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A comparison of the labile disulfide bonds of rabbit γG-immunoglobulin fragments
.4RCHIVF,S
OF
BIOCHEMISTRY
AND
BIOPHYSICS
A Comparison Rabbit MICHAEJ,
113, 709-717 (1966)
of the Labile
Disulfide
+yG-lmmunoglobulin G. MAGE
of
Fragments
EDWARD
AND
Bonds
T. HARRISOK
C’.S. Department of Health, Education, and M*elfare, Public Health Service, AVational In.sfitutes of Health, Xational Institute of Dental Research, Bethesda, Maryland Received
October
20, 1965
The Fab fragment from insoluble papain digestion and mild redllction of rabbit rGimmunoglobulin was compared with the F(ab’)* fragment from peptic digestion of rG, Insoluble papain digestion of F(ab’)z, when preceded or followed by mild reduction and alkylation with iodoacetic acid-1-‘4C, residts in the liberation of S-carboxymethylcysteine (CMC)-peptides cont,aining a total of slightly more than one mole of CMC per Fab fragment formed. Fab has less proline and less leucine than Fat)‘. On mild reduction and alkylation, Fah has a lower content of CMC than either Fab’ or yG per half molecule. These data lend quantitative support to the hypothesis that papain and pepsin may split on opposite sides of a disulfidc bond between the heavy chains of rabbit q/G.
Rabbit immunoglobulins of the rG class have been postulated (25) t,o consist of two heavy (or “A”) and t’wo light (or ‘3”) polypeptide chains. In keeping with the findings of Cecil and Wake (4) t,hat interchain disulfide bonds of several prot,eins are more easily reducible t’han intraahain bonds, the 5 cystine residues of -yG-immunoglobulin found to be reducible under mild conditions were postulated by Port’er (25) t,o form interchain bonds. Palmer and Nisonoff (22) have suggested, on the basis of their studies on reduction and acidification of rabbit rG, that’ there is only one disulfide bond between the heavy chains, the other two bonds suggested by Porter possibly being intrachain bonds of the heavy chains. A variety of high molecular weight fragments have been isolated after limited proteolysis of yG. Limited peptic digestion of rabbit rG products the “5 S” F(ab’)z fragment, which is postulated to consist of t#wo halves (designated Fab’), linked by a highly labile disulfide bond (18). The molecular weight of the F(ab’)2 fragment is approximat,ely 90,000 (7, 28). The Fab fragment, is produced by l)a~)ain digestion and simultaneous reduction (24), or by digestion with 709
insoluble papain (WIP) and subsequent reduction (2) of rabbit rG. Pain (21) found t’he molecular weight of Fab to be approximately 41,000. Unlike the Fab’ fragment produced by peptic digestion and reduction of rabbit rG, the Fab fragment from papain digestion and reduction of rabbit rG dots not reoxidize t’o form a “5 S” fragment. “Fragment I dimer, ” isolated after incubation of WIP-treated -yG wit’h low concentrations of sodium dodecylsulfatc (3, 7), is similar in molecular weight to F(ab’)z. However, on mild reduction, “Fragment I dime? is split into two halves bhat share with Fab t,he inability to rcoxidize to a “5 S” fragment. In t,his report, t,he labile disulfide bonds (i.e., reducible in t,he absence of denat,uring agent) of F(ab’)s and Fab were studied in an effort to underst,and the relat,ionship of these fragments to each other and to the parent rG molecule.’ MATERIALS
AND
p-Aminophenylalanine-leucine the gift of Dr. David Givol.
METHODS co-polymer was Sheep anti-rabbit
1 A portion of the data has been presented preliminary communication (12).
in a
$10
MAGE
AND
FIG. 1. Gel-diffusion reactions between yG fragments and a sheep anti-rabbit -& antiserum specific for Fc determinants, illustrating the purification of F(ab’)l. The ant,iserum (sheep 33) is in t.he cent,er well. Peripheral wells have: a, rC ; b, unfractionated peptic digest of yG; c, F(ab’)? isolated by NanSod precipitation; d, F(ab’)i purified on CM-Sephadex; e, material containing Fc determinants, eluted from CM-Sephadex by RI sodium acetate pII 5.5; f, Fab prepared from WIP-treated F(ab’)z (peak 8, Fig. 4). TG antisera were the gift of Dr. Rose Mage. Rabbits homozygous for the ,4al and Ab4 allotypes were obtained from Dr. Sheldon Dray. SCarboxymethylcysteine (CMC)” was obtained from Mann Laboratories, Inc. Iodoacetic Acid-l14C was purchased from New England Nuclear Corp. 2-Mercaptoethanol came from Eastman Organic Chemicals, and dit.hiothreitol (DTT) came from Calbiochem, Inc. Twice-crystallized pepsin and crystalline mercuripapain were purchased from Worthington Biochemical Corp. Column effluents containing ‘4C-labelled materials were counted on a Packard Tri-Carb liquid scintillation spectromet,er with the counting solution of Kinard (1957). Paper chromatograms and electrophoretograms were counted in a Vanguard model 880 automatic chromatogram scanner. Sedimentation velocities were measured at -~. 2 Abbreviations used: DTT, dithiothreitol; CMC, S-carboxymethyl cysteine; WIP, waterinsoluble papain.
HARRISON
FIG. 2. Gel-diffusion react,ions between +yG fragments and a sheep anti-rabbit ?G antiserum. The ant,iserum (sheep 42) is in the center well; peripheral wells have the same materials as in Fig. 1. 59,780 or 52,640 rpm. Samples were dissolved in 0.1 fit glycine-0.01 12 sodium phosphate buffer at pH 7.3. q/G-Immunoglobulin was prepared from rabbit serum by precipitation with sodium sulfate (9), followed by chromatography on DEAE-cellulose (26), in 0.01 M sodium phosphate buffer at pH 7.3. Water-insoluble papain (WIP) was prepared by coupling mercuri papain t,o diazotized p-aminophenylalanine-leucine co-polymer, according to Cebra et al. (2). Preparation of the Fab fragment. Fab fragments were prepared by WIP-digestion of whole yG (2), followed by reduction in 0.1 !lf mercaptoethanol, and incubation at 4” for 3 days, during which time the bulk of the Fc fragment precipitated. The supernatant solution was dialyzed against sodium acetate bufler, pH 5.5 (0.02 111 in acetate) and chromatographed on CM Sephadex C25 equilibrated with the same buffer. I’reparation of the F(ab’)z fragment. Rabbit rG-immunoglot~uliu in pyridine acetate buffer, at pH 4.0 (0.1 Jf in acetate), was incubated with pepsin at room t,emperature for 24 hours; enzyme to substrate ratios ranging from 1:175 to 1:200 were used. The high molecular weight material was isolated by sodium sulfat,e precipitation (14). Further purification of this fragment is described under Results.
DISULFIDE
BONDS OF rG FRAGMENTS
Reduction and alkylation of yG, Fab, and F(ab’)z. rG, Fab, and F(ab’)z were reduced at pH 8.3-8.5 in various concentrations of 2-mercaptoethanol for 1 hour, as described in Results (Fig. 3), and alkylated by adding a quantity of iodoacetic acid equimolar to the mercaptoethanol, maintaining the pH between 8.3 and 8.5 by additions of NaOH. After 1 hour at room temperature, the reaction mixtures were dialyzed exhaustively against distilled water and lyophilized. When iodoacetic acid-lJ4C was used for alkylation, the alkylation reaction mixtures were fractionated on a 1.5 X 57-cm Sephadex G25 column equilibrated with 0.1 M NHdHCOz. Amino acid analyses. Protein and peptide samples were hydrolyzed for 24 hours in constantboiling HCl by the procedure of Moore and Stein (17). Ion-exchange chromatography and the ninhydrin reaction were done by a modification (11) of the method of Piez and Morris (23). The values of the amino acids were corrected for decomposition and incomplete hydrolysis by using factors
determined
‘711
from 24- and 72-hour hydrolyses
whole rG. When fragments
containing
S-carboxymethylcysteine were analyzed, the CMC content was also calculated from the ratio of cpm to OD~~O,,,~, using an E%, of 16.0 (Ref. 16). RESULTS
PuriJicahn of the F(ab’)g product of peptic digestion. The F(ab’)z fragment prepared by sodium sulfate precipitation still reacted with sheep antibody specific for the rabbit Fc fragment in double-diffusion in agar (20) (Fig. 1). Consequently, it was further purified by passage through carboxymethyl Sephadex C25 equilibrated with sodium acetate buffer, 0.02 M acetate, at pH 5.5. The F(ab’)z fragment was not bound to the ion exchanger under these conditions, and did not react with the anti-Fe antiserum. The bound material was eluted with 1 M
A YG/2 0 Fab’ 0 Fob
IO
0 Fob from F(ab’&
n CMC Peptides
9 8 7
0
0.02
of
KXabelled
0.04 0.06 0.08 0.10 MERCAPTOETHANOL
0.12 0.14 0.16 CONCENTRATION
0.16 0.20 (M)
FIG 3. S-Carboxymethylcysteine content of rG and fragments as a function of reducing agent concentration. Residues of CMC were calculated from amino acid analysis, based on an estimated molecular weight of 72,500 per half-rG molecule (15) and on an assumed molecular weight of 45,000 for Fab and Fab’.
712
MAGE TABLE
AND
I
SEDIMENTATION COEFFICIENTS OF yG AND F(ab’)z BEFORE AND AFTER TREATMENT WITH WATER INSOLUBLE PAPAIN .\ND/OR REDUCING AGENTS sm. w Material
7G WIP-treated F(ab’)z WIP-treated
yG F(ab’),
Unreduced
Dithiothreitol, o,ol w j
h’e~~~Pethanol, 0.14 M
6.3 6.3 4.7 4.7
6.0 3.3 3.2
6.1 3.7 3.0 3.1
sodium acetate buffer at pH 5.5, and reacted with the anti-Fc antiserum (Fig. 1). Another anti-rabbif rG antiserum of broader specificity reacted with the F(ab’)z fragment, and showed it to lack the Fc antigenic determinants present in the unfractionated digest (Fig. 2). CMC content of rG and fragments alkylated after reduction with %mercaptoethanol. In a series of experiments, rG and its fragments were reduced with mercaptoethanol at concentrations ranging from 0.01 to 0.14 M. In Fig. 3, CMC content of the reduced alkylated materials (which is equivalent to content of SH groups on reduction), is plotted vs. mercaptoethanol concentration during reduction. For both rG and ibs proteolytic fragments, the number of cysteine sulfhydryl groups increases with increased concentration of mercaptoethanol. -yG and F(ab’)z have a very similar content of CMC residues The Fab fragment, per half-molecule. whether prepared from rG direct,ly, or from the F(ab’)z fragment, appears to have from 1.5 to 3 fewer CMC residues (depending upon mercaptoethanol concentration during reduction) per assumed 45,000 molecular weight, than either the Fab’ fragment (per 45,000 MW), or the G per half-molecule (assumed 72,500 MW). Sedimentation velocity measurements of yG and the F(ab’)z fragment before and after WIP-treatment and/or reduction. The sedimentation coefficient of TG-immunoglobulin was not appreciably affected by reduction (with either mercaptoethanol or dithiothreitol) or by WIP-treatment, but on combined WIP-treatment and reduction, the
HARRISON
sedimentation coefficient fell to 3.7 S (Table I). The 4.7 S F(ab’)* fragment remained unchanged on WIP-t,reatment. On reduction of F(ab’,Jz, the sedimentation coefficient fell to 3.0-3.3 S, and WIP-treat’ed F(ab’)z had a value of 3.1-3.2 S on reduction. Water-insoluble papain digestion, reduction with mercaptoethanol, and alkylation of the F(ab’)z fragllzent. Purified 4.7 S F(ab’)n fragment was incubated with water-insoluble papain (2) for 10 minutes at room temperature, enzyme substrate ratio 1:250, in 0.03 M sodium phosphate buffer at pH 7.8, containing 0.002 J!1 EDTA. The enzyme was removed by centrifugation. The result,ant 4.7 S WIP-treated F(ab’)z fragment was reduced for 1 hour in 0.03 M mercaptoethanol and alkylated with a quantity of iodoacetic acid-l-14C, equimolar to the mercaptoethanol. The pH was maintained between 8.3 and 8.5 by additions of NaOH. After one hour at room temperature, the reaction mixture was fractionated on a 1.5 X 57-cm Sephadex G25 column equlibrated with 0.1 dl NHdHCOs. The elution diagram is shown in Fig. 4. Peak A contained a 3.1 S component which had the same reactivit,y toward sheep an&rabbit, rG antisera as undigested F(ab’)z fragment (see Figs. 1 and 2); Peak B, with a high ratio of radioactivity to absorbance at. 280 rnp, had approximately ${o of t,he total cpm in the first 2 peaks. High voltage paper electrophoresis of an aliquot of peak B, in pyridine acetate buffer, pH 3.7 (Ref. 8), showed it to contain a mixture of radioactive peptides. In order to be able to relate the radioactive label to S-carboxymet,hylcysteine content, it was necessary to verify that t’he alkylation with iodoacetate-14C was specific for cysteine sulfhydryl groups. Aliquots from peaks A and B were analyzed for radioactivity by paper electrophoresis and paper chromatography of their acid hydrolyzates. Only one radioactive component, was found and it had the mobility of S-carboxymet,hylcyst eine (Table II). The amino acid composition of peak A (Fab fragment) and of peak B (peptide mixture) is shown in Table III, columns 1 and 2, respectively. The peak B peptide mixture (column 2) is limited in amino acid
DISULFIDE
713
BONDS OF -/G FRAGMENTS
-c” 7000 v 6000
b
5000 4000
FIG. 4. Separation on a Sephadex G25 column of the products of reduction and alkylation of WIP-treated F(ab’)z. The vertical lines indicate the portions of peaks A and B pooled for amino acid analysis.
TABLE
II
COMPARISON WITH S -CARBOXYMETHYLCYSTEINE OF THE RADIOACTIVE COMPONENT IN ACID HYDROLYZATES OF RCM-WIP-TREATED
drolyzed aliquots of peak C showed no CMC, and traces of other amino acids totaling less than one tenth of the total amino acid residues present in peak B.
F(ab’h Radioactive component
Paper electrophoresis? Rlyaine Paper chromatography:” Rf
S-Carboxymethylcysteine
-0.26
-0.26
0.22
0.22
QWhatman No. 1 paper, 60 V/cm, 50 minutes, pyridine acetate buffer, pH 3.7. 1,Whatman No. 1 paper, ascending chromatography; butanol-acetic acid-water, 4: 1: 1. composition, and contains 1.1 CMC residues, compared with 1.7 CMC residues in the concomitantly produced Fab fragment (peak A, Fig. 4). In addition to CMC, the amino acids present in highest amounts in the peptide mixture were proline, leucine, and glutamic acid. Peak C contained the low molecular weight by-products of reduction and alkylation. Amino acid analyses of hy-
Amino Acid Analyses of Fab’ and Fab Column 3 of Table III shows the mean of 14 analyses of various preparations of F(ab’)z and Fab’, expressed as residues per Fab’ fragment of assumed 45,000 MW. Column 4 of Table III shows the mean of 13 analyses of several preparations of Fab, whether prepared directly from rG, or from F(ab’)z. The composition of Fab is also expressed as residues, based on the assumption of an alanine content equal to that of Fab’. Calculated on this basis, Fab would have a molecular weight of 43,600, which is higher than the molecular weight value of 40,700 reported by Pain (21). When compared on the basis of the same alanine content as Fab’, Fab has significantly less proline and leucine (p < .OOl) and possibly less glutamic acid (p = .013) than Fab’. For 11 of the remaining amino acids, the differences between Fab’
il4
MAGE
AND
HARRISON
TABLE GINO
ACID
COMPOSITION
III OF
yG
RABBIT
FRAGMENTS
Fib’
Compound id
Sh
-___ 36
sh
5 Residue differences (Fab’ - Fab)
FL
S-Carboxymethylcysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cystine/2
l&(1.7”’ 31.1 Gl.6 50.3 32.2 31.1 38.4 28.4 40.0 9.4
1.1 0.3 0.7 0.6 1.1 2.3 0.3 0.4 0.4 0.0
11.8f 31.4 60.3 50.5 32.7 34.1 39.1 28.1 40.8 4
2.3 1.0 1.9 3.4 0.8 2.5 1.5 0.8 2.7 -
10.5’ 31.1 GO.0 48.0 31.8 29.3 38.8 28.1 41.2 -
1.0 1.1 2.4 2.2 0.7 1.5 1.3 0.5 2.0 -
1.3, 0.3 0.3 2.5 0.9 4.8 0.3 O.oi -0.4 -
Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
2.1 11.0 26.5 18.0 11.9 16.5 2.5 8.9
0.0 0.1 1.1 0.1 0.1 0.4 0.0 0.0
2.3 11.5 28.0 18.2 12.0 17.1 2.8 9.6
0.2 1.2 0.7 1.0 0.7 0.4 0.4 0.5
2.2 11.5 26.4 18.1 12.0 16.6 2.8 9.3
0.2 0.7 0.8 0.6 0.4 0.6 0.4 0.3
0.1 0.0 1.6 0.1 0.0 0.5 0.0 0.3
a Residues, based on an assumed molecular weight of 45,000 for peak A + peak B. 6 Residue calculations for the peptide material are based on the CMC content. The value of 1.1 CMC residues in the peptide material is based on the 2:3 ratio of peak “B” cpm to peak “A” cpm. c The CMC value in parentheses is based on the ratio of cpm to OD280mp. d Mean of 14 analyses; included are analyses of both Fab’ and F(ab’),. The data are expressed as residues per Fab’ molecule of 45,000 MW. *Mean of 13 analyses; included are analyses of Fab from rG, and Fab from F(ab’)z. Residues are calculated on the basis of an alanine content equal to that of Fab’. J Because the sample includes unreduced preparations as well as preparations reduced under a variety of conditions, the values given are the sum of S-carboxymethylcystine and cystine/2. Q Included with S-carboxymethylcysteine. h Standard deviation. i By definition.
and Fab are less than 0.5 residue per univalent fragment. CMC content of fragments formed by WIPtreatment of F(ab’)z, preceded or followed by reduction with 0.01 M DTT, and alkylation with iodoacetic acid-l -14C. Several subsequent experiments, summarized in Table IV, show that from 1.1 to 1.2 residues of CMC were liberated as peptides, when WIP-treatment of F(ab’)z was preceded or followed by reduction with 0.01 2M DTT, alkylation with iodoacetic acid-1-‘JC, and separation on Sephadex G25 by the same procedure as that shown in Fig. 4. Reduction of the F(ab’)z fragment with 0.01 M DTT, followed by alkylation with a 1: 1 molar ratio of iodoacetic acid-l-l% to DTT
sulfhydryl, yielded an Fab’ fragment containing 5.9 CMC residues per assumed molecular weight of 45,000 with less than 0.1 CMC residue present in peptides (Experiment 1, Table IV). When F(ab’)z was treated with WIP and then re-isolated on Sephadex G25, it retained its sedimentation coefficient of 4.7 S; less than 1% of the total amino acid residues were present as peptides. Subsequent reduction and alkylation of this 4.7 S WIP-treated F(ab’)Z fragment yielded a 3.2 S Fab fragment containing 4.9 CMC residues per 45,000 MW, with a total of 1.1 residues of CMC present in peptides (Experiment 2, Table IV). When F(ab’)z was reduced with 0.01 M DTT, and alkylated with but a 3:4 ratio of iodoacetic acid-1-14C
DISULFIDE
BONDS
715
OF q/G FRAGMENTS
TABLE
IV
S-CARBOXYMETHYLCYSTEINE COXTENT OF THE PRODUCTS OF WATER-INSOLUBLE FRAGMENT DIGESTION AND/OR REDUCTION AND ALKYLATION OF THE F(ab’), 0.01 M DITHIOTHREITOL AND IODOACETIC ACID-I-X Alkyla-
CMC content of products after separation on Sephadex G25 (residues)b
tion
(ratio of moles of Experimerit No. lodoace- Starting material tate-‘C added to DTT sulfhydryljc
1 2
1:l 1:l
F(ab% F(ab’h
3 4 5
3:4 3:4
F(ab’)z CM-Fab’d F(ab’)n
PAPAIN WITH
Procedures’ CM-Fab’
Reduction and alkylation Treatment with WIP? followed by reduction and alkylation Reduction and alkylation Treatment with WIP Treatment with WIP, followed by reduction and alkylation
5.9(5.8)’
5.0(5.0) -
CM-Fab
CMpeptides
4.9(4.5)
0.07 1.1
(3.;” 3.5(3.6)
0.2 1.2 1.1
a WIP-digestion and Sephadex G25 chromatography were done as in the experiment illustrated in Fig. 4. b CMC content of the Fab’ and Fab fragments is calculated from duplicate amino acid analyses based on an assumed molecular weight of 45,000. Peptide CMC values are calculated from the ratio of total peptide cpm to Fab or Fab’ cpm. c Reductions were done with 0.01 -11 DTT, i.e., 0.02 M with respect to sulfhydryl. d The CM-Fab’ fragment was prepared in Experiment 3. 6 The WIP-treated F(ab’)s in this experiment was isolated on Sephadex G25 prior to reduction and alkylation. f CMC values in parentheses are calculated from the ratio of cpm to OD,,,,. 0 (-) not applicable. h Not measllred by amino acid analysis.
to DTT sulfhydryl (Experiment 3, Table IV), the resultant Fab’ fragment had 5.0 residues of CMC per 45,000 MW (as compared with 5.9 residues in Experiment 1, Table IV) with 0.2 residues of CMC present as peptides. Nonetheless, treatment of this Fab’ fragment with WIP, followed by passage through Sephadex G25, yielded an Fab fragment containing 3.9 CMC residues per 45,000 MW, with 1.2 residues of CMC present as peptides (Experiment 4, Table IV). Similarly, on WIP-treatment of F(ab’)z, followed by reduction with 0.01 M DTT and alkylation with a 3:4 molar ratio of iodoacetate to DTT sulfhydryl, 3.5 residues of CHIC were present in the resultant Fab fragment, and 1.1 residues of CMC were in peptides (Experiment 5, Table IV). DISCUSSION
The Fab fragment, on mild reduction and alkylation, has (depending on concentration of reducing agent) from 1.5 to 3 less residues
of S-carboxymethylcysteine than rG per half molecule or the univalent Fab’ fragment (Fig. 3). Insoluble papain treatment of F(ab’)z, when followed or preceded by mild reduction and alkylation with iodoacetic-14C acid, results in the liberation of 14C-labelled CMC-peptides totaling slightly more than 1 residue of CMC per Fab’ molecule of 45,000 MW (Table IV). Even though the CMC content of the concomitantly produced Fab fragment varied from 1.7 residues on very mild reduction (i.e., with 0.03 M mercaptoethanol), to 5.0 residues (with 0.01 M DTT), the total CMC content of the liberated peptides remained relatively constant (1.1 and 1.2 residues, respectively). In addition to the difference in CMC content, Fab contains significantly less proline and leucine than Fab’. Taken together, these findings lend quantitative support to the hypothesis that papain and pepsin may split on opposite sides of the disulfide bond between the heavy chains (7, 19). Inman
716
MAGE
AND
and Nisonoff (6) have st,rengthened this hypothesis with their finding that reduction and acidification of the Fc fragment from papain digestion of rG causes a halving of its molecular weight, indicating that the half-cystine residues that form the disulfide bond between the heavy chains may be in the Fe fragment, rather than in the Fab fragment. Alternatively, the data in the present study are equally compatible with the recent suggestion by Utsumi and Karush (28) that the heavy chains of rabbit yG are connected not by one, but by two disulfide bonds that cross each other. These authors postulate that insoluble papain may split the heavy chains between these bonds. According to this hypothesis, one mole of CMC per univalent fragment would be liberated as peptides when insoluble papain treatment of F(ab’)z is preceded or followed by mild reduction and alkylation. In the present study, 1.1-1.2 moles of CMC were liberated as peptides by these procedures. Indeed, these authors have recently reported that on soluble papain digestion of F(ab’)z in the presence of an extremely low concentration of reducing agent (0.001 M mercaptoethanol), the resultant 3 S fragments contained no CMC, whereas an unspecified amount of this constituent was present in their “low molecular weight product” (28). This hypothesis (i.e., of two interchain disulfide bonds that cross each other) offers an explanation for the failure of rG (2) or F(ab’)z (7) to fall in molecular weight on digestion with insoluble papain. Another possible arrangement of heavy chain disulfide bonds is that, after insoluble papain digestion of -rG or F(ab’)z, intrachain disulfide bonds of the heavy chains may connect the latent Fab fragments to the rest of the molecule. An additional finding of this study is that the F(ab’)s fragment from peptic digestion of +yG can be purified by the same method used to isolate the Fab fragment (i.e., chromatography on CM Sephadex C25). The purified F(ab’)s fragment no longer reacts in double diffusion in agar with antiserum specific for the Fe fragment (Fig. 1). This method of purification of F(ab’)z is an alternative to that of Jacquet and Cebra (7),
HARRISON
who used chromatography on Sephadex GlOO to purify the F(ab’)z fragment. The data in Fig. 3, showing CMC content of both reduced, alkylated rG and its proteolytic fragments as a function of reducing agent,, are in keeping wit,h the SH values found for undigested yG by Hong et. al. (5) and by Utsumi and Karush (27). The fragments prepared in this study were characterized by several criteria: Amino acid analyses of the F(ab’)z and Fab fragments (Table III) are in good agreement with the analyses of Mandy et. al. (14) and Porter (24), respectively. The ~20,~values of 4.7 S and 3.3 S for the F(ab’)z and Fab’ fragments (Table I), are similar to those reported by Mandy and Nisonoff (13), and the s20,wvalues of 4.7 and 3.1 S for unreduced and reduced WIP-treated F(ab’)z respectively, are comparable to the values found by Jacquet and Cebra (7). In considering the possible arrangements of disulfide bonds in rabbit rG, it should be noted that this protein may be heterogeneous with respect to lability of the disulfide bonding between the heavy chains (22). Furthermore, disulfide interchange may be a source of heterogeneity with respect to the specific pairing of the disulfide bonding between the latent fragments after WIP-digestion of rG. Cebra (3) and Jacquet and Cebra (7) have postulated that disulfide interchange may be responsible for t’he gradual formation of the ‘(Fragment I dime? found by them when WIP-treated rG was incubated with low concentrations of sodium dodecylsulfate. The concept of disulfide interchange following limited proteolysis, leading to a new arrangement of disulfide bonds, has been substantiated recently by Anfinsen (l), who has shown that, after limited proteolysis of a native protein, the native configuration is no longer thermodynamically most favored. Incubation of such an altered protein with a microsomal enzyme that catalyzes disulfide interchange, results in a rapid loss by the altered protein of its original conformation and of its activity. Experimental efforts now being made to examine cystine peptides and CMC peptides derived from the Fab, F(ab’)z and Fc
DISULFIDE
BONDS
fragments, may help to determine the nature of the disulfide bonding between the heavy chains, and of the bonds holding the latent fragments together after WIP digestion of rG or F(ab’)s. REFERENCES 1. ANFINSEN, C. B., Abstr. 150th Meeting Am. Chem. Sot., September 1965. 2. CEBRA, J. J., GIVOL, D., SILMAN, H. I., AND KATCHALSHI, E., J. Biol. Chem. 336, 1720 (1961). 3. CEBRA, J. J., J. Immunol. 92, 977 (1964). 4. CECIL, R., AND WAKE, R. G., Biochem. J. 82, 401 (1962). 5. HONG, R., DIXON, D. J., AND NISONOFF, A., Federation Proc. 24, Part I, 200, Abstr. No. 401 (1965). 6. INMAN, F. P., AND NISONOFF, A., Federation Proc. $4, Part I, 200, Abstr. No. 402. (1965). 7. JACQUET, IL, AND CEBRA, J. J., Biochemistry 4, 954 (1965). .&;4 8. KATZ, A. RI., DREYER, W. J., AND ANFINSEN, C. B., J. Biol. Chem. 234, 2897 (1959). 9. KEKWICK, R. A., Biochem. J. 34, 1248 (1940). 10. KINARD, F. E., Rev. Sci. Ins&. 28, 293 (1957). 11. KRICHEVSKY, M. I., SCHWARTZ, J., AND MAQE, M., Anal. Biochem. 12, 94 (1965). 12. MAGE, M. G., AND HARRISON, E. T., Abstr. 150th Meeting Am. Chem. Sot., September, 1966.
OF yG FRAGMENTS
717
13. MANDY, W. J., AND NISONOFF, A., J. Biol. Chem. 338, 206 (1963). 14. MANDY, W. J., RIVERS, M. M., AND NISONOFF, A., J. Biol. Chem. 236, 3221 (1961). 15. MARLER, E., NELSON, C. A., AND TANFORD, C., Biochemistry 3, 279 (1964). 16. MCDUFFIE, F. C., AND KABAT, E. A., J. Immunol. 77, 193 (1956). 17. MOORE, S., AND STEIN, W. H., Methods in Enzymology 6, 819 (1963). 18. NISONOFF, A., MARKUS, G., AND WISSLER, F. C., Nature 189, 293 (1961). 19. NISONOFF, A., AND DIXON, D. J., Biochemistry 3, 1338 (1964). 20. OUCHTERLONY, O., in “Progress in Allergy,” Vol. 5, p. 1. Karger, Base1 (1958). 21. PAIN, R. H., Biochem. J. 68, 234 (1963). 22. PALMER, J. L., AND NISONOFF, A., Biochemistry 3, 863 (1964). 23. PIEZ, K. A., AND MORRIS, L., Anal. Biochem. 1, 187 (1960). 24. PORTER, R. R., Biochem. J. 73, 119 (1959). 25. PORTER, R. R., in “Symposium on Basic Problems in Neoplastic Disease,” (A. Gellhorn and E. Hirschberg, eds.), p. 177. Columbia Univ. Press, New York (1962). 26. SOBIER, H. A., AND PETERSON, E. A., Federation Proc. 17, 1116 (1958). 27. UTSUMI, S., AND KARUSH, F., Biochemistry 3, 1329 (1964). 28. UTSUMI, S., AND KARUSH, F., Biochemistry 4, 1766 (1965).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
A Comparison Rabbit MICHAEJ,
113, 709-717 (1966)
of the Labile
Disulfide
+yG-lmmunoglobulin G. MAGE
of
Fragments
EDWARD
AND
Bonds
T. HARRISOK
C’.S. Department of Health, Education, and M*elfare, Public Health Service, AVational In.sfitutes of Health, Xational Institute of Dental Research, Bethesda, Maryland Received
October
20, 1965
The Fab fragment from insoluble papain digestion and mild redllction of rabbit rGimmunoglobulin was compared with the F(ab’)* fragment from peptic digestion of rG, Insoluble papain digestion of F(ab’)z, when preceded or followed by mild reduction and alkylation with iodoacetic acid-1-‘4C, residts in the liberation of S-carboxymethylcysteine (CMC)-peptides cont,aining a total of slightly more than one mole of CMC per Fab fragment formed. Fab has less proline and less leucine than Fat)‘. On mild reduction and alkylation, Fah has a lower content of CMC than either Fab’ or yG per half molecule. These data lend quantitative support to the hypothesis that papain and pepsin may split on opposite sides of a disulfidc bond between the heavy chains of rabbit q/G.
Rabbit immunoglobulins of the rG class have been postulated (25) t,o consist of two heavy (or “A”) and t’wo light (or ‘3”) polypeptide chains. In keeping with the findings of Cecil and Wake (4) t,hat interchain disulfide bonds of several prot,eins are more easily reducible t’han intraahain bonds, the 5 cystine residues of -yG-immunoglobulin found to be reducible under mild conditions were postulated by Port’er (25) t,o form interchain bonds. Palmer and Nisonoff (22) have suggested, on the basis of their studies on reduction and acidification of rabbit rG, that’ there is only one disulfide bond between the heavy chains, the other two bonds suggested by Porter possibly being intrachain bonds of the heavy chains. A variety of high molecular weight fragments have been isolated after limited proteolysis of yG. Limited peptic digestion of rabbit rG products the “5 S” F(ab’)z fragment, which is postulated to consist of t#wo halves (designated Fab’), linked by a highly labile disulfide bond (18). The molecular weight of the F(ab’)2 fragment is approximat,ely 90,000 (7, 28). The Fab fragment, is produced by l)a~)ain digestion and simultaneous reduction (24), or by digestion with 709
insoluble papain (WIP) and subsequent reduction (2) of rabbit rG. Pain (21) found t’he molecular weight of Fab to be approximately 41,000. Unlike the Fab’ fragment produced by peptic digestion and reduction of rabbit rG, the Fab fragment from papain digestion and reduction of rabbit rG dots not reoxidize t’o form a “5 S” fragment. “Fragment I dimer, ” isolated after incubation of WIP-treated -yG wit’h low concentrations of sodium dodecylsulfatc (3, 7), is similar in molecular weight to F(ab’)z. However, on mild reduction, “Fragment I dime? is split into two halves bhat share with Fab t,he inability to rcoxidize to a “5 S” fragment. In t,his report, t,he labile disulfide bonds (i.e., reducible in t,he absence of denat,uring agent) of F(ab’)s and Fab were studied in an effort to underst,and the relat,ionship of these fragments to each other and to the parent rG molecule.’ MATERIALS
AND
p-Aminophenylalanine-leucine the gift of Dr. David Givol.
METHODS co-polymer was Sheep anti-rabbit
1 A portion of the data has been presented preliminary communication (12).
in a
$10
MAGE
AND
FIG. 1. Gel-diffusion reactions between yG fragments and a sheep anti-rabbit -& antiserum specific for Fc determinants, illustrating the purification of F(ab’)l. The ant,iserum (sheep 33) is in t.he cent,er well. Peripheral wells have: a, rC ; b, unfractionated peptic digest of yG; c, F(ab’)? isolated by NanSod precipitation; d, F(ab’)i purified on CM-Sephadex; e, material containing Fc determinants, eluted from CM-Sephadex by RI sodium acetate pII 5.5; f, Fab prepared from WIP-treated F(ab’)z (peak 8, Fig. 4). TG antisera were the gift of Dr. Rose Mage. Rabbits homozygous for the ,4al and Ab4 allotypes were obtained from Dr. Sheldon Dray. SCarboxymethylcysteine (CMC)” was obtained from Mann Laboratories, Inc. Iodoacetic Acid-l14C was purchased from New England Nuclear Corp. 2-Mercaptoethanol came from Eastman Organic Chemicals, and dit.hiothreitol (DTT) came from Calbiochem, Inc. Twice-crystallized pepsin and crystalline mercuripapain were purchased from Worthington Biochemical Corp. Column effluents containing ‘4C-labelled materials were counted on a Packard Tri-Carb liquid scintillation spectromet,er with the counting solution of Kinard (1957). Paper chromatograms and electrophoretograms were counted in a Vanguard model 880 automatic chromatogram scanner. Sedimentation velocities were measured at -~. 2 Abbreviations used: DTT, dithiothreitol; CMC, S-carboxymethyl cysteine; WIP, waterinsoluble papain.
HARRISON
FIG. 2. Gel-diffusion react,ions between +yG fragments and a sheep anti-rabbit ?G antiserum. The ant,iserum (sheep 42) is in the center well; peripheral wells have the same materials as in Fig. 1. 59,780 or 52,640 rpm. Samples were dissolved in 0.1 fit glycine-0.01 12 sodium phosphate buffer at pH 7.3. q/G-Immunoglobulin was prepared from rabbit serum by precipitation with sodium sulfate (9), followed by chromatography on DEAE-cellulose (26), in 0.01 M sodium phosphate buffer at pH 7.3. Water-insoluble papain (WIP) was prepared by coupling mercuri papain t,o diazotized p-aminophenylalanine-leucine co-polymer, according to Cebra et al. (2). Preparation of the Fab fragment. Fab fragments were prepared by WIP-digestion of whole yG (2), followed by reduction in 0.1 !lf mercaptoethanol, and incubation at 4” for 3 days, during which time the bulk of the Fc fragment precipitated. The supernatant solution was dialyzed against sodium acetate bufler, pH 5.5 (0.02 111 in acetate) and chromatographed on CM Sephadex C25 equilibrated with the same buffer. I’reparation of the F(ab’)z fragment. Rabbit rG-immunoglot~uliu in pyridine acetate buffer, at pH 4.0 (0.1 Jf in acetate), was incubated with pepsin at room t,emperature for 24 hours; enzyme to substrate ratios ranging from 1:175 to 1:200 were used. The high molecular weight material was isolated by sodium sulfat,e precipitation (14). Further purification of this fragment is described under Results.
DISULFIDE
BONDS OF rG FRAGMENTS
Reduction and alkylation of yG, Fab, and F(ab’)z. rG, Fab, and F(ab’)z were reduced at pH 8.3-8.5 in various concentrations of 2-mercaptoethanol for 1 hour, as described in Results (Fig. 3), and alkylated by adding a quantity of iodoacetic acid equimolar to the mercaptoethanol, maintaining the pH between 8.3 and 8.5 by additions of NaOH. After 1 hour at room temperature, the reaction mixtures were dialyzed exhaustively against distilled water and lyophilized. When iodoacetic acid-lJ4C was used for alkylation, the alkylation reaction mixtures were fractionated on a 1.5 X 57-cm Sephadex G25 column equilibrated with 0.1 M NHdHCOz. Amino acid analyses. Protein and peptide samples were hydrolyzed for 24 hours in constantboiling HCl by the procedure of Moore and Stein (17). Ion-exchange chromatography and the ninhydrin reaction were done by a modification (11) of the method of Piez and Morris (23). The values of the amino acids were corrected for decomposition and incomplete hydrolysis by using factors
determined
‘711
from 24- and 72-hour hydrolyses
whole rG. When fragments
containing
S-carboxymethylcysteine were analyzed, the CMC content was also calculated from the ratio of cpm to OD~~O,,,~, using an E%, of 16.0 (Ref. 16). RESULTS
PuriJicahn of the F(ab’)g product of peptic digestion. The F(ab’)z fragment prepared by sodium sulfate precipitation still reacted with sheep antibody specific for the rabbit Fc fragment in double-diffusion in agar (20) (Fig. 1). Consequently, it was further purified by passage through carboxymethyl Sephadex C25 equilibrated with sodium acetate buffer, 0.02 M acetate, at pH 5.5. The F(ab’)z fragment was not bound to the ion exchanger under these conditions, and did not react with the anti-Fe antiserum. The bound material was eluted with 1 M
A YG/2 0 Fab’ 0 Fob
IO
0 Fob from F(ab’&
n CMC Peptides
9 8 7
0
0.02
of
KXabelled
0.04 0.06 0.08 0.10 MERCAPTOETHANOL
0.12 0.14 0.16 CONCENTRATION
0.16 0.20 (M)
FIG 3. S-Carboxymethylcysteine content of rG and fragments as a function of reducing agent concentration. Residues of CMC were calculated from amino acid analysis, based on an estimated molecular weight of 72,500 per half-rG molecule (15) and on an assumed molecular weight of 45,000 for Fab and Fab’.
712
MAGE TABLE
AND
I
SEDIMENTATION COEFFICIENTS OF yG AND F(ab’)z BEFORE AND AFTER TREATMENT WITH WATER INSOLUBLE PAPAIN .\ND/OR REDUCING AGENTS sm. w Material
7G WIP-treated F(ab’)z WIP-treated
yG F(ab’),
Unreduced
Dithiothreitol, o,ol w j
h’e~~~Pethanol, 0.14 M
6.3 6.3 4.7 4.7
6.0 3.3 3.2
6.1 3.7 3.0 3.1
sodium acetate buffer at pH 5.5, and reacted with the anti-Fc antiserum (Fig. 1). Another anti-rabbif rG antiserum of broader specificity reacted with the F(ab’)z fragment, and showed it to lack the Fc antigenic determinants present in the unfractionated digest (Fig. 2). CMC content of rG and fragments alkylated after reduction with %mercaptoethanol. In a series of experiments, rG and its fragments were reduced with mercaptoethanol at concentrations ranging from 0.01 to 0.14 M. In Fig. 3, CMC content of the reduced alkylated materials (which is equivalent to content of SH groups on reduction), is plotted vs. mercaptoethanol concentration during reduction. For both rG and ibs proteolytic fragments, the number of cysteine sulfhydryl groups increases with increased concentration of mercaptoethanol. -yG and F(ab’)z have a very similar content of CMC residues The Fab fragment, per half-molecule. whether prepared from rG direct,ly, or from the F(ab’)z fragment, appears to have from 1.5 to 3 fewer CMC residues (depending upon mercaptoethanol concentration during reduction) per assumed 45,000 molecular weight, than either the Fab’ fragment (per 45,000 MW), or the G per half-molecule (assumed 72,500 MW). Sedimentation velocity measurements of yG and the F(ab’)z fragment before and after WIP-treatment and/or reduction. The sedimentation coefficient of TG-immunoglobulin was not appreciably affected by reduction (with either mercaptoethanol or dithiothreitol) or by WIP-treatment, but on combined WIP-treatment and reduction, the
HARRISON
sedimentation coefficient fell to 3.7 S (Table I). The 4.7 S F(ab’)* fragment remained unchanged on WIP-t,reatment. On reduction of F(ab’,Jz, the sedimentation coefficient fell to 3.0-3.3 S, and WIP-treat’ed F(ab’)z had a value of 3.1-3.2 S on reduction. Water-insoluble papain digestion, reduction with mercaptoethanol, and alkylation of the F(ab’)z fragllzent. Purified 4.7 S F(ab’)n fragment was incubated with water-insoluble papain (2) for 10 minutes at room temperature, enzyme substrate ratio 1:250, in 0.03 M sodium phosphate buffer at pH 7.8, containing 0.002 J!1 EDTA. The enzyme was removed by centrifugation. The result,ant 4.7 S WIP-treated F(ab’)z fragment was reduced for 1 hour in 0.03 M mercaptoethanol and alkylated with a quantity of iodoacetic acid-l-14C, equimolar to the mercaptoethanol. The pH was maintained between 8.3 and 8.5 by additions of NaOH. After one hour at room temperature, the reaction mixture was fractionated on a 1.5 X 57-cm Sephadex G25 column equlibrated with 0.1 dl NHdHCOs. The elution diagram is shown in Fig. 4. Peak A contained a 3.1 S component which had the same reactivit,y toward sheep an&rabbit, rG antisera as undigested F(ab’)z fragment (see Figs. 1 and 2); Peak B, with a high ratio of radioactivity to absorbance at. 280 rnp, had approximately ${o of t,he total cpm in the first 2 peaks. High voltage paper electrophoresis of an aliquot of peak B, in pyridine acetate buffer, pH 3.7 (Ref. 8), showed it to contain a mixture of radioactive peptides. In order to be able to relate the radioactive label to S-carboxymet,hylcysteine content, it was necessary to verify that t’he alkylation with iodoacetate-14C was specific for cysteine sulfhydryl groups. Aliquots from peaks A and B were analyzed for radioactivity by paper electrophoresis and paper chromatography of their acid hydrolyzates. Only one radioactive component, was found and it had the mobility of S-carboxymet,hylcyst eine (Table II). The amino acid composition of peak A (Fab fragment) and of peak B (peptide mixture) is shown in Table III, columns 1 and 2, respectively. The peak B peptide mixture (column 2) is limited in amino acid
DISULFIDE
713
BONDS OF -/G FRAGMENTS
-c” 7000 v 6000
b
5000 4000
FIG. 4. Separation on a Sephadex G25 column of the products of reduction and alkylation of WIP-treated F(ab’)z. The vertical lines indicate the portions of peaks A and B pooled for amino acid analysis.
TABLE
II
COMPARISON WITH S -CARBOXYMETHYLCYSTEINE OF THE RADIOACTIVE COMPONENT IN ACID HYDROLYZATES OF RCM-WIP-TREATED
drolyzed aliquots of peak C showed no CMC, and traces of other amino acids totaling less than one tenth of the total amino acid residues present in peak B.
F(ab’h Radioactive component
Paper electrophoresis? Rlyaine Paper chromatography:” Rf
S-Carboxymethylcysteine
-0.26
-0.26
0.22
0.22
QWhatman No. 1 paper, 60 V/cm, 50 minutes, pyridine acetate buffer, pH 3.7. 1,Whatman No. 1 paper, ascending chromatography; butanol-acetic acid-water, 4: 1: 1. composition, and contains 1.1 CMC residues, compared with 1.7 CMC residues in the concomitantly produced Fab fragment (peak A, Fig. 4). In addition to CMC, the amino acids present in highest amounts in the peptide mixture were proline, leucine, and glutamic acid. Peak C contained the low molecular weight by-products of reduction and alkylation. Amino acid analyses of hy-
Amino Acid Analyses of Fab’ and Fab Column 3 of Table III shows the mean of 14 analyses of various preparations of F(ab’)z and Fab’, expressed as residues per Fab’ fragment of assumed 45,000 MW. Column 4 of Table III shows the mean of 13 analyses of several preparations of Fab, whether prepared directly from rG, or from F(ab’)z. The composition of Fab is also expressed as residues, based on the assumption of an alanine content equal to that of Fab’. Calculated on this basis, Fab would have a molecular weight of 43,600, which is higher than the molecular weight value of 40,700 reported by Pain (21). When compared on the basis of the same alanine content as Fab’, Fab has significantly less proline and leucine (p < .OOl) and possibly less glutamic acid (p = .013) than Fab’. For 11 of the remaining amino acids, the differences between Fab’
il4
MAGE
AND
HARRISON
TABLE GINO
ACID
COMPOSITION
III OF
yG
RABBIT
FRAGMENTS
Fib’
Compound id
Sh
-___ 36
sh
5 Residue differences (Fab’ - Fab)
FL
S-Carboxymethylcysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cystine/2
l&(1.7”’ 31.1 Gl.6 50.3 32.2 31.1 38.4 28.4 40.0 9.4
1.1 0.3 0.7 0.6 1.1 2.3 0.3 0.4 0.4 0.0
11.8f 31.4 60.3 50.5 32.7 34.1 39.1 28.1 40.8 4
2.3 1.0 1.9 3.4 0.8 2.5 1.5 0.8 2.7 -
10.5’ 31.1 GO.0 48.0 31.8 29.3 38.8 28.1 41.2 -
1.0 1.1 2.4 2.2 0.7 1.5 1.3 0.5 2.0 -
1.3, 0.3 0.3 2.5 0.9 4.8 0.3 O.oi -0.4 -
Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
2.1 11.0 26.5 18.0 11.9 16.5 2.5 8.9
0.0 0.1 1.1 0.1 0.1 0.4 0.0 0.0
2.3 11.5 28.0 18.2 12.0 17.1 2.8 9.6
0.2 1.2 0.7 1.0 0.7 0.4 0.4 0.5
2.2 11.5 26.4 18.1 12.0 16.6 2.8 9.3
0.2 0.7 0.8 0.6 0.4 0.6 0.4 0.3
0.1 0.0 1.6 0.1 0.0 0.5 0.0 0.3
a Residues, based on an assumed molecular weight of 45,000 for peak A + peak B. 6 Residue calculations for the peptide material are based on the CMC content. The value of 1.1 CMC residues in the peptide material is based on the 2:3 ratio of peak “B” cpm to peak “A” cpm. c The CMC value in parentheses is based on the ratio of cpm to OD280mp. d Mean of 14 analyses; included are analyses of both Fab’ and F(ab’),. The data are expressed as residues per Fab’ molecule of 45,000 MW. *Mean of 13 analyses; included are analyses of Fab from rG, and Fab from F(ab’)z. Residues are calculated on the basis of an alanine content equal to that of Fab’. J Because the sample includes unreduced preparations as well as preparations reduced under a variety of conditions, the values given are the sum of S-carboxymethylcystine and cystine/2. Q Included with S-carboxymethylcysteine. h Standard deviation. i By definition.
and Fab are less than 0.5 residue per univalent fragment. CMC content of fragments formed by WIPtreatment of F(ab’)z, preceded or followed by reduction with 0.01 M DTT, and alkylation with iodoacetic acid-l -14C. Several subsequent experiments, summarized in Table IV, show that from 1.1 to 1.2 residues of CMC were liberated as peptides, when WIP-treatment of F(ab’)z was preceded or followed by reduction with 0.01 2M DTT, alkylation with iodoacetic acid-1-‘JC, and separation on Sephadex G25 by the same procedure as that shown in Fig. 4. Reduction of the F(ab’)z fragment with 0.01 M DTT, followed by alkylation with a 1: 1 molar ratio of iodoacetic acid-l-l% to DTT
sulfhydryl, yielded an Fab’ fragment containing 5.9 CMC residues per assumed molecular weight of 45,000 with less than 0.1 CMC residue present in peptides (Experiment 1, Table IV). When F(ab’)z was treated with WIP and then re-isolated on Sephadex G25, it retained its sedimentation coefficient of 4.7 S; less than 1% of the total amino acid residues were present as peptides. Subsequent reduction and alkylation of this 4.7 S WIP-treated F(ab’)Z fragment yielded a 3.2 S Fab fragment containing 4.9 CMC residues per 45,000 MW, with a total of 1.1 residues of CMC present in peptides (Experiment 2, Table IV). When F(ab’)z was reduced with 0.01 M DTT, and alkylated with but a 3:4 ratio of iodoacetic acid-1-14C
DISULFIDE
BONDS
715
OF q/G FRAGMENTS
TABLE
IV
S-CARBOXYMETHYLCYSTEINE COXTENT OF THE PRODUCTS OF WATER-INSOLUBLE FRAGMENT DIGESTION AND/OR REDUCTION AND ALKYLATION OF THE F(ab’), 0.01 M DITHIOTHREITOL AND IODOACETIC ACID-I-X Alkyla-
CMC content of products after separation on Sephadex G25 (residues)b
tion
(ratio of moles of Experimerit No. lodoace- Starting material tate-‘C added to DTT sulfhydryljc
1 2
1:l 1:l
F(ab% F(ab’h
3 4 5
3:4 3:4
F(ab’)z CM-Fab’d F(ab’)n
PAPAIN WITH
Procedures’ CM-Fab’
Reduction and alkylation Treatment with WIP? followed by reduction and alkylation Reduction and alkylation Treatment with WIP Treatment with WIP, followed by reduction and alkylation
5.9(5.8)’
5.0(5.0) -
CM-Fab
CMpeptides
4.9(4.5)
0.07 1.1
(3.;” 3.5(3.6)
0.2 1.2 1.1
a WIP-digestion and Sephadex G25 chromatography were done as in the experiment illustrated in Fig. 4. b CMC content of the Fab’ and Fab fragments is calculated from duplicate amino acid analyses based on an assumed molecular weight of 45,000. Peptide CMC values are calculated from the ratio of total peptide cpm to Fab or Fab’ cpm. c Reductions were done with 0.01 -11 DTT, i.e., 0.02 M with respect to sulfhydryl. d The CM-Fab’ fragment was prepared in Experiment 3. 6 The WIP-treated F(ab’)s in this experiment was isolated on Sephadex G25 prior to reduction and alkylation. f CMC values in parentheses are calculated from the ratio of cpm to OD,,,,. 0 (-) not applicable. h Not measllred by amino acid analysis.
to DTT sulfhydryl (Experiment 3, Table IV), the resultant Fab’ fragment had 5.0 residues of CMC per 45,000 MW (as compared with 5.9 residues in Experiment 1, Table IV) with 0.2 residues of CMC present as peptides. Nonetheless, treatment of this Fab’ fragment with WIP, followed by passage through Sephadex G25, yielded an Fab fragment containing 3.9 CMC residues per 45,000 MW, with 1.2 residues of CMC present as peptides (Experiment 4, Table IV). Similarly, on WIP-treatment of F(ab’)z, followed by reduction with 0.01 M DTT and alkylation with a 3:4 molar ratio of iodoacetate to DTT sulfhydryl, 3.5 residues of CHIC were present in the resultant Fab fragment, and 1.1 residues of CMC were in peptides (Experiment 5, Table IV). DISCUSSION
The Fab fragment, on mild reduction and alkylation, has (depending on concentration of reducing agent) from 1.5 to 3 less residues
of S-carboxymethylcysteine than rG per half molecule or the univalent Fab’ fragment (Fig. 3). Insoluble papain treatment of F(ab’)z, when followed or preceded by mild reduction and alkylation with iodoacetic-14C acid, results in the liberation of 14C-labelled CMC-peptides totaling slightly more than 1 residue of CMC per Fab’ molecule of 45,000 MW (Table IV). Even though the CMC content of the concomitantly produced Fab fragment varied from 1.7 residues on very mild reduction (i.e., with 0.03 M mercaptoethanol), to 5.0 residues (with 0.01 M DTT), the total CMC content of the liberated peptides remained relatively constant (1.1 and 1.2 residues, respectively). In addition to the difference in CMC content, Fab contains significantly less proline and leucine than Fab’. Taken together, these findings lend quantitative support to the hypothesis that papain and pepsin may split on opposite sides of the disulfide bond between the heavy chains (7, 19). Inman
716
MAGE
AND
and Nisonoff (6) have st,rengthened this hypothesis with their finding that reduction and acidification of the Fc fragment from papain digestion of rG causes a halving of its molecular weight, indicating that the half-cystine residues that form the disulfide bond between the heavy chains may be in the Fe fragment, rather than in the Fab fragment. Alternatively, the data in the present study are equally compatible with the recent suggestion by Utsumi and Karush (28) that the heavy chains of rabbit yG are connected not by one, but by two disulfide bonds that cross each other. These authors postulate that insoluble papain may split the heavy chains between these bonds. According to this hypothesis, one mole of CMC per univalent fragment would be liberated as peptides when insoluble papain treatment of F(ab’)z is preceded or followed by mild reduction and alkylation. In the present study, 1.1-1.2 moles of CMC were liberated as peptides by these procedures. Indeed, these authors have recently reported that on soluble papain digestion of F(ab’)z in the presence of an extremely low concentration of reducing agent (0.001 M mercaptoethanol), the resultant 3 S fragments contained no CMC, whereas an unspecified amount of this constituent was present in their “low molecular weight product” (28). This hypothesis (i.e., of two interchain disulfide bonds that cross each other) offers an explanation for the failure of rG (2) or F(ab’)z (7) to fall in molecular weight on digestion with insoluble papain. Another possible arrangement of heavy chain disulfide bonds is that, after insoluble papain digestion of -rG or F(ab’)z, intrachain disulfide bonds of the heavy chains may connect the latent Fab fragments to the rest of the molecule. An additional finding of this study is that the F(ab’)s fragment from peptic digestion of +yG can be purified by the same method used to isolate the Fab fragment (i.e., chromatography on CM Sephadex C25). The purified F(ab’)s fragment no longer reacts in double diffusion in agar with antiserum specific for the Fe fragment (Fig. 1). This method of purification of F(ab’)z is an alternative to that of Jacquet and Cebra (7),
HARRISON
who used chromatography on Sephadex GlOO to purify the F(ab’)z fragment. The data in Fig. 3, showing CMC content of both reduced, alkylated rG and its proteolytic fragments as a function of reducing agent,, are in keeping wit,h the SH values found for undigested yG by Hong et. al. (5) and by Utsumi and Karush (27). The fragments prepared in this study were characterized by several criteria: Amino acid analyses of the F(ab’)z and Fab fragments (Table III) are in good agreement with the analyses of Mandy et. al. (14) and Porter (24), respectively. The ~20,~values of 4.7 S and 3.3 S for the F(ab’)z and Fab’ fragments (Table I), are similar to those reported by Mandy and Nisonoff (13), and the s20,wvalues of 4.7 and 3.1 S for unreduced and reduced WIP-treated F(ab’)z respectively, are comparable to the values found by Jacquet and Cebra (7). In considering the possible arrangements of disulfide bonds in rabbit rG, it should be noted that this protein may be heterogeneous with respect to lability of the disulfide bonding between the heavy chains (22). Furthermore, disulfide interchange may be a source of heterogeneity with respect to the specific pairing of the disulfide bonding between the latent fragments after WIP-digestion of rG. Cebra (3) and Jacquet and Cebra (7) have postulated that disulfide interchange may be responsible for t’he gradual formation of the ‘(Fragment I dime? found by them when WIP-treated rG was incubated with low concentrations of sodium dodecylsulfate. The concept of disulfide interchange following limited proteolysis, leading to a new arrangement of disulfide bonds, has been substantiated recently by Anfinsen (l), who has shown that, after limited proteolysis of a native protein, the native configuration is no longer thermodynamically most favored. Incubation of such an altered protein with a microsomal enzyme that catalyzes disulfide interchange, results in a rapid loss by the altered protein of its original conformation and of its activity. Experimental efforts now being made to examine cystine peptides and CMC peptides derived from the Fab, F(ab’)z and Fc
DISULFIDE
BONDS
fragments, may help to determine the nature of the disulfide bonding between the heavy chains, and of the bonds holding the latent fragments together after WIP digestion of rG or F(ab’)s. REFERENCES 1. ANFINSEN, C. B., Abstr. 150th Meeting Am. Chem. Sot., September 1965. 2. CEBRA, J. J., GIVOL, D., SILMAN, H. I., AND KATCHALSHI, E., J. Biol. Chem. 336, 1720 (1961). 3. CEBRA, J. J., J. Immunol. 92, 977 (1964). 4. CECIL, R., AND WAKE, R. G., Biochem. J. 82, 401 (1962). 5. HONG, R., DIXON, D. J., AND NISONOFF, A., Federation Proc. 24, Part I, 200, Abstr. No. 401 (1965). 6. INMAN, F. P., AND NISONOFF, A., Federation Proc. $4, Part I, 200, Abstr. No. 402. (1965). 7. JACQUET, IL, AND CEBRA, J. J., Biochemistry 4, 954 (1965). .&;4 8. KATZ, A. RI., DREYER, W. J., AND ANFINSEN, C. B., J. Biol. Chem. 234, 2897 (1959). 9. KEKWICK, R. A., Biochem. J. 34, 1248 (1940). 10. KINARD, F. E., Rev. Sci. Ins&. 28, 293 (1957). 11. KRICHEVSKY, M. I., SCHWARTZ, J., AND MAQE, M., Anal. Biochem. 12, 94 (1965). 12. MAGE, M. G., AND HARRISON, E. T., Abstr. 150th Meeting Am. Chem. Sot., September, 1966.
OF yG FRAGMENTS
717
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