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Spectrophotometric determination of thiols and disulfides with N-ethylmaleinimide
\NALYTICAL
~IOCI~E~LIS’IXY
Spectrophotometric
15, M--1.l
(19661
Determination of Thiols with N-Ethylmaleinimide
Receiveti October
and
Disulfides
4, 1065
TIIC tlrtection and qunntitntivc determination of disulfide compounds i:: usually bnscd upon their reduction and further eatimat,ion as sulfhydryl~. Jn this way tllcb more commonly used nit~ropruseide test (1, 2) and Folin-Mnrenzi reaction (3-G) have been adapted for disulfide dctcction. I\‘~~~crtlwlw ~~-1lcn botll thiols and disulfidcs are present in the solut’ion to be analyzed, the nitroprusside test is practically useless, and the data obtained with the Folin-Mxrcnzi reaction, with and mit,hout added bisulfite, arc oftcln unreliable. Tt is not always possible, indeed, to have a direct correlation bctjwecn tlw 1~1ol:tr extinction of sulfides and clisulfides; that, is. a moldy of di*ulfide does not giTc. under certain experimental conditions, Ihc xmie :Ibl;orbancy :w :L mole of tliiol; moreover. tdle molar absorbnncy produced by thiols varks in the prcwnce or absence of bisulfite. The Folin-illarcnzi reaction, which is uwful in thcl quantitnt,ivc estimat,ion of thiola and disulfidcw xhcn prwcnnt indivirllwlly, is thus of small value for mixtures. Recently we had the opportuniiy to test the reliability of the spectrophotometric rlctcrmin:~tion of thiol:: wit11 .\--ctl~glulnl(~irli~~ii(le (NEM), described by Rotjtbrt:: xnd I~o~wc~~ (7), nhc~n :~l)plic~~l to thcl tletcction of disulfider. Owing to the good result;; oht:Gnctl and tlrr knp1icit.y of the mcthotl, we lwlicw that, it will be uwful to clcwril)ca it in drt:Gl.
.V-Ettlylmnlcininrillc wah p~~rch:~wtl from Schwarz Laboratoriw. Incs.. .\Iount Vernon. N. 5’. Cystcinc h;\-tlrochloritlc nns a l)roduct, of Merck. acre ohtninc~cl from E’luka. anal Cystinc, cyk5teaininc~. and cykamincl oxidized glut,nthione frown 13oc~hringrr uncl Soc~lmc~. All the other coinpoun~l~ XYVI’(Jco~llnicwk~l product*, analytical grade.
~lescrilwcl by Eoberts and Rouser (5) gave \.cry goocl, 11igl11~ IYI)YO~lucihle t~wlt.~;. Furt,hcr we ha~c tried to adapt the reaction to the :inalysis of tlrcl corresponding disulfitles nftcr sulfitolybis or cpanolysis, :rcwn~tlin~ to t II(’ folln&ig equations: I: s--s
1: -~ Sl ),,
1: -,3--s ~1: - (‘1
. I: -s
$0.. + 1:s
4 1(--S- C’S + 1:s
Kincr the 1)It optimuiil for tlic K\;Ei\I-lllioi rc,actiori i.G alwllt 6, 0111‘ fip.., effort. in\:olved t.lic llh(’ of l&ulfite :I< rc~ciucing :lgc’nt, \vitli tllc :tini (Jf
CmTyiIlg
out,
hOth
tllc’
hlllfit(JlysiS
:111t 1 tllP
COU~~hg
TT.ith
xk:lI
ii1
the
same t~uffcwtl mo(liulli. ‘I‘lic r<+lilt+. ot)t:~inc~ti w(‘r( c*ol~ll)l~+t~ly nc~gative, sinr(l bisulflte c;111~c’d ;I ch~l’c%W ill tllcb :i\Js;cJl’lJ:tllC’y Of slulfitc \w:: tliwc~forc~ rcjcctcd, ancl TV(’ t,ricd, more succcs~fully, tile use 0C cy:mitl(~ :I:: rc’(lwing :Igwt. ~Oml~l~tC! ~)WddO1vll Ol lhdfifics I)y I\-(-‘1 rcxcluircs an nlkalinc pH (8) and therefor? the cbtiiiration of ;I clisulfilk t:tlic>s l)lxce in two Steps: first tllc cyunoly&, n-hicli is allon-ctl to oc(011r in 1 L\. :~nimonin; second the coupling with NE11 of the rcwlting tlliol, for Tv-llicll rcnc.tion n pI1 6 buffered medium is necewrry to avoid :~lBalinc liytli~olyxis of SEAl. The o\-er-all reaction ~ws tlicteforc pcrformcd :w follow::: 0.1 to 0.5 ,Lmolc of disulfide disl;ol\-cd in 0.3 1111I[,0 w:w nd~le~d wit11 0.5 ml of 5% KCN in 1 LY NH,. After stunding 30 min at roo~n tcmlwr:lturc, 1 1111of 0.5 A7 ncet,ic acid and 2 nil of 3 ,y 10 ,’ Al K\;EAI tlissolvctl in 0.5 JI phwphate buffer, pH 6, were added in that order. ‘l’lle OD of t11~ sollltion at 300 nip was then recordecl. The final conwntr:ltion of NE11 ( 1.5 X 10 ‘I J1) was chodxi to giw an OD value of around 1 ~vhcn clctcnnincd in l-cm light p:~tll cells (Beckman DU spectrophotometer) in the abwncc of thiolr. Preliminary checks clcmonstrntcd th:lt tlic molar c5tinrtion of NISi\I in 0.1, 0.23, and 0.5 M plloq&ate bufic~r, pH ti, and in tire boll&on containing also KCX ant1 :min~oniuni acetate, remnincd unchanged ; the value of 620 (9) was cc~nfirmcd un~kr our conditione with nccuratcly weighed samples. In regard to the cyanolysis of the tlisulfitlc~, prc~lilriinnry c~spc’riinciit> tlint thr mlction rcquirc(l with 1% KCK solution in 1 -\- XII,: shon-ccl times to go to romplction as long :lF :tbout 90 inin t’or rystnminc> an<1 3 hr for cystine. Increasing tlic concc~nt1rat8ion of th(s xltlcd cyanide up to 5% resultrd in :t shortenin, cr of the rcnction tilllc; :~ntl, :IS i5 shown iii Fig. 1, 30 min is sufficient for the cyanolysis of all thrtc disulfidw tested, which show different reaction rates. Figure 2 show the lincnrity hctwcn the values of A OD and micromoles of clisulfitlcs. :w x--cl1 :w thy mnlal correspondance between thiole and disulfides.
42
FIG. 1. Time course reaction-Reduction of disulfides by KCN: (0) cystnminc ; (0) glutathione; (0) cystine. To 5 prnoles cli.wlfide in 0.5 ml were added 0.5 ml .5% KCN in 1 N NH,, and-at the indicated times-1 ml 0.5 N acetic acid and 2 ml 3 X lO~“Jif NEM in 0.5 IV phosphate buffer, IIH 6. 01). xt 300 mp (l-cm light path ~11s) was subtracted from OD of a solution 1.5 x ‘lo-“111 NEM to obtain LIOD. For glutatjhione, reduction by KCS ~IVCS riw to a alight increase in OD at 300 mp; lhewforc: the vnluw wporled hnve bcrn correct4 for nbsorbancy recorded in the absence of NJXM (~~hosphntc bufffrr added instead of SEM).
Bccausc the methotl l~rovctl reliable for the detcct,ion of disulfides it was also t’cdtcd for their detection in the presence of thiols. For this purpose, from stock solutions containing 10-‘111 cyatinc or cysteine, aliquots were mixed to obtain varying amounts of both compounds (Table 1). In thcsc solutions the t,liiol was determined by ntlding, to 0.5 ml, 1.5 ml of H,O and 2 ml of 3 X lo-” M KEAI, and the disulfide by adding as described above, to another 01.5-ml aliquot, 5% IiCN and after 20 min acetic acid and NEM. From the following equations the amounts of sulfides and disulfides may then br calculated: gmolcs ---SH/ml pmoles -S--S--/ml
=
AOD (-KCN, 620 X lo-”
=
AOD (+IiCN) - A011 (-IiCN) 620 x lo-”
in which AOD (--EiC?u’) = difference in optical density recorded between a blank containing the same quantity of NEM and no thiol, and the test performed in the absence of KCN; and Aon (+E;CNl = dif-
DETERMIX:1TIOK
OF
THIOLS
4ND
43
DISCLFIDICS
0,8-
0.5
1
p moles/ml
(FIN
cone)
FIG. 2. Molar rorrcsllontl(,111,c hetlvveen thiols and disulfides-A(~) D = thcrease in atxorbancy of -UEM solution Cl..5 >( IO-“~11) as function of cysteinc (0). cystine CO), or cystnminc (9) conccntr:ktion. For erpcrimentnl conditions see test.
TABLE DETERRIIS~TIOS
OF TIIIOLS
1 .INI)
0.5 ml of solui ion containing the specifietl amomlts with (A) 1.5 ml Hz0 and 2 ml 3 x 1OF 11 SE21 in the determination of 1he thic~l: (RI 0.5 ml 5y0 Kc’Ii room temperature, I ml O..i S avrlic arid and 2 ml in OD in respect to a solution 1.5 X 10-3 31 ?;15?ll in
s-s
(+I&,
5
0
.-I 1 : !
0.00
0.3
0, i!l.i 0.il.i
O.%O
4.5
07S.i
4 60
0.45
4 :: 5 :;
1
O.ti3,i
0.7sn
4.0s
0 .!I3
1 5 2 2 5
0. ,541)
0. i!lO
3.4s
1.60
0.43~ 0.400
0,775
2 09
1 .!)O
0. ii5
‘2.5s
3 35 4
0.320 0.235
0.235 O.iS5
0. 1FO
0. ii0
2.06 1.5s 1.03
4 5
0 .06.i
0
0.41
5
0.005
U.7iO
2.41 3.00 3.54 3.93 4.60 4.9G
2 5 2
1.5 1 0.5 0 .~
.\ c -1iCS)
DISVLFIDES
of qsteine and cystine were added (EI ,I1 phosphate buffer, pH 6, for in 1 S SHJ anti, after 30 min at SI5hI solution; AOD = difference the presence of t,he same reagents.
-____
is0
0 no
ference in optical density hctmen the blank and the test performed in the lmxnce of KCN. The results obtained in these terts inclicate that the method is reliable for the detection of cliwlficlc!: in the prmcnce of even n greater amount of eulfitles. anI1 Tier vcrs:~.
The WC of KEhI for tile qunntitati~~c ~~~cct~~ol~hotornetric estimation of thiols has :rlrcatly been mpha~~izetl. In thr Imxnt pnpcr it is shown that this methotl may be successfully nd:tpted to 111~c&nation of rlisulfides, even when in the presence of thiols.
h sirnplc ant1 rapid slmtrophotoinetric technique for the quantitation of thiols and tlisulfitlcs has been clexribcd. The method involves the reartion with ,\‘-ctl~yln~:~leinirnidc before :incI after trentnwnt, n-it11 KCN, and is suitable for qunntitntive rccox-cry of both thiolr ant1 diwlficles from solutions in which they nre present together. REFERENCES
~IOCI~E~LIS’IXY
Spectrophotometric
15, M--1.l
(19661
Determination of Thiols with N-Ethylmaleinimide
Receiveti October
and
Disulfides
4, 1065
TIIC tlrtection and qunntitntivc determination of disulfide compounds i:: usually bnscd upon their reduction and further eatimat,ion as sulfhydryl~. Jn this way tllcb more commonly used nit~ropruseide test (1, 2) and Folin-Mnrenzi reaction (3-G) have been adapted for disulfide dctcction. I\‘~~~crtlwlw ~~-1lcn botll thiols and disulfidcs are present in the solut’ion to be analyzed, the nitroprusside test is practically useless, and the data obtained with the Folin-Mxrcnzi reaction, with and mit,hout added bisulfite, arc oftcln unreliable. Tt is not always possible, indeed, to have a direct correlation bctjwecn tlw 1~1ol:tr extinction of sulfides and clisulfides; that, is. a moldy of di*ulfide does not giTc. under certain experimental conditions, Ihc xmie :Ibl;orbancy :w :L mole of tliiol; moreover. tdle molar absorbnncy produced by thiols varks in the prcwnce or absence of bisulfite. The Folin-illarcnzi reaction, which is uwful in thcl quantitnt,ivc estimat,ion of thiola and disulfidcw xhcn prwcnnt indivirllwlly, is thus of small value for mixtures. Recently we had the opportuniiy to test the reliability of the spectrophotometric rlctcrmin:~tion of thiol:: wit11 .\--ctl~glulnl(~irli~~ii(le (NEM), described by Rotjtbrt:: xnd I~o~wc~~ (7), nhc~n :~l)plic~~l to thcl tletcction of disulfider. Owing to the good result;; oht:Gnctl and tlrr knp1icit.y of the mcthotl, we lwlicw that, it will be uwful to clcwril)ca it in drt:Gl.
.V-Ettlylmnlcininrillc wah p~~rch:~wtl from Schwarz Laboratoriw. Incs.. .\Iount Vernon. N. 5’. Cystcinc h;\-tlrochloritlc nns a l)roduct, of Merck. acre ohtninc~cl from E’luka. anal Cystinc, cyk5teaininc~. and cykamincl oxidized glut,nthione frown 13oc~hringrr uncl Soc~lmc~. All the other coinpoun~l~ XYVI’(Jco~llnicwk~l product*, analytical grade.
~lescrilwcl by Eoberts and Rouser (5) gave \.cry goocl, 11igl11~ IYI)YO~lucihle t~wlt.~;. Furt,hcr we ha~c tried to adapt the reaction to the :inalysis of tlrcl corresponding disulfitles nftcr sulfitolybis or cpanolysis, :rcwn~tlin~ to t II(’ folln&ig equations: I: s--s
1: -~ Sl ),,
1: -,3--s ~1: - (‘1
. I: -s
$0.. + 1:s
4 1(--S- C’S + 1:s
Kincr the 1)It optimuiil for tlic K\;Ei\I-lllioi rc,actiori i.G alwllt 6, 0111‘ fip.., effort. in\:olved t.lic llh(’ of l&ulfite :I< rc~ciucing :lgc’nt, \vitli tllc :tini (Jf
CmTyiIlg
out,
hOth
tllc’
hlllfit(JlysiS
:111t 1 tllP
COU~~hg
TT.ith
xk:lI
ii1
the
same t~uffcwtl mo(liulli. ‘I‘lic r<+lilt+. ot)t:~inc~ti w(‘r( c*ol~ll)l~+t~ly nc~gative, sinr(l bisulflte c;111~c’d ;I ch~l’c%W ill tllcb :i\Js;cJl’lJ:tllC’y Of sl
42
FIG. 1. Time course reaction-Reduction of disulfides by KCN: (0) cystnminc ; (0) glutathione; (0) cystine. To 5 prnoles cli.wlfide in 0.5 ml were added 0.5 ml .5% KCN in 1 N NH,, and-at the indicated times-1 ml 0.5 N acetic acid and 2 ml 3 X lO~“Jif NEM in 0.5 IV phosphate buffer, IIH 6. 01). xt 300 mp (l-cm light path ~11s) was subtracted from OD of a solution 1.5 x ‘lo-“111 NEM to obtain LIOD. For glutatjhione, reduction by KCS ~IVCS riw to a alight increase in OD at 300 mp; lhewforc: the vnluw wporled hnve bcrn correct4 for nbsorbancy recorded in the absence of NJXM (~~hosphntc bufffrr added instead of SEM).
Bccausc the methotl l~rovctl reliable for the detcct,ion of disulfides it was also t’cdtcd for their detection in the presence of thiols. For this purpose, from stock solutions containing 10-‘111 cyatinc or cysteine, aliquots were mixed to obtain varying amounts of both compounds (Table 1). In thcsc solutions the t,liiol was determined by ntlding, to 0.5 ml, 1.5 ml of H,O and 2 ml of 3 X lo-” M KEAI, and the disulfide by adding as described above, to another 01.5-ml aliquot, 5% IiCN and after 20 min acetic acid and NEM. From the following equations the amounts of sulfides and disulfides may then br calculated: gmolcs ---SH/ml pmoles -S--S--/ml
=
AOD (-KCN, 620 X lo-”
=
AOD (+IiCN) - A011 (-IiCN) 620 x lo-”
in which AOD (--EiC?u’) = difference in optical density recorded between a blank containing the same quantity of NEM and no thiol, and the test performed in the absence of KCN; and Aon (+E;CNl = dif-
DETERMIX:1TIOK
OF
THIOLS
4ND
43
DISCLFIDICS
0,8-
0.5
1
p moles/ml
(FIN
cone)
FIG. 2. Molar rorrcsllontl(,111,c hetlvveen thiols and disulfides-A(~) D = thcrease in atxorbancy of -UEM solution Cl..5 >( IO-“~11) as function of cysteinc (0). cystine CO), or cystnminc (9) conccntr:ktion. For erpcrimentnl conditions see test.
TABLE DETERRIIS~TIOS
OF TIIIOLS
1 .INI)
0.5 ml of solui ion containing the specifietl amomlts with (A) 1.5 ml Hz0 and 2 ml 3 x 1OF 11 SE21 in the determination of 1he thic~l: (RI 0.5 ml 5y0 Kc’Ii room temperature, I ml O..i S avrlic arid and 2 ml in OD in respect to a solution 1.5 X 10-3 31 ?;15?ll in
s-s
(+I&,
5
0
.-I 1 : !
0.00
0.3
0, i!l.i 0.il.i
O.%O
4.5
07S.i
4 60
0.45
4 :: 5 :;
1
O.ti3,i
0.7sn
4.0s
0 .!I3
1 5 2 2 5
0. ,541)
0. i!lO
3.4s
1.60
0.43~ 0.400
0,775
2 09
1 .!)O
0. ii5
‘2.5s
3 35 4
0.320 0.235
0.235 O.iS5
0. 1FO
0. ii0
2.06 1.5s 1.03
4 5
0 .06.i
0
0.41
5
0.005
U.7iO
2.41 3.00 3.54 3.93 4.60 4.9G
2 5 2
1.5 1 0.5 0 .~
.\ c -1iCS)
DISVLFIDES
of qsteine and cystine were added (EI ,I1 phosphate buffer, pH 6, for in 1 S SHJ anti, after 30 min at SI5hI solution; AOD = difference the presence of t,he same reagents.
-____
is0
0 no
ference in optical density hctmen the blank and the test performed in the lmxnce of KCN. The results obtained in these terts inclicate that the method is reliable for the detection of cliwlficlc!: in the prmcnce of even n greater amount of eulfitles. anI1 Tier vcrs:~.
The WC of KEhI for tile qunntitati~~c ~~~cct~~ol~hotornetric estimation of thiols has :rlrcatly been mpha~~izetl. In thr Imxnt pnpcr it is shown that this methotl may be successfully nd:tpted to 111~c&nation of rlisulfides, even when in the presence of thiols.
h sirnplc ant1 rapid slmtrophotoinetric technique for the quantitation of thiols and tlisulfitlcs has been clexribcd. The method involves the reartion with ,\‘-ctl~yln~:~leinirnidc before :incI after trentnwnt, n-it11 KCN, and is suitable for qunntitntive rccox-cry of both thiolr ant1 diwlficles from solutions in which they nre present together. REFERENCES