Download The Amino Acid Sequences of the Fd Fragments of Two Human y1

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.
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