Loss of cryoprecipitability following proteolytic cleavage of the VH domains from a human IgG cryoglobulin

0161-5890/80/01014001 $02.00/O

Moleclllar Immunology, vol.11,pp.1-7. in Great Bruin. ~Pergmon Press Ltd.1980.Printed

LOSS OF CRYOPRECIPITABILITY FOLLOWING PROTEOLYTIC CLEAVAGE OF THE VH DOMAINS FROM A HUMAN IgG CRYOGLOBULIN DOROTHY

M. PARR and THEO HOFMANN

Department of Biochemistry, University of Toronto. Toronto. Ontario. Canada M5S IA8 (Firsr received ?I

November 1978; in

revisedform

19 March

1979)

Abstract-A human lgG3/~ cryoglobulin, IgG Pav, which had been stored at 4’C for several months was found to have lost its property of gelling when cooled. Electrophoresis in starch gel in the presence of 8 M urea showed that 1gG Pav had broken down during storage into two fragments. Digestion of freshly prepared, intact 1gG Pav with plasmin or trypsin produced identical fragments. Electrophoresis, immunodiffusion and sequence analysis were used to identify these fragments as the variable regions of the heavy chains and the remainder of the molecule. IgG Pav is only the third human immunoglobulin described in the literature from which V, domains have been isolated by enzymatic digestion of the intact molecule; all three are cryoglobulins. Proteolytic cleavage in the fourth hypervariable region of the heavy chains completely removes the ability of IgG Pav to gel when cooled.

domains by enzymatic digestion of intact human immunoglobulins. The first described a fragment found in a papain digest of human IgG3/K cryoglobulin KUP (Dammacco et al., 1972), and the second described a fragment obtained by tryptic digestion of human IgG2/K cryoglobulin Zie (Laschinger & Connell, 1978). In both cases, cleavage occurred close to the C-terminus of the V, region, and appeared to be a property of the particular immunoglobulin, rather than a. property of immunoglobulins in general. A similar fragment has now been isolated from a sample of human IgG3/K cryoglobulin Pav, which had been stored at 4°C. It has also been prepared from intact IgG Par by digestion with plasmin or trypsin. This observation is of interest as it may indicate a structural difference between cryoglobulins and other immunoglobulins. Cryoglobulins are globulins which precipitate or gel reversibly in the cold. They have been known since 1933, when Wintrobe & Buell described a cold-precipitable globulin in the serum of a patient with multiple myeloma. Since then numerous studies of their properties have been made. It has been shown that they may be monoclonal or polyclonal immunoglobulins, or immune complexes, in which a monoclonal immunoglobulin has reacted with an antigen, frequently a polyclonal immunoglobulin, to form a cold-precipitable complex (Klein et al., 1972). Cryoprecipitation has been shown to be dependent upon pH, ionic strength and protein concentration, as well as temperature, but no

Ih’TRODUCTION

Fc* and Fab fragments following papain cleavage of rabbit IgG (Porter, 1959), fragments produced by enzymatic and/or chemical cleavage have been widely used in the elucidation of immunoglobulin structure. Mole et al. (1975) described a method for the preparation of fragments, consisting of most of the V, domain, by papain cleavage of rabbit Fd fragments, and Rosemblatt & Haber (1978) prepared similar fragments by tryptic digestion of rabbit heavy chains. A method for the production, by chemical cleavage, of a fragment consisting of the N-terminal 139 residues of human p chains has also been published (Rodwell & Karush, 1978), and Fv fragments, consisting of the variable regions of both heavy and light chains, have been prepared from mouse IgA (Inbar er al., 1972; Sharon & Givol, 1976) and human IgM (Kakimoto & Onoue, 1973; Lin & Putnam, 1978). Until now, however, there have been only two reports of the preparation of fragments corresponding to essentially complete VH Since

the

characterization

of

l Abbreviations used: SDS, sodium dodecyl sulphate; PBS, phosphate buffered saline: 0.05 M sodium phosphate buffer, pH 7.3, containing 0.14 M NaCI; EDTA, disodium ethylene diamine tetraacetate; abbreviations used for classes, chains, fragments and domains of immunoglobulins are in accordance with World Health Organization recommendations for human immunoglobulins [( 1972) Biochemistry 11, 331 I].

M.IMM. 17/i-~

1

DOROTHY

2

M. PARR and THEO HOFMANN

association has yet been found between cryoprecipitability and primary sequence or conformation, and the physicochemical basis of the phenomenon is unknown, MATERIALS

AND METHODS

When the serum of multiple myeloma patient Pav is cooled, IgG Pav, and IgG3/K immunoglobulin, separates out in the form of a gel. This property was used to prepare IgG Pav. Serum was chilled in ice for 30 min and a dense layer of a gel-like precipitate was obtained. The upper liquid layer was poured off the gel which was then dissolved in PBS by warming at 37°C. This procedure was repeated until examination by electrophoresis on cellulose acetate strips showed a single component. Reduction and alkylation of disulphide bonds was done in 0.5 M Tris-HCl, pH 8.2, using dithiothreitol at 30 mM for 1 hr at 23°C followed by iodoacetic acid at a final concentration of 90 m&f. Gamma and K chains were separated by gel filtration through a column (2.5 x 90 cm) of Sephadex GlOO (Pharmacia (Canada) Ltd., Dorval, Quebec), eluted with 1 M propionic acid. Proteolytic digestion

preparative scale were separated by gel filtration through a column (1.6 x 90 cm) of Sephacryl S200, using 0.1 M sodium acetate buffer, pH 5.1, containing 6 M urea as eluant. Alternatively, a column (5 x 90 cm) of Sephadex G 100 where the eluant was 1 M propionic acid was used. Protein peaks were located by monitoring the column eluates with a Uvicord II absorptiometer (LKB Produkter AB, Bromma 1, Sweden). Fractions were pooled as required, and when necessary, urea was removed by stepwise dialysis into 4 mM sodium acetate buffer, pH 5.4, prior to concentration in a Diaflo cell (Amicon Corp., Lexington, MA, U.S.A.). Dialysis steps were in this order: (i) 4 M urea/60 mM sodium acetate, pH 5.1; (ii) 2 M urea/40 mM sodium acetate, pH 5.0; (iii) 1 Murea/20 mM sodium acetate, pH 4.9; (iv) & (v) 4 mM sodium acetate, pH 5.4. Electrophoresis Cellulose acetate electrophoresis was done the Beckman Microzone system. using Electrophoresis in urea-formate starch gels and SDS polyacrylamide gels was done by the methods of Smithies et al. (1962) and Weber & Osborn (1969) respectively, as described by Parr et al. (1976).

IgG Pav ( 10 mg/ml) was digested with plasmin (AB Kabi, Stockholm, Sweden), trypsin (Type XI, Sigma Chemical Co., St. Louis, MO, U.S.A.) Worthington (mercuripapain, or papain Biochemical Corp., Freehold, NJ, U.S.A.) using enzyme: substrate ratios of 1:100 (w:w). Plasmin digests were done in PBS or in 0.1 h4 phosphate buffer, pH 7.0, trypsin digests in 0.05 M Tris-HCl, pH 8.2, and papain digests in 0.1 M phosphate buffer, pH 7.0, containing 2 mM EDTA, and, in some cases, 10 mM cysteine. All digestions were done at 37°C. Plasmin and trypsin digestions were stopped by the addition of soybean trypsin inhibitor (type I-S, Sigma Chemical Co., St. Louis, MO, U.S.A.) in not less than 10% excess by weight over the enzyme concentration. Papain digestions were stopped by the addition of 1 M iodoacetamide to give a final concentration of 25 mM. Optimal times for preparative digestions were determined by starch gel examining, by urea-formate electrophoresis, samples withdrawn at various times from digestions done under standard conditions.

Sequence determinations Amino-terminal residues were determined by the method of Percy & Buchwald (1972). Carboxy-terminal determinations were done using penicillocarboxypeptidase-S, (Hui et al., 1974). For the latter, samples were dissolved in 0.2 ml of 0.2 M pyridine acetate buffer, pH 4.7; enzyme was added to give an enzyme: substrate ratio of about 1: 100 (mol:mol). Digestion was done at 37°C and was stopped by immersing the tubes in boiling water for 5 min. Released Cterminal residues were identified using a Beckman 12OCamino acid analyser. A Beckman 890C Sequencer was used for automatic sequence determinations, using the programme described by Hermodson et al. (1972). Phenylthiohydantoin derivatives of amino acids were silylated and identified by gas-liquid chromatography. A histidine residue was identified by recovering the amino acid from its thiazolinone, using hydrogen iodide hydrolysis followed by analysis in a Beckman 121 M amino acid analyser. Some residues identified by g.1.c. were confirmed by this method, or by amino acid analysis following alkaline hydrolysis.

GelJiitration The products of proteolytic

Immunodiffusion All samples

digestions on a

for

immunodiffusion

were

Loss of IgG Cryoprecipitability

dialyzed into 4 mM sodium acetate buffer, pH 5.4. Gels were made of 1% agarose in buffer containing 24 mM glycine and 24 mM sodium chloride, pH 8.4.

RESULTS

IgG Pav freshly isolated from Pav serum gelled

readily when cooled, and showed a single component when examined by electrophoresis on cellulose acetate or in a urea-formate starch gel. A sample of IgG Pav which had been stored at 4°C however, had lost the property of gelling in the cold, and although it appeared to be homogenous when examined by cellulose acetate electrophoresis, it showed two components in a urea-formate starch gel (Fig. 1). Gel filtration of the stored IgG using dilute aqueous buffers did not separate the two components, but they could readily be separated in 1 M propionic acid or in buffers containing 6 M urea, as shown in the elution profile illustrated in Fig. 2(a).

3

after Proteolysis

Digestion of fresh IgG Pav with plasmin resulted in the formation of a fragment with electrophoretic mobility in urea-formate starch gels identical to that of the fragment in the stored IgG (Fig. 1). The yield of the plasmin fragment was at a maximum after 30 min digestion and appeared to remain unchanged up to 24 hr digestion. For further characterization the products of a 30 min plasmin digest were separated by gel filtration in 1 M propionic acid. The elution profile is il!ustrated in Fig. 2(b). When examined by urea-formate starch gel electrophoresis, peak A appeared to be normal IgG and peak B the small fragment of high mobility [Fig. 3(a)]. However, electrophoresis in polyacrylamide gels, both before and after reduction and alkylation, showed that peak A contained normal light chains joined by disulphide bonds to y chains that were shorter than those obtained from intact IgG Pav [Fig. 3(b)]. Molecular weights obtained by SDS-polyacrylamide gel electrophoresis, using cytochrome c, ribonuclease, ovalbumin and a monoclonal y 1 heavy chain as standards, were found to be 62,000 for intact y chain Pav and 50,000 for y chain Pav after plasmin digestion. The molecular weight of the small fragment was about 10,000, and was unchanged by reduction. The small fragment gave no precipitin arcs when tested by immunodiffusion against antisera to Fc fragments or to Cu3 fragment; Fc fragments in the same buffer gave positive reactions against both antisera. Both the small fragment and the intact y chain had amino-terminal glutamic acid; the Nterminal sequences of both chains are given below, and are compared with the sequence of the V,III region from protein Tei (Capra & Kehoe, 1974~). 5 Vu111 Tei y chain Pav small fragment

Glu-Val-Gln-Leu-Val-GluSer-Glyaly-Gly/ Glu-Val-Gln-Leu-Ala-Glu(+GlY-GlY-GlY/ Glu-Val-Gln-Leu-Ala-GluSer-Gly-Gly-Gly/

Origin

Fig. 1. Starch gel electrophoresis in urea-formate buffer, pH 3.6, of (A) stored IgG Pav; (B) freshly isolated IgG Pav; and (C) plasmin digest of B.

These data identify the small fragment as the Vu region of IgG Pav. Immunodiffusion, electrophoresis and Nterminal determinations showed no differences between the small fragment produced by plasmin digestion and that isolated from the stored IgG.

DOROTHY M. PARR and THEO HOFMANN

a

Fraction

number

Fig. 2 (a). Gel filtration of stored IgG Pav through a column of Sephacryl S200, eluted with 0. I M sodium acetate buffer, pH 5.1, containing 6 M urea. Column size: 1.6 x 90 cm, fraction size: 1.3ml. (b) Gel filtration of plasmin digest of freshly isolated IgG Pav. The column of Sephadex GlOO was eluted with 1A4propionic acid. Column size: 1 x 110 cm, fraction size: 1.0 ml.

In an attempt to determine the site of plasmin cleavage, the Vu fragment was digested with penicillocarboxypeptidase-S 1. Only arginine and serine, in approximately equal amounts, were released. The sequence analysis of the Nterminal region of the shortened y chain from plasmin-treated IgG Pav gave the following results for residues l-15: (?)-Ser-AspTyr-His-Tyr-Tyr-Ala-MetAsp-Val-TrpGly-Gln-Gly/. This sequence overlaps that of peptide III obtained previously from IgG Pav by urea-pepsin digestion (Parr, 1977): Asp-Val-TrpGly-Gln-Gly-Thr-Thr-Val-Thr/. We have been unable to make a definite identification of the amino acid in position 1 of this sequence. At least one possibility, that of a cysteine residue, has not been ruled out. The Vu domain was not produced by digestion with papain. However, tryptic digestion gave

results similar to those obtained with plasmin. Yields of Fc and Fab-related fragments after 30 min digestion with plasmin were low, but increased with longer digestion. In addition to Fc and Fab-related fragments, over-digestion with trypsin gave a small amount of another fragment that resembled V, in size and electrophoretic mobility. This fragment, however, gave positive reactions with antisera to Fc and Cn3 fragments, and alanine and threonine as amino-terminal residues. These results indicate that some of the tryptic Fc fragment had broken down further to give the fragment tFc’ described by Medgyesi (197 1). DISCUSSION

In 1960, Skvaril demonstrated that changes which occurred in stored y-globulin resembled the fragmentation caused by treatment with

Loss of 1gG Cryoprecipitability

5

after Proteolysis

intact H chain

origin +

A

B‘

C

+

Fig. 3 (a). Starch gel electrophoresis in urea-formate buffer, pH 3.6, of (A) freshly isolated IgG Pav; (B) plasmin digest of A; (C) peak A from Fig. Z(b); (D) peak B from Fig. 2(b). (b) SDS polyacrylamide gel electrophoresis of (A) undigested freshly isolated 1gG Pav: (B) peak A from Fig. 2(b): (C) peak B from Fig. 2(b). All samples in (b) were reduced and alkylated before electrophoresis. enzymes. Connell & Painter (1966) isolated and characterized Fc and Fab fragments from stored normal human IgG, and showed that they were produced by the action of traces of contaminating plasmin. The ‘spontaneous’ breakdown of IgG Pav which we observed was presumably a similar phenomenon. The data presented here lead to the conclusion that the first product of plasmin digestion of IgG Pav is a fragment consisting of almost all of the Vu region, released by cleavage at a site in the fourth hypervariable region. This site in IgG Pav must be extremely sensitive to plasmin, as shown by the fact that after 30 min digestion the yield of V, from IgG Pav was close to 100x, whilst the yields of Fc and Fab-related fragments were still low. The N-terminal sequences of the small fragment and of the intact y chain show that the Vn domain of IgG Pav belongs to the Vu111 subgroup. Inspection of the N-terminal sequence of the plasmin-treated y chain indicated that residue Trp-12 corresponds to the almost invariant tryptophan residue found at position 103 in both Vi-&II and VnII sub-groups (Kabat et al., 1976). Since residue 94 (the numbering system of Kabat ef al., 1976, is used throughout) is arginine in 11 of the 16 known Vu111 sub-group sequences, it seems unlikely that cleavage should occur here only in IgG Pav; also, the proximity of the Cterminal end of the Vn domain intrachain proteoiytic

disulphide loop, at residue Cys-92, may prevent interaction of enzyme with the heavy chain. In 4 of the 16 Vu111 sequences, residue 96 is also arginine, and it seems likely that the C-terminal a&nine of the V, fragment of IgG Pav corresponds to Arg-96. This would result in the C-terminal sequence of the Vu fragment being: Cys-(Ala?HArg?)-Ser-Arg/. However, there is no evidence to prove that only a single site was cleaved and a small peptide could have been lost if cleavage had occurred at two or more sites. Thus it cannot be assumed that the N-terminal residue of the short heavy chain is residue 97. The fourth hypervariable region of heavy chains (approximately residues 95-102) can vary in length as well as in sequence, and Poljak ef al. (1974) have shown that this region projects into the antigen-binding cleft. Laschinger & Connell (1978) suggested that a long hypervariable region, which would extend further into the antigen-binding cleft, would be more susceptible to enzymatic cleavage than a shorter one. If we assume that IgG Pav is cleaved at a single site between residues 96 and 97, then the shortest possible length of the fourth hypervariable region of y chain Pav is equal to that of the fourth hypervariable region of heavy chains Tei, Lay, Porn, Ga and Tro (Capra & Kehoe, 1974a; Capra & Kehoe, 19746; Florent et al., 1974; Kratzin et al., 1975) which are the longest V,III

6

DOROTHY M. PARR and THEO HOFMANN

hypervariable regions so far reported, having 15 residues between Cys-92 and Trp-103. None of these five proteins has arginine at position 96. Since the first enzymatic cleavage of IgG by Porter with papain (1959) studies of enzymatically produced fragments of immunoglobulins have led to much information about the structure of these complex molecules. In spite of the great number of such studies that have been done, IgG KUP, IgG Zie and IgG Pav appear to be the only proteins from which an essentially intact Vn region has been released by enzymatic digestion of the intact molecule. These three proteins are all cryoglobulins. The cryoprecipitability of some immunoglobulins is a very interesting property that has so far defied an explanation in molecular terms. It is not associated with a particular class or sub-class of immunoglobulins. Grey & Kohler (1973) have suggested that the part of the molecule that is responsible for cryoprecipitability may be located in the variable region(s). This is supported by the observation that cryoprecipitability has been retained by peptic F(ab’), fragments (Saha et al., 1970; Pruzanski et al., 1973; Ely et al., 1978), but not by Fc or Fc-related fragments (Saha et al., 1970; Middaugh et al., 1976). Similarly, the amino acid composition and N-terminal residues of cyanogen bromide fragments from IgG Cal, an IgGZ/,l cryoglobulin, indicated that the heavy chain was entirely normal from residue methionine 252 to the C-terminus, and the light chain was also normal (D.M. Parr, unpublished observations). A close connection between the property of cryoprecipitability and the integrity of the heavy chain variable region(s) has been shown clearly in IgG Pav, since the plasmin-treated IgG no longer gelled on cooling. An interesting observation was that cryoprecipitability was lost even before the Vn regions were dissociated from the remainder of the molecule. We propose that cryoprecipitability in some cases, and possibly in all cases, is related to the same structural feature in the Vn domain that is responsible for the sensitivity to proteolytic enzymes and that allows the formation of essentially intact Vu as shown by IgG KUP, IgG Zie and IgG Pav.

REFERENCES Capra J. D. & Kehoe J. M. (1974~) Variable region sequences of five human immunogiobuhn heavy chains of the V,III sub-group: definitive identification of four heavy chain hypervariable regions. Proc. nuns. Acad. Sci. U.S.A. 71, 845-848.

Capra J. D. & Kehoe J. M. (19746) Structure of antibodies with shared idiotypy: The complete sequence of the heavy chain variable regions of two immunoglobulin M antigamma globulins. Proc. natn. Acad. Sci. U.S.A. 71, 4032-4036.

Connell G. E. & Painter R. H. (1966) Fragmentation of immunoglobulin during storage. Can. J. Biochem. 44, 371-379. Dammacco F., Franklin E. C. & Frangione B. (1972) An unusual papain fragment containing the Vu region of an lgG3 myeloma protein. J. fmmun. 109, 565-569. Ely K. R., Colman P. M., Abola E. E., Hess A. C., Peabody D. S., Parr D. M., Connell G. E., Laschinger C. A. & Edmundson A. B. (1978) Mobile Fc region in the Zie IgG2 cryoglobuhn: Comparison of crystals of the F(ab’), fragment and the intact immunoglobulin. Biochemistry 17, 820-823.

Florent G., Lehman D. & Putnam F. W. (1974) The switch point in II heavy chains of human InM immunoalobulins. Biochemistry lj, 2482-2498. Grev H. M. & Kohler P. F. (1973) Crvoimmunonlobuhns. dmin. Haemar. 10, 87-112. ’ ’ Hermodson M. A., Ericsson L. H., Titani K.. Neurath H. & Walsh K. A. (1972) Application of sequenator analyses to the study of proteins. Biochemistry 11, 44934502. Hui A., Rao L., Kurosky A., Jones S. R., Mains G., Dixon J. W., Szewczuk A. & Hofmann T. (1974) The use of penicillocarboxypeptidase-S, in amino acid sequencing. Archs Biochem. Biophys. 160, 577-587. Inbar D., Hochman J. & Givol D. (1972) Localization of antibody-combining sites within the variable portion of heavy and light chains. Proc. nam. Acad. Sci. U.S.A. 69, 2659-2662.

Kabat E. A., Wu T. T. & Bilofsky H. (1976) Variable regions of immunoglobulin chains. Bolt, Beranek and Newman, Cambridge, MA. Kakimoto K. & Onoue K. (1973) Characterization of the Fv fragment isolated from a human immunoglobulin M. /. Immun. 112, 1373-1382. Klein M., Danon F., Bruet J. C., Signoret Y. & Seligmann M. (1972) Etude immunochimique de 130 cryoglobulines humaines. Eur. J. clin. Biol. Res. 17, 948-957. Kratzin H., Altevogt P., Ruban E., Kortt A., Staroscik K. & Hilschmann N. (1975) The primary structure of a monoclonal IgA immunogiobulin (IgA Tro.), II The amino acid sequence of the heavy chain, a type, subgroun III, structure of the complete- IgA mol&le. H;ppeSeyler ‘s 2. phvsiol. Chem 356. 1337-I 342.

Laschinger C.-A: & Connell G. E. (1978) An unusual tryptic cleavage of a myeloma protein. Immunochemistry 15, 119-123.

Lin L.-C. SCPutnam F. W. (1978) Cold pepsin digestion: A novel method to produce the Fv fragment from human immunoglobulin M. Proc. nam. Acud. Sci. U.S.A. 75, 2649-2653.

Medgyesi G.A., Jakab M., Nagy M. C. & Gergely J. (1971) Susceptibility of human yG immunoglobulins to tryptic fragmentation. Acta Biochim. Biophys. Acad. Sci. Hung. 6, 405414.

Acknowledgemems-We

should like to thank Dr. W. Pruzanski of the Wellesley Hospital, Toronto, for his gift of serum from patient Pav, Dr. G. E. Connell for his critical reading of the manuscript, and Mrs. A. Leung, Mrs. A. Cunningham and Mr. S. Rhee for technical assistance. This work was supported by grant No. MRC MT 694 from the Medical Research Council of Canada.

Middaugh C. R., Prystowsky M. B., Gerber-Jenson B., Oshman R. G., Kehoe J. M. & Litman G. W. (1976) Requirements for cryoprecipitability of an IgM monoclonal cryoglobulin. Fedn Proc. Fedn Am. Sots exp. Biol. 35, 274.

Mole L. E., Geier M. D. & Koshland M. E. (1975) The isolation and characterization of the V, domain from

Loss of IgG Cryoprecipitability rabbit heavy chains of different a locus allotype. J. Immun. 114, 1442-1448. Parr D. M., Connell G. E., Kells D. 1. C. & Hofmann T. (1976) Fb’,, a new peptic fragment of human immunoglobulin G. Biochem. J. 155, 31-36. Parr D. M. (1977) Fragments produced by digestion of human immunoglobulin G subclasses with pepsin in urea. Biochem. J. 165, 303-308. Percy M. E. & Buchwald B. M. (1972) A manual method of sequential Edman degradation followed by dansylation for the determination of protein sequences. Ann&. Eiochem. 45, 60-67. Poljak R. J., Amzel L. M., Chen B. L., Phizackerley R. P. & Saul F. (1974) The three-dimensional structure of the Fab’ fragment of a human myeloma immunoglobulin at 2.0 A resolution. Proc. nam. Acad. Sci. U.S.A. 71, 344&3444. Porter R. R. (1959) The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem. J. 73, 119-126. Pruzanski W., Jancelewicz Z. & Underdown B. (1973) Immunoglobulin and physiochemical studies of IgAiL cryogelglobulinaemia. Clin. exp. Irnmw. 15, 181-191. Rodwell J. D. & Karush F. (1978) A general method for the isolation of the V, domain from IgM and other immunoglobulins. J. Zmmun. 121, 1528-1531.

7

after Proteolysis

Rosemblatt M. S. & Haber E. (1978) Isolation of an active variable-domain fragment from a homogeneous rabbit antibody heavy chain. Physiochemical and immunological properties. Biochemistry 17, 3877-3882. Saha A., Chowdhury P., Sambury S., Smart K. & Rose B. (1970) Studies on cryoprecipitation-IV. Enzymic fragments of a human cryoglobulin. J. biol. C/rem. -245, 27362736. Sharon J. & Givol D. (1976) Preparation of Fv fragment from the mouse myeloma XRPC-25 immunoglobulin possessing anti-dinitrophenyl activity. Biochemisrr?: 15, 1591-1594. Skvaril ,F. (1960) Changes in outdated human p-globulin preparations. Nature, Land. X5,475-476. SmithiesO., Connell G. E. &Dixon G. H. (1962) Inheritance of haptoglobin subtypes. Am. J. hum. Genet. 14, 14-21. Weber K. & Osborn M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. bhi. Che& 244, 44b6-&1i. Wintrobe M. M. & Buell M. V. (1933) Hvoeroroteinaemia .. associated with multiple myeloma: with report of a case in which an extraordinary hyperproteinaemia was associated with thrombosis of the retinal veins and svmotoms m*mm-r ~~-~ suggesting Reynaud’s disease. Bull. Johns Hopkins Hosp. 52, 156165. 1