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641 Biochem. J. (1970) 117, 641-660 Printed in Great Britain The Amino Acid Sequences of the Fd Fragments of Two Human y 1 Heavy Chains By E. M. PRESS AND N. M. HOGG Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. (Received 27 November 1969) The amino acid sequences of the Fd fragments of two human pathological immunoglobulins of the immunoglobulin GI class are reported. Comparison of the two sequences shows that the heavy-chain variable regions are similar in length to those of the light chains. The existence of heavy chain variable region subgroups is also deduced, from a comparison of these two sequences with those of another y 1 chain, Eu, a ,t chain, Ou, and the partial sequence of a fourth y I chain, Ste. Carbohydrate has been found to be linked to an aspartic acid residue in the variable region of one of the y 1 chains, Cor. The chemical complexity of the immunoglobulins is in part related to antibody specificity, but some of the complexity can be ascribed to the many classes and subclasses into which the light and heavy chains can be grouped. Amino acid-sequence studies on light chains have shown that the Cterminal half of all light chains of a single class and allotype have an identical sequence, but that the sequence of the N-terminal half of the chain varies from one light chain to another. On the basis of 'fingerprint' studies heavy chains were also assumed to have constant and variable regions, each class and subclass being characterized by a different constant-region sequence. Antibody specificity is believed to be related to the variable part of the chains, and, in order to discover the extent of the variable region of the heavy chain, the amino acid sequences of Fd fragments of two pathological immunoglobulins of yl subclass have been determined. The sequence of the N-terminal 84 residues of one heavy chain, Daw, has been reported previously (Piggot & Press, 1967; Press, 1967), as has the mid-chain sequence 211-253 (Steiner & Porter, 1967). Preliminary data on the chemical structure of the other heavy chain, Cor, have also been given (Press & Piggot, 1967). In this paper we describe the sequence studies on Daw heavy chain for residues 85-225, and on Cor heavy chain for residues 1-225. A report of some of these results was published by Press & Hogg (1969). MATERIALS Enzymes. The enzymes used were described by Press, Piggot & Porter (1966). Other materials. Dithiothreitol was obtained from Cal21 biochem, Los Angeles, Calif., U.S.A. Iodo[1-'4C]acetamide was obtained from The Radiochemical Centre, Amersham, Bucks., U.K. The other reagents were as described by Piggot & Press (1967). Myeloma IgG* (Cor). This was isolated by precipitation from the plasma with Na2SO4 (18%) and purified by fractionation on a column of DEAE-Sephadex A-50 by using a 17-200mM concentration gradient of sodium phosphate buffer, pH 6.2. Pathological IgO (Daw). This was prepared as described by Press et al. (1966). METHODS 'Cor' Fab fragment. IgG (Cor) was digested with papain and fractionated on a column of Sephadex G-100 as described by Press et al. (1966). The Fab and Fc fragments were separated by chromatography on DEAE-cellulose at pH 8 as described by Deutsch, Thorpe & Fudenberg (1963). 'Cor' heavy chain. This was prepared by reduction of IgG with 5mM-dithiothreitol, alkylation with iodoacetamide and separation of the heavy chains from the light chains on a column of Sephadex G-100 in M-acetic acid. 'Daw' Fd fragment 2a'. Daw IgG was digested with papain and the Fab and Fe fragments were separated from undigested IgG on a column of Sephadex G-100 as described by Press et al. (1966). The mixture of Fab and Fc fragments was then digested with cyanogen bromide and chromatographed on a column of Sephadex G-100 in 6M-urea-0.2M-sodium formate, pH3.3. The first peak to be eluted was the Fab fragment (the constituent eyanogen bromide cleavage products are disulphide bonded; Piggot & Press, 1967). This fraction was then totally reduced and alkylated with iodo[l-'4C]acetamide *Abbreviations: IgG, immunoglobulin G; PCA, Cmc, Hsr and CHO (in sequences and tables), pyrrolid-2-one-5carboxylic acid, S-carboxyamidomethylcysteine, homoserine and carbohydrate respectively. Bioch. 1970, 117 1970 E. M. PRESS AND N. M. HOGG 642 as described by Cebra, Givol & Porter (1968) and refractionated on the same column. The light chain, which contains no methionine residues and was therefore not cleaved by cyanogen bromide, was eluted first, followed by fragments 2a', 4 and 2b (see Piggot & Press, 1967). Cyanogen bromide dige8tion. This was as described by Press et al. (1966). C- Terminal analy8i8. C-Terminal amino acids of peptides were determined by digestion of 0.02,umol of peptide with carboxypeptidase A for various times and by hydrazinolysis as described by Press et al. (1966). N- Terminal analy8i8. N-Terminal amino acids of peptides and fragments were determined by either the fluorodinitrobenzene method of Sanger (Porter, 1957) or by the 'dansyl' method of Gray (1967). 'Dan8yl'-Edman technique. This was used, as described by Gray (1967), to determine the amino acid sequences of peptides. The acid and amide forms of aspartic and glutamic residues were distinguished by determining the mobility at pH6.5 of the peptides containing these residues (Offord, 1966). Enzymic dige8tion. This was done in 50mM-NH4HCO3 at pH 8.1-8.3, with a peptide concentration of 3-5mg/ml. Meeaurement of radioactivity of 14C-labelled peptides. Aqueous samples were dissolved in the scintillation fluid described by Kinaird (1957), or were adsorbed on glassfibre paper (Whatman G F/A) and placed in a scintillation fluid consisting of 0.6g of 2,5-diphenyloxazole (PPO) and 12mg of 1,4-bis-(4-methyl-5-phenyloxazol-2-yl)benzene (dimethyl-POPOP) in 100ml of toluene and the radioactivity was measured in a Nuclear-Chicago liquidscintillation counter. RESULTS Sequence of the Fd fragment of IgG (Cor) The Fab fragment was cleaved with cyanogen bromide, totally reduced and alkylated with iodo[1-14C]acetamide as described by Cebra et al. (1968) and fractionated on a column of Sephadex G-100 in 6M-urea-0.2M-sodium formate, pH3.3. Five fractions were obtained: the first was whole light chain (there are no methionine residues in Cor light chain); the other four fragments 2a', 5a, 5b and 6a are derived from the Fd fragment (Press & Piggot, 1967). The amino acid composition of each of these fragments was determined (Tables 1, 2, 5 and 6) and their alignment in the Fd fragment was deduced by comparison with the amino acid sequence of Daw Fd fragment (Fig. 10). Fragment 5b. This was digested with chymotrypsin (80,ug/,umol at 37°C for 3h) and the digest was fractionated by gel filtration on a column of Sephadex G-25 in 0.02M-ammonia and paper electrophoresis at pH 1.9. Five peptides were isolated that accounted for the composition of fragment 5b (see Table 1), and the amino acid sequences of these peptides were determined as shown in Fig. 1. Fragment 5b had been assumed to be the N-terminal fragment of the heavy chain, because of its very similar amino acid composition Table 1. Amino acid composition and isolation procedures of the chymotryptic peptides of Cor fragment 5b Composition (mol of amino acid/mol of peptide) Amino acid Lys Arg Thr Ser Glu Pro Gly Ala Val Leu Phe Cmc Hsr Total G-25 elution volume* Mobility at pH l.9t Peptide C2.a Peptide C2.b Peptide Peptide C3.a C3.c 0 0 1.0 0.9 1.0 1.6 1.3 2.1 1.8 1.3 1.2 0.9 1.9 0 0 0 0 0 0.9 0 1.1 0 0 0 1.0 1.0 0 0 0 0 0 0 1.0 0 0 1.1 0 0 0 0.9 0 0 14 4 +0.50 3.0 0 0 1.1 0 0 1.0 0 0 0.9 7 1.2 +0.35 1.2 1.4 0 0 2.9 0 0 0 0 0 0 1.2 1.2 0.7 0 6 1.4 +0.53 +0.10 +0.40 * Relative to exclusion = Peptide C4.b 3 1.6 1.0. t Mobility relative to lysine = +1.0. Sum of peptides 1 1 7 5 3 2 3 1 2 5 2 1 1 34 Fragment 5b 1.0 1.0 6.6 5.1 3.6 2.0 3.2 1.4 2.0 4.7 1.8 0.9 0.7 34 Vol. 117 AMINO ACID SEQUENCES OF HUMAN HEAVY CHAINS to Daw fragment 2b (Press & Piggot, 1967). The heavy chain has the N-terminal sequence PCA-ValThr (Press & Piggot, 1967), and peptide C3.a from fragment 5b is an extension of this sequence and confirms that this fragment is indeed N-terminal. Peptide C2.a must be the C-terminal peptide of fragment 5b, as it contains homoserine. The other three peptides, C2.b, C3.c and C4.b, have been aligned by homology with the sequence of Daw fragment 2b (Piggot & Press, 1967); there is only one difference, at position 13, between Daw and Cor in the sequence for residues 5-27 (see Figs. 1 and 10). The glutamic residue at position 6 was deduced 643 to be in the acid form from the mobility of peptide C2.b and from the fact that the other glutamic residue near the C-terminal end of peptide C2.b was cleaved by carboxypeptidase A as glutamine. Fragment 5a. This was digested with trypsin (300,ug/,mol at 370C for 21h) and the digest was fractionated by gel filtration, paper electrophoresis and chromatography. The seven tryptic peptides, whose composition is given in Table 2, account for the total composition of fragment 5a. Two other tryptic peptides, TI.b and TI.c, were also isolated and represent the N- and C-terminal parts of peptide TI.a. Hexosamine was found in peptides 10 20 PCA-Val-Thr- Leu-Arg-Glu-Ser-Gly-Pro-Ala- Leu-Val- Lys-Pro-Thr-Gin-Thr- Leu-Thr-Leu-Thr-Cmc-Thr-Phe- Thr-Leu Arg-Glu-Ser-Gly-Pro-Ala-Leu-Val-Lys-Pro-Thr-Gln-Thr-Leu Thr-Leu-Thr-Cmc-Thr-Phe 30 Ser-Gly-Phe-Ser-Leu-Ser-Ser-Thr-Gly Hsr 4-C4.b-* 4 - c2.a -- Ser-Gly-Phe Ser-Leu-Ser-Ser-Thr-GIy Fig. 1. Amino acid sequence of Cor fragment 5b showing the chymotryptic peptides derived from it. Sequence determination for the N-terminal end was by the 'dansyl'-Edman technique (-+) and from the C-terminal end by carboxypeptidase A digestion (*-). Table 2. Cor fragment 5a and tryptic peptides derived from it The tryptophan composition of fragment 5a is calculated from the extinction at 280nm. The tryptophan content of the peptides was assumed to be 1 for those peptides that stained with the Ehrlich reagent on paper. The S-earboxyamidomethylcysteine composition of the peptides was based on the radioactivity. Hexosamine was determined by a modification of the Elson-Morgan method as described by Fleischman, Porter & Press (1963). Composition (mol of amino acid residue/mol of peptide) Amino acid Lys Arg Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Cme Hsr Trp Total Hexosamine Peptide T1.a 0.9 0.7 4.8 1.9 1.4 1.2 0 0 0 0 1.1 1.2 1.8 0 0 1 16.0 1.5 Peptide T2.b5 0 0.9 1.0 0.9 1.2 0 0 0 0 0 0 0 0 0 0 0 4.0 Peptide T2.c4 0 0 1.4 0.8 0 1.2 0 0 0 1.8 0 1.0 0 0 0.8 0 7.0 Peptide T3.a 1.2 0 0 0.8 1.1 0 0 0 0 0 1.0 0.9 0 0 0 0 5.0 Peptide T3.c 0.9 0 0 0 0 1.0 2.0 1.1 0 0 0 0 0 0 0 0 5.0 Peptide T4.a 0 0.7 0 0 0 1.1 0 1.3 1.1 0 0 1.8 0 0 0 1 Peptide T5.a 0 0.9 0 0 0 0 0 1.2 0 1.2 0.7 0 0 1 0 1 Sum of peptides 3 4 7 5 3 4 2 3 1 3 3 5 2 1 1 3 7.0 6.0 50 Fragment 5a 2.9 3.1 6.8 5.3 4.1 3.6 1.6 3.3 1.4 2.3 3.0 4.6 1.7 0.9 1.4 2.9 48.9 1.7 E. M. PRESS AND N. M. HOGG Table 3. Chymotryptic peptides of Cor fragment 5a 644 1970 The tryptophan content was assumed to be 1 for those peptides that stained with the Ehrlich reagent on paper. Hexosamine was calculated from the amino acid analyser trace, without correction for recovery. Composition (mol of amino acid/mol of peptide) Amino acid Lys Arg Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Cmc Hsr Trp Total Hexosamine Peptide C2.c 1.1 0 3.2 0 0 0 0 0 0 0 0 0 1.7 0 0 0 6 Peptide C5.a 1.0 1.2 2.1 1.8 2.2 1.3 0 0 0 Peptide C5.d 0 0 0 1.1 0 1.4 0 2.0 0 1.0 0 0 0 0 0 0.9 0 Peptide C3.c 0 0.9 1.1 1.8 1.1 1.1 0 0 0 0 0 0 8 1.0 1.0 0 0 0 0 13 0 0 0 0 0 2 Peptide C6.a2 1.2 0.7 0 0 0 1.8 2.2 2.2 0 0 0.9 1.0 0 0 0 1 11 Peptide C6.b 0 0.8 1.3 0 0 0 0 0 1.1 0 0.9 0.9 0 0 0 1 6 Peptide C7.e(C7.b) 0 0 0 0 0 0 0 1.2 0 1.1 0 0 0 0.7 0 1 4 shown to be attached to the aspartic residue of this peptide, presumably involving the ,B-carboxyl group, and as sialic acid is unlikely to be present for 3h) and the digest was fractionated. The on the peptide (see section below on carbohydrate compositions of the chymotryptic peptides are of Cor Fd fragment), the glutamic residue in given in Table 3 and the methods used in the isola- peptide M3.c is probably present in the acid form. tion of the tryptic and chymotryptic peptides are The sequence of fragment 5a is complete as shown shown in Table 4. The sequence of fragment 5a in Fig. 2. Fragment 6a. This was digested with trypsin was determined, as shown in Fig. 2. Tryptic peptide T4.a was digested with chymotrypsin and (100lg/,umol at 370C for 2h) and two peptides, the two peptides T4.a.Cl and T4.a.C2 were purified 6a.T1 and 6a.T2, were isolated from the digest. by paper electrophoresis at pH3.5. Chymotryptic Peptide 6a.T1 was further digested with chymopeptides C7.b and C7.e had the same composition, trypsin, and three peptides were separated by gel but peptide C7.e is acidic and its a-amino group is filtration. The amino acid composition and isolation blocked. Tryptic peptide T5.a also has a blocked procedures of these peptides are given in Table 5, a-amino group, and the N-terminal residue of together with the amino acid composition of fragment 5a could not be detected by either the fragment 6a. The sequence of this fragment was 'dansyl' method or the method of Edman degrada- determined, as shown in Fig. 3. The mobility of tion. A blocked N-terminal alkylated cysteine peptide 6a.T1 (see Table 5) established that both residue had previously been found in Daw fragment aspartic residues are present as aspartic acid. Fragment 2a'. This has an N-terminal aspartic 4 (Press, 1967) and presumably arises as a result of condensation of the a-amino group with the residue, and its composition is given in Table 6, carboxamidomethyl side chain. Peptides T2.b1 which shows that it contains no homoserine and is and T2.b5 were neutral at pH 6.5. Peptide T3.c was therefore the C-terminal fragment of the Fd fragbasic and peptide T4a.C2 was acidic. Peptide T1.b ment. It was digested with trypsin (200,ug/,umol was acidic with a mobility of -0.7, from which it at 370C for 5h). A precipitate formed and this was was deduced that the four aspartic residues were separated by centrifugation and found to contain present as aspartic acid and this was confirmed by about half the total digest. The precipitate repthe mobilities of peptides C6.b (neutral) and C2.c resents a large insoluble peptide, 2a'.T1. The (-0.65). Peptide C3.c, which contained hexosamine, soluble portion of the tryptic digest was fractionated was neutral at pH6.5. As the carbohydrate was on columns of Sephadex G-50 and G-25 and eight Ti.a and Ti.c; it was also detected in the chymotryptic peptide C3.c (see below). Fragment 5a was digested with chymotrypsin (200,ug/,tmol at 370C Vol. 117 AMINO ACID SEQUENCES OF HUMAN HEAVY CHAINS Table 4. Isolation procedures for the component peptides of Cor fragment 5a 645 Paper electrophoresis Sephadex-gel filtration or pH chromatography T2.bl Type (a) G-25 (b) G-50 (a) G-25 (b) G-50 (a) G-25 (b) G-50 G-25 Solvent 0.02m-NH3 0.05M-NH3 0.02M-NH3 0.05M-NH3 0.02M-NH3 0.05M-NH3 0.02M-NH3 Elution vol.* 1.0 1.2 1.0 1.3 1.0 1.5 1.2 T2.b5 G-25 0.02M-NH3 1.2 T2.C4 G-25 0.02M-NH3 1.2 3.5 BAW: T3.a T3.c T4.a T5.a Chymotryptic peptides C2.c G-25 G-25 G-25 G-25 0.02M-NH3 0.02M-NH3 0.02m-NH3 0.02M-NH3 1.4 1.4 1.8 1.9 BAWT BAWt 3.5 3.5 C5.d C6.a2 G-50 G-50 G-50 G-50 G-50 0.05M-NH3 0.05M-NH3 0.05m-NH3 0.05M-NH3 0.05m-NH3 1.5 1.7 1.9 1.9 2.2 3.5 3.5 3.5 3.5 3.5 C6.b G-50 0.05M-NH3 2.2 C7.b G-50 G-50 0.05M-NH3 0.05m-NH3 2.8 2.8 3.5 6.5 3.5 3.5 Tryptic peptides Tl.a Tl.b Tl.c system C5.a -0.3 6.5 -0.7 6.5 3.5 0 +0.1 3.5 BAW$ 1.9 C7.e * Relative to exclusion volume = RF 6.5 BAW$ C3.c Mobilityt or 0.8 +0.1 0.1 0 0.2 0.5 0.1 +0.2 0 0 +0.1 +0.6 +0.2 +0.5 +0.6 +0.3 0 0 -0.6 1.0. t Mobility relative to lysine = +1.0 and to aspartic acid -1.0. I Chromatography in butan-l-ol-acetic acid-water (12:3:5, by vol.). tryptic peptides were isolated as shown in Table 7. The composition of the nine tryptic peptides given in Table 6 accounts for the composition of the whole of fragment 2a'. Peptide 2a'.T1 had aspartic acid as the N-terminal residue and it was digested with chymotrypsin (570,ug/,gmol at 370C for 2jh). The soluble digest was fractionated on a column of Sephadex G-50 in 0.05M-ammonia. Nine peptides were isolated, which together accounted for the composition of 2a'.T1. (see Tables 8 and 7 for composition and methods of purification of these peptides). The amino acid sequences of the peptides were determined as shown in Fig. 4. Peptide TI.C2a was shown to be the N-terminal peptide of peptide TI by isolation of a peptide 2a'C3.6a from a chymotryptic digest of peptide 2a' (see next section and Fig. 7). Peptide Ti .C6h is the only lysine-containing peptide, and must therefore be C-terminal. Peptides TI.C4e and TI.C4b were overlapped by another peptide, C3.5d, isolated from the chymotryptic digest of peptide 2a' (see Fig. 7). Peptide TI.C5a4 was an extension of peptide TI.C6d and overlapped with peptide TI.C6h. Peptide Tl.C6g is the Cterminal tripeptide of peptide TI and its amino acid sequence was determined as shown in Fig. 4. The other chymotryptic peptides of peptide TI were aligned in the order shown in Fig. 4, by the similarity in sequence between this human heavy chain and rabbit heavy chain in this region (see Fig. 5) (Fruchter, Jackson, Mole & Porter, 1970). The full stops in the sequence of Cor peptide 2a'.Tl indicate where overlapping peptides were not available and the sequence was deduced by homology with the rabbit heavy chain. The aspartic and glutamic residues in peptide TI.C2a must be present in the acid form, since the chymotryptic peptide 2a'C3.6a referred to above had a mobility of -0.13 at pH6.5. All the other aspartic and glutamic residues of peptide TI are in the amide form, since peptides Tl.C5b2, Tl.C4d, TI.C4b and TI.C5a4 are neutral at pH6.5 and peptide TI.C6g is basic. The method of sequence determination of the E. M. PRESS AND N. M. HOGG fi46 -T5.am 10 4 T3.c -T4.a- 10 4' Gln-Pro-Pro-Gly 1970 l.a )- 4 I le-Asp -)I. Gly-Leu-Glu 4-T4.a.C2--* *-T4.a.Cl-* 4- T1.b- * Leu-Ala-Arg lie-Asp 4 -4 --)2c -) -4-*. 6.- Cmc-Vail lIe-Arg-Gln Trp Asp-Asp-Asp -4-. -4o. 4- - -4l. .4-- --* -4- --)I Val-Gly-Trp *4+ 4- 4-- CHO Tyr-Tyr-Asx-Thr-Ser- Leu-Glu-Thr-Arg-Leu-Thr- I le-Ser- Lys-Asp-Thr-Ser-Arg-Asn-Gin-Val-Val- Leu-Thr-Hsr Tl.a 4 +. -T1.c- T3.a # 4 T2.b5- T2.bl- 4. Asp-Thr-Ser-Arg Asn >Leu-Thr-lie-Ser -Ty -4y. -x -T. --* -4-) --* --) --* Tyr-Tyr-Asx-Thr Asn C5.d* Val-Val-Leu Thr Tyr-Tyr Asx-Thr-Ser- Leu-Glu-Thr-Arg-Leu Thr-Ile-Ser-+p --) -+. -+0 -4,* 4- -_* -), -.* 4-- 4- -+ .-4 4-4+-4*- --* 4~- Fig. 2. Amino acid sequence of Cor fragment 5a. Sequence determination from the N-terminal end was by the 'dansyl'Edman technique (-*) and from the C-terminal end by carboxypeptidase A digestion (-) or by hydrazinolysis (+-i). Table 5. Amino acid composition and isolation procedures of Cor fragment 6a and tryptic and chymotryptic peptide8 derived from it Composition (mol of amino acid residue/mol of peptide) Amino acid Arg Asp Thr Pro Gly Ala Val Ile Tyr Cmc Peptide 6aT1 0.9 2.0 2.0 1.3 0.6 1.9 1.1 0 1.8 Peptide 6aT2 0 0 1.3 . 1.5 2.2 0.8 1.7 0.9 0 Hsr 0 Total Residues Sephadex G-25 12 1.1 0.02m-NH3* Mobility at -0.2 0.9 11 1.2 Peptide 6aTl.C1 0 2.2 2.0 0.9 0 1.1 0.9 0 1.0 Peptide 6aTl.C2 0.9 0 0 0 0 1.1 0 0 0 Peptide 6a.Tl.C3 1.0 0 1 0 0 0 8 3 1.8 4 1.2 0 pH6.5t * 0 0 0 0 1.2 0 0 0.8 Elution volume given relative to exclusion volume. t Mobility relative to aspartic acid -1.0. = 2.0 Fragment 6a 0.8 2.0 3.5 3.2 1.5 3.8 1.9 1.9 2.6 0.7 1.1 23 AMINO ACID SEQUENCES OF HUMAN HEAVY CHAINS Vol. 117 647 Asp-Pro-Val-Asp-Thr-Ala-Thr-Tyr-Tyr-Cmc-Ala-Arg- I le-Thr-Val- Ile-Pro-Ala-Pro-Ala-Gly-Tyr-Hsr Asp-Pro-Val 6a.T1 6a.T2 lIe-Thr-Val-lle-Pro-Ala-Pro-Ala-Gly-Tyr-Hsr Asp-Pro-Val-Asp-Thr-Ala-Thr 4 1- -+-0 - -0+ -+* -+*-_- (- 6aT1.C1 - Asp-Pro ~-~444 -6aTl .C24 Cmc-Ala Ala Thr Tyr 6aTl.C3-* 4 Tyr Fig. 3. Amino acid sequence of Cor fragment 6a. Sequence determination from the N-terminal end was by the 'dansyl'Edman technique (-÷) and from the C-terminal end by carboxypeptidase digestion (+-). Table 6. Tryptic peptides of Cor fragment 2a' The S-carboxyamidomethylcysteine composition of fragment 2a' is calculated from amino acid analysis. S-Carboxyamidomethylcysteine residues are based on the radioactivity of the peptides. The tryptophan composition of fragment 2a' is calculated from the extinction at 280nm. The number of tryptophan residues in the peptides was assumed to be 1 when the peptide stained with Ehrlich reagent on paper. The composition of fragment 2a' is based on 6 residues of alanine/molecule of fragment 2a'. The composition of peptide Ti is taken from the sum of the constituent residues of its chymotryptic peptides (Table 8, column 10). Composition (mol of amino acid residue/mol of peptide) Amino acid Lys His Arg Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe Cmc Trp Total residues Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Ti T5 T4.1c T4.1d T4.2a T4.2b T4.2d T4.2e T4.3f 2 2 0 5 7 11 3 5 4 2 8 1 5 3 2 1 1 62 1.0 0 0 0 2.1 2.4 0 0 2.9 1.9 1.0 0 1.8 0 0 1 0 14 0.9 0 0 0 2.6 3.1 0 1.0 1.0 1.1 1.9 0 0 0 0 0 0 12 1.1 0 0 1.0 0 0.9 0 0 0 0 0 0 0 0 0 1 0 4 1.0 0 0 1.1 0 0 0 0 0 0 0.9 0 0 0 0 0 0 3 eight smaller tryptic peptides of fragment 2a' is shown in Fig. 6. Peptides T4.1c, T4.1d and T5 were digested with chymotrypsin and the amino acid sequences of the chymotryptic peptides determined as shown. The mobilities of the four peptides containing glutamic and aspartic residues are also 1.0 0 0 0 0 3.1 0 2.8 1.2 1.0 1.1 0 1.0 0 0.9 0 0 12 1.7 0 0 0 0 0 1.0 1.2 0 0 0.9 0 0 0 0 0 0 5 0 0.9 0 0 1.1 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0.9 1.1 0 0 0 0 1.2 0 0.8 0 0 0 0 0 1 5 Sum of peptides 9 3 1 8 13 20 4 10 10 6 15 1 8 3 3 3 2 119 Fragment 2a' 7.9 2.4 1.1 8.4 13 20 4.3 10 9.6 6.0 13 1.2 7.9 3.0 2.9 2.0 2.0 115 given in Fig. 6, and establish that these residues are all present in the acid form. The tryptic peptides of peptide 2a' were aligned by isolation and partial sequence-determination of nine chymotryptic peptides. Fragment 2a' was digested with chymotrypsin (500,ug/,umol at 370C E. M. PRESS AND N. M. HOGG 648 1970 Table 7. Isolation procedures for component peptides of Cor fragment 2a' Paper electrophoresis Sephadex-gel filtration Tryptic peptides T4.1c T4.ld T4.2a T4.2b T4.2d Solvent 0.05M-NH3 0.02M-NH3 0.05M-NH3 0.02M-NH3 0.05M-NH3 0.02M-NH3 0.05M-NH3 0.02M-NH3 Type G-50 G-25 G-50 G-25 G-50 G-25 G-50 G-25 0.05M-NH3 0.02M-NH3 GE-50 G-25 Elution volume* 1.8 1.0 1.8 1.0 1.8 1.2 1.8 1.2 1.8 1.2 pH or chromatography system +0.2 6.5 +0.3 6.5 -0.04 6.5 3.5 +0.5 6.5 PAWt +0.25 0.35 6.5 +0.4 6.5 +0.5 -0.6 0.3 0.45 0.65 +0.05 0.4 +0.2 +0.25 0.5 +0.5 +0.2 +0.7 +0.8 0.05M-NH3 0.02M-NH3 0.05M-NH3 0.02M-NH3 0.05M-NH3 1.8 1.2 1.8 1.3 2.5 G-50 G-50 G-50 G-50 G-50 0.05M-NH3 0.05M-NH3 0.05M-NH3 0.05M-NH3 3.5 0.05im-NH3 1.3 1.7 1.7 1.7 1.9 TI.C5bl Tl.C5b2 G-50 G-50 0.05M-NH3 0.05M-NH3 1.9 1.9 3.5 3.5 TI.C5e TI.C6d TI.C6g TI.C6h Chymotryptic peptides C3.5d G-50 G-50 0.05M-NH3 0.05m-NH3 G-50 G-50 0.05M-NH3 0.05M-NH3 1.9 2.3 2.3 2.3 BAW: 3.5 3.5 3.5 3.5 G-50 0.05M-NH3 1.8 3.5 C3.6a G-50 0.05M-NH3 1.8 C3.6b.c G-50 0.05M-NH3 1.8 C3.7d G-50 0.05M-NH3 1.8 T4.3f T5 Chymotryptic peptides of peptide TI TI.C2a TI.C4b TI.C4d TI.C4e Tl.C5a4 C3.11 C4.2.3c C4.2.9b C4.3f5 C5.1 0.05M-NH3 0.05M-NH3 0.02M-NH3 1.8 2.0 1.2 G-50 G-25 0.05M-NH3 0.02M-NH3 2.0 1.2 0.05M-NH3 G-50 G-25 0.02 M-NH3 BAW$ BAWT BAWt 3.5 BAWt BAWt G-50 G-50 G-25 3.5 6.5 3.5 6.5 0 -0.1 0.5 0 -0.1 BAW: 0 -0.05 0.35 3.5 6.5 3.5 +0.1 +0.8 3.5 6.5 +0.6 +0.3 3.5 0 0.2 BAWt 2.0 1.4 RF 6.5 G-50 G-25 G-50 G-25 G-50 T4.2e Mobilityt or +0.1 3.5 +0.45 2.5 +-0.5on lo -0.5 2.2 3.5 0.05M-NH3 G-50 * Relative to exclusion volume 1.0. t Mobility relative to lysine = +1.0 and to aspartic acid = -1.0. $ Chromatography in pyridine-3-methylbutan-1-ol-water (7:7:6, by vol.) (PAW) or in butan-l-ol-acetic acid-water (12:3:5, by vol.) (BAW). = Vol. 117 AMINO ACID SEQUENCES OF HUMAN HEAVY CHAINS 649 Table 8. Chymotryptic peptides of Cor tryptic peptide 2a'TI The composition of peptide TI is based on 2 alanine residues/molecule of peptide. The S-carboxyamido. methylcysteine composition of peptide TI is calculated from the amino acid analysis. S-Carboxyamidomethylcysteine residues are based on the radioactivity of the peptides. Composition (mol of amino acid residue/mol of peptide) Amino Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide acid TlC2a TlC4b TlC4d TlC4e TlC5bl T1C5b2 TlC5e TlC6d TlC6h 2.0 0 0 0 0 0 0 0 0 Lys 0 0 0.8 0.9 0 0 0 0 His 0 0 0 0 0 0 0 0 0 0 Arg 0 1.1 1.9 0.8 0 0 0 0 1.1 Asp 0 1.0 0 1.0 0 1.0 1.0 1.9 Thr 1.0 1.1 1.2 1.2 0 2.0 5.5 0 1.1 0 Ser 0 0 0 0 0 0 1.0 1.0 1.0 Glu 0 0 1.1 0 0 1.1 1.0 0 Pro 1.9 1.2 1.2 0 0 1.2 0 0 1.2 0 Gly 0 0 0 1.0 1.0 0 0 0 0 Ala 1.0 0 0.8 0 2.5 1.0 1.9 0 0 Val 0 0 0 0.9 0 0 0 0 0 Ile 0 0 0 1.2 0.8 1.0 1.9 0 0 Leu 0 0 0 0.8 0 0 0 0.9 1.0 Tyr 0 0.8 0 0 0 0 0 1.0 0 Phe 0 1.0 0 0 0 0 0 0 0 Cmc 0 0 0 0 0 0 0 1.0 0 Trp 5 5 3 9 6 6 12 5 11 Total residues Sum of peptides 2 2 0 5 7 11 3 5 4 2 8 1 5 3 2 1 1 62 Peptide Ti 1.9 1.4 0.1 4.1 6.3 10.0 2.9 4.2 4.0 2.0 6.3 0.9 4.7 2.6 2.0 0.7 1.0 55 for 2 h) and the digest was fractionated on a colunm complete, as shown in Fig. 7, except for the order of Sephadex G-50 in 0.05M-ammonia. The methods of the five chymotryptic peptides of peptide TI, of purification of the relevant peptides are shown which have been aligned by comparison with the in Table 7 and their compositions are given in rabbit heavy chain, as described above. Alignment of thefourfragments of Cor Fdfragment. Table 9. Fig. 7 shows the partial-sequence data on the chymotryptic peptides and the consequent Fragment 5b has been shown to be the N-terminal alignment of the tryptic peptides. The N-terminal fragment of the Fd fragment and fragment 2a' residue of fragment 2a' is aspartic acid, which is in the C-terminal fragment. Fragment 5a is assumed agreement with the sequence shown in Fig. 7. to follow fragment 5b, and fragment 6a to be the A chymotryptic peptide overlapping tryptic pep- third fragment from the N-terminal end, by the tides T4.2d and T4. Ic was not found, but two similarity in sequence of Cor fragment 5a to Daw peptides, C4.2.9b and C3.6bc, were isolated and, fragment 4 (Press, 1967) and the similarity of Cor as shown in Fig. 7, these peptides come from this fragment 6a to the N-terminal sequence of Daw part of the sequence. The latter peptide results fragment 2a' (see next section). The complete from cleavage by chymotrypsin (or contaminating sequences of Daw and Cor Fd fragments are comtrypsin) of the Lys-Ser bond. The chymotryptic pared in Fig. 10. peptide C4.2.3c must occur at the C-terminal end of fragment 2a', as this is the only position in the Sequence of Dawfragment 2a' sequence where it can be placed. Papain presumThe amino acid sequence of the first 84 residues ably digested the Cor heavy chain at this histidine residue. Steiner & Porter (1967) reported that of Daw heavy chain has already been determined papain digested Daw heavy chain at a histidine (Piggot & Press, 1967; Press, 1967). The fragment residue in an identical sequence consisting of 2a' comprised the C-terminal part of Daw Fd peptides C3.1 1-C4.2.3c. Peptides derived from the fragment from residue 85 onwards. This fragment tryptic peptide TI were also found in the chymo- has an N-terminal asparagine residue (see mobility tryptic digest and one of these, C3.5d, overlapped of peptide T2.5 given below) and its sequence has two of the peptides derived from the chymotryptic been established by isolating the tryptic and digest of peptide TI (T1.C4e and TI.C4b). The chymotryptic peptides. The sequence becomes sequence of Cor fragment 2a' is therefore almost identical with that of Cor heavy chain from residue E. M. PRESS AND N. M. HOGG 650 1970 ~T1 Asp-Tyr-Phe-Pro-Glu-Pro-Val-Thr-Val-Ser-Trp.Asn-Ser-Gly-Ala- Leu.Thr-Ser-Gly-Val-His. Tl.C2a 4 Tl.C5b2 'o 4 )- 4- -)O -]O -* 4). 4-. -lo T.C5bl- 4 ). 41I- --) --) -+* -0* -* ), - 1T.C4d 10 Leu Thr-Ser-Gly-Val-His Asp-Tyr-Phe-Pro-Glu-Pro-Val-Thr-Val-Ser-Trp Asn-Ser-Gly ,.-* 4-T1.C5e T1.C4e 4 T1.C4b----- Thr-Phe-Pro-Ala-Val-Leu Gln-Ser-Ser-Gly-Leu-Tyr Ser-Leu-Ser-Ser-Val-Val-Thr-Val-Pro-Ser-Ser-Leu-Gly-Thr-Gln-Thr-Tyr --lo --)O --. --O --O -4. -+ --* --* --, *- -- -+> -. -0. -+ -+ -+. -0. -0. -+. -0. 4- --)I --o --* -+O --)O 4-- 4- 4-4 Ser-Leu-Ser Tyr -a-I 4- -a-) Ile-Cmc-Asn-Val-Asn-His ( Lys,Pro,Ser)Asn-Thr- Lys 4-Ti.C6d-* Tl.C6h 4---- lIe-Cmc-Asn Val-Asn-His -a-. -a-* -a-l 4-T I.C6g-- 4-T1.C5a4- Asn-Thr-Lys Fig. 4. Amino acid sequence of Cor tryptic peptide 2a'.Tl. Sequence determination from the N-terminal end was by the 'dansyl'-Edman technique (-+) and from the C-terminal end by carboxypeptidase A digestion (-) or by hydrazinolysis (e+). 170 160 150 Cor 2a'T1 Asp-Tyr-Phe-Pro-Glu-Pro-Val-Thr-Val- Ser-Trp.Asn-Ser-Gly-Ala-Leu.Thr-Ser -Gly-Val-His.Thr-Phe-Pro-Ala-Val- Rabbit heavy chain Gly-Tyr-Leu-Pro-Glu-Pro-Val-Thr-Val-Thr-Trp-Asn-Ser-Gly-Thr-Leu-Thr-Asp-Gly-Val-Arg-Thr-Phe-Pro-Ser-Val Cor 2a'T1 Leu.Gln-Ser-Ser-Gly-Leu-Tyr.Ser-Leu-Ser-Ser-Val-Val-Thr-Val-Pro-Ser-Ser-Leu-Gly-Thr-Gln-Thr-Tyr. Rabbit heavy chain* Arg-Gln-Ser-Ser-Gly-Leu-Tyr-Ser-Val-Pro-Ser-Thr-Val-Ser-VaI 180 190 -- 200 lie -Cys-Asn-Val Ser-Glx-Pro(Pro,Ser)Thr-Cys-Asx-Val Fig. 5. Comparative sequences of Cor heavy chain peptide 2a'.T1 and the rabbit heavy chain for residues 150-203 (Daw numbering). Identical residues are underlined. * Fruchter, Jackson, Mole & Porter (1970). 115 onward, and so the sequence-determination work will only be given in detail for residues 85-123, but the composition of all the constituent tryptic peptides of Daw fragment 2a' are given in Table 10. In those instances where an identical tryptic peptide was also isolated from Cor fragment 2a', the number of the Cor peptide is put in parentheses. Tryptic peptides. Fragment 2a' was digested with trypsin (400,ug/,umol at 37°C for 4h). The digest was centrifuged, and the precipitate contained more than half the total digest and was found to be a large insoluble peptide Ti of 62 residues (Table 10). This peptide had a composition identical with that of Cor peptide 2a'.Tl, and its amino acid established by isolating the constituent chymotryptic peptides, and found to be sequence was AMINO ACID SEQUENCES OF HUMAN HEAVY CHAINS Vol. 117 651 Ser-Thr-Ser-Gly-Gly-Thr-Ala-Ala-Leu-Gly-Cmc-Leu-Val-Lys T4. 1 c --Io --Io --) --). T4.1c.C2- 4 *--T4.1c.Cl-- - Ser-Thr-Ser Gly-Gly-Thr Ala-Ala-Leu Gly-Cmc-Leu Val T-S4-Al -- T4.1d Gly-Thr-Pro-Val-Thr-Val-Ser-Ser-Ala-Ser-Thr-Lys Ser-Ser-Ala-Ser-Thr -+)N -5). -5l. --)O Mobility at pH 6.5 = 0 T4.2a Ser-Cmc-Asp-Lys V-A -L -T2 T4.2b -5.p Mobility at pH 6.5 = 0 Val-Asp-Lys -5*. -5l. T4.2d Gly-Pro-Ser-Val-Phe-Pro-Leu-Ala-Pro-Ser-Ser-Lys -5). -5). -5O. --). T4.2e ,. --5. -.O. ~-5. Mobility at pH 6.5 = + 0.35 Lys-Val-Glu-Pro-Lys -5*. -5O. -5). T4.3f -5. Thr-His -T5 As --T. T5 Mobility at pH 6.5 = 0 Asp-Val-Trp-Gly-Arg ---b -5'. +-T5.C1-* *T5.C2* Asp Gly-Arg Fig. 6. Amino acid sequence of the tryptic peptides of Cor 2a' (except peptide TI, shown in Fig. 4). Sequence determination from the N-terminal end was by the 'dansyl'-Edman technique (-*) and from the C-terminal end by carboxypeptidase A digestion (-<-) or hydrazinolysis (-i). Table 9. Chymotryptic peptides of Cor fragment 2a' The S-carboxyamidomethylcysteine residues are calculated from the radioactivity of the peptides. The tryptophan residues are assumed to be 1 when the peptide stained with Ehrlich's reagent. Composition (mol of amino acid residue/mol of peptide) Amino acid Lys His Arg Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe Cmc Trp Total residues Peptide C3.5d Peptide C3.6a 0.8 Peptide C3.6bc Peptide C3.7d 0.8 Peptide C3.11 3.3 Peptide C4.2.3c Peptide C4.2.9b Peptide C4.3f 5 Peptide C5.1 0 0 0 0 0 1.0 0.9 0 0 0 0 0 0 0 0 0 0 0 0 0.8 0 0 0 1.2 0 1.0 1.0 3.8 0 1.0 0.9 1.2 0 1.6 2.0 0 1.8 0 2.1 0 0 0 0 1.1 1.1 0 0 0 0 0 0.9 0 0 0 0 1.2 2.4 0 0 1.0 0 0 2.2 0 0 1.8 1.0 0 0 0 0 0 0 0 2.2 5.8 0.9 1.4 1.7 1.2 1.1 1.7 0.9 2.0 0.9 0 0 2.3 2.2 2.2 2.7 0 2.2 1.3 1.4 1.0 0 0 0 0 2.1 0.7 0.8 0.8 0.9 1 1 0 0 0 0 17 16 0 0 0 0 1.1 0.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0.7 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 9 13 9 6 4 8 1 3 1970 E. M. PRESS AND N. M. HOGG 652 Asp-Val-Trp-Gly-Arg-Gly-Thr-Pro-Val-Thr-Val-Ser-Ser-Ala-Ser-Thr-Lys-Gly-Pro-Ser-Val-Phe-Pro- Leu-Ala-Pro-Ser-Ser-Lys- ---* C3.7 C4.3f55 .4-C.5.1--+ 4 Ser-Ser-Ala Gly-Arg Asp-Val Ala -4'O -4* + C3.7d.T2 C3.7d.Tl Ser-Thr-Ser-Gly-Gly-Thr-Ala-Ala- Leu-Gly-Cmc- Leu-Val- Lys-Asp-Tyr-Phe-Pro-G lu-Pro-Val-Thr-Val-Ser-Trp.Asn-Ser-Gly-Ala- Leu. Ser-Thr Ala-Ala-Leu 4-C3.6a.T2- -4)I -+* 4- 4- Gly 316 C Asp Thr-Ser-GIy-Val-His.Thr-Phe-Pro-Ala-Val- Leu.GIn-Ser-Ser-Gly- Leu-Tyr.Ser- Leu-Ser-Ser-Val-Val-Thr-Val-Pro-Ser-Ser- Ser- Leu-Ser Leu-Gly-Thr-Gln-Thr-Tyr. lle-Cmc-Asn-Val-Asn- His( Lys,Pro,Ser)Asn-Thr- Lys-Val-Asp- Lys- Lys-Val-Glu-Pro- Lys-Ser-Cmc-Asp-Lys-Thr Tl C3.5d +-T4.2b4 Lys-Val-Asp Tyr T4.2e C3.11 T4.2a His 4*T4.3f* -C 4-C4.2.3c- Ser-Cmc-Asp- Lys-Thr - H is 4- Fig. 7. Amino acid sequence of Cor fragment 2a', showing partial-sequence data on the chymotryptic peptides and the alignment of all the tryptic peptides. The sequence determination was by the 'dansyl'-Edman technique (-*) and by carboxypeptidase A digestion (-). Peptides C3.7d.Tl and C3.7d.T2 were tryptic peptides of peptide C3.7d; peptides C3.6a.Tl and C3.6a.T2 were tryptic peptides of peptide C3.6a. identical with that of Cor peptide 2a'.T 1. The it was found impossible to purify it by paper soluble portion of the tryptic digest was fractionated electrophoresis since it could not be eluted from by gel filtration and paper electrophoresis and the paper after electrophoresis in more than 10% yield. method of isolation and the compositions of the It was, however, possible to precipitate it from the relevant tryptic peptides are shown in Tables 11 gel-filtration fraction concentrated to 0.5[Lmol of and 10 respectively. Seven of the nine tryptic peptide/ml by adding acetic to a concentration of peptides were identical with seven of the tryptic 1 M and leaving at 2°C overnight. The precipitate peptides isolated for Cor fragment 2a' (Table 6); was centrifuged and washed with O.1M-acetic acid their amino acid sequences were determined and no at 0°C, and on analysis was found to be pure (see differences were detected. The amino acid sequences Table 10). It was recovered in 60% yield by this of the other two peptides, T2.5 and T2.p, were procedure, and peptide T2.5 was isolated from the supernatant by electrophoresis (Table L1). Peptide determined as shown in Fig. 8. Peptide T2.5. This was digested with chymo- T2.p has serine as the N-terminal residue. It was trypsin. The isolation and compositions of the digested with chyinotrypsin (800 Lg/pnmol at 37°C chymotryptic peptides, T2.5.C1, T2.5.C2, and for 4h) and six peptides were isolated from the T2.5.C3, are shown in Table 12 and their sequence digest by paper electrophoresis at pH 3.5 (see Table is given in Fig. 8. Peptide T2.5 was neutral at 12). The amino acid sequences of these chymopH 6.5 and after one step of the Edman degradation tryptic peptides of peptide T2.p were determined as the residual 'peptide was also neutral; therefore shown in Fig. 8. Peptide T2.p.C3 must be the N-terminal peptide, the N-terminal residue must be asparagine and the since it has serine as the N-terminal residue. Peptide other aspartic residue is present as aspartic acid. Peptide T2.p. This had very low soluibility, and T2.p.C4 contains lysine and mulst therefore be the a Vol. 117 AMINO ACID SEQUENCES OF HUMAN HEAVY CHAINS Table 10. Tryptic peptides of Daw fragment 2a' 653 The S-carboxyamidomethylcysteine composition of fragment 2a' is calculated from the amino acid analysis. The S-carboxyamidomethylcysteine residues in the tryptic peptides are calculated from the radioactivity of the peptides. The tryptophan content in fragment 2a' is calculated from the extinction at 280nm. The tryptophan content of the peptides is based on staining of the peptides on paper with Ehrlich's reagent. The composition of fragment 2a' is based on 8 alanine residues/molecule of fragment 2a'. The composition of peptide Ti is based on the sum of the residues of its constituent chymotryptic peptides (Table 8). The numbers of the equivalent Cor 2a' tryptic peptides are given in parentheses under the numbers of the appropriate Daw tryptic peptides. Composition (mol of amino acid/mol of peptide) Amino acid Lys His Arg Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe Cmc Trp Total residues Peptide Peptide Peptide Peptide Peptide Peptide Peptide T.4 T3.6 T3.4 T3.3 T3.2 Ti Peptide Peptide T3.1 T2.p (T4.2a) (T4.2b) (T4.1c) (T4.2d) (T4.2e) (T4.3f) T2.5 (TI) 1.2 2.1 0 1.1 1.1 1.1 1.0 2 0 0 0 1.0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 1.1 0 0 0 0 1.0 1.0 1.3 5 1.8 0 0 1.0 0 1.9 1.9 0 7 2.4 0 3.2 0 5.1 1.0 0 2.2 11 0 0 0 0 0 1.2 0 0 2.0 3 2.4 0 0 0 0.8 0 0 5 1.3 1.2 0 0 0 3.2 4 2.9 0 2.2 1.1 0 0 0 1.8 2 2.2 1.3 0 0 2.0 0 1.0 1.0 0.9 8 0.9 1.2 0 0 0 0 0.9 0 0 1 0 0 0 5 1.0 0 0 1.8 1.0 0 0 0 0 0 0 1.6 0 3 1.8 0 0 0 0 0 0.9 2 0 0.9 0 1 1 0 1 0 0 1 1 0 0 1 0 0 1 0 0 0 12 2 4 3 14 5 24 62 15 C-terminal peptide. Peptide T2.p.C5 is a shorter part of peptide T2.p.C4. The three peptides, T2.p.C3, Cl and C4, together account for the composition of peptide T2.p. Peptide T2.p.C3 is neutral at pH 6.5 and peptides T2.p.Cl and T2.p.Cla both have one net negative charge, and therefore the glutamic residue in peptide T2.p.C1 must be glutamine and the aspartic must be present as aspartic acid. A peptide 2a'C6d, Phe-Asp-Tyr-Trp, overlapping peptides T2.p.C3 and T2.p.C1 was isolated from a chymotryptic digest of the whole of Daw fragment 2a' (see next section), thus confirming the sequence of peptide T2.p shown in Fig. 8. Chymotryptic peptides. Fragment 2a' was digested with chymotrypsin (500,ug/,umol at 37°C for 4jh). The digest was fractionated on a column of Sephadex G-50 in 0.05M-ammonia. The constituent chymotryptic peptides of the tryptic peptide Ti were isolated, together with the chymotryptic peptides overlapping the tryptic peptides that are common to Cor and Daw 2a' fragments. In addition the chymotryptic peptides derived from the N-terminal part of Daw fragment 2a', comprising the tryptic Sum of peptides 9 3 1 10 15 22 6 10 13 8 15 2 9 7 4 5 2 141 Fragment 2a' 8.0 2.4 0.8 10.0 14.0 21.0 6.0 10.0 13.0 8.0 14.0 1.9 9.0 6.0 3.7 3.4 2.0 133.2 peptides T2.5, T2.p and T3.4, were isolated by the procedures shown in Table 11 and their analyses are given in Table 13. Peptide C3.a. This has the same sequence as the N-terminal 11 residues of tryptic peptide T2.5 (see Fig. 9). Peptide T2.5 must therefore be the Nterminal peptide of fragment 2a', which fragment has asparagine as N-terminal residue. This is further confirmed by the comparison of the sequences of Cor and Daw heavy chains (Fig. 10). Peptide C5.gl. The partial amino acid sequence of this peptide was determined as shown in Fig. 8. Peptides C5.gl.Tc and Ta are tryptic peptides of peptide C5.gl. This peptide overlaps the two tryptic peptides, T2.5 and T2.p, of fragment 2a' (see Fig. 9). Peptide C4.f. The partial amino acid sequence of this peptide was determined as shown in Fig. 8. It contains the C-terminal part of tryptic peptide T.2p and the N-terminal part of peptide T3.4 (which has a sequence identical with that of Cor peptide 2a'.T4.2d; see Fig. 6). Another chymotryptic peptide, C4.e, also overlaps the two tryptic peptides (see Fig. 9). Peptide C4.f has an identical E. M. PRESS AND N. M. HOGG 1970 654 Table 11. Isolation procedures for the tryptic peptides and some chymotryptic peptides of Daw fragment 2a' Paper electrophoresis Sephadex G-50 (0.05 m-NH3) elution volume* 1.5 1.5 2.0 2.0 2.0 2.0 2.0 2.7 Tryptic peptides T2.5 T2.p T3.1 T3.2 T3.3 T3.4 T3.6 T4 Chymotryptic peptides C3.a C4.fd Mobilityt pH or chromatography RF 3.5 +0.3 Precipitation (see the text) 6.5 -0.04 6.5 0 6.5 +0.2 6.5 +0.3 6.5 +0.4 3.5 +0.5 3.5 3.5 1.9 2.1 -0.3 +0.45 PAWt C5.a C5.d9 2.2 2.2 6.5 6.5 C5.e8 2.2 6.5 C5.gl C6.cl 2.2 2.7 6.5 3.5 C6.c2 2.7 3.5 C6.d 2.7 3.5 0.4 -0.25 0 0.8 0 0.7 +0.3 -0.2 0.4 -0.2 0.6 -0.1 BAWt BAWt BAW+ BAWt 0.8 BAW+ * Relative to exclusioni volume = 1.0. t Mobility relative to lysine = +1.0, to aspartic acid -1.0. + Chromatography in pyridine-3-methylbutan-l-ol-water (7:7:6, by vol.) (PAW) or in butan- l-ol-acetic acid-water (12:3:5, by vol.) (BAW). counterpart, in Cor 2a' chyinotryptic peptide C3.7d, and from this point the sequence of Daw fragment 2a' is identical with that of Cor fragment 2a'. The identity in sequence was established by determining the amino acid sequences of all the constituent tryptic and chymotryptic peptides of Daw fragment 2a' (N M. Hogg, unpublished work; this work forms part of a Thesis to be presented by N. M. H. in partial fulfilment of the requirements for a Ph.D.). Peptides C6.d, C5.e8, C5.d9 and C5a. These were also isolated (see Tables 11 and 13) and Fig. 9 shows that these peptides are derived from tryptic peptide T2.p and confirm the sequence of the latter peptide. Free tyrosine and phenylalanine (C6.cl and C6.c2) were also present in the chymotryptic digest of fragment 2a' and are presumably derived from this part of the sequence (see Fig. 9). The complete amino acid sequence of Daw fragment 2a' (residues 85-225) is shown in Fig. 10. Intrachain disulphide bridges chains in Cor and Daw heavy The heavy chain of Cor was digested with cyanobromide and fractionated on a column of gen Sephadex G-100 in 6M-urea-0.2M-sodium formate described by Piggot & Press (1967) for the cleavage products of Daw heavy chain. Fraction 5 (elution volume 2.5 relative to exclusion volume = 1.0) was totally reduced as described by Cebra et al. (1968) and alkylated with iodo[L-14C]acetamide and re-run on the same column of Sephadex G- 100. The three fragments 5a, 5b and 6a, described above, were separated. Fragments 5b and 6a were radioactive, but fragment 5a was not radioactive although it contains an alkylated cysteine residue (see Fig. 2). Presumably the cysteine residue in fragment 5a was alkylated during the mild reduction and alkylation of the IgG before the isolation of the heavy chain. The cysteine residues in fragments 5b and 6a must be linked by a disulphide bond, and these cyanogen bromide fragments, joined by a disulphide bond, were eluted in the same elution volume as fragment 5a on the first column. The two cysteine residues in Cor fragment 2a', which occur in peptides T4. lc and TI.C6d (Figs. 4 and 7), are presumably also linked by a disulphide bond. This arrangement of two intrachain disulphide bonds in the Fd fragment is in agreement with the work of Frangione & Milstein (1967), and the sequences around cysteine as 655 AMINO ACID SEQUENCES OF HUMAN HEAVY CHAINS Table 12. Method8 of i8olation and composition of the chymotryptic peptides of Daw fragment 2a' tryptic peptide8 T2.5 and T2.p Vol. 117 Mobility is given relative to lysine = +1.0, aspartic acid -1.0. The S-carboxyamidomethylcysteine content of the peptides is based on radioactivity. The tryptophan content of the peptides is based on the staining of the peptides on paper with Ehrlich's reagent. Composition (mol of amino acid/mol of peptide) Amino Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide acid T2.5.Cl T2.5.C2 T2.5.C3 T2.p T2.p.Cl T2.p.C3 T2.p.C4 T2.p.C5 T2.p.Cla T2.p.Clb T2.5 0 0 1.2 1.1 0 0 1.1 0 0 0 0 Lys 0 0 0 0 0 0 1.1 1.0 1.0 0 0 Arg 0 0 0 0 1.0 0 1.4 0 1.3 1.8 1.9 Asp 0.7 0 0 0 1.7 0 0 1.9 2.4 2.6 0 Thr 0 2.2 0 2.4 3.0 0 0 0 5.1 0 0 Ser 0 1.1 0 0 0 1.0 0.9 0 2.0 0 0 Glu 0 0 0 0 0 0 0 0 0 Pro 1.3 1.2 0 0 0 2.2 0 2.2 1.2 2.9 0 2.2 2.3 Gly 1.2 1.0 0 0 0 0 1.1 1.0 1.3 2.2 1.1 Ala 0 0 0 0 0 1.9 2.0 0 1.1 0 1.2 Val 0.7 0 0 0 0 0.7 0 0 0 0.9 0 Ile 0 1.0 0 0 0.8 0 0 0 1.0 Leu 0 0 0 0 1.0 0 0 0.7 1.6 0.9 1.8 0.9 1.8 Tyr 0 0 0 0 0 0.8 0 0 0.9 Phe 0 0 0 0 1 0 0 1 1 0 1 0 1 Cmc 0 1 0 0 0 1 1 0 0 0 0 Trp 5 24 7 9 5 4 8 3 12 3 15 Total at 3.5 Electrophoresis. Mobility pH 0 +0.36 -0.3 0 +0.14 -0.5 -0.33 +0.55 +0.65 +0.3 T2.5 Asn-Thr-Val-Gly-Pro-Gly-Pro-Gly-Asp-Thr-Ala-Thr-Ty!--Tyr-Cmc-Aa-Arg -5. -4 . .4 -0* .4-4.4.4.-4o-10. -04.4 .4-T2.5.C3-* T2.5.Cl Asn-Thr -~~~ -~~ Ala-Thr-Tyr-Tyr-Cmc-Ala-Arg 4- 4- 4-4-~~~~*_ -10 --)O -30 4 T2.5.C2-- Tyr-Cmc T2.p 10 < T2.p.Cl T2.p.C- I T2.p.C3 p < Ser-Cmc-Gly-Ser-GIn-Tyr-Phe-Asp-Tyr-Trp-Gly-Gln-Gly-Ile-Leu-Val-Thr-Val-Ser-Ser-Ala-Ser-Thr-Lys -5 - 4' - -+) -4-b. -+)-4- 4--46-4-45.1*45. -4+ +T2.p.Cla-5. 4-T2.p.Clb--- P -T2.p.C5.-5 Ser-Ala-Ser-Thr Asp-Tyr-Trn Gly-GIn-Gly-Ile-Leu 0- -s' -+-+ -4. ---P --P 46- C5.gi Cmc-Ala-Arg-Ser(Cmc,Gly)Ser-GIn-Tyr -+0.44-_4- 44-C5.g1.Tc-. .4Ser C4.f T5.g1.Ta -- Ser Gin-Tyr 4-4-44- Ser-Ser-Ala-Ser(Thr,Lys,Gly,Pro,Ser,Val,Phe,Pro) Leu Fig. 8. Amino acid sequence of some tryptic and chymotryptic peptides of Daw fragment 2a'. Sequence determination from the N-terminal end was by the 'dansyl'-Edman technique (-*) or by digestion with leucine aminopeptidase (-) and from the C-terminal end by digestion with carboxypeptidase A (-) or by hydrazinolysis (4--). are very similar to those reported for other y7 heavy chains by these authors. The cysteine residue in fragment 5a must be present as a free thiol group or perhaps linked to a cysteine residue, as found in a light chain (Milstein, Clegg & Jarvis, 1968). Daw heavy chain has cysteine residues at positions E. M. PRESS AND N. M. HOGG 656 1970 Table 13. Chymotryptic peptides of Daw fragment 2a' S-Carboxyamidomethylcysteine residues are based on the radioactivity of the peptides. The tryptophan content of the peptides is based on staining of the peptides with Ehrlich's reagent. Composition (mol of amino acid/mol of peptide) Peptide Amino acid Peptide C4.f C3.a Lys 0 1.1 0 0 0 0 His Arg Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe Cmc Trp Total residues 2.0 2.6 0 0 1.0 2.1 1.0 1.3 0 1.0 3.1 0 2.3 1.4 1.1 1.0 0 1.2 0 0 1.( 0 0.8 0 0) 0 13 I11 Peptide C5.a Peptide Peptide C5.d9 C5.e8 0 0 0 0 0 0 0 0 1.0 0 0 1.2 0 2.0 0 0 0.8 1.0 1.0 0 0 0.9 0 0 0 0 0.8 0 0 2.3 1.0 0 1.2 1.0 0 0 0 1 0 8 3 0 0 0 0 0 1.1 0 1.9 0 0 0.9 1.1 0 0 0 0 5 0 0 2.1 0 0 0 0 90 85 Peptide C5.gl Peptide C6.d 0 0 0 1.2 0 0 0 0 0 0 0 0 0 0.9 0.9 0 0.7 0 2 0 0 1 4 9 100 Asn-Thr-Val-G ly-Pro-G ly-Asp-Thr-Ala-Thr-Tyr-Tyr-Cmc-Ala-Arg-Ser-Cmc-G ly-Ser-GlIn-Tyr-Phe-Asp-Tyr-TrpC3.a 4 )C6.cl C5.g1 4 4- C6.d-+ C6.c2*-C5.ao10. Gly-G n-G y- 120 130 le- Leu-Val-Thr-Val-Ser-Ser-Ala-Ser-Thr- Lys-Gly-Pro-Ser-Val-Phe-Pro- Leu- -T.3.4- T.2p 4 C5.e8 C.5a 4-C5.d9 -* 4 0 -( C4.f C4.e Fig. 9. Sequence of the N-terminial part of Daw fragment 2a', showing tryptic and chymotryptic peptides. 22, 35, 97, 101, 146 and 201 (Fig. 10). The cysteine at position 221 in Daw heavy chain was shown by Steiner & Porter (1967) to be in disulphide linkage with the light chain. Presumably cysteine-22 is joined to cysteine-97, and cysteine-146 to cysteine201, as shown for Cor heavy chain. The cysteine residues at positions 35 and 101 must also be linked by a disulphide bond, since it was shown by Piggot & Press (1967) that fragment 4 (residues 35-84) was linked to fragment 2a' (residues 85-225) in the Fab fragment. Carbohydrate on Cor Fab fragment HIexosamine was found in Cor fragmnent 5a, the corresponding region of Daw, fragment whereas 4, contained no hexosamnine. The hexosamiine content of whole IgG of Cor was 13.5mol/mol, of which 6 nol/mol was on the Fc fragment and 7 mol/ mol on the Fab fragment. However, only 1.5mol of hexosamine/mol was found in fragment 5a, and so the other fragments of Cor Fd (5b, 6a and 2a') and the light chain were also analysed, and the hexosamine content was nil in all three fragments and in the light chain. We assume therefore that Fd fragment 5a had a similar amount of carbohydrate to that on the Fc fragment (Franklin, 1960, found 5mol of hexosamine/mol of normal Fc fragment), but that during digestion with cyanogen bromide in 70%o formic acid and subsequent fractionation in 6m-urea on a column of Sephadex G-100 most of the carbohydrate was destroyed. 657 AMINO ACID SEQUENCES OF HUMAN HEAVY CHAINS Vol. 117 The neutral sugar content was not investigated. The carbohydrate was located on the chymotryptic peptide C3.c of fragment 5a (see section on the sequence of Cor fragment 5a). The residue involved in the linkage was determined by digesting totally reduced Fab fragment with chymotrypsin (1 mg/ ,umol at 37°C for 4h) and with pronase (8mg/,tmol at 370C for 48h). The digest was fractionated on a column of Sephadex G-25 in 0.02M-ammonia, which separated the glycopeptide from the peptides, and refractionation on a column of Sephadex G-50 in 0.05m-ammonia to separate the glycopeptide from the enzymes. The only amino acid present in the glycopeptide was aspartic acid, and therefore the carbohydrate must be attached to the aspartic acid residue at position 62 in Cor Fd fragment. Presumably the linkage involves the P-carboxyl group of aspartic acid and N-acetylhexosamine. DISCUSSION The entire amino acid sequences of Daw and Cor Fd fragments are compared in Fig. 10. The numbering is based on Daw heavy chain, and to maximize the homology between the two a deletion of three residues (88-90) and an insertion of four residues after position 104 have been placed in Cor heavy chain. There is only one difference between the two chains in the first 30 positions, namely a substitution of a lysine for an arginine residue at position 13. Then for residues 31-33 there are no residues in common between the two chains and thereafter there are blocks of identical residues, e.g. 38-44 and 64-69, interspersed with regions of differing residues. Altogether, 77 % of the positions between 1 and 99 are occupied by the same residue in the two chains. The incidence of identical residues between positions 100 and 114 is very much less, only five or 30%, but from position 115 to the C-terminal residue the two chains have an identical sequence. The amino acid sequence of another y 1 heavy chain, Eu, has been determined (Edelman et al. 1969) and this also has the same sequence as Daw and Cor from position 115; but a fourth yl chain, He, for which a sequence of 21 residues was reported (Edelman et at. 1969), has an alanine residue instead of threonine at position 116. The variable region of the heavy chain must therefore extend at least to position 116. The variable regions of the light chains extend to positions 108 (K chains) and 109 (A chains), and there Daw Cor 10 20 30 PCA-Val-Thr- Leu-Arg-Glu-Ser-Gly-Pro-Ala- Leu-Val -Arg- Pro-Thr-GIn-Thr-Leu-Thr- Leu-Thr-Cys-Thr-Phe-Ser-Gly-Phe-Ser-Leu-SerPCA-Val-Thr- Leu-Arg-Glu-Ser-Gly-Pro-Ala-Leu-VaI -Lys- Pro-Thr-Gln-Thr-Leu-Thr-Leu-Thr-Cys-Thr-Phe-Ser-Gly-Phe-Ser-Leu-Ser- Daw Cor Gly-Glu-Thr-Met-Cys-Val-Ala-Trp-lle-Arg-Gln-Pro-Pro-Gly- Glu-Ala- Leu-Glu-Trp-Leu-Ala-Trp-Asp-lle-Leu-Asn-Asp-Asp-Lys-TyrSer-Thr-Gly-Met-Cys-Val-Gly-Trp- le-Arg-Gln-Pro-Pro-Gly- Lys-Gly- Leu-Glu-Trp-Leu-Ala- Arg-Ile-Asp-Trp-Asp- Asp-Asp-Lys-Tyr- Daw Cor Tyr-Gly-Ala-Ser-Leu-Glu-Thr-Arg-Leu-Ala-Val -Ser-Lys-Asp-Thr-Ser -Lys -Asn-Gln-Val-Val-Leu -Ser-Met-Asn-Thr-Val-Gly-Pro-GlyTyr-Asx-Thr-Ser-Leu-Glu-Thr-Arg-Leu -Thr -lie -Ser-Lys-Asp-Thr-Ser -Arg -Asn-Gln-Val-Val-Leu -Thr-Met-Asp-Pro-Val- - Daw Asp-Thr-Ala-Thr-Tyr-Tyr-Cys-Ala-Arg -Ser-Cys-Gly-Ser-Gin Cor Asp-Thr-Ala-Thr-Tyr-Tyr-Cys-Ala-Arg -lie -Thr -Val -Ile -Pro -Ala-Pro-Ala-Gly-Tyr-Met-Asp-Val-Trp-Gly-Arg-Gly-Thr-Pro- Daw Cor 120 130 140 Val-Ser-Ser-Ala-Ser-Thr- Lys-Gly-Pro-Ser-Val-Phle-Pro- Leu-Ala-Pro-Ser-Ser-Lys-Ser-Thr-Ser-G ly-Gly-Thr-Ala-Ala- Leu-G Iy-Cys- Daw Cor 150 160 170 Leu-Val- Lys-Asp-Tyr-Phe-Pro-Glu-Pro-Val-Thr-Val-Ser-Trp.Asn-Ser-Gly-Ala- Leu.Thr-Ser-Gly-Val-His.Thr-Phe-Pro-Ala-Val- Leu. Daw Cor 180 190 200 Gln-Ser-Ser-Gly- Leu-Tyr.Ser- Leu-Ser-Ser-Val-Val-Thr-Val-Pro-Ser-Ser- Leu-Gly-Thr-Gln-Thr-Tyr. le-Cys-Asn-Val-Asn-His( Lys, Daw Cor Pro,Ser)Asn-Thr- Lys-Val-Asp- Lys- Lys-Val-Glu-Pro- Lys-Ser-Cys-Asp- Lys-Thr-His 50 40 60 80 70 CHO 100 210 90 110 220 - 115 Tyr-Phe-Asp-Tyr-Trp-Gly-Gln -Gly -lle-LeuVal-Thr- 225 Fig. 10. Comparative sequences of the Fd fragments of the two yI heavy chains Daw and Cor. Identical are underlined for positions 1-114; for positions 115-225 the two fragments have an identical sequence. residues E. M. PRESS AND N. M. HOGG 658 is a sequence near the beginning of the constant region common to both classes of light chain, Ala-Pro-Ser-Val-, which is very similar to a sequence, Gly-Pro-Ser-Val-, occurring near the beginning of the heavy-chain constant region. The highly variable region between positions 100 and 114 referred to above has also been found to occur in an analogous position in K and A chain sequences, just after the second intrachain cysteine residue (Milstein, 1967, 1969; Langer, Kayne & Hilschmann, 1968; Putnam, 1969). Position 31 in K chains is also highly variable (Milstein, 1969), and since Daw and Cor heavy chains have different sequences for positions 31-33 this region of heavy chains may also prove to be highly variable when more sequences are available for comparison. In all these respects the pattern of sequence variation, revealed by the study of heavy chains Cor and Daw, is very similar to that observed for light chains. Comparison of the sequence of the y 1 chain Eu (Edelman et al. 1969) with that of Daw shows that in only 29 positions between 1 and 99 does the same residue occur as in Daw heavy chain. This is in contrast with the 76 identical residues between Cor and Daw referred to above. The sequence of the first 105 residues of the heavy chain of another class of immunoglobulin, a ,u chain Ou, has been reported by Wikler, Kohler, Shinoda & Putnam (1969), and, rather surprisingly, the sequence is very similar to those of Daw and Cor; 70% of the positions between 1 and 99 are occupied by the same residue (see Press & Hogg, 1969). As a result ofthe extensive sequence studies on light chains, it has been suggested that both K and A chain variable regions can be divided into subgroups, such that within a subgroup the extent ofthe variability is very much more limited than between subgroups. A comparison between the sequences of the four heavy chains, of Daw, Cor, Eu and Ou, prompts the suggestion that the variable regions of heavy chains may also be divided into subgroups. A partial sequence of another y l chain, Ste, has also been reported (Fisher, Palm & Press, 1969), and in this case 18 out of the first 24 residues are identical with those in Eu heavy chain. It therefore appears that Daw, Cor and Ou heavy chains may belong to one variable- region subgroup, and Eu and Ste to another subgroup. However, the division into subgroups is not apparent in the highly variable region positions 100-104, but for positions 105-114 division into subgroups may again be detected (Fig. 11). Since the heavy chains of Daw and Cor proteins are y 1 chains and the heavy chain of Ou is a ,u chain, it is apparent that the same variable-region subgroup may occur in heavy chains of either the y or ,u class. Oudin & Michel (1969), using anti-idiotypic sera, have found cross-reaction between IgG and IgM antibodies of the same specificity from a single animal. These results might also be interpreted as indicating that heavy-chain variable regions may be shared between classes of immunoglobulins. The existence of variable-region subgroups common to different classes of heavy chain supports the view that at least two genes are involved in the synthesis of a heavy chain. Two genes are probably also involved in the synthesis of a single light chain, but in contrast with the heavy chains the variableregion subgroups of the light chains are peculiar to the class of light chain (K or A); this could be related to the fact that, in the rabbit at least, the genes controlling the synthesis of K and A chains are not closely linked (Mage, Young, Rejnek, Reisfeld & Appella, 1969), whereas the genes controlling the synthesis of the different classes of heavy chains are linked (see review by Herzenberg, McDevitt & Herzenberg, 1968; also Kunkel, Smith, Joslin, Natvig & Litwin, 1969). The allotypic markers Gm (z+) and (f+) are on the Fd fragment of the heavy chain, and Cor and Daw are both Gm (z+) but Eu is Gm (f+). The only difference between Cor and Daw and Eu in the constant region of the Fd fragment is a substitution of an arginine at position 215 in Eu for the lysine residue at this position in Daw and Cor. The variable-region subgroups are unlikely to be correlated with the Gm groups because although Daw and Cor are Gm (z+) and Eu and Ste are both Gm (f+), the ,u chain does not express Gm determinants and it has a different constant-region sequence from the y 1 chains, but can be grouped with Cor and Daw on the basis of its variable-region sequence. Antigen binding activity is known to be a property of the Fab fragments of the immunoglobulins, and 100 Daw Cys Cor Cys-Ala jArg- lie -Thr-Val- Ile -Pro-Ala-Pro-Ala-G *Eu Arg-Ser-Cys-Gly-Ser-Gln -- Cy s Ile -AGly-Gly-Tyr-Gly- 1970 105 110 yr 115 Asp TyrTrp-Giy Gin Gly le-Leu jij Phe Met Val i yArjGly Thr-Pro Val TyI Ser Pro-Glu-G lu-Tyr-Asn Gly GIy-LeutVal - Fig. 11. Comparison between the sequences of three y l chains in the variable region, showing the highly-variable region (positions 100-104) and the probable persistence of subgroup sequences for positions 105-114. * Edelman et al. (1969). Vol. 117 AMINO ACID SEQUENCES OF HUMAN HEAVY CHAINS since there is also good evidence that antibody specificity is related to primary structure it is reasonable to assume that the antigen binding site is within the variable regions of the Fd fragment and light chain. XVithin the variable region of light chains there are relatively constant regions and also positions that are apparently highly variable (Milstein, 1969). The comparative sequence study of Daw and Cor Fd fragments reveals a high incidence of variation for positions 100-104, in an analogous position to one of the light-chain hypervariable regions, and perhaps also around positions 31-33, as noted above. These highly variable regions might be involved in antigen binding, or alternatively residues from many different positions in the primary sequence may contribute to form an antigen binding site in the three-dimensional struicture. The solution of this problem will, however, require the application of soine such technique as affinity labelling, in conjunction with sequence studies of antibodies. The Fd fragment of Cor heavy chain has a carbohydrate moiety attached at position 62, which is unusual for a y1 heavy chain, although Abel, Spiegelberg & Grey (1968) also have evidence for the presence of carbohydrate on the Fd fragments of three gamIna heavy chains. The attachmnent of carbohydrate on the variable region of light chains has also been reported in a few instances (Edinundsori et al. 1968; Melchers, 1969; Hood, Grant & Sox, 1969). The sequence around the site of attachment of the carbohydrate on Cor Fd fragment is Tyr-Asx-Thr-Ser, which is very similar to the sequence around the carbohydrate oIn the Fc f ragment of human and rabbit heavy chains, namely Tyr-Asn-Ser-Thr and Phe-Asx-Ser-Thr (Edelinan et al. 1969; Hill, Delaney, Fellows, & Lebovitz, 1966) and on the moutse light chain, Gln-Asx-Ile-Ser (Melchers, 1969). Comparison of glycopeptides from heavy chains of several species is consistent with the sequence -Asx(CHO)-X-Ser (Thr)- as the site of attachment of carbohydrate, where X can be one of several amino acids (Howell, Hood & Sanders, 1967), and it was suggested by Neuberger & Marshall (1968) that this sequence might be a recognition site for an enzyme involved in the attachment of hexosamine to proteins. However, bovine ribonuclease B contains a carbohydrate moiety attached to an aspartic residue, whereas in ribonuclease A the sarne sequence occurs, namely Asn-Leu-Thr-Lys, without any carbohydrate attached (Plummer & Hirs, 1964). Presumably, therefore, sequence is not the only requirement for carbohydrate attachment to protein chains. We thaink Professor R. R. Porter, F.R.S., for his advice and encouragement throughout this work and Dr V. 659 Wynn and Dr H. G. Kunkel for the generous supply of pathological sera. Our thanks are also due to Miss C. Poole for excellent technical assistance and to Mr T. Gascoyne for efficient operation of the amino acid analyser. We thank the AMedical Research Council for their financial support and N.M.H. acknowledges a studentship from the Medical Research Council of Canada. REFERENCES Abel, C. A., Spiegelberg, H. L. & Grey, H. M. (1968). Biochemistry, Easton, 7, 1271. Cebra, J. J., Givol, D. & Porter, R. R. (1968). Biochem. J. 107, 69. Deutsch, H. 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