Introduction of sulfhydryl groups into proteins using acetylmercaptosuccinic anhydride

ARCHIVES

OF

BIOCHEMISTRY

AND

Introduction Using

BIOPHYSICS

96, 605-612 (1962)

of Sulfhydryl

Groups

Acetylmercaptosuccinic

October

Proteins

Anhydride

From the Department of ChernistrU, Sodwestern Receircd

into

University, El,an.ston, 11linoi.s

2, 1961

Protected mercaptan groups may be introduced into a variety of proteins under very mild conditions with S-acetylmercaptosuccinic anhydride as reagent. The effects of pH, temperature, type of protein, and other I-nriables on the extent of react,ion are described. The protecting acetyl group may bc removed and the free -SH generated with the aid of a nuc*leophilic rengcnt, hydrosyluminc being palticularly effective in this respect. INTROIKCTIOK

The introduction of thiol groups into proteins, beyond merely t’he reduction of P-S linkages, has been attempted by numerous investigators for nearly 30 years (l-4). These efforts have had many aims, some technical, some fundament’al. Technical object,ires have been largely the manipulation of keratin fibers, particularly of wool and hair, and the control of propc,rties of other fibrous proteins. From 21more academic viewpoint, a wide range of IAL‘nomena have been recognized as tlependcnt’ on sulfur-containing side chains. The configuration of globular proteins, for example, is influenced by sulfur linkages. In these globular macromolecules, furthermore, thiol groups may play a critical chemical role bcpond the control of configuration. Many cnzymes (e.g., papain, glyceraldehydcphosphatc dehydrogenase) maintain activity only if their thiol groups arc free. In other proteins (e.g., hemerythrin, carboxypcptidase), the active site seems to involve :I tnrrcaptan group t’o which a metal is attached. More subtle effects of a thiol group :UY shown in hemoglobin, where interactions between oxygen-carrying sites, as well as sickling phenomena, are influenced bg the state of the mrrcaptan group. For technical purposes the conditions allowable for thiolation, such a8 :t warn1 telu-

peruture or high pH, are not as restricted as muat be imposed for reactions with biologically active proteins. In the latter case one must devise a procedure which operates rapidly at room temperature, or lower, and at pIi’s and other condit,ions near physiological. The thioglycolidc or thiolactone rcagcnts used (3-6) for the introduction of nwrcaptm groups suffer some deficicnries in out or 111orcof t’licse respects. On the other h:md, :I suitably substituted anhydride, aucll a:: S-ncetyllllercaptosuccinic anhydride (7)) operates very well within the rcatrirtions ideally dcsir:tblc for thiolation of l)rotcins. The course of the mercaptosuccinylation reaction may be represented by the schematic diagram of Equation (1). Depcnding on t,lic ratio of molts of reagent,, X, to protein P- (NH-), , the number of groups ?I introduced may vary from a small number all the way up to 72,i.e., up to complete coverage of amino groups of the protein. During the reaction some acetyl group:: may be removed by hydrolysis of (II) so t’hat (III) is obtained with a few free thiol groups but most’ly acetylthio groups. Also, during the reaction some of the anhydride reagent (VI is hydrolyzed to (VI) :md subsequently all the way to mercnptosuccinic acitl (VII 1. Throughout t,he reaction tlic 1)II is maintained nbove 5 so t’hnt all the

KLOTZ

3

AXD HEINEY

SITI,FHYDR\-I,

GROI’PS

carboxylic acids shown are actually present as carboxylate ions, II-COO-. As salts, therefore, the small molecules are caeily separated from the macromolecules by ion exchangers, dialysis, or an ion-retardationtype resin. Thereaft’cr, the wmaining acetyl groups may hc easily rcino~-cd from (III I n-it11 an appropriate nuclcophilic rcxgvnt~ and the thiolatcd protein (IV1 is thereby gtincratcxd. ESI’ERIMESTAI, ,%ik!ETYLMERC4PTOSCCCIKIC

-AXHYDRIDE

Tllie

cornpound has been prepawl lxeviouul! by Holmbcrg and SchjHnberg (8) by the addition of ttliolncetic acid to mnlcic~ anhydride. However. a more convenient method, recommended to ui by Dr. I,. De Mytt of the Toni I,aboratoriw, is an adaptation of that described for the prepawtion of succinic anhydride (9), Alusing of the :rp1lropriatc~ acitl with acetyl chloritlc: HSCH-COOH + PCH,COCl

+

&I-COOH

CH,CO-S-CH-C I

I

(:H,-C

(2)

\ ‘0

+ CH,COOH

/ \

+ 2HCl 0

.I commerical sntnple of mercaptosuczinic acid was purified by extracting it from an aqueous solution with ether. Evaporation of the ethereal solulion gnvc a solid wiih mp. 147.5-149” [lit. (10) 148”]. -1 loo-ml. flask was fitted with a reflus condenser and a gas trap. and into it mere placed 14.; g. mercapt,osuccinic acid and 28 ml. acrtyl chloride. The mixture was rcfluxed on a steam bath. *ilftel 1 hr. the solid acid dissolved completely, giving :I 1& yc~llow solution. Thr reaction flask was removed, stoppered, allowed to cool slowly to room temperature. and then immersed in an ice bath. The crystals obtained were collected by filtration, wnshc~tl four times with lo-ml. portions of col(l c,thcr, and dried in a vacuum desiccator. The yield was 12.5 g. of white crystals, m.p. 75-76”. Recrytallization from benzene raised the m.p. to 76-ii” [lit. (8) 71-73”].

WT

ISTROI>I’(‘TIOK

1~rtrchasc~tl from Armour and Company, ornlbuniin fwnl tllc, F{-orthington Biochemical Corporation. OTHER

REAGEKTS

Inorganic chemicals were all of reagent grade. Tris(l~?-drox~t~iet~~~l):~tni~~ot~~~~t~~~~nc TWS primary standard grntle from the Sigma Chemical Com1,:my. Imidazolc was 1~urc~hawd from Distillation Products Industries, hisl.amine hydrochloride from thcs California Coq>oration for Riochcmical Rcscwc~tl , and t,-histidine nronohydroclllori(Ic from Merck and Company. Ion-exchange resins. Arnbtst,litc IRA400 in ihe chloridr form and Amberlite MB-l, were analytical grade tnatrrials from the Fisher Chetnical Cotnpany. Srphndcs G-25 was obt ainctl from I’h:~rmaci:t J,abornt orics, In(a. ?LIERCAPTOSUCCI~L~-L.~TI~~

REACTIOS

A benkrr WLS fitted wilh a thin rubber stolywt c*ontaining liol~s for the accommodation of the following: an mlet, for nitrogen gas. glass and cAomc1 clectrodcs. lhcrmometw, l)nret. small funnel as inlrt port for solid anhydridc reagent. A stirring bar was Ihrn placed in the beaker, and this container w-as placed on top of a magnetic &wr. Dry protein was pl:wcd iwide the bcwlic~ and tliwolvc~d in added tieoxygenated watu wllile a Arcam of nit,rogen flowed grntly 01-w the liclllitl s\lrfucr within the beaker. The solution was atljusted io operaiing pH. Solid anhydridc was adtl~l in small portions to the st irlcd protein solution. The acid lib(xrat(d during the reaction was neutralized by ad&d sodium hydroxide. and the pH was maintainotl within a fwi tenths of a unit of its starting value. Addition of the reagent took from 15 min. to 1 hr., the time tlcpentling on the (11i;mt ity of anhydride adtlcd. The modified protein was freccl of stnall-tnolecult impurities by one of three procedulcs. In the first. th? wctyl mercaptoswcina?te (VI) and rric~rc,a1)toauccirl:~te (VII) anions wcrc first, t’etno\-cxd, and replaced by chloride. t)y passage through :I11 dtnberlitc IR.X400 anion-cxvhange c~>l~nm. Then all salts were remored by dialysis, unclcr nitrogen, for sevrral days. A swond 1,rowtlnre wnio\-e(l all salts by passage of the solttt ion tht,orlgh a mixt,cl lwd, .Amberlitc MB-l, rsxc~h:mgc~i~csin. -4 third approach wed gel filtration with .Sephadcx (II). The c~o11mm methods FWW mow rapid and mow r~fkicnt in the t~ry~~or:~l of salt than was dialysis. Salt-free, motlifird prokins were Iislulty 1;cpt in tlry form after lyophilization.

PROTEIN Two Litlantic Bovine

diffrrent gelatins wcrc Ilsed. from the and Grayslake companics, respectively. serum albumin and ribonllr~lense were

L)WERMISA'TIOS

OF EXTEST

The rnodificd protein (III) form+ w HS- and CH:,CO-S--.

OF CWPLISC: has sulfur in two Analysis for free

608

KLOTZ

AND

HEINEY thereby the progress of the dencetylation was followed.

reaction

EFFECT OF PH

01

I 4

I 5

I 6

I 7

I 6

I 9

I IO

I

PH FE. 1. Dependence of mercaptosuccinylation on pH. Reaction carried out with Grayslake gelatin, reagent being added within 20 min. -SH was made by two methods, amperometric silver titration (12) and reaction with a disulfide dye (13). Analysis for total sulfur introduced was rarried out, by the same two methods, after the protein had been exposed to a pH of about 11.5 for 1 hr.,’ during which time acetyl groups were split off from CH,CO-S--. The number of CHXO-Sgroups initially present was taken as the differenre between titration aftrr and before exposure to base.

DEACYLATION OF ACETTLMERCAPTO GROUPS To convert (III) into a completely HSprotein it is necessary to remove the (u-2) acetyl groups from the acctylthioprotein. Of course this result can be accomplished by dilute base, as used for the annlrsrs described above. but the rsH’s attain4 might be damaging to some proteins. In vicar of the well-known catalysis of hydrolysis by in the nitrogcnorls bases (14, 15), deacetylation presence of several such compounds was examined. In each case the nitrogen base was added to a solution of controlled pH near 7-8. At suitable intervals an aliquot of this solution was removed and titrated amperometrically with silver to determine the number of -SH groups liberated, and ‘The rate of hydrolysis of the thiol ester linkage at pH 11.5 was followed in a separate set of experiments. Hydrolysis was essentially complete in 1~~sthan 1 hr.

Since protons are liberated during the coupling reaction, one would expect a marked pH dependence in some region of pH. Figure 1 summarizes a series of experiments wit,h Grayslake gelatin and a fixed amount of S-acetylmercaptosuccinic anhydride, the reaction being carried out over a period of 20 min. at pH’s from 5 to 9. Although the reaction is markedly pH dependent below pH 7, the extent of introduction of sulfur is essentially constant’ in the pH range of 7-9. Maximum speed is attained in t’he physiological pH region. Similar pH effects have been reported in the reaction of succinic anhydride itsrlf (161. EFFECT OF REAGENT COIYCENTRATION

=2 variety of experiments was carried out’ with both gelatins and with bovine serum albumin, and these arc summarized in Fig. 2. As the ratio of acetylmercaptosuccinic anhydride to protein is increased, the ext,ent of thiolation increases for all proteins. With gelatin, however, a plateau is soon reached at about 32-36 groups per 10” g.; this figure corresponds closely with the number of lysine groups in gelatin (17). With serum albumin the moles of anhydridc used were not carried to as high a ratio, but it, seems likely (Fig. 2i that with t,his protein, t,oo, a plateau would be reached when the lysine content is approached. Such coincidences, of course, do not prove the specificity of the anhydride reagent. Ncrerthclcss, the question of specificity has not been pursued, for the ol-jjective of this investigation has been the dcvelopmcnt of a reagent for the introduction of sulfur into proteins and not the design of a specific reagent for amino groups. EFFECT OF TEMPERATURE

The extent of thiolation of hoth bovine albumin and gelatin is hardly affected by a change in temperature from 30” down to 0”. The results of a pair of experiments for

SULFHYDRYL

609

GROUPS INTRODUCTIOK

dfur introduced

r0td

Protein

Bovine serum iLlbllI~lill

2 2

1. 1

a The reaction was run in a phosphate buffer (0.125 M) which maint~ained the pH in the range indicated. TABLE @OYPARISO.U

I 2. Variation in extent of thiolation with quantity of X-acetylmercaptosuccinic anhydride added. Reaction carried out at room temperature, pH 7-8. Crossed areas represent total lysine content of each protein. Square point is for different gelatin sample. FIG.

cac.11of these proteins are summarized in Table I. In the gela.tin case the concentration of protein as well as t’emperature was changed, but it is still obvious that, the reaction rate is not lowered in the cold. Again these observations are similar to those reported in the reaction of unsubstituted succinic anhydride (16).

COMPARISON OF PROTEINS Thiolation has been achieved easily with four proteins of different size and nature: ribonuclease, ovalbumin, bovine serum albumin, and gelatin. The results of some typical experiments are assembled in Table II. It is clear that the reaction proceeds readily with native proteins as well as with a denatured one such as gelatin. Likewise, molecular weight or isoelectric point is not a controlling variable.

SPECIFICITY As was mentioned above in the reaction wit.h gelatin, high concentrations of reagent

II

OF PROTEIUV& L

I

Moles introduced/W g. protein

Protein

Gelatin (Atlantic)

0.8 18

(

7 30

Bovine serum :tlbumin

3 16 46

Rihonrwleaw

44

Ovnlbumin

29

QAll react,ions were c*arriecl out. at room t.enpesat we. b The reaction was run in a phosphate ~&AI* (0.0625 -11) which maintained the pH.

introduce an amount of sulfur corresponding to the total lysine groups in this protein. It seems, therefore, that the reagent couples with strong preference for amino groups and that’ little or no 0-acylation OCCUF L. Such a conclusion has also been reached in st’udies with other anhydrides

and proteins (16, 18). For purposes of represcntation of the reaction> we have assuni~l, therefore, that. amide formation is n-hat occurs. It should be noted in passing, nc~vcrtltrless, that with niacrotnolecules containing no amino groups but having a large number of ltydroxyl groups, thiolation may still bc effected i19) by the acctylmercaptosuccinic anhydride through t)he formation of ester linkages. In the tnercaptosuccinylation reaction, E:q. ( 1 ) , amide forma,tion has been assumed to occur with the anhydride bond closest to the acctylthio substituent. This choice has bwn made on the basis of estimated inductive cfferts of the acet,ylthio group. HOWe\-cr, we hare not, attempted an cxperimental lwoof since tltc question is only lwri1)hcral to our main aim with this rcaction. the introduction of thiol groups into tuacrot~~olecules. This objective is attained no matt,cr which side of the anhydride forms the presumed amide linkage, and no ttlnttcr whether an amide or rater linkage is tlw chemical bond. Moles SH 2O

Idgm.Protein i

I

0.5M

I

I

lmidarole

I

60

I20 MINUTES

VIG. 3. Catalysis of hydrolysis of wetylfhio linliagc by various nucleophilic reagents. Sodium hydroxide and hydrosylamine were 0.01 M, histidint 0.1 :W. All solutions were at pH 7.2-8, except SuOH.

EFFECT

OF PROTEIR.

~ONCESTRATIOS

iYo systctttatic stttdy was made in which only this variable was changed, all others being hold constant. Nevertheless, it slio~tl~l lw nwntioncd tliat in the course of many thiolation reactions, protein conccntrat)ions from 1 to 85 \v(‘ro used successfully. KFFECT

OF BUFFER

Again no systematic study was made, but in the course of sewral preparations 0.125 M phosphate buffer of pH 6.8 was used and no sodium hydroxide added to maintain the pH. During the reaction the pH dropped a fen tenths of a unit, but the extent of tliiolation n-as cotn1~arat~le to t.liat in a nonbuffered medium tttaintained at the santo pH by the addition of sodium hydroxide.

For analytical purposes tlw acctyl group n-as cleaved by mild basic hydrolysis of the succinylated protein in 0.01 AV SaOH at pH 11.5. Clewragc of the acctyl group was wscntially cotq)lctc in less than 60 min. at room tctuperaturc as is shown in Fig. 3 for a gelatin preparation. For prcparatirc purpow basic cleavage migltt bc ltarmful to the protein, and hence a variety of nitrogenous nucleophilcs n-erc examined. Some rcsuits with imidazole under tliffcrtnt contlitions are shown in Fig. 3 in comparison with sodium hydroxide. The rate of cleavage with imidazole is muclt slower t,han with aodiunt ltydroxitlc, but csscntially contpletc liberation of tnercaptan groups could 1~ obtained, at room tetnpcraturc and probably lower, in about 6 hr. Histatnine and hi&dine are about as effective as imidazole; aniline and p-nitrophenol arc ineffertive. On the other ltand, 0.01 X liydroxylaminc, at 1)H 7.2-7.8, is wry much better than the othrr nucleophilcs (Fig. 31, bring comparablc in action t,o sodium hydroxide at the same concentration. Furthermore, 0.1 M hydroxylamine produced essentially irnmediate removal of the acctyl group, tlir full complement, of tncrcaptan being drtcctablc

STJLFHTDRYL

GROUPS

within the first few minutes’ required to carry out an analysis. In general, the proteins have been kept with their original complement of acetylniercaptosuccinyl groups and the acetyl groups cleaved with 0.01 M NH,OH immediately prior to experiments in which fully liberated mercapt,an groups were desired. STABILITY

OF ACETYLTHIO ON PROTEIN

GROUP

group in S-acet~ylmcrcaptosuccinic anhydride keeps the thiol protect,ed during the coupling to the macromolecule and thereafter until one wishes to expose the mcrcaptan for further reactions. It is also possible to prepare a number of derivatives of succinic anhydride with other sobstituents in the CLpositions. These may provide routes for the introduction of other sulfur-cont’aining groups into proteins and Illac~ol~~olcclllcs.

In aqueous solution at pH 7.8 there was no significant cleavage of acetyl-S linkages on a gelatin preparation. Likewise, dialysis for several days versus water adjusted to pH 9 showed no hydrolysis. Significant hydrolysis was detect,able at pH’s above 10 and, as shown above, rapid cleavage was observed at pH 11.5. The masked behavior of mercaptan groups in proteins has often been attributed to their existence in some chemically modified form within the macromolecule, e.g., as a thiol ester (20). It seemed of interest, therefore to see if the acetylt,hio group was dcacylated by a denaturing medium, 8 &f urea. A sample of thiolated bovine strum albumin cont,aincd 2.6 free SH and 13 acctylthio groups per 10” g., determined by silver titrations in aqueous tria ( hydroxymethylj aminomet~hane buffer, still showed only 2.5 free SH groups in 8 31 urea, and the: number did not change over a period of an hour. It seems unlikely, therefore, that thcl kind of masking which can be stripped by urea is due t,o the presence of an arttyl group on a mercaptan side chain. These experiments, however, do not rule out the possibility of masking involving an acylthio linkage with a carboxyl group from an ainino acid rcsicluc within the same ljrotcin 11101cc111e.

It i:: apparent that this type of reaction is not limited to derivatives of succinic anhydride. 0th~ anhydrides readily suggest themselves. Some further possibilities are being examined, hut so far nothing superior in act,ual practice t’o t,he succinir anliydritlc has been obtained. Specific physical and chemiral properties of individual anhydrides l)lny an important, role in establishing the practical conditions undri \vhich react,ion will take place rapidly. Hcnrc it is difficult to predict whether a 1)articular anhydride will work, and one must carry out the actual coupling reaction to estnblisli its feasibility.

I.

E.,

3. 4. 5.

i.

8.

exposure

of thfx protein

to 0.1 JI hyc‘on-

tent. about 10% in 1 hr.

by.,

wnd. 201, 966 (1935). ~LlW!FIM.4SK, F., .~SD p,.
Ii., J.

(1042). Y~TI~~BERL,-1.. hrycw. Cl~cm. 60, 7 (1948). B~I~:scH, R.. .JSD BESKXH, R. E.. Z’,YIC. Sn(l. drritl. Sri. I‘. S. 44, 848 (1958). SIXER, s. J., ~OTISXtOILL, .J. E., .asn SFI.AISOF~, ,J. R., J. Am. C’I/(~m. Ser. 82, 565 (1960). .%BAI)I, D. hr., ASD \1'1i.IHERG, B., .4x) &!HJ%SBERG, E.. B&%T Kot~i, .Jli~~c).ul. Gwd. 14A, So. 7 (1’340).

cl. FIRsER, filr~ws.

drosplamine resulted in n slow tlrop of -SH

&EDERLE,

WISKLER.

6.

A suitably substituted succinic anhydride thus provides a convenient method for the introduction of mcrcapt~an groups into proteins under mild condit’ions. The acctyl

J., (‘urnld.

I~OISELEUH,

2. FHASZ,

CONCI,USIOS

‘Longer

61 I

IKTRODUCTIOK

10.

~~IlLX4SS,

11.

~'ORATH,

(l(350).

1,.

F..

AS11

&fARTIS,

p;.

I,.,

(jig.

15, 93 ( 1935). E., aA,r,f. 339, 371 (190.5). J.. ASII FLOI)IS, I'.. Sr/tir,~c~ 183,

,%//c-

1657

12. ~~SSC~II, R. E., hRDY, H. A., AS” BEKESC~I. R., J. Hiol. Chcm. 216, 663 (1955). 13. KLOTZ. I. M., Amas, J.. Ho, J. X7. C., HOROWITZ, M. G., AKD HEIYEY, R. E., .I. Am. Chcm. Sot. 80, 2132 (1958). 14. BENDER, M. L., .~ND TURXVQUEST, B. W., b. Am. Chem. Sot. 79, 1652 (1957). 15. BRUICE, T. C., AKD SCHWR. G. I,., J. Am. Chem. b’oc. 79, 1663 (1957). 16. HABEER, A. F. s. ak., CASSIDY, H. G., AND ,%NGER,

19. IiLoTz, 1. M.. AKD STRPKEH. V. H., Rio&em. Biophys. Kcsearch Cornn~~ms. 1, 119 (1959). 20. SJIITIT, E. L., J. Bid. Chem. 233, 1392 (1958).