetic barrier, A g ; this includes the dynamic slowing of molecules in their movement toward the interface and chemical association as a depletion mechanism of monomers; (2) structural barrier, AG*. These combine to provide the

264

ROBERT E. FEENEY AND YIN YEH

enthalpic and entropic effects that affect this free energy of cluster formation. 2. Heterogeneous Nucleation Currently there is no one definitive theory that deals with nucleation processes in the presence of foreign substrate. The classical approach, as formulated by Fletcher (1960), is basically similar to the case of homogeneous nucleation, except for the introduction of a new parameter, y, which is a measure of the disparity between the solid embryo and the foreign nucleating substance. However, Zettlemoyer (1968) has more recently shown that such a macroscopic treatment is inadequate to explain the nucleation of ice by well known nucleators, such as AgI. Conceptually, Zettlemoyer’s results show that hydrophilic sites invariably present on hydrophobic surfaces are necessary for initiation of the crystalline phase. In the present review, we first discuss the classical treatment as more recently developed by Hobbs (1974). Here y provides a quantitative measure of the differences in the surface interaction energies between the ice embryo-foreign substrate, uB,ice embryo-liquid, ~ 1 2 and , liquid-foreign substrate, uI3,in the following form: Y

=

[a12

-

b13

+ (+B)l/~lZ

(42)

In the subsequent derivation of the critical free energy for nucleation of this type, y enters as a significant parameter for the critical free energy change: AG*

=

(4mblZ3y)/(~L -

(43)

where 5, a geometric factor, replaces the density, ps, of the homogeneous solid phase. Correspondingly, the rate of nucleation for a system of n, liquid particles in contact with a spherical foreign substrate of radius R , is given by JF

=

(n,kT/h) 4mRn2e x p [ - ( A g / k T ) ] exp [ - ( A G * / k T ) ]

(44)

Thus we show here that should ~ 2 and/or 3 ( ~ 1 3be large, y is decreased and nucleation is favored. It must be emphasized, however, that mi of Eq. (24) must be compared with y here. Thus, an increase of y or ui leads to unfavorable nucleation or antifreezing action, whereas a decrease in y or uileads to a favorable nucleation condition. Since the kinetic parameter is unaltered from that of the homogeneous nucleation, the difference betweenJF andJ of the homogeneous nucleation is governed by the structural condition, which in turn is related to the lattice misfit factor, y .

ANTIFREEZE PROTEINS FROM FISH BLOODS

265

On the microscopic scale, the action of a foreign substrate in promoting nucleation is primarily through ordering brought about by interactions across the interface. For example, substrates exhibiting strong interaction lead to high density of solvent in its neighborhood, but can lead to solidification only if the molecular configuration of the liquid phase is appropriate for nucleation growth. On the other hand, weakly interacting substances will cause low interfacial liquid density and hinder nucleation. There has been only one major attempt to examine this problem in some detail. Turnbull and Vonnegut (1952) proposed a model whereby there are certain substrate sites that are capable of local ordering. These regions are treated using the classical nucleatiqn concepts. The degree of ordering is imposed by the relationship between the lattice arrangement of the substrate and the nucleated solid phase. On a thermodynamic scale, this relationship produces a modification in the free energy of the nucleating species. Here, if ~ 2 is3 the interfacial free energy between the foreign substrate and the embryo phase, one has an expression: U B = ~2

+

~3

-

Xu23

(45)

uZ3being the interaction energy of the atoms on one side of the solid-solid interface with those on the other side. When the two lattices do not match, the lattice distortion will tend to decrease the surface energy, leading to a less efficient nucleating agent. Furthermore, the mismatch between the two phases also results in a build-up of strain energy, E 0~ 6', where 6 = (a3 - a2)/a2is the lattice mismatch factor between the substrate lattice (a3)and the nucleating embryo lattice (a2). In the treatment of Turnbull and Vonnegut, this strain energy decreases the volume free energy of a cluster and therefore further increases the energy barrier to nucleation. Another related effect is the entropic contribution to the interfacial tension. Consider the specific case of a water molecule in ice: Since this is a highly polar molecule, the binding to a nucleating surface is basically electrostatic. This favors ionic material for the nucleating site. Assume that an ionic material does serve as the nucleating agent; the OH dipoles will interact strongly with the electric field from such a nucleating substrate. It is well known that energy of the interface is minimized if all the dipoles of water molecules orient parallel to the electric E-field direction. However, such ordering of the dipoles may produce a lower entropy, and therefore higher free energy, in the bulk of the ice well away from the interface. From analysis of the relationship between interfacial energy and bulk entropy, Fletcher (1959) has concluded that any substance that orients the ice

266

ROBERT E. FEENEY AND YIN YEH

dipoles at its surface parallel to one another will be a poor nucleating agent. In the case of hexagonal materials that have lattice dimensions close to those of ice, this theory predicts that the basal planes are poor ice-nucleating surfaces and that the activity should be confined to the prism faces. Large steps on basal planes will expose prism faces, and for this reason alone they might provide good nucleating sites. The idea of hydrophobic surfaces playing a major role in heterogeneous nucleation has been advanced by Zettlemoyer (1968). Basically, Zettlemoyer finds that even the best ice nucleants, such as AgI, are very much hydrophobic, and consequently a really pure solid surface would not be expected to adsorb water. He proposes that even though the ability to adsorb water onto speci$c hydrophilic sites is relatively weak and the adsorbed molecules are highly entropic, it is the hydrophobic regions that finally are responsible for driving the water molecules together toward the neighborhood of this hydrophilic site. Consequently, hydrophobic interaction is postulated to play a major role in nucleation. In the next two sections, we shall draw closer the relationship between water and biological macromolecules. In particular, the important roles that hydrogen bonding and hydrophobic interaction play in both ice formation and macromolecular conformations will be described.

3. The H20System The theories outlined above are general and pertain to any nucleation processes within a melt. The H 2 0 system is of particular interest to us for a number of reasons: (1)the hydrogen-bonding capabilities of tliese molecules, (2) the hydrophobic and hydrophilic interactions of biomaterials, and (3) the absolute requirements for water in living systems. Consequently, freezing of H 2 0 from the liquid phase is of special significance for biological systems. A unique feature of H 2 0 is the formation and sharing of hydrogen bonds with other molecules. Such bonds play a major role in determining the structure of both liquid and solid phases of H 2 0 . It is believed that for intermolecular spacings of less than 2 hi, the two water molecules exert strong repulsive forces on each other. As such, there exists a hard sphere radius of little interpenetration of the molecules. Usually, the repulsive part of Lennard-Jones 6-12 potential can be considered appropriate to describe these repulsive characteristics. At distances of separation greater than 5 A, dipole-dipole interaction plays a dominant role. This is reasonable, because each H 2 0 molecule has a large dipole moment, p = 1.84 D. Within the intermediate range, 2 A c R < 5 A, a particular sharing

ANTIFREEZE PROTEINS FROM FISH BLOODS

267

of the hydrogen atoms may take place. When HzO is in the ice Ih form along each of the 0-0lines, there is one H atom located 1 A from one of the 0 atoms and the other 1.76 A away. This phenomenon produces bonding energies intermediate between typical energies of nonpolar molecules and those of covalent or ionic bonds. Hydrogen bonding is also highly orientation-dependent. The strength of hydrogen bonds is estimated from the heats of sublimation as the energy necessary to take one HzO molecule from the solid to the vapor phase. This corresponds to the breaking of two hydrogen bonds, or approximately 6 kcal/mol. Owing to the formation of hydrogen bonds, the ice Ih crystal possesses configuration degeneracy which leads to the “residual entropy” of the ice. Pauling’s formulation is as follows (Ben-Na’im, 1974): Consider a perfect ice lattice, containing N H 2 0 molecules. There are N oxygens, 2N hydrogens, and 2N 0-0 axes. How many ways can we distribute these 2N hydrogen atoms in such a way so as to satisfy (1) the restrictions of tetrahedral bonding and (2) the nonsymmetric placement of the hydrogen atoms? Consider a particular oxygen and count all the possible arrangements of hydrogens in its immediate environment. There are altogether 16 such arrangements distributed in the following manner: (1) one arrangement in which all the hydrogens are close to the oxygen; (2) four arrangements in which three hydrogens are close and one is far; (3)six arrangements in which two hydrogens are close and two are far; (4) four arrangements in which one is close and three are far; (5) one arrangement in which all four hydrogens are far from the oxygen. In the Pauling approximate solution the 6 out of 16 acceptable arrangements about each oxygen provides a probability of 6/16 that a given oxygen has the correct arrangement around it. Furthermore, assuming that all the oxygens are independent, we get (6/16)N as the probability of finding simultaneously all of the oxygens in an arrangement consistent with the second condition. Hence the total number of accepted configurations is W

=

(6/16)”22N= (3/2yY

=

(1.5)”

(46)

This number has been used to estimate the residual entropy of ice, So

=

k In W

=

0.805 e.u.

(47)

which is in remarkable agreement with the corresponding experimental value of 0.82 e.u. Any model that is to be used to describe ice formation from the liquid phase must then accommodate this degree of disorder. In the early theories of liquid water, Pople (1951) suggested that

268

ROBERT E. FEENEY AND YIN YEH

only very few hydrogen bonds are broken upon the melting of ice. He proceeded to suggest that the liquid phase accommodates more disorder by having many distorted hydrogen bonds. On the other hand, advocates of the mixture model (Nemethy and Scheraga, 1962), extending the model initially suggested by Frank and Wen’s (1957) “flickering cluster” picture of water molecules, postulate that clusters arise from cooperative formation of hydrogen bonds. Owing to this cooperative nature, the probability of finding large clusters of hydrogen-bonded molecules is high. The molecular theory of structural change from low-temperature liquid water to that of ice Ih structure is yet incomplete. Eisenberg and Kauzmann (1969) suggested that the observed temperature dependence of the molar volume of water can be explained qualitatively based on two competing effects: (1) continuation of the strengthening of the four-coordination of molecules keeps the ice form in an open structure; (2) the expected decrease in all the anharmonic vibrations will lead to a decrease in the ice volume. Probably, it is the dominance of the first effect that keeps ice Ih in a low density state. In calculated intermolecular frequencies specifically carried out to point to the differences of cluster and liquid structural mismatch, Plummer (1973) showed that the free energy change for a 20-molecule clathrate is 57 kcal/mol, while the ice Ih of 20-molecule size has a 72 kcal/mol difference. The conclusion that this may be a reason for difficulties associated with Ih ice formation via homogeneous nucleation is worthy of further consideration. Biologically active materials, such as polypeptides and nucleic acids, are composed of many of these polar groups and many apolar residues. Not only are the conformations of the macromolecule influenced by the presence of water as the solvent, but the presence of these macromolecules is able to influence the solvents’ behaviors. Thus we might look at a system and decide if it could serve as an antifreeze or a good nucleating substrate by first obtaining an understanding of its solution conformations. 4 . The Hydrophobic Interaction Even though theories concerning liquid H 2 0 differ on the nature of hydrogen bonds, in general these bonds are known to exist. The presence of any other molecule in water must then lead to competition for hydrogen bonds with the solvent hydrogen bonds. Should the solute be an ionic species or a strongly polar species, solute competition for hydrogen bonding of water is favorable, and the ion or

ANTIFREEZE PROTEINS FROM FISH BLOODS

269

polar molecule is considered soluble. However, an apolar molecule has no potentially strong bonds to compete against the hydrogen bonds of water. As a result, these molecules do not dissolve in water readily. Some of the simplest substances of this type, called hydrophobic substances, include inert gases and hydrocarbons or other organic substances. Biological molecules typically have both hydrophilic groups and apolar groups, such as the aliphatic hydrocarbons. As such they are termed amphiphilic. One can see that when such a molecule is in water, the bonding groups will tend to associate with water while the apolar residues are forced away from water. Hydrophobic interaction is then the shielding of those specific apolar molecules due to their absence of interaction with water. Quite often a macromolecule in a helical conformation will “open up” in solution owing to the presence of water-polar hydrophilic interactions. On the other hand, proteins with extensive hydrophobic areas, e.g., lipoproteins, could become globular in water due to the need for the shielding of hydrophobic groups, thus exposing only polar groups. Therefore, solution conformation is affected by hydrophobic interaction. Conversely, the existence of hydrophilic bonds and the conformation that a macromolecule has taken in solution affects the configuration of the surrounding water molecules appreciably. The presence of water of hydration about ionic groups can disrupt the original icelike water lattice. Therefore, it is easy to see that when macromolecules are present in solution, a noncolligative mechanism exists proportional to the number of polar or ionic groups in solution, regardless of their location on the macromolecule, to affect the water structure SO as to either promote or disrupt the formation of ice. As such, the macromolecule could provide either effective heterogeneous nucleation sites or effective antifreezing sites. Furthermore, these molecules may contribute collectively or cooperatively in the promotion or disruption of the water structure. Since the nature of the cooperativeness is bond specific and molecule specific with respect to the macromolecules themselves, such a process constitutes another noncolligative phenomenon. An interesting example has been the work by Warner (1962) on possible relationships of carbohydrates and scyZZo-inositol with the water-liquid phase structure. It was observed that inositol can maintain good contact with the icelike layers above and below it. However, Warner suggested that the rigid covalently bonded inositol ring has a different temperature-lattice spacing relationship from that of the hydrogenbonded water system. One expects then that the fitting is better at certain temperatures than at other temperatures. This result can lead

270

ROBERT E. FEENEY AND YIN YEH

to the interesting idea that solubility of a substance in water is related to structural mismatch. Similarly, Duman and DeVries (1972) suggested that should the glycoproteins acquire a helical pattern in solution, the sugar residues might all align on one side of the helix at regular intervals. Such an effect could conceivably result in water-sugar OH binding and prevent ice Ih formation. In our search for antifreeze mechanisms, we must (1) be examining the competitive nature of bonds of molecules with water hydrogen bonds, and (2) be examining the appropriateness of the fitting of the ice structure to that of the molecules, considering both entropic and enthalpic effects. Ix. DISCUSSIONS O F MODELS FOR MECHANISMS OF ACTION OF ANTIFREEZE SYSTEMS In any discussions of postulated models, one must be able to utilize the basic concepts set forth in Section VIII, at least to make some qualitative assessment of the origin and magnitude of the enthalpic and entropic contributions. More specifically, x and C between the molecule in solution and the solvent liquid/solid phase must be examined. Furthermore, there must be checks of the model against known experimental results. The suggestion has been made that the function of AFGP may simply be to shield the normally present heterogeneous nucleation sites. Such a phenomenon, called poisoning of nucleation sites, has been shown to be inadequate as the cause of AFGP activity. Feeney and Hofmann (1973) have convincingly shown that AFGP actually functions in the presence of already seeded ice crystals. From a careful examination of Fig. 17, the A T lowering effect for low solute concentrations is very efficient. The comparison with other proteins, such as lysozome, and of other glycoproteins, such as ovomucoids, shows that indeed the colligative effect is negligible (Feeney and Hofmann, 1973). This result implies that the term for the surface interaction energy of active species is very strong. Both large u and small embryo dimension I would tend to render K / k T >> l / m in Eq. (27). This increase in F or y as defined by Eq. (42) will in turn increase AG of formation, and accordingly, decrease nucleation probability, Eq. (44), while increasing AT of Eq. (27). The entropic effect, which must play a large role in the waterhydrogen bonded system, is nonetheless much more difficult to assess. One can expect that, in general, if the binding of the substrate molecule to the ice surface were very strong, it may, as indicated in

ANTIFREEZE PROTEINS FROM FISH BLOODS

271

Section VIII, cause appreciable distortion of local order, hence increasing local entropy. If it were possible for these antifreeze systems to create far more local disorder than the polymer systems studied by Solms and Rijke (1971) or by Jellinek (1972), then a total contribution to x, mainly due to the entropic term, h,could exceed the value of 4. Once that were achieved, we could expect the observed trend of Fig. 34 effecting “saturation” of the antifreeze function. The two possible schemes that we postulate will both lead to large values of a and xs,and will thus be qualitatively consistent with the experimental data. A. A Model Involving Hydroryl Interactions The glycoproteins from the Antarctic fishes and the Arctic polar cod have repeated amino acid sequences of Ala-Ala-Thr in their backbone polypeptide structure. Furthermore, disaccharide groups are regularly attached to all the threonine residues through a @1+3 linkage. Both of these regularities must be accounted for in any model for antifreeze action. In this section we consider the model where the regular spacing of the backbone is as vital as the hydrophilic disaccharide side chains. Experimental results have shown that the hydrodynamic conformation of AFGP corresponds to an extended structure (Ahmed et al., 1975). Raman spectroscopy on this system (Tomimatsu et al., 1976) revealed that the solution spectra in the amide I region is dominated by extended conformations, consistent with the earlier results (DeVries et al., 1970), where circular dichroism also showed no extensive regions of helices. Such an open structure in turn leads to open exposure of the disaccharide groups. We have suggested in Section VIII that the carbohydrate-water structural comparisons show remarkable similarity, even though the nature of the bonds is different. It is indeed possible to envision the placement of a carbohydrate ring on the hexagonal face of the ice Ih lattice. Suggett (1975) further discussed this problem in terms of glucose fitting in the tridymite structure of water. In our model, let us suppose that the equilibrium position acquired by the covalently linked carbohydrate chain is only minimally affected by thermal changes. On the other hand, we suppose that the weaker hydrogen bonds forming the hexagonal ice lattice is such that the zero point motion is far greater. We assume that, at the temperatures immediately below T o = O”C,the ice lattice dynamic structure is easily arranged to be compatible with the more rigidly fixed carbohydrate groups. This is consistent with ideas that ice Ih may have much lattice anharmo-

272

ROBERT E. FEENEY AND YIN YEH

nicity at temperatures immediately below O"C, and consequently a broad region of zero-point lattice motion. When the temperature is lowered, these oscillations about the equilibrium would be much smaller owing to a continuing decrease of the contributions of these anharmonic lattice vibrations (Eisenberg and Kauzmann, 1969). Consequently, even very slight misalignments between the lattice spacing of the ice Ih and the carbohydrate ring will cause the intermolecular hydrogen bonding to be stressed. We propose that at a temperature of approximately - 0.8"C, this decrease in zero point motion is sufficient to cause the carbohydrate-H,O hydrogen bonds to break in favor of H20-H,0 hydrogen bonds. As a result, the molecule, which previously created a large surface interaction energy through its hydrogen bonds becomes either partially released or completely released from the H 2 0 structure owing to excessive structural mismatch. Since the larger AFGP molecules are more flexible, their release from the ice surface bonds is more likely to be incomplete. As a result, ice growth is inhomogeneous. AFGP can in fact be entrapped (Tomimatsu et al., 1976) between grain boundaries. Figure 36 illustrates schematically the surface compatibility of the AFGP and nucleated ice crystal. Such a model is also consistent with the fact that the ice, once formed, is normal ice and its melting temperature is To = 0°C.

0

h

FIG. 36. Schematic representation of disaccharide-HzO binding. (a) T > T,: AFGP hydrogen bonds to water molecules with ease, leading to a hydration layer. (b) T , > T 4 Tf: Ice lattice at this temperature has large zero-point motion. Therefore the lattice spacing, represented by the vertical spacings within the lower-region, can vary with relative ease. Surface HzO molecules easily form hydrogen bonds with AFGP with only minor distortions. (c) T Tf: The decreased zero-point motion of the ice lattice leads to its stiffening. This results in very much stressed hydrogen bonds between surface HzO molecules and the AFCP molecule. (d) T < Tf: Further decrease in temperature increases the lattice rigidity to the point of breaking all but a few AFCP-HZO hydrogen bonds.

-

ANTIFREEZE PROTEINS FROM FISH BLOODS

273

1 . Functional Additivity of AFGP and Colligative Salts As long as AFGP exists in solution in a fairly low concentration where salt-AFGP interaction is at a minimum compared with either salt-H20 or AFGP-HzO interactions, the independence of these effects of the freezing temperature lowering is reasonable. We consider the AFGP of molecular weight 10,500. At a concentration of 10 mg/ml, this corresponds to 6 x lo" AFGP molecules per cubic centimeter of solution. The center-to-center spacing, S, is then = lop6cm. Since the average size of an active sample has Re = 30 A (Ahmed et al., 1975), the average linear spacing between molecules is S - 2R, = 40 A. At NaCl concentrations of 0.05 M , there can be only about four Na+ or C1- in the volume of (40 A)3. This means the interaction between AFGP and salt is indeed not significant. In a more extreme situation, since in the blood serum almost 1.3"C of the freezing temperature depression is due to smaller sized dialyzable compounds, corresponding to a total salt or small molecule concentravolume must contain nearly 24 ions. tion of about 0.3 M , the (40 For ionic diameters of 1.5 A, there should still be enough space for a hydration sheath ( - 3 A) to develop around each of these ions and not interfere greatly with the position of the antifreeze molecules.

2. The Necessity of the Disaccharide Side Chains As indicated in this model, the disaccharide group, galactose and N-acetylgalactosamine, could be the center of HzO binding in this system. Suggett (1975) pointed out that the most important interaction mechanism between carbohydrates and water is through hydrogen bonding with the sugar hydroxyls. Furthermore, this bonding would be expected to be highly orientationally dependent. As a result, the precise spatial arrangement of the many hydroxyls in a solute molecule could greatly influence the nature and extent of this solute-solvent interaction. In particular, are certain spatial arrangements of carbohydrate groups more favorable for hydration than others? The answer seems to lie with the size of the carbohydrate group. For the monomeric unit, the competition for OH groups of a glucose molecule by HzO hydrogen bonds is highly favorable when HzO is in a tridymite lattice (Suggett, 1975). For larger groups, other interactions begin to be of importance. It was shown by Neal and Goring (1970) that, for the disaccharide maltose in HzO, intramolecular hydrophobic interaction begins to shield certain OH sites. In fact, Shiio (1958) found that the number of bound HzO molecules decreases rapidly as the degree of polymerization increases beyond the disac-

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charide configuration, which has capacity for binding approximately ten H 2 0 molecules. Recently, Haschemeyer et al. (1977), reported that nuclear magnetic resonance (NMR) -proton resonance of AFGP indeed revealed 0.66 g of bound water per gram of carbohydrate. If these carbohydrates were disaccharides with a molecular weight of approximately 300, then about 10 H 2 0 molecules bound per disaccharide group is again inferred. These workers also indicated NMR results showed about 5 % of “additional unfrozen water” at -5°C. The relationship of this latter observation to antifreeze function is unclear at this time. The need for the carbohydrate side chain for antifreeze action of AFGP has been indirectly proved. In almost all studies where the disaccharide group is either removed or modified, the glycoprotein becomes functionally inactive (Komatsu et al., 1970a; Shier et al., 1972; Ahmed et al., 1973, 1976). In such a hydrogen-bonded carbohydrate-H20 system, one envisions a large interfacial term 5 leading to antifreeze function and a substantial value for x. Should x > 3 be the case, functional saturation can be observed at higher concentrations. While functioning in the capacity of an antifreeze molecule, these carbohydrate groups do not allow the embryonic ice nucleus to become critical. As a result, these unstable embryos will redisperse into the solution, and these molecules experience basically the environment of supercooled water within the temperature range of interest. B . Models Emphasizing the Roles of the Methyl Groups

Even though the above model is consistent with the antifreeze function of the AFGP species, the recent surge of evidence that the protein antifreeze without sugar side chains also functions in a noncolligative capacity requires other modifications of the above model or a new model. Vandenheede et al. (1972) initially suggested that hydrophobic groups could be important in the antifreeze activity. They pointed out that the presence of alanine and threonine, instead of glycine and serine, in the AFGP could be significant in a functioning capacity. The basic difference between the former and the latter pair of amino acid residues lies with methyl groups replacing the hydrogens on the carbons. These hydrophobic methyl groups can indeed affect solvent-solute interaction and consequently antifreeze function in many ways. In the simplest use of these methyl groups, the previously suggested polymer adsorption model can be taken over completely.

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Here, however, instead of disaccharides serving as hydrophilic links to the embryonic ice-lattice sites, one can postulate that certain hydrophilic groups of the flounder, e.g., Glu and Asp residues (Duman and DeVries, 1976), can attach to the ice surface. Then the surface disruption mechanism and temperature dependence of this disruption as previously discussed for the disaccharide group may be carried over to the Asp and Glu residues. As the temperature is lowered, gradual incompatibility of the bonding sites for the helical protein would lead to the sa-turation of the antifreeze activity. The problem associated with this model, where Ala are used only as " spacers," is that the regular sequencing of either Asp or Glu is not evident in any of the observed protein species or the synthetic antifreeze protein. The exact need for regularity or irregularity of the hydrophilic groups is yet unclear. A second use of methyl groups is that of a hydrophobic barrier. By assuming that either the disaccharide or the many hydrophilic amino acids are the only molecules that can form hydrogen bonds with the hydroxyls of the ice interface, these bonds may be strong enough to overcome the hydrophobic interaction of the methyl groups. Consequently, methyl groups could be exposed (F. Franks, unpublished data). Such a hydrophobic barrier may be sufficient to provide an additional kinetic barrier, A g , to keep the liquid H 2 0 molecules from reaching the ice crystal surface. If the enhanced kinetic barrier, A g , results in the observed antifreeze function, then according to Eq. (44),the continued growth of the nucleus is a rate phenomenon. In experiments conducted with seed crystals of ice nuclei held at - 0.2"C,there does not seem to be a time-dependent growth of the seed nucleus, even over extended periods (Feeney and Hofmann, 1973; Ahmed et d.,1975). The fact that no rate effects have been observed, however, is insufficient reason to eliminate a kinetic mechanism. As a third alternative way of using the methyl groups, one can imagine these groups to serve as a hydrophobic matrix. The infrequently occurring hydrophilic residues are again assumed to be bound to the ice-lattice sites. If one assumes that these sites are sufficiently disrupted by the presence of these hydrophilic residues, antifreeze activity can indeed take place by the postulated surface adsorption phenomenon. However, the hydrophobic regions of the methyl groups are simultaneously forcing the water molecules to seek its own for binding. A bifunctional pattern could consequently develop. Since the previous embryo has been bound to the hydrophilic residue, antifreeze activity via surface adsorption has already taken place.

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However, the hydrophobic groups are forcing new H 2 0 clusters to form within the hydrophobic voids. When these clusters become large and integrated with the original embryos, nucleation and growth commences. This idea is consistent with the postulate that a hydrophilic site within a hydrophobic matrix is the key to nucleation (Zettlemoyer, 1968). Finally, it is necessary to emphasize that, owing to the scarcity of data on the physical characterization of the flounder and the synthetic AFP systems, all these ideas are still awaiting more conclusive evidence. In particular, the degree of antifreeze activity in the AFGP, the AFP, and the synthetic AFP are different. Is there a variation of degrees of one antifreeze mechanism, or is there more than one basic mechanism of antifreeze and saturation of the antifreeze function?

x. SUMMARY AND

CONCLUSIONS

Glycoproteins of molecular weights ranging from approximately

10,500 to 27,000 isolated from the blood sera of the two Antarctic fishes, Trematomus borchgrevinki and Dissostichus mawsoni, exhibit antifreezing properties in pure H,O or salt solutions. Since the melting point is not affected, a hysteresis exists between the freezing and melting temperatures, and the glycoproteins are considered to lower the freezing temperature rather than the freezing point. The information obtained on the basic structure of these glycoproteins includes the following points: 1. The primary sequence of amino acids of the active species is Ala-Ala-Thr in repeated units. A disaccharide, galactosyl-hracetylgalactosamine, with a p 1+ 3 linkage, is glycosidically linked to all the threonines. 2. The secondary structure of the species indicates a predominance of expanded regions, although Raman spectra indicate that some ahelical sections do indeed exist in solution. 3. Tertiary structure, as determined by sedimentation and quasielastic light-scattering studies, indicates that the solution conformation is highly hydrated. A radius of gyration of approximately 30 A was estimated for the component with a molecular weight of 21,500. Functionally, the active molecules are capable of lowering the freezing temperature of pure water by approximately 0.8"C. Activity can be nullified when chemical modifications are made on the disaccharide groups. Additionally, lower molecular weight components exhibit more structural rigidity and show little functional capacity. Even though the hydrodynamic conformations of the active glycoproteins do not seem to change significantly in the active temperature

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range (Ooto -8OoC), there are significant detailed structural changes, as seen by the spectral region sensitive to COH bonds among the active species in solution, active species trapped in ice, and the inactive species. Models of antifreeze mechanism have been discussed. In these models the polypeptide is assumed to form a nonideal, nonregular solution in H20. In one model, the ability of the hydrophilic sites of these molecules to enter into favorable competition for hydrogen bonds between the H 2 0 molecules themselves provides for a mechanism of lowering the freezing point. The gradual incompatibility with ice Ih structure as a function of temperature and/or the prevalence of hydrophobic side chains forcing water molecules together near specific hydrophilic sites may be the cause for the saturation of antifreeze activity and growth of normal ice crystals. In the other models, the hydrophobic methyl groups of the polypeptide backbone are envisioned to play more major roles. Generally, methyl groups can assume the role of either spacer molecules, barrier molecules, or hydrophobic matrices for heterogeneous nucleation and propagation. The recent findings that the northern flounder, the sculpin, as well as synthesized hydrophobic polypeptides also exhibit anomalous freezing-point lowering suggests that the role of the hydrophobic methyl groups on each amino acid residue must be significant. So far the only overriding similarity between these antifreezing molecules is the predominance of alanine. It may be that it is this feature which really has primary and critical importance in antifreeze function observed in these fishes. The role of these groups must be examined in much more detail. One must also be willing to develop a yet undescribed model to bring about a consistent picture of all these molecular antifreezing systems. The following is a short list of some possible areas for future studies: 1. Comparative studies of the primary, secondary, and tertiary structure for both types of antifreeze proteins. 2. Additivity of function with mixtures of glycoprotein and protein systems and additivity with other substances that normally function in a colligative manner. 3 . Synthesis of structures or analogous structures, including replacement of some, or all, of the methyl groups by hydrogen (i.e., conversion of alanines to glycines), and replacement of the carbohydrates in the glycoproteins by different carbohydrates or other substances.

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4. Study of antifreezing properties of different-length segments of the polypeptide chains-determination of the minimum lengths of polymers required. 5 . Chemical modifications of both types of proteins, including, particularly, the carbohydrate side chains of the glycoproteins. 6. Further examinations of interactions of the antifreeze proteins by examination of the growth patterns of ice crystals and examining the local structure of the ice through such techniques as NMR and Raman with selective deuterations. 7. Precise calorimetric studies of the heats of fusion and melting. Since these two quantities should be the same (in absolute value) for a pure H 2 0 system, differential measurement should indicate the excess energy required to break the interactions between the antifreeze systems and HzO. 8 . A selective study of the function of the antifreeze proteins in solvents other than water. 9. The general biology and metabolism, including such diverse subjects as the biosynthesis, adaptive synthesis, presence or absence in other areas of the body of the fish, and possible biological roles of the smaller inactive proline-containing glycopeptides. The brevity of this list will be obvious to anyone reading this review, and scientists in different fields will find many alternative directions for the studies. We hope that the breadth and importance of the subject will stimulate others to pursue such alternative approaches. ACKNOWLEDGMENTS The authors would like to thank Dr. A. C. Zettlemoyer, Dr. Donald G. Knox, and Dr. Terrell Hill for critical reviews of the manuscript, David T. Osuga, Chris Howland, and Laura Hayes for editorial assistance, and Clara Robison for typing the manuscript. Appreciation is also due to the National Institutes of Health (Grant No. HD 00122 for R.E.F.) and the National Science Foundation (Grant No. GA-12607 for R.E.F. and BMS 7506918 for Y.Y.) for their financial support pertaining to this research. Particular appreciation is due to the National Science Foundation for advice and logistics in research in the Antarctic and for support in research on other proteins from Antarctic species. The authors are also thankful to the Royal Norwegian Council for Scientific and Industrial Research for financial support and the Norwegian Institute for Marine Biology, Bergen, for their hospitality and assistance in obtaining materials and performing research on shipboard for the Arctic work mentioned in this article.

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Haschemeyer, A. E. V., Guschlbauer, W., and DeVries, A. L. (1977). Nature (London) 269,87-88. Hemmingsen, E. A., Douglas, E. L., and Griggs, G. C. (1969). Comp. Biochem. Physiol. 29,467-470. Henderson, D., and Leonard, P. J. (1971). Phys. Chern. SB, 413-510. Hew, C. L., and Yip, C. (1976). Biochem. Biophys. Res. Commun. 71,845-850. Hobbs, P. V. (1974). “Ice Physics.” Oxford Univ. Press (Clarendon), London and New York. Home, R. A., ed. (1972). “Water and Aqueous Solutions; Structure, Thermodynamics and Transport Processes.” Wiley (Interscience), New York. Jellinek, H. H. G. (1972). In “Water and Aqueous Solutions; Structure, Thermodynamics and Transport Processes” (R. A. Home, ed.), pp. 65-107. Wiley (Interscience), New York. Komatsu, S. K. (1969). Ph.D. Thesis, University of California, Davis. Komatsu, S. K., and Feeney, R. E. (1970). Biochim. Biophys. Acta 206,305-315. Komatsu, S. K., DeVries, A. L., and Feeney, R. E. (1970a). J. Biol. Chem. 245, 2909-2913. Komatsu, S. K., Miller, H. T. DeVries, A. L., Osuga, D. T., and Feeney, R. E. (1970b). Comp. Biochem. Physiol. 32, 519-527. Kuhn, W. (1956). Helo. Chim. Acta 39, 1071-1086. Lee, H. S., Osuga, D. T., Nashef, A. S., Ahmed, A. I., Whitaker, J. R., and Feeney, R. E. (1977). J. Agric. Food Chem. 25, 1153-1158. Leivestad, H. (1965). Int. Comm. Northwest Atlantic Fish., Spec. Publ. No. 6, pp. 747-752. Lin, Y., Duman, J. G., and DeVries, A. L. (1972). Biochem. Biophys. Res. Commun. 46,87-92. Mason, B. J. (1957). “Physics of Clouds.” Oxford Univ. Press (Clarendon), London and New York. Means, G. E., and Feeney, R. E. (1971). “Chemical Modification of Proteins.” Holden-Day, San Francisco, California. Morris, H. R., Thompson, M. R., Osuga, D. T., Ahmed,A. I., Chan, S. M., Vandenheede, J. R., and Feeney, R. E. (1978). J. Biol. Chem. 253 (in press). Neal, J. L., and Coring, D. A. I. (1970). Can. J. Chem. 48,3745-3747. Nemethy, G., and Scheraga, H. A. (1962). J. Chem. Phys. 36,3382-3400. Osuga, D. T., and Feeney, R. E. (1978). J . Biol. Chem. 253 (in press). Pathria, R. K. (1972). “Statistical Mechanics.” Pergamon, Oxford. Plummer, P. L. M. (1973). In “Physics and Chemistry of Ice” (E. Whalley, S. J. Jones, and L. W. Gold, eds.), p. 109. Royal Society, Ottawa. Pople, J. A. (1951). Proc. R. Soc. London, Ser. A 205, 163-178. Rakusa-Suszczewski, R., and McWhinnie, M. A. (1976). Comp. Biochem. Physiol. A 54,291-300. Raymond, J. A., and DeVries, A. L. (1972). Cryobiology 9,541-547. Raymond, J. A., and DeVries, A. L. (1977). Proc. Natl. Acad. Sci. U . S. A. 74, 2589-2593. Raymond, J. A., Lin, Y., and DeVries, A. L. (1975). J. E x p . Zool. 193, 125-130. Scholander, P. F., and Maggert, J. E. (1971). Cryobiology 8,371-374. Scholander, P. F., Flagg, W., Walters, V., and Irving, L. (1953). Physiol. Zool. 26, 67-92. Scholander, P. F., VanDam, L., Kanwisher, J. W., Hammel, H. T., and Gordon, M. S. (1957). J. Cell. Comp. Physiol. 49, 5-24.

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Schroder, B. (1970). “Wasser.” Suhrkamp Verlag, Frankfurt (in German). Shier, W. T., Lin, Y., and DeVries, A. L. (1972). Biochim. Biophys. Acta 263,406-413. Shier, W. T., Lin, Y., and DeVries, A. L. (1975). FEBS Lett. 54, 135-138. Shiio, H. (1958). J . Am. Chem. Soc. 80, 70-73. Sjoquist, J., Blomback, B., and Wallen, P. (1960). Ark. Kemi 16, 4 2 5 4 3 6 . Smith, R. N. (1972). Br. Antarct. Sum. Bull. 30,81-93. Solms, D. J., and Rijk6, A. M. (1971). J. Phys. Chem. 75, 2623-2631. Sprenger, I., Uhlenbruck, G., and Pardoe, G. I. (1970). Haematologia 4,373-378. Suggett, A. (1975). In “Water, a Comprehensive Treatise” (F. Franks, ed.), Vol. IV, pp. 519-567. Plenum, New York. Tanford, C. (1961). “Physical Chemistry of Macromolecules.” Wiley, New York. Tanford, C. (1973). “The Hydrophobic Effect: Formation of Micelles and Biological Membranes.” Wiley, New York. Tomimatsu, Y., Scherer, J. R., Yeh, Y., and Feeney, R. E. (1976). J. B i d . Chem. 251, 2290-2298. Turnbull, D., and Vonnegut, B. (1952). Znd. Eng. Chem. 44,1292-1298. Vandenheede, J. R. (1972). Ph.D. Thesis, University of California, Davis. Vandenheede, J. R., Ahmed, A. I., and Feeney, R. E. (1972). J. Biol. Chem. 247, 7885-7889. Walton, A. G. (1969). In “Nucleation” (A. C. Zettlemoyer, ed.), pp. 225-307. Dekker, New York. Warner, D. T. (1962). Nature (London) 196, 1055-1058. Wenzel, J., Linderstr0m-Lang, C. U., and Rice, S. A. (1975). Science 187,428-430. Wohlschlag, D. E. (1964). In “Antarctic Research Series” (M. 0. Lee, ed.), Vol. I, pp. 33-62. Am. Geophys. Union, Washington, D. C. Zettlemoyer, A. C. (1968). J. Colloid Interface Sci. 28,343-369. NOTE ADDED

IN

PROOF

In recent studies in our laboratories, progress has been made on two of the subjects recommended for future study in the last section of this article. These studies have concerned suggestion number 9 (page 278) for obtaining information on possible properties and functions of the smaller AFGP glycopeptides 7 and 8 and suggestion number 2 (page 277) for determining the possible additive effects of mixtures of active AFGP with AFY. Our studies on the possible function of the small AFGP 7 and 8 glycopeptides have now shown that they are capable of potentiating the activity of the larger active AFGP (D. T. Osuga, Y. Yeh, F. Ward, and R. E. Feeney, unpublished data, 1978). When relatively large amounts of AFGP 8 (5-10 mg) are added to small amounts of active AFGP 1-5 (1 mg), there is a stimulation of the active antifreeze activity which is more than five times the antifreeze activity obtained with AFGP 1-5 alone. The smaller AFGP 7 and 8 glycopeptides have been termed inactive in previous publications, and their inactivity when tested alone was confirmed in this study. Either AFGP 7 or 8 from T. borchgrevinki or B . saida potentiated the antifreeze activity of the active AFGP proteins from either species. We have also observed in these studies that the effect of increasing concentration of active AFGP 1-5 on the lowering of the freezing temperature differs somewhat from the older data of DeVries et al. (1970) shown in Fig. 17. Present data show the plot to bemore linear, and even sigmoidal, at low concentrations of the large glycoproteins, whereas the older data showed a convex function versus concentration dependence at low concentrations. Although none of these old preparations

of active AFGP is available for analysis by current, more definitive methods, it would seem possible that the samples used for these old determinations contained a sufficient amount of the small AFGP glycopeptides to give some potentiation at low amounts of active AFGP, and hence gave a more convex response than is currently obtained. With levels of active AFGP near the saturation amounts (over 10 mg/ml), the effects of added AFGP 7 or 8 are much less than at the very low levels (1mg) of aChVe AFGP. Consequently, plateaus are still obtained. The potentiation of the antilectin activity of the larger active AFGP by the smaller AFGP glycopeptide 8 has not been obtained. This observation that the potentiation only occurs for antifreeze functioning could be considered evidence for a direct effect in the antifreeze mechanism. Possible molecular interactions are under investigation. In studies on the additive effects of the glycoprotein and nonglycoprotein antifreezes, mixtures of active AFGP and AFP exhibit, at least in part, additive lowerings of the freezing temperature and similar plateaus at higher concentrations (D. T. Osuga, Y. Yeh, C. L. Hew and R. E. Feeney, unpublished data, 1978). Thus, when concentrations of the two different antifreezes are adjusted to give the same lowering of the freezing temperature, mixtures of the two solutions give similar lowering when the concentrations of either are increased correspondingly. This was considered additional evidence that the two different antifreeze proteins function by similar mechanisms. The potentiation of active AFGP glycoproteins by the smaller AFGP glycopeptides, however, does not seem to be inconsistent with any of the mechanisms discussed in this article.

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.

By F MACRITCHIE CSIRO Wheat Research Unit. North Ryde. New South Wales. Australia

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Experimental Techniques . . . . . . . . . . . . . . . . . . . . . . .

111. Kinetics and Mechanism of Adsorption

283

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. . . . . . . . . . . . . . . . . 286

A . Diffusion to Interfaces . . . . . . . . . . . . . . . . . . . . . . . B. Interfacial Pressure Barrier . . . . . . . . . . . . . . . . . . . . . C . Electrical Potential Barrier . . . . . . . . . . . . . . . . . . . . . D . Transition State in Adsorption . . . . . . . . . . . . . . . . . . . IV . Conformation of Protein Molecules at Interfaces . . . . . . . . . . . . A . Interpretation of Interfacial Pressure, Potential, and Viscosity . . . . B. Configurational Changes in Response to Compression and Expansion . C . Measurement of Helix Contents . . . . . . . . . . . . . . . . . . D . Reversibility of Conformational Changes During and Following Desorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Equilibrium Aspects of Adsorption . . . . . . . . . . . . . . . . . . . A . Adsorption Isotherms . . . . . . . . . . . . . . . . . . . . . B. Thermodynamics of Adsorption . . . . . . . . . . . . . . . . . . C . Reversibility and the Problem of Desorption of Large Molecules . . . . . VI . Reactions at Interfaces . . . . . . . . . . . . . . . . . . . . . . A . Effects of High Concentration and Orientation on Reaction Rate . . . B. Bonding and Crosslinking Following Exposure of Reactive Groups . . . . C . Interactions with Lipids . . . . . . . . . . . . . . . . . . . . . . . D . Interfacial Coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . VII . Proteins at Interfaces in Biological Systems A . Biological Membranes . . . . . . . . . . . . . . . . . . . . . . . B. Cell Surfaces and Recognition Processes . . . . . . . . . . . . . . C . Thrombus Formation . . . . . . . . . . . . . . . . . . . . . . . D. Immobilized Enzymes . . . . . . . . . . . . . . . . . . . . . . . E . Miscellaneous Topics . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

286 288 290 292 293 293 296 299 300 301 303 308 3 10 312 312 313 315 316 319 319 320 321 322 322 323

I . INTRODUCTION The strongly amphipathic nature of proteins. resulting from their mixture of polar and nonpolar side chains. causes them to be concentrated at interfaces . As a result of their great stability in the adsorbed state. it is possible to study them at fluid interfaces by the classical techniques of insoluble monolayers . A review of early work along these lines in this series (Bull. 1947) serves as an excellent introduction to the subject . The effect of adsorption on the biological activity of proteins was treated by Rothen (1947) in the same volume . Further 283

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developments were dealt with by Cheesman and Davies (1954) in a later volume. The subject of proteins at interfaces is an extremely broad one, encompassing a very wide range of apparently unrelated phenomena. Apart from the fundamental subject of protein adsorption and its effects, many technological processes, especially in the pharmaceutical and food industries, utilize proteins as stabilizers for foams and emulsions. However, perhaps the most exciting areas for development at present are in the fields of biology and medicine. There is an increasing awareness of the major role that surface chemical processes play in these areas. As a result, it is becoming evident that an interdisciplinary branch of science needs to develop in which both the surface and colloid physical chemist and the biologist participate. Many of the problems cannot be adequately tackled by either specialist alone. In accordance with this view, the present article aims to first present protein adsorption and its ramifications, such as the effects of adsorption on conformation and reactivity, from a fundamental physicochemical approach insofar as present knowledge allows. Mention is made of features relevant to biological systems at appropriate points. Following this, some of the many areas where interface studies of proteins are pertinent to important problems in biology are discussed.

11. EXPERIMENTAL TECHNIQUES Methods used in the study of proteins at interfaces are discussed in previous reviews in this series and in standard texts (Alexander and Hibberd, 1971; Davies and Rideal, 1961). Only some recent developments of interest are mentioned here. A striking feature is that, although many proteins are very soluble in water, adsorption leads to stable monolayers that are extremely difficult to desorb. Unlike the common substances studied as insoluble monolayers, quantitative spreading of proteins requires that none is able to dissolve into the bulk solution and diffuse away. This problem was considered by Trurnit (1960), who devised methods for spreading proteins from purely aqueous solutions under conditions where practically 100% of the protein molecules would be assured of diffusing to the interface. The technique depends on allowing a very thin layer of the solution to flow down a glass rod before reaching the water surface. The influence of the flow rate, protein concentration, and glass rod length and diameter was investigated, and results from spreading experiments were compared with theoretical predictions. Satisfactory quantitative spreading has also been achieved with 60% propanol containing 0.5 M sodium acetate, introduced by Stallberg and Teorell (1939).

PROTEINS AT INTERFACES

285

The method of Trurnit avoids problems that may arise with a spreading solvent. However, a factor that was not studied was the effect of the electric charge carried by the protein and the accompanying electrical potential barrier produced at the interface, as discussed in Section II1,C. For this reason, it is preferable, when using the Trurnit method, to have the protein at a p H close to its isoelectric point to ensure complete spreading. Adsorption may b e followed at fluid/fluid interfaces by measuring changes in interfacial pressure, potential, or viscosity, using spread monolayers for calibration purposes. The most accurate method for measuring rates of adsorption is by the rate of increase of interfacial area at constant interfacial pressure. If (1/A) (dA/dt)is the fractional rate of increase of interfacial area expressed in sec-' and n is the interfacial concentration in g found from measurements on spread monolayers, then the rate of adsorption d n / d t in g cm-2 sec-' is given by

dn/dt

=

(1/A) ( d A / d t ) n

(1)

The rate may thus be evaluated from a plot of log A vs t . If measurements of the interfacial pressure, II, are made at a fixed interfacial area (i.e., II - t curves are obtained), the rate of adsorption at a given value of II is dn/dt

( d n / d I I )( d I I / d t ) (2) where dII/dt is evaluated from the II - t curve and dn/dII from the =

n - I1 curve of the spread monolayer. Some workers are critical of the use of interfacial pressure measurements for following adsorption, believing that the structure of protein films formed by spreading and by adsorption from solution are different. This view is not shared by the author and is discussed further in Section IV,A. One of the most useful experimental methods to be applied to protein adsorption in recent years is the radiotracer technique (Muramatsu, 1973). Proteins labeled with I3lI and lZ5I(Brash et al., 1974) and [14C]acetylderivatives of proteins (Phillips et al., 1975) have been used as tracers. As well as measuring adsorption directly, this method has the great advantage that it can detect exchange between interface and bulk even when the total amount adsorbed does not vary. A technique that has been used to obtain independent measurements of the amount of protein adsorbed b y measuring film thickness is ellipsometry (Trurnit, 1953). The application of infrared difference spectrometry to the measurement of protein adsorption at solid/liquid interfaces is potentially

286

F. MACRITCHIE

valuable (Morrissey and Stromberg, 1974). Binding of carbonyl groups to a solid interface is accompanied by a shift in the absorption frequency. This is made use of not only to measure the amount adsorbed, but to estimate the number of carbonyl attachments and thus give information about the molecular conformation in the adsorbed state. 111. KINETICS AND MECHANISM OF ADSORPTION It has become customary to refer to the action of surfaces on proteins as surface denaturation. This is an unfortunate term in some ways. First, it does not satisfy the requirements of a scientific term that needs to be simple and unambiguous. Second, it tends to discourage further interest in the 'subject whereas, in natural systems, proteins must be continually interacting with interfaces and undergoing changes in conformation and properties. It therefore seems preferable to examine protein adsorption, adhering to strict terminology in order to describe the processes which occur.

A. Diffusionto lnterfaces Before a protein molecule can adsorb and exert its influence at a phase boundary or take part in an interfacial reaction, it must arrive at the interface by a diffusion process. If we assume there is no barrier to adsorption other than diffusion, simple diffusion theory may be applied to predict the rate of adsorption. Under these conditions, after formation of a clean interface, all the molecules in the immediate vicinity will be rapidly adsorbed. The protein concentration in a sublayer, adjacent to the interface.and of several molecular diameters in thickness, will thus be depleted to zero. A diffusion process then proceeds from the bulk solution to the sublayer. The rate of adsorption, d n / d t , will be simply equal to the rate of this diffusion step given by classical diffusion theory (Crank, 1956) as

dn/dt

=

C, ( D / T ) ' / ~ ~ - ' / ~

(3)

where C, is the bulk concentration, D is the diffusion coefficient, t the time, and T is 3.14. Integration of Eq. (3) gives the number of molecules that adsorb in a time t

n

=

2c, (Dt/T)"2

(4)

Equation (4)has been applied to the adsorption, at an air/water interface, of bovine serum albumin (BSA) from solutions of different concentrations (MacRitchie and Alexander, 1963a). The times were measured for monolayers to build up to a surface pressure of 0.1 milli-

287

PROTEINS AT INTERFACES

newton per meter (mN m-l) (corresponding to a surface concentration of 7 x lo-' g cm-e) after creation of a new surface. In Table I, the measured times are compared with times calculated from Eq. (4). For the higher concentrations, close agreement is observed. As the bulk concentration is decreased, measured times become smaller than those predicted. This is readily explained by the effect of convection. Equation (4)assumes a diffusion-controlled process. This will be valid providing the diffusion gradient occurs close to the surface because for all fluid phases there is an undisturbed layer adjacent to the interface where mass transport occurs only by diffusion. This is called the stationary layer or boundary layer for mass transfer. Its width depends on the nature of the fluid and on conditions such as temperature, amount of stirring, and the properties of the interfacial film. The width in aqueous solutions generally varies from about 0.1 cm for unstirred systems (Ter Minassian-Saraga, 1955) to 3 x lop3cm where vigorous stirring is used (Moelwyn-Hughes, 1947). Widths of the concentration gradient, calculated from diffusion theory, for each predicted time are included in Table I. For the lowest concentration used, it can be seen that the stationary layer width is obviously exceeded. Trurnit (1954) has described two closely related methods for determining diffusion coefficients of proteins based on measurement of the rate of adsorption of protein from a dilute solution on to a BlodgettLangmuir slide. One method employs stirred conditions so that the TABLEI Measured and Calculated Times for Bovine Serum Albumin Films To Reach 0.1 mN m-l at a Fresh Interfacea** Width of stationary layer at t calculated

Concentration (g liter-')

t Measured

t Calculated

0.03 0.02 0.01 0.005 0.003 0.002 0.001

6 f 1 sec 15 ? 2 sec 55 2 5 sec 3.5 f 0.2 min 9.0 ? 0.2 min 19 2 1 min 45 f 5 min

7 16 sec 64 sec 4.2 min 11.9 min 26.7 min 107 min

(4 0.001 0.005 0.011 0.022 0.037 0.055 0.11

From MacRitchie and Alexander (1963a), reproduced with permission. Note on units of surface pressure: Surface pressure has the dimensions of force divided by length. The most convenient unit, in the SI system, is millinewton per meter (mN m-l). Note that 1 mN m-I = 1 dyne cm-'.

F. MACRITCHIE

288

concentration gradient occurs across the stationary layer. In the other method, no stirring is used and the diffusion coefficient is evaluated from Eq. (4). Agreement was found between the rates of adsorption and the predicted rates of diffusion at the solidlwater interface. However, results of MacRitchie and Alexander (1963a) for the airlwater interface show that once a close-packed monolayer is present at the interface, the rate of adsorption progressively falls below the rate of diffusion, indicating the presence of an energy barrier to adsorption (Table 11). We may assume, nevertheless, from the close agreement of experimental and predicted times shown in Table I that for isolated protein molecules adsorbing at the airlwater interface in the absence of an appreciable interfacial pressure, there is no energy barrier to the adsorption step.

B . Interfacial Pressure Barrier Two experimental results indicate that there is an adsorption energy barrier related to the interfacial pressure. First, the presence of an energy barrier becomes evident only after an interfacial pressure of 0.1 mN m-l is attained (Table 11). In the second experiment, different compounds were spread at the airlwater interface and the rate of adsorption of pepsin and lysozyme were measured under conditions where charge effects were minimized (MacRitchie and Alexander, 1963a). I t was found that the rates of adsorption for these proteins were independent of the nature of the surface film and depended only on the surface pressure. A theory, first introduced by Ward and Tordai (1952) was applied to explain the adsorption behavior. This theory assumes that, in order TABLEI1 Measured and Calculated Times for Bovine Serum Albumin Films to Reach Various Pressures"

C,

n

=

0.02 g liter-'

Co = 0.03 g liter-'

t measured (set)

t calculated

t measured

(mN m-')

(set)

(sec)

t calculated (set)

0.1 0.2 0.3 0.5 1.0 2.0

15 22 32 40 65 135

16 17.5 18.5 19.5 23 26.5

6 10 15 22 50 120

7 7.5 8 8.5 10 11.5

From MacRitchie and Alexander (1963a), reproduced with permission.

PROTEINS AT INTERFACES

289

for a molecule to adsorb, it must compress molecules already adsorbed against the interfacial pressure II to create an area of interface AA, equal to that required for the molecule to move into. The amount of work required is then equal to

lo""

IIdA. If the adsorption step is

sufficiently rapid that it takes place while II remains effectively constant, this amount of work is simply IIAA. The rate of adsorption is then given by the number of molecules reaching the interface per unit time multiplied by the fraction having energies in excess of IIAA, i.e.,

dnldt

=

k,C, exp

-

[llAA/kTl

(5)

where k, is the rate constant for adsorption, C , is the bulk concentration of protein, k is Boltzmann's constant, and T the absolute temperature. Equation ( 5 ) assumes that there is no desorption. This is a valid assumption for moderate surface pressures (below 15 mN m-l) as discussed in Section V,C. Taking logarithms of both sides of Eq. ( 5 )and plotting log (dn/dt) against II has been found to give straight lines for a number of proteins (MacRitchie and Alexander, 1963b). Values of AA have been calculated from the slopes. When similar plots are made for interfacial processes in the case of simple molecules, values of AA are found to correspond closely with the limiting areas of molecules evaluated by extrapolation of II - A curves to zero II (MacRitchie, 1976a). For the proteins, values of AA are considerably smaller than those expected from the areas of molecules in their interfacial configurations. They fall within relatively narrow limits of 100-175 Az for those proteins studied and show no relation to the molecular size of the protein (Table 111). For example, AA was found to be 135A' for BSA. This protein, from its dimensions in solution (Oncley et al., 1947), has a smallest molecular cross-sectional area of about 1100 hi'. TABLE111

Values of Mf o r Various Proteins"

Protein Myosin Human y-globulin Human albumin Ovalbumin Lysozyme

Concentration (g liter-l)

0.03 0.01 0.02 0.03

0.01

M

(tf*) 145 130 100 175 100

From MacRitchie and Alexander (1963b), reproduced with permission.

Molecular weight x 10-3 600 180 70 44

15

F. MACFUTCHIE

290

From the I1 - A curve of a BSA monolayer, the area per molecule in the surface configuration is of the order of 10,000 k. The conclusion that can be drawn is that only a small portion of the protein molecule needs to enter the interface in order for adsorption to then proceed spontaneously.

C . Electrical Potential Barrier When a protein adsorbs from a solution in which the pH is close to its isoelectric point, the rate of adsorption at mobile interfaces is controlled by the rate of diffusion to the interface and the interfacial pressure barrier. However, when the protein molecule takes on a net electrical charge, an additional barrier to adsorption appears, owing to the electrical potential set up at the interface by the adsorbed protein. In order to adsorb, a charged molecule must do work equal to

qd$,

where q is the charge on the molecule and $ is the electrical potential in the plane of the charged groups at the interface. The rate of adsorption (11, $ constant) is then given b y

dnldt = k,C, exp

-

[(IIAA

+ q+)/kT]

(6) This barrier was investigated by spreading a variety of compounds at the air/water interface in order to vary the electrical potential (MacRitchie and Alexander, 1963~).The rate of adsorption of protein, dissolved in the subphase, was measured at constant surface pressure by the increase of interfacial area with time. As protein adsorbs,

I 5

10

I 15 Time ( m i n )

20

FIG.1. Adsorption of lysozyme plotted as log areavs time. 0,Adsorption into cephalin monolayer; 0 , adsorption into lysozyme monolayer. From MacRitchie and Alexander (1963c), reproduced with permission.

29 1

PROTEINS AT INTERFACES

forming a mixed monolayer, the electrical potential changes, thus causing the adsorption rate to alter. The behavior is illustrated in Fig. 1 for the adsorption of positively charged lysozyme into a negatively charged monolayer of cephalin at a surface pressure of 6 mN m-l. By evaluating the initial slopes of the logA - t graphs, it was possible to obtain relative rates of adsorption into the different monolayers, taking the rate of adsorption into lysozyme itself as 1.0. Approximate values of J, were obtained by measuring [-potentials of oil droplets to which the monolayer substances were adsorbed under identical conditions to those used in the adsorption experiments (Hartley and Roe, 1940). The adsorption and 5-potential data are summarized in Table IV. Equation (6) was tested by plotting the logarithm of the adsorption rate against the 5-potential. An approximately linear relation was found and an effective charge, q , of + 1.1 electronic units was calculated from the slope. Lysozyme, under the conditions of the experiment (pH 6.5, p 0.01) has a net charge of + 9 1 electronic units. There are two reasons why the effective charge of lysozyme in the adsorption step is appreciably less than the net charge. First, it will be less owing to the concentration of negatively charged counterions near the surface of the molecule, so that it is the electrophoretic charge that is possibly more relevant. Second, as has been seen, only a small portion of the molecule needs to penetrate the interface to create the transition complex for adsorption. Because of the large size of protein molecules relative to the range of electrical forces, only that part of the molecule near the point of contact with the interface will be involved in the activation step. Similar measurements have been made by Arnold and Pak (1962) for the adsorption of proteins into films of other proteins. In some

*

TABLEIV Relatiue Initial Rates of Adsorption of Lysozyme into Different Monolayersa

I-Potential Monolayer

Rate

(mv)

Cephalin Polyglutamic acid Pepsin Serum albumin Octadecanol Trypsin Lysozyme Polylysine

34.5

-68.1

13.2 5.4 4.5 5.0

-51.5 -16.7 -14.1 Not measured

+

3.0

~

~

~

~~~~~

3.2

+ 12.2

(1.0) 0.3

+38.5 ~~~

~~~

~~~

From MacRitchie and Alexander (1963c), reproduced with permission.

292

F. MACRITCHIE

cases, the results are somewhat contrary to those expected from the isoelectric points of the three proteins studied, namely a-globulin, bovine albumin, and insulin. However, 9 will depend on the isoelectric point of the protein in the adsorbed state, and this may not be the same as that in the solution.

D. Transition State in Adsorption Adsorption at a fluid/fluid interface is pictured to occur as follows. The protein molecule, in aqueous solution, since it contains roughly equal numbers of polar and nonpolar side chains, will try to form a micellelike structure; i.e., with nonpolar residues interacting within the molecule and polar ones at the periphery. The resemblance to a micelle will, however, be only approximate owing to steric restrictions on chain folding and disrupting effects of segmental thermal motion. On reaching an interface, the molecule now has the opportunity to lower the free energy of the system by orienting its nonpolar residues toward the nonaqueous phase. The area, AA (Section III,B), is analogous to the critical radius in nucleation theory. Molecules having sufficient energy to penetrate the interface, but only enough to clear holes smaller than AA, are unstable and return to solution. Once an area of AA has been cleared by a molecule, it becomes anchored in the interface and its area grows spontaneously until the whole molecule has unfolded into its interfacial configuration. Since the side-chain composition varies over the periphery of the dissolved protein molecule, values of AA, calculated from adsorption experiments, represent average values for all orientations of the adsorbing molecule. Variations in composition might b e expected to lead to different AA values for different proteins. Insufficient data are available to verify this, but it has been shown that the magnitude of AA is sensitive to the conditions. Table V summarizes data for the effects of different conditions on values of AA, calculated for the adsorption of ovalbumin at the air/water interface (F. MacRitchie and N. F. Owens, unpublished results, 1962). An increase of AA occurs with increasing bulk concentration, increasing temperature, and increasing electrical charge. By analogy with nucleation theory, there must be two free-energy contributions, one positive and the other negative, when a protein molecule penetrates an interface. When the penetrated area is less than the critical area PA, the positive term outweighs the negative one but is equal to the negative term once an area of AA is cleared. At this point, we can equate the two terms

AA (7.3 -

Yb) =

G,* - G,

(7)

PROTEINS AT INTERFACES

293

TABLEV Variation of M with Concentration, Temperature, and p H f o r Adsorption of Ovalbumin"

a

Conc. (g liter-')

Temperature ("C)

PH

(AZ)

0.001 0.01 0.10 1.00 1.00 10.00 10.00 10.00 40.00

20 20 20 20 20 20 5 20 20

6.8 6.8 6.8 6.8 10.2 6.8 6.8 10.2 6.8

75 65 70 100 200 175 150 220 210

AA

From F. MacRitchie and N. F. Owens (unpublished results, 1962).

where ya is the initial interfacial free energy, yb the interfacial free energy between nonpolar groups of the protein and the nonpolar phase, G,* and G , are the free energies of the protein molecule in the activated state and in solution, respectively. The term on the left-hand side will be negative since the polar/nonpolar interactions are replaced by interactions between nonpolar groups of the protein and the nonpolar phase. The term on the right-hand side is a positive contribution. It will probably be made up largely of a negative entropic term resulting from some loss of translational freedom. Increase of temperature should decrease ya and increase G,*,both effects leading to a larger value for AA. Increase of charge causes an increase of AA, possibly by decreasing G,. The increasing values of AA as the bulk concentration is increased above a certain value must arise from an increase in the term on the right-hand side of Eq. (7). Providing protein molecules are relatively uncharged, their free energies will be decreased when surrounded by neighboring molecules. On diffusing to the interface, their free energies increase owing to the reduced interaction with neighbours. I v . CONFORMATION OF PROTEIN MOLECULES AT INTERFACES

A. Interpretation of Interfacial Pressure, Potential, and Viscosity At fluid/fluid interfaces, it is well established that proteins lose their tertiary structure. Measurements of II - A isotherms give areas that can be ascribed to unfolded polypeptide chains; e.g., limiting

294

F. MACRITCHIE

areas are invariably close to 1 m2 mg-' for all proteins that have been quantitatively spread. A number of workers have interpreted their results to mean that two types of film may be formed depending on whether the protein is spread at high areas/molecule and then compressed or spread at constant area against an increasing interfacial pressure (Augenstine et al., 1958; Mussellwhite and Palmer, 1968). An elementary fact that is overlooked is that, when proteins are spread against an interfacial pressure, the resulting barrier to adsorption (Section II1,B) prevents all the protein from becoming adsorbed, so that the areas per molecule, calculated on the assumption of quantitative spreading, are in error. The resulting II - A isotherms appear different although, if correction could be made for loss of protein by dissolution, they would probably be found to coincide. Furthermore, there appears to be no a priori reason why films formed by spreading and those formed by adsorption from an adjacent bulk phase should be fundamentally different. There has been a reluctance to use interfacial pressure measurements to follow adsorption; this does not appear to be warranted except perhaps in cases where interfacial coagulation occurs (Section V1,D). Interfacial pressure is a direct measure of the change of interfacial energy and, as such, is the most fundamental quantity that can be measured at an interface. Conclusions reached about proteins that do not readily unfold are also doubtful. One example is the protein lysozyme. This protein is atypical in that it has an unusually high isoelectric point, close to 11. As a result, the molecule carries a high positive charge at neutral pH so that a high electrical potential barrier to adsorption (Section II1,C) will prevent quantitative spreadiqg unless precautions are taken to minimize the barrier. Where this is done, lysozyme spreads well and behaves similarly to other proteins. Another suggestion that has frequently been made, and which should be criticized, is that protein films may contain unfolded, partly folded, and globular molecules, a concept that ignores certain well established principles of statistical mechanics. The situation in a monolayer at equilibrium is a dynamic one in which each molecule varies its energy about the mean value, the latter determining the stable interfacial pressure. Only if the energy differences between different configurations are small, is it possible to have significant numbers of molecules in configurations different to the average one. On the basis of the model described in Section IV,B in which segments distribute themselves between interface (trains) and bulk phase (loops), energy differences between configurations are relatively large. For example, the work required to remove one segment of area 60 from an interface at an interfacial pressure of 20 mN

w2

PROTEINS AT INTERFACES

295

m-l is about 3 kT. The Maxwell-Boltzmann distribution law tells us that, at a given instant, the ratio of molecules with one segment more, or one less, in the interface to molecules having the average number of segments adsorbed is 1 in 20 at an interfacial pressure of 20 mN m-l. For molecules with two segments more or less than the average, the ratio becomes 1 in 400. Interfacial potential, although not a fundamental quantity like interfacial pressure, can be interpreted to give useful information about the orientation of dipoles in the film. The technique has been used mainly to study spread monolayers. The surface potential of protein monolayers is high at low pH and progressively decreases with increasing pH (Harrap, 1954). The pH dependence has been shown to be due to contributions from ionogenic side chains (Glazer and Dogan, 1953). At pH values near the isoelectric point, the surface potential fluctuates wildly at high areas per molecule as the electrode is moved across the surface (Cockbain and Schulman, 1939; Harrap, 1954), indicating a monolayer phase change. The very low surface potentials at areas higher than the range over which instability is observed, suggest the presence of a gaseous-type film with side chains possibly lying flat at the interface. On compression to higher surface pressures, the surface moment of protein monolayers remains effectively constant over a wide range of compression (Hughes and Rideal, 1932). An apparent decrease occurs at high compression, but this is merely caused by displacement of segments from the interface (Section IV,B), not by a change of orientation. Protein monolayers can attain very high interfacial viscosities and elasticities. Moore and Eyring (1938) have developed a theory of interfacial viscosity, based on the theory of absolute reaction rates. In this theory, the flow of a molecule in a monolayer is treated as a movement of flow units, normally molecules, from one equilibrium position to another, passing over an intermediate activation energy barrier. The equation for the interfacial viscosity, qs,which is derived is q, = h / A exp [(AG

+ IIA)/kTl

(8)

AG is the free energy of activation for flow, h is Planck’s constant, and A is the area per flow unit. This theory was applied to the surface viscosity of a number of proteins, measured as a function of surface pressure (MacRitchie, 1970). Plots of log vs against II were found to be linear, enabling AG and A to be evaluated. The data, which are summarized in Table VI, show that the flow unit for all the proteins and poly-DL-alanine is a segment of approximately 6-8 amino acid residues and also that the free energy of activation for flow is similar for all

296

F. MACRITCHIE TABLEVI Calculated Values of A and AG for Proteins"

~~

Protein

Molecular weight x 10"

(A*)

AG (kJ per mole of segment-')

Poly-DL-alanine y-Globulin Pepsin Serum albumin Lysozyme

1.5 160 34 70 15

105 110 120 100 115

69 69 68 66 65

A

From MacRitchie (1970). Reprinted from J . Macromol. Sci.-Chem. by courtesy of Marcel Dekker. Inc.

proteins under the conditions studied. This agrees with the conclusions of Joly (1948) and is consistent with the bulk viscosity behavior of flexible polymers.

B . Configurational Changes in Response to Compression and Expansion Studies of polymer adsorption have shown that an adsorbed flexible polymer molecule may be divided into three parts: (1) the trains, which are segments adsorbed at the interface; ( 2 )the loops, which are segments of the chain that extend into the adjacent bulk phase(s); (3) the tails, the two segments at the two ends of each polymer chain; these generally have lower free energies of adsorption than other segments and thus also tend to extend into the bulk phase. The distribution between trains, loops, and tails depends on the free energy of adsorption of segments of the polymer molecule and the flexibility of the chain. The importance of the tail segments decreases with increase of molecular weight. Most of the information has come from adsorption studies at solid/liquid interfaces, and theoretical treatments have been developed over recent years for polymer adsorption (Hoeve, 1971; Silberberg, 1971). Although most of the important characteristics of polymer adsorption have been accounted for, the great variety of polymers and interfaces make it difficult to arrive at one general theory that covers all situations (Eirich, 1977). One of the advantages of the fluid/fluid type of interface is that the distribution between adsorbed (trains) and displaced segments (loops) may be quantitatively studied by carrying out measurements with a film balance. This approach has been used for spread monolayers of protein (MacRitchie, 1963). The high free energy of adsorption of proteins results in practically all segments being at the inter-

297

PROTEINS AT INTERFACES

face at low interfacial pressures. Compression of a monolayer causes displacement of segments, which is made evident by a decrease of interfacial pressure with time at constant area or alternatively a decrease of area with time at a fixed interfacial pressure. This is an equilibrium process since a constant value for the decrease in area is obtained for a given interfacial pressure and, if the film is now expanded, the return of segments is manifested by an increase of area with time (constant interfacial pressure). The rate of displacement and return of segments are sufficiently slow to enable the kinetics to be measured. Figure 2 shows results for the net rate of displacement of segments for a BSA monolayer at 16 mN m-l. A plot of log [x,/(x, - x)] vs t is linear as required for a reversible first-order process. Here x, is the area at equilibrium and x is the area at any time t. The surface concentration of segments is assumed to be proportional to the interfacial area. IfNi is the number of segments of a molecule that are adsorbed, and Nb is the number that are displaced from the interface at equilibrium, then the distribution should follow the equation

Ni/Nb = exp [(AG - n A A ) / k T ]

(9)

where AG is the free energy difference between absorbed and displaced segments at II = 0. Here, AA is the area occupied at the interface by the unit segment of the displacement process. Plots of Time ( m i n )

2

L

6

8

30

LO

- 30 E -E

$1 5

20

m 0

X

_1

20

10

2

6

10 1L Time ( m i n )

18

FIG.2. Kinetics of displacement of segments from a bovine serum albumin monolayer at 16 mN m-'; x is the change of distance between barriers and is proportional to the change of the area; x, is the value of x at which equilibrium is established. From MacRitchie (1963).

298

F. MACRITCHIE

o.o/

, 10

,

,

20

30

TT (rnNrn-')

FIG.3. Plots of log (Ni/Nb)vs II for protein monolayers; 0, BSA on 25%ammonium sulfate solution at 25°C; 0 ,pepsin on distilled water at 20°C. From data of MacRitchie (1963).

log (Ni/Nb),vs II,made from data for pepsin spread on distilled water at 20°C and BSA on 25% ammonium sulfate solution at 25"C, are shown in Fig. 3. The slopes are similar for the two proteins, leading to values of 63 A2 for AA. The intercepts at II = 0 give values of 5.4 kT and 6.4 kT for AG. The area of 63 hi2 is equivalent to the area occupied at the interface by 3-4 amino acid residues. It seems possible from this that the displacement process might involve the rotation of bonds in the polypeptide chain with formation of helices. For example, the 3.6 residues that participate in one turn of an a-helix would occupy precisely the required area. The mechanism of the process would then involve the breaking of intermolecular hydrogen bonds between adsorbed segments with formation of new intramolecular hydrogen bonds as displaced segments form into helices. More detailed study of this equilibrium process is required to further test this possibility. Distribution between trains and loops in molecules adsorbed at solid/liquid interfaces is also possible and has been shown to occur for flexible polymers. There are some indications that protein molecules at solid/liquid interfaces do not always undergo the drastic conformational changes that occur at fluid/fluid interfaces. At a solid/liquid interface, an adsorbing molecule cannot penetrate the solid phase. Furthermore, adsorption may be confined to sites and thus be localized. Using infrared difference spectroscopy, Morrissey and Stromberg (1974) found a bound fraction (number of carbonyl surface

PROTEINS AT INTERFACES

299

attachments) of 0.11 for prothrombin and BSA adsorbed on silica. The fraction remained constant for all values of the amounts adsorbed, indicating that these proteins underwent no significant changes from their conformations in solution. However, similar measurements by Fenstermaker and Morrissey (1976) showed that for y-globulin and P-lactoglobulin, the fraction of adsorbed C=O groups decreased as the amount adsorbed increased. This behavior is consistent with changes in the distribution of segments brought about by compression of the adsorbed film. C . Measurements of Helix Contents It had been thought that proteins adsorbed at fluid/fluid interfaces lose all secondary as well as tertiary structure. They have been believed to exist in extended configurations with hydrophobic side chains oriented toward the nonaqueous phase and hydrophilic side chains directed toward the aqueous phase. Work in recent years, mainly by Malcolm (1973) has challenged this concept and has accumulated a large amount of evidence which suggests that secondary structure, in particular the a-helix, is retained at the interface. The approach of Malcolm has been principally to remove surface films of a range of polypeptides after compression and examine the removed material by a number of techniques, including hydrogen exchange and infrared spectroscopy. These studies have usually confirmed the presence of the a-helix in the film material. Apart from the experimental evidence, other reasonably sound arguments have been put forward to justify the presence of the helical configuration. For example, the area per molecule is not greatly different to that for an extended chain structure so that either model could be acceptable. Determination of the configuration of a polymer at an interface is a difficult problem and one which is not easily resolved with certainty. The method of compressing films and examining the coagulated material (fibers) after removal is probably sound in the sense that the interfacial configuration at the moment of transformation to a threedimensional structure is preserved. However, by this time, the film has been considerably compressed and much of the molecule, by necessity, will have been expelled from the interface, forming loops in the adjacent bulk phase. It seems probable that these loops would form into helices. Thus, the subsequent experiments might not be confirming that the adsorbed portions (trains) are in fact in the form of helices. A substantial amount of evidence has accumulated which suggests that protein chains have a high degree of flexibility at the interface. This has been discussed by Cheesman and Davies (1954).

300

F. MACRITCHIE

Results from interfacial viscosity, adsorption isotherms, and the adsorbed-displaced segment equilibrium are difficult to reconcile with a rigid helical structure. Of course, most proteins have only a limited amount of their molecules in a helix form even in the so-called native state, and there are sequences of amino acid residues that cannot form into helices. On energetic grounds, a polar/nonpolar interface is a good solvent for proteins. Since nonpolar side chains can interact with the non polar phase and hydrogen bonding can be satisfied intermolecularly, there appears to be little driving force for energy minimization by helix formation. Nevertheless, the opposing views have stimulated the subject of protein conformation at interfaces and should help to clarify the real situation.

D . Reversibility of Conformational Changes During and Following Desorption

The conformational changes that accompany desorption from an interface have direct bearing on the important subject of protein biosynthesis. The evidence suggests that synthesis begins at the NH2 termini of the chains and amino acids are added in a stepwise procedure (Anfinsen and Scheraga, 1975). After chain termination, the protein molecule leaves the site of synthesis and folds into its “native” globular configuration. Just how this latter step occurs has been a fascinating area for conjecture. It is presently believed that the unfolded chain follows the path that leads to a final configuration of lowest free energy for the system. During this process, nucleation sites on the molecule may aid in guiding the chain to its correct unique conformation. A similar situation exists for desorption of protein molecules from interfaces. Experiments demonstrating that proteins may be desorbed and may exchange between interface and bulk solution are mentioned in Section V,C. Whether protein molecules return to a unique configuration in solution on removal from the interface is an intriguing question. Results of experiments designed to test enzymic activity after removal from the interface are conflicting on this point, as discussed in Section VI1,D. It has been shown that solubility and serological activity are recovered after removing spread monolayers of proteins from the interface (Reisner and MacRitchie, 1962). Other evidence comes from vigorous shaking of protein solutions, conditions under which protein molecules must be continuously being interchanged between interface and bulk solution. Providing interfacial coagulation is avoided, many protein solutions can be shaken for long periods without any insoluble protein appearing. I t therefore

PROTEINS AT INTERFACES

30 1

appears that unfolded protein molecules can readily return to soluble folded configurations, Study of the adsorbed-displaced segment equilibrium (Section IV,B) can, in theory, give us useful information on the path that a molecule takes from the unfolded to the folded state. Folding will begin with the more easily displaced segments such as the tail segments, those rich in polar groups and possibly segments that readily form helices. Proteins containing long sequences of nonpolar side chains such as are believed to occur in some transmembrane proteins (Section VILA) are a special case. It seems possible that the nonpolar segments would be displaced preferentially into the nonpolar phase. Such a molecule, containing a distribution of displaced segments in both phases would be extremely difficult to desorb.

v. EQUILIBRIUM ASPECTS OF

ADSORPTION

The question of whether protein adsorption can be considered as an equilibrinm phenomenon has been controversial. The reasons that have led to a widespread belief in the irreversibility of protein adsorption may be summarized as follows. 1. Changes in the surface tension of protein solutions occur over long periods of time and this makes it difficult to know with certainty whether constant values are reached. 2. Globular protein molecules undergo drastic unfolding to give films having the thickness of a single polypeptide chain at the interface. This has often been termed “surface denaturation.” 3. Interfacial films of protein coagulate under certain conditions, the properties of the coagulated protein being quite different from those ofthe original crystalline protein. For example, solubility is lost. 4. Proteins are found either not to desorb or to desorb only with great difficulty from quiescent interfaces. Langmuir and Schaefer (1939) calculated, on the basis of the Gibbs adsorption equation, that compression of a monolayer of protein of molecular weight 35,000 by 15 mN m-l should increase its solubility by a factor of los5. This results from the large area occupied by the molecule at the interface and the resultant large pressure increment of solubility. The failure of protein monolayers to desorb readily on compression was thus taken as an indication of irreversibility. A critical examination of the above four criteria reveal that none is necessarily a proof of irreversibility. The changes in surface tension of protein solutions over long periods can be attributed to slowness in adsorption due to the relatively high activation energy barriers (Sections III,B and 111,C). Slowness in approaching adsorption equilib-

302

F. MACRITCHIE

rium has been observed even for simple compounds (Ward and Tordai, 1952). Because of the relatively large area of interface required by the transition complex, the interfacial pressure barrier becomes quite high for protein molecules, especially at higher interfacial pressures. For example, a protein for which AA is 100 hi2 (Table 111) adsorbing at an interfacial pressure of 20 mN m-l experiences an activation energy barrier (apart from any electrical barrier) of about 5 kT. This means that only one in about 150 of the molecules striking the interface will have sufficient energy to adsorb. Many common surfactants have AA = 20 A2. For these compounds, a barrier of about 1 kT would be encountered at 20 mN m-l so that about one in three of the molecules colliding with the interface would adsorb. The slowness' of adsorption places great demands on the experimenter if he wishes to establish whether constant interfacial pressures or concentrations are reached. Since proteins have diffusion coefficients of the order of ten times less than those of common surface active compounds and have appreciably higher activation energy barriers, strict precautions need to be taken to ensure that measurements are free from effects of surface active impurities. The unfolding that occurs at an interface is the natural result of the protein molecule assuming a configuration of lowest free energy for the system. There is no reason to believe that the original folded configuration, again that of minimum free energy for the system, is not recovered on desorption, always providing no new chemical bonds are formed while in the adsorbed state (Section V1,B). Interfacial coagulation results when the initial interfacial free energy is exceeded (Section V1,D). Since the coagulum is formed by precipitation of unfolded molecules, it has very different properties to the original crystalline protein. However, coagulation is a process that may be superimposed on adsorption but is not directly relevant to the question of reversibiiity of the adsorption process. Difficulty of desorption is a characteristic feature of the adsorption of high polymers. There is no reason to expect that in the transition from a monomer to a high polymer, there should be a point where reversible adsorption changes to irreversible. What appears to occur is that the activation energy barrier to adsorption remains fairly constant as the molecule grows in size but the activation energy barrier to desorption increases and becomes very large as discussed in Section V,C. One of the strongest arguments in favor of protein adsorption being an equilibrium process is that adsorption isotherms have been reported by a number of workers. What this work shows is that stable interfacial pressures are reached and these increase in response to increases of chemical potential in solution.

PROTEINS AT INTERFACES

303

A. Adsorption lsotherms 1. Fluid/Fluid lnterfaces Benhamou and Guastalla (1960) were the first to question the assumption of irreversibility with an analysis of the adsorption of insulin, p-lactoglobulin, and ribonuclease. They investigated whether the Gibbs adsorption equation was obeyed. This basic equation, applicable to reversible adsorption, is firmly based on thermodynamics and has been amply verified experimentally. It can be written in the simple form C, = (l/kT) (dn/dlnCb)

(10)

where C, is the surface concentration of the adsorbed species, Cb the bulk concentration, and II the equilibrium interfacial pressure. Because an adsorbed monolayer consists of closely packed molecules, it generally has a low compressibility so that C, changes only slightly as II is varied. As a result dn/d1nCb is usually found to be approximately constant. The slope of the 11 - lnCb plot can then be used to evaluate C s , the surface concentration in molecules cm-2. If the II C, relations of a protein are determined from a spread monolayer under the same conditions, a comparison can then be made between the two values of C,. By expressing the C, measured from the spread monolayer as g cm-2, Benhamou and Guastalla were able to calculate values for the molecular weights of their proteim from M = C, (g cm-2)/C, (mol ern-"). This procedure has been applied to the adsorption of lauric acid by Ter Minassian Saraga (1955), good agreement being obtained between the calculated (205-210) and true (200) molecular weights. Benhamou and Guastalla found a sensible value for the molecular weight of insulin (SOOO), but the calculated values for p-lactoglobulin (3000) and ribonuclease (6500) were very much smaller than their true values. It was concluded that the adsorption of insulin obeyed the Gibbs equation and was therefore reversible. No conclusions could be inferred regarding the reversibility of the other proteins. By substituting C, = 1/A where A is the area per molecule in the adsorbed monolayer, Eq. (10) can be written as dII/dlnCb

=

kT/A

(11)

A similar equation to Eq. (11)was derived b y Joos (1969) using the statistical mechanical approach of Frisch and Simha (1957) for linear flexible polymer chains. In the equation of Joos, A = u c0where uois the limiting area of one amino acid residue and u is the number of resi-

304

F. MACRITCHIE

dues present at the surface. Table VII summarizes data for adsorption isotherms of proteins in which II - log Cbplots were linear over a certain range of measurements. Values ofA, calculated from Eq. (11) are included in the table. Although there is variation in theA values, the following generalizations may be made: (1)There appears to be no relation between the values ofA and the molecular size of the protein. An average value of 265 A2 is found from all the results. This corresponds to the area occupied by about 15 amino acid residues. (2)The areas are considerably less than the areas per molecule found from the II - A curves of spread monolayers. For example, the molecular area of BSA is about 10,000 A2 in the pressure range studied. It would appear that, at fluid/fluid interfaces, proteins give adsorption isotherms for which interfacial pressure is a linear function of the logarithm of the bulk concentration over appreciable ranges as has been found for simpler compounds. What has not been satisfactorily explained is the reason for the very low values of A. Joos has interTABLEVII Adsorption Data f o r Proteins at FluidlFluid Znterfaces

Protein

Conditions

BSA'

Air/water, pH 4.9, 0.1 N NaCl BSA Air/water, pH 4.9, no salt BSA Air/water, pH 5.5, no salt BSA Heptane/water, pH 4.9, 0.1 N NaCl Pepsin Air/water, pH 1.57 Trypsin Air/water, pH 3.0 a-Chymotrypsin Airlwater, pH 3.0 Insulin Airlwater, 0.01N HCI P-Lactoglobulin Air/water, pH 5.2, 0.01 N salt Lysozyrne Air/water, pH 7 Joos (1969). Gonzalez and MacRitchie (1970). ' Benhamou and Guastalla (1960). Phillips et al. (1975). Bovine serum albumin.

Interfacial pressure range of linearity (mN m-l)

(A')

per segment

Reference

0-20

158

19

a

0-12

227

18

a

14-18

250

20

b

0-30

55

16

a

12-18 9-18 5-12 12-18 12-16

672 209 96 290 330

38 21 14 21 23

a a c c

0-20

140

17

d

AGdkT hA

U

PROTEINS AT INTERFACES

305

preted it to mean that these values represent the area of that part of the molecule which is attached at the surface. This, however, requires the assumption that the structure of the adsorbed film is different to that of a spread monolayer. The reason for the lack of dependence on molecular weight is also not apparent. Possibly the small values ofA reflect the property that each adsorbed molecule is sufficiently flexible to behave as a series of largely independent kinetic units. The adsorption is therefore a function of a segment and is independent of molecular size. 2. SolidlLiquid Znterfaces Traditionally the study of adsorption at solid/liquid interfaces has been experimentally more difficult than at fluid/fluid interfaces. The usual method for obtaining isotherms has been to monitor changes of concentration in the liquid phase by, for example, measuring the absorbance near 278 nm following equilibration and centrifugation of the suspended solid (Bull, 1956; MacRitchie, 1972; Norde, 1976). Apart from the unknown effects of centrifugation on the equilibrium, the interfacial area and changes of interfacial energy are not known precisely as they are at fluidlfluid interfaces. In recent years, however, there has been a great impetus to the study of protein adsorption at solid/liquid interfaces, largely motivated by biomedical problems. More diversified techniques have been applied, which yield a wider range of information and allow measurement of accurate adsorption isotherms at these interfaces. The radiotracer technique and differential infrared spectroscopy have been mentioned (Section 11). Other useful methods are electron microscopy (Eberhart et al., 1977), infrared internal reflection spectroscopy (Lee and Kim, 1974), and ellipsometry (Vroman, 1964). Methods such as ellipsometry measure thicknesses as distinct from interfacial concentrations. Early work (Kemp and Rideal, 1934; Lindau and Rhodius, 1935) showed that the concentration dependence of adsorption followed a Langmuir-type isotherm, and recent studies have confirmed this in many cases (Dillman and Miller, 1973; Brash and Lyman, 1969). Thus the adsorbed film is believed to be monomolecular and its concentration is frequently found to approach a plateau value as the solution concentration is increased, consistent with the saturation of available sites. In addition to the plateau value concentration (capacity), two other quantities, the thickness of the adsorbed film and the initial slope of the isotherm are often used to characterize polymer adsorption. The thickness data supplements interfacial concentrations to give clues about molecular conformation. For proteins, the interfa-

306

F. MACRITCHIE

cia1 concentrations, considered alone, can, in most cases, be rationalized either in terms of adsorbed globular molecules or extended molecules with a fraction of the segments attached to the interface. The initial slope reflects the distribution tendency of a polymer between interface and bulk and therefore can be related to the free energy of adsorption, as discussed in Section V,B. Table VIII contains a summary of selected data in terms of the plateau value of interfacial concentrations for a number of proteins and types of interface where conditions have been well controlled. Although the isotherms often correspond to the Langmuir type, there has been a tendency to doubt the applicability of this model of adsorption because of the apparent irreversibility. Similarly to fluid/fluid interfaces, desorption is usually very difficult at solid/liquid interfaces. However, desorption of albumin from glass and polystyrene in contact with blood plasma has been demonstrated recently (Coleman et al., 1976), and the work of Brash et al. (1974)and Brash and Sam& (1978) leaves little doubt that a dynamic process is occurring in adsorption, whereby there is a turnTABLEVIII Adsorption Data for Proteins at SolidlLiquid Interfaces

Protein

Conditions

BSAa BSA Bovine prothrombin Human albumin Human albumin Rihonuclease Ribonuclease BSA BSA

Glass, pH 4.6 Silica, pH 7.4 Silica, pH 7.4 Polystyrene, pH 7.0 Polystyrene, pH 4.7 Polystyrene, pH 4.0 Polystyrene, pH 7.0 Silica, pH 5.5 Pol yeth y lene/pol ystyrene sulfonic acid, pH 7.4 Polyethylene, pH 7.3 Polyethylene, pH 7.3 Glass, pH 7.3 Glass, pH 7.4

Human fibrinogen Human albumin Human fibrinogen Human albumin a

Bull (1956).

* Morrissey and Stromberg (1974). Norde (1976). MacRitchie (1972). Dillman and Miller (1973). Brash and Davidson (1976). Bovine serum albumin.

Plateau concentration (mg m-Y 4 -5 0.88 4.36 1.1 2.0

Reference

a b

b C

c

0.9

C

1.0 2.3 1.0

C

11.8 2.0 7.4 0.4

d e

f f f f

307

PROTEINS AT INTERFACES

100 80

60 LO

20

.,

20

40

60

80

100

120

Time ( h o u r s )

FIG.4. Turnover at steady state of human albumin adsorbed on polyethylene. 0 , [1251]Albumin, [l3II] albumin. albumin concentration, 0.8 mg m1-I; shear rate, 250 sec-'. 0, 1Z51-albumin; 0 ,1311-albumin: albumin concentration, 2.0 mg ml-l; shear rate, 2500 sec-I. From Brash and Samak (1978), reproduced with permission.

over of protein at the interface under steady-state conditions. The latter workers followed the adsorption on polyethylene tubing by first 1''' to esflowing through a solution of human albumin labeled with tablish steady-state conditions. This solution was subsequently replaced with a solution of the albumin labeled with 1311. The loss of lZ5I and gain of 1311 from the interface was then measured. The results are shown in Fig. 4 for two sets of experimental conditions. They indicate, first, that a constant exchange of protein occurs between interface and solution. Second, the leveling off of the loss and gain curves indicate that there is a fraction of the surface layer which is exchangeable and the remainder is not and that this fraction varies with the conditions. The authors suggest that the behavior may reflect heterogeneity in the binding energies of different sites. Similar conclusions have been reached by Dillman and Miller (1973). They obtained results indicating that adsorption of the proteins BSA, yglobulin, and fibrinogen on to a variety of polymer membranes takes place in two separate and distinct ways, evidently a result of two types of adsorption sites. Apart from those mentioned, other general features of protein adsorption at solid/liquid interfaces are as follows: (1) Adsorption is sensitive to pH, as it is for fluid/fluid interfaces, a maximum usually being observed near the isoelectric point of the protein (Dillman and Miller, 1973; Norde, 1976). (2) Greater adsorption occurs at hydrophobic interfaces than at hydrophilic ones (MacRitchie, 1972; Brash and

F. MACRITCHIE

308

Davidson, 1976) evidently resulting from a higher free energy of adsorption. As a result, desorption occurs more easily from hydrophilic solids. Theories of macromolecular adsorption predict that film thickness should increase as the square root of the molecular weight (Frisch and Simha, 1957), and this has frequently been verified (Stromberg et al., 1965; Rowland and Eirich, 1966). The sparse data for proteins do not permit conclusions regarding molecular weight dependence of film thickness although the limited results in Table VIII indicate that the capacities do appear to be greater for the larger proteins.

B . Thermodynamics of Adsorption The free energy of adsorption can be expressed as where AHad,and ASad,are, respectively, the net changes of enthalpy and entropy in the adsorption process. Each of the three componentsinterface, protein, and solvent-make their contributions to m a d s and ASad,. For adsorption to occur, all contributions must add up to a negative value for AGads,and the larger this negative value, the greater the driving force for adsorption. Doubts concerning the reversibility of the adsorption process for proteins has tended to discourage attempts to evaluate thermodynamic parameters. 1 . FluidIFluid Interfaces The distribution between interface and bulk solution is determined by ci/cb

=

exp (AGadshT)

(13)

where Ci and Cb are the concentrations at the interface and in solution, respectively, and AGad,refers to the free energy per molecule at zero interfacial pressure. To apply this equation, we need to assume a thickness for the interfacial layer in order to express both concentrations in the same units. A value of 10 h; can be taken as the approximate thickness of a protein monolayer, and since the IT - A curves extrapolate to values close to 1.0 mg rnp2at II = 0, the interfacial concenSubstituting this value in Eq. tration of protein becomes 1 g (13) and using the values of Cb found by extrapolation of the linear I3 - log Cb plots for adsorption, it is possible to evaluate AGad,. The values calculated (Table VII) correspond to segments of the molecules, the areas of which are found from the slopes. To take a specific example, the isotherm of BSA for which AA = 250 Az extrapolates to a

PROTEINS AT INTERFACES

309

log C b value of -8.8 ( c b expressed in g cm-2). A value of 20 kT is calculated from Eq. (13) for the free energy of adsorption of the 250 A2 segment. Since a whole BSA molecule occupies an area of 10,000A2 at IT = 0, the total free energy of adsorption will be of the order of 800 kT per molecule. The reason for the absence of desorption from protein monolayers at low values of II is immediately obvious. Lack of data prevents generalizations about factors that affect the magnitude of A G a d s - It does appear, however, that it is larger at oil/water than at air/water interfaces if compared per molecule.

2. SolidlLiquid Interfaces More data are available for solid/liquid interfaces although the diversity of types of interface increases the difficulty of arriving at generalities. Calorimetric measurements have been made to evaluate m a d s . A thorough investigation has been made by Norde (1976), who has measured the enthalpies of adsorption of human albumin and ribonuclease on polystyrene latices over a range of pH, interfacial electrical charge, and temperature. The behavior is complex. The adsorption process is endothermic (AHadspositive) over an intermediate pH range reaching maximum values as high as 4-5 m] m-2 in this region. At pH values on either side of the maxima, the enthalpy decreases, eventually becoming negative. In the region where AH& is positive, there must be a large positive contribution from A s a d s to make AGadsnegative. This is confirmed by the effect of temperature on the initial slopes of the adsorption isotherms. In general, the higher the temperature, the steeper the slope, indicating that entropy gain is the dominant factor governing protein adsorption at this type of interface. From an analysis of the possible factors that contribute to a positive A s a d s , it is concluded that two are of major importance (1)dehydration of hydrophobic parts of the polystyrene surface. (2) structural changes in the adsorbing protein molecules including changes in hydration. At a hydrophilic glass surface, the adsorption of hyman y-globulin was found to be exothermic ( m a d s negative) by Nyilas et al. (1974). Maximum values of 4200 kJ mol-' of y-globulin were measured. The adsorption isotherms were not of the Langmuir type. The interesting experiments of Dillman and Miller (1973), mentioned in Section V,A, may throw light on the reasons for some of the apparently conflicting results obtained for protein adsorption. It was found that the isotherms for adsorption of proteins on a series of polymer membranes could be separated into two distinct isotherms. One, called type 1, was characterized by being easily reversible, hydrophilic, and exother-

310

F. MACRITCHIE

mic with AHad, values of the order of -40 kJ mol-', typical of a condensation process. The other, type 2 adsorption, was characterized as being tightly bound, of a hydrophobic nature, and endothermic with a heat of reaction varying from 20 to 80 kJ mol-', characteristic of chemical bond formation. The heats were estimated from the temperature dependence of the adsorption, not measured directly. It seems clear from the results discussed that adsorption characteristics are different depending on whether adsorption occurs at a hydrophilic or hydrophobic solid. A thermodynamic approach, along the lines of Norde, Nyilas et al., and Dillman and Miller appears to be the best way of increasing knowledge of protein adsorption. C . Reversibility and the Problem of Desorption of Large Molecules

Whereas only a small portion of a protein molecule needs to penetrate the interfacial film for adsorption to occur (Section III,B), desorption necessarily requires that all segments of the molecule leave the interface together. As a result, desorption of large molecules is a process with a very high activation energy. At an air/water interface, proteins can only be removed by compressing monolayers to relatively high interfacial pressures (Adams et al., 1971). Langmuir and Waugh (1940) studied pressure displacement and pressure solubility of proteins. Pressure displacement refers to the reversible displacement of segments discussed in Section IV,B while pressure solubility is essentially desorption. It was found that as the molecular size of insulin was reduced by enzymic digestion, the rate of desorption increased. Rates of desorption into a protein free-subphase at constant interfacial pressure were measured by Gonzalez and MacRitchie (1970)for BSA. The high activation energy barrier was manifested by a very large value for the interfacial resistance at a given interfacial pressure. When a protein molecule adsorbs, an area of interface of the order of 100 Az has to be cleared for adsorption to occur (Section 111,B). It seems reasonable to assume that once an adsorbed molecule has been compressed until its area in the interface, due to pressure displacement of segments, falls below this critical value, it will be unstable in the adsorbed state and will desorb. This transition state for desorption may be reached in two ways: (1)at constant interfacial pressure and total area, by fluctuations in energy of the adsorbed molecules about the mean value, resulting in certain molecules achieving the transition state configuration; (2) by compression of the film, thus increasing the interfacial pressure and decreasing the molecular area until the latter has been reduced to the critical value.

PROTEINS AT INTERFACES

311

In any reversible process, the free-energy difference between two states is independent of the path. Since the compression step, with accompanying displacement of segments, can be camed out reversibly, we can use this procedure to evaluate the free energy difference between a protein molecule in its most probable configuration at a given equilibrium interfacial pressure, II,, and the molecule in its transition-state configuration. This will be equal to

AdII, which

can be evaluated by integration of the equilibrium II - A curve. Here II, is the interfacial pressure at which the area per molecule has decreased to the critical value of about 100 A2. This method was applied to calculate the free energies of activation for desorption of BSA from an air/water interface (MacRitchie, 1977a). The results are summarized in Table IX. The equilibrium II - A curve of BSA from which the calculations were made is shown in Fig. 5. The changes in the calculated activation energies for desorption with increasing interfacial pressure are consistent with the limited data available for rates of desorption and show that desorption can become significant only at higher interfacial pressures. It also follows from the theory that

FIG.5. II - A curve of bovine serum albumin on distilled water at 20°C. Curve on left is equilibrium curve, assuming no desorption. Curve on right is dynamic curve, which would be obtained if no expulsion of segments from interface occurred. From MacRitchie (1977a),reproduced with permission.

F. MACRITCHIE

312

TABLEIX Calculated Free Energies of Activation (AGdes)and Rate Constants f o r Desorption of Monolayers at Different Surface Pressures

n (mN m-l) 0 20 22.4 24.0 25.6 27.2 28.8

AGdes(l (pJmolecule-') 2.6 x 4.3 x 2.6 x 1.7 x 1.0 x 5.8 x 3.6 x

lo-" 10-13 10-13 10-13 10-13 10-14 10-14

AGdes/kT

650 106 64 42 24 14 9

Rate constant for desorptionb (g cm-* sec-1'2 x

0.42 1.83 3.50 7.50 9.17

From MacRitchie (1977a), reproduced with permission.

* Calculated from data of Gonzalez and MacRitchie (1970). molecular size will be an important factor in the desorption of a protein. It is reassuring to note that the increase in free energy required to desorb a BSA molecule at ll = 0 is of the same order of magnitude as the 800 kT estimated for AGadsin Section V,B. VI. REACTIONSAT INTERFACES

A. Effects of High Concentration and Orientation on Reaction Rate There are two principal ways in which interfaces influence chemical reaction. First, reactants may achieve very much higher concentrations at interfaces than in bulk solution if they have amphipathic characteristics. Second, interfaces orient molecules so that the entropy of activation (probability factor) for a reaction may be considerably reduced. Interfacial concentrations of 1 g in adsorbed protein films (Section V,B) are obviously very much greater than can be attained in any bulk solution phase. A direct effect of the high concentrations is that the interfacial pressure and electrical potential may subject molecules to forces not normally experienced in bulk solution. This can have profound effects on reactivity (MacRitchie, 1977b). Assuming a monolayer thickness of 10 di, an interfacial pressure of 10 mN m-l is equivalent to a bulk pressure of 10s mN mP2,or about 100 atmospheres. Havinga (1954), from studies of model systems, has suggested that the electrical potential at the catalytically active patches of the interfaces within the living cell, set u p by regularly arranged charged groups, could b e of the order of 100 or 200 mV. Apart

PROTEINS AT INTERFACES

313

from the effects mentioned, the rather unique energy fields that surround an interface can tend to orient molecules so that strain may be placed on certain bonds, thus causing them to become more susceptible to reaction. This factor is probably more important at unidimensional and zero-dimensional interfaces. Because of the mosaic nature of cell membranes, such interfaces appear to be prevalent in biological systems and would be highly specific just as active centers, their counterpart in nonbiological catalysis (Taylor, 1925), have been shown to be. Chemical reactions in biological systems proceed with a speed and specificity that cannot usually be matched by laboratory methods, even employing extreme conditions. This has led to the view that many reactions occur at the extensive interfaces which result from the microheterogeneous nature of the living cell. Relatively few studies of the reactions of proteins at interfaces have been made, and there appears to be a fertile field for study here.

B . Bonding and Crosslinking Following Exposure of Reactive Groups In addition to the specific action of an interface, which affects chemical reactions of all compounds, proteins are known to undergo a drastic unfolding from the globular molecule in solution to the polypeptide chain at the interface. Consequently, many of the reactive groups buried in the interior of the molecule, where they could either not interact or interact only intramolecularly, may now be exposed. The high concentrations at interfaces promote conditions favorable for these groups to react, and they may do so intermolecularly or with other molecules in solution. The bond which is most numerous in proteins is the keto-imino hydrogen bond. Each amino acid residue can form two of these, but since each hydrogen bond is shared by two residues, the maximum number that can form is equal to the number of residues (neglecting terminal residues). Although comparatively weak, this bond can have a large influence on the properties of proteins because of its frequency and regularity. Other noncovalent bonds that need to be considered are the salt links and hydrophobic bonds. From a study of the surface viscosity of protein monolayers, using polyamino acids for comparison (MacRitchie, 1970), some information was obtained about the intermolecular bonding present. Poly-L-proline, in which no keto-imino hydrogen bonds can form, was the only polyamino acid that did not give a detectable surface viscosity. This suggested that the high surface viscosity of proteins and polypeptides arises from the breaking of these hydrogen bonds causing easier flow of segments relative to one

314

F. MACRITCHIE

another. Surface viscosity of protein monolayers is a maximum near the isoelectric point, falling off on either side. At the isoelectric point, the number of salt links would be a maximum. However, poly-i-lysine and poly-L-glutamic acid, which cannot form salt links alone, gave high surface viscosities in the uncharged states, but ionization caused the surface viscosity to disappear. This behavior suggests that the decrease of surface viscosity with increasing charge is caused by the breaking of hydrogen bonds as a result of the electrical repulsive forces set up in the surface. The role of hydrogen bonds in protein monolayers and their breaking b y electrical forces has been stressed by Miller (1954) and Llopis and Albert (1959). An alternative interpretation of the results is that changes in surface viscosity result from conformational changes. For example, in solution, ionization of polyamino acids brings about the helix-coil transition. The surface viscosity behavior, however, appears to reflect a flexible chain configuration rather than the rigid rodlike properties of a helix. Of greater importance to protein adsorption is the possibility of forming covalent disulfide bonds by the disulfide-sulfhydryl interchange reaction. This reaction has been shown to proceed between unfolded molecules in the bulk state (Halwer, 1954; MacRitchie, 1976b). It would therefore appear that conditions are appropriate for the same reaction at an interface although no studies have been directed toward establishing this possibility. Whether disulfidesulfhydryl interchange occurs in monolayers is important to the question of reversibility of protein adsorption, since such a polymerization would necessarily destroy reversibility. This could be the explanation of at least some of the irreversible effects that have been observed. It may be significant that in membrane proteins, in which crosslinking would not be desirable, disulfide and sulfhydryl groups are virtually absent (Benson, 1968). Crosslinking of protein monolayers by mercuric ion (MacRitchie, 1970)and silicic acid (Miiiones et al., 1973) has been reported. These studies are relevant to poisoning b y heavy-metal ions and to silicosis, effects that seem likely to result from attack on the cell membrane proteins. Crosslinking by mercuric ion was detected by a spectacular increase in surface viscbsity and a decrease in compressibility when a number of proteins (BSA, insulin, ovalbumin, and hemoglobin) were spread on 0.001 M mercuric chloride solution. Poly-DL-danine was unaffected whereas poly-L-lysine and poly-L-glutamic acid were affected in a similar manner to the proteins, indicating that mercuric ion interacts with the ionizable carboxyl and amino groups on the protein side-chains. Silicic acid similarly caused protein monolayers

PROTEINS AT INTERFACES

315

(human albumin, collagen, and gelatin) to become less compressible and behave as rigid gels. The effect was greatest in the isoelectric region and increased with increase of ionic strength. Poly-DL-alanine behaved similarly to the proteins, but only at ionic strengths above 0.5. It was concluded that the interaction is due to the formation of hydrogen bonds between the silanol (SiOH) groups of polymerized silicic acid and the carboxyl, amino, and ketoimino groups of the protein. Crosslinking of collagen by vegetable tannin has also been attributed to hydrogen-bond formation between phenolic hydroxyl groups of the tannin and ketoimide groups of the protein (Ellis and Pankhurst, 1954).

C . lnteractions with Lipids Proteins and lipids are the principal surface-active compounds of nature. In association, they produce the properties required for the regulation of 'reaction rate and permeability in living systems. This has stimulated a large number of studies of mixed protein-lipid films with the hope that such model systems will illuminate the nature of biological components and processes. Unfortunately, the experimental approach in much of the work has been rather unimaginative, and little useful knowledge has emerged. The practice of injecting protein below a lipid film or vice versa and measuring changes in interfacial pressure has too often been interpreted without consideration of the appropriate theoretical treatment, expounded by Fowkes (1961). Attention has been focused in recent times on the fluidity of cell membranes and the lateral diffusion that can occur in the plane of the membrane. An interesting observation, originally due to Schulman and Rideal (1937), is that mixed monolayers of protein and lipid, when compressed, show unusual and relatively large changes in fluidity. As the mixed film is compressed, the surface viscosity at first increases normally but then goes through a maximum, thereafter decreasing. The behavior is reversible. The same effect has been shown for a mixed monolayer of poly-DL-alanine and lecithin (MacRitchie, 1970). This is illustrated in Fig. 6. The lipid molecules are thought to act by disrupting interpeptide hydrogen bonds as their relative field of influence increases with compression. The experimental study of lipid bilayers (Mueller et al., 1962) has been an important innovation, which promises to help in the understanding of membrane structure and function. The incorporation of small peptides and membrane-active antibiotics into planar bilayers containing hydrocarbon solvents has advanced knowledge about the carrier and channel mechanisms of ion transport across membranes

3 16

F. MACRITCHIE

1 1

8

lT

12 (mNrn-’ )

16

20

FIG.6. Log qSvs II for mixed nlonolayers of poly-DL-ahnine and lecithin at pH 5.5. 0, Polyalanine; 0 , polyalanine-lecithin 4:1 by weight; A, polyalanine-lecithin 2:1 by weight. From Macfitchie (1970).

(Haydon and Hladkly, 1972; Eisenman et aZ., 1973) and electrical excitability (Mueller and Rudin, 1969). Further developments may be anticipated when well defined membrane proteins are incorporated into the bilayers.

D. Interfacial Coagulation When protein solutions are shaken, insoluble protein is often seen to separate out (Bull and Neurath, 1937). The coagulation occurs at the interface and may be observed when protein is allowed to adsorb from solution at a quiescent interface (Cumper and Alexander, 1950) or when spread protein monolayers are compressed (Kaplan and Frazer, 1953). This is an interesting type of phase separation in which a three-dimensional coagulum is formed from the twodimensional monolayer, once a certain critical value of the interfacial pressure is exceeded. The concentration of protein in the monolayer when the critical pressure is reached may be thought of as the solubility in the interface under those conditions. When this concentration is exceeded, precipitation occurs. A simple model may help to illustrate how free energy considerations govern the coagulation. A substance will be stable as a monolayer at an interface provided it lowers the initial free energy of the interface, ya,and will continue to dissolve (spread) in the interface until ya = y e where y e is the interfacial free energy in the presence of the monolayer. y e may be interpreted

317

PROTEINS AT INTERFACES

approximately by treating the protein monolayer as a special case of a duplex film (Harkins, 1952) and splitting y e into three terms such that, at equilibrium Ya

= Yb

+ Yab + Hc

(14)

where Yb is the interfacial free energy between the nonpolar face of the monolayer and the nonaqueous phase, Yab is the interfacial free energy between the polar face of the monolayer and the aqueous phase, and II, is the critical coagulation pressure. Every compound has a critical interfacial pressure at a given interface, usually termed the equilibrium spreading pressure, and will precipitate once this is exceeded. Many monolayers may be compressed to higher interfacial pressures where they are supersaturated, but once nucleation occurs, crystallization or condensation causes the pressure to drop to its equilibrium value (Brooks and Alexander, 1962). It is not known whether this can occur for proteins. It can be seen from Eq. (14) that, other things being equal, a lowering of ya produces a lowering of II,. This explains why proteins coagulate more readily at oil/water than at air/water interfaces. Some values for the coagulation pressures of ovalbumin at different interfaces together with estimated values for Yb and Yab are given in Table x. A comparison between the ease of coagulation by shaking of a number of proteins has been carried out by Henson et al. (1970). Several different conditions were used and a measure of the amount of coagulum formed under conditions of vigorous shaking for 1hour was obtained by nephelometry. The results are summarized in Table XI. It can be seen that the amount of coagulation is sensitive to the conditions. Since the isoelectric points of the proteins vary over a fairly large range (e.g., pepsin 2, lysozyme 11),this needs to be taken into account when making comparisons under a fixed set of conditions. However, it does appear that there is a difference in the ease of coagulation TABLEX Coagulation Pressures of Ovalbumin at Different Interfaces' 'Ya

Interface

(mN m-')

Air/water Air/5 M ammonium acetate Heptane/water Benzene/water

72 67.5 50 35

a

K (mN m-')

27 17 16 12

Yb (mN rn-')

35 35 24 13

From MacRitchie and Owens (1969),reproduced with permission.

Yab

(mN m-')

10 15.5 10 10

318

F. MACRITCHIE

between different proteins. Ovalbumin is especially susceptible, and p-lactoglobulin, y-globulin, and hemoglobin also coagulate readily at the air/water interface. There are a number of factors that might be expected to'influence the rate of coagulation by shaking. First, the rate of adsorption and the compressibility of the monolayer determine whether the coagulation pressure is reached during the brief time a given area of interface is exposed during shaking. In addition, the magnitude of the coagulation pressure and also the rate at which coagulation proceeds once the coagulation pressure is exceeded, will both be important. The reason that interfacially coagulated protein is insoluble is that it is formed from unfolded molecules similarly to heat precipitation. A large activation-energy step provides an effective barrier to dissolution (MacRitchie, 1973), although this may be lowered by using solutions in which the protein has a high charge or concentrated urea solutions, allowing coagulated protein to pass into solution. Irreversible changes such as disulfide-sulfhydryl interchange may also be superimposed, as in heat precipitation.

TABLEXI Mean Zncrease in Turbidity on Shaking Protein Solutions" Phosphate buffer, pII 7.0

Acetate buffer, pH 5.5

Water, pH 5.5

Protein

Mean value

SEM

Mean value

P-Lactoglobulin Cytochrome c a,-Casein a-Lactalbumin Bovine serum albumin Pepsin K-Casein p-Casein Lysozyme M yoglobin Hemoglobin y-Globulin Ovalbumin

0.3b 0.4b 0.4b 0.8 0.9 1.0 1.9 1.9 7.0 7.4 31.0 39.0 51.0

0.18 0.24 0.36 0.19 0.20 0.14 0.84 0.35 2.1 2.1 6.6 7.4 3.7

4.5 115.0 22.6 104.0 10.0 118.0 0.03 0.8 0.18 0.2 0.2 0.04 0.16 1.6 0.35 0.5 1.2 0.35 0.6b 0.22 0.8 0.26 7.6 24.4 4.5 0.5 0.13 16.7 1.4 -0.2b 1.5b 0.49 0.3 2.6 8.5 3.9 3.9 0.4 1.4 5.0 1.2 0.13 6.0 1.8 4.9 25.4 9.5 2.1 3.1 28.8 3.3 27.0 29.0 5.0 6.5 54.0 154.0 12.7 109.0 14.0 144.0 16.8

SEM

Mean value

0.1 M KCI pH 5.5

From Henson et al. (1970),reproduced with permission. Change in turbidity not significant at the 5% level.

SEM

Mean value

SEM

PROTEINS A T INTERFACES

VII. PROTEINS

AT

INTERFACES

IN

319

BIOLOGICAL SYSTEMS

The variety of natural, both inorganic and biological, as well as technological processes in which protein adsorption plays a role is so great that it would be pointless to attempt to mention them all. It is proposed to select a few research areas from biology and briefly emphasize how they are developing and how a surface and colloid chemical approach is being applied or could be relevant. It is realized that cellular processes are interconnected and the division of research areas is made only to accentuate certain features. All the physicochemical phenomena that have been treated in previous sections, including adsorption, desorption, changes of conformation and reactivity, and interfacial coagulation may occur in biological systems and the challenge is to translate this knowledge, limited as it is, to the in vivo situation.

A. Biological Membranes The concerted attack on the difficult subject of the structure and function of membranes has been rewarded by significant advances. The presently held general picture of a membrane is a fluid lipid bilayer in which proteins are dispersed (Singer, 1977). The polar groups of the lipids are at the outer surfaces of the membranes, and the nonpolar chains interact within the membrane. The membrane is compositionally asymmetric with respect to both lipid and protein, evidently an important requirement for its specific function. Patient study of the nature and position of the membrane proteins has led to a number of discoveries. The proteins may be divided into two classes, the peripheral and integral proteins, each class differing markedly in composition and properties. Peripheral proteins are soluble and bind specifically to one surface of the membrane. Integral proteins, on the other hand, are proteins that span the membrane, and thus may have one part of their chain in contact with the cytoplasmic fluid and another protruding into the extracellular fluid. Structural analysis of a number of these integral proteins has revealed that they exhibit linear amphipathy; i.e., the linear amino acid sequence consists of a hydrophilic part and a hydrophobic part. One example, which has been well studied, is the glycoprotein glycophorin from the erythrocyte membrane (Tomita and Marchesi, 1975). The polypeptide chain has 131 amino acid residues. The first 80 residues from the amino terminus contain the carbohydrate, covalently bound, and this portion is known to protrude from the external surface of the membrane. This hydrophilic segment is followed by a linear stretch of 22 amino acid residues, none of which are ionic, which is believed to span the

320

F. MACRITCHIE

thickness of the membrane, probably as a single a-helical segment. This hydrophobic part is then followed by a hydrophilic sequence up to the carboxyl terminus of the chain. This is present at the interior surface of the membrane. The existence of membrane-spanning proteins provides possible mechanisms for transmission of signals across the membrane as well as permeation channels for membrane transport (Dutton et aZ., 1976). It would be most informative to study the conformation and behavior of these unusual integral proteins in the form of monolayers at selected interfaces.

B . Cell Surfaces and Recognition Processes It has become clear that much of the behavior of the living cell, including recognition and adhesion functions, is mediated by the cell surface. As a result, intensive studies of cell-surface topology are in progress. A greater understanding of the molecular architecture and function of the cell surface has come about by the use of a class of proteins called lectins, which are mostly found in plants (Sharon, 1977). Lectins are believed to bind specifically, by multiple sites, to sugar groups of glycoproteins and glycolipids at cell membrane surfaces and cause selective aggregation (agglutination) of cells. For example, they can distinguish between red blood cells of various blood groups. They also readily distinguish malignant cells from normal cells by preferentially agglutinating malignant cells. An interesting result is that the total number of lectin molecules bound to the surface is the same whether cells are agglutinated or not. When normal cells are treated with proteolytic enzymes to remove their surface proteins and glycoproteins, they become as sensitive to agglutination by lectins as transformed cells, although the proteolytic treatment does not change the amount of binding by the lectin. One theory that is currently favored is that the membrane of a malignant cell is more fluid than that of a normal cell. This idea arises from the result that lectin molecules, labeled with fluorescent dyes, are found to induce redistribution of the surface receptors and become bunched into clusters (capping) on the surface of malignant cells. This may facilitate the formation of multiple bridging between the cancer cells, thereby producing agglutination. As the study of membrane surface topology proceeds, more facts are emer ing. A correlation between tumorigenicity and the absence of the ce 1-surfaceglycoprotein (LETS) has been reported recently (Chen et al., 1977). Addition of LETS protein partially restores normal morphology, adhesion, and contact inhibition of movement to transformed cells. Using a surface chemical approach, Read et al. (1977) studied the inhibition by sugars to binding of the

!

PROTEINS AT INTERFACES

32 1

lectin concanavalin A to lipid monolayers. I t was found that, for penetration of the lectin to occur, specific receptor carbohydrates were not necessary at low film pressures but were needed at higher film pressures. When foreign particles such as bacteria enter the blood stream, they are removed by cells, located largely in the liver and spleen, which are capable of recognizing them and causing them to enter the cell by a process called phagocytosis. Also, human red blood cells have a lifetime of the order of 100 days, after which they are recognized as different and removed by engulfment. Wilkins (1967),in a paper that is important because of the pioneering approach to a challenging problem, has attempted to point out both the biological and surface chemical problems involved in the recognition of nonnative particles. Two approaches were outlined: (1) detailed analysis of surfaces of bacteria and other cells, known to be recognized as foreign or not, in the hope that certain characteristics would emerge; (2) use of model particles, the surface properties of which are designed and known, and to measure the interactions of these colloids with the living system. Other areas where recognition processes are important are immune reactions, membrane receptors for hormones and drugs (Cuatrecasas and Bennett, 1974), and the general subject of the movement and positioning of cells in embryogenesis (Burger, 1974).

C . Thrombus Formation When blood comes into contact with foreign surfaces, a complex series of events occurs that may ultimately lead to thrombus formation. It is believed that the initial event is the adsorption of a layer of plasma proteins that conditions the surface such that platelet adhesion and coagulation can subsequently take place. Of the plasma proteins, albumin reduces, and fibrinogen greatly enhances, platelet adhesion, and y-globulin activates the release reaction (Packham et al., 1969). With the increasing use of medical implant devices, much effort is being applied to characterize the thrombogenic properties of different polymer surfaces to provide a sound basis for the choice of materials (Lyman et al., 1974). The groups who are active in this area are making fundamental contributions to the understanding of protein adsorption at solid interfaces, some of which have been discussed in Section V,A. It has been shown that platelets begin to adhere to foreign surfaces only after about 30-60 seconds of contact time (Petschek et al., 1968) even with a glass surface, which is one of the strongest procoagulants. In the intervening time, proteins become adsorbed and possibly undergo changes to become adhesive to

322

F. MACRITCHIE

normal platelets. Brash and Uniyal (1978) studied the adsorption of fibrinogen and albumin at surfaces varying in degree of hydrophilicity and thrombogenicity. It was found that all surfaces showed a preference for fibrinogen and the degree of preference correlated with thrombogenic tendency and platelet reactivity. The dynamic character of protein adsorption, shown by Brash and Samak (1978) and illustrated in Fig. 4,is believed by the authors to have two possible consequences: (1) Compositional changes could create time-dependent thrombosis effects. (2) The protein released from the surface may be altered and become available for activating one of the steps in the coagulation process.

D. Immobilized Enzymes Except for extracellular enzymes, most enzyme systems are believed to function at surfaces of membranes or in ordered assemblages within the cell, such as mitochondria. It seems likely that some enzymes remain in an inactive state and become activated by appropriate stimuli. It has also become clear that a catalytically active site of an enzyme may correspond to only a small portion of the molecule in a specific topological arrangement. Considerable progress is now being made in characterizing enzyme topology. To take one example, cytochrome b, is an integral protein of the endoplasmic reticulum. It is believed to have a hydrophobic tail which dissolves in the fluid hydrocarbon region of the membrane bilayer. The catalytically active head, which is separated by a neck portion, is free to interact with other molecules at the membrane surface and with molecules diffusing through the aqueous medium outside the membrane (DePierre and Ernster, 1977). Surface chemical approaches to studying enzymic activity at interfaces are difficult to assess because of the large number of apparently conflicting results. It seems doubtful if testing the activity after removal of coagulated protein can lead to valid conclusions about the effects of adsorption. It does appear, however, that enzymic activity in some cases is greater in a compressed film than in an expanded one (Skou, 1959). Possibly, segments displaced from the interface by compression could form the required active conformation, while the molecule is firmly anchored at the interface by the nonactive adsorbed segments. This is essentially similar to the example of cytochrome bs described.

E . Miscellaneous Topics A surfactant is present in the lung which is believed to play a role in the stability of the alveoli. Its main component is lipid, but protein is

PROTEINS AT INTERFACES

323

also present. Among numerous studies, Blank and Britten (1970) have measured the surface rheological properties of the surfactant and Tabak and Notter (1977) have recently investigated the dynamic surface pressure characteristics of model phospholipid-protein films. Adenosine triphosphatases (ATPases) are enzymes responsible for the maintenance of high K+ inside and high Na+ outside cells. One of the essential properties is the requirement of lipid for their activity. Deleers et al. (1977) have spread mixed ATPase-lipid films at the air/water interface. They used Il - A measurements to characterize the protein in its lipid environment and tritium-hydrogen exchange and surface viscosity to assess the orientation of ATPase and its influence on lipid fluidity. Preliminary studies of the surface pressure and potential of monolayers of the apoprotein A of the Folch-Lees proteolipid, a structural protein involved in the building of myelin in the central nervous system, have been reported by Ter Minassian Saraga (1976). One area that perhaps has not received the attention merited has been synthesis involving peptides at interfaces (Davies, 1954). The concentrating and orienting effects of interfaces, possibly aided by ultraviolet radiation, appear likely to have played a role in both the synthesis of prebiotic protein precursors and the early evolution of organisms.

REFERENCES Adams, D. J., Evans, M. T. A., Mitchell, J. R., Phillips, M. C., and Rees, P. M. (1971). J. Polym. Sci. C34, 167. Alexander, A. E., and Hibberd, G. E. (1971). In “Physical Methods of Chemistry” (A. Weissberger, ed.), Vol. 1, Part V, p. 557. Wiley, New York. Anfinsen, C. B., and Scheraga, H. A. (1975). Ado. Protein Chem. 29,205. Arnold, J. D., and Pak, C. Y. C. (1962). J . Colloid Sci. 17,348. Augenstine, L. G., Ghiron, C. A., and Nims, L. F. (1958). J. Phys. Chem. 62, 1231. Benhamou, N., and Guastalla, J. (1960). J. Chim. Phys. 57, 745. Benson, A. A. (1968). In “Membrane Models and the Formation of Biological Membranes” (L. Bolis and B. A. Bethica, eds.), p. 190. North-Holland Publ., Amsterdam. Blank, M., and Britten, J. S. (1970). J . Colloid Interface Sci. 32, 62. Brash, J . L., and Davidson, V. J. (1976). Thromb. Res. 9,249. Brash, J. L., and Lyman, D. J. (1969). J . Biomed. Mater. Res. 3, 175. Brash, J. L., and Samak, Q. (1978). J. Colloid Interface Sci. (in press). Brash, J. L., and Uniyal, S. (1978). J . Polym. Sci. (in press). Brash, J. L., Uniyal, S., and Samak, Q. (1974). Trans. Am. Soc. Artif. Intern. Organs 20, 69. Brooks, J. H., and Alexander, A. E. (1962). J. Phys. Chem. 66, 1851. Bull, H. B. (1947). Ado. Protein Chem. 3,95. Bull, H. B. (1956). Biochim. Biophys. Acta 19,464. Bull, H. B., and Neurath, H. (1937). J . Biol. Chem. 125, 113. Burger, M. M. (1974). In “Perspectives in Membrane Biology” (S. Estrada-0 and C. Gitler, eds.), p. 509. Academic Press, New York.

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Lyman, D. J., Metcalf, L. C., Albo, D., Richards, K. F., and Lamb, J. (1974). Trans. Am. SOC.Artif. Intern. Organs 20,474. MacRitchie, F. (1963). J . Colloid Sci. 18, 555. MacRitchie, F. (1970). J. Macromol. Sci., Chem. 4, 1169. MacRitchie, F. (1972). J. Colloid Interface Sci. 38,484. MacRitchie, F. (1973). J. Colloid Interface Sci. 45,235. MacRitchie, F. (1976a). J. Colloid Interface Sci. 57,393. MacRitchie, F. (1976b). J. Polym. Sci., Polym. S y m p . 55, 139. MacRitchie, F. (1977a). J. Colloid Interface Sci. 61,223. MacRitchie, F. (1977b). I n “Interfacial Synthesis” ( F . Millich and C. E. Carraher, Jr., eds.), Vol. I, p. 103. Dekker, New York. MacRitchie, F., and Alexander, A. E. (1963a). J. Colloid Sci. 18,453. MacRitchie, F., and Alexander, A. E. (196313). J. Colloid Sci. 18,458. MacRitchie, F., and Alexander, A. E. (1963~).J . Colloid Sci. 18,464. MacRitchie, F., and Owens, N. F. (1969). J. Colloid Interface Sci. 29,66. Malcolm, B. R. (1973). Prog. Surf. Membr. Sci. 7, 183. Miller, I. R. (1954). J. Colloid Sci. 9, 579. Miiiones, J., Garcia Fernandez, S., Iribamegaray, E., and Sanz Pedrero, P. (1973). J. Colloid Interface Sci. 42, 503. Moelwyn-Hughes, E. A. (1947). “The Kinetics of Reactions in Solutions,” 2nd ed. Oxford Univ. Press, London and New York. Moore, W. J., and Eyring, H. (1938). J. Chem. Phys. 6,391. Morrissey, B. W., and Stromberg, R. R. (1974). J. Colloid Interface Sci. 46, 152. Mueller, P., and Rudin, D. 0. (1969). Cum. Top. Bioenerg. 3, 157. Mueller, P., Rudin, D. O., Tien, H. T., and Wescott, W. C. (1962). Nature (London) 194,919. Muramatsu, M. (1973). Surf. Colloid Sci. 6, 101. Mussellwhite, P. R., and Palmer, J. (1968). J. Colloid Interface Sci. 24,310. Norde, W. (1976). “Proteins at Interfaces.” Commun. Agric., Univ. Wageningen, The Netherlands. 76-6. Nyilas, E., Chiu, T.-H., and Herzlinger, G. A. (1974). Trans. A m . SOC. A d i f . Intern. Organs 20,480. Oncley, J. L., Scatchard, G., and Brown, A. (1947). J. Phys. Colloid Chem. 51, 184. Packham, M. A,, Evans, G., Glynn, M. F., and Mustard, J. F. (1969). J . Lab. Clin. Med. 73, 686. Petschek, H., Adamis, D., and Kantrowitz, A. R. (1968). Trans. A m . SOC. Artif. Zntern. Organs 14,256. Phillips, M . C., Evans, M. T. A., Graham, D. E., and Oldani, D. (1975). Colloid Polym. Sci. 253,424. Read, B . D., Demel, R. A,, Wiegandt, H., and Van Deenen, L. L. M. (1977). Biochim. Biophys. Acta 470, 325. Reisner, A. H., and MacRitchie, F. (1962). Aust. J. Biol. Sci. 15,592. Rothen, A. (1947). Adu. Protein Chem. 3, 123. Rowland, F., and Eirich, F. (1966). j . Polym. Sci., Part A-1 4,2401. Schulman, J. H., and Rideal, E. K. (1937). Proc. R. SOC. London, Ser. B 122,29. Sharon, N. (1977). Sci. Am. 236, 108. Silberberg, A. (1971). Pure Appl. Chem. 26,583. Singer, S. J. (1977). J . Colloid Znterface Sci. 58,452. Skou, J. C. (1959). Biochim. Biophys. Acta 31, 1. Stallberg, S., and Teorell, T. (1939). Trans. Faraday SOC. 35, 1413. Stromberg, R. R., Tutas, D. J., and Passaglia, E. (1965). J. Phys. Chem. 69, 3955.

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Tabak, S. A., and Notter, R. H. (1977). J. Colloid Interface Sci. 59,293. Taylor, H. S. (1925). Proc. R . S O C . London, Ser. A 108, 105. Ter Minassian Saraga, L. (1955). /. Chim. Phys. 52, 99, 181. Ter Minassian Saraga, L. (1976). /. CoZloid Interface Sci. 56,412. Tomita, M., and Marchesi, V. J. (1975). Proc. Natl. Acad. Sci. U.S. A. 72,2964. Trurnit, H. J. (1953). Arch. Biochem. Biophys. 47,251. Trumit, H. J. (1954). Arch. Biochem. Biophys. 51, 176. Trurnit, H. J. (1960). J. Colloid Sci. 15, 1. Vroman, L. (1964). Thromb. Diath. Haemorrh. 10,455. Ward, A. F. H., and Tordai, L. (1952). Rec. Trau. Chim. Pays-Bas 71, 572. Wilkins, D. J. (1967). /. Colloid Interface Sci. 25,84.

AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed.

A

Avey, H. P., 32, 73 Avery, 0. T., 6,71 Avrameas, S., 6,69 Awdeh, 2. L., 180,188

Abola, E. E., 32, 33, 34,46, 70 Abu Samra, S., 180,188 Acree, T. E., 244,278 Adams, D. J., 310,323 B Adamis, D., 321,325, Babers, F. H., 6, 71 Adetugbo, K., 64,73 Baglioni, C., 4, 19, 69, 73 Agnello, V., 50, 51, 72 Baldwin, T. O., 168,185 Ahmed, A. I., 197, 209, 210, 211, 212, Ballou, D. P., 152,185 213, 214,215, 216, 217, 218, 219, Balny, C., 103, 104, 107, 117, 124, 125, 230, 235, 238, 239, 240, 241, 242, 126, 135, 138, 139, 141, 148, 153, 243, 244, 245, 271, 273, 274, 275, 154, 155, 156, 159, 160, 161, 162, 278,279,280,281 163, 164, 165, 166, 167, 169, 170, Ainsworth, C. F., 15,71 171, 172, 173, 178, 184,185, 186, Air, G. M., 65, 74 187 Akerlof, G., 86,185 Bardawill, G. J., 147,187 Akesson, A., 150, 177,189 Barnikol, H. W., 52,74 Albert, A., 314,324 Barnstable, C., 18, 74 Albo, D., 321,325 Alexander, A. E., 284,286,287,288, 289, Baron, J., 147,188 Barrell, B. G., 65, 74 290,291,316,317,323,324,325 Barry, C. D., 37, 74 Allen, J. C., 3,69 Bayne-Jones, S., 4,69 Aloni, Y.,65,69 Beam, A. G., 18,69 Alvares, A. P., 152,188 Becker, L., 138, 139,185 Amdur, B. H., 193,279 Becvar, J. E., 168,185 Ames, 8. N., 152,185 Bender, M. L., 174,186 Amzel, H. P., 32, 73 Benhamou, N., 303,304,323 Amzel, L. M., 20, 32,40,45,69, 73, 74 Ben-Na’im, A., 267,279 Ananthanarayanan, V. S., 246, 249, 250, Bennett, V., 321,324 251,252,253,279 Benson, A. A., 314,323 Anderson, B., 51,70 Benyamin, Y.,185 Andersson, K., 185 Berg, D., 7, 72 Andrade, J. D., 306,324 Berget, S. M., 65,69 Anfinsen, C. B., 37,69, 300,323 Berggard, I., 18,69, 73 Ansari, A. A., 22,69 Bernard, O., 67, 74 Antonini, E., 178,185 Bemhard, S. A., 174,486 Apella, E., 22, 23, 45,69 Bienenstock, J., 16, 74 Arnold, J. D., 291,323 Bilofsky, H., 3, 6, 20, 22, 23, 25, 26, 29, Aronoff, S., 244,279 30, 31, 37, 41, 44, 45, 46, 47,48, 50, Ashworth, J . N., 124, 128,186 52, 53, 54, 57, 58, 59, 60, 61, 63, 64, Atwood, A. I., 295, 306,324 65, 66, 67, 72, 75 Augenstine, L. G., 294,323 327

328

AUTHOR INDEX

Blank, M., 323 Blomback, B., 206,281 Blomberg, B., 24,70 Bodmer, W. F., 18, 74 Boyd, W. C., 6, 71 Bode, W., 20, 32, 52,69, 70 Boyse, E. A., 18,69 Brack, C., 65, 67, 74 Brandts, J. F., 78,186 Brash, J. L., 285, 305, 306, 307, 322,323 Braun, D. G., 3, 45, 47,69 Brewer, C. F., 43,69 Britten, J. S., 323 Brooks, J. H., 317,323 Brown, A., 289,325 Brown, N. L., 65, 74 Brown, W. D., 194,200,279 Bruice, T. C., 171, 173,187 Brunori, M., 178,185 Bull, H. B., 283, 305, 306, 316,323 Burger, M. M., 321,323 Butler, P. J. G., 117,186

C

Cameron, D. J., 44,69 Campbell, D. H., 17,72 Cannon, L. E., 111, 44, 61, 72 Cannon, L. E., 3, 25, 71 Cantor, H., 18,69 Capra, J. D., 19, 21, 22, 25, 29, 30, 35, 50, 51, 52, 72 Caravaggio, T., 177,188 Carta-Sorcini, M., 22,69 Casenave, P.-A., 6,69 Caspers, J., 323,324 Castaneda-Agullo, M., 79,186 Cebra, J. J., 31,69 Cecka, J. M., 18, 74 Cesari, I. M., 23, 75 Chan, S. M., 207, 209,210, 214, 216, a79,280 Cheesman, D. F., 284,299,324 Chen, A. B., 320,324 Chen, K. C. S., 45,69 Chen, B. L., 20, 32, 40, 45, 73, 320,324 Chen, T. C., 244,279 Chersi, A., 45,69 Chesebro, B., 31,69 Cheveldayoff, M., 244,279 Childs, R., 6,69

Chilson, 0. P., 183,186 Chiu, T.-H., 309,325 Chothia, C., 77, 129,186 Chou, P. Y.,37,69, 74 Chrambach, A., 179,186 Chuba, J. V., 219, 220, 221, 222, 234, 235,252,279 Ciani, S., 316,324 Cioli, D., 4, 19, 69, 73 Cisar, J. O., 3, 7, 8, 9, 10, 11, 12, 13, 14, 68,69 Cline, T. W., 173,186 Cockbain, E. G., 295,324 Cohen, G. H., 32, 35,44,45,48, 55, 74 Cohn, E. J., 124, 128,186 Cohn, M., 7, 8, 23, 24,69, 72, 75 Coleman, D. L., 306,324 Colman, P. M., 32, 52, 70 Coon, M. J., 147, 152,185,188 Cormier, M. J., 171,186 Costello, L. A,, 183,186 Coulson, A. R., 65, 74 Cowan, N. J., 64,73 Craig, L. C., 18, 71 Crank, J., 286,324 Cross, R. J., 4, 73 Crumpton, M. J., 18, 74 Cuatrecasas, P., 321,324 Cumper, C. W; N., 316,324 Cunningham, B. A., 17, 18, 70, 73 D

David, M. M., 147,187 Davidson, V. J., 306,307,323 Davies, D. R., 18, 20, 32, 35, 37, 40, 41, 43, 44, 45, 46, 48, 55, 70, 73, 74 Davies, J. T., 284, 299, 323,324 Davies, B. J., 143, 144, 145,186 Davis, R. C., 43, 70 Dayhoff, M. E., 2, 27, 45, 49, 67, 70 Debey, P., 85, 138, 141, 148, 153, 154, 155, 156, 159, 160, 161, 162, 163, 164, 165, 166,186 Debrunner, P. G., 156, 157, 158, 166, 187,188 Del Castillo, L. M., 79,186 Deleers, M., 323,324 Delwiche, C. V., 165,187 Demel, R. A., 320,325 DePierre, J. W., 322,324 Determan, H., 175,186

329

AUTHOR INDEX Deutsch, H. F., 32,46,70 DeVries, A. L., 192, 194, 195, 196, 201, 202,203,204,205,206,207, 208, 209, 210, 213, 214, 215, 216, 223, 225, 227,236, 237, 238, 239, 241, 246,247,249,250,251,252,259, 270,271,274,275,279,280,281 Dhar, R., 65,69 Dickerson, R. E., 2, 74 Dillman, W. J., 305, 306, 307, 309,324 Doerr, P., 179,186 Dogan, M. Z., 295,324 Dorner, M., 3, 7, 8, 10, 11, 12, 13, 68,69, 75 Douglas, E. L., 194,280 Douzou, P., 78, 79, 80, 82, 83, 84, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 101, 103, 104, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 132, 135, 136, 137, 140, 152, 153, 156, 159, 160, 161, 162, 163, 164, 165, 166, 167, 169, 170,178, 184,185,186,187,188, 189 Dover, S., 40, 41, 43, 45,46,70 Dower, S. K., 43, 70 Dreyer, W. J., 19, 20, 71 Drysdale, R. G., 222,279 Duane, W., 167,187 Dubin, D. T., 152,185 Duman, J. G., 195, 209, 225, 249, 250, 251,270,275,279,280 Durham, A. C. H., 117,186 Dutton, A., 320,324 Dwek, R. A., 32, 40, 41, 43, 44,45, 46, 68,70, 74 E

Eberhard, A., 167, 168, 170,186,187 Eberhart, R. C., 305,324 Ebisu, S., 14, 15, 68, 75 Edelman, G. M., 3, 4, 16, 17, 18, 70, 73 Edgell, M. H., 65,72 Edman, P., 19, 20.73 Edmundson, A. B., 25,32, 33, 34,46,69, 70, 74 Edsall, J. T., 128,186 Eggers, D. F., Jr., 255,279 Eirich, F. R., 296,308,324,325 Eisen, H. N., 31, 41,44, 71, 73

Eisenberg, D., 192, 268, 272,279 Eisenman, G., 316,324 Ellis, S. C., 315,324 Ely, K. R., 32, 33, 34, 46, 70, 74 Enquist, L. W., 65,72 Epp, O., 32, 52, 70 Erlanger, B. F., 44,69 Ernster, L., 147, 186, 322,324 Estabrook, R. M., 147, 156,188 Eulitz, M., 50, 70 Evans, G., 321,325 Evans, M. T. A,, 285,304, 310,323,325 Eyring, H., 295,325

F Farber, L., 79,186 Farr, A. L., 147,188 Fasman, G. D., 37,69 Fausch, M. D., 32, 46,70 Farnsworth, V., 61,71 Fasman, G. D., 37, 74 Fawcett, J. S., 175,186 Feeney, R. E., 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 219, 220, 221, 222, 223, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 252, 270, 271, 272, 273, 274, 275,278, 279,280, 281 Fehlhammer, H., 20, 32, 52,69, 70 Feizi, T., 6, 51, 52, 69, 70 Feldberg, R., 170,188 Feldman, R. J., 37, 70 Fenstermaker, C., 299,324 Fiddes, J. C., 65,74 Fink, A. L., 174, 175,186 Finkelstein, A. V., 37,74 Finlayson, G. R., 179,186 Flagg, W., 193,280 Fleischman, J., 16,70 Fletcher, N. H., 192, 264, 265,279 Flory, P. J., 257,279 Foust, G. P., 170,188 Fowkes, F. M. 315,324 Franek, F., 21, 31, 70 Frank, H. S., 268,279 Franks, D., 222,279 Franks, F., 192,279

330

AUTHOR INDEX

Franzen, J. S., 129,187 Frater, R., 142,186 Frauenfelder, H., 158,187 Frazer, M. H., 316,324 Freed, R. M., 43, 70 Freed, S., 93, 138, 140,186, 187 Friedland, J., 167, 168,187 Friedman, J., 37, 74 Frisch, H., 303, 308,324 Fudenberg, H. H., 51, 52,70

G Gall, W. E., 17, 70 Gally, J. A., 4, 16, 70 Garcia Fernandez, S., 314,325 Gavish, M., 32,43,44, 70 Gaylor, J. L., 152, 165,187, 188 Geckeler, W., 24,69 Gelzer, J., 7, 70 Gergely, J., 51, 52, 70 Gettins, P., 40,41,43,45,46, 70, 74 Ghiron, C. A., 294,323 Gibson, Q. H., 167, 168, 169, 173,187 Gilbert, W., 65,67, 73, 74 Gillette, J. R., 147,187 Ginsburg, V., 68,75 Giphart, M., 18,74 Girling, R. L., 32, 33, 34, 46, 70, 74 Givol, D., 30,31,32,37,40,41,43,44, 45,46,68,70, 71,73, 74, 75 Glaudemans, C. P. J., 13, 74 Glazer, J., 295,324 Glockner, W. M., 210,279 Glynn, M. F., 321,325 Goebel, W. F., 6, 71 Goetzl, E. J., 30, 71 Goldstein, I. J., 11, 14, 15, 68, 71, 74, 75 Gonzalez, G., 304,310,312,324 Goodfellow, P., 18, 74 Goodman, J. W., 7, 71 Gordon, M. S., 193,279,280 Goring, D. A. I., 273,280 Gomall, A. G., 147,187 Gottlieb, P. O., 17, 70 Graham, D. E., 285,304,325 Gram, T. E., 147,187 Grantham, R., 26,29,71 Gray, W. R., 19,20, 71, 206,279 Green, A. A., 184,187 Greenbaum, E., 158,187 Greene, F. C., 195,279

Greenwood, C., 168, 173,187 Greer, J., 2, 71 Gregory, N. W., 255,279 Griggs, G. C., 194,280 Grollman, A., 43,69 Grossberg, A. L., 48, 71 Gudor, R. C., 320,324 Gueffroy, D. E., 178,187 Guinand, S., 132,187 Gunsalus, A., 167, 168,187 Gunsalus, I. C., 80,83,84,86, 87,89, 90, 91, 92, 93,94,95,96, 108, 111, 117, 118, 120, 122, 123, 156, 157, 158, 160, 162, 166,187,188 Gunsalus-Miguel, A., 167,187 Guschlbauer, W., 274,280 Gustalla, J., 303, 304,323 Gutfreund, H., 79,187 Gutman, A. B., 4,71, 73 Gutman, E. B., 4, 71

H Haber, E., 3, 25,33,44, 61, 71, 72, 74 Hadler, N., 31, 69 Haimovitch, J., 31, 41, 71 Halsey, G. D., Jr., 255,279 Halwer, M., 314,324 Hammel, H. T., 193,280 Hammond, B. R., 79,187 Hardman, K. D., 15,71 Hargens, A. R., 195, 223, 224, 246,279 Harkins, W. D., 317,324 Harrap, B. S., 295,324 Hartley, G. S., 291,324 Hartree, E. F., 82,187 Haschemeyer, A. E. V., 274,280 Hastings, J. W., 167, 168, 169, 170, 171, 172, 173,185,186,187,188 Havinga, E., 312,324 Haydon, D. A,, 316,323 Hemmingsen, E. A,, 194,280 Henderson, D., 256,280 Henry, J. P., 168,187 Henson, A. F., 317, 318,324 Herrick, P. R., 222,279 Herzlinger, G. A., 309,325 Hess, G. P., 174,187 Hew, C. L., 31, 71, 195, 246, 247, 249, 250, 251,252,253,279,280 Heyda, A,, 185

33 1

AUTHOR INDEX Hibberd, G. E., 284,323 Hilschmann, N., 18, 52, 71, 74 Hirst, J. W., 7, 8, 72 Hjerten, S., 141, 142, 143, 176, 177,187, 188 Hladky, S. B., 316,324 Hobbs, P. V., 192, 260,264,280 Hoeve, C. A. J., 296,324 Hofmann, R., 197, 198,223,224,225, 270,275,279 Hood, L., 18, 19, 20, 61, 71, 74 Hooker, S. B., 6, 71 Hopper, J. E., 6,71 Horne, R. A., 192,280 Horowitz, M., 65,69 Howell, L. G., 170,188 Hozumi, N., 65,67,74 Hsia, J. C., 68, 71 Huber, R., 20, 32, 52,69, 70 Hughes, A. H., 295,324 Hughes, W. L., 124, 128, 184,186,187 Hui Bon Hoa, G., 108, 109, 110, 112, 113, 114, 115, 116, 117, 119, 121, 125, 132, 135, 137, 152, 156, 186, 187,188 Hurwitz, E., 31, 41, 71 Huser, H., 3,45, 47, 50, 66,69 Hutchison, C. A., 111, 65, 74 Hwang, P. T. R., 165,188 Hysert, D. W., 169,188

I

Ichikawa, Y., 153,187 Irving, L., 193,280 Irbarnegaray, E., 314,325 Ishimura, Y., 147,188

J Jackson, R., 43, 70 Janin, J., 77, 129,186 Jaton, J.-C., 3, 44, 47, 69, 71 Jeanes, A., 7, 71 Jellinek, H. H. G., 271,280 Jernigan, R. L., 37, 75 Johnston, M. F. M., 44, 71 Joly, M., 296,324 Jones, P., 182,187 Jones, R. W., 7, 71

Joos, P., 303,304,324 Joslin, F. G., 50, 51, 72 K

Kabat, E. A., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 26, 29, 30, 31, 37, 41, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54,57, 58, 59, 60, 61, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75 Kallai, 0. B., 2, 74 Kantrowitz, A. R., 321,325 Kanwisher, J. W., 193,280 Kaplan, J. G., 316,324 Kaplan, N. O., 183,186 Kappas, A., 152,188 Kaufman, J. F., 18,74 Kaufman, S., 77, 127,187 Kauzmann, W., 129,187, 192,268,272, 2 79 Kay, R. L., 102, 105,188 Kehoe, J. M., 19, 22, 29, 30, 35, 51, 52, 69, 72 Keilin, D., 82,187 Kemal, C., 171, 173,187 Kemp, I., 305,324 Khoury, G., 65,69 Kim, S. W., 305,324 Kindt, T. J., 3, 21,45, 69, 74 Klapper, D. G., 50, 52,72 Kley, H.-P., 50,70 Klotz, I. M., 129,187 Klug, A,, 117,186 Kochwa, S., 3, 72 Kohler, G., 64, 72, 73 Kolin, A., 141, 175,188 Komatsu, S. K., 195, 196, 197, 203, 204, 205, 206, 213, 214, 215, 216, 223, 236, 237, 238, 239, 247, 271, 274, 279,280 Konigsberg, W. H., 31,40,43, 71, 73, 74 Koo, P. N., 31,69 Korngold, L., 4, 16, 72 Koshlund, M. E., 16, 72 Krasne, S., 316,324 Krause, R. M., 3,45,69, 72 Kuhn, W., 259,280 Kuhns, W. J., 219,220,221, 222,234, 235, 252,279 Kunkel, H. G., 3, 6, 50, 51, 52,69, 72, 73, 75

332

AUTHOR INDEX

1

Lamb, J., 321,325 Lampert, K., 175,186 Landsteiner, K., 6,72 Langmuir, I., 301, 310,324 Lanni, F., 17, 72 Larroque, C., 103,104, 107, 117, 124, 125, 126, 178,187 Lattman, E. E., 32,70 Lau, S. J., 174,186 h u b , O., 65,69 Lavelle, F., 168,187 Lawrence, S., 154,188 Leder, A., 65,72 Leder, P., 65, 72 Lee, H. S., 210, 211,212, 213,280 Lee, R. G., 305,324 Leivestad, H., 193,280 Leon, M. A., 7, 8,72 Leonard, P. J., 256,280 LePeuch, C., 138, 141, 148, 153, 154, 155, 167, 169, 170,185,186,187 Levine, H., 7, 74 Levitt, M., 37, 74 Liao, J., 3, 7, 8, 9, 10, 11, 12, 13, 14, 68, 69 Licht, A., 43, 74 Lifter, J., 31, 71 Lim, V. I., 37,74 Lin, Y., 195, 209, 210,239, 241, 246, 249, 274,280,281 Lindau, G., 305,324 Linderstr0m-Lang, C. U., 192,281 Lipari, R., 4, 16, 72 Lipscomb, J. D., 156, 157, 158, 166,187, 188 Llopis, J., 314,324 Lowry, 0. H., 147,188 Lu, A. Y. H., 147, 152,188 Lundahl, P., 141, 142, 176, 177,188 Lundblad, A., 7, 8, 72 Luner, S. J., 141, 175,188 Lyman, D. J., 305,321,323,325 M

McCabe, W. J., 182,188 McCapra, F., 169,188 McClellan, V., 4,71 McConnell, D. G., 152,188 McIntire, K. R., 7, 8,72

McKean, D., 61,71 McLaughlin, S., 316,324 McLennan, D. M., 152,188 McMillan, M., 18, 74 MacRitchie, F., 286,287, 288, 289,290, 291,292,293,295,296,297,298, 300,304,305,306,307,310,311, 312, 313,314,315, 316, 317, 318, 324,325 McWhinnie, M. A., 194,280 Mage, R. G., 22,35,44,68,69, 72 Maggert, J. E., 195,246,280 Mair, B. J., 165,188 Maitland, G. C., 129,188 Malcolm, B. R., 299,325 Mann, D. L., 18,74 Marchesi, V. J., 319,326 Marcus, D. M., 43, 51, 69, 72 Margolies, M. N., 3, 25, 33, 44, 61, 71, 72, 74 Marsh, D., 43, 68, 74, 75 Marsh, W. L., 51, 70 Marshall, V., 158, 166,187,188 Mason, B. J., 263,280 Mason, H. S., 152,188 Massa, J., 167,187 Massey, V., 170,188 Mattews, B. W., 37,72 Mathews, R. G., 170,188 Matthyssens, G., 65, 74 Maurel, P., 103, 104, 107, 108, 109, 110, 112, 113, 114, 115, 116, 117, 119, 121, 124, 125, 126, 136, 137, 140, 178,187, 188 Maurer, P. H., 7, 72 Maxam, A. H., 65, 67, 73, 74 Mayhew, S. G., 170,188 Means, G. E., 233,280 Meeks, J. R., 156, 157, 158,187 Meighen, E. A., 167, 168, 173,187, 188 Melin, M., 124, 128,186 Metcalf, L. C., 321,325 Metzger, H., 30, 31,69, 71 Meyerhof, O., 79,188 Michaelides, M. C., 44, 73 Michelson, A. M., 168,187 Migita, S., 4, 73 Miles, L. E. M., 179,186 Miller, H. T., 195, 201, 202,280 Miller, I. F., 305, 306, 307, 309,324 Miller, I. R., 314,325

333

AUTHOR INDEX Milstein, C., 19,20,21, 64,72, 73 Miiiones, J., 314,325 Mitani, F.,152,188 Mitchell, G. W., 167, 168,187, 188 Mitchell, J. R., 310, 317, 318,323, 324 Miyake, A., 4, 74 Miyake, Y., 152,188 Moelwyn-Hughes, E. A., 287,325 Moore, C., 65,69 Moore, D. H., 4,71, 73 Moore, W. J., 295,325 Moms, H. R., 209,210,214,216,280 Momssey, B. W., 286,298, 299, 306, 325,325 Mosca, A., 185 Mosesson, M. W., 320,324 Mueller, P., 315, 316,325 Mulford, D. L., 124, 128,186 Muller, F., 170,188 Miinck, E., 156, 157, 158, 166,187,188 Muramatsu, M., 285,325 Mussellwhite, P. R., 294, 317, 318,324, 325 Mustard, J. F., 321,325

N Nagano, K., 37, 74 Nakashima, Y., 40,73 Nakhleh, E. T., 180,188 Namtvedt, H. J., 158,188 Nashef, A. S., 210,211,212, 213,280 Natvig, J. B., 6, 73 Neal, J. L., 273,280 Nealson, K. H., 167,187 Ne'methy, G., 268,280 Neurath, H., 316,323 Newman, R. A., 210,279 Niall, H. D., 19,20,73 Nicoli, M. Z., 168, 173,185, 188 Nigrelli, R. F., 219, 220,221, 222, 234, 235,252,279 Nims, L. F., 294,323 Nishibayashi, H., 147, 156, 188 Nisonoff, A., 6, 16, 17, 50,69 71, 73 Noller, H., 174,186 Norde, W., 305,306, 307, 309,325 Norman, B., 65,72 Notter, R. H., 323,326 Nyilas, E., 309,325

0 Ohlmeyer, P., 79,188 Oldani, D., 285,304,325 Omura, T., 147, 156,188,189 Oncley, J. L., 289,325 Omstein, L., 144, 145,188 Osserman, E., 4, 73 Osuga, D. T., 194, 195, 197,201,202, 207, 209, 210, 211, 212, 213, 214, 215, 216, 217, 219, 220, 221, 222, 223,230,233,234,235,238,239, 241, 242, 243, 244,245, 246, 247, 248, 249, 252, 271, 273, 274, 275, 278,279,280 Oudin, J., 6, 73 Owens, N. F., 292,293,317,325 P Packham, M. A., 321,325 Padlan, E. A., 20, 26, 27, 29, 30, 32, 35, 37,40,41, 43, 44,45, 46, 48, 50, 70, 73, 74 Pain, R. H., 16, 70, 182,187 Pak, C. Y. C., 291,323 Palade, G. E., 147,186, 188 Palmer, J., 294,325 Panagiotopoulos, N., 33, 34, 70 Pankhurst, K. G. A., 315,324 Pantaloni, C., 132,187 Pappenheimer, A. M. Jr., 15, 73 Pardoe, G. I., 221,281 Parfentiev, I. A., 15, 73 Passaglia, E., 308,325 Pathria, R. K., 256,280 Pecht, I., 37, 40, 41,43,73 Perkins, S. J., 40, 41, 43, 45, 46, 70 Perrella, M., 185 Petermann, M. L., 15, 73 Peterson, J. A,, 147,188 Peterson, P. A., 18, 73 Peterson, T., 80, 83, 84, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 108, 111, 117, 118, 120, 122, 123, 162,189 Petschek, H., 321,324 Petsko, G. A., 152,186 Phillips, D. C., 11, 73 Phillips, M. C., 285, 304, 310,323,325 Phizackerley, R. P., 20, 32,40, 45, 73 Piette, L. H., 68, 71

334

AUTHOR INDEX

Pink, J. R. L., 21,73 Pirotta, V., 65, 67, 74 Plummer, P. L. M., 268,280 Poljak, R. J., 20, 32,40,45,69, 73, 74 Polsky, F. I., 65, 72 Pope, C. G., 15,73 Pople, J. A., 267,280 Poretz, R. D., 11, 71 Porter, R. R., 15, 16,70, 73 Potter, M., 4, 5, 6, 7, S; 9, 12, 13, 14, 20, 22, 32,35,44,45,48, 55, 58,69, 71, 73, 74 Pressman, D., 45, 48,69, 71 Prokop, L. D., 305,324 Ptitsyn, 0. B., 37, 74 Putnam, F. W., 4, 73

Q Quattrocchi, R., 4,73

R Rabinovitch, B. S., 255,279 Radola, B. J., 176, 180,188 Radzimski, G., 45,69 Rakusa-Suszczewski, R., 194,280 Randall, R. J., 147, 188 Raschke, W., 24,69 Raub, W. F., 23,73 Ray, A., 31,69 Raymond, J. A., 195, 227, 246, 249,259, 280 Read, B. D., 320,325 Rees, E. D., 320,324 Rees, P. M., 310,323 Reisner, A. H., 300,325 Rhodius, R., 305,324 Riblet, R., 24,25,69, 75 Rice, S. A., 192,281 Richards, K. F., 321,325 Richards, F. F., 31, 32,43,69, 71, 74 Rideal, E. K., 284, 295, 305, 315, 324,325 Riesen, W. F., 3,45,69 Righetti, P. G., 177,188 Rijke, A. M., 259,271,281 Rilbe, H., 175,188 Rist, C. E., 7, 71 Rivers, M. M., 17, 73 Robson, B., 37, 74 Rockey, J. H., 43,70 Rodbard, D., 179,186

Roe, J. W., 291,324 Rohdewald, M., 79,189 Roholt, 0. A., 45,48,69, 71 Rosebrough, N. J., 147,188 Rosemblatt, M. S., 3, 25, 71 Rosenfield, R. E., 51, 72 Rossi-Bernardi, L., 185 Rothen, A., 283,325 Rowland, F., 308,325 Ruberto, R. G., 165,188 Ruckel, E. R., 11, 74 Rudikoff, S., 20,22,32, 35,44,45,48, 55, 73, 74 Rudin, D. O., 315,316,325 Rutishauser, U., 17, 70 Ruysschaert, J. M., 323,324

s Sack, R., 140,187 Samak, Q., 285,306,307,322,323 Samejima, T., 182,188 Sanders, B. G., 19,20, 71 Sangar, F., 65, 74 Sanz Pedrero, P., 314,325 Sarma, V. R., 18,74 Sassa, S., 152,188 Sato, R., 147, 156,188 Saul, F., 20,32,40,45,69, 73 Saul, F. A., 20, 74 Scatchard, G., 289,325 Schaefer, V. J., 301,324 Schechter, I., 43, 74 Schellman, J. A., 129,188 Scheraga, H. A., 37,69, 268,280, 300, 323 Scherer, J. R., 217, 226, 227, 228,229, 230,231,232,233,234,271,272, 281 Schiffer, M., 32,33, 34, 46, 52, 70, 74 Schlossman, S. F., 7, 74 Scholander, P. F., 193, 195, 246,279, 280 Schonbaum, G. R., 174,186 Schramm, H. J., 32, 70 Schroder, B., 191,281 Schuerch, C., 11,74 Schuller, R., 65, 74 Schulman, J. H., 295,315,324,325 Schulz, G., 37,74 Schuman, M., 170,188 Schwager, P., 32,70 Secher, D. S., 64,73

335

AUTHOR INDEX

Segal, D. M., 32,35,44,45, 48, 55, 70, 74 Seidman, J. G., 65, 72 Shankar Iyer, P. N., 15, 74 Sharon, Pi.,320,325 Sharp, P. A., 65,69 Sharrock, M., 166,188 Shedlovsky, R., 102, 105,188 Sherins, R., 179,186 Shier, W. T., 210,239,241, 274,281 Shiio, H., 273,281 Siddoway, L. A., 18, 74 Siekevitz, P., 147,186, 188 Silberberg, A., 296,325 Silver, J., 18, 74 Silverton, E. W., 18, 32, 35, 44,45,48, 55, 74 Simha, R., 303,308,324 Singer, S. J., 128, 130, 189, 319, 320, 324,325 Sireix, R., 156,186 Sjiiquist, J., 206,281 Skaer, H. Le B., 192,279 Skou, J. C., 322,325 Sligar, S. G., 158,188, 189 Slocombe, P. M., 65, 74 Smith, B. B., 129,188 Smith, J. F., 206,279 Smith, M., 65, 74 Smith, R. N., 194,281 Snary, D., 18,74 Sneath, P. H. A., 26, 29, 74 So, L. L., 11,71 Solms, D. J., 259, 271,281 Somero, G. N., 194,279 Sophianopoulos, A. J., 119,189 Sprenger, I., 221,281 Springer, T. A., 18, 74 Stallberg, S., 284,325 Steigemann, W., 32, 70 Steller, R., 7, 8, 72 Sternlicht, J., 43,69 Strausbach, P. H., 31,41, 71 Streefkirk, D. G., 13, 74 Stringer, C. S., 7, 71 Strittmatter, P., 154,188 Stromberg, R. R., 286,298, 306, 308,325 Strominger, J. L., 18, 74 Strong, L. E., 124, 128,186 Strosberg, A. D., 33,44, 61, 72, 74 Suggett, A., 182,187,271,273,281 Sullivan, P. A., 170,188 Sun, T., 320,324

Sunderland, C. A., 43, 68, 70, 75 Suter, L., 52, 74 Sutton, B., 40,41,43,45, 46, 68, 70, 74, 75 Svensson, H., 141,189 Swanson, R., 2,74 Szabo, G., 316,324 szu, s. c.,37,75 T

Tabak, S. A., 323,326 Takano, T., 2,74 Takesue, S., 147,189 Tanford, C., 79, 121, 149,189, 216, 257, 258,281 Taylor, H. L., 124, 128,186 Taylor, H. S., 313,326 Teorell, T., 284,325 Terhorst, C., 18, 74 Ter Minassian Saraga, L., 287,303, 323, 326 Ternynck, T., 6,69 Terry, W. D., 18, 74 Theorell, H., 150, 177,189 Thompson, M. R., 209,210, 214,216, 280 Thunberg, A. L., 3,45, 74 Tiemeier, D. C., 65, 72 Tien, H. T., 315,325 Tilghman, S. M., 65, 72 Tizard, R., 67, 74 Tomasi, T. B. Jr., 16, 74 Tomimatsu, Y., 217,226, 227, 228, 229, 230,231, 232, 233, 234, 271, 272, 281 Tomita, M., 319,326 Tonegawa, S., 65,67, 74 Tordai, L., 288,302,326 Totter, J., 171,186 Travers, F., 80, 83,84, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 108, 111, 117, 118, 120, 122, 123, 125, 135, 136, 137, 140, 156, 162,186,188, 189 Trurnit, H. J ,284,285,287,326 Tsai, C.-M., 68, 75 Tsuchiya, H. M., 7, 71 Tung, A. S., 50,69 Turnbull, D., 265,281 Tutas, D. J., 308,325 Turvey, J. R., 7, 74 Tyson, C. A., 158,189

336

AUTHOR INDEX

Tzagoloff, A., 152,188

U Uhlenbruck, G., 210,221,279,281 Uniyal, S., 285, 306, 322,323 V

VanDam, L., 193,280 Van Deenen, L. L. M., 320,325 Vandenheede, J. R., 194,200,206,207, 208, 209, 210,214,216,219, 220, 221,222,223,234,235,236,237, 238, 239,240, 241, 252, 274,279, 280,281 Van der Hoeven, T. A., 152,185 van der Scheer, J., 6,72 Varga, J . M., 32, 69 Veeger, C., 152,185 Vesterberg, O., 141,189 Vicari, C., 51, 70 Vonnegut, B., 265,281 Voss, E. W., Jr., 44, 75 Vrana, M., 22, 74 Vroman, L., 305,326

Wiegandt, H., 320,325 Wilchek, M., 31, 41, 71 Wilde, C. D., 64,73 Wilham, C. A., 7, 71 Wilkins, D. J., 321,326 Wilkinson, K. D., 15, 74 Wilkov, M. A., 305,324 Willan, K. J., 68,75 Willan, S., 43,74 Williams, R. C., Jr., 51, 52, 69 Willstatter, R., 79 189 Wilson, D. W., 4 , 6 9 Winchester, R. J., 50, 51, 52,69, 72 Winkelhake, J. L., 44,75 Wissenger, J., 305,324 Wissler, F. C., 16, 73 Woernley, D. L., 16,73 Wohlschlag, D. E., 194, 195, 196,279,281 Wood, C., 14, 15, 68,75 Wright, C., 37,40,41,43, 70, 73 Wu, A., 11, 37,75 Wu, T. T., 3, 5, 6, 17, 20, 21, 22,23, 24, 25, 26, 29, 30, 31, 37, 41, 44, 45, 46, 47, 48, 50,52,53,54,57,58,59,60,61,63, 64,65, 66,67, 72, 74, 75 Wynne, A. M.,?'9,186

W

Wagner, M. L., 121, 149, 177,189 Wain-Hobson, S., 40,41,43,45,46,68, 70, 74, 75 Waldenstrom, J., 4,75 Wall&, P., 206,281 Walters, V., 193,280 Walton, A. G., 261,262,281 Wang, A. C., 51,52,70 Ward, A. F. H., 288, 302,326 Warner, D. T., 269,281 Warner, N. L., 4, 75 Watanabe, S., 52, 74 Waugh, D. F., 310,324 Waxdal, M. J., 17,70 Weber, C., 167,187 Weber, K., 167, 168,187 Weigert, M. C., 7, 8, 11, 23, 24, 25, 37,69, 72, 75 Wen, W. Y., 268,279 Wenzel, J., 192,281 Wescott, W. C., 315,325 Westholm, F. A., 32,46, 70 Whelan, W. J., 7, 74 Whitaker, J. R., 210,211, 212, 213,280

Y

Yamano, T., 153,187 Yang, J. T., 182,188 Yang, Y., 11,71 Yaron, A., 7, 74 Yeh, Y., 214, 215, 216, 217, 219, 226,227, 228,229,230,231,232,233,234,241, 242,243,244,245,271; 272,273,274, 275,278,279,281 Yip, C., 195, 247, 250,252, 253,280 Yonkevich, S. J., 23, 75 Yoshioka, M., 31,71 Young, N. M., 7, 8, 72 Yount, W. J., 3, 75 Yu, C. A., 158, 160,189 Z

Zerner, B., 174,186 Zettlemoyer, A. C., 264,266,276,281 Ziegler-Nicoli, M., 167,187 Ziv, E., 43, 74 Zopf, D. A., 68,75

SUBJECT INDEX A

B

Adsorption, by proteins, kinetics and mechanism of, 286-293 Antarctic fishes, freezing resistance of, 194-195 Antibody(ies) affinity labeling data on, 30-32 combining sites of immunochemistry, 6-15 predictive studies, 3 7 4 5 complementarity of, 1-75 correlation with amino acids in chains, 2 6 3 0 fragments and chains of, 15-18 hypervariable regions in, 21 3 0 statistical analysis of sequences in, 4561 variable regions in, 18 -21 Antifreeze proteins, from fish bloods, 191-281 Antifreeze glycoprotein (AFGP) antilectin activity of, 219-222 carbohydrate structure and properties of, 210-212 chemical composition of, 204-205 chemical derivatives of, 233-246 covalent modification of carbohydrate chain, 238-241 disaccharide side chains of, 273-274 effects on freezing, 222-233 functional additivity of, 273 inactivation of, 24 1-246 mechanism of, 254 -260 methyl group role in, 274 -276 models for, 270-276 from northern cod, 246-249 nucleation and propagation of, 260270 peptide chain shortening, 234 -238 physical properties of, 212-219 preparation and properties of, 198-204 sequence of, 206-210 from sculpin, 219 Antilectin activity, of antifreeze glycoprotein, 219-221

Binary solvents, use in low temperature protein fractionation, 80 Biological membranes, protein interfaces in, 319-320 Bonding, at protein interfaces, 313315 Buffer electrodes, for low-temperature isoelectric focusing, 144 treatment effects on properties of, 235

C Cell surfaces, protein interfaces in, 32032 1 Chromatography, of proteins, at low temperatures, 146-175 Cod, antifreeze protein from, 246-249 Column chromatography, of proteins, at low temperature, 141 -142 Crosslinking, at protein interfaces, 313315 Cytochrome c, isoelectric focusing of, at low temperatures, 180-181

D Density, of proteins, at low temperatures, 82-85 Dimethylformamide-water mixtures, dielectric constant of, 95 Dimethyl sulfoxide-water mixtures, dielectric constant of, 41 E

Electrofocusing tube, for low-temperature isoelectric focusing, 143-144 Electrophoresis, of proteins, at low temperatures, 181-183 Enzymes immobilized, protein interface role in, 321 3 2 2 isolation and purification of, at low temperatures, 156-175 Ethylene-glycol-methanol-water mixture, dielectric constant of, 96 337

338

SUBJECT INDEX

Ethylene glycol-water mixture, dielectric constant of, 89

F Fish bloods antifreeze proteins from, 191-281 antifreeze glycoprotein, 198-219 freezing resistances of, 193-198 freezing temperatures of, 195-198 Flounder, antifreeze protein from, 249251 Fructose, trisaccharides of, 14 G

Generators, low-temperature, for protein fractionation, 135-140 Glycerol-water mixtures, dielectric constant of, 93 Glycoprotein, antifreeze type, see Antifreeze glycoprotein

I lmmunoglobulins fragments and chains of, 15-18 of myeloma, 4, 6-13 Interfaces, proteins at, 2 8 3 3 2 6 Isoelectric focusing, of proteins, at low temperatures, 141-144, 175-181

L Lipids, protein interface interaction with, 3 1 5 3 1 6 Luciferase-flavin complex, isolation of, at low temperatures, 167-175 M

Methanol-water mixture, dielectric constant of, 90 2-Methynyl-2,4-pentandiol-water mixture, dielectric constant of, 91 Myeloma immunoglobulins, 4, 6-13 X-ray crystallography of, 3 2 3 7 Myogloblin, isoelectric focusing, at low temperatures, 178-180

P Polar cod, antifreeze protein from, 248249

Polar fish blood sera, freezing resistances of, 193-198 Polar fishes, freezing resistance of, 195 1,2-Propanediol-water mixture dielectric constant of, 92 Protein fractionation at subzero temperatures, 77 -189 acid-base equilibria, 100-122 applications, 146-185 column chromatography, 140-141 density and viscosity changes, 82-85 dielectric constant variations, 85-99 general principles, 135-140 isoelectric focusing, 141-144 methods, 140-146 physicochemical data, 78 -134 protein dissociation, 129-134 protein titration, 116-122 solubility of salts and solutes, 122129 structural aspects, 78 -134 Proteins adsorption by, 286-293 at interfaces, 283 3 2 6 adsorption isotherms, 3 0 3 3 0 8 adsorption kinetics, 286-293 adsorption thermodynamics, 308310 in biological systems, 319-323 bonding and crosslinking, 313-315 coagulation at, 3 1 6 3 1 8 conformation at, 293 3 0 1 desorption of large molecules, 310312 diffusion to, 286-288 electrical potential barrier, 290-292 equilibrium aspects of, 3013 1 2 fluidlfluid interfaces, 303-305 helix contents and, 299-300 immobilized enzymes and, 321 -322 interfacial pressure barrier, 288-290 reactions at, 312-318 solidfliquid interfaces, 305-308 thrombus formation and, 321-322 transition state in adsorption, 292-293

s Saffron cod, antifreeze protein from, 246 Sculpin, antifreeze protein from, 249

339

SUBJECT INDEX Statistics, use in antibody structure studies, 45-61 Subzero temperatures, protein fractionation at, 77-189

Thermal gradient unit, for low-temperature isoelectric focusing, 143 Thrombus, formation of, protein interface role in, 321322

T

V

Temperatures, subzero, protein fractionation at, 77-189

Viscosity, of proteins, at low temperatures, 82 -85

CONTENTS OF PREVIOUS VOLUMES Volume 1 Lipoproteins ERWINCHARGAFF Structural Proteins of Cell and Tissues FRANCIS0. SCHMITT Some Contributions of Immunology to the Study of Proteins HENRYP. TREFFERS The Interaction between the Alkali Earth Cations, Particularly Calcium, and Proteins DAVIDM. GREENBERC

Terminal Amino Acids in Peptides and Proteins SIDNEYW. Fox The Copper Proteins C. R. DAWSONAND M. F. MALLETTE Mucoids and Glycoproteins KARL MEYER The Reactions of Formaldehyde with Amino Acids and Proteins AND JOHNT. EDSALL DEXTERFRENCH Wheat Gluten M. J. BLISH

The Purification and Properties of Certain Protein Hormones BACONF. CHOW

Protein Denaturation and the Properties of Protein Groups M. L. ANSON

Soybean Protein in Human Nutrition DONALDS. PAYNEAND L. S. STUART

X-Ray Diffraction and Protein Structure L. FANKUCHEN

Nucleoproteins JESSE P. GREENSTEIN The Proteins of Skeletal Muscle KENNETH BAILEY AUTHOR INDEX-SUBJECT

INDEX

Volume 2

AUTHOR INDEX-SUBJECT

INDEX

Volume 3 Transamination and the Integrative Functions of the Dicarboxylic Acids in Nitrogen Metabolism ALEXANDER F. BRAUNSTEIN Ferritin and Apoferritin LEONORMICHAELIS

Adsorption Analysis of Amino Acid Analytical Chemistry of the Proteins Mixtures A. J. P. MARTINAND R. L. M. SYNGE ARNE TISELIUS The Microbiological Assay of Amino Spread Monolayers of Protein Acids HENRYB. BULL ESMONDE. SNELL Films of Protein in Biological Processes The Amino Acid Composition of Food ALEXANDRE ROTHEN Proteins The Chemical Determination of Proteins RICHARDJ. BLOCK PAUL L. KIRK The Relationship of Protein Metabolism Reactions of Native Proteins with to Antibody Production and Resistance Chemical Reagents to Infection ROGERM. HERRIOTT PAUL R. CANNON

340

341

CONTENTS OF PREVIOUS VOLUMES

The Amino Acid Requirements of Man ANTHONY A. ALBANESE

Biological Evaluation of Proteins JAMESB. ALLISON

The Use of Protein and Protein Hydrolyzates for Intravenous Alimentation ROBERTELMAN

Milk Proteins THOMASL. MCMEEKINAND B. DAVIDPOLS

Plant Proteins The Preparation and Criteria of Purity J. W. H. LUGG of the Amino Acids MAXS. DUNNAND LOUIS B. ROCKLAND Synthetic Fibers Made from Proteins HAROLDP. LUNDGREN The Plasma Proteins and Their Fractionation Some Protein-Chemical Aspects of TanJOHN T. EDSALL ning Processes AUTHOR INDEX-SUBJECT INDEX

K. H. GUSTAVSON Proteins, Lipids, and Nucleic Acids in Cell Structures and Functions ALBERT CLAUDE

Volume 4

AUTHOR INDEX-SUBJECT

Protein Gels JOHN D. FERRY

INDEX

The Interactions of Proteins and Synthetic Detergents FRANKW.PUTNAM Proteins of Pathogenic Bacteria A. M. PAPPENHEIMER, JR. The Plasma Proteins in Disease ALEXANDER B. GUTMAN Preparative Electrophoresis and Ionophoresis HARRYSVENSSON Stereochemistry of Amino Acids A. NEUBERGER X-Ray Studies of Amino Acids and Peptides ROBERT B. COREY Heme Proteins JEFFRIESWYMAN,

The Electron Microscopy of Macromolecules RALPH W. G. WYCKOFF Light Scattering in Protein Solutions PAUL DOTY AND JOHN T. EDSALL Poly-c~-Amino Acids EPHRAIMKATCHALSKI Egg Proteins HARRYL. FEVOLD Natural and Artificial Iodoproteins MICHEL JEANROCHEAND RAYMOND Glutamic Acid and Cerebral Function HEINRICHWAELSCH

JR.

AUTHOR INDEX-SUBJECT

Volume 6

INDEX

Volume 5 The Synthesis of Peptides JOSEPH S. FRUTON Amino Acid Composition of Purified Proteins G. R. TRISTRAM

Cross Linkages in Protein Chemistry JOHANBJORKSTEN The Relation of Protein Metabolism to Disease HERBERTPOLLACK AND SEYMOURLIONELHALPERN AUTHOR INDEX-SUBJECT

INDEX

CUMULATIVE SUBJECT INDEX FOR VOLUMES

1-5

342

CONTENTS OF PREVIOUS VOLUMES

Volume 9

Volume 7 The Arrangement of Amino Acids in Proteins F. SANGER The Structure of Collagen Fibrils RICHARDS. BEAR Muscle Contraction and Fibrous Muscle Proteins HANSH. WEBERAND HILDEGARDPORTZEHL The Proteins of the Mammalian Epidermis K. M. RUDALL

The Metabolism of Clycine H. R. V. ARNSTEIN The Digestion of Protein and Nitrogenous Compounds in Ruminants MARGARETI. CHALMERS AND R. L. M. SYNGE The Resolution of Racemic a-Amino Acids JESSE P. CREENSTEIN Naturally Occurring Trypsin Inhibitors M. LASKOWSKI AND M.LASKOWSKI, JR.

Infrared Analysis of the Structure of Amino Acids, Polypeptides, and Proteins G. B. B. M. SUTHERLAND

The Formation, Composition, and Properties of the Keratins WILFRED H. WARD AND HAROLDP. LUNDGREN

Ultraviolet Absorption Spectra of Proteins and Amino Acids C . H. BEAVENAND E. R. HOLIDAY

The Molecular Structure of Simple Substances Related to Proteins SAN-ICHIROMIZUSHIMA

AUTHOR INDEX-SUBJECT

INDEX

Volume 8 Naturally Occurring Peptides E. BRICASAND CL. FROMAGEOT

Protein-Protein Interactions DAVIDF. WAUGH Physiochemical and Biological Aspects of Proteins at Interfaces D. F. CHEESMANAND J. T. UAVIES AUTHOR INDEX-SUBJECT

INDEX

Peptide Bond Formation HENRYBORSOOK Bacteriophages: Nature and Reproduction FRANKW. PUTNAM Assimilation of Amino Acids by GramPositive Bacteria and Some Actions of Antibiotics Thereon ERNESTF. GALE Peanut Protein: Isolation, Composition, and Properties JETT C. ARTHUR,JR. Rotational Brownian Motion and Polarization of the Fluorescence of Solutions CKECORIOWEBER Zone Electrophoresis ARNE TISELIUSAND PER FLODIN AUTHOR INDEX-SUBJECT

INDEX

Volume 10 The Nature of Phosphorus Linkages in Phosphoroproteins GERTRUDEE. PERLMANN Metabolism of the Aromatic Amino Acids C. E. DALGLIESH Hydrogen Ion Equilibria in Native and Denatured Proteins AND JACINTOSTELNHARDT ETHEL M. ZAISER Fish Proteins G. HAMOIR The Sea as a Potential Source of Protein Food LIONEL A. WALFORDAND CHARLESG. WILBER

343

CONTENTS OF PREVIOUS VOLUMES Zinc and Metalloenzymes BERT L. VALLEE AUTHOR INDEX-SUBJECT

INDEX

Volume 1 1 Protein Structure in Relation to Function and Biosynthesis CHRISTIAN B. ANFINSEN AND ROBERT R. REDFIELD Hormones of the Anterior Pituitary Gland. Part I. Growth and Adrenocorticotropic Hormones CHOH HAO LI Column Chromatography of Peptides and Proteins STANFORD MOOREAND WILLIAMH. STEIN Countercurrent Distribution in Protein Chemistry P. VON TAVELAND R. SIGNER Complex Formation between Metallic Cations and Proteins, Peptides, and Amino Acids FRANKR. N. GURDAND PHILIP E. WILCOX Measurement and Interpretation of Diffusion Coefficients of Proteins LOUIS J. COSTING AUTHOR INDEX-SUBJECT INDEX CUMULATIVE SUBJECT INDEX F O R VOLUMES

6-10 Volume 12

and Lactogenic Hormones CHOH HAO Lr The Activation of Zymogens HANS NEURATH The Chemical Nature of Antibodies HENRYC. ISLIKER The Synthesis of Peptides MURRAYGOODMAN AND G. w. KENNER AUTHOR INDEX-SUBJECT

INDEX

Volume 13 The Use of Immunochemical Methods in Studies on Proteins PIERRE GRABAR Protein-Carbohydrate Complexes F. R. BETTELHEIM-JEVONS The Silk Fibrions F. LUCAS,J. T. B. SHAW, S. G. SMITH

AND

Synthesis and Chemical Properties of Poly-a-Amino Acids EPHRAIMKATCHALSKl AND MICHAELSELA AUTHOR INDEX-SUBJECT

INDEX

Volume 14 Some Factors in the Interpretation of Protein Denaturation W. KAUZMANN Zone Electrophoresis in Starch Gels and Its Application to Studies of Serum Proteins 0. SMITHIES

The Fibrinogen-Fibrin Conversion HAROLDA. SCHERAGA AND MICHAELLASKOWSKI, JR.

The Specificity of Protein Biosynthesis MARTHA VAUGHAN AND DANIEL STEINBERG

X-Ray Analysis and Protein Structure F. H. C. CRICKAND J. C. KENDREW

Structural Aspects of Tobacco Mosiac Vims H. FRAENKEL-CONRAT AND L. K. RAMACHANDRAN

The Human Hemoglobins: Their Properties and Genetic Control HARVEYA. ITANO Hormones of the Anterior Pituitary Gland. Part 11. Melanocyte-Stimulating

The Serum Proteins of the Fetus and Young of Some Mammals R. A. KEKWICK

344

CONTENTS OF PREVIOUS VOLUMES

Volume 17

The Sulfur Chemistry of Proteins R. CECIL AND J. R. MCPHEE Biological Properties of Poly-or-Amino Acids MICHAEL SELA AND EPHRAIMKATCHALSKI AUTHOR INDEX-SUBJECT INDEX

The Properties of Proteins in Nonaqueous Solvents S. J. SINGER The Interpretation of Hydrogen Ion Titration Curves of Proteins CHARLESTANFORD Regularities in the Primary Structure of Proteins F. SORM AND B. KEIL

Volume 15 Protamines KURT FELIX

Cross-Linked Dextrans as Molecular Sieves JERKERPORATH

Osmotic Pressure D. W. KUPKE Protein Malnutrition in Man J. c. WATERFLOW, J. CRAVIOTO, JOANM. L. STEPHEN

AND

Reactive Sites and Biological Transport HALVORN. CHRISTENSEN Crystallized Enzymes from the Myogen of Rabbit Skeletal Muscle R. CZOK AND TH. BUCHER

Elastin S. M. PARTRIDGE Ultraviolet Spectra of Proteins and Amino Acids D. B. WETLAUFER AUTHOR INDEX-SUBJECT

INDEX

Volume 18

AUTHOR INDEX-SUBJECT INDEX

Volume 16

Recent Studies on the Structure of Tobacco Mosaic Virus F. ALFRED ANDERER

The Structure of Collagen and Gelatin AND WILLIAMF. HARRINGTON PETER H. VON HIPPEL

Assembly and Stability of the Tobacco Mosaic Virus Particle D. L. D. CASPAR

The Proteins of the Exocrine Pancreas P. DESNUELLEAND M. ROVERY

The Dissociation and Association of Protein Structures F. J. REITHEL

Enzyme Fractionation by Salting Out: A Theoretical Note MALCOLMDIXON AND EDWINC. WEBB Nonenzymatic Methods for the Preferential and Selective Cleavage and Modification of Proteins B. WITKOP The Viscosity of Macromolecules in Relation to Molecular Conformation JEN TSI YANG Optical Rotation and the Conformation of Polypeptides and Proteins PETER URNESAND PAUL DOTY AUTHOR INDEX-SUBJECT

INDEX

The Amino Acid Composition of Some Purified Proteins G . R. TRISTRAMAND R. H. SMITH AUTHOR INDEX-SUBJECT

INDEX

Volume 19 The Hemoglobins G. BRAUNITZER, K. HILSE, V. RUDLOFF, AND N. HILSCHMANN Hemoglobin and Myoglobin ALESSANDRO ROSS FANELLI,ERALDO ANTONINI,AND ANTONIO CAPUTO

345

CONTENTS OF PREVIOUS VOLUMES Linked Functions and Reciprocal Effects in Hemoglobin: A Second Look JEFFRIESWYMAN,JR. Thermodynamic Analysis of Multicomponent Solutions EDWARDF. CASASSA AND HENRYKEISENBERG AUTHOR INDEX-SUBJECT

INDEX

Volume 20 Thrombosthenin, the Contractile Protein from Blood Platelets and Its Relation to Other Contractile Proteins M. BETTEX-GALLAND AND E. F. LUSCHER Hydrolysis of Proteins ROBERTL. HILL The Unusual Links and Cross-Links of Collagen JOHNJ. HARDING

Volume 22 Covalent Labeling of Active Sites S. J. SINGER Milk Proteins H. A. MCKENZIE Crystal Structure Studies of Amino Acids and Peptides RICHARD E. MARSHAND JERRYDONOHUE Crystal Structures of Metal-Peptide Complexes HANSC. FREEMAN AUTHOR INDEX-SUBJECT

INDEX

Volume 23 Relaxation Spectrometry of Biological Systems GORDONG. HAMMES The Preparation of Isomorphous Derivatives C. C. F. BLAKE

The Chemistry of Keratins Protein Denaturation W. G. CREWTHER,R. D. B. FRASER, CHARLESTANFORD F. G. LENNOX,AND H. LINDLEY Conformation of Polypeptides and AUTHOR INDEX-SUBJECT INDEX Proteins G . N. RAMACHANDRAN AND V. SASISEKHARAN

Volume 21 Naturally Occurring Peptides S. G. WALEY

AUTHOR INDEX-SUBJECT

INDEX

Volume 24

Cytochrome c E. MARGOLIASH AND A. SCHECTER

Protein Denaturation Part C. Theoretical Models for the Mechanism of Denaturation Hydrogen Exchange in Proteins CHARLESTANFORD AASE HVIDTAND SIGURD 0. NIELSEN Selective Cleavage and Modification of Selenium Derivatives in Proteins Peptides and Proteins J. JAUMGUI-ADELL T. F. SPANDE,B. WITKOP,Y. DEGANI, AUTHOR INDEX-SUBJECT INDEX AND A. PATCHORNIK CUMULATIVE AUTHOR INDEX FOR VOLUMES

1-21

CUMULATIVE TITLE INDEX FOR

VOLUMES

1-21

Recent Developments in Chemical Modification and Sequential Degradation of Proteins GEORGER. STARK

346

CONTENTS OF PREVIOUS VOLUMES

Partition of Cell Particles and Macromolecules in Polymer Two-Phase Systems PER-AKEALBERTSON Analytical Gel Chromatography of Proteins GARYK. ACKERS Nuclear Magnetic Resonance Spectroscopy of Amino Acids, Peptides, and Proteins G . c . K. ROBERTSAND OLEG JARDETZKY AUTHOR INDEX-SUBJECT

Rate of Conformational Transitions in Biological Macromolecules and Their Analogs HERBEHTMORAWETZ Insulin: The Structure in the Crystal and Its Reflection in Chemistry and Biology TOM BLUNDELL,GUY DODSON, DOROTHYHODGKIN,AND DAN MERCOLA AUTHOR INDEX-SUBJECT

INDEX

INDEX

Volume 27 Volume 25 Carboxypeptidase A: A Protein and an Enzyme FLORANTEA. QUIOCHOAND WILLIAMN . LIPSCOMB The Structure of Papain J. DRENTH,J. N. JANSONIUS, R. KOEKOEK,AND B. G. WOLTHERS Protein Malnutrition in Children: Advances in Knowledge in the Last Ten Years J. c . WATERLOW AND G. A. 0. ALLEYNE The Chemistry and Structure of Collagen WOLFIE TRaus AND KARL A. PIEZ AUTHOR INDEX-SUBJECT

INDEX

Structural Aspects of the Fibrinogen to Fibrin Conversion R. F. DOOLITTLE The Structure and Chemistry of Keratin Fibers J. H. BRADBURY The Elongation Reactions in Protein Synthesis PHILIP LEDER Peptide Chain Termination c. T. CASKEY Structure of Bacterial Ribosomes ROGERA. GARRETTAND H. G. WITTMANN Glycoproteins ROBERTG.

SPIRO

AUTHOR INDEX-SUBJECT

INDEX

Volume 26 Glutamine Synthetase of Escherichia coli: Some Physical and Chemical Properties ANN GINSBURG The History of the Discovery of the Amino Acids. 11. A Review of Amino Acids Described Since 1931 as Components of Native Proteins HUBERTBRADFORDVICKERY Interferons: Physicochemical Properties and Control of Cellular Synthesis MUN H. NG AND JAN VILCEK

Volume 28 Phosphoproteins GEORGETABORSKY The Mechanism of Interaction of Red Cell Organic Phosphates with Hemoglobin

RUTH E. BENESCH AND REINHOLD BENESCH Hydration of Proteins and Polypeptides I. D. KUNTZ,JR., AND w. KAUZMANN

CONTENTS OF PREVIOUS VOLUMES Molecular Orbital Calculations on the Conformation of Amino Acid Residues of Proteins BERNARDPULLMAN AND ALBERTE PULLMAN AUTHOR INDEX-SUBJECT INDEX

347

HAROLDEDELHOCHAND JAMESc. OSBORNE,JR. Membrane Receptors and Hormone Action PEDROCUATRECASAS AND MORLEYD. HOLLENBERG AUTHOR INDEX-SUBJECT INDEX

Volume 29 Energetics of Ligand Binding to Proteins GREGOFUO WEBER Avidin N. MICHAEL GREEN

Volume 31

Carbonyl-Amine Reactions in Protein Chemistry ROBERTE. FEENEY,GUNTER BLANKENHORN, AND HENRYB. F. DIXON Experimental and Theoretical Aspects of Protein Folding C. B. ANFINSEN AND H. A. SCHERAGA AUTHOR INDEX-SUBJECT

INDEX

Volume 30 Repressors AND SUZANNE BOURGEOIS MAGNUS PFAHL

The c-(y-G1utamyl)lysine Crosslink and the Catalytic Role of Transglutaminases J. E. FOLKAND J. S. FINLAYSON Thin Film Dialysis KENT K. STEWART Tobacco Mosaic Virus Protein Aggregation and the Virus Assembly P. JONATHAN, G. BUTLER,AND ANTHONYC. H. DURHAM The Plasma Lipoproteins JAMES c . OSBORNE, JR., AND H. BRYANBREWER,JR. Nerve Growth Factor ALFRED C. SERVER AND E ~ u c M. SHOOTER

Bovine Liver Glutamate Dehydrogenase AUTHOR INDEX - SUBJECT INDEX HENRYK EISENBERG, ROBERTJOSEPHS, CUMULATIVE AUTHOR INDEX FOR AND EMIL REISLER VOLUMES 22-31 The Thermodynamic Basis of the StabilCUMULATIVE TITLE INDEX FOR VOLUMES ity of Proteins, Nucleic Acids, and 2231 Membranes

A

B

C

8

E

O

D 9

F 1

6 2 H 3

1 4

J 5