C0V A LENT S T R U C T U R E OF
G
- I M M U N O G L 0 B U L IN.
Bennett, C., Konigsberg, W. H., and Edelman, G. M. (1970), Biochemistry 9,3181. Cunningham, B. A+, Gottlieb, P. D., Konigsberg, W. H., and Edelman, G. M. (1968), Biochemistry 7,1983. Edelman, G. M., and Gall, W. E. (1969), Annu. Rev. Biochem. 38,415. Edelman, G. M., Gall, W. E., Waxdal, M. J., and Konigsberg, W. H. (1968), Biochemistry 7,1950. Edelman, G. M., and Gally, J. A. (1962), J . Exp. Med. 116, 207. Gottlieb, P. D., Cunningham, B. A., Waxdal, M. J., Konigsberg, W. H., and Edelman, G. M.(1968), Proc. Nut. Acad. Sci. U. S.61,168. Gray, W. R. (1967a), Methods Enzymol. 11,139. Gray, W. R. (1967b), Methods Enzymol. 11,469. Hilschmann, N . (1967), Hoppe-Seyler's 2. Physiol. Chem. 348,1077. Hilschmann, N., and Craig, L. C. (1965), Proc. Nut. Acad. Sci. U. S.53,1403. Hood, L., Gray, W. R., Sanders, B. G., and Dreyer, W. J. (1967), ColdSpring Harbor Symp. Quant. Biol. 32,133.
VII
Hood, L., andTalmage, D. W. (1970), Science 168,325. Milstein, C. (1966), Biochem.J . 101,352. Milstein, C.(1967), Nature (London)216,330. Milstein, C. (1969), FEBS (Fed. Eur. Biochem. Soc.) Lett. 2,301. Niall, H. D., and Edman, P. (1967), Nature (London) 216, 262. Putnam, F. W. (1969), Science 163,633. Schroeder, W. A. (1967), Methods Enzymol. 11,361. Schwartz, J . H., and Edelman, G. M. (1963), J . Exp. Med. 118,41. Singer, S. J., and Thorpe, N. 0. (1968), Proc. Nut. Acad. Sci. U.S.60,1371. Titani, K., Whitley, E. J., and Putnam, F. W. (1968), J . Biol. Chem. 244,3521. Waxdal, M. J., Konigsberg, W. H., and Edelman, G. M. (1968a), Biochemistry 7,1967. Waxdal, M. J., Konigsberg, W. H., Henley, W. L., and Edelman, G. M. (1968b), Biochemistry 7,1959. Woods, K. R., and Wang, K.-T. (1967), Biochim. Biophys. Acta 133,369.
The Covalent Structure of a Human yG-Immunoglobulin. VII. Amino Acid Sequence of Heavy-Chain Cyanogen Bromide Fragments HI-H," Bruce A. Cunningham, Urs Rutishauser, W. Einar Gall, Paul D. Gottlieb, Myron J. Waxdal, and Gerald M. Edelman
ABSTRACT: The amino acid sequence of the cyanogen bromide fragments H1-H4 from the y chain of the immunoglobulin Eu has been determined. These four fragments contain the amino-terminal 252 residues of the heavy chain and include the entire Fd(t) portion of the molecule. Comparisons of the sequence with that of the K chain from the same protein and with the reported sequences of portions of other immunoglobulins indicate that: (1) heavy chains, like light chains, are composed of a variable region and a constant region. (2) The variable region of y chains (V,) begins at
S
tudies on the amino acid sequences of light chains (Hilschmann and Craig, 1965;Titani et al., 1967;Cunningham et al., 1968; Gottlieb et a[., 1970) support the conclusion that they are composed of regions of variable and constant amino acid sequences. A variety of data (Frangione and ~~
* From The
Rockefeller University, New York, New York 10021. Received December 8, 1969. This work was supported in part by grants from the National Science Foundation (GB 8371) and the U . S. Public Health Service (AM 04256 and AI 09273). Preliminary reports of these results have appeared elsewhere (Gottlieb et al., 1968; Edelman et al., 1969).
the amino terminus and extends for at least 114 residues; thus the VL and VH regions are similar in length. (3) There are at least two subgroups of heavy-chain variable regions. (4) The variable region of the Eu heavy chain (VH)is homologous to the variable region of the light chain (VL), but there is no obvious, special relationship between the VX and VL regions from the same immunoglobulin molecule. (5) The beginning of the constant (CH)region (CHI, residues 119-220) of the heavy chain is homologous in sequence to the constant region of the Eu light chain (CL).
Franklin, 1965; Press and Piggot, 1967; Gottlieb et ai., 1968) suggested that heavy chains also have variable (V) and constant (C) regions. Confirmation of this hypothesis requires the determination of the sequences of several immunoglobulin heavy chains. In this and the subsequent paper in this series (Rutishauser et al., 1970), we present the determination of the complete amino acid sequence of the 7 chain (446 residues) from protein Eu. All of the residues of the heavy chain of this immunoglobulin are accounted for by CNBr fragments HI-H7 (Waxdal et al., 1968b). Here we report the amino acid sequence of CNBr fragments H1-H( which comprise the first 252 residues
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161 (271 Hi1
HT6 IHO-ll
H4 11711
Trypsin 1
HTB
HT17
IHO-21
(HO-31
FIGURE 1: CNBr fragments H1-H4 of the heavy chain of the immunoglobulin Eu. Numbers in parentheses indicate the number of amino acid residues in each fragment. The vertical arrow designates the lysyl residue at which trypsin cleaves the heavy chain to produce Fab(t) and Fc(t). Methionyl residues at positions 48, 54, 81, and 252 are indicated by vertical lines.
of the heavy chain. This sequence includes the entire Fd(t)' region as well as the first 30 residues of the Fc(t) region of the molecule. Materials and Methods The preparation of CNBr fragments H I , HP, H3, and H 4 has been described (Waxdal et al., 1968b). Fragment H1 was further purified by ion-exchange chromatography on CM-cellulose (Whatman CM-52, W. and R. Balston, England) in 0.02 M sodium acetate (pH 4.8) using an exponential gradient from 0 to 0.1 M KC1. Fragment Ha was most readily prepared in large amounts by gel filtration of a mixture of H 3 and H1 (Waxdal et al., 1968b) on Sephadex G-50 in n-propyl alcohol-acetic acid-water (3 :1 :8, v/v). Fragment H4 was purified by gel filtration on Sephadex G-100 in 1 N propionic acid or on Sephadex G-75 in 1 N propionic acid which was also 6 M in urea. Enzymatic digests were performed as described previously (Gottlieb et al., 1968) except that tryptic digests of H4 were routinely performed at 25" for 1-2 hr. Procedures used for the isolation of peptides by gel filtration and ion-exchange chromatography have been reported (Cunningham et al., 1968). High-voltage paper electrophoresis was carried out at pH 1.8 and 4.7 (Schwartz and Edelman, 1963). The fractionation of peptides by ion-exchange chromatography on DEAE-cellulose (Whatman DE-52, W. and R. Balston, England) was carried out at 25" in 0.02 or 0.05 M Tris-HC1 buffer (pH 8.0) using a linear gradinet from 0 to 0.2 M KCl. The column was then washed with the same buffer made 1.0 M in KC1. Fractionation of peptides on CM-cellulose (Whatman CM-52, W. and R. Balston, England) was carried out in 0.02 M sodium acetate, (pH 5.0) using a linear gradient from 0 to 0.3 M KC1. The tripeptide containing the NHz terminus of the heavy chain was isolated both from a pronase digest of 300 mg of heavy chains and from a pronase digest of 50 mg of HI (Gottlieb et al., 1968). T o determine the amino acid sequence of this peptide, 1 X 10-7 mole was dissolved in 100 p1 of 1 N NaOH and allowed to stand 17 hr at room temperature (see Bennett, 1968). To this solution was added 300 p1 of pyridine. After centrifugation the upper phase was removed and evaporated to dryness. The dried sample was dissolved 1 Abbreviations used that are not listed in Biochemistry 5, 1445 (1966), are : Fab(t), Fc(t), Fd(t), tryptic fragments corresponding to Fab, Fc, and Fd (World Health Organization, 1964); dansyl, l-dimethylaminonaphthalene-5-sulfonyl; Asx, aspartic acid or asparagine; Glx, glutamic acid or glutamine.
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e t ai.
PCA~al-Gln-Leu-Vol~Gl~-Ser-Gly-Aia-Glx-V~l-ty~-tys-P~a G l ~ ~ S e r - S e r - ' ~ o l - L ~ i - V o l - S e r - C y s -Clo-Sei Lyi Gly-Gly-
33 40 Thr-PteSer-Arq S e i Ala-Ile-;le-Trp ' W A r g G l n ~ A , aFro
48
G y-Slx-ily-le;iSll-Trp-lvlel-
FIGURE 2 : Amino acid sequence of CNBr fragment HI. Solid lines beneath the sequence designate peptides isolated from enzymatic digests of HI. HTl-HT6a are tryptic peptides and HlCl-HlC3 are chymotryptic peptides. Peptides HlClP1-HlClP3 were obtained from a peptic digest of HlC1. Pronase peptide HPrl was isolated from a digest of intact heavy chains and from a digest of HI. Arrows indicate the method used for determining the amino designates the dansyl-Edman proacid sequences of peptides: cedure, and designates digestion with carboxypeptidase A. The symbol - denotes positions which did not yield a detectable dansylamino acid.
-
-
in 50% pyridine-water and the sequence of the peptide determined by the dansyl-Edman procedure (Cunningham et al., 1968). Procedures employed for amino acid analysis (Edelman et al., 1968), the determination of the amino acid sequence of peptides (Cunningham et al., 1968), and partial acid hydrolysis (Gottlieb et al., 1968) have been reported. The presence of tryptophan in peptides was determined spectrophotometrically or by staining with p-dimethylaminobenzaldehyde (Easley, 1965). Results The order of the CNBr fragments of the heavy chain has been established (Waxdal et al., 1968a). The determination of the amino acid sequence of fragments H1-H4 (Figure 1) is described below. Tryptic peptides were isolated from digests of HI, Ha, and H4 and their order was established by the isolation and characterization of chymotryptic and peptic peptides. Fragment H z contains six amino acid residues and its sequence was determined directly. The sequence of the COOH-terminal 38 residues of H 4 has been reported (Gall et al., 1968); this region includes Lys-222 at which trypsin cleaves the heavy chain to produce the Fab(t) and Fc(t) fragments. Tryptic peptides (HT1-HT17a) are numbered from the NHz terminus of the heavy chain. Other peptides are named according to the CNBr fragment or the larger peptide in which they are contained. Tryptic peptide HT6 (Figure 1) is equivalent to the previously reported peptide HO-1 (Waxdal et a[., 1968a) and contains the COOH-terminal tryptic peptide (HT6a) of fragment H1, all of fragment Hz (HT6b), and the NHz-terminal tryptic peptide (HT6c) of fragment H 3. Peptide HT8 is equivalent to peptide HO-2 (Waxdal et al., 1968a) and contains the COOH-terminal tryptic peptide (HT8a) of fragment H s and the NHs-terminal tryptic peptide (HT8b) of fragment H4. The COOH-terminal tryptic peptide
C 0 V A L E N T S T R U C T U R E 0 F ')'G - I M M U N 0 G L O B U L I N .
TABLE I:
VII
Amino Acid Composition of Tryptic and Chymotryptic Peptides from H1.a
LYS Arg Hsr CMCys ASP Thr Ser Glu Pro GlY Ala Val Ile Leu Phe TrP Total residues Yield (%)"
HTl
HT2
HT3
1.o (1)
2 . 0 (2)
0.9 (1)
HT4
HT5
HTSa
1.2(1)
1,0(1)
HlCl
H1C2
H1C3
3.5 (4) 0.8(1)
1.0(1)
0.7 (1)
1 . 1 (1) 0.9 (1)
1 . 1 (1) 4.1 (4) 1 . 2 (1) 0 . 9 (1) 2 . 8 (3)
1.7(2)
1.0(1)
1 . 2 (1) 1.o (1) 1,0(1)
0 . 9 (1) 1.7(2)
1 . 9 (2) l.O(l) 1.2(1)
0.9(1) 2.9 (3) 1.0(1) 1 . 9 (2) 1.1 (1)
0.9(1) 1 . 0 (1) 1 . 3 (2)
0.7 (1) 0.2 0 . 7 (1) 4 . 3 (5) 4 . 3 (4) 1 . 1 (1) 3.4 (4) 2.0(2) 4 . 9 (5)
1 . 8 (2) 3.1 (3) 1 .o (1) 2 . 1 (2) 1 . 1 (1) 1 . 1 (1)
l.O(l) 1 . 4 (2)
1 .o (1)
0 . 9 (1) 0 . 9 (1) (1)
1 .o (1) 0.8 (1)
1 . 2 (1)
(1)
(11
(1)
12
7
4
8
7
10
29
7
12
39
23
28
21
12
25
42
29
21
Values reported are amino acid residues. Amino acids present at a level of 0.1 residue or less are omitted. Assumed integral numbers of residues are given in parentheses. * Yields are based on micromoles of peptides isolated compared with the micromoles of CNBr fragment H1 originally digested. 0
(HT17a) of Hd is equivalent to the tetrapeptide (HO-3a) obtained from the tryptic peptide (HO-3) which overlaps fragments H4 and H5 (Waxdal et al., 1968a). Amino Acid Sequence of HI. Fragment HIcontains 48 amino acid residues, has no detectable NHz-terminal residue, and includes tryptic peptides HTl-HT6a (Figures 1 and 2). The amino acid compositions of these tryptic peptides (Table I) account for all of the residues of H1. Extraction of a tryptic digest of Hl with n-propyl alcohol-acetic acid-water (1 :1:3, v/v) yielded peptide HTl as a solid residue. Gel filtration of the soluble extract gave three major fractions (Figure 3). Additional HT1 was obtained as a solid residue from fraction A (Figure 3) by extraction of the lyophilized material with 0.05 M pyridine-acetate buffer (pH 3.1). Peptides HT2, HT4, and HT5 were isolated from fraction B by ion-exchange chromatography on AG50X4. Similar treatment of fraction C gave HT3 and additional HT4. Peptide HT6a (Table I) was isolated at alkaline pH to prevent cyclization of its NHzterminal glutaminyl residue to pyrollidonecarboxylic acid. Extraction of a tryptic digest of HI with 0.015 M N H 4 0 H which was 10% in n-propyl alcohol, and gel filtration of the soluble material on Sephadex G-25 in the same solvent gave a single tryptophan-containing fraction. Peptide HT6a was isolated from this fraction by chromatography on DEAE-cellulose in 0.05 M Tris (pH 8.0). The amino acid sequences of peptides HT2-HT5 (Figure 2) were determined directly. Peptide HTl had a blocked NHzterminal residue and therefore its sequence could not be determined by dansyl-Edman analysis. The determination of the amino acid sequence of this region of HIwas obtained by analysis of chymotryptic, peptic, and pronase peptides.
Chymotryptic peptides HlC1, HlC2, and H1C3 (Table I) were obtained from a digest of H1. Gel filtration of this digest gave two major fractions (Figure 4). Ion-exchange chromatography of fraction A on AGlX4 gave peptides HlC1 and HlC3. Gel filtration of fraction B on Sephadex G-25 in 0.015 M NHaOH which was 10% in n-propyl alcohol yielded peptide HlC2. Peptide H l C l had no detectable
2 .o
1
A , I
8 , C HT3
i
i
1
1
'
1.5-
A 1.0
-
0.5
40
80
I20
Tube number FIGURE 3: Gel filtration of tryptic peptides of HIsoluble in npropyl alcohol-acetic acid-water (1 :1:3, v/v) on a column (2.5 X 100 cm) of Sephadex G-25 in the same solvent. Each tube contained 1.6 ml of effluent. The absorbance of effluent fractions at 280 mp is indicated by the solid line. The ninhydrin color yield was determined automatically (Cunningham et ai., 1968) and is 'represented on an arbitrary scale by the dashed line; ninhydrin analysis was not performed on the effluent fractions in tubes 73-1 20.
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TABLE 11:
Amino Acid Composition of Peptic Peptides from
HlC1:
Tube n u m b e r
4: Gel filtration of a chymotryptic digest of HI on a column (2.2 X 100 cm) of Sephadex G-50 in 0.015 M NHlOH which was 10% in n-propyl alcohol. Each tube contained 3.2 ml of effluent.
FlGURE
The solid line indicates the absorbance at 280 mp and the dashed line the absorbance at 230 mu of emuent fractions.
NHZ-terminal residue. The sequences of H1C2 and H1C3 were determined by the dansyl-Edman procedure (Figure Peptic peptides H l C l P l and HlClP3 (Table 11) were obtained by ion-exchange chromatography of a digest of H l C l on AG50X4. A peptide identical with H l C l P l was isolated from a peptic digest of H1 by the same procedure used to isolate pronase peptide HPrl (see below). Peptide HlClP2 (Table 11) was obtained by gel filtration of a peptic digest of H l C l on Sephadex G-25 in 0.015 M NHIOH which was 10% in n-propyl alcohol. The amino acid sequences of HlClP2 and H l C l P 3 were determined directly (Figure 2). Peptide H l C l P l had a blocked NHZ-terminal residue. Treatment of this peptide with carboxypeptidase A released 1 mole equiv each of leucine and glutamine (Figure 2). The peptide HPrl (Figure 2) was obtained from a pronase digest of heavy chains and from a pronase digest of CNBr fragment HI (Gottlieb et af., 1968). The amino acid composition of this peptide was Glx(2.0) and Val(l.0). Peptide HPrl had no detectable NHderminal residue, but after exposure to 1 N NaOH to open the ring of the pyrrolidonecarboxylic acid residue (Bennett, 1968; Dekker et ai.,1949) this peptide had an NHderminal Glx residue and was analyzed by the dansyl-Edman procedure (Figure 2). These data indicate that the amino acid sequence of H l C l P l is PCA-Val-Gln-Leu. The amino acid sequence of CNBr fragment H1 is summarized in Figure 2. The intact heavy chain and peptides HT1, HlC1, HlClP1, and HPrl all had blocked NH2terminal residues. This finding and the amino acid compositions of these peptides suggest that they are all derived from the NHZ-terminal portion of HI. Peptides HT6a and
49
54
Gly-Gly-Ile-Val-Pro-Met-
Amino acid sequence of cyanogen bromide fragment H2 as determined by the dansyl-Edman procedure. FIGURE 5 :
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HlClPl
HlClP2
2.1 (2)
0 . 9 (1) 2 . 0 (2)
0.3 1 . o (1) 0 . 9 (1) 4 17
HlClP3
1 . 1 (1) 1 .o (1) 1 .o (1)
2 . 9 (3) 1 . 8 (2) 0.2 1 . o (1) 1 . 2 (1) 0.2 1 . 9 (2)
6
9
26
22
a Values reported are amino acid residues. Amino acids present at a level of 0.1 residue or less are omitted. Assumed integral numbers of residues are given in parentheses. * Yields are based on micromoles of peptides isolated compared with micromoles of H l C l originally digested.
H1C3 (Table I) each contain one residue of homoserine, placing them at the COOH terminus of HI. The chymotryptic peptides HlC1, HlC2, and H1C3 (Table I) account for all the residues of fragment HI. Peptide H1C3 orders HT5 and HT6a, and peptide H1C2 overlaps HT4 and HT5 (Figure 2). From the amino acid compositions and sequences of peptides HT2, HT3, and HT4 (Table I and Figure 2), we deduce that peptide H l C l contains H T l , HT2, HT3, and the first six residues of HT4. Peptic peptide HlClP3 (Table 11) contains HT2 preceded by a Val-Lys sequence (Figure 2). The sequence of HT3 is Val-Ser-CysLys. Therefore, HT2 must follow HT1, and HT3 is located between HT2 and HT4. The sequence of the first four residues of peptide HT1 is provided by the sequence of H l C l P l (Table 11). The remaining residues of HT1 are accounted for by HlClP2 and the sequence of this peptide completes the determination of the sequence of CNBr fragment H I . Amino Acid Sequence of H2. The amino acid sequence of fragment Hz was determined by the dansyl-Edman procedure and is presented in Figure 5. Amino Acid Sequence of Hs. CNBr fragment H a contains 27 residues and includes tryptic peptides HT6c, HT7, and HTSa (Figure 1). These peptides (Table 111) were isolated from a tryptic digest of H3by ion-exchange chromatography on AGlX4 (Figure 6). Fraction A yielded HT7, fraction C gave HT6c, and fraction D gave HT8a. Fraction B contained no peptides. The sequence of HT6c, HT7, and the first seven residues of HT8a (Figure 7) were determined directly. Peptide HT8a was treated with leucine aminopeptidase and free amino acids were removed from the digest by gel filtration on Sephadex G-25 in 0.015 M NHIOH which was 10% in n-propyl alcohol. The partial sequence of the remaining peptide (HT8a-LAP, Table 111) was then determined by the dansyl-Edman procedure (Figure 7). Peptides H3C1, H3C2, and H3C3 (Table 111, Figure 7) were obtained from chymotryptic digests of H Bby gel filtration
COVALENT STRUCTURE OF YG-IMMUNOGLOBULIN.
TABLE III:
VI1
Amino Acid Composition of Tryptic and Chymotryptic Peptides from H3.a HT6c
LYS Arg Hsr ASP Thr Ser Glu Pro GlY Ala Val Ile TYr Phe Total residues Yield (%)"
HT7
1 . 1 (1)
HT8a
1.o (1) 0 . 8 (1) 2 . 0 (2) 4 . 0 (4) 0 . 9 (1) 1.0(1)
1 .o (1) 1 . 1 (1) 2 . 0 (2) 1.o (1) 0 . 9 (1)
0 . 9 (1) 0 . 9 (1) 9 34
HT8a-LAP
l.O(l)
H3C1
1.o (1) 2 . 0 (2) 2 . 2 (2) 0 . 9 (1) 1 .o (1)
H3C2
H3C3
1.0(1)
0.4 0 . 7 (1) 0.7 (1) 2 . 0 (2) 3.3 (4) 1 . 1 (1) 2.0(2) 0.4 1 . 2 (1) 1 . 8 (2) 1.o (1) 0 . 9 (1) 0 . 8 (1) 0.3 17
1 .o (1) 0.3 1,0(1) 1 . 8 (2) 1 . 3 (1)
0 . 9 (1) 1 . 9 (2) 1 . 1 (1) 1 .o (1) 1 .o (1)
1 . 2 (1) 0.2
0 . 9 (1)
0 . 8 (1)
0 . 9 (1) 0 . 8 (1) 6 42
1.o (1) 4 50
14 37
9 75.
1,0(1) 4 63
56
H3C3-Hf
0 . 9 (1) 1.a (1)
1.1 (1) 0 . 9 (1) 4 12d
a Values reported are amino acid residues. Amino acids present at a level of 0.1 residue or less are omitted. Assumed integral numbers of residues are given in parentheses. b Yields are based on micromoles of peptides isolated compared with the micromoles of CNBr fragment Ha originally digested. c Yield based on micromoles of peptide isolated compared with micromoles of HT8a originally digested. d Yield based on micromoles of peptide isolated compared with the micromoles of H3C3 hydrolyzed.
on Sephadex G-25 in n-propyl alcohol-acetic acid-water (1 :2:97, v/v), and by high-voltage electrophoresis at pH 1.8. Partial acid hydrolysis of H3C3 followed by gel filtration on Sephadex G-25 in n-propyl alcohol-acetic acid-water (1 :2 :97, v/v) gave a single tyrosine-containing fraction. Gel filtration of this fraction on Sephadex G-10 in 2 % acetic acid-water (v/v) yielded the tetrapeptide H3C3-HC, the sequence of which completes the sequence of HT8a (Figure 7). The sequence of H a is summarized in Figure 7. The amino acid composition (Table 111) and sequence of HT6c and the amino acid composition of HT6 (HO-1, see Waxdal et af.,1968a) places HT6c at the NH2terminus of Ha. Peptides
HT8a and H3C3 (Table 111) each contain one residue of homoserine and, therefore, are placed at the COOH terminus of Ha. Because HT6c, HT7, and HT8a account for all of the residues of fragment Ha, HT7 is placed between HT6c and HT8a. The compositions of H3C1 and H3C2 (Table 111) are consistent with their assigned positions (Figure 7). Amino Acid Sequence of H4. Fragment H1 contains 171 amino acid residues and includes tryptic peptides HT8bHT17a (Figure 1). The sequence of the COOH-terminal 38 residues (peptides HT14-HT17a, Figure 8) has been reported (Gall et af., 1968). The amino acid compositions of peptides HT8b-HT14a are given in Table IV. These peptides were initially fractionated by gel filtration of a tryptic digest of H4 (Figure 9). Fraction A contained peptide HT12 (Table IV and Figure 8) which was purified by repeated gel filtration. Fraction B contained a mixture of HT9 and HT16 from which HT9 was isolated by ion-exchange chro-
55 60 70 80 PheCly-ProPro-Arr;Tyr~Ato~GIx-Lyr-Phe Glx-GIpArg-Val -Thr-Ile~Thr-Aladsx.Clx-Ser-Thr-Arx-~r-Alo-Tyr-Mel-
____---
_----------
'
HTBC
HT7
'
HTBo ------
1 4
o'6b HTBP-LPP
I
25
50
75
I
H3C2
H3C3
100
Tube number FIGURE 6 : Ion-exchange
H3CI
I I
chromatography of a tryptic digest of Ha
on a column (0.9 X 6.0 cm) of AGlX4 at 38". The solid line represents the ninhydrin color yield on an arbitrary scale ( A ~ , o *which )
was determined automatically. Each tube contained 1.3 ml of effluent.
1
m
7: Amino acid sequence of CNBr fragment Ha. Peptides H T k , HT7, and HT8a were obtained from tryptic digests of Hs, and H3Cl-H3C3 were isolated from chymotryptic digests of this fragment. Peptide HT8a-LAP was obtained after treatment of HT8a with leucine aminopeptidase. Partial acid hydrolysis of H3C3 yielded peptide H3C3-Hf. Arrows designate sequence determination by the dansyl-Edman procedure.
FIGURE
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,
B
,
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HT 12
H4P2
Tube number
I
h4PZa
,e:
(7C
189
Leu.T~r-Ser.;!y-Vol.Hr -Ti'r-Phe Prs-Alo-Va1-Leu.Glr-Sei-Ser-Gly-Le~.Tyr-Ser-ieu-Ser-Ser-Val-Val -Thr-Lol Pro-
220 230 24c Proiyr-Ser-Cys-Aspiys-Thr-HIs-Thr-Cysf ro-Pro-Cyi-Pro-Aia-ProGx-Leu LeuGly-Cly.Pro.Ser.Val-Phe-Leu-DheI
HT14'
I
HT15
HTI6
H4P3
-
I
HTl6
Hlila
L
M
H4P4
'
H4P4
FlGURE 8: Amino acid sequence of CNBr fragment Ha. Peptides HTBb through &IT17a were obtained from tryptic digests of Ha. Peptides H4Pt, H4P2a, H4P3, and H4P4 were isolated from a peptic digest of H4, and peptide H4P2 was obtained from a peptic digest of the CNBr fragment complex H1-H4-LI. Sequence determination by the dansyl-Edman technique is indicated by -. The symbol denotes digestion with carboxypeptidase A. To complete the sequences of HT9, HT11, and HT12, chymotryptic and peptic digests were carried out. Chymotryptic peptides are indicated with a C (e.g., HT9Cl) and peptic peptides with a P (e.g., HTl2P1). The determination of the sequence of HT14HT17a has been presented elsewhere(Gall et a)., 1968).
-
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9: Gel filtration of a tryptic digest of H4on a column (2.2 X 100 cm) of Sephadex G-75in 1 N propionic acid which was also 6 M in urea. The absorbance of effluent fractions at 280 mp is indicated by the solid line. Each tube contained 2.2 ml of effluent. FIGURE
matography on DEAE-cellulose. Fraction C contained the remaining tryptic peptides of H4. Peptide HT8b was isolated by ion-exchange chromatography of a tryptic digest of H 4 on AG50X4. To obtain peptides HTlO and HT11, a tryptic digest of Hd was extracted with 0.015 M N H 4 0 H which was 10% in n-propyl alcohol and the soluble material was filtered on a column of Sephadex G-25 in the same solvent. The first fraction eluted from the column was submitted to ion-exchange chromatography on DEAE-cellulose as well as to high-voltage paper electrophoresis at pH 4.7 to yield HTlO and HT11. The large peptides in a tryptic digest of H 4 were removed by gel filtration on Sephadex G-25 in 0.015 M N H 4 0 H which was 10% in n-propyl alcohol. Peptides HT13, HT13a, and HT14a (Figure 8) were obtained from fractions containing the small peptides by ion-exchange chromatography on AG50X4 or by high-voltage paper electrophoresis at pH 4.7. The amino acid sequences of peptides HT8b, HT10, HTl3, HT13a, HT14a, and the first nine residues of HTl1 (Figure 8) were determined by the dansyl-Edman procedure. The sequence of peptide HTll was completed by the determination of the sequence of peptide HT11P1 (Table IV and Figure 8). This peptide was isolated from a peptic digest of H T l l by high-voltage paper electrophoresis at pH 4.7. The determination of the amino acid sequences of peptides HT9 and HT12 required special attention. The sequence of the first nine residues of HT9 (Figure 8) was determined directly. Ion-exchange chromatography of a chymotryptic digest of HT9 on AG50X4 yielded peptides HT9C1, HT9C2, and HT9C3 (Figure 8 and Table V). The sequences of HT9C1, HT9C2, and the first seven residues of HT9C3 were determined directly. Edman degradation of peptide HT9C3 stopped abruptly at the Asx-Gly sequence (residues 109-1 10). Peptide
C O V A L E N T S T R U C T U R E OF Y G - I M M U N O G L O B U L I N .
TABLE IV
VI1
: Amino Acid Composition of Tryptic Peptides from H4:
HT8b LYS His Arg CMCys ASP Thr Ser Glu Pro GlY Ala Val Ile Leu TYr Phe TrP Total residues Yield
1 . 3 (1)
1 . 7 (2) 1 . o (1) 0.3
1 . 9 (2)
HT9
HTlO
HT11
HT11P1
HT12
HT13
HT13a
HT14a
0 . 9 (1)
0 . 9 (1)
1 . 1 (1)
0.9(1)
2.3(2) 1 . 8 (2)
l.O(l)
l.O(l)
l.O(l)
0 . 8 (1)
3 , l (3)
0 . 8 (1) 0.2 1 . 8 (2) 1 . 9 (2)
2 . 5 (3) 1 .o (1) 1 . 2 (1) 1 . 2 (1)
0.2 2 . 5 (3) 1 . 6 (2) 1 . 1 (1)
1.1(1)
1 . 1 (1)
2 . 1 (2)
1 . 2 (1)
0 . 7 (1) 4.7(5) 6 . 0 (7) 9 . 1 (12) 3.3(3) 4.4 (5) 4 . 0 (4) 2 . 1 (2) 7.5(8) 0 . 9 (1) 4 . 3 (5) 2 , 5 (3) 1 . 7 (2) (11 63
1 . o (1) 1 . 4 (2) 3 , 4 (3) 4.3(5) 3 . 1 (3) 1 . 1 (1) 4.6 (5) 2 , 6 (3) 1 . 8 (2) 1 . o (1) 1 . 1 (1) 3 . 5 (4) 1 . 8 (2)
l.O(1)
0 . 9 (1)
6
34
12
14
(x)” 11
20
29
26
5
15.
1 .o (1)
0 . 9 (1)
l.O(l)
1 . 1 (1)
0.2 0.2
1 . 1 (1) 0 . 9 (1)
0.2 1.0(1)
l.o(l)
i.o(i)
4
3
5
15
16
12
0.2
38
~
a Values reported are amino acid residues. Amino acids present at a level of 0.1 residue or less are omitted. Assumed integral numbers of residues are given in parentheses. * Yield is based on micromoles of peptide isolated compared with micromoles of CNBr fragment H 4 originally digested. c Yield is based on micromoles of peptides isolated compared with micromoles of HT11 digested.
HT9C4 (Table V and Figure 8) was isolated by ion-exchange chromatography of a chymotryptic digest of HT9 on DEAEcellulose. The composition of this peptide indicates that the COOH-terminal nine residues of HT9 (and HT9C3) are identical with the first nine residues of H4P2 (Figure 8). A peptide (HT9P1, Table V) identical with HT9C4 in composition was obtained by high-voltage electrophoresis of a peptic digest of HT9 at pH 4.7. Therefore the sequence of residues 111 and 112 was assigned as Gly-Leu because both chymotrypsin and pepsin cleaved the bond between residues 112 and 113. Peptide HT9C1 is placed by the direct sequence analysis of HT9; HT9C2 is placed between HT9C1 and HT9C3 because HT9C3 contains lysine and must be derived from the COOH terminus of the tryptic peptide. The amino acid sequence of peptide HT12 (Figure 8) was completed by isolating chymotryptic and peptic peptides obtained from digests of HT12. The fractionation of the chymotryptic peptides (Table VI) by gel filtration is shown in Figure 10. Ion-exchange chromatography of fraction A on AG50X4 yielded HT12C5 and HT12C6 (Figure 8). Peptide HT12C2 was obtained from fraction B (Figure 10) by ionexchange chromatography on AG50X4. Fraction C contained HTl2C2a and HT12C3 which were separated by ionexchange chromatography on AG50X4. Peptides HT12C4 and HT12C1 were obtained directly from fractions D and E, respectively. The amino acid sequences of chymotryptic peptides HT12C2, HTl2C2a, HT12C3, HT12C4, HT12C5, and the partial sequence of HTl2C6 (Figure 8) were determined by the dansyl-Edman procedure. The remaining se-
v: Amino Acid Composition of Chymotryptic and Peptic Peptides from HT9:
TABLE
HT9C1 HT9C2 LYS 0 . 8 (1) CMCys ASP Thr Ser Glu Pro 2 . 1 (2) GlY 1 . o (1) Ala Val Ile Leu 0 . 9 (1) TYr 0 . 7 (1) Phe 6 Total residues Yield ( % ) b 14
HT9C3 1 . 1 (1)
0.7(1) 1 . o (1) 1 . O (1) 3 28
HT9C4
HT9P1
0.9(1)
1.0(1)
0 . 8 (1) 2 . 2 (2) 3.7(4) 2 , 3 (2) 1 . 1 (1) 2 . 1 (2) 1 . o (1) 1.7(2)
1 . 9 (2) 3 . 1 (3)
2 . 0 (2) 2 . 9 (3)
1 . o (1) 2.0(2)
1 . o (1) 2 . 1 (2)
1 . o (1) 0 . 9 (1) 0.3 18
9
9
15
25
34
.Values reported are amino acid residues. Amino acids present at a level of 0.1 residue or less are omitted. Assumed integral numbers of residues are given in parentheses. b Yields are based on micromoles of peptides isolated compared with micromoles of HT9 originally digested.
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TABLE VI: Amino Acid Composition of Chymotryptic and Peptic Peptides from HT12.. -
-. ._._. ~_.
HTl2Cl HT12C2 HT12C2a HT12C3 HT12C4 HT12C5 HT12C6 HT12P1 HT12P2 HT12P3 HT12P2,3 2 . 0 (2) 2 . 0 (2) LYS His 1.0(1) 0.7(1) 0 . 9 (1) 0 . 8 (1) 1 . o (1) CMCys 0 . 8 (1) 0 . 7 (1) 1 . o (1) 1 . o (1) 0.3 3 . 0 ( 3 ) 1 . 3 (2) 0 . 3 3 . 0 (3) ASP Thr l.O(1) l . O ( l ) 0 . 9 ( 1 ) l . 0 ( 1 ) 0 . 3 2.9(3) l . O ( l ) 2.9(3) 2.9(3) l . O ( l ) Ser 1 . 1 (1) 2 . 1 (2) 1.3(1) 0 . 2 1 . 9 ( 2 ) 5.9(6) 1.0(1) 3 . 0 ( 3 ) 5 . 5 ( 6 ) 1 . 1 ( 1 ) Glu 1 . 1 (1) 0.2 1 . 2 ( 1 ) 1.0(1) 0 . 2 1 . 2 ( 1 ) 1 . 1 (1) Pro 1 . 7 (2) 1,0(1) l . O ( l ) 0.9(1) 2.5(3) 0 . 9 ( 1 ) l . O ( l ) 0.2 2.0(2) 1 . 1 (1) 0 . 2 1.1 (1) 1 . 1 (1) 2 . 2 (2) 1 . 2 (1) GlY Ala 1 .o (1) 0 . 9 (1) 2 . 1 (2) Val 2 . 1 (2) 1 . 1 (1) 0.8(1) l . O ( l ) 0.2 3 . 1 (3) l . O ( l ) 4 . 1 (4) 2.8(3) 1 . 2 ( 1 ) Ile 0 . 9 (1) 0 . 8 (1) Leu 1 .o (1) 1 . 0 (1) 1 . o (1) 2 . 2 (2) 1 . 8 (2) 2 . 0 (2) 1 .o (1) 0 . 9 (1) 0 . 8 (1) 0 . 9 ( 1 ) 0 . 5 (1) 0 . 5 (1) TYr Phe 1 . o (1) 0.9(1) 0.2 2 . 0 (2) TrP (1) (1) Total 11 10 5 6 6 18 12 27 18 13 residues 44 22 17 20 33 26 22 26 8 10 Yield
(z)”
31 16
a Values reported are amino acid residues. Amino acids present at a level of 0.1 residue or less are omitted. Assumed integral numbers of residues are given in parentheses. * Yields are based on micromoles of peptides isolated compared with micromoles of HT12 originally digested.
quence of HT12 and the order of the chymotryptic peptides were determined by the isolation and characterization of peptic peptides. Gel filtration of a limited peptic digest (OSZ pepsin, 30 min, 25’) of HT12 on Sephadex G-25 in 0.015 M NH4OH which was 10 in n-propyl alcohol gave two major fractions. The first fraction eluted from the column was resolved into peptides HT12P2, HTl2P3, and HT12P2,3 (Table VI and
,
A , B , C ,D
E
,
HTIZCS HTIZC6
0.4
0.2
Tube number FIGURE 10: Gel filtration of a chymotryptic digest of HT12 on a column (2.2 X 110 cm) of Sephadex G-25 in n-propyl alcoholacetic acid-water (1 :2:97, v/v). Each tube contained 2.0 ml of effluent. The absorbance of effluent fractions at 230 mp is indicated by the solid line.
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Figure 8) by high-voltage electrophoresis at pH 4.7. Electrophoresis of the second fraction at pH 1.8 yielded peptide HT12P1. The amino acid compositions and sequences of HT12 (Table IV), H4P2 (see below), and the chymotryptic and peptic peptides derived from HT12 (Table VI) establish the amino acid sequence of this large tryptic peptide (Figure 8). Although no peptide overlapping HT12C3 and HT12C4 was isolated, comparison of the sequence of HT12 with its amino acid composition (Table IV) indicates that all residues have been accounted for. The order of the tryptic peptides in CNBr fragment H 4 was established by direct sequence analysis of the fragment and by the isolation and characterization of peptic peptides H4P1, H4P2, H4P2a, H4P3, and H4P4 (Table VII). Peptide H4P1 was obtained from a peptic digest of H 4by high-voltage electrophoresis at pH 4.7. Gel filtration of a peptic digest of H 4 on Sephadex G-50 in 0.015 M N H 4 0 H which was 10% in n-propyl alcohol gave one major fraction. Chromatography of this fraction on CM-cellulose yielded peptides H4P2a, H4P3, and H4P4. Peptide H4P2 was isolated from a limited peptic digest of the CNBr fragment complex H1-H4-L3 (Waxdal et al., 1968b) by gel filtration and ion-exchange chromatography on AG50X4. Direct sequence analysis of the CNBr fragment H4 places tryptic peptides HT8b and HT9 (Figure 8). The composition (Table VII) and partial sequence (Figure 8) of peptide H4P1 agree with this sequence. The sequence of the first 12 residues of H4P2 was determined directly, placing peptide HTlO adjacent to HT9 (Figure 8). The position of peptide H T l l is suggested by the amino acid composition (Table VII) and partial sequence (Figure 8) of peptide H4P2a. Peptides
COVALENT STRUCTURE OF yG-IMMUNOGLOBULIN.
TABLE VII:
Amino Acid Composition of Peptic Peptides from
VI1
I IO 20 XI PCNol-Gin-LPvVol ~Gln.Ser.Gly~Alo-GluYoI-Lys-Lyr-Pio-Gly-Ser-Ser.Vol~Lys-Vol-Ser-tyr-Lyr-Alo-Ser-Gly-Gly-Thr~S~i~
40 50 €0 Arq-Serdlo~Ile-IleTp-Vol~ArgGln~Alo.Pro.Gly~Gln.Gly-Leu6lu -Tip-Met-Gly-Gly -1le.Vol-Pro-Met-PheCly-Pio-Pro~As~Tyr-
H4.a
_H4P1
H4P2
H4P2a
H4P3
H4P4
2.7(3) 1 . 9 ( 2 ) 5 . 0 ( 5 ) 2 . 1 (2) LYS 2 .o (2) His 0 . 8 (1) 1 .o (1) Arg 0 . 9 (1) Hsr 0 . 8 (1) 3 . 6 (4) CMCys 5.0(5) l.O(l) 1 . 1 (1) 1 . 1 (1) ASP 0 . 9 (1) 4 . 3 ( 5 ) 3 . 3 (4) 2 . 6 (3) 0 . 7 (1) Thr 1.1(1) 7.0(8) 7.1(8) 1.8(2) 0 . 2 Ser 1 . O (1) 0 . 9 (1) 2 . 1 (2) 0 . 2 Glu 4 . 9 ( 5 ) 2 . 6 (3) 5 . 8 (6) 2 . 6 (3) Pro 3.9 (4) 3 . 1 (3) 0.2 GlY 1 . 0 ( 1 ) 3.9(4) 4.0(4) 1 . 1 (1) Ala 4 . 6 ( 5 ) 2.5 (3) 2 . 8 (3) Val 0 . 9 (1) Ile 2 . 9 (3) 2 . 1 (2) 0 . 9 (1) 1 . 8 (2) Leu 0 . 5 (1) 0 . 9 ( 1 ) 1 . 1 (1) TYr 0 . 9 (1) 1 . 6 (2) 0 . 8 (1) Phe 7 43 30 37 11 Total residues Yield (%)* 17 6 45 25 30 *Values reported are amino acid residues. Amino acids present at a level of 0.1 residue or less are omitted. Assumed integral numbers of residues are given in parentheses. b Yields are based on the micromoles of peptides isolated compared with the micromoles of CNBr fragment H4 or fragment complex H1-H4-L3originally digested.
HTlO, HT11, and a peptide containing the NHAerminal eight residues of HT12 were isolated from a tryptic digest of H4P2, establishing the order HT10-HT11-HT12 (Figure 8). Direct dansyl-Edman analysis of peptide H4P3 gave the sequence of the first 14 residues (Figure 8). This sequence and the amino acid composition of H4P3 (Table VII) establish the order of peptides HT12, HTl3, and HT13a. The position of peptide HT14a is established by its sequence and the sequence of HT14 (previously designated H4Tnl; Gall et al., 1968). Evidence for the placement of peptides HT14-HT17a has been presented (Gall et al., 1968) and the compositions of peptides H4P3 and H4P4 (Table VII) are in accord with this sequence (Figure 8). These data complete the determination of the sequence of CNBr fragment HI. Discussion The amino acid sequence of the 252 residues of the heavy chain of the immunoglobulin Eu that make up CNBr fragments H1-H4 is presented in Figure 11. Determination of the positions of asparaginyl and glutaminyl residues is reported in another paper in this series (Bennett et al., 1970). With two notable exceptions, the sequence of residues 113-214 (Figure 11) has been confirmed by studies of the yG1immunoglobulin Daw (Press and Hogg, 1969). Both of these differences may be associated with allotypic differences in
a0
70
90
Ala-Gln~Lyr-PheGln.Gly-ArqVol-Thr.Ile~Thr.Ala~A~pGlu-Ser-Thr-AsnThr-Alo-Tyr-MelClu-Leu.SerS’er-leu.lrp.Ser.CI~-ASp. IW ll0 1% lhr.Alo~Phe-Tyr-PheCyr-Alo-Gly-Gly-Tyi-Gly-Ile-Tyr-Ser-Pro.Glu-Glu-~yr-As~Gly~G1y-Leu-Vol -Thr-Val-Ser-Ser-Alo-Ser.ThrI30 140 153 LysGly-Pro-Ser-Vol-Phe-Pro~Leu-Ala-Pio~Ser-Ser-Lys-Ser-Thr.Ser-Gly-Gly -Thi-AIo-Alo-Leu-Gly-Cys-Leu-Vol~Lys~As~Tyf~e~ 160
Pro-Glu-Pro-Val-Thr.Vol-Ser-Trp.Ain-Sei-Gly
I70 180 -Alo-Leu.Thr.Sei.Gly~Vol~His~Thr-PhePra~Alo-Vol-Leu-Gln-Ser~Ser.Gly~Le~lyi-
190 200 210 Ser-Leu-Ser-Sei-Val-Vol-Thr-Vol-Pio-Ser-Ser~Se~Leu.Gly-lhr-Gln-Thr~Tyr-IIe-Cyr.Prn~Voi-Aswn~i-tys~Pro~Ser-Asn.Th~Ly~~
220 239 240 Vol-AipCyr-Arg-Vol-Glu-Pro-Lyr-Se~Cyi.Aspty~Thi~Hii-Thr-Cyi.Pro-Pro.Cyi-Pio~Alo-Pro-Glu-Leu.LeuGly-Gly-Pro-Sa-Vol250 PheCe~9ro-Pro.Lyr.Pro-Lyi.AspTh~Leu.Mel.
FIGURE 11: Amino acid sequence of residues 1-252 of the heavy chain of the yGl-immunoglobulin Eu.
the two proteins. Residue 214 in the Eu heavy chain is lysine, but in Daw the corresponding position is occupied by arginine. Positions 190-192 in the sequence of Eu are occupied by three consecutive seryl residues. Only two seryl residues are found in this portion of Daw. Detailed studies (M. N. Pfiumm, U. Rutishauser, B. A. Cunningham, and G . M. Edelman, manuscript in preparation) on the sequence of this region of another human yG1-immunoglobulin (designated He and of the same antigenic type as Eu) indicate that the sequence is identical with that of Eu. Together with earlier results (Gall et al., 1968) the present studies show that the Fd(t) region of the molecule extends from the NH2terminus of the heavy chain to Lys-222 (Figure 11). All of the half-cystines of the heavy chain that contribute to the interchain disulfide bonds are located between residues 220 and 229 (Gall et al., 1968). The four additional half-cystines (positions 22, 96, 144, and 200) form intrachain disulfide bonds (Gall and Edelman, 1970). Comparison of the results of recent studies on the y chain of another human yGl-immunoglobulin (Cunningham et al., 1969) with the present studies, and with those on CNBr fragments Hs-H7 (Rutishauser et al., 1970) substantiates the idea that heavy chains, like light chains, have an NH2terminal variable region followed by a constant region. Moreover, the sequences of heavy-chain variable regions fall into subgroups (Gottlieb et al., 1968; Cunningham et al., 1969). The VH region of subgroup I, to which protein Eu belongs, may be slightly shorter (114 residues) than the VH regions of subgroup I1 to which protein He (118 residues; Cunningham et al., 1969), Daw (Press and Hogg, 1969), and Cor (Press and Hogg, 1969) belong. All of the available evidence indicates that proteins Eu and He have different VH subgroups but identical CH regions. This finding strongly supports the translocation hypothesis (see Edelman and Gall, 1969). There are a number of features of heavy-chain variable regions which indicate that they are similar to VL regions. Thus VH and VI.regions have comparable lengths, a single highly conserved intrachain disulfide bond, and subgroups which appear to differ in length. There is some suggestion that there is a greater number of
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variable positions in VE regions than in VL regions (Cunningham et al., 1969). Definitive estimates of the relative variability of Va and VLdepend, however, upon the determination of additional heavy-chain sequences. Examination of the types of interchanges shows that the variable positions of heavy and light chains are similar in nature and distribution. This indicates that the mechanism of variation must be the same for both chains, despite the fact that separate genes specify VH and VL regions. Moreover, there is no evidence to suggest that known heavy-chain subgroups are restricted in their interaction with known light-chain subgroups. The sequence of the variable region of the Eu heavy chain has been compared in detail with the sequence of the variable region of the Eu light chain (Gottlieb et a[., 1968; Edelman, 1970). While the two variable regions are homologous in sequence, the Vu region of Eu resembles the VL region of Eu no more closely than any other heavy-chain variable region resembles the variable region of the Eu light chain. The constant regions of the heavy and light chains show more homology in sequence than do VH and VL, and a comparison of residues 119-220 (CB1) with CL shows strong homology (Edelman et ai., 1969; Edelman, 1970). Additional homologies have been revealed by studies of the amino acid sequence of the remainder of the constant region of the Eu heavy chain. These studies are presented in the next paper in this series. Acknowledgments We wish to thank Miss Joan Low and Mrs. Helvi Hjelt for their excellent technical assistance. We also thank Mrs. Helen Papen for her assistance. References Bennett, C., Konigsberg, W. H., and Edelman, G . M. (1970), Biochemistry 9, 3181. Bennett, J. C. (1968), Biochemistry 7,3340. Cunningham, B. A., Gottlieb, P. D., Konigsberg, W. H., and Edelman, G. M. (1968), Biochemistry 7,1983. Cunningham, B. A., Pflumm, M. N., Rutishauser, U., and
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Edelman, G. M. (1969), Proc. Nut. Acad. Sci. U . S . 64,997. Easley, C. W. (1965), Biochim. Biophys. Acta 107,386. Dekker, C. A., Stone, D., and Fruton, J. S. (1949), J . Biol. Chem. 181,719. Edelman, G. M. (1970), Biochemistry 9,3197. Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottlieb, P. D., Rutishauser, U., and Waxdal, M. J. (1969), Proc. Nat. Acad. Sci. U.S. 63,78. Edelman, G . M., and Gall, W. E. (1969), Annu. Rev. Biochem. 38,415. Edelman, G . M., Gall, W. E., Waxdal, M. J . , and Konigsberg, W. H. (1968), Biochemistry 7, 1950. Frangione, B., and Franklin, E. C. (1965), J . Exp. Med. 122,l. Gall, W. E., Cunningham, B. A., Waxdal, M. J., Konigsberg, W. H . , and Edelman, G . M. (1968), Biochemistry 7, 1973. Gall, W. E., and Edelman, G. M. (1970), Biochemistry 9, 3188. Gottlieb, P. D., Cunningham, B. A., Rutishauser, U . , and Edelman, G . M. (1970), Biochemistry 9,3155. Gottlieb, P. D., Cunningham, B. A., Waxdal, M. J . , Konigsberg, W. H., and Edelman, G. M. (1968), Proc. Nut. Acad. Sci. U . S. 61,168. Hilschmann, N., and Craig, L. C. (1965), Proc. Nut. Acad. Sci. U . S . 53,1403. Press, E. M., and Hogg, N. M. (1969), Nature (London) 223, 807. Press, E. M., and Piggot, P. J. (1967), Cold Spring Harbor Symp. Quant. Biol. 32,45. Rutishauser, U., Cunningham, B. A., Bennett, C., Konigsberg, W. H., and Edelman, G. M. (1970), Biochemistry 9, 3171. Schwartz, J. H., and Edelman, G . M. (1963), J . Exp. Med. 118,41. Titani, K . , Wikler, M., and Putnam, F. W. (1967), Science 155,828. Waxdal, M. J.. Konigsberg, W. H., and Edelman, G. M. (1968a), Biochemistry 7, 1967. Waxdal, M. J., Konigsberg, W. H., Henley, W. L., and Edelman, G. M. (1968b), Biochemistry 7,1959. World Health Organization (1964), Bull. World Health Organ. 30,447.
