- Email: [email protected]
Application of a specific-ion electrode to the determination of disulfide groups in proteins
.\NALYTICAL
BIOCHE~~WRY
Application
42, 398-404
of a Specific-Ion
Determination
of Disulfide
23. S. HARRAP Division
(1971)
of Protein
Electrode Groups
in Proteins
ASD L. C. GRUEN
Chemistry, CSIRO, Parkville Victoria 305.2, Austmlia Rereivcd
to the
Octohcr
(Melbourne),
30. 1970
There are now numerous met’hods available for the determination of -S-Sand -SH groups in proteins, based on spectrophotometry and amperometry (see comprehensive reviews by Benesch and Benesch (1) and Leach (2)). One of the simplest of these methods involves the amperometric titration of the reduced thiol group with silver ions, either at the rotating platinum electrode or at the dropping mercury electrode, but this method fell somewhat into disrepute in recent years becauseof doubts of the specificity and stoichiometry of the reaction between -SH and Ag+ (l-3). These doubts are mainly based on the variable stoichiometry of the reaction between silver ion and cysteine (4-6). However, it now seemsthat the Tris or ammonia buffers customarily used in investigations of this reaction may have an influence on its stoichiometry since Sluyterman (7) has shown t’hat the cspected I : 1 stoichiometry between Ag+ and cysteine can be obtained using imidazole buffers. Moreover, we have shown (8) that cyst’eine can be determined by argentometric titration using a specific-ion electrode as indicator, provided Tris or ammonia buffers are avoided. In view of these findings it seemed worthwhile reinvestigating the reaction between Ag+ and thiol groups in proteins following reduction of their disulfide group s, ancl of exploring the use of t’he Ag/S specific-ion electrorlc in this analysis. EXPERIMENTAL
Standard AgN03 solutions (O.lOOM) were prepared from B.D.H. ampules. Mercaptoethanol (Koch-Light), NaBH, (Metal Hydrides Inc.), and all other chemicals were either of SnalaR grade or the best available commercially and were used without further purification with the exception of tri-n-butylphosphine (Fluka purum) which was redist,illed prior to use. Deionized water was employed throughout. 398
The proteins used were bovine trypsin (General Biochemicals, 2X cry&, salt-freej , bovine pancreatic ribonuclcast~ (Sigma, cliromatographic grade type 2)) bovine P-lartoglohulin (Pentcs. tryst. 1, egg-white lysozymc (Sigma, 3 X tryst., and Armour, cry&), borinc, tu-chymotrypsin (Worthington Biochemic;ll Corp.), bovine Zn-insulin (C’ommonwcaltll Strum I,ahoratorics, cry&) and boviiic strum nllnmiin (Clommonwewltll Serum Laboratories, crpst.!. (0 j Jiefhod.~ (1) Protein solutions. All proteins wcrc dissolved in water (with the exception of insulin, which was dis;;olrcd in dilute HNC),) to give SO~Utions of concentration 2-2.5s. The concentrations of these solut’ions were calculated from their extinction coefficients in the 280 nm region using the following estinction coefficients (Eiz;,) hrllaY: trypsin (I 5.6) y;p (9) ; rihonuclease (7.1 I ziY (9) ; /3-lactoglol~ulin (9.4) z;x (IO) ; 1yWeymr (26.9)242 (11) ; tu-chymotryl&n (20.1)zhLl (12); insulin (I l.O),Y; (131 ; bovine serum albumin (6.6) ziq (9) (2) Redrtcfiorl of disulfide. In order to &mate t.he -S-Scontent of proteins hy tit8rntioii with -4%’ it il; first ncctlssary to convert the rlisuifide to the thiol form by rctluctiou. Commonly use~l reductants such as 2-iiierca~~toetliaiiol an11 tliio&colic acitl cannot, be ~niployed since the . I excess retluctant itself reacts wit11 -AR+. Previous workers in this field (14,15) hare used NaHSO, (in the prcsencc of urea) as reductant. We have observed t,hat, when an Ag;‘S qxxific-ion clcctrode is immersed in a solution of 0.4M NaHSO$3 M urea, the potential is about -100 rnV,l suggesting that t’he HSO,- was nlqxtciably lowcriug the activity of the Ag+ (in 8M urea the potent,ial was about +80 mY) ; for this reason we decided to investigate the use of other reducing agents. Furt’hermore, reduction with NaHSO,, producr~ only one --8H group from every -S-S-, anI1 to int,r(‘as(l the scnGtivity of the rnethocl it would clearly he preferable to choose :L retluctant which gencrnterl two tliiol group from each -S-Sgroup. Rcccnt ytudie:: in t.his laboratory (16-18) haye intlicntecl the u~cfulnc~s of tri-n-hutylphosphine (Bu,,P) as n recluctntlt for the disulfitlc !)onrl~ in proteins according to the equation: I: --S---S-
1: + lh:J
+ lT,O
-+ 2 RSH
+ BN~PO
However, iti model expcrirnt~uts itlvolving t,ho titration of simple t.hiols with -4g+, the presence of tri-n-I-~ut.pl~~hosphirle was found to interfere with tlitb location of the c11t1 1joint and the gt~ncr:~l sl~upe of the titration cilrvc. J1oi.covcrl sirict~ tri-)r-l~rit~l~~l~osl,liiiie is ininkihle IYitll t81icaqua’ This
suggests that the AC/~ clrc~lr~~tleis not cntirc,ly qx&ic
for
S
ous protein solution, frequent shakin g is necessary during t,he rctluction period, lessening t.he convenience of the method. The use of sodium borohydride, NaBH,, was introduced (I 9,20) as an attractive reductant for -S-Sgroups in proteins, yielding two -SH groups for each -S-Sand having the advantage that the excess rcduct’ant could be simply clestroyed by the addition of acid or of acetone. Although the method has disadvantages in preparative protein chemistry in that some peptidc bond hydrolysis occurs during the reduction procedure (21)) this is immaterial in the analysis for -S-Sgroul)s and has been so used for both ampcrometric (22) and calorimetric (23) clcterminat.ion of disulfide groups in proteins. It therefore seemed worthwhile to investigate the use of this reductant in the present study with a specific-ion electrod?. (3) Apparatus. An Orion model 94-16 ,4g/S specific-ion electrode was used in conjunction with a double-junction sleeve-type Ag/AgCl clect’rode (filled with 1 M IWO, as t’he bridging electrolyte) as a reference electrode. Potentials were mcasuretl with a Beckman Expandomatic pH meter. An Agla micrometer syringe (Burroughs Wellcome Ltd.) u-a:: used as a microburet. (4) Procedwre. Except as otherwise noted in the text, the -S-S+ -SH content of a protein was estimatetl by t,he following method, tcrmecl the %vo-stage reduct.ion procedure”: An aliquot of protein solution corresponding to about 5 ,kmoles -S-Swas added to 20 ml 8 X urea cont,aincd in a 50 ml beaker. To this was added 0.2 ml 0.1 M EI>TA and 0.05 gm KaBH, and 2 drops of capryl alcohol to prevent frothing. This mixture was incubated at 40°C in a thermostatted water bath for 30 min. Oxygen-free nitrogen ww passedthrough the solution during this period. After incubaCon t,he solution was acidified to about. pH 3 wit’111 M HNO, (the procedure up to t.his point would correspond to a “single-stage reduction”), and a measured volume of 0.1 M AgNO,, (about 50-80% of bhat required for stoichiometric reaction of Ag+ with the expected --8H groups) was added. The pH of the solut,ion was then increased to about, 9.0 by addition of 1 M KOH and a further 0.05 gm NaBH, was ad&d and allowed to react at room temperature for a further 15 min. -4 mixturct of 1 M HNO,/acetone (1: I by volume) was added to destroy excess NaBH, and to reduce the pH to about 3.0 and the solution was t,itrnted with 0.1 M ,4gNO,, using the spcicific-ion electrode a!: an indicator. RESULTS
AND DISCUSSION
Th(x influcncc of tcmprraturc anal time of incubation, quantity of NaBH I) and number of rccluction steps necessary for complete reaction of
-SH with XgNO,, llnvc~ b(s~n inr-cstig:ttkvl using l)oxinc serum albumin, which has 17 -S-Sant1 1 -SH Iwr n~olcculc of 1111’ 69,000 (14,15), as a model protein. X11 rwctioii~ were pcrformr~l in 5-8 M urea in order to minirnizt~ possible ma,~-king of librratcvl -SH groups and to maintain tliv rcrlurc~ll proteins iii wlution. Initial ~spcriiiiwt~ iiir-olving a single-stage reduction were lwrformed over times ranging front 15 to 60 min. The maximum number of titratablc --SH groups was founcl after 30 min, beyond which time fen-er -SH groups n-care available for titration with AgYO,, but even after 30 min only about 70980% of the cxpectcd number of -SH groups were titrated. Incubation was carried out at 25”, 40”, 50”, and 70”. The recovery of --SH groulw at 25” md 70” was ~onsidcrably 1~s than at 40” or 50”. Therrforc further cspcrimc~nts were conduct~ed at. 40°C. The decrease in the number of -SH groups formed at the higher temperatures or longer time.5 is prol~ably due to hydroly& of the excess NaBH, and reoxidation of some of the -SH groulw liberated. The maximum nunlber of titrntable --SH groulis w:w obt,ained when the concentration of XaBH., in the reduction solution was 0.25% (i.e., 0.05 gm in 20 ml solution). Beyond this amount there ww no increase in the number of titratable --SH groups. A xlight iiicreas:c in thr number of titratable --8H groups was obtained if a mensurccl exws of 0.1 X AgSO:, wt.‘: nddc~l at the conclusion of the reduction t after acidification) am1 thv csc~w XgNO,, back-titrated with standardized mercaptoethnnol solution. t,It has recently been shown (8) th:it mercal~toethanol may be stoichiomct8rically tikatecl with AgNO,, wing the Ag,‘S tslectrotlc :IS indicator.) This observation suggested that some --8H groups may bcl lost bctwccn completion of the reduction and the t’itration. An attempt was made to overcome this by a two-stage rcductioii in which an alicluot of -1gNO,,, equivalent to about, two-thirds of the expectcvl -SH groups, was added to the reduced anal acidified soluCon; this was then reincuhatecl with a further 0.05 gm of XaBH, after adjusting the pH to about 9.0. By taking 1.00 ml of 2.11% bovine serum albumin solmion and adding 0.070 ml of 0.100 JI ,4gNO, after the first reduction, a titration curve after the second reduction is obtained, as shown in Fig. 1. The total Xg+ con~:und (0.070 + 0.037 ml) corresponds to 35.0 [ (--S-S-),;21 + SH groups lwr molecule, which is in excellent agreement, with data obtained by other mctlioclx (14,15 1. This esperiment has becw rcprated swcral time. giving rcwlts within 5% of this value. A further experiment8 was pcrforine(l in orcler to check the c>ffect of a t,liree-stagcl reduction cyc~l~~011 bo\Tiiw 6(‘runi all)umin. Taking the same :uiionnt~ of protein aA nbo\-(1. tlw ~oluni~:: of 0.100 M L\gNO,: nclcled nft,er the first and second rccluction steps new 0.07 and 0.02 ml, reqwtively. Using 0.05 gm NaBH, in each of the three retlurtion steps, the second and
402
I OO
0.05
0.10 Vol 0.100 M AgNO,(mi
0.15 1
FIG. 1. Titration of thiol groups from reduced bovine serum albumin with O.lOOM AgKO,. A f.00 ml aliquot, of 2.11% bovine serum albumin was subjected to a twostage reduction (see text) and 0.07 ml 0.10044 AgN03 was added after the first. reduction stage.
third steps being carried out at room temperature, the final end point was 0.025 ml of O.lOOM .%gNO:,, giving a total volume of AgNOe of 0.115 ml, which is not significantly different from the value obtained in the two-stage reduction described above. (2) Two-Stage Reductions on Several Proteins The two-stage reduction procedure as outlined under “Esperimental” has been usetl to determine the number of -S-Sgroups in several proteins, giving the results outlinecl in Table 1. Wit,h the exrqjtion of lysozymc, the values are all within 73%of the Iiterakc values and indicate that, this is a feasible n&hod for the determination of disulfide in a number of proteins. The only difficulty that we have encountered is in the determination of the disulfide content of lysozyme, for which an apparent disulfide content of about 5 -S-Sgroups per mole was found, i.e., 25% higher than the expected value (28). The actual value determined was variable and appeared to depend on the quantity of AgNO, added at the end of the first reduction stage. Only marginal changes could bc t4ffrctrd 13~variation of ~rh paramett~t’s as incubation t,ime, number of reduction stages, use cif bisulfite instead of NaBH., as reductant, replacement, nf 8 dl
urea
as prot,ein
denat,urant.
by 15% sodium
dodecyl
sulfate
:; so 5.20 17.4 2.s-l
1: ibonuclense &kymot,rypain Serum nlhllmili Insulin Trypsin /3-Lactoglohulin Lysozgme
.i TS 2. tis (‘it. r-6
solution, and different, batches of lysozyme. Thrw tlwta suggest cithcr that, the silver ions react llor~stoiclliolllctric;zll~ with thcx thiol groulw or that t,hcy reac.t less specificxlly at other site:: 011 the lysozynw molecule. In order to test the lnttcr 1)ossihility we have compared the cft’ert 011 the potential at the Ag/S electrotle of the adrlition of unreduccll lysozyme 01 of unreduced bovine semn albumin (about 2.5% aqueous solution) to 0.1 ml of 0.1 M AgNO., in 20.0 ml of 8 JI urea. \~~YYW the ad(lition of bovine serum albumin had no effect (ot’her than that, cause11 by tlilutioll) 011 the potent,ial arising from the ,lgXC)., holution, the nd(lition of ly-ozyrne raused a marl4 change in potential. Thus it seems likely that the binding of silver iorls to other th:w --SH sites 011 l~wzy~~w m:~y account for the diflkultics ~xperienccd iI1 cjuantitntivcly assessing the --S--Smid --SH contciit, of this 1)rotriu. ACBSOWLEDCMENT The brown.
autllors
wisll
to acknowledge
tile
capable
technical
asAatnnc~(~
of blr.
A. 1~.
REFERENCES 1.
2.
3. 4.
5. 6. 7.
8.
R.. .ATI) &XXI~II. I~. b:.. i/c “~1etllori:: of l3iocll~mical Analysis” ((:livk. I>., cd.). Vol. 10. p. 43. Interscience, Sew York. 1962. Iz.\c~r. S. .J., &I “:\n:d,vtic,al Mvtllwls of Protein Cllc-nlistry” (.U~~ln(l~~r. I’.. ;111rl Imdprcw. H. I’.. c~ls.). Vol. 1. ,I. :2. Prrg:imon Pre*s, Elmsford, K. T., :1nti Oxford, 1966. CECIL, R., AND MCPHEE, J. It., Advctn. Z’roteir~ Chem. 14, 255 (1959). QLUYTERMAN, L. A E., Biochim. Biophys. Actn 25, 402 (1957). BLXTOS. H.. Biochir,~. Riophj/s. 9clrr 29. 193 (19jS). KOLTHOFF, I. M.. ASD EISENHTKIWKH. .I.. Arr~l. Chirn. Acfn 24, S3 (1961). ~I,IIYTlr:RM.~N. I,. zi. E:.. dart/. f~ir,chrr,/. 14, 317 (1966). (~RLIEN. I,. c‘.. AND HARHW, 13. S.. AM/. Hior/~c,nl. 42, 377 (1!)71). Ib:XESCH,
13.. i/c ‘LOpl id l~ol;~iory Ihpcb3ion of Pwteins and Olhor M:1rro9. .IlKc:lrssoss. 1~1olcc111rs.” 11. 32, Sllrillr(,r-V[,rln~. Krw York, 1969. lo. ~OZARI. ‘I’.. ~~1TNVILI.I.:. I,. C;.. AND T.INFOIW, C.. J. il/rcr,/.. (‘hvvz. Sot. 81, 5523 I 1959 1 11. HAYASHI. Ii.. IMOTO, II., AKIJ F~~K~sII, M., J. Biochem. (Il’okyo) 54, 281 (1963). 12. WINZOR. D. J.. .\SD SCIIERAGA, H. A.. Biochemistry 2, 1263 (1963). 13. HUMAN. J. P. E.. ANLI IXACH, S. J.. Aust. ,f. Chem. 14, 169 (1961). 14. CARTER. J. R.. J. Sol. (‘hem. 234, 1705 (1959). 15. KOLTI-~OFF. I. M.. AS.IST.MI. A.. I~~~ TIN, B. H., J. Amer. Chem. Sot. 80, 3235 (1958). 16. SWEICT~~AN, 13. J.. AA-II MACLAREN, J. A., Aust. J. Chem. 19, 2347 (1966). 17. MACLAKIGK, J. A.. ASD SWEETMAN, 13. J.. AM. J. Chew 19, 2355 (1966). 18. MA(~LAREP;, J. .-2.. KILPATRICK, D. J.. AND KIRKPATRICK. A., Aust. J. Biol. sci. 21, SO5 (1968). 19. MOORE. S.. COLE, R. I)., GUIYDLACH, H. G., AND STEIN, W. H., Proc. Int. Congr. Biochem. ,jfh, 1.953 8, 52. 20. GILLESPIE. J. M.. Nature 183, 322 (1959). 21. GILIXWIF:. .J. M.. O’DONNELL, I. J., THOMPSON, E. 0. P., AND WOODS, E. F., J. Test. Inst. 51, T 703 (1960). 22. BROWX. UT. I).. Binchim. Biophys. Acln 44, 365 (1960). 23. ~AV.~I,I,IYI, D.. (:RAZI.~SI, M. T.. AXD DUPRB. S.. Nature 212, 294 (1966). 24. HIRS. C. H. W., MOOHI~. 8.. AND STEIN, W. H.. J. Biol. Chem. 235, 633 (1960). 25. WILCOX. P. E.. COHKK, E.. .~ND TAN, W., J. Biol. Chem. 228, 999 (1957). 26. RYLE, A. P.. SINGER. F.. SMITH. L. E’.. BND RITAI. R., Biochem. J. 60, 541 (1955). 27. WEIL, L.. ASD SEIBLI;~. T. S., Arch. Biochem. Biophgs. 84, 244 (1959). 28. JOLL$S. J.. BPRNNR. I., JAUREG~I. J., .~ND JOLLES. P., C. R. Acnd. Sci. Pariq 250, 413 (1960).
BIOCHE~~WRY
Application
42, 398-404
of a Specific-Ion
Determination
of Disulfide
23. S. HARRAP Division
(1971)
of Protein
Electrode Groups
in Proteins
ASD L. C. GRUEN
Chemistry, CSIRO, Parkville Victoria 305.2, Austmlia Rereivcd
to the
Octohcr
(Melbourne),
30. 1970
There are now numerous met’hods available for the determination of -S-Sand -SH groups in proteins, based on spectrophotometry and amperometry (see comprehensive reviews by Benesch and Benesch (1) and Leach (2)). One of the simplest of these methods involves the amperometric titration of the reduced thiol group with silver ions, either at the rotating platinum electrode or at the dropping mercury electrode, but this method fell somewhat into disrepute in recent years becauseof doubts of the specificity and stoichiometry of the reaction between -SH and Ag+ (l-3). These doubts are mainly based on the variable stoichiometry of the reaction between silver ion and cysteine (4-6). However, it now seemsthat the Tris or ammonia buffers customarily used in investigations of this reaction may have an influence on its stoichiometry since Sluyterman (7) has shown t’hat the cspected I : 1 stoichiometry between Ag+ and cysteine can be obtained using imidazole buffers. Moreover, we have shown (8) that cyst’eine can be determined by argentometric titration using a specific-ion electrode as indicator, provided Tris or ammonia buffers are avoided. In view of these findings it seemed worthwhile reinvestigating the reaction between Ag+ and thiol groups in proteins following reduction of their disulfide group s, ancl of exploring the use of t’he Ag/S specific-ion electrorlc in this analysis. EXPERIMENTAL
Standard AgN03 solutions (O.lOOM) were prepared from B.D.H. ampules. Mercaptoethanol (Koch-Light), NaBH, (Metal Hydrides Inc.), and all other chemicals were either of SnalaR grade or the best available commercially and were used without further purification with the exception of tri-n-butylphosphine (Fluka purum) which was redist,illed prior to use. Deionized water was employed throughout. 398
The proteins used were bovine trypsin (General Biochemicals, 2X cry&, salt-freej , bovine pancreatic ribonuclcast~ (Sigma, cliromatographic grade type 2)) bovine P-lartoglohulin (Pentcs. tryst. 1, egg-white lysozymc (Sigma, 3 X tryst., and Armour, cry&), borinc, tu-chymotrypsin (Worthington Biochemic;ll Corp.), bovine Zn-insulin (C’ommonwcaltll Strum I,ahoratorics, cry&) and boviiic strum nllnmiin (Clommonwewltll Serum Laboratories, crpst.!. (0 j Jiefhod.~ (1) Protein solutions. All proteins wcrc dissolved in water (with the exception of insulin, which was dis;;olrcd in dilute HNC),) to give SO~Utions of concentration 2-2.5s. The concentrations of these solut’ions were calculated from their extinction coefficients in the 280 nm region using the following estinction coefficients (Eiz;,) hrllaY: trypsin (I 5.6) y;p (9) ; rihonuclease (7.1 I ziY (9) ; /3-lactoglol~ulin (9.4) z;x (IO) ; 1yWeymr (26.9)242 (11) ; tu-chymotryl&n (20.1)zhLl (12); insulin (I l.O),Y; (131 ; bovine serum albumin (6.6) ziq (9) (2) Redrtcfiorl of disulfide. In order to &mate t.he -S-Scontent of proteins hy tit8rntioii with -4%’ it il; first ncctlssary to convert the rlisuifide to the thiol form by rctluctiou. Commonly use~l reductants such as 2-iiierca~~toetliaiiol an11 tliio&colic acitl cannot, be ~niployed since the . I excess retluctant itself reacts wit11 -AR+. Previous workers in this field (14,15) hare used NaHSO, (in the prcsencc of urea) as reductant. We have observed t,hat, when an Ag;‘S qxxific-ion clcctrode is immersed in a solution of 0.4M NaHSO$3 M urea, the potential is about -100 rnV,l suggesting that t’he HSO,- was nlqxtciably lowcriug the activity of the Ag+ (in 8M urea the potent,ial was about +80 mY) ; for this reason we decided to investigate the use of other reducing agents. Furt’hermore, reduction with NaHSO,, producr~ only one --8H group from every -S-S-, anI1 to int,r(‘as(l the scnGtivity of the rnethocl it would clearly he preferable to choose :L retluctant which gencrnterl two tliiol group from each -S-Sgroup. Rcccnt ytudie:: in t.his laboratory (16-18) haye intlicntecl the u~cfulnc~s of tri-n-hutylphosphine (Bu,,P) as n recluctntlt for the disulfitlc !)onrl~ in proteins according to the equation: I: --S---S-
1: + lh:J
+ lT,O
-+ 2 RSH
+ BN~PO
However, iti model expcrirnt~uts itlvolving t,ho titration of simple t.hiols with -4g+, the presence of tri-n-I-~ut.pl~~hosphirle was found to interfere with tlitb location of the c11t1 1joint and the gt~ncr:~l sl~upe of the titration cilrvc. J1oi.covcrl sirict~ tri-)r-l~rit~l~~l~osl,liiiie is ininkihle IYitll t81icaqua’ This
suggests that the AC/~ clrc~lr~~tleis not cntirc,ly qx&ic
for
S
ous protein solution, frequent shakin g is necessary during t,he rctluction period, lessening t.he convenience of the method. The use of sodium borohydride, NaBH,, was introduced (I 9,20) as an attractive reductant for -S-Sgroups in proteins, yielding two -SH groups for each -S-Sand having the advantage that the excess rcduct’ant could be simply clestroyed by the addition of acid or of acetone. Although the method has disadvantages in preparative protein chemistry in that some peptidc bond hydrolysis occurs during the reduction procedure (21)) this is immaterial in the analysis for -S-Sgroul)s and has been so used for both ampcrometric (22) and calorimetric (23) clcterminat.ion of disulfide groups in proteins. It therefore seemed worthwhile to investigate the use of this reductant in the present study with a specific-ion electrod?. (3) Apparatus. An Orion model 94-16 ,4g/S specific-ion electrode was used in conjunction with a double-junction sleeve-type Ag/AgCl clect’rode (filled with 1 M IWO, as t’he bridging electrolyte) as a reference electrode. Potentials were mcasuretl with a Beckman Expandomatic pH meter. An Agla micrometer syringe (Burroughs Wellcome Ltd.) u-a:: used as a microburet. (4) Procedwre. Except as otherwise noted in the text, the -S-S+ -SH content of a protein was estimatetl by t,he following method, tcrmecl the %vo-stage reduct.ion procedure”: An aliquot of protein solution corresponding to about 5 ,kmoles -S-Swas added to 20 ml 8 X urea cont,aincd in a 50 ml beaker. To this was added 0.2 ml 0.1 M EI>TA and 0.05 gm KaBH, and 2 drops of capryl alcohol to prevent frothing. This mixture was incubated at 40°C in a thermostatted water bath for 30 min. Oxygen-free nitrogen ww passedthrough the solution during this period. After incubaCon t,he solution was acidified to about. pH 3 wit’111 M HNO, (the procedure up to t.his point would correspond to a “single-stage reduction”), and a measured volume of 0.1 M AgNO,, (about 50-80% of bhat required for stoichiometric reaction of Ag+ with the expected --8H groups) was added. The pH of the solut,ion was then increased to about, 9.0 by addition of 1 M KOH and a further 0.05 gm NaBH, was ad&d and allowed to react at room temperature for a further 15 min. -4 mixturct of 1 M HNO,/acetone (1: I by volume) was added to destroy excess NaBH, and to reduce the pH to about 3.0 and the solution was t,itrnted with 0.1 M ,4gNO,, using the spcicific-ion electrode a!: an indicator. RESULTS
AND DISCUSSION
Th(x influcncc of tcmprraturc anal time of incubation, quantity of NaBH I) and number of rccluction steps necessary for complete reaction of
-SH with XgNO,, llnvc~ b(s~n inr-cstig:ttkvl using l)oxinc serum albumin, which has 17 -S-Sant1 1 -SH Iwr n~olcculc of 1111’ 69,000 (14,15), as a model protein. X11 rwctioii~ were pcrformr~l in 5-8 M urea in order to minirnizt~ possible ma,~-king of librratcvl -SH groups and to maintain tliv rcrlurc~ll proteins iii wlution. Initial ~spcriiiiwt~ iiir-olving a single-stage reduction were lwrformed over times ranging front 15 to 60 min. The maximum number of titratablc --SH groups was founcl after 30 min, beyond which time fen-er -SH groups n-care available for titration with AgYO,, but even after 30 min only about 70980% of the cxpectcd number of -SH groups were titrated. Incubation was carried out at 25”, 40”, 50”, and 70”. The recovery of --SH groulw at 25” md 70” was ~onsidcrably 1~s than at 40” or 50”. Therrforc further cspcrimc~nts were conduct~ed at. 40°C. The decrease in the number of -SH groups formed at the higher temperatures or longer time.5 is prol~ably due to hydroly& of the excess NaBH, and reoxidation of some of the -SH groulw liberated. The maximum nunlber of titrntable --SH groulis w:w obt,ained when the concentration of XaBH., in the reduction solution was 0.25% (i.e., 0.05 gm in 20 ml solution). Beyond this amount there ww no increase in the number of titratable --SH groups. A xlight iiicreas:c in thr number of titratable --8H groups was obtained if a mensurccl exws of 0.1 X AgSO:, wt.‘: nddc~l at the conclusion of the reduction t after acidification) am1 thv csc~w XgNO,, back-titrated with standardized mercaptoethnnol solution. t,It has recently been shown (8) th:it mercal~toethanol may be stoichiomct8rically tikatecl with AgNO,, wing the Ag,‘S tslectrotlc :IS indicator.) This observation suggested that some --8H groups may bcl lost bctwccn completion of the reduction and the t’itration. An attempt was made to overcome this by a two-stage rcductioii in which an alicluot of -1gNO,,, equivalent to about, two-thirds of the expectcvl -SH groups, was added to the reduced anal acidified soluCon; this was then reincuhatecl with a further 0.05 gm of XaBH, after adjusting the pH to about 9.0. By taking 1.00 ml of 2.11% bovine serum albumin solmion and adding 0.070 ml of 0.100 JI ,4gNO, after the first reduction, a titration curve after the second reduction is obtained, as shown in Fig. 1. The total Xg+ con~:und (0.070 + 0.037 ml) corresponds to 35.0 [ (--S-S-),;21 + SH groups lwr molecule, which is in excellent agreement, with data obtained by other mctlioclx (14,15 1. This esperiment has becw rcprated swcral time. giving rcwlts within 5% of this value. A further experiment8 was pcrforine(l in orcler to check the c>ffect of a t,liree-stagcl reduction cyc~l~~011 bo\Tiiw 6(‘runi all)umin. Taking the same :uiionnt~ of protein aA nbo\-(1. tlw ~oluni~:: of 0.100 M L\gNO,: nclcled nft,er the first and second rccluction steps new 0.07 and 0.02 ml, reqwtively. Using 0.05 gm NaBH, in each of the three retlurtion steps, the second and
402
I OO
0.05
0.10 Vol 0.100 M AgNO,(mi
0.15 1
FIG. 1. Titration of thiol groups from reduced bovine serum albumin with O.lOOM AgKO,. A f.00 ml aliquot, of 2.11% bovine serum albumin was subjected to a twostage reduction (see text) and 0.07 ml 0.10044 AgN03 was added after the first. reduction stage.
third steps being carried out at room temperature, the final end point was 0.025 ml of O.lOOM .%gNO:,, giving a total volume of AgNOe of 0.115 ml, which is not significantly different from the value obtained in the two-stage reduction described above. (2) Two-Stage Reductions on Several Proteins The two-stage reduction procedure as outlined under “Esperimental” has been usetl to determine the number of -S-Sgroups in several proteins, giving the results outlinecl in Table 1. Wit,h the exrqjtion of lysozymc, the values are all within 73%of the Iiterakc values and indicate that, this is a feasible n&hod for the determination of disulfide in a number of proteins. The only difficulty that we have encountered is in the determination of the disulfide content of lysozyme, for which an apparent disulfide content of about 5 -S-Sgroups per mole was found, i.e., 25% higher than the expected value (28). The actual value determined was variable and appeared to depend on the quantity of AgNO, added at the end of the first reduction stage. Only marginal changes could bc t4ffrctrd 13~variation of ~rh paramett~t’s as incubation t,ime, number of reduction stages, use cif bisulfite instead of NaBH., as reductant, replacement, nf 8 dl
urea
as prot,ein
denat,urant.
by 15% sodium
dodecyl
sulfate
:; so 5.20 17.4 2.s-l
1: ibonuclense &kymot,rypain Serum nlhllmili Insulin Trypsin /3-Lactoglohulin Lysozgme
.i TS 2. tis (‘it. r-6
solution, and different, batches of lysozyme. Thrw tlwta suggest cithcr that, the silver ions react llor~stoiclliolllctric;zll~ with thcx thiol groulw or that t,hcy reac.t less specificxlly at other site:: 011 the lysozynw molecule. In order to test the lnttcr 1)ossihility we have compared the cft’ert 011 the potential at the Ag/S electrotle of the adrlition of unreduccll lysozyme 01 of unreduced bovine semn albumin (about 2.5% aqueous solution) to 0.1 ml of 0.1 M AgNO., in 20.0 ml of 8 JI urea. \~~YYW the ad(lition of bovine serum albumin had no effect (ot’her than that, cause11 by tlilutioll) 011 the potent,ial arising from the ,lgXC)., holution, the nd(lition of ly-ozyrne raused a marl4 change in potential. Thus it seems likely that the binding of silver iorls to other th:w --SH sites 011 l~wzy~~w m:~y account for the diflkultics ~xperienccd iI1 cjuantitntivcly assessing the --S--Smid --SH contciit, of this 1)rotriu. ACBSOWLEDCMENT The brown.
autllors
wisll
to acknowledge
tile
capable
technical
asAatnnc~(~
of blr.
A. 1~.
REFERENCES 1.
2.
3. 4.
5. 6. 7.
8.
R.. .ATI) &XXI~II. I~. b:.. i/c “~1etllori:: of l3iocll~mical Analysis” ((:livk. I>., cd.). Vol. 10. p. 43. Interscience, Sew York. 1962. Iz.\c~r. S. .J., &I “:\n:d,vtic,al Mvtllwls of Protein Cllc-nlistry” (.U~~ln(l~~r. I’.. ;111rl Imdprcw. H. I’.. c~ls.). Vol. 1. ,I. :2. Prrg:imon Pre*s, Elmsford, K. T., :1nti Oxford, 1966. CECIL, R., AND MCPHEE, J. It., Advctn. Z’roteir~ Chem. 14, 255 (1959). QLUYTERMAN, L. A E., Biochim. Biophys. Actn 25, 402 (1957). BLXTOS. H.. Biochir,~. Riophj/s. 9clrr 29. 193 (19jS). KOLTHOFF, I. M.. ASD EISENHTKIWKH. .I.. Arr~l. Chirn. Acfn 24, S3 (1961). ~I,IIYTlr:RM.~N. I,. zi. E:.. dart/. f~ir,chrr,/. 14, 317 (1966). (~RLIEN. I,. c‘.. AND HARHW, 13. S.. AM/. Hior/~c,nl. 42, 377 (1!)71). Ib:XESCH,
13.. i/c ‘LOpl id l~ol;~iory Ihpcb3ion of Pwteins and Olhor M:1rro9. .IlKc:lrssoss. 1~1olcc111rs.” 11. 32, Sllrillr(,r-V[,rln~. Krw York, 1969. lo. ~OZARI. ‘I’.. ~~1TNVILI.I.:. I,. C;.. AND T.INFOIW, C.. J. il/rcr,/.. (‘hvvz. Sot. 81, 5523 I 1959 1 11. HAYASHI. Ii.. IMOTO, II., AKIJ F~~K~sII, M., J. Biochem. (Il’okyo) 54, 281 (1963). 12. WINZOR. D. J.. .\SD SCIIERAGA, H. A.. Biochemistry 2, 1263 (1963). 13. HUMAN. J. P. E.. ANLI IXACH, S. J.. Aust. ,f. Chem. 14, 169 (1961). 14. CARTER. J. R.. J. Sol. (‘hem. 234, 1705 (1959). 15. KOLTI-~OFF. I. M.. AS.IST.MI. A.. I~~~ TIN, B. H., J. Amer. Chem. Sot. 80, 3235 (1958). 16. SWEICT~~AN, 13. J.. AA-II MACLAREN, J. A., Aust. J. Chem. 19, 2347 (1966). 17. MACLAKIGK, J. A.. ASD SWEETMAN, 13. J.. AM. J. Chew 19, 2355 (1966). 18. MA(~LAREP;, J. .-2.. KILPATRICK, D. J.. AND KIRKPATRICK. A., Aust. J. Biol. sci. 21, SO5 (1968). 19. MOORE. S.. COLE, R. I)., GUIYDLACH, H. G., AND STEIN, W. H., Proc. Int. Congr. Biochem. ,jfh, 1.953 8, 52. 20. GILLESPIE. J. M.. Nature 183, 322 (1959). 21. GILIXWIF:. .J. M.. O’DONNELL, I. J., THOMPSON, E. 0. P., AND WOODS, E. F., J. Test. Inst. 51, T 703 (1960). 22. BROWX. UT. I).. Binchim. Biophys. Acln 44, 365 (1960). 23. ~AV.~I,I,IYI, D.. (:RAZI.~SI, M. T.. AXD DUPRB. S.. Nature 212, 294 (1966). 24. HIRS. C. H. W., MOOHI~. 8.. AND STEIN, W. H.. J. Biol. Chem. 235, 633 (1960). 25. WILCOX. P. E.. COHKK, E.. .~ND TAN, W., J. Biol. Chem. 228, 999 (1957). 26. RYLE, A. P.. SINGER. F.. SMITH. L. E’.. BND RITAI. R., Biochem. J. 60, 541 (1955). 27. WEIL, L.. ASD SEIBLI;~. T. S., Arch. Biochem. Biophgs. 84, 244 (1959). 28. JOLL$S. J.. BPRNNR. I., JAUREG~I. J., .~ND JOLLES. P., C. R. Acnd. Sci. Pariq 250, 413 (1960).