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A method for pH-dependent selective reduction of disulfide bonds in human immunoglobulin M using heterocyclic compounds
286
BIOCHIMICAET BIOPHYSICAACTA
BBA 35561 A METHOD F O R p H - D E P E N D E N T S E L E C T I V E R E D U C T I O N OF D I S U L F I D E BONDS IN HUMAN I M M U N O G L O B U L I N M USING H E T E R O C Y C L I C COMPOUNDS
t;RITZ D O L D E R W.H.O. International Reference Center for Irnmunoglobulins, Institut de Biochimie, Universit~ de Lausanne, Lausanne (Switzerland) (Received December I2th, 1969)
SUMMARY
The ability of various substituted pyridines and pyrimidines to dissociate immunoglobulin M (IgM) from patients with Waldenstr6m's disease was investigated. 2-Thiopyridine (2-TP) dissociated IgM into subunits with a molecular weight of 2o3 ooo. These subunits did not split into their constituent polypeptide chains at acid pH and were therefore regarded as having intact interchain disulfide bonds. At physiological p H values (8.o) first-order kinetic constants k for this cleavage ranged from 3.5" lO-6 to 1.6. IO -5 sec -1 at 25 °, i.e. IOOOtimes smaller than the values obtained with the routinely used aliphatic thiols such as dithiothreitol (DTT). The dissociation with 2-TP was found to be highly p H dependent, in that higher yields and increased reaction rates were achieved at alkaline p H values ranging from 9.0 to 12.o. This allows selection of an appropriate p H value for this dissociation of a given protein substrate or selection of a particular reaction rate of the dissociation, if the substrate is not pH sensitive. Attempts to S-carboxymethylate 2-TP-treated IgM subunits with iodoacetamide invariably failed, thus confirming that the interchain disulfides were left intact. The IgM subunits possessed antigenic determinants characteristic of both # and/¢ chains and were immunochemieally identical with subunits obtained by reduction of IgM with DTT. The lack of S-carboxymethylated eysteine residues is explained by a disulfide interchange mechanism, in which intersubunit disulfide bonds are converted into intrasubunit inter-# disulfide bonds.
INTRODUCTION
Since MILLER AND METZGER1,2 have reported on the distribution of SS bonds in immunoglobulin M (IgM), part cular efforts have been made to determine the exact Abbreviations: IgM, immunog,obulin M in the native state; IgMs, subunits of IgM prepared by reduction with aliphatic thiols and containing cleaved interchain disulfide bonds; DTNB, dithiobisnitrobenzoic acid; CM-cysteine, S-carboxymethylated cysteine; 2-TP, 2-thiopyridine; DTT, dithiotreitol. Biochim. Biophys. Acta, 207 (197 o) 286 296
p H - D E P E N D E N T REDUCTION OF
IgM
287
number of SS bonds which account for intersubunit, interchain, and intrachain bonding, respectively. Various authors have found that out of a total of 70-9 ° SS bonds in the IgM molecule 24 disulfides account for all of the interchain bonding, whereas the remaining disulfides are apparently intrachain bonds, located in either the heavy (#) or the light (K, ~) polypeptide chains. Special interest has been focused, furthermore, on determining how m a n y of these 24 interchain disulfides account for intersubunit linking, in other words, have to be cleaved to obtain a complete subunit dissociation of IgM. Interest in the subunit linking in IgM has also grown due to the reports that low-molecular-weight IgM occurs naturally in some disease states in man. In consequence, IgM subunits prepared with minimal SS cleavage would appear to offer an ideal material for comparing high-molecular-weight IgM with low-molecular-weight IgM and could possibly contribute to the eventual elucidation of the pathways of biosynthesis and metabolism of IgM. These investigations deal with some features of the dissociation of human Waldenstr6m IgM into minimally denatured subunits b y means of different heterocyclic compounds of the pyridine and pyrimidine class. MATERIALS AND METHODS
Preparation of IgM M macroglobulins of Waldenstr6m from four macroglobulinemic sera (Man, Eld, Lan, Hul) were prepared by four subsequent euglobulin precipitations against distilled water. The final precipitate was redissolved in 0.25 M Tris-NaC1 buffer (pH 8.o) and filtered through columns of Sephadex G-2oo. The final IgM preparations were homogeneous in terms of ultracentrifugal and immunochemical analysis.
Dissociation of IgM with heterocyclic compounds Appropriate concentrations of the heterocyclic compounds* were added to a 1% protein solution in the following different buffer solutions : for p H 5 and 6 solutions of glycine-HC1 (o.I M) were chosen, for pH 7 and 8 Tris-NaC1 solutions of I = 0.25, and for p H 9-12 glycine-NaOH solutions of o. I M. Care was taken to avoid air contact of the system, by either layering the reaction mixture with paraffin oil or b y flushing it with N 2.
Carboxymethylation procedure Three different methods were used for S-carboxymethylation of protein-bound - S H groups formed during the dissociation process. (I) lodoacetamide was added in a lO% excess over the molarity of the dissociating reagent. The p H was adiusted at the initial value by adding appropriate amounts of solid Tris to the mixture, and the reaction was allowed to continue for 15 rain. (2) In order to ensure a previous reduction of the molarity of the dissociating agent, I ml of the reaction mixture containing IO mg of protein was dialyzed against 2 1 of a I mM solution of the heterocyclic compound for 2 h. Alkylation was then performed by adding o.I ml of a 16 mM solution of * All c h e m i c a l s were p u r c h a s e d f r o m F l u k a Inc., Buchs, Switzerland, a n d were of r e a g e n t g r a d e quality. 2-Thiopyridine, however, h a s been recrystallized 3 t i m e s f r o m light p e t r o l e u m benzene.
Biochim. Biophys. Acta, 2o 7 (197 o) 286-296
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v, I)OLDER
iodoacetamide to give a final concentration of 1.6 mM iodoacetamide in the reaction mixture. In this case, alkylation was allowed to continue for 9o min at room temperature. (3) Alternatively, the reaction mixture was passed through a column of Sephadex G-25 which had been previously equilibrated with a I mM solution of the heterocyclic compound. The protein fractions were then concentrated by evaporation and alkylated by adding an equivalent amount of 1.6 mM iodoacetamide. In all three cases, samples were subsequently dialyzed against Tris-NaC1 buffer for 24 h in order to eliminate all low-molecular-weight reagents.
Titration of protein-bound SH groups The titration was performed using dithiobisnitrobenzoic acid (DTNB) following the modification of MILLER AND METZGER1,2 of the original method of ELLMANN3.
Isolation of IgM subunits from partially dissociated IgM Material sedimenting at 8 S was separated from unreacted I9-S material by several subsequent passages of the reaction mixtures through columns of Sephadex G-2oo equilibrated with 0.25 M Tris-NaC1 buffer (pH 8.0) (Fig. 4). IgMs* control material was prepared with I mM dithiothreitol (DTT) and alkylated with an equivalent amount of 1.6 mM iodoacetamide.
Characterization of IgM subunits Analysis of amino acid composition. Analysis was performed on a Beckman Model 12o C amino acid analyzer. For optimal separation of S-carboxymethylcysteine, the modification of the basic technique described by MORRIS AND INMAN4 was used. Polyp@tide chain composition of IgM subunits. The different subunit samples were dialyzed overnight against formate buffer (pH 3.50, I = 0.02) and passed subsequently on a column of Sephadex G-Ioo equilibrated against the same buffer. The different fractions were tested for /,- and K-chain determinants by Ouchterlony double-diffusion analysis. Determination of molecular weights. Molecular weights were determined by the approach to sedimentation equilibrium method of ARCHIBALD5 on 1% protein samples previously dialyzed against a 0.15 M KC1 solution. optical rotatory dispersion measurements. The optical rotatory dispersion of the different subunits was measured between 210 and 400 m# on a Perkin-Elmer P 22 spectropolarimeter, using cells of 2- and 0.2-cm path length. The observed rotations as were converted to specific rotations [az] and then into [R']z by the formulas: (~A " IOO
c (g/Ioo ml) [R']~. .
3 3t . . . [a]~. n2 + 2 IOO
where the refractive index, n, was taken as 1.37 below 260 m# and as 1.33 from 260 to 4o0 m#. A mean residue weight, M, of lO8 was used for IgM and its subunits. Subunits of IgM prepared by reduction with aliphatic thiols and containing cleaved interchain SS bonds.
Biochim. Biophys. Acta. 207 (i97 o) 286-296
pH-DEPENDENT
REDUCTION
OF IgM
289
Synthesis of 2-pyridyl-fl-cysteinyl disulfide, CsHloN202S~ All a t t e m p t s to synthesize the mixed disulfide of cysteine and 2-thiopyridine (2-TP) at physiological conditions invariably failed. Only under strong alkaline or acid conditions could it be produced by the following reaction (Pyr = C~HaN-) : Pyr-SH
+ H O O C _ C H N H 2 _ C H 2 _ _ S S _ C H 2 _ C H N H 2 _ C O O H - - - ~O HH + 'Pyr-SS-CH2-CHNH,.-COOH
+ HSH2C-CHNH2-COOH
After fractionation of the reaction mixture on columns of Dowex 50 W-X8 and by high-voltage paper electrophoresis, the mixed disulfide was identified by means of performic acid oxidation and subsequent chromatographic characterization of the oxidized cleavage products. RESULTS
Dissociation of IgM with heterocyclic compounds IgM (Man) was treated with various concentrations of various derivatives of pyridine and pyrimidine in the hope of recovering IgM subunits having their inter/z-chain and inter-/zK-chain SS bonds intact. Table I shows the effect of 0.2 M concentrations of different heterocyclic compounds on IgM. The only reasonable yields of subunit material were obtained with 2-TP, a sulfur derivative of pyridine known to exist in two tautomeric forms. Only trace amounts of subunit material were obtained with other derivatives, and, in particular, the unsubstituted pyridine did not result in subunit formation. TABLE I DISSOCIATING EFFECT OF DIFFERENT HETEROCYCLIC COMPOUNDS ON MAN IGM AT p H 8.0
Substance
2,4-Dithi°pyrimidine Thiouracil 2 - T h i o p y r i d i n e (2-TP) 2-Pyridone (2-hydroxypyridine) 4-Pyridone (4-hydroxypyridine) Pyridine
Number of tautomeric forms
Yield of I g M subunits (%) at p H 8.0 and 25 °)
4 4 2
< 2 %2 2o-24
2
Trace
2 -
Trace -
(0.2 M reagent, 24-h dissociation
Substrate dependency of the kinetics of 2-TP reduction of IgM at physiological pH Since a 0.2 M concentration of 2-TP resulted in the highest yields of slowsedimenting fragments of IgM, this reagent was chosen for further investigations. The rate of formation of material sedimenting at approx. 8 S and the rate of disappearance of the I9-S component of four different IgM native preparations were studied. In all cases, no components sedimenting between 19 and 8 S were observed. When the logarithm of the residual I9-S component is plotted against time of treatment with 2-TP, a straight line is obtained with all four different Waldenstr6m macroglobulins during the first 24 h. This indicates that the formation of subunits is a first Biochim. Biophys. Acta, 207 (197 o) 286-296
290
F. DOLDER Ela
I~(18S)
100- S
pH11 ,---
HuL
~I0
°--o
#-16
o--o
~5
60
~
60
~
"
n -55
~
~
96
108
h
12
~
36
48
60
"/'2
hours
Fig. i. The rate of breakdown of IgM by 2-TP at pH 8.0 and 25 ° depending on the particular protein substrate (protein samples Man, Lan, Hul, Eld). Fig. 2. The rate of formation of IgM subunits by 2-TP depending on the pH value of the systenl (protein sample Man).
order rate reaction. The rate constants (sec-1) were calculated to be of the order of 3.23" IO-~ for Man, 8.80. IO-n for Lan, 1.19 • lO -5 for Hul, and 1.65 • lO-5 for Eld at pH 8.0 and 25 °. It is a striking feature, however, that these values are smaller by a factor of IOOOthan the rate constants calculated for reductions with mercaptoethanol or similar aliphatic thiols n. The rate constants were found to be different for each protein, although they were of similar order. This finding is not unexpected in view of the known heterogeneity among Waldenstr6m proteins ~. The procedure of alkylation, however, did not essentially affect the rate constant or the yield of subunit material. Thus, the yield of subunit material levelled off at at 35-4o% of the total protein material in sample Man, at 7o-8o% in sample Eld, regardless of the method of alkylation.
pH dependency of the dissociation kinetics of IgM with 2-TP Since 2-TP is known to exist in a pH-dependent tautomerism equilibrium 7, dissociation experiments on Man macroglobulin were performed at different p H levels ranging from 5 to 12. The results in Fig. 2 indicate that the dissociation properties of 2-TP depend in fact on the p H value of the protein substrate solution in the expected way. Whereas at p H 5.0 no formation of 8-S material took place, the rate of formation of IgM subunits increased with increasing p H values. The m a x i m u m speed of the reaction was achieved at pH II.O. In that pH region, the first-order rate constants are comparable, though still 2-4 times smaller, with the reaction rates achieved with the commonly used aliphatie thiols like DTT. Higher p H values were not suitable for this investigation, since at p H 12.o IgM (Man) dissociated spontaneously into a polydisperse system of smaller fragments. Control samples were subjected to the identical conditions omitting the 2-TP. IgM (Man) was entirely stable at all p H values used in this investigation even after a prolonged incubation of lO6 h.
Attempts to S-carboxymethylate cysteine residues in 2-TP-treated lgM subunits Although the only heterocyclie compound able to dissociate IgM with reasonable yields was a thiol, 2-TP, only trace amounts of protein-bond - S H groups were demonBiochim. Biophys. Acta, 207 (I97 o) 286-296
pH-DEPENDENT REDUCTION OF IgM
291
8
0.2M 2-TP
o.
•
:
.
t
*l
Fig. 3. Rate of f o r m a t i o n of protein - S H g r o u p s in IgM (Man) u p o n t r e a t m e n t with o.ooi M D T T and 0.2 M 2-TP. Fig. 4- Ultracentrifugal analysis of a purified 8-S p r e p a r a t i o n f r o m dissociation of the p r e p a r a t i o n Man w i t h 2-TP. The picture was t a k e n 48 min after the r o t o r had reached c o n s t a n t speed (63 56o rev./min).
strable during the reaction with IgM ; whereas the control material reduced with I mM D T T yielded about 8- 9 - S H groups per subunit formed (Fig. 3) as well as 8- 9 residues of S-carboxymethylated cysteine (CM-cysteine) (Table III), which is consistent with the model of 24 interchain SS bonds per mole of IgM 1,~. This finding was confirmed by the complete failure to S-carboxymethylate the cysteine residues in any of the subunits produced b y 2-TP treatment as evidenced by amino acid analysis (Table III). These findings strongly suggest that during the course of the dissociation process the interchain disulfides in 2-TP-treated IgM subunits have been left intact. In some but not all experiments, a third protein fraction apart from the I9-S and 8-S material eluting over a broad range after the IgM subunit on Sephadex G-2oo, but accounting for only 2 % of the total protein material, was detected. This fraction (III) did not contain CM-cysteine residues either, which rules out the possibility that this small fraction could account for the subunit linking in IgM. TABLE II SOME PHYSICOCHEMICAL PROPERTIES OF I G M SUBUNITS (MAN)
All experiments were staged o n i ~ o protein solutions in o.15 M KC1 previously equilibrated with the same buffer. 3Ira, calculated f r o m the meniscus of the ultracentrifuge cell; Mb, calculated from the b o t t o m of the ultracentrifuge cell.
Mol. wt. and sedimentation rate
,~I m Mb Av. S2o,w (cf. Fig. 5)
IgM8 treated with D T T
178 ooo 174 5oo 176 ooo 7.14 S
I g M subunits treated with 2 - T P A lkylation
A Ikylation
(I)
(2)
2o8 ooo 198 ooo 203 ooo
198 500 211 5oo 2o 5 ooo 7.92 S
Biochim. Biophys. Acta, 207 (197 o) 286-296
292
u. DOLDER
80-
o;o x
~c
o
d, Fig. 5. C o n c e n t r a t i o n d e p e n d e n c i e s of t h e s e d i m e n t a t i o n r a t e of Ma n IgM s u b u n i t s ; © - - © , IgMs r e d u c e d w i t h o.ooi M D T T ; A - - A , IgMs r e d u c e d w i t h o. i M m e r c a p t o e t h a n o l a n d a l k y l a t e d w i t h 2 mM i o d o a c e t a m i d e ; O---O, IgM s u b u n i t s t r e a t e d w i t h o.2 M 2-TP.
Formation of the mixed disulfide 2-pyridyl-fl-cysteinyl disulfide It was virtually excluded that 2-pyridyl-fl-cysteinyl disulfide could have been formed under the physiological conditions used in the preparation of 2-TP subunits of IgM (see MATERIALS AND METHODS). Furthermore, no chromatographic evidence was found for the presence of that mixed disulfide on the basis of the amino acid analysis date of the sample hydrolysates.
Physicochemical characterization of 2-TP-treated IgM subunits The data in Table I I and in Fig. 5 indicate that all the subunits prepared with 2-TP and then "alkylated" display rather high sedimentation rates compared with T A B L E 111 CM-cYSTEINE
ANALYSIS
OF IGM
IgM protein
Mode of dissociation
Man
Native I m M DTT, I h, 25 ° 0.2 M 2-TP, 48 h ' * 8-S s u b u n i t s Fraction III
Hul
Native I mM DTT, I h, 25 ° 0.2 M 2-TP, 48 h 8-S s u b u n i t s Fraction III
SUBUNITS
Molesof CM-cysteine per mole of IgMs* 8.45
8.34
* These v a l u e s refer to a m e a n m o l e c u l a r w e i g h t of t h e p e p t i d e n i o i e t v of 158 ooo in IgMs. ** 2 - T P - t r e a t e d s u b u n i t s of IgM h a v e b e e n a l k y l a t e d a c c o r d i n g t o P r o c e d u r e (i) as i n d i c a t e d in
MATERIALS
AND METHODS.
Biochim. Biophys. Acta, 207 (197 o) 286-296
pH-DEPENDENT
R E D U C T I O N OF
IgM
293
the subunits with intact intrasubunit disulfides isolated by MORRIS AND INMAN4. Reduction with 2-TP resulted in the formation of subunits of molecular weights of 203 ooo and 205 ooo, respectively, whereas those subunits obtained by D T T treatment had significantly lower molecular weights of the order of 176 ooo (refs. 6, 8). Since 2-TP-treated IgM subunits have sedimentation rates of 7.95 S at infinite dilution, they are physicochemically similar to those subunits described b y SUZUKI AND DEUTSCH6 which showed also sedimentation coefficients of around 8 S. The essential difference between these two species lies in the fact that the 8-S subunits described by SUZUKI AND DEUTSCH6 were readily alkylated to yield smaller subunits sedimenting at 7 S, whereas 2-TP-treated IgM subunits could not be alkylated. A representative optical rotatory dispersion spectrum for I9-S IgM (Man) and several of its subunits is shown in Fig. 6. A single Cotton effect minimum is present at 233 m y and a weak but consistent Cotton effect is seen in the region of aromatic amino acid absorption. Within experimental error the spectra of native IgM and its subunits are identical.
Antigenic analysis of 2-TP-treated IgM subunits In double diffusion tests a line of identity was observed between all three 0.3 0.2
03 0
Onti-~i
0[
Clr~i-y
/~'iI /
i ~
U i'~/
...... ~,lve Man - - 2-TP ) - - - DTT .')recluceO
%',fl
--
.Ill
=
r4E
L/ 200
65 m[ -
I lonti-~(
40
I t~--.
65 rat
I anti-x:
0.1
f
-2~
l
~0"21
i
-I000
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0
250
3~0 Amp
350
40 a~i-)~
65 rat
--- v.......~ctr'#.i- ~.
F i g . 6. O p t i c a l r o t a t o r y d i s p e r s i o n o f : , M a n I g M ; -- -- , I g M s r e d u c e d w i t h I m M D T T ; - - • - - , I g M s r e d u c e d w i t h o. I M m e r c a p t o e t h a n o l (ME) ; - - , I g M s u b u n i t s d i s s o c i a t e d w i t h 2 - T P . T h e s p e c t r a h a v e b e e n m e a s u r e d in T r i s N a C 1 b u f f e r (I = 0 . 2 5 , p H 8.0) in cells w i t h a p a t h l e n g t h o f 0 . 0 2 d m ( 2 1 5 - 2 6 o m/A) a n d 0.2 d m ( 2 6 0 - 3 5 0 m , , ) . F i g . 7. C h a i n s e p a r a t i o n e x p e r i m e n t s o n d i f f e r e n t I g M s u b u n i t s o f p r o t e i n p r e p a r a t i o n M a n . C o n d i t i o n s a r e i n d i c a t e d i n MATERIALS AND METHODS. i - c m l i g h t p a t h . A. I g M s o b t a i n e d b y r e d u c t i o n o f I g M w i t h I m M D T T ; B. I g M s u b u n i t s o b t a i n e d b y d i s s o c i a t i o n o f I g M w i t h 2 - T P ; C. I g M s u b u n i t s f r o m B b u t r e d u c e d in a s e c o n d s t e p w i t h i m M DTT.
Biochim. Biophys, Acta, 2 0 7 (197 o) 2 8 6 - 2 9 6
F. DOLDER
294
• 2 2-TP
ix
r-t:f-, t',
.2 2-TP
/\
II •
22TP
"2
2-TP
"22-TP
r-It--, Fig. 8. Diagrammatic representation of a disulfide exchange reaction upon treatment of IgM with 2-TP. Details are given in DISCUSSION.2-DPS = 2-dipyridine disulfide.
compounds IgM native, IgMs, and 2-TP-treated IgM subunits, when tested against specific anti-# and anti-K antisera. Thus, 2-TP-treated subunits retained at least a portion of their #-chain and K-chain antigenic determinants. The IgM, IgMs prepared with DTT, and the 2-TP IgM subnnits were examined by immunoelectrophoresis in agarose plates. Antiserum against whole human serum and specific anti-/, antisera revealed only one single line of precipitation. This line was cathodically displaced in the case of both IgMs and 2-TP-treated IgM subunits as compared to the native I9-S preparation. Resistance of I g M subunits to acid treatment The purified 8-S material from protein preparation Man was dialyzed exhaustively against 0.02 M formate buffer (pH 3.50). lO-15 mg of dialyzed material were passed through a Sephadex G-ioo column equilibrated against the same formate buffer, and the different fractions were assayed for/~- and K-chain determinants by double-diffusion analysis. The control material consisting of DTT-treated IgMs eluted in two peaks. The first and larger peak contained #-chain determinants only, the second peak K-chain determinants only (Fig. 7A). The 2-TP-treated subunit material eluted as one single peak still containing both #- and K-chain determinants (Fig. 7B). However, when 2-TP-treated subunits were reduced in a second step with I mM D T T and subsequently dissociated with formate buffer, an almost identical elution pattern resulted as encountered with the original one-step DTT-treated IgMs (Fig. 7 C) which gives additional evidence that the 2-TP-treated IgM subunit contained intact intrasubunit SS bonds. DISCUSSION
Previous studies on the dissociation of IgM with aliphatic thiols such as mercaptoethanol or dithiothreitol have shown that the cleavage of 24-25 SS bonds results not only in a complete splitting into subunits but is also sufficient to separate subunits into their constituent polypeptide chains without any further reducing process1,% Biochim. Biophys. Acta, 207 (197o) 286 296
pH-DEPENDENT REDUCTION OF IgM
295
Hence, it has been argued that these 24 disulfides per IgM, or five per IgMs, account for all of the interchain bonding in the IgM molecule 1. A t t e m p t s to determine whether fewer than these 24 disulfides could be cleaved and nevertheless result in a complete dissociation of IgM into subunits have previously been discouraging 1. Encouraging results, however, have been obtained in this field by MORRIS AND INMAN4, who were able to reduce IgM into subunits with O.Ol5 M mercaptoethylamine, which invariably yielded two residues of CM-cysteine per subunit. Recently, a selective reduction of IgM was described by BEALE AND FEINSTEIN9 and by BEALE AND BUTTRESS 1°, who were able to dissociate IgM into its subunits by means of low concentrations of dithiothreitol, ranging from 0.125 mM to 0.275 mM. The data reported here m a y be accounted for in the following way: 2-Thiopyridine reduces immunoglobulin M in a selective way leaving intrasubunit disulfide bonds intact. The procedure results in the formation of IgM subunits which display molecular weights of around 200 000 and are identical in their polypeptide chain composition with DTT-reduced IgMs, as judged by immunochemical analysis. The interchain SS bonds of 2-TP-treated IgM subunits can be split by a subsequent reduction with stronger reagents such as DTT. Since it is virtually ruled out that 2-TP forms mixed disulfides with cysteine residues of the protein substrate, the failure to alkylate cysteine residues of 2-TPtreated IgM subunits m a y be explained as follows: The oxidized form of 2-TP, dipyridine disulfide, is known to be a strong oxidizing reagent for protein - S H groups 7. Hence, it m a y be assumed that protein SH groups released during the treatment of IgM with 2-TP are reoxidized by means of the equivalent amount of dipyridine disulfide formed simultaneously. Since this oxidation process is known to be fast 7, it should be locally restricted to the site of the initial protein SS cleavage. It seems conceivable, therefore, that intersubunit disulfide bonds might be converted in this oxidation step into intrasubunit inter-#-chain disulfides, thereby giving rise to an equivalent amount of 8-S IgM subunits (Fig. 8). In this case, one would actually observe the overall equilibrium reaction between reoxidation of the - S H form of IgM subunits (II) into the initial I9-S sedimenting IgM (I) and reoxidation into the SS form of the 8-S subunit (IIl). In a general way, 2-thiopyridine offers interesting properties for the cleavage of SS bonds in proteins. At physiological p H its weak reducing properties result in a selective reduction of the most reactive SS bonds in a given protein substrate. On the other hand, to explain the significant pH dependency of its dissociating properties, one has to consider that 2-TP displays a tautomerism between - S H and - S speciestn. This tautomerism favors form I I at neutral pH, whereas acid or alkaline p H ' s shift the equilibrium to form I (ref. II).
R Acid and alkaline pH
Neutrol pH
Since only form I is appropriate for reduction of SS bonds, one should theoretically expect an increase in the p H value of the reaction mixture to result in the increase in the reaction rate as reported in our experiments (Fig. 2). This significant p H deBiochim. Biophys. Acta, 207 (197 o) 286~296
296
E. DOLDER
pendency makes 2-TP a more versatile reagent for the controlled dissociation of SS bonds in proteins. It enables one to choose an optimal pH value for dissociation of a particular protein substrate or to select a definite reaction rate or a desired yield of fractionated material by varying the pH value of the system. ACKNOWLEDGMENTS
I gratefully acknowledge the constant advice and many helpful discussions of Professor H. Isliker and Professor D. S. Rowe. Plasma samples and specific antisera were a generous gift from Professor D. S. Rowe. This investigation was supported in part by U.S. Army Grant No. DAJA 37-68-C-oi36. REFERENCES I 2 3 4 5 6 7 8 9 IO II
F. MILLER AND H. METZGER, J. Biol. Chem., 240 (I965) 474 o. F. MILLER AND H. METZGER, J. Biol. Chem., 240 (1965) 3325. G. L. ELLMANN, Arch. Biochem. Biophys., 82 (1959) 7 o. J. E. MORRIS AND F. P. INMAN, Biochemistry, 7 (196,8) 2851. W. J. ARCHIBALD, J. Phys. Colloid Chem., 51 (1947) 12o4. T. SuzuKI AND H. F. DEUTSCH, J. Biol. Chem., 242 (1967) 2725 . H. GRASSETTI AND B. MURRAY, Arch. Biochem. Biophys., 119 (1967) 41. F. W. PUTNAM, M. KOZURU AND C. W. EASLEY, .[. Biol. Chem., 242 (1967) 2435. D. BEALE AND A. FEINSTEIN, Biochem. J., 112 (1969) 187. D. BEALE AND N. BUTTRESS, Biochim. Biophys. Acta, 181 (1969) 25 o. B. MARKWALDER, Chem. Ber., 33 (19 °°) I557.
Biochim. Biophys. Acta, 207 (197 o) 286-296
BIOCHIMICAET BIOPHYSICAACTA
BBA 35561 A METHOD F O R p H - D E P E N D E N T S E L E C T I V E R E D U C T I O N OF D I S U L F I D E BONDS IN HUMAN I M M U N O G L O B U L I N M USING H E T E R O C Y C L I C COMPOUNDS
t;RITZ D O L D E R W.H.O. International Reference Center for Irnmunoglobulins, Institut de Biochimie, Universit~ de Lausanne, Lausanne (Switzerland) (Received December I2th, 1969)
SUMMARY
The ability of various substituted pyridines and pyrimidines to dissociate immunoglobulin M (IgM) from patients with Waldenstr6m's disease was investigated. 2-Thiopyridine (2-TP) dissociated IgM into subunits with a molecular weight of 2o3 ooo. These subunits did not split into their constituent polypeptide chains at acid pH and were therefore regarded as having intact interchain disulfide bonds. At physiological p H values (8.o) first-order kinetic constants k for this cleavage ranged from 3.5" lO-6 to 1.6. IO -5 sec -1 at 25 °, i.e. IOOOtimes smaller than the values obtained with the routinely used aliphatic thiols such as dithiothreitol (DTT). The dissociation with 2-TP was found to be highly p H dependent, in that higher yields and increased reaction rates were achieved at alkaline p H values ranging from 9.0 to 12.o. This allows selection of an appropriate p H value for this dissociation of a given protein substrate or selection of a particular reaction rate of the dissociation, if the substrate is not pH sensitive. Attempts to S-carboxymethylate 2-TP-treated IgM subunits with iodoacetamide invariably failed, thus confirming that the interchain disulfides were left intact. The IgM subunits possessed antigenic determinants characteristic of both # and/¢ chains and were immunochemieally identical with subunits obtained by reduction of IgM with DTT. The lack of S-carboxymethylated eysteine residues is explained by a disulfide interchange mechanism, in which intersubunit disulfide bonds are converted into intrasubunit inter-# disulfide bonds.
INTRODUCTION
Since MILLER AND METZGER1,2 have reported on the distribution of SS bonds in immunoglobulin M (IgM), part cular efforts have been made to determine the exact Abbreviations: IgM, immunog,obulin M in the native state; IgMs, subunits of IgM prepared by reduction with aliphatic thiols and containing cleaved interchain disulfide bonds; DTNB, dithiobisnitrobenzoic acid; CM-cysteine, S-carboxymethylated cysteine; 2-TP, 2-thiopyridine; DTT, dithiotreitol. Biochim. Biophys. Acta, 207 (197 o) 286 296
p H - D E P E N D E N T REDUCTION OF
IgM
287
number of SS bonds which account for intersubunit, interchain, and intrachain bonding, respectively. Various authors have found that out of a total of 70-9 ° SS bonds in the IgM molecule 24 disulfides account for all of the interchain bonding, whereas the remaining disulfides are apparently intrachain bonds, located in either the heavy (#) or the light (K, ~) polypeptide chains. Special interest has been focused, furthermore, on determining how m a n y of these 24 interchain disulfides account for intersubunit linking, in other words, have to be cleaved to obtain a complete subunit dissociation of IgM. Interest in the subunit linking in IgM has also grown due to the reports that low-molecular-weight IgM occurs naturally in some disease states in man. In consequence, IgM subunits prepared with minimal SS cleavage would appear to offer an ideal material for comparing high-molecular-weight IgM with low-molecular-weight IgM and could possibly contribute to the eventual elucidation of the pathways of biosynthesis and metabolism of IgM. These investigations deal with some features of the dissociation of human Waldenstr6m IgM into minimally denatured subunits b y means of different heterocyclic compounds of the pyridine and pyrimidine class. MATERIALS AND METHODS
Preparation of IgM M macroglobulins of Waldenstr6m from four macroglobulinemic sera (Man, Eld, Lan, Hul) were prepared by four subsequent euglobulin precipitations against distilled water. The final precipitate was redissolved in 0.25 M Tris-NaC1 buffer (pH 8.o) and filtered through columns of Sephadex G-2oo. The final IgM preparations were homogeneous in terms of ultracentrifugal and immunochemical analysis.
Dissociation of IgM with heterocyclic compounds Appropriate concentrations of the heterocyclic compounds* were added to a 1% protein solution in the following different buffer solutions : for p H 5 and 6 solutions of glycine-HC1 (o.I M) were chosen, for pH 7 and 8 Tris-NaC1 solutions of I = 0.25, and for p H 9-12 glycine-NaOH solutions of o. I M. Care was taken to avoid air contact of the system, by either layering the reaction mixture with paraffin oil or b y flushing it with N 2.
Carboxymethylation procedure Three different methods were used for S-carboxymethylation of protein-bound - S H groups formed during the dissociation process. (I) lodoacetamide was added in a lO% excess over the molarity of the dissociating reagent. The p H was adiusted at the initial value by adding appropriate amounts of solid Tris to the mixture, and the reaction was allowed to continue for 15 rain. (2) In order to ensure a previous reduction of the molarity of the dissociating agent, I ml of the reaction mixture containing IO mg of protein was dialyzed against 2 1 of a I mM solution of the heterocyclic compound for 2 h. Alkylation was then performed by adding o.I ml of a 16 mM solution of * All c h e m i c a l s were p u r c h a s e d f r o m F l u k a Inc., Buchs, Switzerland, a n d were of r e a g e n t g r a d e quality. 2-Thiopyridine, however, h a s been recrystallized 3 t i m e s f r o m light p e t r o l e u m benzene.
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v, I)OLDER
iodoacetamide to give a final concentration of 1.6 mM iodoacetamide in the reaction mixture. In this case, alkylation was allowed to continue for 9o min at room temperature. (3) Alternatively, the reaction mixture was passed through a column of Sephadex G-25 which had been previously equilibrated with a I mM solution of the heterocyclic compound. The protein fractions were then concentrated by evaporation and alkylated by adding an equivalent amount of 1.6 mM iodoacetamide. In all three cases, samples were subsequently dialyzed against Tris-NaC1 buffer for 24 h in order to eliminate all low-molecular-weight reagents.
Titration of protein-bound SH groups The titration was performed using dithiobisnitrobenzoic acid (DTNB) following the modification of MILLER AND METZGER1,2 of the original method of ELLMANN3.
Isolation of IgM subunits from partially dissociated IgM Material sedimenting at 8 S was separated from unreacted I9-S material by several subsequent passages of the reaction mixtures through columns of Sephadex G-2oo equilibrated with 0.25 M Tris-NaC1 buffer (pH 8.0) (Fig. 4). IgMs* control material was prepared with I mM dithiothreitol (DTT) and alkylated with an equivalent amount of 1.6 mM iodoacetamide.
Characterization of IgM subunits Analysis of amino acid composition. Analysis was performed on a Beckman Model 12o C amino acid analyzer. For optimal separation of S-carboxymethylcysteine, the modification of the basic technique described by MORRIS AND INMAN4 was used. Polyp@tide chain composition of IgM subunits. The different subunit samples were dialyzed overnight against formate buffer (pH 3.50, I = 0.02) and passed subsequently on a column of Sephadex G-Ioo equilibrated against the same buffer. The different fractions were tested for /,- and K-chain determinants by Ouchterlony double-diffusion analysis. Determination of molecular weights. Molecular weights were determined by the approach to sedimentation equilibrium method of ARCHIBALD5 on 1% protein samples previously dialyzed against a 0.15 M KC1 solution. optical rotatory dispersion measurements. The optical rotatory dispersion of the different subunits was measured between 210 and 400 m# on a Perkin-Elmer P 22 spectropolarimeter, using cells of 2- and 0.2-cm path length. The observed rotations as were converted to specific rotations [az] and then into [R']z by the formulas: (~A " IOO
c (g/Ioo ml) [R']~. .
3 3t . . . [a]~. n2 + 2 IOO
where the refractive index, n, was taken as 1.37 below 260 m# and as 1.33 from 260 to 4o0 m#. A mean residue weight, M, of lO8 was used for IgM and its subunits. Subunits of IgM prepared by reduction with aliphatic thiols and containing cleaved interchain SS bonds.
Biochim. Biophys. Acta. 207 (i97 o) 286-296
pH-DEPENDENT
REDUCTION
OF IgM
289
Synthesis of 2-pyridyl-fl-cysteinyl disulfide, CsHloN202S~ All a t t e m p t s to synthesize the mixed disulfide of cysteine and 2-thiopyridine (2-TP) at physiological conditions invariably failed. Only under strong alkaline or acid conditions could it be produced by the following reaction (Pyr = C~HaN-) : Pyr-SH
+ H O O C _ C H N H 2 _ C H 2 _ _ S S _ C H 2 _ C H N H 2 _ C O O H - - - ~O HH + 'Pyr-SS-CH2-CHNH,.-COOH
+ HSH2C-CHNH2-COOH
After fractionation of the reaction mixture on columns of Dowex 50 W-X8 and by high-voltage paper electrophoresis, the mixed disulfide was identified by means of performic acid oxidation and subsequent chromatographic characterization of the oxidized cleavage products. RESULTS
Dissociation of IgM with heterocyclic compounds IgM (Man) was treated with various concentrations of various derivatives of pyridine and pyrimidine in the hope of recovering IgM subunits having their inter/z-chain and inter-/zK-chain SS bonds intact. Table I shows the effect of 0.2 M concentrations of different heterocyclic compounds on IgM. The only reasonable yields of subunit material were obtained with 2-TP, a sulfur derivative of pyridine known to exist in two tautomeric forms. Only trace amounts of subunit material were obtained with other derivatives, and, in particular, the unsubstituted pyridine did not result in subunit formation. TABLE I DISSOCIATING EFFECT OF DIFFERENT HETEROCYCLIC COMPOUNDS ON MAN IGM AT p H 8.0
Substance
2,4-Dithi°pyrimidine Thiouracil 2 - T h i o p y r i d i n e (2-TP) 2-Pyridone (2-hydroxypyridine) 4-Pyridone (4-hydroxypyridine) Pyridine
Number of tautomeric forms
Yield of I g M subunits (%) at p H 8.0 and 25 °)
4 4 2
< 2 %2 2o-24
2
Trace
2 -
Trace -
(0.2 M reagent, 24-h dissociation
Substrate dependency of the kinetics of 2-TP reduction of IgM at physiological pH Since a 0.2 M concentration of 2-TP resulted in the highest yields of slowsedimenting fragments of IgM, this reagent was chosen for further investigations. The rate of formation of material sedimenting at approx. 8 S and the rate of disappearance of the I9-S component of four different IgM native preparations were studied. In all cases, no components sedimenting between 19 and 8 S were observed. When the logarithm of the residual I9-S component is plotted against time of treatment with 2-TP, a straight line is obtained with all four different Waldenstr6m macroglobulins during the first 24 h. This indicates that the formation of subunits is a first Biochim. Biophys. Acta, 207 (197 o) 286-296
290
F. DOLDER Ela
I~(18S)
100- S
pH11 ,---
HuL
~I0
°--o
#-16
o--o
~5
60
~
60
~
"
n -55
~
~
96
108
h
12
~
36
48
60
"/'2
hours
Fig. i. The rate of breakdown of IgM by 2-TP at pH 8.0 and 25 ° depending on the particular protein substrate (protein samples Man, Lan, Hul, Eld). Fig. 2. The rate of formation of IgM subunits by 2-TP depending on the pH value of the systenl (protein sample Man).
order rate reaction. The rate constants (sec-1) were calculated to be of the order of 3.23" IO-~ for Man, 8.80. IO-n for Lan, 1.19 • lO -5 for Hul, and 1.65 • lO-5 for Eld at pH 8.0 and 25 °. It is a striking feature, however, that these values are smaller by a factor of IOOOthan the rate constants calculated for reductions with mercaptoethanol or similar aliphatic thiols n. The rate constants were found to be different for each protein, although they were of similar order. This finding is not unexpected in view of the known heterogeneity among Waldenstr6m proteins ~. The procedure of alkylation, however, did not essentially affect the rate constant or the yield of subunit material. Thus, the yield of subunit material levelled off at at 35-4o% of the total protein material in sample Man, at 7o-8o% in sample Eld, regardless of the method of alkylation.
pH dependency of the dissociation kinetics of IgM with 2-TP Since 2-TP is known to exist in a pH-dependent tautomerism equilibrium 7, dissociation experiments on Man macroglobulin were performed at different p H levels ranging from 5 to 12. The results in Fig. 2 indicate that the dissociation properties of 2-TP depend in fact on the p H value of the protein substrate solution in the expected way. Whereas at p H 5.0 no formation of 8-S material took place, the rate of formation of IgM subunits increased with increasing p H values. The m a x i m u m speed of the reaction was achieved at pH II.O. In that pH region, the first-order rate constants are comparable, though still 2-4 times smaller, with the reaction rates achieved with the commonly used aliphatie thiols like DTT. Higher p H values were not suitable for this investigation, since at p H 12.o IgM (Man) dissociated spontaneously into a polydisperse system of smaller fragments. Control samples were subjected to the identical conditions omitting the 2-TP. IgM (Man) was entirely stable at all p H values used in this investigation even after a prolonged incubation of lO6 h.
Attempts to S-carboxymethylate cysteine residues in 2-TP-treated lgM subunits Although the only heterocyclie compound able to dissociate IgM with reasonable yields was a thiol, 2-TP, only trace amounts of protein-bond - S H groups were demonBiochim. Biophys. Acta, 207 (I97 o) 286-296
pH-DEPENDENT REDUCTION OF IgM
291
8
0.2M 2-TP
o.
•
:
.
t
*l
Fig. 3. Rate of f o r m a t i o n of protein - S H g r o u p s in IgM (Man) u p o n t r e a t m e n t with o.ooi M D T T and 0.2 M 2-TP. Fig. 4- Ultracentrifugal analysis of a purified 8-S p r e p a r a t i o n f r o m dissociation of the p r e p a r a t i o n Man w i t h 2-TP. The picture was t a k e n 48 min after the r o t o r had reached c o n s t a n t speed (63 56o rev./min).
strable during the reaction with IgM ; whereas the control material reduced with I mM D T T yielded about 8- 9 - S H groups per subunit formed (Fig. 3) as well as 8- 9 residues of S-carboxymethylated cysteine (CM-cysteine) (Table III), which is consistent with the model of 24 interchain SS bonds per mole of IgM 1,~. This finding was confirmed by the complete failure to S-carboxymethylate the cysteine residues in any of the subunits produced b y 2-TP treatment as evidenced by amino acid analysis (Table III). These findings strongly suggest that during the course of the dissociation process the interchain disulfides in 2-TP-treated IgM subunits have been left intact. In some but not all experiments, a third protein fraction apart from the I9-S and 8-S material eluting over a broad range after the IgM subunit on Sephadex G-2oo, but accounting for only 2 % of the total protein material, was detected. This fraction (III) did not contain CM-cysteine residues either, which rules out the possibility that this small fraction could account for the subunit linking in IgM. TABLE II SOME PHYSICOCHEMICAL PROPERTIES OF I G M SUBUNITS (MAN)
All experiments were staged o n i ~ o protein solutions in o.15 M KC1 previously equilibrated with the same buffer. 3Ira, calculated f r o m the meniscus of the ultracentrifuge cell; Mb, calculated from the b o t t o m of the ultracentrifuge cell.
Mol. wt. and sedimentation rate
,~I m Mb Av. S2o,w (cf. Fig. 5)
IgM8 treated with D T T
178 ooo 174 5oo 176 ooo 7.14 S
I g M subunits treated with 2 - T P A lkylation
A Ikylation
(I)
(2)
2o8 ooo 198 ooo 203 ooo
198 500 211 5oo 2o 5 ooo 7.92 S
Biochim. Biophys. Acta, 207 (197 o) 286-296
292
u. DOLDER
80-
o;o x
~c
o
d, Fig. 5. C o n c e n t r a t i o n d e p e n d e n c i e s of t h e s e d i m e n t a t i o n r a t e of Ma n IgM s u b u n i t s ; © - - © , IgMs r e d u c e d w i t h o.ooi M D T T ; A - - A , IgMs r e d u c e d w i t h o. i M m e r c a p t o e t h a n o l a n d a l k y l a t e d w i t h 2 mM i o d o a c e t a m i d e ; O---O, IgM s u b u n i t s t r e a t e d w i t h o.2 M 2-TP.
Formation of the mixed disulfide 2-pyridyl-fl-cysteinyl disulfide It was virtually excluded that 2-pyridyl-fl-cysteinyl disulfide could have been formed under the physiological conditions used in the preparation of 2-TP subunits of IgM (see MATERIALS AND METHODS). Furthermore, no chromatographic evidence was found for the presence of that mixed disulfide on the basis of the amino acid analysis date of the sample hydrolysates.
Physicochemical characterization of 2-TP-treated IgM subunits The data in Table I I and in Fig. 5 indicate that all the subunits prepared with 2-TP and then "alkylated" display rather high sedimentation rates compared with T A B L E 111 CM-cYSTEINE
ANALYSIS
OF IGM
IgM protein
Mode of dissociation
Man
Native I m M DTT, I h, 25 ° 0.2 M 2-TP, 48 h ' * 8-S s u b u n i t s Fraction III
Hul
Native I mM DTT, I h, 25 ° 0.2 M 2-TP, 48 h 8-S s u b u n i t s Fraction III
SUBUNITS
Molesof CM-cysteine per mole of IgMs* 8.45
8.34
* These v a l u e s refer to a m e a n m o l e c u l a r w e i g h t of t h e p e p t i d e n i o i e t v of 158 ooo in IgMs. ** 2 - T P - t r e a t e d s u b u n i t s of IgM h a v e b e e n a l k y l a t e d a c c o r d i n g t o P r o c e d u r e (i) as i n d i c a t e d in
MATERIALS
AND METHODS.
Biochim. Biophys. Acta, 207 (197 o) 286-296
pH-DEPENDENT
R E D U C T I O N OF
IgM
293
the subunits with intact intrasubunit disulfides isolated by MORRIS AND INMAN4. Reduction with 2-TP resulted in the formation of subunits of molecular weights of 203 ooo and 205 ooo, respectively, whereas those subunits obtained by D T T treatment had significantly lower molecular weights of the order of 176 ooo (refs. 6, 8). Since 2-TP-treated IgM subunits have sedimentation rates of 7.95 S at infinite dilution, they are physicochemically similar to those subunits described b y SUZUKI AND DEUTSCH6 which showed also sedimentation coefficients of around 8 S. The essential difference between these two species lies in the fact that the 8-S subunits described by SUZUKI AND DEUTSCH6 were readily alkylated to yield smaller subunits sedimenting at 7 S, whereas 2-TP-treated IgM subunits could not be alkylated. A representative optical rotatory dispersion spectrum for I9-S IgM (Man) and several of its subunits is shown in Fig. 6. A single Cotton effect minimum is present at 233 m y and a weak but consistent Cotton effect is seen in the region of aromatic amino acid absorption. Within experimental error the spectra of native IgM and its subunits are identical.
Antigenic analysis of 2-TP-treated IgM subunits In double diffusion tests a line of identity was observed between all three 0.3 0.2
03 0
Onti-~i
0[
Clr~i-y
/~'iI /
i ~
U i'~/
...... ~,lve Man - - 2-TP ) - - - DTT .')recluceO
%',fl
--
.Ill
=
r4E
L/ 200
65 m[ -
I lonti-~(
40
I t~--.
65 rat
I anti-x:
0.1
f
-2~
l
~0"21
i
-I000
t,O
0
250
3~0 Amp
350
40 a~i-)~
65 rat
--- v.......~ctr'#.i- ~.
F i g . 6. O p t i c a l r o t a t o r y d i s p e r s i o n o f : , M a n I g M ; -- -- , I g M s r e d u c e d w i t h I m M D T T ; - - • - - , I g M s r e d u c e d w i t h o. I M m e r c a p t o e t h a n o l (ME) ; - - , I g M s u b u n i t s d i s s o c i a t e d w i t h 2 - T P . T h e s p e c t r a h a v e b e e n m e a s u r e d in T r i s N a C 1 b u f f e r (I = 0 . 2 5 , p H 8.0) in cells w i t h a p a t h l e n g t h o f 0 . 0 2 d m ( 2 1 5 - 2 6 o m/A) a n d 0.2 d m ( 2 6 0 - 3 5 0 m , , ) . F i g . 7. C h a i n s e p a r a t i o n e x p e r i m e n t s o n d i f f e r e n t I g M s u b u n i t s o f p r o t e i n p r e p a r a t i o n M a n . C o n d i t i o n s a r e i n d i c a t e d i n MATERIALS AND METHODS. i - c m l i g h t p a t h . A. I g M s o b t a i n e d b y r e d u c t i o n o f I g M w i t h I m M D T T ; B. I g M s u b u n i t s o b t a i n e d b y d i s s o c i a t i o n o f I g M w i t h 2 - T P ; C. I g M s u b u n i t s f r o m B b u t r e d u c e d in a s e c o n d s t e p w i t h i m M DTT.
Biochim. Biophys, Acta, 2 0 7 (197 o) 2 8 6 - 2 9 6
F. DOLDER
294
• 2 2-TP
ix
r-t:f-, t',
.2 2-TP
/\
II •
22TP
"2
2-TP
"22-TP
r-It--, Fig. 8. Diagrammatic representation of a disulfide exchange reaction upon treatment of IgM with 2-TP. Details are given in DISCUSSION.2-DPS = 2-dipyridine disulfide.
compounds IgM native, IgMs, and 2-TP-treated IgM subunits, when tested against specific anti-# and anti-K antisera. Thus, 2-TP-treated subunits retained at least a portion of their #-chain and K-chain antigenic determinants. The IgM, IgMs prepared with DTT, and the 2-TP IgM subnnits were examined by immunoelectrophoresis in agarose plates. Antiserum against whole human serum and specific anti-/, antisera revealed only one single line of precipitation. This line was cathodically displaced in the case of both IgMs and 2-TP-treated IgM subunits as compared to the native I9-S preparation. Resistance of I g M subunits to acid treatment The purified 8-S material from protein preparation Man was dialyzed exhaustively against 0.02 M formate buffer (pH 3.50). lO-15 mg of dialyzed material were passed through a Sephadex G-ioo column equilibrated against the same formate buffer, and the different fractions were assayed for/~- and K-chain determinants by double-diffusion analysis. The control material consisting of DTT-treated IgMs eluted in two peaks. The first and larger peak contained #-chain determinants only, the second peak K-chain determinants only (Fig. 7A). The 2-TP-treated subunit material eluted as one single peak still containing both #- and K-chain determinants (Fig. 7B). However, when 2-TP-treated subunits were reduced in a second step with I mM D T T and subsequently dissociated with formate buffer, an almost identical elution pattern resulted as encountered with the original one-step DTT-treated IgMs (Fig. 7 C) which gives additional evidence that the 2-TP-treated IgM subunit contained intact intrasubunit SS bonds. DISCUSSION
Previous studies on the dissociation of IgM with aliphatic thiols such as mercaptoethanol or dithiothreitol have shown that the cleavage of 24-25 SS bonds results not only in a complete splitting into subunits but is also sufficient to separate subunits into their constituent polypeptide chains without any further reducing process1,% Biochim. Biophys. Acta, 207 (197o) 286 296
pH-DEPENDENT REDUCTION OF IgM
295
Hence, it has been argued that these 24 disulfides per IgM, or five per IgMs, account for all of the interchain bonding in the IgM molecule 1. A t t e m p t s to determine whether fewer than these 24 disulfides could be cleaved and nevertheless result in a complete dissociation of IgM into subunits have previously been discouraging 1. Encouraging results, however, have been obtained in this field by MORRIS AND INMAN4, who were able to reduce IgM into subunits with O.Ol5 M mercaptoethylamine, which invariably yielded two residues of CM-cysteine per subunit. Recently, a selective reduction of IgM was described by BEALE AND FEINSTEIN9 and by BEALE AND BUTTRESS 1°, who were able to dissociate IgM into its subunits by means of low concentrations of dithiothreitol, ranging from 0.125 mM to 0.275 mM. The data reported here m a y be accounted for in the following way: 2-Thiopyridine reduces immunoglobulin M in a selective way leaving intrasubunit disulfide bonds intact. The procedure results in the formation of IgM subunits which display molecular weights of around 200 000 and are identical in their polypeptide chain composition with DTT-reduced IgMs, as judged by immunochemical analysis. The interchain SS bonds of 2-TP-treated IgM subunits can be split by a subsequent reduction with stronger reagents such as DTT. Since it is virtually ruled out that 2-TP forms mixed disulfides with cysteine residues of the protein substrate, the failure to alkylate cysteine residues of 2-TPtreated IgM subunits m a y be explained as follows: The oxidized form of 2-TP, dipyridine disulfide, is known to be a strong oxidizing reagent for protein - S H groups 7. Hence, it m a y be assumed that protein SH groups released during the treatment of IgM with 2-TP are reoxidized by means of the equivalent amount of dipyridine disulfide formed simultaneously. Since this oxidation process is known to be fast 7, it should be locally restricted to the site of the initial protein SS cleavage. It seems conceivable, therefore, that intersubunit disulfide bonds might be converted in this oxidation step into intrasubunit inter-#-chain disulfides, thereby giving rise to an equivalent amount of 8-S IgM subunits (Fig. 8). In this case, one would actually observe the overall equilibrium reaction between reoxidation of the - S H form of IgM subunits (II) into the initial I9-S sedimenting IgM (I) and reoxidation into the SS form of the 8-S subunit (IIl). In a general way, 2-thiopyridine offers interesting properties for the cleavage of SS bonds in proteins. At physiological p H its weak reducing properties result in a selective reduction of the most reactive SS bonds in a given protein substrate. On the other hand, to explain the significant pH dependency of its dissociating properties, one has to consider that 2-TP displays a tautomerism between - S H and - S speciestn. This tautomerism favors form I I at neutral pH, whereas acid or alkaline p H ' s shift the equilibrium to form I (ref. II).
R Acid and alkaline pH
Neutrol pH
Since only form I is appropriate for reduction of SS bonds, one should theoretically expect an increase in the p H value of the reaction mixture to result in the increase in the reaction rate as reported in our experiments (Fig. 2). This significant p H deBiochim. Biophys. Acta, 207 (197 o) 286~296
296
E. DOLDER
pendency makes 2-TP a more versatile reagent for the controlled dissociation of SS bonds in proteins. It enables one to choose an optimal pH value for dissociation of a particular protein substrate or to select a definite reaction rate or a desired yield of fractionated material by varying the pH value of the system. ACKNOWLEDGMENTS
I gratefully acknowledge the constant advice and many helpful discussions of Professor H. Isliker and Professor D. S. Rowe. Plasma samples and specific antisera were a generous gift from Professor D. S. Rowe. This investigation was supported in part by U.S. Army Grant No. DAJA 37-68-C-oi36. REFERENCES I 2 3 4 5 6 7 8 9 IO II
F. MILLER AND H. METZGER, J. Biol. Chem., 240 (I965) 474 o. F. MILLER AND H. METZGER, J. Biol. Chem., 240 (1965) 3325. G. L. ELLMANN, Arch. Biochem. Biophys., 82 (1959) 7 o. J. E. MORRIS AND F. P. INMAN, Biochemistry, 7 (196,8) 2851. W. J. ARCHIBALD, J. Phys. Colloid Chem., 51 (1947) 12o4. T. SuzuKI AND H. F. DEUTSCH, J. Biol. Chem., 242 (1967) 2725 . H. GRASSETTI AND B. MURRAY, Arch. Biochem. Biophys., 119 (1967) 41. F. W. PUTNAM, M. KOZURU AND C. W. EASLEY, .[. Biol. Chem., 242 (1967) 2435. D. BEALE AND A. FEINSTEIN, Biochem. J., 112 (1969) 187. D. BEALE AND N. BUTTRESS, Biochim. Biophys. Acta, 181 (1969) 25 o. B. MARKWALDER, Chem. Ber., 33 (19 °°) I557.
Biochim. Biophys. Acta, 207 (197 o) 286-296