THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 243, No. 16, Issue of August 25, pp. 4206-4215, 1908

Printed in U.S.A.

The Amino Acid Sequence of the Fc Fragment

of Rabbit Immunoglobulin G

II. CLEAVAGE OF THE Fe FRAGMENT WITH CYANOGEN BROMIDE*

(Received for publication, April 25, 1968)

ROBERT DELANEY$. AND ROBERT L. HILL

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27706

SUMMARY

Reaction of S-carboxymethyl-Fc fragment from rabbit immunoglobulin G with cyanogen bromide cleaves the fragment into five unique peptides, each of which has been isolated by chromatographic methods. The amino acid composition of the five peptides accounts closely for the amino acid composition of the Fc fragment. On the basis of the amino acid composition of the tryptic peptides derived from each of the five peptides and the composition of the four unique methionine-containing peptides from Fc frag- ment, it is possible to obtain a partial sequence of the Fc fragment. These results suggest that this may be a major sequence for the rabbit Fc fragment.

The preceding paper (1) describes the purification and partial characterization of the peptides obtained from tryptic hydroly- sates of S-carboxymethyl-Fc fragment’ from nonspecific rabbit immunoglobulin G. Although a set of tryptic peptides was isolated, the composition of which accounted closely for the amino acid composition of the Fc fragment, some tryptic peptides were difficult to purify and many were obtained in low yields. Thus, in order to circumvent the difficulties encountered in isolating peptides in high yields from complex tryptic digests of FC fragment, additional studies have been made on the cleavage of Fc fragment with cyanogen bromide. As shown earlier (2-5), cyanogen bromide cleaves the peptide bonds formed by the carboxyl groups of the 4 methionyl residues in the Fc fragment to give five unique polypeptides. Each of the unique polypeptides should give a set of tryptic peptides which are much easier to

* This study was supported by a research grant from the Na- tional Heart Institute, National Institutes of Health.

$ Postdoctoral Fellow, National Institutes of Health, 1964 to 1966. Present address, Department of Biochemistry, University of Oklahoma Medical School, Oklahoma City, Oklahoma.

l The Fd fragment represents the first 220 to 240 residues from the NH2 terminus of heavy chain, and the Fc fragment the re- maining 215 residues in the chain.

purify than the tryptic digests of the Fc fragment described in the preceding paper. With knowledge of the four methionine- containing tryptic peptides from Fc fragment, it should also be possible to order each cleavage product from the amino to the carboxyl terminus of the fragment, and thus assign each of the tryptic peptides to a limited region of fragment.

We wish to describe here the isolation and characterization of the five peptides produced on cleavage of the S-carboxy- methyl-Fc fragment with CNBr. Each of the five fragments, obtained in yields from 37 to 52% has been digested with trypsin and the resulting tryptic peptides have been isolated and partially characterized. Although all of the tryptic peptides from the CNBr fragments correspond to those described in the preceding paper (1)) most were obtained in considerably higher yields. In addition, many of the problems encountered in puriiication of the tryptic peptides from the Fc fragment (1) were solved only with knowledge of the composition of the tryptic peptides derived from the cyanogen bromide cleavage products.

EXPERIMENTAL PROCEDURE

Carboxymethyhtion of Fc Fragment-Fc fragment was prepared and carboxymethylated as described earlier (1).

Cleavage with Cyanogen Bromide-Treatment with CNBr was performed essentially as described earlier (6). S-Carboxy- methyl-Fc fragment (390 mg) was dissolved in 24 ml of 97% formic acid, and 780 mg of cyanogen bromide were then added. After 24 hours at 25”, 360 ml of water were added to the mixture. The reagents were removed by lyophilization, and 380 mg of the dried cleavage products were recovered.

Separation of Cyanogen Bromide Cleavage Products--The peptides (380 mg) obtained after cleavage of Fc fragment with cyanogen bromide were dissolved in 20 ml of 1 N acetic acid and applied to a column, 125 x 2.5 cm, of Sephadex G-100 which had been equilibrated with 1 N acetic acid. The columnwas developed at a flow rate of 50 to 60 ml per hour at room temperature. The effluent fractions (6 ml) were collected automatically. The peptides in each fraction were detected by measurement of the absorption at 235 rnp. Appropriate fractions were pooled and concentrated almost to dryness by rotary evaporation under reduced pressure. The resulting samples were diluted to a

4206

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known volume with water and stored frozen. Further purifica- tion of the fractions was achieved by rechromatography on the same column of Sephadex or by ion exchange chromatography on Dowex-50 as described below.

Puri$cation of Tryptic Peptides-The tryptic peptides were separated by ion exchange chromatography on columns of Dowex 50-X8 maintained at 50” by methods described earlier (7). The peptides were eluted from the columns at a flow rate of 60 to 80 ml per hour by gradient elution with volatile pyridine- acetic acid buffers. Portions of the column eluates were con- tinuously removed prior to collection in an automatic collector (Beckman-Spinco model 132) and, with the aid of a Technicon automatic analyzer, allowed to react with ninhydrin after alkaline hydrolysis. The resulting ninhydrin color was recorded auto- matically. Columns of Dowex 50-X8 (Beckman-Spinco resin type 50; designed for use with the model 120 amino acid analyzer) were used throughout. Three different linear gradients were used. Gradient 1 was formed with 500 ml of 0.2 N pyridine- acetic acid, pH 3.1, as the starting buffer and 500 ml of 2 N

pyridine-acetic acid, pH 5.0, as limit buffer. Gradient 2 was formed with 500 ml of 0.05 N pyridine-acetic acid, pH 2.75, as starting buffer and 500 ml of 0.5 N pyridine-acetic acid, pH 3.8, as limit buffer. Gradient 3 was formed with 500 ml of 4.9 M

acetic acid as starting buffer and 500 ml of 0.2 N pyridine-acetic acid, pH 3.1, as limit buffer. The column was equilibrated in each case with the appropriate starting buffer. The peptides were recovered from the volatile buffers by rotary evaporation under reduced pressure.

of the cyanogen bromide cleavage products. Cleavage products MC IV and MC V (0.05 to 0.1 pmole) were carbamylated in urea as described earlier (9) and the urea was removed by gel filtration in 50% acetic acid on columns of Sephadex G-25. Cleavage products VII-d and VII-I (0.05 to 0.1 pmole) were carbamylated as described for peptides (9). Glutamic acid, which is formed from pyrrolidone carboxylic acid during the procedure, was not removed prior to amino acid analysis. The yields of the end groups were calculated with the correction factors given earlier (9). The NHz-terminal groups of MC VII-b and the tryptic peptides derived from each cleavage product were determined by the Edman method as modified by Konigsberg and Hill (10).

Amino Acid Composition-Peptides (0.04 to 0.2 pmole) were hydrolyzed in 6 N HCl in evacuated, sealed tubes at 110” for 24 hours (11). Acid was removed by rotary evaporation under re- duced pressure. The amino acid composition of the resulting hydrolysate was determined on a Spinco model 120 amino acid analyzer with the modification of Benson and Patterson (12). Correction was not made for losses of specific amino acids during acid hydrolysis.

Materials and Reagents-Guanidine hydrochloride was purified by the method of Anson (13). Iodoacetic acid was recrystallized from petroleum ether (b.p. 66-75”) prior to use. All other com- pounds were of reagent grade.

RESULTS

PuriJication of Tryp&insoluble Peptides-After tryptic digestion of one of the cyanogen bromide cleavage peptides (MC IV), a portion of the digest was insoluble at pH 2 to 5. This fraction was soluble at pH 8 and was purified on columns (4.5 x 46 cm) of Sephadex G-75 equilibrated with 0.1 M triethylam- monium carbonate, pH 9.9. The insoluble fraction was applied to the column in 10 ml, and the column was developed at room temperature at a flow rate to 15 to 20 ml per hour. Fractions (5 to 6 ml) were collected automatically and monitored at 235 mp. Triethylammonium carbonate was removed by repeated rotary evaporation from water under reduced pressure.

Cleavage of Fc Fragment with Cyanogen Bromide

Tryptic Hydrolysis-Each of the cyanogen bromide cleavage products was digested with trypsin as follows. The cleavage product (2.5 pmoles) was dissolved in 3.0 ml of water and the solution was adjusted to pH 9 with NaOH. Trypsin (3 mg) was added as a 1% solution in 0.001 N HCl. The mixture was incubated at 37” and maintained at pH 8 to 9 by frequent addi- tions of 0.01 N NaOH. After no further change in pH was observed (2 to 3 hours), the mixture was acidified to pH 2 to 3 with glacial acetic acid. All digests were soluble at this pH except that from the cyanogen bromide cleavage product, MC IV. In this case the insoluble fraction was removed by centrifu- gation.

Cleavage of S-carboxymethyl-Fc fragment with cyanogen bromide in 70% formic acid was almost complete after 24 hours of reaction. Amino acid analysis of a 24-hour acid hydrolysate showed that less than 5% of the methionine in PC fragment, or less than 0.2 residue per molecule, remained unmodified. The cleavage products from 380 mg of fragment were fractionated on Sephadex G-100 in 1 N acetic acid as shown in Fig. 1A. Although four peaks were obtained in major amounts, the eluate fractions were combined to give eight different pools, as indicated. The large, asymmetric peak which emerged between 450 and 890 ml was arbitrarily split into three fractions, designated MC II, MC III, and MC IV. Three other fractions, designated MC V, MC VI, and MC VII, corresponded to the other major peaks. The peaks designated MC I and MC VIII did not contain significant amounts of peptide and were not studied further. In the remainder of the paper, each of the cleavage products will be designated by the symbol MC and the appropriate Roman numeral, given in Fig. 1.

Chymotryptic activity in some trypsin preparations was re- moved as described earlier (8). A solution of trypsin (1 To) was treated at room temperature for 23 hours with L-(1-tosylamido-2- phenyl)ethyl chloromethylketone in 0.01 M Tris-succinate buffer, pH 6.5, containing 0.005 M CaCl2. It was assumed that 10% of the weight of the trypsin was chymotrypsin, and sufficient inhibitor was used to provide a lo-fold molar excess per mole of chymotrypsin.

The cleavage products MC III, MC IV, MC V, and MC VI were repurified on Sephadex G-100 under the same conditions as those described for Fig. 1A. The behavior of these fractions on rechromatography is shown in Fig. 1, B to E. The amino acid compositions and NHt-terminal groups of five of the cleavage products are given in Table I. The composition of these five fractions accounts closely for the amino acid composition of the intact Fc fragment. Considerable insight into the nature of the fragments was obtained when the tryptic peptides from each fragment were isolated, characterized, and compared with the tryptic peptides isolated earlier (1). The methods for charac- terization of each cleavage product were as follows.

Amino End Group Analysis-The cyanate method of Stark Peptides MC III and MC IV-These peptides rechromato- and Smyth (9) was used to determine the NHt-terminal groups graphed as asymmetric peaks (Fig. 1, B and C) similar to the

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4208 Amino Acid Sequence of Fc Fragment of Rabbit IgG. TI Vol. 243, No. 16

400 800 1200 Volume-ml

800 1000

800 1000 Volume-ml

FIG. 1. Purification of the peptides from cyanogen bromide-treated Fc fragment. A, S-carboxymethyl-Fc fragment treated with cyanogen bromide. The peptides were applied to a column (2.5 X 125 cm) of Sepha- dex G-100 equilibrated with 1 N acetic acid. The effluent fractions were monitored at 235 mp. The volume between urrows indicates which portions of the effluent frac- tions were pooled. Fractions III, IV, V, and VI were repurified on the same column of Sephadex G-100 to give the patterns shown in B to E. B, Fraction III; C, Fraction nT; D, Fraction V; E, Fraction VI. The arrows in B to E indicate those fractions from each column which were combined.

TABLE I

Amino acid composition of peptides from cyanogen bromide-treated S-carboxymethyl-Fe fragment

Amino acid

--

Lysine .................................. Hiitidine ............................... Arginine ............................... Aspartic acid. ......................... Threonine ............................. Serine ................................. Glutamic acid. ........................ Proline ................................ Glycine ................................. Alanine ................................. Carboxymethylcysteine ................. Valine .................................. Methionineb ............................ Isoleucine .............................. Leucine ................................. Tyrosine ................................ Phenylalanine .......................... Tryptophanc ........................... Glucosamined........................... >4

MClV MC VI

Peptide

MC VII-b MC-VII-d

residues/molecule

fragient MC VII-l

8.0 (8) 1.92 (2) 7.80 (7) 8.02 (8) 8.06 (8) 6.44 (5)

16.7 (16) 9.9 (10) 2.48 (2) 6.38 (5) 2.06 (2) 9.0 (9) 0.78 (1) 6.13 (6) 6.68 (6) 2.04 (2) 3.84 (3) 2 (21

Total no. of residues.. . . . . . . . . . . . . 102 Percentage yield. . . . . . . . . . 52 Percentage yield, NHI-terminal residue.. IIe (77),

Glu (34), Pro (24)

4.12 (4) 1.88 (2)

0.91 (1) 8.60 (8) 4.36 (4) 7.80 (8) 6.34 (5) 8.88 (3) 4.29 (4) 1.83 (2) 1.H (1) 4.97 (5) 0.8 (1) 2.18 (2) a.34 (3) 3.76 (4) a.24 (3) 2 (2)

1.17 (1) 1.00 (1) 1.14 (1) 1.39 (1) 6.7 (6) 1.87 (2)

0.67 (1) 1.16 (1) 0.3 (1) 0.93 (1) 8.62 (3)

1.71 (2)

60 23 17 18 44 37 50 48

CM-Cysa Gly (87) His (79) (60)

-

0.96 (1) 3.01 (3)

l.YO (2) 0.88 (1) 1.01 (1)

1.0 (1) 1.01 (1) 5.66 (4) 8.79 (3) 2.63 (2) 2.07 (2) 8.21 (2) 0.90 (1) 0.90 (1) 1.07 (1)

0.99 (1) 1.01 (1) 0.97 (1) 0.6 (1)

1.06 (1) 2.00 (2) 1.06 (1)

0.47 (1)

15 15 5 5

11 12 18 19 15 18 21 21 26 26 22 23 10 11 8 8 5 5

16 19 4 4

10 10 15 14 7 6 8 8 4 5

220

D Composition is given as molar ratio (in italics). The numbers in parentheses indicate the assumed number of residues per molecule. b Determined as homoserine lactone. 0 Estimated by spectral analysis at 289 nm (14). d Determined on amino acid analyzer; not quantitative. 0 Carboxymethylcysteine.

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0.2

0

0.4

0

.2

I

e . v) ’ I E 5 0

ly MC -YI C

T-3a-5a

MCXII -d

- I

- - - II W -

MCP E

0.2

0

0.2

0 s s

MC XII-1 T-l

G

.05

0.1

0 MCPt-3a -5a

T-5a

- - I

5a MC E-5a F

T-5a L ” I

T-21 H

Volume -m I FIG. 2. Chromatographic separation of tryptic digests of peptides from CNBr-treated S-carboxymethyl-Fc fragment on

Dowex 50-X8. The comnosition of the buffers and the details of oneration of the columns are eiven in the text. The bars indicate the fractions which were pooled. A, MC VII-d; B, MC VII-b*; C, MC VI; D, Peak Tp-3a-5ufrom MC VIC; E, MC V; F, Peak Tp-5a from MC V; G, MC VII-l; H, MC IV.

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4210 Vo.. 243, No. 16 Amino Acid Xeguence of Fe Fragment of Rabbit IgG. II

TABLE II Amino acid composition of tryptic peptides from MC IV

Amino acid Tryptic peptide

Tp-1Oa ( Tp-11 1 Tp-12 1 Tp-13 1 Tp-14 1 Tp-15 1 Tp-16 1 Tp-17 1 Tp-18 1 Tp-19 1 Tp-20 1 Tp-21 1 Tp-23 1 Tp-25b

Lysine .................. Histidine ................ Arginine ................ Aspartic acid ............ Threonine. .............. 1.4 (1) Swine ................... Glutamic acid. Proline. ................. Glycine ................. Alanine ................. Carboxymethyleysteine Valine ................... f.8 (1) Methionir&. ............ 0.86 (1) Isoleucine. .............. La&e. ................ Tyrosine ................ 0.91 (1: Phenylalanine ........... Tryptophand ............ Glucosaminee: ..........

0.87 (1)

0.16

8.01 (2) Q.04 (2) 0.99 (1)

0.18 0.08

1.0 (1) 0.10

Total no. of residues. ... 4 7 Percentage yield ........ 23 100 Amino-terminal residue. Val GUY

01 (1

99 (1

2 81

Ala

1

)

0 1

1

0

-

.or (1

.95 (1

.oo (1

.98 (1

4 84

Thr

1.01 (1) 0.89 (1 0.70 (1

1.0s (1 0.38

1.01 (1) l.Q4 (2)

I.82 (2)

0.40 0.66 (1 0.19 0.98 (1) 1.00 (1) 0.18

,913 (1) 00 (1

05 (1

.04 (1:

2 96

CM- Cysf

.G5 (I

3 92

GlU

.GS (1

2 14 68 95

GUY Vsl

0.84 (1) f.Od (1) 0.84 (1) 0.98 (1) 1.0s (1) 1.17 (1) 0.97 (1)

1.00 (1)

s.oob (2)

0.88 (1) 2.02 (2)

e (1)

2.00 (1) G.04 (2) O.Gd (1) 1.so (1) 4.98 (5) 1.18 (1) 0.96 (1) 1.9.G (2) 1.10 (1) 1.90 (1) 3.46 (3) 6.88 (6) o..so 2.09 (2) d.18 (2) 0.18

1.00 (1) 1.14 (1)

0.51 6.64 (‘3)

1.01 (1) 1.00 (1) 0.18 0.90 (1)

0.89 (1) 2.09 (1) 0.94 (1)

e (1) 2

--

9 7 28 100 86 23 Glu Thr Val

00 (1

84 (1

0.3 (I

3 69 Ile

1

)

.I

-_

-

7 8 2 2 7 7 8 8 7 8 5 5

14 16 9 10 2 2 6 5 2 2

10 9 1 1 6 6 5 6 2 2 3 3 2 2

97 102

(1 Reported &8 molar ratios (in it&m): the values in parentheses are the assumed number of residues. b 72-hour hydrolysis. c Determined .m homoserine b&one. d Estimated by spectral analysis at 280 rnr (14). e Not included in the total. f Carboxymethyloysteine.

peak from which they were derived, as shown in Fig. 1A. How- ever, each of the rechromatographed fractions, when pooled as indicated, possessed the same amino acid composition, which is given in Table I for MC IV. This fraction contained 102 resi- dues plus amino sugars, which are derived from the carbohydrate prosthetic group of Fc fragment. The yield of this cleavage product was 52’%, with 30% of the peptide being derived from MC IV and the remainder from MC III. Yields of all peptides from the CNBr-treated fragments were calculated from their amino acid compositions. End group analysis by the cyanate method (9) revealed three different NHz-terminal residues. Isoleucine was obtained in 77% yield, glutamic acid in 34% yield, and proline in 24% yield. The glutamic acid may have been derived from pyrrolidone carboxylic acid, since special pre- cautions were not taken to remove this compound and it is known to be produced as an artifact during end group analysis (9). Although the proline end group cannot be explained, the stoichiometry of the molar ratios among the amino acids in MC IV (Table I) suggests that this peptide is a unique segment of the Fc fragment. The NHz-terminal isoleucine may be expected in one peptide formed on cleavage of Fc fragment with CNBr, since only two of the methionine-containing tryptic peptides, Tp-25 and Tp-8, also contain isoleucine (1).

A tryptic hydrolysate of 4.5 Mmoles of MC IV was completely soluble at pH 8 to 9, but on acidification to pH 2 to 3 an insoluble precipitate formed. The precipitate was removed by centrifu- gation, then washed once with 0.01 N HCl, and the washings were combined with the soluble fraction. The soluble peptides were chromatographed on a column (2.5 x 17 cm) of Dowex 50-x8 with Gradient 1 and gave the elution pattern shown in Fig. 2H.

Fourteen peaks were obtained, each of which contained a unique peptide. The amino acid composition and the NHz-terminal groups of these peptides are given in Table II. In general, each of the peptides was obtained in a high yield, with the exception of peptide Tp-lOa, which contained methionine, determined as homoserine lactone, and must represent the 4 residues at the COOH terminus of MC IV. Tp-23 was not found in the soluble tryptic fraction, but was obtained by purification of the in- soluble residue on a column (4.5 x 46 cm) of Sephadex G-75 with 0.1 M triethylamine carbonate buffer, pH 9.9, as shown in Fig. 3. The first peak, which emerged between 300 and 400 ml, proved to have the composition given in Table II for Tp-23. The second peak, indicated earlier (2) as Tp-24, proved to be a mixture of small peptides from the soluble fractions. This peak is not believed to represent a unique segment of MC IV, and has not been characterized further.

A small peak inFig. 2H, designatedTp-22, had the composition, Ile, Asps, Gluz, Val, Arg, and was thought originally to represent a unique sequence in MC IV (2). Recent studies have shown that this peptide is derived on hydrolysis of Tp-23 by chymotryp- sin2 Its presence in tryptic digests is believed to arise from the chymotryptic activity of the trypsin used in this experiment. Thus, thii peptide is not included in Table II as a unique tryptic peptide from MC IV. When this peptide is omitted, the com- position of MC IV is closely accounted for by the composition of the tryptic peptides given in Table II.

Peptie MC VI--The amino acid composition of peptide MC VI, given in Table I, indicated that it contained 60 residues, in-

z H. E. Lebovitz, unpublished observation.

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Issue of August 25, 1968 R. Delaney and R. L. Hill 4211

eluding 1 residue of homoserine lactone. Although end group analysis was not performed, it appeared to be of high purity on the basis of the stoichiometric ratio of its constituent amino acids, as well as on the basis of the peptides isolated from tryptic di- gests. The tryptic hydrolysates of MC VI were soluble over a

041 I I J 350 500

Volume ml FIQ. 3. Purification of tryptic-insoluble residue on Sephadex

G-75. The insoluble residue from the MC IV tryptic digest was applied to a column (4.5 X 46 cm) of Sephadex G-75 equilibrated with 0.1 M triethylamine carbonate buffer, pH 9.9. The flow rate was 15 to 20 ml per hour. Fractions (5 to 6 ml) were collected automatically and monitored manually at 235 rnp.

Amino:acid

Lysine ......................... Histidine ...................... Arginine ....................... Aspartic acid. ................. Tbreonine ..................... Serine. ........................ Glutamic acid. ................ Proline. ....................... Glycine ........................ Alanine ........................ Carboxymethylcysteine ........ Valine ......................... Methionine” .................... Isoleucine. ..................... Leucine ........................ Tyrosine ....................... Phenylalanine ................. TryptophanC. ..................

Total No. of residues. ......... Percentage yield. .............. Amino-terminal residue. .......

-

-

_-

--

-

wide pH range, and when chromatographed on columns of Dowex 50-X8 (2.5 x 17 cm) with Gradient 1 gave the elution pattern shown in Fig. 2C. Six major peaks were observed, all proved to be pure except Peak Tp-3a-5a, which emerged near the void volume of the column. This peak, rechromatographed on the same column with Gradient 3, gave two major peaks as shown in Fig. 2C. The amino acid composition of the tryptic peptides from MC VI are given in Table III. Seven peptides were ob- tained which accounted exactly for the amino acid composition of MC VI. Peptide Tp-3a contained homoserine lactone, and its composition suggests that it represents a portion of the sequence contained in tryptic peptide Tp-3, which contains a single residue of methionine (1). The NH&erminal group of peptide Tp-3a is glycine, the same end group as in tryptic peptide Tp-3. Peptide Tp-5a contains neither lysine nor arginine, and since MC VI does not represent the COOH-terminal residue of the Fc fragment, it is probable that peptide Tp-5a arises as the result of splitting by chymotrypsin in the trypsin preparations used in this experiment. In addition, peptide Tp-Bb, seryllysine, had heretofore not been found in tryptic digests of Fc fragment (1); thus it and peptide Tpdaappeared to be produced by chymotryptic cleavage of a sin- gle tryptic peptide. Proof that Tpda and Tp-5b are contained in a single tryptic peptide was obtained in a separate experiment. Peptide MC V (0.7 pmole), which is a mixture of the cleavage products MC VI and MC VII-l and -d (see below), was hydro- lyzed in 1 to 2 ml at pH 8 to 9, 37”, for 1 hour with 0.015 mg of trypsin which had been treated with n-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Chromatography of this hydroly-

TABLE III Amino acid composition of tryptic peptides from MC VI

Tp-3a Tp-4 Tp-sa

Peptide=

Tp-Sb

1.01 (1) 1.01 (1) 0.99 (1)

1.00 (1)

0.99 (1) I.76 (2) 0.3 (1)

0.86 (1)

9 35

GUY -

0.36

0.98 (1)

0.40 1.00 (1) 1.93 (2) 8.14 (2) 1.11 (1) 0.27 0.07

1.07 (1)

0.14 1.00 (1) 0.17 0.09 0.79 (1)

10 94

LeU

2.17 (2) 2.20 (2) 2.00 (2)

1.08 (1) 1.01 (1) 0.76 (1)

1.06 (1)

1.88 (2) 1.87 (2) 0.96 (1)

15 80

Thr

--

-

1.0 (1)

2

Ser

1.08 (1) 4 4

9.04 (2)

1.02 (1)

0.91 (1)

1.00 (1)

1.11 (1)

2.32 (2) 0.23 2.88 (2) 2.27 (2) 1.30 (1) 1.39 (1)

0.99 (1)

1.81 (1)

0.88 (1)

8.08 (2) 0.13 1.00 (1) 1.00 (1) 0.60 (1)

6 3 15 80 120 52

Ala ASP Ile -

_-

-

1 1 8 8 4 4 8 8 5 5 3 3 4 4 2 2 1 1 5 5 1 1 2 2 3 3 4 4 3 3 2 2

60 60

0 Compositions are given as molar ratios (in italics) ; the values in parentheses are the assumed number of residues. b Determined as homoserine lactone. c The amounts of tryptophan indicated in Tp-4 and Tp-8b were recovered from acid hydrolysates and determined on the short column

of the amino acid analyzer.

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4212 Amino Acid Sequence of Fe Fragment of Rabbit IgG. II Vol. 243, No. 16

1.0

I

E e! z X $ 0.5 - MC-m-b 24 I MC-YU-d

1

0 Volume -ml

FIG. 4. Chromatographic purification of MC VII. The pep- tides from CNBr-treated Fc fragment were purified on columns (1.9 X 17 cm) of Dowex 50-X8 as described in the text with Gradi- ent 1. The fractions which correspond to each peptide are indi- cated by the dashed bars.

sate on Dowex 50-X8 gave all the peaks shown in Fig. 2E except those designated Tpda and Tp-5b. A new peak, indicated by the dashed line in Fig. 2E, was found, which possessed a composition equal to the sum of compositions of peptides Tp-5a and Tp-5b.

Finally, peptide Tp-8b, which was recognized to contain NHa- terminal isoleucine, was also not found in tryptic digests of Fc fragment (1). However, it possessed a composition very similar to that of tryptic peptide Tp-8 (1)) which contains a single residue of methionine. For this reason peptide Tp-8b is thought to rep- resent the NHa-terminal sequence of MC VI.

Peptides MC VII-b, MC VII-d, and MC VII-Z-These peptides were found together in Peak VII on chromatography of the cyanogen bromide cleavage products of Fc fragment (Fig. 1). They were readily soluble between pH 2 and 5, and because of their apparent small size were purified by chromatography on columns of Dowex 50-X8 (1.9 X 17 cm), as shown in Fig. 4. Only three major peaks were observed; these have been desig- nated arbitrarily MC VII-b, MC VII-d, and MC VII-l. The composition of each peptide is given in Table I. The composi- tion of these three peptides and those of MC VI and MC IV account for the composition of Fc fragment within the accuracy of the analytical methods.

MC VII-b possesses NHz-terminal carboxymethylcysteine and contains 20 residues, including 1 of homoserine lactone (see Table I). Its high prolme content (7 residues per molecule) is particularly unique. A tryptic hydrolysate of this peptide on a column (1.9 x 17 cm) of Dowex 50-X8 gave three peaks, as shown in Fig. 2B. Two of the peaks, peptides Tp-25a and Tp-26, were pure and gave the compositions shown in Table IV. Clearly, the composition of MC VII-b is accounted for by the composition of peptides Tp-25a and Tp-26. Peptide

Tp-25a contains 1 residue of homoserine lactone, and appears to be derived from that region of the sequence contained in tryptic peptide Tp-25, which was isolated from tryptic digests of Fc frag- ment (1). It was apparently produced by cyanogen bromide cleavage at the single residue of methionine in peptide Tp-25. Peptide Tp-26 had NHa-terminal carboxymethylcysteine, the same end group as peptide MC VII-b; thus it represents the NH*-terminal sequence of MC VII-b.

MC VII-d contained 17 residues, including a single residue of homoserine lactone. Its single NHt-terminal residue was gly- tine, in 87% yield. Tryptic digests of the peptide, purified on columns (1.9 x 17 cm) of Dowex 50-X8 with Gradient 1, gave the chromatographic pattern shown in Fig. 2A. Four major peaks were observed; these were pure peptides as judged by paper chromatography and the stoichiometry of their constituent amino acids (Table IV). The amino acid composition of the four pep- tides, given in Table IV, accounted exactly for the composi- tion of MC VII-d. Tryptic peptide Tp-8a, which contained 1 residue of homoserine lactone, represented the COOH-terminal sequence of MC VII-d. Tryptic peptide Tp-lob possessed NHa- terminal glycine, the same residue as unhydrolyzed MC VII-d. It appears that tryptic peptide Tp-lob was formed by cleavage of the methionyl bond in tryptic peptide Tp-10, which was isolated from tryptic hydrolysates of Fc fragment (1).

MC VII-l contained 18 residues with a single, NHa-terminal histidyl residue. Its composition is identical with that reported earlier by Givol and Porter (3) for a peptide isolated from cyanogen bromide-treated heavy chain from rabbit IgG.3 The complete absence of homoserine lactone indicated that it represented the 18 residues from the COOH terminus of Fc fragment and heavy chain, as suggested earlier (3). Tryptic hydrolysates of MC VII-l, purified on columns of Dowex 50-X8 with Gradient 1, gave the pattern shown in Fig. 2G. Three pure peptides were obtained; their compositions, listed in Table IV, accounted exactly for the composition of whole MC VII-l.

Peptide MC V-This peptide was judged to be impure by NHa- terminal end group analysis. By the cyanate method, isoleucine was found in 92% yield and glycine was in 50% yield. Each of these residues is the single end group in other peptides from cyanogen bromide-treated Fc fragment. Isoleucine is the end group in MC IV and MC VI, and glycine is the NHz-terminal residue of MC VII-d. Furthermore, on amino acid analysis methionine was detected. These results suggested that MC V represents a mixture of some of the five major peptides from cyanogen bromide-treated Fc fragment (Table I). The nature of MC V was clarified by hydrolysis with trypsin and chromatog- raphy of the resulting peptides under the same conditions as shown in Fig. 2C. Fig. 2E shows the chromatographic pattern obtained with the tryptic peptides from 1 pmole of MC V. Twelve distinct peptides were obtained; they had compositions identical with those of peptides derived from either MC VII-d, MC VII-l, MC VI. Peptides T-l and T-2 were derived from MC VII-l; peptides Tp-4, Tp-Sa, Tp-5b, Tp-6, Tp-7, and Tp-8b were from MC VI; and peptides Tp-9 and Tp-lob were from MC VII-d. In addition, peptide Tp-3 (I), which is found par- tially in MC VI-p as Tp-3a and partially in MC VII-l as peptide Tp-3b, was found as a single peptide. This suggests that CNBr cleavage did not proceed completely at the single methionyl

8 The abbreviation used is: IgG, symbol for immunoglobulin recommended by a conference on human immunoglobulins sponsored by the World Health Organization (15).

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Issue of August 25, 1968 R. Delaney and R. L. Hill 4213

TABLE IV Amino Acid Composition of Tryptic Peptides MC VII-b, MC VIZ-d, and MC VII-l

MC VII-b I MC VII-d I MC VII-I

-

-_

Peptide

Tp-26 I

Tp-251~

Amino acid

Lysine. ............... Histidine .............. Arginine .............. Aspartic acid .......... ThROllin.3 ............. Sfhle ................. Glutamic acid ......... Proline. ............... Glycine ............... AlaIlin.3 ............... Carboxymethyl-

cysteine. ............ Vl%liIle ................. Methionim?. .......... Isoleucine ............. LeUCiUqi ...............

Tyro&ne .............. Phenylalanine .........

1.08 (2)

0.47 0.46 1.09 (1) 1.08 (1) 6.80 (7) a.os (2)

1.08 (1) 1.00 (1)

0.89 (1) 6.56 (2)

1.7s (2)

2

1 1 1 1 7 2

1 1 1 1 3

2

2

2

24 23

1.08 (1)

1.06 (1) O.fi8 (1)

1.08 (1) 1.06 (1) 1.86 (2)

1.88 (2) 8.07 (2)

1.04 (2) 1.00 (1)

1.81 (2) a.06 (2) a.10 (2) 1.08 (1)

1.04 (1) 1.08 (1)

6 4 10 66 20

GUY Gly

2

1 4 2 2 1

1 1 1

2

1.03 (1) 0.96 (1)

0.97 (1)

1.00 (1)

4 74

ASP

0.88 (1) 1.86 (2)

1.06 (1) 1.81 (1) 0.s (1)

1.08 (1)

P.95 (1)

l.9Ll (1) ‘.08 (1)

_-

-

_-

-

-_

18 ( -

20 44

xcyd

17 17 3 38

SIX

11 27 Hi8

18 18 7 33

Ser

Total No. of residues. . Percentage yield.. . . Amino-terminal residu

- a Compositions me given 88 molar ratios (in it&m). The number in parentheaea is the ammed number of residues. b Tp-9,lOb is not included in the total number of r&dues. a Muurcd as homoseFine laotone. d Carboxymethylcysteine.

(T-S)Ser(Val,Ser,Leu,Thr,CMC) Met-Ile(Asp~Ser~Glu~Pro,Gly,Val,Ile,Tyr,Phe,Try,Lys)(T-7,T-6,T-5,T-4)Gly(Asp,Val,Phe,Thr,CMC,Ser,~al)Met- - MCVlld .L MCVI WA+

+T-8 T-8- VT-3 W-T-80 .L T-8a- b-T-3a T-3a 4

His(Glu,Ala,Leu,His,Asp,His,Tyr,Thr,Glu,Lys)(Ser,Ile,Ser,Arg)(Ser,Pro,Glu)-COOH c MCVII I-

T-3-T-2-T-I+

FIQ. 5. Alignment of cyanogen bromide cleavage peptides of Fe fragment. The tryptic peptides Tp3, Tp-8, Tp-10, and Tp-25 which are the four methionine-containing peptides in Fc fragment, were isolated as described earlier (1). The other peptides were isolated as described in the text. The alignment of peptides Tp-1, Tp-2, and Tp-3 is in accord with that given by Givol and Porter (3). CMC, carboxymethylcysteine.

residue in the segment of Fc fragment represented by Tp-3 and that MC V contains, in part, MC VI and MC VII-l joined to- gether through Tp-3. This structure provides the NH&erminal isoleucine residue in MC V. Furthermore, the presence of Tp-9 and Tp-lob suggests that MC VII-d is joined to MC VI through incomplete splitting of the single methionyl residue in Tp-8, and that MC VII-d provides theNHz-terminal glycine. Thus, MC V is not one of the five unique CNBr cleavage products from Fc fragment, but is composed of MC VI and MC VII-l,

joined through a single methionine residue, and MC VII-d and MC VI, joined through another methionyl residue in Tp-8. Characterization of this complex peptide was not required to place the five CNBr cleavage products in a unique order, but confirmed the order that was deduced as described below.

Alignment of Cyanogen Bromide Peptides in Fc Fragment

From the composition of the four methionine-containing tryp- tic peptides in Fc fragment (1) and the composition of the tryptic

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Amino Acid Xequence of Fc Fragment of Rabbit IgG. II Vol. 243, No. 16

peptides in each of the five CNBr peptides, it was possible par- tially to order the sequence of Fe fragment. This alignment is shown in Fig. 5. MC VII-I, which contains neither methionine nor homoserine lactone, represents the last 18 residues from the carboxyl terminus of Fc fragment, as shown earlier by Givol and Porter (3). Tp-3b represents the sequence from its NHz-terminal histidine residue, which was derived from the sequence contained in Tp-3 by cleavage of a methionylhistidine bond. Tp-Sa, which was found only in MC VI, contains the remainder of Tp-3 not found in MC VII-l. Thus, MC VI precedes MC VII-1 in the se- quence of Fc fragment. The NHz-terminal sequence of MC VI is represented by peptide Tp-8b, which contains NHz-terminal iso- leucine, the same end group as MC VI. Tp-Sb was produced by cleavage of the methionylisoleucyl bond contained in the se- quence represented by peptide Tp-8. Tp-8b, which was derived uniquely from MC VII-b, contains the remainder of the compo- sition of Tp-8. Thus, MC VII-d precedes MC VI in the sequence of Fc fragment. The NHz-terminal sequence of MC VII-d is rep- resented by Tp-lob, which contains the last 4 residues of Tp-10 and possesses NHz-terminal glycine, the same end group as MC VII-d. Tp-lob was obtained as the result of cleavage of the me- thionylglycine bond in Tp-10. Tp-lOa, which contains the first 3 residues of Tp-10, is uniquely found in MC IV; thus MC IV precedes MC VII-d in the sequence of Fc fragment. The NH2 terminus of MC IV is isoleucine, which would be produced by cleavage of the methionylisoleucyl bond contained in the se- quence of Fe fragment represented by Tp-25. The tripeptide Tp-25b was found in MC IV, and Tp-25a, which represents the remainder of the sequence of Tp-25, was found only in MC VII-b. Thus, MC VII-b precedes MC IV and contains the remainder of the sequence of PC fragment.

DISCUSSION

Treatment of X-carboxymethyl-Fe fragment with CNBr re- sults in cleavage of peptide bonds at the 4 methionyl residues in the molecule and gives rise to five unique peptides. The com- position of the five peptides accounts closely for the composition of the Fc fragment. Earlier studies have shown that CNBr treatment of heavy chain gives rise to the expected number of peptides. Givol and Porter (3) found six unique zones on starch gel electrophoresis of CNBr-treated heavy chains from rabbit IgG. One peptide, which corresponds to MC VII-l, was isolated in 80% yield and corresponds to the COOH-terminal 18 residues in heavy chain. A similar peptide, isolated by Piggot and Press (4) from the heavy chain of normal human IgG, was found to differ from the corresponding peptide of rabbit heavy chain by only 2 residues. An identical peptide was isolated from the heavy chain of a pathological human IgG by Press, Piggot, and Porter (5). Each of these studies gives strong support to the view that heavy chain of immunoglobulin G is a single peptide chain and that at least the 18 residues from the COOH terminus of the chain have a common sequence.

The work reported here is in accord with the studies on CNBr cleavage of heavy chains, but provides evidence that most, if not all, of the sequence of heavy chain represented by the FC frag- ment is the major sequence in this region of rabbit heavy chain. First, the amino acid composition of the peptides from CNBr- treated Fc fragment accounts closely for the composition of the fragment. Second, the compositions of the tryptic peptides derived from each of the CNBr peptides account for the compo- sition of the CNBr peptide. If considerable sequence hetero-

geneity existed in Fc fragment but was not associated with the 4 methionyl residues, then five CNBr peptides, as shown in Table I, would be produced. On the other hand, sequence heter- ogeneity of this kind would be shown by isolation of a variety of tryptic peptides, which would contain more residues than ex- pected on the basis of the composition of the CNBr peptide. Since this was not observed, much of the sequence of Fc chains is common to all heavy chains,

The good evidence for sequence homogeneity in Fc fragment does not preclude the existence of a modest amount of sequence heterogeneity. First, the yields of each of the CNBr peptides varied from 37 to 52%. Clearly, the fact that yields were less than theoretical raises the possibility that some portions of Fc fragment are not represented by the five unique CNBr fragments. On the other hand, the yields given in Table I are minimal values, uncorrected for losses which occur on handling of the samples. The amount of peptide which could be lost on purification and characterization varies from peptide to peptide, but may be as much as 15 to 20%. Indeed, Givol and Porter (3) isolated MC VII-l in 80% yield, in contrast to a yield of 48% in this study. Perhaps as much as 15% of this peptide was found in MC V, which is a mixture of MC VI, MC VII-d, and MC VII-l. Thus, the actual yield of MC VII-l may approach that reported by Givol and Porter (3). Second, sequence variations may be associated with different subclasses of the Fc fragment. The fragment used in this study was prepared from IgG of unknown allotype isolated from pooled, normal rabbit serum. Two pep- tides, isolated in low yields from PC fragment, have been found to diier by 1 residue from a corresponding peptide which was ob- tained in high yield. A tryptic peptide isolated from MC III was identical with Tp-20 plus Tp-21, but contained 2 residues of valine and only 1 residue of arginine.4 A chymotryptic peptide from MC IV was found to be identical with a sequence derived from Tp-15, Tp-IS, and Tp-17, but contained 1 residue of aspartic acid replacing a lysine residue in Tp-17.6 Further studies will be required to establish the exact sequence of those peptides and their position in the sequence of Fc fragment, but it is possible they are derived from heavy chains of a different subclass.

The partial sequence of PC fragment, given in Fig. 5, was con- structed on the basis of the five unique peptides from CNBr digests of Fc fragment, knowledge of the methionine-containing tryptic peptides from Fc fragment (I), and the amino acid com- position of the tryptic peptides from each of the CNBr peptides. It is of interest that MC VII-b, which contains NHz-terminal carboxymethylcysteine, represents the NHz-terminal sequence of Fc fragment. End group analyses of whole Fc fragment by the cyanate method revealed a mixture of end groups, none of which was in major yield.2 Carboxymethylcysteine was not detected, although special precautions to identify this residue were not taken. Several different peptides in low yield, which correspond closely to Tp-26 (from MC VII-b), were isolated from tryptic digests of Fc fragment (1). This observation suggests that the NH2 terminus of Fc fragment is heterogeneous, probably as the result of cleavage of several different peptide bonds by papain. Recent studies by Cebra (16) and Porter (17) confirm the view that Tp-26 represents the NHz-terminal sequence of the Fc frag- ment and that the sequence contained in Tp-26 is that portion of the heavy chains at which the proteolytic enzymes (17) and

4 R. Delaney, unpublished observation. 5 R. E. Fellows, Jr., unpublished observation.

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Issue of August 25, 1968 R. Delaney and R. L. Hill 4215

CNBr (18) act. This sequence must be on the surface so as to allow the large sized proteolytic enzymes to come in contact with the IgG molecule. The single disulfide bond formed by the half- cystinyl residue at the NH, terminus of Tp-26, as well as the high proline content of Tp-26, may be responsible for a unique orienta- tion of this region of heavy chain so as to place it close to the surface of the molecule. It is of interest that CNBr attacks the heavy chains in IgG at one methionyl residue to form an active fragment somewhat smaller than the pepsin fragment but larger than the active papain fragment (18). Thii is exactly as ex- pected if the methionine residue in Tp-25 forms the peptide bond most susceptible to CNBr. Pepsin, on the other hand, can hydrolyze heavy chain to give a fragment approximately half the size of the Fc fragment. This pepsin fragment has been found by Prahl (19) to yield tryptic peptides essentially identical with peptides Tp-1 through Tp-13 described in this and the preced- ing paper.

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Robert Delaney and Robert L. HillII. CLEAVAGE OF THE Fc FRAGMENT WITH CYANOGEN BROMIDEThe Amino Acid Sequence of the Fc Fragment of Rabbit Immunoglobulin G:

1968, 243:4206-4215.J. Biol. Chem. 

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