Specific reduction of human immunoglobulin electroactive disulfide bridges by dithiothreitol: a redox reduction

Specific Reduction of Human Immunoglobulin Disulfide Bridges by Dithiothreitol:

-by *

Unite

>I. FOXTAIXE de

Humaines. **

Institut

Manuscript

*,

Recherches 543,

chemin

U+

received

C. RIVAT*, C. HAMET** de

1’IXSERM

de la Breteque.

UnilFersitaire

de

November

A Redox

‘76230

sur

and

C.

CAULLET**

des

GCnCtique

Bois-Guillaume

76130

Technologie,

la

Electroactive

Reduction

ProtCines

(France)

Mont-Saint-Aignan

(France)

1976

3rd

Special symboIs DTT

Dithiothreitol

(HS-CH,-CH-CH-CH,-SH)

I

OH SDS

DithioerythritoI Sodium dodecyl

PAGE H-H

Polyacrylamide gel Peptide containing

DTE

I

OH

sulfate electrophoresis a cysteine involved

in

an

inter_ heax-v

chain

di-

sulfide H-L x,x x\i-.

Peptide containing the cysteine of the -inter heavy light chain disulfide Peptides containing the cysteine of the in the inter heavy light chain disulfide JLoIecular

heav>light

chain chain

invol\-ed

in the

(i’- or A) invoked

weight

Summary

This paper describes a method for reducing and titrating in a short time the electroactive disulfide bridges and for labelling with a radioactive alkylating reagent some particular sites of the immunoglobulin molecules in a specific way_ The reducing agent emplo_yed was dithiothreitoh The mechanism of this reduction was not a functronal eschange mechanism but an osydoreductive one. Furthermore the study on IgA immunogIobulins permitted to clarify some aspects of the lability of disulfide bridges_

Tmmunoglobulin Electroacti\*c Disulfide Bridges

?43

Introduction Xanv authors were interested in the reduction of the disulfide bonds of human immunoglobulins in order to obtain information about the structure of these proteins and about thei; assembly during the biosynthesis of the different chains. Therefore the conditions ~of reduction varied greatlv in the work of the various authors. For example. VIRELLA' stud&d the lability of the different interchain disulfides of the IgG immunoglobulins of the four subclasses by means of dithioerythri-~ to1 (DTE) at various concentrations_ KOWGATZKI~ described the kinetics of release of Ig31 fragments in terms of the concentration of dithiothreito1 (DTT). In our previous research, we studied the electrochemical properties of IgG and TgA immunoglobulips 3- 5 and defined the redox characteristics of the electroactive disulfide bridges. This paper describes a simple method of polarographic control of the immunoglobulin reduction by a reducing reagent such as a thiol. The results show that immunoglobulin disulfides are reduced according to two different mechanisms : an osvdo-reduction and a furrctional The beginning of the reduction is governed by the oxydoexchange. We called this peculiar point of the reduction reduction mechacism. redox

The

reihctio~z,

reductions

and it could be controlled by polarographic analysis. commonly achieved in laboratory on immunoglobulins

were called classical mfzcctions to distinguish the two t_ypes of experiments.

Experimental Proteins IgG and IgA immunoglobulins were obtained from patients affected IgG immunoglobulins were isolated by preparwith multiple myeloma. ative starch block electrophoresis (24 h at 4 OCin 0.025 M barbital buffer, 5 V/cm) and then purified by ion eschange chromato,oraphy on DE_=cellulose (EATSM_+ ORGASIC CHEMICALS, Rqchester N-I’_ ; 0.75 meq[g) in o.005 dd sodium phosphate buffer.pH 6.5. After dialysis against polvethylene glvcol (Carbowvas 20 M), the IgG containing fractions were dialyzed agahst distilled water and lyophilized. IgA immunoglobulins were isolated by the method devised by FIXE and STEIX_JYNJCH:~ Pure monomeric and polymeric forms of IgA were obtained by gel The IgG and IgA subclasses were deterfiltration on Sephades G-200. mined by serological methods as already described’ and by *specific antisera_ The purity of isolated protein was determined by immunoelectrophoresis and immunodiffusion using the following antisera : horse antihuman serum (NETHERLASDSRED CROSS, -Amsterdam), and specific antiIgG, anti-IgA and anti-IgM antisera prepared in our laboratory-

Fontaine, Rix-at, Hamet and Caullet

‘-l-i

The protein concentration was determined by spectrophotometric ments at zSo nm with E1y$, = 14.

mea-

Xeasurements were performed with the three-electrode cE_c_and superimposed n_c_ polarographv system : TACUSSEL PRG 3. The equipment consisted of a rack con&-kg a PRT 30-01 potentiostat, a UAP 3 adapting unit -and a EPL z potentiometric recorder. The classical arrangement with three electrodes was used : a reference electrode S-C-E_. a platinum counter electrode. and a D_M_E. Reductions Redox reduction- - Fifteen mg of protein were dissolved in five cm3 of a solution of L M, KC1 and 0.05 AI phosphate buffer pH = S.Z. The reduction was performed by adding IO mm3 aliquots of 5 x IO-~ AC dithiothreitol (2~3~4) _ Oxygen was removed by bubbling nitrogen passed over copper at 400 OC through the solutions_ During the experiment an atmosphere of nitrogen was maintained in the polarographic cell over the solution surface. Classical reduction - IgG and IgA immunoglobulins (IO tig[cm”) in 0.5 M Tris, HCl buffer pH = S _z were reduced by various amounts of DTT (o-5 mild to 5 mM) durin,D I h at 37 OC under nitrogen. Electrochemical reduction formed as described previously_” [IX]

- LnbeEling the reduced

Electrochemical

reduction was per-

disttlfide bridges

Reduced immunoglobulins were incubated for an hour with [LK} iodoacetic acid (2.5 &i/ymole ; at a IO y0 equivalent escess, Amersham, England) - The material of low molecular weight was removed by dialysis_ The specific radioactivity of the labelled proteins was determined with a liquid scintillation counter (IXTER~ECHNIQUESL 40). The identification of the labelled disulfides was performed by autoradiography after electrophoresis of peptic-tryptic digests according to the method described by FRASGIONE et n1.8 PoZyncryZizmide

gel

electrophoresis

Sodium dodecyl. sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed using a 5 o/o acrylamide concentration ; protein mobilities were calculated according to WEBER and OSBORM.~

Cm-rent-tioltage

curves of the titrai%Lg reage4zt.

Many- reducing reagents -were s&died by .polardgraphyi_.to::.de’t;~~~-. mine their redox potentials (DTE, DTT, cysteine; ~th$@@.i~& _&n&. captoethanol...) . For example,. Fig. I- and. --2 .$oti~ the_‘oxyd~~~~~~~~~~~~~=: of DTT and cysteine respectively._ -G.&s 16, rc;:-rd,;r? ~W~r~‘~~b%&l?l$Y~. adding increasing amounts- of. a; 5 x IO LS -jj& Dm;~ ‘~l&-j&iy:,~~~ _Fi~~;$:_~_~~&&:-; recorded

In

for_ vay-jable

-these

_t\vo

amounts’

cases,

the

-of

a

limiting

103

-&f -c~~teine-gol;ition_~~-~:~~

;;>:~x-z I ;,i-dC (Ii) $@&r~:@@$&Z ; ~-As’th~~-heights:9f;=th~-_~_~~sz .M cysteine, +-ik- two_ sulfh$ :

&ff&&j~.~&-&~~~~

tional to the reducing reagent concentratibk were identical for 5 x IO --3 M DTT and’-Io_rf dry1 groups of DTT were involved in .the ri$dox:-_$%t&~. -: -._c-~;~:‘~:‘_~~:-1_~__:~_-_ i ; -2::._. ,; I.__.

-.- .-I

..

. . . ;:. :__i_

Fontaine,

Rivat.

Hamet

and

Caullet

Polarograms of cysteine obtained for different amounts of a IO-= M cysteine soIution. n - supporting electrolyte ; 6 - IO mm3 of IO-% 211 cysteine ; c - 20 mm= of IO-? -11 cysteine ; d - 40 mm= cf IO-~ ~11 cl-steine ; d - 60 mm3 of IO-’ ~11 cl-steine ; II - limiting diffnsion cm-rent.

Polnrogra~hic

control

oj the reduction

This control was performed by recording a polarogram of the reactive medium after each addition- of an aliquot of the reducing reagent. Fig. 3 represents the different current-voltage curves recorded during the reduction of an IgG I immunoglobulin by DTT. As it was proved in our previous works, 3-5 the reduction wave of immunoglobulins became an osydation wave at the end of the reaction. Curve 36 was recorded at the end point. Curve 3c was obtained for an excess of DTT equal to the quantity necessary to reach the end point. Two titrating methods can be deduced from Fig. 3. A first method consists in measuring the potential differerrce between the mercury electrode and the reference electrode which vary with the titrating reagent volume (potentiometry). A second one consists in measuring the current crossing the cell at a fised

Fig.

3.

El-olution of the polarogram of an IgG I during the assay. n - polarogram of an IgG I ; b - poiarogram recorded at the end point ; c - polarogram obtained for an excess of DTT.

Immunoglobulin

Electroactive

Disulfide

Bridges

‘$7

potential (0.5 V vs. S.C.E.) and in plotting it in relation to the volume of DTT solution (Amperometry). Typical titrating curves, obtained for the reduction of an IgG I (ZEG) are shown in Fig. 4 and 5.

600 -

0 Fig.

I 10

I

I ’ Y(mm%DT 5~10-~M ,I, I I, I 30

50

70

_+_

Typical potentiometric curve of the assay of IgG I disulfide bridges by DTT (5Xx0-~ M solution)_ The reference electrode is a S.C.E. The working electrode is a D.3I.E.

Fig.

5.

Typical amperometric curve of the assay of IgG I disulfide bridges by DTT (5 XIO-” M soiution). The controlled potential is --o-s V vs. S.C.E. The reference electrode The working electrode is a is a S.C.E. D.M.E. The counter electrode is a platinum The potential was fixed at electrode. -x1.5 V 2s. SC-E.

U7hen the reducing reagent at an excess equivalent to 400 oh waS added directly to the protein solution, the control of the reduction could be achieved by recording the current passing through the mercury electrode whose potential was fixed (-o.s V vs. S.C.E.). A typical curve is shown in Fig. 6. It corresponds to the reduction of the IgG I (ZEG). by

Fig. 6. Decrease of DTT wave height during the reduction of an IgG I (ZEG) (3 mg/cm=) with Wave heights have been an excess of DTT. calibrated in disulfide equivalent.

30

60

90

Fontaine,

2@

Rivat,

Hamet

and

Caullet

In the first step, corresponding to the part of the an excess of DTT. curve showing a high slope, disulfide bridges were quickly reduced ; in the second step (esponential curve), the reduction of the remaining labile disulfides became slower and slower. For IgG I (ZEG), two disulfide bonds were reduced during the first part of the reaction. Deternti9rntio9t redox

of

the

9t2cntber

of

i9ra9~~2~9co~~ob211i9~

Hiszcdfides

zirvolved

i9t iY2e

redmtion

Polarograms and titrating curves (amperometry and potentiometry) were plotted for three subclasses of IgG (IgG I, IgC; 2, IgG 4) and two sublcasses of IgA (IgA I,IgA 2). For IgA I, the monomeric and dimeric forms were studied separately. The results are summarized in Tab. I. In this table we compared these results with those obtained by coulometry in our previous works.3-5 The number of coulombs necessary for the reduction of the electroactive species were measured coulometrically. As (96,500 x 2) coulombs are necessary to reduce one disulfide per mole of immunoglobulin, we can calculate the number of disulfides reduced by the electrochemical reduction. Moreover, after each redox reduction , the free cysteines were alkylated by [iK]-iodoacetic acid and counted by liquid scintillation. The values are reported in Tab. r. Two disulfides were reduced by the redos reduction for TgG I and IgG 4 and four for IgG 2. These values corresponded to those obtained by coulometry. For IgA immunoglobulin, it could be noticed that only one disulfide was reduced for the monomeric form, whereas two disulfides were broken for the dimeric form. We had obtained the same results for the electrochemical reduction of IgA.-I*5 Identificatio92.

oi

tke

dis2clfide

bridges

reduced

by

the

redo-t- reihcctio9z

AS the free cysteines were alkylated by [l”CJ-iodoacetic acid after each experiment, we identified the [14C]-carbosymethyl cysteine peptides by autoradiographies according to the chemical typing of immunoglobulins described by FRAWGTONEand FRASRLIN.lo ‘g. 7 shows schematicallv the peptide map obtained for an IgG I. Autor%ographv 7 A was obtained for IgG I (ZEG) reduced bv 5 mM DTT in 0-5 _M -Tris, HCl buffer pH = S-2 and followed by radioactive alkylationTwo. main radioactive bands were observed which corresponded to tne peptides involved in the inter-heavy chain (H-H) and interheavy-light chain (H--L) bonds. The position of peptides was given by FRAXGIONE~~~ FRXSKLIN-10 Autoradiographv 7 B was obtained for IgG I (ZEG) reduced by the redox reduction ; the alkylation was performed at the end point. Cn this autoradiography, only one radioactive band was observed which corresponded to the peptide of the hin.ge region containing the cysteines involved in the inter-heavy chain bonds. The peptide maps of an IgA I: (monomeric form) are represented schematically in Fig_ S_ Autoracliography S A was obtained for IgA I (MET) reduced by 5 mM DTT. Autoradiography S B was obtained for

Immunoglobulin

Electroactive

Disulfide

Bridges

-

-

I I i

a. ca

i

,I iI

1:

n

-

-

CI 1

-4

i

1

1 J I

1

:

>f-

1:. i

) : J

i : :

I i

I

-

.

I

2 ci

7-l

/

cl

0

t-4

-5

6 s

I

c

I

H

N

0%

c\

-

l-4

0 H

.

r=?

% 2 is

0

i cr

/

c3

cn

w

-1 k

CJ to

c

I c; m

2 t

i

i

I

N

N

ri

Fontaine, Rivat,

250

?--H-L =

-l-i-!-l

E-~INP-LYS

GW

m-H-ii-1

-

Hamet

Fig.

and Canllet

T_

Autoradiographies after electrophoresis (3000 V. 60 min. pH 3.5) of a peptic-tryptic disest of the heavv-chains of an IgG I (ZEG). A - IgM I chemical& reduced 15 mM DTT) and alkplated with 0.1 M 1X-iodoacetic acid ; I3 - IgG I reduced by the redos reduction. The position of peptides is given by FRAXGIOSE and FRASRLIS.~O H-H, H-L see abbreviations.

IgA I (LEBI) reduced by the redos reduction ; the radioactive alkylation was achieved at the end point_ Fig_ S C represents the autoradiography of IgA I (LEU) electrochemically reduced and [Z4CJ-labelled as described previously_ * On Fig_ S A, three radioactive bands corresponded to the cysteines involved in the hinge region (H-H inter-heavy chain bonds). Two peptides (a cathodic and an anodic one) corresponding to the cysteine of ihe light chain involved in the H-L bond (pep&de 2.) and the H-L peptide of the heavy chain, were also present_

H-H-+-

Fig.

S.

Autoradiographies after electrophoresis (3000 V. 60 min. pH 3-5) of a peptic-tryytic digest of an &_A I (MET). (Monomeric form) _ _-f - Q-1 I reduced by 5 m31 DTT : B - IgX I reduced by redos redution ; C - Ig_1 I electrochemically reduced on a mercuqpool (I V VS. S.C.E.). The position of peptides is g-i\-eu by FRAXGIOSE and FRASKLIS.'" See abbrel-iations.

NH-+H-H-m -E-ONP-LYS xfZ=

x1-B

B

~3lc-PTl -GLU

A

C

markers

Two other radioactive bands (XI, Xz) were observed. but not identified_ The less cathodic one (XI) corresponded to the unique radioactive peptide obsen-ed (PTI) on the autoradiographies S B and S C. The purification of this peptide permitted us to obtain an aminoacid analysis_ The results are reported in Tab. 2.

Immunoglobulin Table

2.

Amino-acid

analysis 1

Electroactive of the

Amino-acid

peptide

I

Thr

Set

Bridges

_

PTr.

i j

Asp

Disulfide

I I

1

i

o.s3

o-gz 1-36

0.5;

Gill

Leu Composition

are reported

I

0.46

as moles of amino-acid

per mole of peptide.

The redos reduction of IgA did not split the interchain disulfide bonds since any fragment could be detected by SDS-PAGE.

To get further information about the cysteine linkages and labilities of disulfide in IgA molecule, Igil I (LEM) was classically reduced by \-arious concentrations of DTT. The reduction was followed by an alkylation with [‘%I-iodoacetjc acid_ The autoradiographies of the labeiled peptides are shown schematically in Fig. 9_- The release of fragments during the reduction was studied by SDS-PAGE electrophoresis (Fig. IO). For 0.5 m&f DTT, two radioactive peptides appeared which car-, responded to the unknown peptides (PTr, X2) described above. I< and H-L peptides were not clearly observed but a fragment with a r55.000 N.W. and another one with a 25,ooo 3I.W. and identified as light chains, could be seen on acrylamide gels. For 0.75 mM DTT, the bands corresponding to the fra,oment described above had a-greater intensity, and on the peptide maps I< and H-L peptides presented strong radioactive bands For I m&f, 1-5 mM and 2.5 m.M, two other fragments were released They were identified as dimeric (Hz) and (M.W. 120,ooo and 60,000). Simultaneously the three monomeric (H) heavy chains respectively. radioactive peptides correspondin g to the H-H peptides appeared on the autoradiographies.

Fontaine,

252

Rivat,

Hamet

and

Caullet

m

c

-EzzJ

H-HH-H x2-m

m

m

PTI-

E-ONP-LYS

tzEzQzz2 +GLU

Ezzza-

KH-L--,5

5

izm-

mm

4

3

2

1

Fig. g-

Autoradiographies after electrophoresis of (3000 V.

60 min. pIi 3.5) of a peptic-tryptic digest of an Q-1 I (LEU) (3Lonomez-k form) _ I - reduced by o-5 r&l DTT ; z - reduced by o-75 md1 DTT ; 3 - reduced by I m&I DTT ; 4 -reduced by I.s-~M_DTT ; 5 - reduced by 2.5 mill DTT_

-

60 000 Fig_

I I -25

060

IO.

SDS. polyacrylamide get eiectrophoresis. I - IgX I (LEN) (Monomeric form) reduced by 0.5 mdf DTT ; 2 - 0-75 mIk1 DTT ; 3 - I rnlM DTT ; _I - 1.5 mX DTT ; 5 - 2.5 mM DTT_

12345

Discussion

Among the sulfhydryl compounds used for the reduction of protein disulfides we chose dithiothreitol for the following reasons : If U, is the redox potential of disulfide bridges RI-S-S-RI

+ 2 e- + 2 H+ f

2 RI-S-H

(1)

Immunoglobulin

Eiectroactiye- D@i&de:-Bridges

: : 253. -.. .:.--’ ._ .. -. _. 11 r : .:+-~--I

_ 1:

-_

and U, the redox potential of the reducing .r+gent -.2

then the constant

RI-S-S-RI

R2-S-H

+ R2_&_R2

of equilib&m

-1_

.- .’

-.

.

+ ;-:H~.~f--;.k’-

of -the red&i&

_ .:_.,:rez&o&

__=

2

+ z R2-S-3T5 _ 2 R&-H-+

;p:,:

:

R&S--S_R2

:-

:::-.;

.-- .-

(ij: T’.--

z-~~:-~;!L-c.~~-:

- .-_-y. _ _-_--.- z-f- : : -t:_i3):

is _

K=

z -__ -._’ ~--_ .-. (& and k, are the reaction &es) _ The- NERN~T’S equation applied to equakik (I). &d:($ to calculate K . , log K =

-v;__ u 0.03

2

:- ‘_I--‘=: &&$i-$$ _:- -

.-

_-

-_

-It appears that if U, is much smaller than Ui,: U@&&ioti :lof-i.& --- .--.. --_- ,_- .y duction (kJ i’s complete and rabid. For DTT. at pH -= S,2 the app&& -~~~~d,d:‘po~e~t~al-( Uo)‘~@$& tion (4)] of the redo-u-system was -_.6S V. vs. IS;C.E:‘: &ider~.th~.-Katie. conditions the UO of immur)ogl~b&s‘ ++vas+$+S -V .@-_~S~$X_J-? -7 -_ I .:. .‘I At. this pH, Vi - U, = 400 mV ‘and thus-K 1! IO~[=_ e,-.-“.::.-. ml-‘Y:m. 171 We can conclude that thie reduction. was Complete. and, _vkry:r%pid,: With cysteine (U,O = 9-5 V US_S.C.E.),‘mK 410?;_3,: t& r&cti~tiwas rapid and complete as well, bnt -the jump in th~‘&ei;ltitil_ dutig.:.apotentiometric assay was ndt impdrtzkt :eno&h gcd- t&e--e$l fp@nt.‘Was not well defined. $+thermore, .for an. amperometrilc_ titrati&,l,.t+% PO_: tential had to be-fixed in _ti intervd.of. potk+tig:wber& ti.sh‘oti~@ttinti&. variation did not cause a- relatively hi~~.v&iation__~f--i&kkit$,.-m ‘&$%dition was -realized wheti the potential d@&nce be&e& ;the’;U$‘df-“the I._q .- : : Aredox systems was high eriougli. ’ _--- : -~ -- _. : ; ? F- :.;_-__;1__ For -the-same reasork. the redox red&io~- was_ p$rfk@L $t- pH- = S-2; the I?‘,, of DTT was-a func$iqn of pK_ _; .__ _ _~‘_._- __l:l._ -,_ :.- .;.

..I : ._:_;..::I_ 11 --_-I:& ~_._.. ...-:. ._ __;:;-where n is the number of electrons -involved- in. the- reaction;-: L-B& ffie Uro of immunoglobulin disulfides did not vary .with!the .pH; SO-th&t;.tiheti : ‘__

:

i

‘5-r

Fontaine,

Ricat.

Hamet

and

Caullet

the pH decreased, the difference in potential between the U’,, of the two redos systems decreased as well. In our previous work, I1 we showed that only the inter-heavy-chains disulfides of IgG immunoglobulins were reduced electrochemically. Autoradiographies performed on ~e&xx~e&wed IgG I, IgG 2 and IgG 4 showed that only the inter-H-H-bonds were split. For esample. Fig_ 7 shows the autoradiographies of an IgG I reduced by 5 mili DTT (7 A) and redox redlsced (7 B). For the last one, only one radioactive band, corresponding to the inter H-H bonds of the hinge region of the IgG I: was observed. 10 The results obtaiiied by amperometry and potentiometry were in good agreement with those obtained bv coulometry and with the theoretical values of the number of inter H-H disulfides of the IgG immunoglobulins: two bonds for IgG I and IgG 4, and four disulfides for IgG 2. Fig. S shows the same evidence for. IgA immunoglobulins. The same radioactive band was obsert-ed on autoradiographies S B and S C which corresponded respectively to the recEo_x re&rced IgA and to the electroreduced Ig_A. The results of potentiometric and amperometric assays were in good apeement with those obtained by coulometry-. We have asserted that only the electroactive disulfide bridges of immunoglobulins were reduced by the redos reduction. As far as IgA are concerned, the location of the electrochemically reduced disulfide bridges is still uncertain_ The only certainty lies in the fact that the reduction is specific to the inter-heavy-chains disulfide bridges. 3*4 The radioactive peptide PTx -obtained after the YerEoxve&ctIon or the electrochemical reduction may correspond to a labile intraheavy-chain disulfide bond. (FR_XXGIOSE, personal communication)_ When we compared the amino-acid composition of PTx peptide with the sequence of the (TRO) a-chain.12 the peptide could be localized on the a-chain between the positions 259-367 : (GlY&er~_,~--Glui-AliZla,, I-Aspo_,-Leu,

,,--Thr,_,-Cys,,--Thr,,)

Cysteine 266 is involved in an intra-chain bond which may have a particular position in the a-chain as represented by KRATZIS et aZ.lc It was shown that the redo_%redwtiost did not release any fragment on monomeric Ig_A molecule as judged by SDS-PAGE.. At a very low molar concentration of DTT (Fig. IO). we observed that cysteine 266 could be one of the most labile cysteines in the a-chain. PTr peptide is one of the two peptides which appeared first when IgA was mildly reduced. The findings illustrated in Fig. 6, showed that the reduction of immunoglobulins by an excess of a reducin g reagent could occur by two different mechanisms. The reduction began rapidly by the reduction of the electroactive disulfides. The first part of the curve cotiesponded always to the reduction of a number of disulfides equivalent to the number of electroactive disulfides (For example. two bonds for an IgG I)_ We can assert that the fir+ part of the reduction was governed by an osydo-reduction mechanism [equation (5)]_

Immunoglobulin

Electroactivti

Protein t_ z H+ + i s

2

Bridges

e' z+ Protein

I -.

Disulfide

1

c

DTT (%I),-~

:

(SH) p Z DTT

-- -.~:

. -I

-. -.

DTT (S-S) +. z H+ + z e-

Protein -j- DTT

255

.-

.

1

SH SH

s

--

(S-S-)

.

,-

(5)

-

+ Protein bH i& -1...i --

L-4

:. The second part of the curve (exljone&a.l curve): represented the reduction of the remaining. labile disulfides (for example; the two -.-H-L _ bonds of an IgG I). This reduction Was -controlled by knother reactionscheme [equation (6)] which might be a functional-exchange as proposed by WHITE :x3 Protein + DTT (SH), z

Protein

I s-s

SH

I

I

s

Protein + DTT (SS>

_

I

S-S-DTT-SH

‘(61

The redox reduction allowed us to reduce and titrate very &i&ly the immunoglobulin disulfide bridges involved in the electrochkmkal _ reduction on a mercury pool, and to label some special sites of the ‘immunoglobulin molecules in a specific -way. This point is of a g&t. inte?est because electrochemical reductions on a mercury pool.. preSented some technological difficulties as -they required ldng experiments,-Furthermore, this work permitted us to clarify some &pe<=ts. df the reduction mechanism of immunoglobulin disulfide bridges and some aspects of the lability of IgA disulfide bridges.

Acknowledgement This work was supported by the h&itut W&ox& de la SantC. etde la Recherche Wdicale and by the Institut Universitaire de Tec$nol?_ logie de Rouen. We thank Dr. R~USSEAUX~ (Faculte de &Gdecine, Lille) -for his a& -sistance in the experiments of amino-acid analysis. 1. .. _

References 1 “-

3

G. VIRELLA and RACE. E. ~ows~~z~~, Scad.

PARKHOUSE, ~Isnmunodtenaisfry 10, gr3 (1973). 2, 433.(1973) >I_FONTAINE. C. RLVAT. C. ROPXRTZ and C. CA~LLET, BM. Sot. ChikFr. 1973.

x573

.-

J_ Immunol-

:.

~

Fontaine,

256 4

5

7

6

Hamet

and

Caullet

M. FOXTAIXE, C. RIVAT, C. ROPARTZ and C. CAULLET. BdL Sm. Chi~rt. Fv. 1974. 15.13 M. FOXTAINE, C. RIVAT, C. HA~IET, C. ROPARTZ and-C. CAUL~.ET. BzcZZ. SOL Clzinz: Fr.

6

Rivat.

1975,

1357

J.M. FIKE and M. STEISBUCH, Rev_ Em-. Etzrd. Ctin. Biob_ 15, 1115 (1970) C. RIVAT, L. RIVAT, J_ BOURGUIGXOX and C. ROPARTZ, I~?zz,zrcnoc~emistr3r S, sI3 (1971) 33. FRAXGTONE,

C_ MILS-CEIS and E-C.

FRASRLIS,

~Votzrve

(London!

221,

149

(1969) PC. WEBER

and M_ OSBO&. J_ Biol. Chelrz- 244, 4406 (1969) 10 B_ FRASGIOXE and E-C. FRANKLIX, FEBS Lett. 20, 331 (1972) 11 C. RIVAT, M. FOXTAIKE, C. ROPARTZ and C. GULLET, Ezra. J. Im~l~zzc?zul. 0

3, 537 (1973) H. KRATZIS,

13

P. ALTEVOGT, E. RUBAN. A. KORTT. I<. STAROSCIK HILSCHJIAXX, Hoppe Seybr’s 2. Physiol. Chent. 356, 1337 (1975) FM_ WHITE. J_ Biot_ Clzetlz. 235, 333 (1960)

and

N.