Structure and Biological Activity of Immunoglobulins

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SYDNEY COHEN AND RODNEY R PORTER

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Department of Immunology. St Mary’s Hospital Medical School. London. Fngland

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I I1. 111.

Introduction ............................................... Physical Studies ............................................. A . Molecular Weight ...................................... B. Electron Microscope Studies ............................. C . Tertiary Structure ...................................... Chemical Properties ........................................ A . Amino Acid Analysis ................................... B Peptide Patterns ....................................... C. Enzymatic Splitting of Immunoglobulins .................... D. Reduction of Immunoglobulins ........................... E. Stability of the Disulfide Bonds in Rabbit IgG .............. F. Structural Relationships of IgG. IgM. and IgA .............. G. Carbohydrate Content of Immunoglobulins ................. H Possibility of Three Types of Peptide Chains ............... I Position of the Antibody-Combining Site ................... J. Heterogeneity of Immunoglobulins ......................... Biological Properties of Immunoglobulins ...................... A. Antigenic Properties .................................... B. Allotypes of Immunoglobulins ............................ C Urinary Excretion of Immunoglobulin Fragments ............ D. Transfer of Antibodies from Mother to Fetus ............... E . Fixation of Antibody to Skin .............................. F. Complement Fixation ................................... G Distribution and Turnover of Immunoglobulins ............. H. Synthesis of Antibodies ................................. Comments ............................................... References ................................................

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287 289 289 290 290 291 291 294 295 297 302 303 30.5 308 309 316 319 319 325 331 332 334 336 338 339 341 342

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1 Introduction

It seems clear that antibody activity is present in three classes of serum proteins . The major component. y2 or 7s y. comprises some 85430% of the total. whereas the second. Y1M. PzM. or 19 S y. has a much higher molecular weight. a higher electrophoretic mobility at pH 8.6, and contains about five times as much carbohydrate as the major component. The third protein in the group. y l A or &A. was not detected until the technique of immunoelectrophoresis was introduced by Grabar and Williams (1953) when it was realized that there was another protein which was antigenically related to 7 S y and 19 S y (Grabar et d.,1956; Heremans et al., 1959) It has been suggested that these proteins be col-

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SYDNEY COHEN AND RODNEY R. PORTER

lectively known as immunoglobulins (Heremans, 1960) and, by analogy with the hemoglobin nomenclature, the 7S, 19s and Y ~ Afractions be referred to as IgG, IgM, and IgA, respectively. Indication of the nature of the constituent peptide chains by a subscript may soon be possible, This terminology will be used throughout this article. The association of antibody activity with IgG and IgM has been recognized for many years (Tiselius and Kabat, 1939; Heidelberger and Pedersen, 1937), but there was some doubt as to whether antibodies were present in IgA. It now seems probable that reagins-the skin-sensitizing antibodies-in human sera are associated with this fraction (Heremans and Vaennan, 1962,; Fireman et al., 1963; Yagi et al., 1963), and Heremans et al. (1963) and Vaerman et al. (1963) showed that IgA prepared from the serum of patients recovering from infection with Brucellu abortus contained antibody activity. IgG and IgM are readily recognized in sera from all species examined, but IgA has only been clearly identified in human serum. However, Schultze (1959) and Heremans (1959) have suggested that the T- or p2-globulin which contains most of the antitoxic activity of serum from a horse strongly immunized with diphtheria or tetanus toxoid (Kekwick and Record, 1941; Van der Scheer et al., 1941) may be the equine equivalent to IgA. This component has a molecular weight of about 150,000, a higher electrophoretic mobility at pH 8.6, and a higher carbohydrate content than IgG from the same serum. Antigenically it is related to IgG and IgM, but is not identical with it and, hence, in all its properties the T-or &!-globulin conforms with IgA. A rather similar protein has been reported present in guinea pig serum after prolonged immunization (Benacerraf et al., 1963; White et d.,1963). No carbohydrate analysis has been made, but from other properties this antibody-containing component also seems likely to be IgA. Rabbit colostrum shows the presence of a component which probably corresponds with IgA, but it was not detected in rabbit serum (Feinstein, 1963). Schwick and Schultze (1961) have drawn attention to the presence, detected by immunoelectrophoresis, of other components in horse antiserum which are antigenically related to IgG. Kunkel and Rockey (1963) have found from ultracentrifuge studies that there are globulins containing anti-red-cell antibodies with sedimentation coefficient &15 S in some human sera and that these may be related to the IgA known to be present. There is, therefore, every prospect of further subdivision of the three main types of immunoglobulin.

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II. Physical Studies

A. MOLECULAR WEIGHT The reported values for the molecular weight of IgG from different species have ranged from 136,000 to 190,000 (Porter, 1960a), but recent measurements favor the lower values. Cammack (1962) estimated the molecular weight of rabbit IgG from measurements of sedimentation rate and diffusion coefficient and found a negative concentration dependence extrapolating to give a value of 137,300. Pain (1963), using similar methods for horse IgG, found no concentration dependence and arrived at a value of 151,000, whereas Marler et aZ. (1964) give 145,000 2 5000 as the molecular weight of rabbit IgG. Small et al. (1963), however, found the molecular weight of rabbit IgG to be 170,000 but this was estimated in solutions containing 6 M guanidine HC1. It was possible that IgG contained a mixture of molecules of differing sizes and that part of the difficulty arose from selection during preparation. However, Pain (1964) could not confirm this. He found that if horse IgG was fractionated on a long Sephadex G-200 column it gave a remarkably symmetrical elution peak except for a barely detectable aggregated component which appeared with the void volume of the column. The molecular weights of the material in the leading and trailing edge of the main peak were the same and both were close to 150,000. Similar results were obtained with rabbit IgG, but the average molecular weight was somewhat lower (140,000), more aggregate was present, and it re-formed when solutions of the main component were allowed to stand for several days at 2°C. It is possible that these small amounts of aggregated material are responsible for the higher molecular weight of IgG reported in earlier papers. Few critical studies have been made of the molecular weight of IgA from normal human serum, as it is very difficult to isolate in appreciable amounts, free of contaminants. Pathological IgA proteins prepared from the serum of patients with myelomatosis may have sedimentation coefficients in the range 7-15 S (Laurell, 1961). Horse IgA (T) has a molecular weight very close to that of IgG (Largier, 1958; Smith and Brown, 1950; Pappenheimer et al., 1940). IgM from horse or human serum has a sedimentation coefficient of 18-20 S and a molecular weight of about 1,OOO,OOO, but smaller amounts of material with a higher sedimentation coefficient are present in most preparations and probably also in the serum from which they were isolated (Kunkel, 1960).

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SYDNEY COHEN AND RODNEY R. PORTER

B. ELECIXON MICROSCOPE STUDIES The shape of antibody molecules has been calculated from hydrodynamic data to be asymmetrical, with a ratio of the long to short axis of 8 or 9 to 1, the dimensions being about 250-300 and 40 A. (Neurath, 1939). Some assumptions have to be made in these calculations and several laboratories have attempted to obtain more direct data from electron microscopy studies. Hall et al. (1959) and Hoglund (1964) used shadow-casting techniques and obtained pictures showing granular moleclues with average dimensions close to those calculated by Neurath. Hoglunds work suggested a dimerlike structure for IgG, whereas IgM appeared as a roughly spherical molecule of diameter about 300 A. Valentine ( 1959), using negative staining, obtained pictures suggesting that rabbit y-globulin molecules were spherical with a diameter of about 70 A., but he now considers that the objects visualized may have been incompletely dissolved particles and that the individual molecules may have been invisible with this technique (Valentine, 1964). Others have photographed antibody bound to tobacco mosaic virus ( Kleczkowski, 1961), human wart virus (Almeida et al., 1963), influenza virus (Lafferty and Oertelis, 1961), and bacterial flagella (Elek et al., 1964). The antibody molecules, in most cases, appeared more elongated than when photographed alone, but the dimensions measured by Almeida et d. (1963) were again close to those calculated by Neurath ( 1939). C. TERTIARY STRUC~URE Jirgensons ( 1958) reported optical rotatory dispersion measurements which suggested that y-globulin contains little or no peptide in a-helical formation. Winkler and Doty (1961) have confirmed this from studies with rabbit y-globulin and the papain digest pieces and have suggested that this may be due to the high content of proline. In an attempt to define the molecular properties of IgG more closely, Edelhoch and collaborators have investigated the changes in a variety of physical parameters brought about by low and high pH and high concentrations of urea or guanidine. The methods used included velocity sedimentation, viscosity, optical rotation, solubility, fluorescence, and ultraviolet spectrophotometry. It was found that, while 8 M urea caused swelling of the molecule, some organization remained and the effect was reversible. Complete disorganization was achieved only in solution of IgG in 8 M urea at pH 11-12 or in solutions of detergent ( trimethyl dodecyl ammonium chloride) at the same pH (Edelhoch et al., 1962; Steiner and Edelhoch, 1962), Similarly, 20 of the 58 tyrosine residues would not

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react with iodine in aqueous solution or in solution in 8.5 M urea at pH 9. If 5.3 M guanidine was used, less than 10 residues were unreactive and there was some evidence that at higher iodine concentration this number could be reduced further (Edelhoch and Schlaff, 1963). Iodination of the easily reactive tyrosine in aqueous solution destroyed the specific affinity of rabbit antithyroglobulin for its antigen as expected from earlier work (Johnson et al., 1960; Grossberg et al., 1962). The unusual stability of IgG in 8 M urea agrees with other observations that, although antibody activity cannot be demonstrated in such a solution, no loss can be demonstrated after the urea has been dialyzed away (Karush, 1958; Nisonoff and Pressman, 1959; Winkler and Doty, 1961). If the antibody is kept in solution in 10 M urea for 5 days, activity is not recovered after dialysis ( Winkler and Doty, 1961), but under such conditions it is possible that reactions, such as that of cyanate with amino groups, occur. More remarkable is the recent demonstration (Buckley et aL, 1963) that after standing in solution of 7.5 M guanidine for 12 hours, papain piece I of rabbit anti-bovine serum albumin will regain its specific affinity for the antigen. Maximum unfolding of the molecule was demonstrated by optical rotation, values being obtained for (m-)365 which were close to those for other completely unfolded molecules. As the authors point out, this ability to regain activity when dialyzed in the absence of antigen is strong evidence that the specific affinity is determined by the covalent structure of the molecule. They argue on statistical grounds that the disuEde bonds present cannot play a major role and, hence, conclude that amino acid sequence must determine antibody specificity. 111. Chemical Properties

A. AMINOACIDANALYSIS Introduction of the automatic amino acid analyzer has led to the publication of accurate analyses of the immunoglobulins from several species, and these are summarized in Table I. The characteristic features that distinguish these figures from those of other proteins are the high values for the hydroxy and dicarboxylic amino acids and, particularly, the proline content which is higher than that of any other globular protein. Analyses of several purified rabbit antibodies have been published (Smith et al., 1955;Fleischer et al., 1961; Askonas et al., 1960), and their amino acid contents were all very close to the figures quoted above for rabbit IgG (Crumpton and Wilkinson, 1963). Since only about 1% of the total molecule may be concerned in the antibody site (Karush, 1962),

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SYDNEY WHEN A M ) RODNEY R. PORTER

it was not surprising that no convincing differences were found even if amino acid sequence does vary with antibody specificity. However, Koshland and Englberger (1963) have now found small differences between rabbit antibodies to an acid hapten (p-azobenzenearsonic acid) and to a basic hapten (pazophenyltrimethylammonium). The antibodies were prepared by dissociation of a specific precipitate and their purity measTABLE I AMINO ACIDANALYSISOF IMMUNOGLOBULINS Amino acid residue (gm.)/lOOgm.protein Rabbit Human Human Horse Horse Amino acid IgGo 1gCa IgMb IgGo IgA (T)o 5.76 Lysine 7.06 4.91 6.77 6.50 Histidine 1.73 2.44 1.98 2.58 2.57 3.02 4.75 3.34 4.02 Arginine 4.42 Aspartic acid 8.08 7.25 7.31 7.77 6.95 Threonine 10.37 8.31 7.13 7.04 6.17 Serine 9.29 8.32 9.13 6.58 9.88 Clutamic acid 11.05 10.27 9.36 11.18 9.92 Proline 6.79 6.40 4.95 6.02 6.52 Glycine 3.68 3.53 3.98 3.37 2.91 Alanine 3.71 3.60 3.28 3.29 3.12 Valine 8.36 5.77 8.14 7.74 7.92 Methionine 1.13 0.78 0.59 0.93 1.02 Isoleucine 3.49 3.14 2.70 2.83 2.16 Leucine 6.73 6.51 6.09 6.63 7.40 5.55 4.93 Tyrosine 6.17 5.44 5.76 Phenylalanine 4.15 3.79 3.47 4.07 3.85 Cystine 2.08 1.90 2.63 2.07 2.30 Tryptophan 2.90 2.63 2.47 2.57 2.47 2.40 2.80 12.30 2.40 4.90 Carbohydrate 101.6 97.4 95.70 97.80 94.80 0 Crumpton and Willrinson (1963). b Chaplin et al. (1965). 0 Weir ( 1964).

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wed by their hapten binding power. Some preparations were from pooled antisera and some from the serum of a single rabbit immunized with both haptens. In the last case the chances of individual variations influencing the result would be reduced, though not necessarily eliminated, as antibodies may be associated with different allotypic groups in one individual (Cell and Kelus, 1962). Multiple analyses were carried out and careful corrections made for all the experimental variables. The results from analysis of the two antibodies prepared from the serum of a single rabbit are given in Table 11. There is close agreement in the

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number of residues per molecule for each amino acid except aspartic acid and arginine. The differences of +4 aspartic acid and -2 arginine for the antibody to the basic hapten are in the direction expected if there are salt linkages in the hapten-antibody combination, as suggested by Grossberg and Pressman ( 1960). In view of the care with which the work was carried out, these results are of great interest, but the authors are cautious about drawing TABLE I1

AMINOAcm ANALYSISOF Two PURIFIED ANTIBODIESISOLATED FROM THE

Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine 0

SAME RABBIT"

Residues/160,000 p. Arsonic Ammonium antibody antibody 69.8 69.4 16.6 16.4 44.7 42.5 106 110 162 162 151 151 125 127 109 110 110 110 81.1 81.4 128 128 13.8 13.5 48.4 46.4 89 91 56.1 56.2 44.3 44.9

Koshland and Englberger (1983).

definite conclusions as to their significance. The difficulty of interpreting the data is shown by the observation that antibodies of a given specificity may have any mobility in the IgG range and yet papain pieces I and I1 have different amino acid compositions related to their mobility (Mandy et UI!., 1963). There is also a variation of the amide content (Feinstein, 1962) and the sialic acid content of whole IgG with charge (Schultze, 1982). Thus, there is a variation in composition unrelated to specificity which is difficult to control. These underlying complexities make it very difficult to get a convincing relation between the properties of the whole molecule and its antibody activity.

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R. PORTER

PEPIlDEPAWS

The demonstration by Ingram (1956) that small differences in amino acid sequence of abnormal human hemoglobins could be detected and identified by the relatively rapid technique of comparing peptide patterns suggested that this method might offer a short cut to deciding whether antibodies of different specificity had the same amino acid sequence or not. With this method the protein is denatured and the disulfide bonds are reduced or oxidized in order to destroy all tertiary structure. An enzymatic digest is prepared (often using trypsin because of the welldefmed specificity) in which all the potentially susceptible bonds are broken. This is difficult to achieve but is essential if reproducible results are to be obtained. The complex mixture of peptides is spread on a sheet of filter paper by electrophoresis in one direction and by chromatography in the other. Such a pattern of peptides can then be revealed by spraying with ninhydrin and other reagents, but Ingram (1961) has stressed the considerable technical difficulties in obtaining satisfactorily clear and reproducible results. With the abnormal hemoglobins it was apparent that, although the majority of the peptides were common to all, one or two were unique to each type. When rabbit antibodies with different specificities were examined, no clear-cut differences could be distinguished (Gitlin and Merler, 1961; Gourvitch et al., 1961b). Reasonably clear patterns were obtained from a tryptic digest of oxidized papain pieces from inert rabbit y-globulin (Seijen and Gruber, 1963) and the number of peptides detected in piece I11 was half that expected, suggesting a duplicate structure. Givol and Sela (1964a) studied digests of reduced rabbit antibodies using several enzymes and reported that after tryptic and chymotryptic digestion of papain pieces I and 11, insoluble, large molecular weight material remained. Pepsin and nagarse gave more complete digestion, and the resulting peptide maps from the digest of two different antibodies were very similar but with differences detectable in a few peptides. Peptide maps of piece I11 were clearer and appeared to be identical whether derived from inert y-globulin or antibody. The most surprising finding to be made with this method has come from a comparison of the peptide patterns of Bence-Jones proteins of different antigenic types. These urinary proteins are excreted by some patients with myelomatosis and appear to correspond with the smaller peptide chain of the abnormal immunoglobulins in the serum of the same patients (Edelman and Gally, 1962). Putnam (1962) also suggested that Bence-Jones protein was a peptide chain of myeloma globulin, on the grounds that there were tryptic peptides common to both. The Bence-

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Jones proteins occur in two antigenic types, and Putnam et al. (1963) found that the peptide patterns of these two types contained very few common peptides, with the implication that they had a different amino acid sequence. Schwartz and Edelman ( 1963) isolated the corresponding B ( L ) chains from myeloma proteins of different antigenic types and found, as expected, the same difference in peptide pattern. The normal human immunoglobulins also occur in these two antigenic types in the ratio of about 2:1, and, if the B chains from these also show the same big digerences, then any preparation of normal human y-globulin will contain equivalent peptide chains with very different sequences. This work has been carried out so far only with pathological human immunoglobulins, but it is possible that a similar complexity occurs in normal immunoglobulin from humans, rabbits, and other species. This might vitiate attempts to relate amino acid sequence to antibody specificity. Such evidence as is available suggests, in agreement with other data, that all antibodies from the same species have a very similar structure, but the position seems to be too complex for any definite conclusion to be drawn about the sequence of amino acids at the combining site. OF IMMUNOGLOBULINS C. ENZYMATIC SPLITTING The size and heterogeneity of immunoglobulins made it unlikely that correlation of structure and function could be achieved from studies of the whole molecule. As it was known from much earlier work that the specific affinity for antigens survived some enzymatic digestion, this method of reducing the size without loss of activity has been studied in some detail. The results of enzymatic digestion of IgG have been reviewed recently (Porter and Press, 1962) and may be summarized briefly as follows. IgG from all species examined is split by papain into approximately thirds with very little production of small peptides. Two of these pieces contain an antibody site and are identical, their range of electrophoretic mobility being related to the IgG from which they were prepared (Palmer et d., 1962). From the rabbit IgG these pieces are known as I, if of higher mobility at neutral pH, and 11, if of lower mobility. From IgG of human and other serum these pieces are known as S (Edelman et al., 19eO), or A (high mobility) and C (low mobility) (Franklin, 1960). The third piece, which contains no antibody site and is of quite different character in all properties, can be crystallized easily from rabbbit IgG and from guinea pig IgG and, with difficulty, if prepared from human IgG (Hershgold et al., 1963). In the rabbit this piece is named I11 and in most other species F or B.

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If, instead of papain, pepsin at pH 5 is used to digest IgG, then one piece of molecular weight approximately 100,000 is obtained, together with smaller peptides. The large fragment, usually referred to as the 5 s fragment, will precipitate with antigerl and can be split into two equal halves by reduction with thiol (Nisonoff et d.,1960). Each half possesses one antibody-combining site and is very similar to papain pieces I and 11. (During peptic digestion the part of the molecule equivalent to I11 is degraded to smaller peptides.) It follows that the two pieces I or I1 obtained by papain digestion were also probably linked by a disulfide bond which is split by the 0.01 M cysteine added to the digestion medium to activate the enzyme. That this was so, was proved by Cebra et al. (1961) who used papain made insoluble by coupling to an amino acid polymer. If this enzyme was activated, washed free of thiol, and used to digest rabbit IgG, 3-4 peptide bonds were split, but there was no reduction of molecular weight and no loss of precipitating power if antibody was used. If cysteine was now added, in the absence of papain, the molecule broke to give three pieces, identical with those prepared with soluble papain in the presence of cysteine. The presumption is that the two pieces, I and 11, are held together by a disulfide bond and that I11 is held to both by noncovalent bonds that break when the other two pieces separate. Putnam et al. ( 1962) examined the action of a variety of other enzymes on human IgG; generally, cysteine was necessary to get high yields of 3.5s components. Rather surprisingly, trypsin was remarkably effective in the presence of only 0.001 M cysteine. Both Skvafil (1960) and Hanson and Johansson (1962) reported considerable degradation of human IgG by trypsin in the absence of cysteine, though no sedimentation studies of the products were made. Schrohenloher (1963) has found that trypsin will split human IgG to give about 50% 3.5 S pieces, the remainder being apparently undigested 7 S globulin. Addition of 0.01M cysteine raised the amount of 3.5S products to about 60%. This would seem to suggest that trypsin can digest half of a preparation of human IgG to 3.5s products without any reducing agent being present and that, even in the presence of cysteine, most of the other material is resistant to digestion. These results are not easily explicable in terms of the rather simple structure for rabbit IgG of two antibody site containing pieces held together by a disulfide bond and attached to the third piece by peptide and noncovalent bonds. There may be a species difference in the placing of the disulfide bonds and possibly differences in individual molecules in this respect. Attempts to split pieces I or I1 further by enzymatic digestion without loss of the power to combine with antigen were unsuccessful (Porter, 1960b), but

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Kulberg and Tarkhanova (1962) reported that this can be achieved using papain in 6 M urea. The molecular weight of the active fragment was given as 13,000 k 1800. Investigations of the biological activities of the papain digest pieces are discussed later, but perhaps the most surprising result is that all the activities investigated survive this drastic splitting of the molecule into thirds. This suggests that the original molecule may have a tripartite structure and that the enzyme is acting at one or two limited sites without affecting the steric structure of the three main sections of the molecule. Support for this idea is given by the observation of Goodman and Gross (1963) that no new antigenic sites are exposed by papain digestion.

D. REDUCTIONOF IMMUNOGLOBULINS Edelman ( 1959) and Edelman and Poulik (1961) showed that human and rabbit IgG could be split into smaller components by reduction in urea solution and, hence, demonstrated that both proteins were probably composed of several polypeptide chains. Equine (Fran&k, 1961) and bovine (Ramel et al., 1961) IgG behaved similarly. These results proved that the IgG of several species had a similar number of peptide chains, contrary to results with N-terminal amino acid assay where in several species the total gave less than one molecule of N-terminal amino acid per molecule of I@. It is clear that several N-terminal acids must be substituted on the amino group or be unreactive for other reasons. Cterminal amino acids, however, may be free, as a total of 4 moles ( 2 glycine, 1 serine, and 0.5 threonine and 0.5 alanine) per mole of rabbit IgG was estimated (Silman et al., 1962). The method used (hydrazinolysis) does, however, require correction for loss which exceeds 50%. Difficulties were met in the fractionation of the products of reduction in urea, as they were insoluble except in urea solution, and the recoveries from chromatographic columns were poor. Since all biological activity was lost, Fleischman et al. (1962; cf. Porter, 1962) re-examined the conditions of reduction in the absence of denaturing agent and found, as previously reported (Porter, 1950), that, although several disulfide bonds were split, there was no apparent change in molecular weight nor, if antibody was used, in the power to precipitate with the antigen. If, after reduction, the ten sulfhydryl groups liberated were reacted with iodoacetamide and the reduction mixture dialyzed against 1 N acetic or 1 N propionic acid, dissociation into two components resulted and these could be separated either by zone electrophoresis or, more simply, on a Sephadex column (Fig. 1). The larger component was named A and the smaller B (Porter, 1962). When examined on starch-gel electrophoresis in 8 M urea

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SYDNEY COHEN AND RODNEY R. PORTER

and pH 3.5 (Fig. 2), conditions used by Edelman and Poulik (1961), it was apparent that these components were equivalent to those obtained by more drastic reduction and named H and L, respectively, by Edelman and Benacerraf ( 1962). It seemed likely that these two components were the peptide chains of IgG, and an investigation of their properties led to the postulation of a diagrammatic structure for the molecule (Fig.

0.6

04

0.2

0

FIG. 1. Fractionation of reduced human IgG on a column (2.5 Sephadex G-75 in 1 N acetic acid (Cohen, 198313).

x

70 cm. ) of

3) (Porter, 1962). More detailed studies have given support to this structure, and the evidence from rabbit IgG (Fleischman et al., 1963; Crumpton and Wilkinson, 1963; Pain, 1963) and human IgG (Cohen, 1963b) may be summarized as follows 1. Complete reduction of all the disulfide bonds of either A or B in 6 M guanidine caused no further reduction in molecular weight. Conditions expected to split esterlike bonds also had no effect, and, hence, it is probable that IgG consists of only two types of peptide chains, unless either A or B is two chains held by an unknown type of linkage. 2. The molecular weights (A, 50,000 and By 20,000) and yields

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(70-75% A and 25-30% B ) of these chains are consistent with a fourchain structure and agree with the weight of the whole molecule (140,OOO-150,000). Small et al. ( 1963) reduced rabbit IgG in 6 M guanidine and separated the A and B chains in 6 M guanidine solution and got

FIG.2. Electrophoresis in 8 M urea, formic acid, starch gel of Iiuman JgC (U), reduced IgC ( R ) , A chain ( A ) , and B chain ( B ) (Cohen, 1963a).

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SYDNEY COHEN AND RODNEY

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higher yields (about 33%) of B. They estimated the molecular weights to be 50,000 for A and 25,000 for B, in agreement with the higher yield of B. Marler et al. (1964) determined the molecular weights of y-globulin reduced in 6 M guanidine and also found values of 50,000 and 25,000 for A and B, close to the values of Small et al. 3. The amino acids of the whole molecule are accounted for by the amino acids present in two A and two B chains. Papain-digestion piece I or II

Papain-dlgertion piece 111 Site of papain digestion

J-

:i; 7 B Chain

I

s I

t

I

I

I s

I 5 II

I

I

S

C

I

I

I I

I

I S I 5

Chain A

I

5

I

Carbohydrate

5

I

I I

Chain A

I

FIG.3. Diagrammatic structure of rabbit IgC (Porter, 1962).

The carbohydrate content of the rabbit IgG A chain equals that of the whole molecule, although there appear to be traces on the B chain. The N-terminal amino acids of rabbit IgG are found almost entirely on the B chain-the small amounts on the A probably arise from contamination with B. In human IgG the N-terminal amino acids are present in both chains. 4. There are four S-carboxymethylcysteine residues per mole of A chain and one per mole of B chain. No dissociation occurs without reduction and it is, therefore, likely that B is held to A by one disulfide bond and, if it is assumed that only interchain bonds have broken, A to A by three disulfide bonds.

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The relation of the peptide chains to the pieces produced by papain was determined simply by testing the reaction of A and B with goat antiserum to rabbit pieces I and 111. Chain A reacted with both antisera and B reacted only with anti4 and, hence, I contained B and part of A, whereas I11 contained only A (Fleischman et ul., 1962). From the known molecular weights of the pieces, the papain must hydrolyze the A chain approximately at the midpoint as shown in Fig. 3. It follows that, if this is true, the molecular weight of 111 should halve on reduction, and this appears to be true (Marler et ul., 1964). Pieces I or 11, on reduction and alkylation, should give B chains, as obtained from the whole molecule, together with the N-terminal half of A named A piece. This has been confirmed, and B from whole IgC and papain piece have been shown to be identical in size, amino acid analysis, antigenic specificity, N-terminal amino acids, and carbohydrate cbntent. The A piece was separated from B by its ability to form a dimer under acid conditions. It differed in amino acid content, antigenic specificity, and in having very little Nterminal amino acid, as does the whole A chain. There were two S-carboxymethylcysteine residues per molecule of A piece, in agreement with the suggested position of the disul6de bonds, it being assumed that one links A piece and B and the other A piece to A piece. The investigation of the antigenic relation of the papain piece to the peptide chain was repeated using human IgG and rabbit antisera (Olins and Edelman, 1962; Cohen, 1963a,b). The results with rabbit IgG were confirmed in that anti-F reacted with A and anti8 with B and, similarly, a n t i d reacted with F and anti-B with S. This suggests that F, as 111, contains A chain, whereas S, as I or 11, contains B, but neither laboratory showed a reaction between anti-S and A or between anti-A and S. There is thus no direct evidence that S contains A piece as well as B, although if the structure shown is correct for human as well as rabbit I@, it must be present. Clearly, investigation and isolation of the components of the reduced S pieces will be necessary. Another observation that does not fit the proposed structure was made by Cebra (1964). He found that if rabbit IgG was digested by insoluble papain in the absence of cysteine, there was no change of molecular weight, although 3 4 peptide bonds were split. If sodium dodecyl sulfate was now added, I11 could be dissociated leaving I and I1 joined by a disuEde bond. However, the release of I11 was slow and could be inhibited by thiol reagents, such as N-ethyl maleimide, suggesting that disuMde interchange was necessary. This finding might be reconciled with the proposed structure if it is assumed that insoluble papain has split a peptide bond within an intrachain disulfide bond of the A chain

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SYDNEY COHEN AND RODNEY R. PORTER

and that with soluble papain this whole sequence is hydrolyzed. Comparative analysis of the products of the two methods of hydrolysis should clarify this discrepancy.

E. STABILITYOF THE DISULFIDE BONDSIN RABBIT IgG The evidence obtained so far is most easily explained on the assumption that the interchain disulfide bonds are much more labile than the intrachain bonds, in agreement with the observation of Cecil and Wake (1962) on a number of other proteins. There are also, however, differences in stability within the two types of bonds. Mandy and Nisonoff (1983) showed that the 5 S pepsin fragment is split into halves by standing in 0.008 M 2-mercaptoethylamine for 1 hour at 37"C., pH 5, with the reduction of 1 disulfide bond, but that under more drastic conditions 5 more disulfide bonds were split without any further change in molecular weight at neutrality. The labile bond is presumably that shown between A piece and A piece (Fig. 3).When reoxidation is allowed to take place, even after breaking 6 disulfide bonds, only a 5 S product results. The other SH groups must be held close to each other so that the original disulfide bonds are re-formed; random formation of disulfide bonds would lead to aggregation. Palmer and Nisonoff (1963) showed that this disulfide bond between the N-terminal sections of A chains is equally labile in the whole molecule, since gentle reduction followed by pepsin digestion also gives 3.5 S fragments. If gentle reduction in 0.03 M mercaptoethanol was followed, not by peptic digestion, but only by acidification, the sedimentation value at pH 2.5 fell to 3.5s and was believed to be due to the formation of half-molecules. Under these conditions, 2-3 disulfide bonds were split with very little release of B chains, and it seems likely that the disulfide bonds between A chains are more labile than those between A and B chains. Further work (Palmer and Nisonoff, 1964) has now shown that when whole rabbit y-globulin is reduced at pH 5 in 0.015M mercaptoethylanime, about two-thirds of the molecule is split into halves and can be separated from undissociated material on a Sephadex G-200 column. If the halfmolecules are reacted with iodoacetamide, analysis shows that there is one S-carboxymethylcysteine and, therefore, one SH group per 75,000. This is strong evidence that in at least some of the molecules only 1 disulfide bond holds the A chains together, not 3, as shown in Fig. 3. This suggests that the 2 interchain disulfide bonds in the C-terminal end of the A chain may, in fact, be intrachain, though, if true, it is not clear why piece I11 cannot be split into halves without prior reduction (Marler et al., 1964). Clearly, it is too early to draw firm conclusions as to the

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

303

position of the interchain disulfides. These discrepancies in the apparent number of interchain disul6de bonds may result from the occurrence of disulfide interchange during fractionation, or may reflect the heterogeneity of any preparation in this respect, as well as many others. When horse IgA (T) is examined (Weir, 1964), quite a different situation is found. Papain digestion in the presence of 0.01 M cysteine gives a divalent 5 s antibody together with smaller peptides. The 5 S antibody can be split into halves but 0.2 M mercaptoethanol is necessary to break this disulfide bond completely. Since horse IgG gives (like rabbit IgG) only 3.5 S pieces on papain digestion, it is clear that the differences in structure between these two immunoglobulins from the same species include the interchain disulfide bonds. In horse IgA present evidence suggests that there are 2 disulfide bonds between the A piece, rather than 1, as in IgG, and this may explain their greater resistance to reduction. It should be stressed that in all the discussion of the position of the disulfide bonds it is assumed that the thiol groups formed on reduction come only from disulfide bonds. Though probable, this is not necessarily correct and, indeed, Karush et d.(1964) have developed a novel technique for the assay of thiol groups in proteins which gives nearly 25% higher values in rabbit IgG than those expected from the cystine content estimated after oxidation (Smith et al., 1955; Crumpton and Wilkinson, 1963), or after reduction (Koshland and Englberger, 1963). As this discrepancy between the different methods is not found in other proteins, it is of considerable interest and it may reflect some peculiar structural features which have not yet been recognized.

F. STRUCTURAL RELATIONSHIPS OF IgG, IgM, AND IgA In the human immunoglobulins, IgG, IgM, and IgA all across react with antisera to any one (see p. 319), but much of the antibody is specific to the homologous globulin. This suggests that parts of the structure are common to all three types and parts are distinct. In agreement with this, one allotypic antigenic marker of the human immunoglobulins, InV, is common to all three types, but the other, Gm, is unique to IgG. It would be expected that one chain, carrying the InV marker, was common to all, and the other, carrying the Gm in IgG, was not (Edelman and Benacerraf, 1962). As the InV factor is also found on papain piece S and the Gm on F, the presence of InV on the B chain and Gm on the A chain was the likely distribution, and this was demonstrated by Lawler and Cohen (1965) who found a clean separation of the two antigenic markers in the two chains. Cohen (1963a,b) also showed that the common B and distinct A chains were apparent when the reduced immunoglobulins were

304

SYDNEY COHEN AND RODNEY R. PORTER

electrophoresed in starch gel in 8 M urea at pH 3.5 (Fig. 4) (see also Carbonara and Heremans, 1963). The B chains of the three types have the same mobility, whereas the A chains have different mobilities. The identity of the B chains is more striking when electrophoresed at alkaline pH

FIG.4. Electrophoresis in 8 M urea, formic acid, starch gel of A chain ( 1 ) and B chain ( 2 ) of normal IgC, of A chain ( 3 ) and B chain ( 4 ) of normal IgM, and of whole, reduced IgC ( 5 ) (Cohen, 1963b).

when the same complex pattern is obtained from the B chain of either IgG or IgM (Cohen and Porter, 1964). Amino acid analysis of the separated chains of human IgG and IgM also shows near identity for the B chains and an obvious difference in the A chains (see Tables 111 and

305

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

N )(Chaplin et al., 1964). Thus it is clear that human immunoglobulins carry a common B chain and a distinct A chain. Similarly, the B chains of horse IgG and IgA ( T ) are identical in all chemical and physical properties, whereas the A chains show differences (Weir, 1964). TABLE 111 AMINO ACID COMPOSITION OF A CHAINS Residues/50,000 gm.

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Half-cystine S-Carboxymethylcysteine Tryptophan Hexose Hexosamine

Rabbit IgG 23 7 16 33 49 49 40 36 33 24 41 5 15 30 17 14 10

Human IgG 29 10 12 33 34 50 39 33 28 19 41 4 8 31 17 13 7

4 8 4.5 4

4 8 4 4.5

Human IgM 20 7 18 34 38 42 40 29 28 26 36 5 13 30 12 16 7 3(74) 7 20 12

Horse IgG 26 10 10 30 37 45 38 31 28 22 46 4 13 28 16 13 3 3(74) 8

5

4

Horse IgA(T) 27 12 10 34 33 46 38 38 28 22 45 3 11 31 16 12 6 4 7 10 7

Rabbit IgG carries allotypic antigenic markers controlled by two independent loci (Oudin, 1956), again suggesting that two peptide chains are present, but both sets of antigenic specificities are present on IgG and IgM (Todd, 1963). The b locus determines the specificity of the B chain and identity between IgG and IgM was expected here. The fact that the A chains of IgG and IgM also carry the same allotypic specificity suggests a partial identity of sequence which could arise if, as discussed below, A was in fact two peptide chains. G. CARBOHYDRATE CONTENT OF IMMUNOGLOBULINS The immunoglobulins of all types and all species contain carbohydrate. It seems unlikely that it plays any part in the antibody-combining

306

SYDNEY COHEN AND RODNEY R. PORTER

site, as papain pieces I and I1 can be obtained almost free of carbohydrate, yet retaining their power to combine specifically with antigen. The carbohydrate may well be an important factor in other biological activities and, clearly, is an important structural feature of, for example, the TABLE IV AMINOAcm COMPOSITION OF B CHAINS Residues/20,000 gm. Rabbit IgG Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Pheny1a1anine Half-cystine S-Carboxymethylcysteine Tryptophan Hexose Hexosamine

8

2

3 16

25

19 18 10 15 13 18 0.9 6 11 10 5 6

1

4

0.3 0.2

Human IgG

Human IgM

Horse IgG

10 3 6 13 15

10

10

5 13 15

4 12 17

24 20 11

11 12 13 0.6 5 13 8 6 3 1 2 0.25 0.2

2

24 19

2

28

11 12 12 13 0.7 5 13 8

16 11 16 12 14 0.8 10 11 0

3

3

5

1 3

0

0.3

4

1 3 0.4 0.1

Horse IgA(T) 10 2

4

13 19 32 16 12 18 13 14

0.4 7

12 6 4 3

1 3 0.6 0.1

A chain of human IgM where it comprises some 15% of the total weight. Analytical procedures for the estimation of carbohydrate are not yet as satisfactory as those used for amino acids, but the data available for the three immunoglobulins from different species are summarized in Table V. In all cases examined sqfar (human IgG and IgM, rabbit IgG, and horse IgG and IgA) the carbohydrate is almost entirely confined to the A chains. The traces present in the B chains may be due to contamination by A, but they are remarkably difficult to remove and might be an integral part of a small percentage of the B chain molecules. When the carbohydrate content of the enzymatic pieces is estimated, the position is not so uniform. In rabbit papain pieces, about two-thirds of the carbo-

307

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

hydrate is firmly bound to piece I11 and the remainder is present in a glycopeptide which adsorbs onto piece I but which can be digsociated by acid (Fleischman et all, 1983). It seems likely that a similar distribution of the carbohydrate occurs in human IgG (Dische and Franklin, 1964) and horse IgG (Weir, 1964). With horse IgA (T) one-third is bound to the nonantibody-containing part equivalent to piece I11 and two-thirds to the equivalent of pieces I or 11, and none of this material can be dissociated from the protein by acid (Weir, 1964). Schultze ( 1963) who used peptic digestion also found that two-thirds of the carbohydrate of horse IgA ( T ) was in the 5 S fragment which, by analogy with rabbit, is TABLE V CARBOHYDRATE COMPOSITION OF IMMUNOGLOBULINS ( %) Carbohydrate Hexose Fucose Hexosamine Sialic acid

b 0

d f

Rabbit IgcD

1.2 1.0 0.2 2.4

Human

Horse

IgGb

IgMC

IgAd

1.2 0.3 1.1 0.2 2.8

6.2 0.7 3.3 2.0 12.2

4.8 0.2 3.8 1.7 10.5

IgCe 1.1

IgA(T)e

1.1 0.2 2.4

1.9 0.9 4.9

2.1

Bovine Igcl

0.9 0.2 1.5 0.3 2.9

Fleischman et al. ( 1963). Miiller-Eberhard et al. (1956). Miiller-Eberhard and Kunkel ( 1959). Heremans ( 1959 ) . Schultze ( 1959). Nolan and Smith (1962a).

equivalent to pieces I and 11. It is clear, therefore, that in IgA there are two distinct fractions of the carbohydrate attached to the A chain. In IgG there is probably only one carbohydrate fraction. Smith and collaborators (Rosevear and Smith, 1961; Nolan and Smith, 1962a,b) have isolated the glycopeptides obtained from a papain digest of heatdenatured IgG from human, bovine, and rabbit serum and concluded that there is only one carbohydrate moiety which is bound to an aspartic acid residue present in the following sequence: Human: Asp NH, Tyr Glu Asp Carbohydrate Bovine: Glu Glu NH, Phe Asp Carbohydrate Rabbit: Glu NH, Glu NH, Phe Asp Carbohydrate

This is not easy to reconcile with the presence of carbohydrate in both a

glycopeptide and piece I11 from rabbit and human IgG. In both cases analysis shows significant differences between the two portions, but it is possible that these conflicting results could arise from the heterogeneity of molecules which might be such that different molecules contain

308

SYDNEY COHEN AND RODNEY R. PORTER

slightly different carbohydrate moieties and were digested by papain in different ways. A fuller discussion of glycoprotein aspects of immunoglobulin structure is given by Press and Porter (1964).

H. POSSIBILITY OF THREE TYPES OF PEPTIDECHAINS The evidence above suggests that there are two pairs of peptide chains in all the immunoglobulins, but some biological evidence would be more easily explained if there were three pairs of chains. First, it is claimed that the antibody site is on the A piece and it is presumably similar in IgG, IgM, and IgA, yet the A chains of the three

s

I

S

s

I S

S

s

S

S

I

I

FIG.5. Possible diagrammatic structure of a six-chain IgG molecule.

types differ. If A were two chains, A and C, as shown in Fig. 5, then both A and B might be common to the three types and only C different. However, the position of the antibody site is still in dispute and, further, the stability of antibody acitvity to gentle reduction digers in IgG and IgM (Jacot-Guillarmod and Isliker, 1962). The latter suggests that the antibody sites of these two types of globulin may not be formed on identical structures. Second, when the allotypic antigenic specificities of the different fractions of rabbit IgG were examined, it was found that A l , A2, and A3, controlled by the a locus, were on the A chain, and A4, A5, and A6, controlled by the b locus, were on the B chain (Stemke, 1964; see also Feinstein et al., 1963). Both antigenic sites are on papain pieces I or I1 and, hence, the a locus must be on the gene controlling the synthesis of

STRUClWRE AND ACTMTY OF IMMUNOGLOBULINS

309

the A piece. Todd (1963) has now found that the a locus phenotypes are present on rabbit IgM as well as I@, and Feinstein (1963) has made a similar finding with what is believed to be rabbit IgA. It, therefore, seems likely that part of A piece is common to IgG, IgA, and IgM, although the whole A chain differs. Again, if only a .C chain differed, this would fit well. Third, there may be three independent loci involved in the synthesis of human IgG: InV, associated with the B chain, Gm, associated with the A chain, and a third sex-linked locus which appears to be concerned in some cases of agammaglobulinemia. Similarly, in rabbit IgG there has been a report of a third locus, P, which may be independent of a and b (Dray et al., 1963b). Although other explanations are possible this evidence could be used as support for a six-chain structure. None of these arguments can be considered conclusive at present, but taken together, suggest that the immunoglobulins may consist of three, rather than two, pairs of peptide chains and that A is really two chains. However, there is good evidence that there is no disulfide interchain bond present within a single A chain and some evidence that there is no ester interchain bond. Further, it would follow that there must be two blocked N-terminal amino acids on rabbit IgG A chain and, so far, none have been identified. Hence, there is at present no chemical evidence in support of a six-chain structure.

I. POSITION OF THE ANTIBODY-COMBINING SITE Several attempts have been reported to place the position of the antibody-combining site in terms of the peptide chains of IgG. Conflicting results have been obtained but, from examination of the papain pieces, it is certain that the combining site is on piece I or piece I1 and, hence, must be on the B chain, the A piece, or be formed jointly by these chains. Edelman et al. (1961, 1963a) found that if guinea pig antibodies prepared by dissociation of specific precipitates were reduced and a b l a t e d and then electrophoresed in starch gel in 8 M urea at pH 3.5, the A chain gave a relatively compact band identical with that from reduced inert y-globulin. The B chain from reduced antibody globulins, however, gave a series of sharp lines, in contrast to the broad smudge shown by reduced inert IgG. Several antihapten and antiprotein antibodies were examined and a correlation was found between the pattern of the banding and the specificity of the antibody used. If guinea pig antidinitrophenyl protein antibodies were fractionated by dissociation of the specific precipitate with dinitrophenol, followed by dinitrophenyl-

310

SYDNEY COHEN AND RODNEY R. PORTER

lysine, two fractions were obtained specific for either one or the other hapten (Fig. 6 ) . It could be shown that some bands were present in the B chain of one, but not of the other, again giving a correlation between the type of banding, under these conditions of electrophoresis, and the specificity of the antibody. The papain fraction S containing the antibody activity was also reduced and alkylated and this showed a series of rather indistinct bands overlapping but slightly faster than the B bands of reduced whole antibody. This correlation between electrophoretic patterns of the reduced B chain with antibody specificity suggested that the B chain contains or contributes to the antibody-combining site. This phenomenon is not observed with rabbit or horse antibodies where the B chains from antibody or inert y-globulin are equally diffuse when electrophoresed under the same conditions (Fleischman et al., 1963). Further, if the B chain from inert IgG of any species is electrophoresed at neutral pH and 8 M urea in starch gel, than all give some ten widely separated bands (Cohen, 1963~;Cohen and Porter, 1964). If B chains from guinea pig antibodies were used, there was a difference in relative intensity of staining of some of the bands, but all were present. As discussed later, there is slight evidence that the bands formed under these conditions are related to the cell type making the IgG molecules. If true, this would imply that different cell types were contributing unequally to the different kinds of antibody, but that there was unlikely to be any direct relation of B chain pattern to the antibody specificity under these conditions of electrophoresis. When a direct attempt to measure antibody activity in the separated chains was made (Fleischman et a,?., 1963), different results were obtained. If reduction in the absence of urea was used, about 20% of the activity survived with rabbit antiprotein antibody as judged by specific coprecipitation. If horse antirabbit IgG was used, about 70% activity was recovered, again measured by coprecipitation, and the same recovery was found with horse antidiphtheria toxoid, but with this flocculating system, measurement was by specific inhibition of precipitation. With these methods, the activity was found to be entirely on the A chain, absent from the B chain, and not significantly increased by addition of B to A. The conclusion was drawn, therefore, that the antibody site, at least in horse antibodies, was situated in the A chain. It presumably was, therefore, in the A piece and, in fact, activity could be demonstrated in this fraction (Weir, 1964). Utsumi and Karush (1963) used rabbit antihapten antibody reduced in aqueous solution and separated the A and B chains on a Sephadex

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

311

FIG. 6. Specifically isolated fractions of dinitrophenyl (DNP) guinea pig albumin ( GPA) antibodies and picryl (Pic) guinea pig albumin antibodies compared after dissociation, reduction, and electrophoresis in starch gel containing 8 M urea and formic acid, pH 3. 1. DNP-lysine fraction, DNP-GPA antibodies, animal 8; 2. DNPOH fraction, 3. DNP-lysine fraction, DNP-GPA antibodies, DNP-GPA antibodies, animal 8; animal 1-3; 4. DNPOH fraction, DNP-GPA antibodies, animal 1-3; 5. DNPlysine fraction, Pic-GPA antibodies, animal 2-1; 6. DNPOH fraction, Pic-GPA antibodies, animal 2-1 (Edelman et al., 1963a).

312

SYDNEY COHEN AND RODNEY R. PORTER

column in 0.03M sodium decyl sulfate. Under these conditions they found a much higher recovery of the activity (measured by equilibrium dialysis) than had been found using 1 N propionic acid as dissociating agent, and it was again entirely in the A chain (Fig. 7).

? 2 x

8-

732

7-

- 28

6-

- 24

5-

.- 20

+

A-PLAC K ~ 2.1 . x to4

4-

u

\ L

1'

0

-16 x

KA. 18 x lo4

k-pLA V

-0

3-

-12

2

\

- 8

+

\O

I- 4

0

0.2

1

I

0.4

0.6

I

0.8

1.0

1.2

\ 1.4

4

1

1.6

I

1.8

2.0

Cebra et al. (1963) also used detergent but the reduction with mercaptoethanol was carried out in the sodium dodecyl sulfate and, hence, more disulfide bonds would be split (Karush, 1957). When rabbit antilysozyme or rabbit antibacteriophage antibody was reduced under these conditions, there was little or no loss of antibody activity, even though the drop in sedimentation coefficient showed that dissociation had

STRUCTURE AND A.(;TMTY OF IMMUNOGLOBULINS

313

occurred. On removal of the detergent with ion-exchange resin, 10-2070 of the reduced antibody remains in solution, the molecular weight is of the order of 50,000, and the activity, measured as inhibition of enzyme, is close to that of native antibody (Cebra et al., 1963). There is some doubt as to the nature of this material but the most recent evidence suggests that it consists of B chain and probably also material equivalent to A piece (Jaquet et al., 1964). Frandk and Nezlin (1963) separated the peptide chains of horse antibodies to diphtheria and tetanus toxoid after splitting the disulfide bonds with sodium sulfite and cupric nitrate to give S sulfo derivatives and fractionation on Sephadex G-100 in 0.05 M formic a c i d 4 M urea. Measurement of activity was made by estimation of the proteins bound to toxoid coupled to cellulose (Gourvitch et al., 1961a), and it was found that under these conditions of reduction and isolation 9%97% of the antibody activity was lost. There appeared to be some activity in the A chains which was increased by addition of the nonspecific B chain and more by addition of the specific B chain. It was concluded that the combining site was on the A chain, but that the B chain was necessary for full activity. In view of the very low recovery of activity, some uncertainty remains as to the significance of these results. Edelman and colleagues (1963b) had similar results using guinea pig antibodies to two bacteriophages and a hapten. In this work the A and B chains were separated according to Fleischman et al. (1962), and there was a heavy loss (80-95%) of activity with the phage antibodies and less ( 5 0 % ) loss with antidinitrophenol antibodies. What activity survived was in the A chain, but it was increased from 1.5- to 10-fold by the addition of the B chain. An indirect approach is to label part of the antibody molecule which is concerned in the combination with antigen, prepare the peptide chains, and find which contains the label. This has been done in two ways. Pressman and Roholt (1961) and Roholt et d. (1963) iodinated a rabbit antihapten antibody with I1sl in the presence of the hapten, and in the absence of the hapten. The two preparations were mixed with 1125 and digested with pepsin, the peptides separated by high-voltage electrophoresis on paper, and the relative labeling with the two isotopes measured. Most of the peptides had the same relative labeling as expected, but three fractions showed preferential labeling with 1126, suggesting that the tyrosyl residues were partly protected from reaction with Ilsl when hapten was bound to antibody. When this was repeated, but the A and B chains separated (according to Fleischman et al., 1962) before digestion, then the unequally labeled peptides were entirely in

314

SYDNEY COHEN AND RODNEY R. PORTER

the B chain, suggesting that these parts of the B chain are closely associated with the combining site or, possibly, are protected in some less direct manner. The second possibility is suggested by the observation that the binding of 2 moles of fatty acid to 1 mole of serum albumin alters considerably the susceptibility of the tyrosine residues to iodination, though direct protection is impossible on steric grounds (Glazer and Sanger, 1963). In the second method, Metzger et al. (1963) used a technique which they named affinity labeling. A hapten is substituted to give a reactive group, and when it reacts with antihapten antibody the specific association is followed by covalent bonding with a reactive amino acid residue in, or close to, the combining site. Thus, rabbit antibenzene arsonic acid was mixed with p-( arsonic acid)benzene diazonium fluoroborate, and it was shown that 2 moles were taken up per mole antibody at a rate some 100 times greater than the nonspecific reaction with other sites in the molecule. With antibody specifically and covalently labeled, A and B chains could be separated and now the label was found to be in both, but about twice as much was in A as in B. Further evidence has been obtained by Metzger and Singer (1963) who used rabbit antibody to the hapten dinitrophenyllysine. The antibody was reduced and fractionated in 1 N propionic acid columns according to the method of Fleischman et al. (1962) and gave the expected yield as judged by absorption at 280 mp of 25% B and 75% A. If the hapten was added before acidification of the reduced antibody, there was almost complete retention of hapten-binding affinity and, when run on the Sephadex column, all the hapten was associated with the A peak. However, the yield of B chain fell from 25 to 16%, i.e., in the presence of hapten, one-third of the B chain was not dissociated from the A chain by acid (Fig. 8). Either some of the B chains were directly involved in the A chain-hapten linkage, or the presence of hapten restricted the unfolding of the A chain by propionic acid and hence was indirectly responsible for the persistent affinity of the A chain for the B chain. Givol and Sela (1964b) have used yet another method based on their observation that if polytyrosyl gelatin forms a specific precipitate with its antisera and this is digested with collagenase, the gelatin is hydrolyzed to peptides and leaves antibody with polytyrosyl groups bound firmly to it. These are the haptenic groups of the original antigen and can only be fully dissociated at pH 1.8. Givol and Sela prepared such antibody with bound C14-polytyrosine and made the A and B chains according to Fleischman et al. (1962) by reduction and separation on a Sephadex column in 1 N propionic acid. Two peaks of radioactivity were

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

1.6 1.4

-

1.0:

3 08 n

0 06: 0.4

c -

11 / \

12

I

-

0.2-

\

t

- - % - . O

+++tt+

' ' . -02 80 IOU 120 140 160 180 200 220 240 260 I

I

I

'

'

'

1

315

316

SYDNEY COHEN A N D RODNEY R. PORTER

found, the largest running very slowly and corresponding to dissociated free polytyrosine and a smaller peak moving at the front of the A chain peak. The B peak was inactive. As 80% of the hapten was dissociated from reduced or unreduced antibody in 1 N propionic acid, the results are not decisive, but it was suggested that the polytyrosine was bound either to an A dimer or possibly to undissociated antibody. Clearly, with this series of conflicting results, firm conclusions are impossible. Direct evidence with high over-all recovery favors A as the location of the antibody-combining site, but this cannot be accepted without reserve until some explanation of the other results has been found. J. HETEROGENEITY OF IMMUNOGLOBULINS The physical, chemical, and biological heterogeneity of immunoglobulins has often been discussed (see Fahey, 1962). The resolution of the three types of immunoglobulins and the definition of the relation between them has helped to reduce the confusion, but it remains abundantly clear that there is a complexity of structure in any one type which is unique in protein chemistry. It is highly probable that this complexity is related to biological function, but there is little convincing evidence as to what the connection is. More detail of the heterogeneity has been obtained by studies of the isolated chains (Cohen and Porter, 1964). The A chain, when electrophoresed in starch gel in 8 M urea at neutral pH, shows a diffuse band whose mobility is related to the mobility of the immunoglobulin from which it was prepared. At acid pH, A appears less heterogeneous than the whole IgG. The B chain gives a broad smudge when electrophoresed in 8 M urea at pH 3.5, except when derived from guinea pig antibodies, as discussed above, or mouse antihapten antibodies (Merryman and Benacerraf, 1963). However, when electrophoresed under the same conditions, but at pH 7-8, the B chain from IgG of all species examined gives about ten well-separated components (Fig. 9) and the mobility of these fractions is unrelated to the mobility of the whole molecule. Similarly, the B chains from IgG from all individuals of one species are indistinguishable and the complexity does not, therefore, seem to arise from genetic variations, as in the haptoglobins (Smithies, 1959). It was possible that this multiplicity arose as an artifact during handling but no support could be found for this and perhaps the best evidence that this was a genuine phenomenon came from examination of the B chains of human myeloma globulins. These globulins have long been known to be much more homogeneous by charge (Putnam, 1960), and Edelman and

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

317

FIG. 9. Electrophoresis in 8 M urea, glycine, starch gel of IgG B chains of rabbit ( 1 ), guinea pig ( 2 ) , bovine ( 3 ) , horse ( 4 ) , baboon ( 5 ) , and human ( 6 ) (Cohen and Porter, 1964).

318

SYDNEY COHEN AND RODNEY R. PORTER

colleagues showed that the R chain was a sharp band when electrophoresed in 8 M urea at pH 3.5 (Edelman and Poulik, 1961). At p H 7.5 myeloma B chains again gave a single main component, but sometimes with a second minor component (Fig. 10). As this material was prepared

FIG. 10. Electrophoresis in 8 A l urea, glycine, starch gel of the B chains of normal human IgC ( 1) and of five myeloma imrnuno~lol~ulins(2-6) ( Cohen ant1 Porter, 1964).

in exactly the same conditions as the B chain of normal :,-globulins, it is unlikely that complexity of the latter is an artifact. The myeloma globulins are thought to be synthesized by a relatively homogenous group of cells, possibly deriving from a single clone, and this raises the possibility

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

319

that the ten forms of B may be derived from a corresponding number of different cell types. Differentiation of the cells would be expected to occur soon after birth, when immunoglobulin synthesis increases very rapidly; hence, the B chain was prepared from the IgG of a colostrum-deprived calf at different time intervals after birth. A progressive increase in complexity was anticipated but, in fact, although there was an obvious change in the relative intensity of the bands from 3 to 6 weeks after birth, there was no increase in complexity. Much more convincing evidence will be necessary to relate cell and B chain types. Further, it is apparent that even the single bands of B chains are not homogeneous since bands of identical mobility prepared from myeloma IgG may be either of antigenic Type 1 or Type 2,' and presumably both are present in the normal subfractions of B. There is already evidence that the A chain may show the same type of multiplicity if handled suitably, and it is probable that both chains exist in many different molecular forms. Until that complexity, which in many cases is unrelated to antibody specificity, is resolved, correlation between specificity and structure will be very difficult. IV. Biological Properties of immunoglobulins

A. ANTIGENIC PROPERTIES It has often been remarked that the antigenic specificity of IgG, in contrast to its many other variable properties, shows a surprising homogeneity in any one species. Thus, analyses using anti-y-globulin sera in gel-diffusion tests usually reveal only a single precipitation line. Recent investigations showed, however, that the antigenic structure of immunoglobulins is far more complex than was originally appreciated. The concept of the immunoglobulins as a family of related proteins arose from the observation that several serum globulins which appear to possess antibody activity also carry common antigenic determinants; the same determinants are present on myeloma proteins, pathological macroglobulins, and Bence-Jones proteins ( Heremans, 1960). In addition, each of the three main classes of immunoglobulins has specific antigenic determinants which can be revealed by immunoelectrophoresis and gel diffusion (Korngold and Lipari, 1956a; Franklin and Kunkel, 1958; Grabar and Burtin, 1960). Recent studies have shown that the common determinants present on all immunoglobulins are of two distinct types. It has been known for some time that Bence-Jones proteins can be divided into two mutually exclusive antigenic types which were called Groups A and B by Korngold and Lipari (1956b) and Groups I1 and I, respectively, by Burtin

320

SYDNEY COHEN AND RODNEY R. PORTER

et al. (1956). In the same way myeloma proteins were differentiated into three antigenic groups termed Group 1 and Group 2, which are present on IgG type proteins, and Group 3, which has since been shown to consist of IgA myeloma proteins (Korngold and Lipari, 1956a). Later studies have shown that the antigenic determinants that differentiate the two types of IgG myeloma proteins are the same as those present on the two types of Bence-Jones proteins (Mannik and Kunkel, 1962; Franklin, 1962); for this reason the latter are now referred to as Types I and I1 Bence-Jones proteins, instead of Groups B and A, respectively, as originally suggested by Korngold and Lipari. On the basis of the same determinants it is possible to divide myeloma proteins belonging to both IgA and IgM fractions into the two distinct types, I and I1 (Franklin, 1962; Mannik and Kunkel, 1962; Fahey and Solomon, 1963). Approximately two-thirds of myeloma proteins have Type I and about one-third, Type I1 determinants (Fahey and Solomon, 1963). In these studies the antigenic classification of human myeloma proteins has been carried out using antisera to isolated Bence-Jones proteins or antisera to normal human IgG absorbed with a myeloma protein of the appropriate group. With the use of the same methods, the Type I and I1 determinants have been demonstrated on normal IgG, IgA, and IgM fractions of human serum (Fig. 11) (Franklin, 1962; Mannik and Kunkel, 1963a; Fahey, 1963a), as well as on the low molecular weight fragments of immunoglobulin present in normal human urine ( Stevenson, 1962; Fahey, 1963a) , Precipitation of immunoglobulin preparations with type-specific antisera has shown that the determinants are carried on separate molecules; approximately 60% of normal IgG molecules have Type I determinants and about 30% carry Type I1 specificity (Mannik and Kunkel, 1963a). The proportion of Type I and I1 molecules appears to be similar in the three normal subfractions of immunoglobulins ( Fahey, 1963a) , All antibody activities investigated have been associated with both antigenic types of immunoglobulin (Mannik and Kunkel, 1963b). In different individuals there is considerable variation in the proportion of Type I and Type I1 molecules present in antibodies of a given specificity. In a single individual the ratio of the two antigenic types varies considerably in different specific antibodies, and these ratios are different from that observed in the total Ig (Mannik and Kunkel, 1963b). Antisera to human IgG produced in rhesus monkeys and absorbed separately with two myeloma sera have been reported to show three distinct antigenic determinants in normal 7 S y-globulin (Dray, 1980). Molecules carrying these determinants have somewhat different mobilities

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321

on immunoelectrophoresis and cannot, therefore, be correlated with Type I and Type I1 molecules which have the same electrophoretic distribution (Mannik and Kunkel, 1963a; Fahey, 1963a). Moreover, both myeloma proteins used for absorption of the antiserum were Type I which indicates that the monkey antisera are revealing an additional antigenic heterogeneity within the Ig system. In the case of IgM, antigenic heterogeneity has been demonstrated by immunoelectrophoresis using rabbit antisera to human macroglobulin (Fessel, 1963).

FIG.11. Demonstration of the presence of

two antigenic types of normal human

I@. Purified, Types I and 11, IgG myeloma ( y mp) proteins were placed in wells

of an Ouchterlony plate adjacent to chromatographically prepared normal IgG (6.0 S y ). Pooled antiserum against Types I and I1 Bence-Jones proteins was placed in the lower well ( A S ) ( Fahey, 1963a).

Recent investigations have established the location of the common and specific antigenic determinants on the fragments of immunoglobulin molecules produced by reduction or enzymatic digestion. The enzyme papain separates parts of the molecule which carry the common and specific determinants. The fragment designated F (Edelman et al., 1960) is associated with the specific determinants of human IgG, whereas the S fragment carries determinants common to normal and pathological IgG, IgA, and IgM, as well as Bence-Jones proteins (Heremans, 1960; Franklin and Stanworth, 1961; Migita and Putnam, 1963). Similar findings have been reported for the immunoglobulins of mouse (Askonas and Fahey, 1962), rabbit (Thorbecke and Franklin, 196l), and guinea pig (Thorbecke et al., 1963). The F fragment obtained by papain digestion is made up of part of the A (or H ) polypeptide chains, whereas the S fragment contains the B chain (Olins and Edelman, 1962). The isolated A chain has now been shown to carry the type-specific, antigenic determinants of normal and pathological immunoglobulin subfractions, whereas the common determinants of Types I and I1 have been identified

322

SYDNEY COHEN AND RODNEY R. PORTER

FIG. 12. Reaction of rabbit anti-( B c h i n of Iiuman IgC) with Rcnce-Jones protein Types I (1) and 11 ( 2 ) , and with B c h i n of 1gG ( B ) , S fr'igmmt of IgC ( S ) , and a mixture of Types 1 und 11 Bencc-Jonc\ protc4n\ ( 1 2 ) . 13otli tlic S fragment and the B chain form slight spurs with the miuture of Typca 1 und 11 Bence-Jones proteins (Cohen, 1963h).

+

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323

on B chains (Fig. 12) (Cohen, 1963a,b; Fahey, 1963b). Thus, antisera against purified immunoglobulin fractions can be made specific for the corresponding fraction by absorption with B chain. Similarly, rabbit antisera against the human B chain of IgG give reactions of identity with other normal immunoglobulin fractions, whereas antisera against the A chain react only with IgG (Fig. 13). Certain antigenic determinants of immunoglobulins appear to be concealed in the whole molecule and may be revealed by enzymatic digestion. Osterland et al. (1963) used anti-Rh antibodies digested with pepsin or with papain at pH 4.1 to coat Rh-positive red cells and showed that these were agglutinated by the 7 S globulin fraction from normal sera and from sera of some patients with rheumatoid arthritis and bacterial endocarditis. These same sera did not, however, agglutinate cells coated with the whole antibody; in addition, the agglutination could be inhibited by fragments obtained by enzymatic digestion at acid pH, but not by native y-globulin or by the isolated S fragment obtained by papain digestion at pH 7.4. Goodman and Gross (1963) also found that no new antigenic determinants were exposed by papain digestion of rabbit IgG at neutral pH. These results indicate that hidden antigenic determinants of the S fragment of IgG are revealed by enzymatic digestion only when this is caried out at acid pH under conditions that destroy the F fragment. Repeated attempts to show that isolated antibodies possess individual antigenic specificity have been unsuccessful (see Kabat and Mayer, 1961). However, such individual specificity has recently been demonstrated in the case of several antibodies. Kunkel et al. (1963) isolated four human antibodies of the 7 S class from specific precipitates and used these with Freund's adjuvant for immunization of rabbits. The rabbit antisera after absorption with normal human IgG or normal human serum reacted with the corresponding individual antibody, but not with antibodies of the same combining specificity obtained from other subjects. Oudin and Michel ( 1963) obtained antisera specific for individual antibody preparations by immunizing rabbits with Salmonella typhosa agglutinated by the antiserum of another rabbit. In some instances antibodies were produced which precipitated with the immunizing antiserum but failed to precipitate with the serum of the same rabbit before immunization with S . typhosu or, subsequently, after antipneumococcal immunization; nor did such antisera precipitate with the immune or nonimmune sera of other rabbits. Deutsch and MacKenzie ( 1964) have shown that individual monkey antisera to Rh saline agglutinins (IgM antibodies) can inhibit the serological activity of some Rh saline agglutinins but not others.

324

SYDNEY COHEN AND RODNEY R. PORTER

FIG.13. Reaction of normal human IgC ( 1) and normal human IgM ( 2 ) in ( a ) , left, with rabbit antihuman IgC ( 7 ) and, right, with the same antiserum after absorption with IgC B chain, in ( b ) , left, with rabbit antihuman IgM (19) and, right, with the same antiserum after absorption with IgC B chain (19); in ( c ) , left, with rabbit anti-IgC B chain ( B ) and, right, with rabbit anti-IgC A chain ( A ) (Cohen, 1963b).

STRUCTURE AND ACTIVITY OF IMMVNOGLOBULINS

325

The fact that some antisera used in these studies reacted only with the antibodies of single individuals suggests that their specificity is not directed against the combining sites of the antibodies used for immunization. Kunkel et al. (1963) found that the individual specificity of the human antibodies was associated with the S fragment isolated after papain digestion; in addition, when the antibodies with individual specificity were reduced, they showed particularly sharp banding of B chains on starch-gel electrophoresis (Kunkel et al., 1963). These findings suggest that the determinants responsible for the observed individual specificity may be localized on B chains, which, as shown above, form part of the S fragment. It is of interest that individual specificity has previously been found on myeloma proteins (Slater et al., 1955), macroglobulins (Habich and Hassig, 1953; Korngold and van Leeuwen, 1957), and “monoclonal y-globulins” ( Mannik and Kunkel, 1963c), all of which are known to have characteristically homogeneous B chains (Poulik and Edelman, 1961; Cohen and Porter, 1964). In considering the implications of these experiments which demonstrate individual specificity on antibodies and pathological immunoglobulins, it is worth bearing in mind that studies on insulin (Berson and Yalow, 1961, 1963) and ribonuclease (Mills and Haber, 1963) have shown that proteins, which are apparently identical in chemical structure, may yet have distinct antigenic properties. It seems likely from the known heterogeneity of the constituent peptide chains that the antigenic structure of all antibody preparations will prove to be extremely complex. The experiments outlined above provide evidence for the antigenic complexity of human B chains which are known to occur in two distinct antigenic forms. In several species, B chains are separable by electrophoresis on urea-glycine starch gels into multiple components which probably differ in amino acid composition and can be expected to have distinct antigenic properties; several BenceJones proteins have recently been shown to possess such antigenic individuality (Stein et al., 1963). The significance of the multiple forms of the B chain is not understood, and it will be a matter of great interest to establish whether the B chain occurs in two fundamentally distinct antigenic forms in species other than man.

B. ALLOTYPESOF IMMUNOGLOBULINS During recent years it has become apparent that in several species, including man, rabbit, mouse, guinea pig (Benacerraf and Gell, 1961 ), baboon (Kelus and Moor-Jankowski, 1962), and chimpanzee (Podliachouk, 1959; Boyer and Young, 196l), there occur individual variants of

326

SYDNEY COHEN AM) RODNEY R. PORTER

immunoglobulin which can be differentiated on the basis of serological differences. These variants, which Oudin ( 1956) named allotypes, were first recognized by injection of specific precipitates of rabbit antibody together with Freund’s adjuvant into other rabbits. When the recipients carried a different allotypic specificity from that of the donor, they responded by producing isoantibodies which precipitated with immunoglobulin from the donor rabbit. With the use of gel-diffusion techniques it has been possible to demonstrate six allotypic specificities in rabbit IgG and, according to an agreed terminology (Dray et al., 1962), these are now referred to as A1 to A6 (Table VI).The specificities Al, A2,and A3 appear to be controlled TABLE VI NOMENCLATURE FOR ALLOTYPIC SPECIFICITIES IN RABBITIgG Previous nomenclature Present nomenclature A1 A2 A3 A4 A5 A6

Dubiski and Kelus

Dray and Young

J

a

E B D F

g f

c

-

Oudin b C

d

A

L

K I1 I

by three allelic genes at one locus ( a ) , and A4, A5, and A6 by three allelic genes at a second locus (b). Specificities which are systematically found together and are, therefore, probably controlled by the same allele or by closely linked genes, are designated with the superscripts “prime” or “double prime,” e.g., Al, Al’, and Al” (Oudin, 1960a,b; Dray and Young, 1961; Dray et a,?.,1963b). The available data indicate that the a and b loci are not closely linked and are not sex-linked (Dray and Young, 1960, 1961; Dubiski et al., 1962; Dray et al., 1963b). Individual rabbit sera always contain at least two allotypes, but animals heterozygous at one or both loci may have three or four allotypes. Dray et al. (1963b) have recently shown that rabbit IgC carries two additional allotypic specificities, P and T. The genetic control of the former was investigated and evidence obtained for the presence of a single gene at a third locus distinct from a and b. The distribution of allotypic specificities on various purified antibodies was studied by Gel1 and Kelus (1962). In the case of a heterozygous rabbit (Al-A3, A4-A5), the specificity A5, which is determined by the b locus, appeared to be eliminated from an antihapten antibody, while

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

327

A3, determined by the a locus, was also probably absent. A similar absence of some allotypic determinants from specific antibodies is indicated by the fact that anti-T2 phage antisera from heterozygous rabbits (A1,4,5) retain phage-neutralizing activity after addition of excess anti-A5 antiserum (Stemke, 1963), but are inactivated by whole anti-B chain antiserum. Rieder and Oudin (1963), on the other hand, found that in heterozygous rabbits purified antibodies against ovalbumin, dinitrophenol, and Type I1 pneumococcal polysaccharide contained all the allotypic specificities of the animal; however, the relative concentrations of the allotypes varied considerably in different antibodies produced by the same rabbits. Oudin (1961, 1962) showed by agar tube analysis of individual sera that the nonallelic A l and A6 were present on the same molecules, whereas the allelic forms, A1 and A3, were on different molecules. The distribution of allotypic specificities on individual IgG molecules has been further clarified by the development of a method of analysis which is based upon the successive precipitation of specific allotypes from Ilal-labeled IgG with monospecific antiallotype sera ( Dray and Nisonoff, 1963). This method was used to study the contribution of allelic genes At,* and Ab6 to the formation of IgG in heterozygous rabbits. In these animals about 65% of 1131-labeled IgG was precipitated by anti-A4 sera and about 25% by anti-A5; since the observed proportions were independent of the order of precipitation it is clear that A4 and AS were carried on separate molecules (Dray and Nisonoff, 1963). About 1040% of I'*l-labeled IgG was not precipitable by either antid4 or anti-AS; from subsequent experiments (Dray et al., 1963a,b) it seems probable that such molecules carry one or more of the allotypic specificities defined at other genetic loci. The contribution that genes at the a and b loci make to the formation of IgG was studied by the same method of immune precipitation and also by gel diffusion (Dray et al., 1963a). From these studies it is clear that in double homozygotes (Al, A5) as well as in double heterozygotes ( Al-A3, A4-A5 ) the two nonallelic, allotypic specificities, A1 and A5, occur on separate molecules as well as on the same molecule. The proportions of A1 molecules associated with A4 and AS, respectively, were found to be sufficiently close to the proportions of A4 and A5 in the total population to suggest that the formation of individual molecules involves a random association of nonallelic specificities. In cases in which A1 and A4 were under the control of genes derived from different parents, the hybrid molecules of the offspring have an antigenic specificity not present in either parent; results previously obtained by Oudin (1962) had suggested this possibility. It is clear from

328

SYDNEY COHEN AND RODNEY R. PORTER

these studies in the rabbit that nonallelic genes contribute to the formation of single antibody molecules, whereas allelic genes do not. Among laboratory animals, mice, which exist in a variety of inbred strains, are likely to prove particularly valuable for studies on the genetic control of antibody synthesis. Kelus and Moor-Jankowski (1961) first demonstrated the presence of an allotypic form of Ig in BALB/c mice; in these experiments antisera were obtained by immunization of C57BL recipients with P r o t m oulgaris coated with BALB/c antibody. The isoantigen present in BALB/c mice was subsequently designated MuAl (Dubiski and Cinander, 1963); this was found to be absent from the immunized strain (C57BL), but was identified in three other strains. By immunizing BALB/c mice, another isoantigen (MuA2) has been found in C57BL and SJLmice (Dubiski and Cinander, 1963). What is probably the same isoantigen (referred to as Gg-2), has been identified by Wunderlich and Hertzenberg (1962) in all the C57BL strains tested, as well as in the sera of several other strains; the study of segregation ratios indicated that this specificity is under the control of a single gene. The specificities MuAl and MuA2 have been identified by Dray et a2. (1963~) using isoantibodies harvested from peritoneal fluid exudates after comparatively brief periods of immunization. A single allotypic specificity was present in each of forty-two inbred strains studied; the inheritance of these specificities appeared to be determined by codominant, autosomal alleles. Individual variants of human Ig have been distinguished from one another by differences in their ability to inhibit the agglutination of sensitized cells by sera containing substances serologically related to rheumatoid factors. Since the time this technique was introduced by Grubb (1956), several genetically determined types of human Ig have been described. Of these Gm( a ) (Grubb and Laurell, 1956), Gm( b ) (Harboe, 1959), Gm(x) (Harboe and Lundevall, 1959), Gm( r ) (Brandtzaeg et al., 1961), and probably also Gm( p ) ( Waller et al., 1963) are determined by genes at one locus (Gm). In Caucasians, genes controlling the production of Gm(a) and Gm(b) behave as alternate alleles (Harboe, 1959), whereas among Negroes, these factors appear to be produced by a single allele ( Gmab) (Steinberg et al., 1960a). Two alleles at an independent locus ( InV) determine the factors InV( a ) and InV( b ) (Ropartz et al., 1961; Steinberg et d.,1962). The seventh factor, Gm-like (Steinberg et al., 1960b) is found in Negroes, but is extremely rare in other racial groups; this locus is independent of the InV locus in population studies (Steinberg, 1962), but its relationship to the Gm locus is uncertain because all Negroes are Gm (a+ b+). The work on human

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

329

allotypes has been impeded by the fact that some typing sera have been derived from individual patients and have, therefore, had a limited and transient availability; interest, therefore, attaches to the attempts which are being made to use experimental animals to raise antisera which will distinguish allotypic variants of human immunoglobulins. There is some evidence that isolated human antibodies may not contain all the genetic characters of the total Ig fraction of the individual; thus, Harboe ( 1 9 0 ) found that many human anti-D sera from Gm( b+) individuals did not appear to carry Gm( b ) specificity. Studies of myeloma proteins which are generally regarded as products of individual cell clones, have provided further information about the probable distribution of allotypic specificities on separate human IgG molecules (Fahey and Lawler, 1961; Franklin et d.,1962; Mlrtensson, 1961; Harboe et al., 1962a). It has been shown that myeloma proteins belonging to the IgG fraction may have both Gm and InV specificities, but in heterozygous individuals these proteins do not carry more than a single allelic form of either specificity (Mlrtensson, 1961; Harboe et al., 1962a); the Gm (a+ b+) myeloma proteins found by Fahey and Lawler (1961) were apparently contaminated with small amounts of normal immunoglobulin present in the serum. Myeloma proteins may be Gm (a+ x+) (Mhtensson, 1961; Harboe et al., 1962a), but this is to be expected since the genetic studies have shown that Gm(a) and Gm(x) are controlled by a single allele or by two closely linked genes. These findings indicate that, as in the case of the rabbit, nonallelic genes contribute to the formation of individual human antibody molecules, whereas allelic genes do not. The structural basis of allotypic specificity is unknown, but in the case of the B chain this specificity is probably not associated with a carbohydrate moiety. The distribution of distinct specificities on different types of immunoglobulin and on molecular subunits has been investigated. Studies on myeloma proteins have shown that the Gm factor is associated only with human I@ and is not found on IgA or IgM, whereas InV specificity occurs on all types of immunoglobulin (Fahey and Lawler, 1961; Mlrtensson, 1961; Franklin et al., 1962; Harboe et al., 1962a). The Gm specificity of IgG is localized on the F fragment of the molecule obtained by papain digestion (see above) and the S fragment carries the InV determinants (Franklin et al., 1962; Harboe et al., 1962b). Fleischman et al. (1962) showed that papain piece I11 of rabbit IgG, which is equivalent to human F, consists only of A chain; this suggested that Gm determinants must be carried by A, and InV specificity by B chains. This distribution of specificities was subsequently confirmed by Lawler and

330

SYDNEY COHEN AM) RODNEY R. PORTER

Cohen (1965) who found that Gm and InV specificities of normal human IgG are confined to A and B chains, respectively (Cohen, 1963a). The distribution of InV specificity on the two distinct antigenic types of normal human B chain has not been established. Studies on Bence-Jones proteins, which appear to be composed of B chains (Edelman and Gally, 1962; Schwartz and Edelman, 1963), showed that InV specificity is frequently present on proteins of Type I, but is very rarely found on Type I1 Bence-Jones proteins (Franklin et al., 1982; Harboe et al., 1962a). However, Harboe et al. (1962a) found that InV factors occur in both Group I and Group I1 myeloma proteins. This indicates that InV specificity is associated with B chains of both antigenic types and that Bence-Jones proteins, in some cases, are not identical with the corresponding myeloma B chains. It is evident from the above discussion that the autosomal locus, InV, determines the synthesis of the polypeptide chain ( B ) which appears to be common to all types of immunoglobulins, whereas the second autosomal locus, Gm, controls the synthesis of the polypeptide chain ( A ) which is specific for IgG. The IgA and IgM fractions also have specific A chains and are presumably determined by two additional genetic loci; polymorphisms corresponding to these have not been identified; however, their separate genetic control is suggested by familial cases of agammaglobulinemia having normal or increased levels of IgM (Burtin, 1961; Rosen et al., 1961; Fudenberg et al., 1963), by the absence of IgA in the relatives of some agammaglobulinemics ( Fudenberg et al., 1963) , and also by cases in which IgA and IgM are absent but IgG is present in normal or increased amount (Barandun et al., 1959; Giedion and Scheidegger, 1957). In contrast to results obtained with human allotypes, the rabbit specificities determined by the two genetic loci, a and b, appear to be present on all types of immunoglobulin. Todd (1963) fractionated rabbit serum by gel filtration on Sephadex G-200and showed that the macroglobulin peak contained A1 and A4 specificities; this peak failed to react with antisera to piece I11 and was, therefore, not significantly contaminated with IgG (Fig. 14). On the other hand, Feinstein et al. (1963) using a gel-diffusion method, were unable to detect A3 determinants and obtained doubtful reactions for A1 and A2 in macroglobulin preparations from rabbit sera which contained all these specificities. An immunoglobulin fraction, which appeared to have a faster electrophoretic mobility than IgM and was antigenically distinct from I@, has been isolated from rabbit colostrum; this fraction, which may correspond to human IgA, was shown to carry allotypic specificities determined by

STRUCTURE AND ACTIVITY OF IMhEUNOGLOBULINS

331

allelic genes at both the a and b loci (Feinstein, 1963). After papain digestion of rabbit I@, the allotypes determined by both genetic loci are found on pieces I and I1 (Kelus et al., 1960; Marrack et al., 1962; Feinstein et al., 1963). The isolated rabbit B chain is associated only with allotypes determined by the b locus (Kelus, 1963; Feinstein et d.,1963). Allotypic specificities determined by the a locus would, therefore, be expected to occur on the A chain; it seems likely that these determinants are on that portion of the A chain ( A piece) which is present in papain 3.5.

61%

2 3.0.

-: 0

2

0 ._ c

f

2.52.0 I .o

0.5

0

400

BOO

1200

1600

2000

2400

Volume of eluate(mls.)

FIG.14. Optical density at 280 mv of the effluent from the G-200 filtration of 15 ml. of A1,4 rabbit serum on a Sephadex G-200 column; bed volume, 2100 ml. Shaded areas indicate fractions that gave positive tests for both allotypic specificities. Percentages at the top of each peak represent the per cent of the total optical density units (OD x volume) found in each peak. The fraction at the center of the first peak give a positive test for A1 at a dilution of 1/4 and for A4 at a dilution of 1/8.It was negative for 111 specificity. The fraction at the center of the second peak gave a positive test for A1 and A4 at a dilution of 1/64 and for 111 specificity at a dilution of 1/256 (Todd, 1963).

pieces I and I1 and may be common to IgG, IgA, and IgM. Experiments reported to date have shown, however, that the A chain contains specscities determined by both a and b loci and this may indicate that preparations of rabbit A are contaminated by B chain (Kelus, 1963; Feinstein et al., 1963). C. URINARYEXCRETION OF IMMUNOGLOBULIN FRAGMENTS The urine of normal subjects contains small amounts of relatively low molecular weight proteins which have antigenic determinants in common with serum immunoglobulins (Webb et d.,1958; Franklin, 1959; Stevenson, 1960; Berggard, 1961; Rowe and Soothill, 1961; Cornillot et ul., 1963). These proteins are antigenically related to the S fragment of IgG

332

SYDNEY COHEN AND RODNEY R. PORTER

and carry determinants corresponding to Types I and I1 Bence-Jones proteins (Hanson and Berggard, 1962; Stevenson, 1962; Fahey, 1963a). These findings suggest that urinary immunoglobulin fragments may be composed of B chains. Berggard and Edelman (1963) have demonstrated the close similarity between immunoglobulin fragments present in normal urine and B chains in regard to antigenic structure and thermosolubility; in addition, the molecular weight of the urinary immunoglobulin fragment determined by equilibrium sedimentation was 25,000, which agrees approximately with the value for the B chain monomer. Using antisera specific for A and B chains of IgG, Cohen (1964) found that a normal subject excreted daily 5-10 mg. of a protein which carried only B chain determinants. What appears to be an unrelated antibody fragment has been recovered from the urine of subjects immunized with poliovirus vaccines or with tetanus toxoid (Remington et d.,1962; Merler et al., 1963). Concentrates of normal urine were fractionated by chromatography on diethylaminoethyl cellulose columns followed by centrifugation in a sucrose gradient. The top fraction of the sucrose gradient contained a protein of molecular weight about 13,000 which was antigenically related to serum IgG and was able to precipitate with the appropriate antigen. This fragment gave a pattern of tryptic peptides which showed little overlap with those of serum IgG, and its relationship to circulating antibody is not clear. Rowe (1963) was unable to confirm these findings in studies on the urine of a normal subject immunized with typhoid vaccine; in this instance fractionation on a Sephadex G-200 column showed that urinary antibody activity was apparently confined to whole immunoglobulin and could not be detected in smaller fragments. The origin of urinary immunoglobulin fragments has been studied in radioactive-labeling experiments. The results of Franklin ( 1959) and Webb et d. (1958) indicated that these fragments were derived from the degradation of normal immunoglobulin. However, other studies (Stevenson, 1962) have shown that the urinary proteins which probably correspond to B chains arise as precursor or by-products of immunoglobulin synthesis and correspond in this respect to Bence-Jones proteins.

D. TRANSFER OF ANTIB~DIES FROM MOTHERTO FETUS In a paper written in 1892, Ehrlich provided detailed experimental evidence for the occurrence of a passive transfer of antibodies from mother to offspring both in utero and during suckling. These early experiments were carried out in mice, and it has since become apparent that the mechanism of antibody transfer from mother to her offspring

STRUCX'URE A N D ACTIVITY OF IMMUNOGLOBULINS

333

varies considerably in different species. In the pig, horse, and goat, immunoglobulin is present only in trace amounts at birth and is absorbed from the colostrum during the first 2 days of life. Human subjects, on the other hand, as well as rabbits, guinea pigs, and monkeys, acquire maternal antibodies only during prenatal development ( Hemmings and Brambell, 1961). Immunoglobulin begins to appear in the human fetal circulation at about the fourth month of pregnancy and the level rises progressively until it reaches that of the mother at term. The transfer of IgG takes place across the pIacenta in the rhesus monkey and probably also in human subjects (Bangham, 1960); in the rabbit, transfer is effected by way of the uterine cavity and the vascular, fetal, yolk sac splanchnopleure. Transmission across the fetal membranes is a highly selective process since IgG is transmitted preferentially to the other serum proteins, homologous IgG is transferred more readily than heterologous protein (BrambeIl, 1958) and human IgG is freeIy transferred, whereas IgA and IgM are not (Hitzig, 1957; Franklin and Kunkel, 1958; Gitlin et d., 1963). This implies that transmission is dependent on specific structural features of the molecule. Investigations using the papain fragments of rabbit IgG have shown that piece 111, isotopically labeled with 1131, is transmitted across the membranes of the fetal circulation of the rabbit eleven times more rapidly than piece I and six times more rapidly than piece 11; piece I11 was transmitted at 70% the rate of labeled IgG (Brambell et d.,1960). Experiments in suckling mice and rats similarly indicate that piece I11 is involved in the transmission of antibodies across the gastrointestinal mucosa during neonatal life (Morris, 1963). The human placenta during the last month of pregnancy is freely permeable to the F fragment, but evidence was also obtained for transmission of the S fragment of IgG (Gitlin et al., 1964). The chemical configuration required for the transmission of intact human antibodies across fetal membranes may be associated primarily with the portion of the molecule equivalent to rabbit piece 111. If this were so, the observation made by Hartley (1951) that peptic digestion of horse diphtheria antiserum destroys its ability to pass from mother to fetus, would be explained, since pepsin destroys piece I11 and leaves an active fragment consisting of I and 11. As mentioned above, piece I11 is composed of part of the A chain which has different properties in the case of IgG, IgA, and IgM. Failure of the latter two human immunoglobulins to cross the fetal membranes may, therefore, be due to the absence of the necessary transmission site from their respective A chains. In the case of the rabbit, the transfer of IgM agglutinins from mother to fetus has been reported

334

S M N E Y COHEN AND RODNEY R. PORTER

(Hemmings and Jones, 1962); this suggests that the transmission site may be present on both IgG and IgM molecules in this species.

E. FIXATION OF ANTIBODYTO SKIN The antibodies that mediate certain hypersensitivity reactions have the property of fixing to skin and other tissues. The technique of passive cutaneous anaphylaxis (PCA) has been used to investigate the skinattaching properties of various antibodies in the guinea pig (Ovary, 1958). In the PCA reaction, antiserum is injected intradermally and is followed by intravenous injection of antigen together with a blue dye; a positive reaction is shown by diffusion of dye through a localized area of increased permeability. Ovary et al. (1980) showed that human 7 S antibodies are able to elicit a positive PCA test in guinea pigs, but 19 S antibodies of the same specificity gave a negative response, apparently owing to an inability to fix to guinea pig tissue. Subsequent studies have shown that normal human IgA and nine different IgA myeloma proteins, belonging to the two major antigenic types, were all unable to sensitize guinea pig tissues (Franklin and Ovary, 1983). These studies indicate that of the human immunoglobulins only IgG can sensitize the skin of the guinea pig. This suggests that the attachment site for guinea pig skin is carried on the IgG A chain, since as shown above, this chain is associated with the antigenic and allotypic determinants specific for IgG. This idea is supported by experiments using the fragments of rabbit antibodies; it was shown by the reverse PCA reaction that the site responsible for attachment to guinea pig skin is localized on piece I11 (Ovary and Karush, 1961) and, as shown above, this part of the molecule consists only of a part of the A chain. Similarly, Liacopoulos et al. (1963) showed that the Schultz-Dale reaction of guinea pig tissues to rabbit antibody can be inhibited by piece I11 of nonimmune IgG, whereas piece I has no inhibitory effect. Thus, as pointed out by Brambell (1983), the same section of the IgG molecule is concerned in skin fixation and in the passive transfer of immunity from mother to offspring. In the case of human IgG the site necessary for skin sensitization appears to be lost during papain digestion, although the in vivo antibody activity of S fragments separated by the same procedure was retained (Franklin and Ovary, 1983). Antibodies that occur spontaneously in the sera of allergic subjects with immediate-type hypersensitivity are distinguished by their apparent ability to become rapidly and firmly attached to human epithelial tissues. These antibodies, which are referred to as reagins, are detected by the passive transfer of wheal and erythema reactivity (the P-K reaction). No

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satisfactory means is available for their quantitative estimation since specific antigens (allergens) do not produce any detectable in uitm interaction with reaginic sera, probably as a result of the extremely low levels of antibody present. Chan (1963) has developed a method for estimating the concentration of reaginic antibody in sera from subjects showing immediate-type hypersensitivity to horse serum albumin. The capacity of reaginic sera to bind highly purified samples of horse albumin labeled with 1131 was determined by electrophoresis on cellulose acetate membranes. The total concentration of albumin-binding antibody was calculated from the weight of antigen bound per unit volume of serum assuming an antibody-antigen weight-combining ratio of 10:1. The concentration of heat-labile (reaginic) antibodies was less than 30 pg./ml. in the subjects tested so that the total circulating plasma always contained less than 65 mg. of antibody; the difficulty of obtaining sufficient human reaginic antibody for detailed structural studies is apparent from these results. In addition, it was found that the degree of sensitivity shown by subjects reacting to horse albumin was unrelated to the concentration of reaginic antibody and was apparently determined by the relative proportions of heat-labile (reaginic ) and heat-stable (blocking) antibodies. For example, two subjects whose sera were equally active, as judged by P-K testing, had concentrations of 0.8 and 29 pg. of reaginic antibody per milliliter serum; the levels of blocking antibody were 1.5 and 66 pg./ml., respectively, so that the ratio of blocking to reaginic antibody was about 2 : l in each case. It will be a matter of great interest to establish whether a similar relationship between clinical reactivity and the ratio of blocking-to-reaginic antibody concentration is observed with other purified allergens. Human reaginic antibodies do not have the ability to sensitize the guinea pig for the PCA reaction (Augustin, 1955) and they do not cross the placental barrier in allergic mothers (Bell and Eriksson, 1931; Sherman et al., 1940). Since human IgG is able to sensitize guinea pig skin and is transmitted freely into the fetal circulation, these findings suggest that reaginic antibodies do not belong to the IgG fraction. Evidence which suggested that reaginic antibody is associated with the IgA fraction of serum (Augustin, 1961; Heremans and Vaerman, 1962) could not be regarded as conclusive because of the difficulty of isolating IgA in pure and uncontaminated form. More recently, however, Fireman et al. (1963) showed that the specific removal of IgA by immune absorption eliminated all detectable skin-sensitizing activity from the sera of three ragweed-sensitive individuals, whereas similar absorption of IgG had no detectable effect. Antibodies to I13'-labeled ragweed

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antigen have been shown by immunoelectrophoresis to be associated with IgG in the sera of eight ragweed-sensitive subjects. Antibody activity was also present in the IgA fraction of seven cases and in the IgM of two subjects; the biological activity of these various antibody fractions was not determined (Yagi et al., 1963). In view of the controversy which exists in regard to the molecular weight and electrophoretic behavior of reagins, it seems probable that the distribution of these antibodies among the immunoglobulin subfractions may vary in different sera. Their association with IgA is of particular interest since this would account for the fact that reagins which often appear to have a sedimentation coefficient of 7 (Stanworth, 1963) fail to cross the placenta and cannot sensitize guinea pig skin for the PCA reaction. The presence in IgA of reagins that sensitize human skin suggests that different molecular sites are involved in the attachment of human antibodies to the tissues of different species. In this connection it is of interest that a study of guinea pig immunoglobulins has shown that a 7 S yz-globulin fraction was unable to elicit the PCA reaction in guinea pigs, whereas a 7 S yl-globulin which may be analogous to IgA of other species effectively sensitized guinea pig skin (Ovary et d.,1963).

F.

COMPLEMENT

FIXATION

Complement fixation is known to occur with antibodies belonging to both IgG and IgM fractions, but the few IgA antibodies which have been studied have been inactive in this respect. Thus, antibrucella agglutinins in human IgG and IgM were able to fix complement, but IgA antibodies of the same specificity lacked this capacity (Heremans et al., 1963). Similarly, complement-fixing activity has been demonstrated on guinea pig 7 S yz-globulins but not on 7 S yl-globulins which may be analogous to the human IgA fraction (Bloch et al., 1963). Complement is fixed by IgG molecules not only upon reaction with antigen, but also after nonspecific aggregation by heat (Taranta and Franklin, 1961; Amiraian and Leikhim, 1961; Ishizaka et al., 1962). IgG treated with 0.1 M mercaptoethanol and subsequently dialyzed against iodoacetamide can still combine with antigen and is aggregated by heat, but cannot fix complement ( Wiedermann et al., 1963). These results indicate the importance of disulfide bonds in the complement fixation reaction; it is also apparent that loss of complement-fixing ability after treatment with sulfhydryl reagents cannot be regarded as a valid means of identifying antibodies as belonging to the IgM fraction. Several attempts have been made to establish which part of the

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immunoglobulin molecule is involved in complement fixation. Taranta and Franklin (1961) found that a peptic digest of rabbit antiovalbumin or anti-bovine serum albumin would not bind complement when precipitated by addition of antigen in the presence of fresh guinea pig serum. Since peptic digestion destroys a part of the molecule equivalent to piece 111, the result suggested that this piece carried complement-fixing sites. In agreement with this is the demonstration that piece 111 aggregated by coupling with bisdiazotized benzidene or by heating is able to bind complement (Ishizaka et aZ., 1962). Similarly, piece I11 has been shown to inhibit immune hemolysis of sheep erythrocytes primarily by interacting with guinea pig complement, whereas peptic digests of normal and antipertussis IgG are not inhibiting ( Amiraian and Leikhim, 1961); however, in the latter experiments some complement fixation occurred with peptic digests of rabbit anti-sheep erythrocyte IgG in the presence of the antigen. Further evidence that the pieces ( I and 11) that survive peptic digestion may be involved in complement fixation comes from the experiments of Schur and Becker (1983a,b); washed specific precipitates obtained by adding antigen to peptic digests of rabbit or sheep antibodies fixed complement when added to fresh guinea pig serum. The 5 S component precipitated with antigen absorbed up to 40% of the complement which was fixed by a precipitate of whole IgG and antigen. However, the absorption of complement did not occur if the precipitation of the 5 S antibody fragments was carried out in the presence of guinea pig serum, and no explanation of this anomalous behavior was found, Reiss and Plescia (1963) provided evidence that complement was present on the papain fragments I and I1 but not on piece I11 when these were separated from ovalbumin antiovalbumin precipitates which had fixed human serum complement prior to enzymatic digestion; localization of complement was based upon the interaction of the papain pieces with a rabbit antiserum to human serum. Direct evidence that all three pieces of the antibody molecule may be involved in complement fixation was provided by Cebra (1963). He showed that rabbit antibody which has been split by insoluble papain, but not dissociated by thiol, precipitates with antigen and binds complement as effectively as the native antibody. When thiol is added to the specific precipitate carrying bound complement, only a partial dissociation occurs suggesting that the antibody is held together by components of complement bound to all parts of the molecule. The conclusion at present, therefore, seems to be that piece 111 is predominantly concerned in the binding of complement to antibody reacted with antigen, but that other parts of the molecule are also involved in this process.

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G. DISTRIBUTION AND TURNOVER OF IMMUNOGLOBULINS

Estimations based on immunological techniques have shown that the immunoglobulins comprise about 20% of the total protein in human serum. The average normal concentrations of IgG and IgM are about 1250 and 125 mg./100 ml. serum, respectively (Soothill, 1962; Fahey and Lawrence, 1963; Chodirker and Tomasi, 1963); estimates of the mean normal level of IgA have varied from about 150 (Heremans, 1960; Chodirker and Tomasi, 1963) to 400 mg./100 ml. serum (Fahey and Lawrence, 1963). The total IgG of the body (about 80 gm.) in healthy adults is distributed equally between the circulating plasma and the interstitial fluids. About 25% of the circulating IgG fraction passes across capillaries into the extravascular fluids each day, and a similar amount is returned to the bloodstream through the main lymphatic ducts (Cohen, 1963d). A fluorescent antibody technique has shown that extravascular immunoglobulin is present in all extracellular fluids as well as in the ground substance of connective tissue (Gitlin et al., 1953). The concentration of immunoglobulin in the interstitial fluids varies considerably in different sites, being highest in the hepatic lymph (about 1 gm./100 ml.) and relatively low (200 mg./100 ml.) in the interstitial fluids of muscle and subcutaneous tissue (Gitlin and Janeway, 1954). The distribution of IgM differs from that of IgG in that only a small proportion of the total pool is present in extravascular tissues (Cohen and Freeman, 1960; Wochner et al., 1963). The IgA fraction appears to be present in relatively high concentration in human milk (Hanson, 196l), as well as in saliva and tears (Tomasi and Zigelbaum, 1963; Chodirker and Tomasi, 1963), suggesting that this protein may have a specific transmission site responsible for secretion into these biological fluids. All plasma protein fractions, including the immunoglobulins, are in a state of dynamic equilibrium undergoing constant degradation and replacement by newly synthesized molecules. A homogeneous protein, such as human albumin, when labeled with 1131has a constant rate of breakdown (measured by urinary excretion of label) over a period of several weeks. On the other hand, the fractional catabolic rate of P31labeled human immunoglobulin prepared by zone electrophoresis falls progressively during the first 1 or 2 weeks after injection, suggesting the presence of a mixed population of molecules having different breakdown rates. This metabolic heterogeneity appears to be attributable mainly to differences in the turnover rates of 19 and 7 S fractions. Thus, human IgM isolated by ultracentrifugation and zone electrophoresis (Cohen and Freeman, 1960) or by electrophoresis and gel filtration (Wochner et al.,

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1963) has a relatively high turnover rate, whereas IgG is catabolized at a slower rate; a similar digerence in the half-lives of small and large molecular weight antibodies has been reported in rabbits (Taliaferro and Talmage, 1956). Little is known about the site and mechanism of immunoglobulin catabolism. On the basis of labeled protein studies it has been suggested that plasma cells which are known to be involved in antibody synthesis may also be responsible for immunoglobulin breakdown (Soons and Westenbrink, 1958).However, perfusion experiments in which biologically screened proteins are used show that the normal rat liver, which is not a site of antibody synthesis, catabolizes IgG at a rate equivalent to 30% of the total breakdown in vivo (Cohen et al., 1962). The fractional breakdown rate of IgG can be increased in the mouse by infusing large amounts of either IgG or piece I11 derived from it, but not by injecting IgA or IgM; the removal of circulating protein by a process such as pinocytosis cannot easily account for such selectivity, and the presence of specific mechanisms controlling immunoglobulin breakdown appears likely (Fahey and Robinson, 1963). Normal subjects synthesize and break down about 2 gm. of IgG and 0.5 gm. of IgM per day, but in pathological conditions associated with hypergammaglobulinemia the absolute rate of immunoglobulin turnover may be increased as much as sevenfold (Cohen, 1963d; Birke et al., 1963; Solomon et al., 1963; Wochner et al., 1963). Increased rates of immunoglobulin formation presumably result from the replication of antibodyproducing cells but are not necessarily associated with an enhanced response to antigenic stimulation; for example, African children with hypergammaglobulinemia often show a relatively poor response to immunization with tetanus toxoid ( McGregor and Barr, 1962).

H. SYNTHESIS OF ANTIBODIES The structural studies described above indicate that the chains of immunoglobulin molecules are extremely heterogeneous. In particular, the polypeptide chain which carries the antibody-combining sites must occur in a very large number of different forms. The question of how many of these immunoglobulin chains can be synthesized by individuaI cells cannot be answered at present. The in vitro production of antibody by singIe lymph node cells indicates that a single cell can synthesize both A and B chains and assemble these into the complete antibody molecule. The possibility that some cells may synthesize B chain only i s suggested by the observation that Bence-Jones proteins sometimes have a difFerent InV speciscity from the corresponding myeloma protein; this

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suggests that at least under pathological conditions the two proteins may originate in different cell clones (Harboe et d.,1962a). Myeloma proteins always belong to one or other of the three immunoglobulin types; since each type has a distinctive A chain, this suggests that the A chains of IgG, IgA, and IgM are synthesized in separate cells. Similarly, the relative homogeneity of myeloma B chains described above (Fig. 10) supports the idea that different forms of the normal B chains are derived from distinct cell types. In addition, analysis of the allotypic specificities of myeloma proteins suggests that individual cells synthesize molecules which have only a single form of allelic specificity (Harboe et al., 1962a). A similar conclusion is suggested by the finding that young rabbits whose mothers were immunized against a given allotypic specificity showed a diminution in molecules of that specificity, but no decrease in the molecules of an allelic specificity (Dray, 1962). On the other hand, immunofluorescent studies of intracellular y-globulin in the rabbit have shown that two allelic forms of the allotypic specificity determined by the b locus (A4 and A5) may be present in the same cell (Colberg and Dray, 1983). Since these allelic forms do not occur on the same molecule, Dray and Nisonoff (1963) suggested that chains controlled by a single gene form pairs immediately after synthesis. There is now a considerable amount of information about the antibody-forming capacity of individual cells, but it has not been possible to reconcile the different results which have been obtained. Nossal and collaborators (Nossal, 1962) found that cultures of single cells taken from rats immunized with a mixture of antigens almost invariably form detectable amounts of only one antibody; less than 2% of the cells tested were found to be doubly active. Attardi et al. (1959, 1984) also found that the majority of individual rabbit lymph node cells produced antibacteriophage antibody of a single specificity. However, in these experiments 10% of the cells studied were shown to form two antibodies; this incidence is considerably greater than would be expected if each diploid cell formed only two different antibodies. Thus, Attardi et al. (1964) point out that if an animal can make a total number of antibodies, S, and each cell can synthesize only two randomly distributed specificities, then the expected ratio of cells responding to one antigen, as compared to those responding to two antigens, would be 1/S. If, as is generally whereas the observed ratio was l O - l , assumed, S > lo4 then 1/S < i.e., the incidence of doubly active cells was at least a thousand times greater than expected. Unless immunization is associated with a selective proliferation of doubly active cells, then these results lend support to the view that individual antibody-forming cells are pluripotential.

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At present, therefore, it is not known whether every antibody-forming cell carries a very large number of genes controlling immunoglobulin synthesis, or has a limited genetic potential. Smithies (1963) has pointed out that a considerable degree of variability in the expression of the immunoglobulin genetic loci could arise by chromosomal rearrangements between sister chromatids or between single chromatids occurring during mitotic divisions of antibody-forming cells. There is evidence that such chromosomal rearrangements have given rise to variants of human haptoglobins (Smithies et al., 1962) and hemoglobins (Baglioni, 1962; Nance, 1963), but it should be pointed out that the complexity of antibodies is of a different order of magnitude from that observed in the case of these proteins. An analysis of the amino acid sequences of individual variants of antibody chains will be required in order to show whether their structural variations could have arisen from somatic rearrangements of genes occurring during the differentiation of immunoglobulin-forming cells. V. Comments

The multichain structure of antibody molecules has been established during the past few years. Correlation of the properties of the separated polypeptide chains with those of fractions obtained by enzymatic digestion has provided a diagrammatic picture of the basic immunoglobulin molecule consisting of two A ( H ) chains and two B ( L) chains. However, certain biological considerations suggest that A is two separate chains, so that the molecule may consist of six, rather than four, peptide chains. The structural relationship between the three types of immunoglobulins has been clarified by the identification of a common pair of peptide chains; if the six-chain structure is correct, then there may, in fact, be two pairs of peptide chains common to all immunoglobulins. There has been a satisfactory allocation of antigenic and genetic markers and of many other biological activities among the different parts of the molecule. However, the position of antibody-combining sites is still in doubt, and no clear concept of the structural basis of any biological activity has been suggested, The function of the carbohydrate of immunoglobulins is entirely unknown as, indeed, is true of most other glycoproteins. The structural complexity of all the immunoglobulins becomes increasingly apparent as further information is gained. The resolution of these multiple forms and the characterization of their chemical variations may be required before the structure of antibodies can be correlated with their activity. An understanding of the biological origin of this complexity

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may well be an essential preliminary to a solution of the mechanism of antibody function. In spite of the great complexity of the problem, it is encouraging that, at last, the accumulation of factual information has become more rewarding than the rephrasing of old theories.

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Spinal cord compression due to brown tumor.

We report a rare case of a vertebral brown tumor causing spinal cord compression and resulting in progressive paraparesis in a 27-year-old female with...
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