Szajewski. Whiresides/ Thiol-Disulfide InterchangeReacrions
I
201I
Rate Constantsand EquilibriumConstants
for Thiol-DisulfideInterchangeReactions
InvolvinsOxidizedGlutathionel
Richard P. Szajewski2 and George M. Whitesides*
Contributionfrom the Department of Chemistry, MassachusettsInstitute of Technology,
. Cambridge, Massachusetts02139. ReceiuedMay 8, 1978
Abstract:The rate of reductionof oxidizedglutathione(GSSG) to glutathione(GSH) by thiolateanions(RS-) followsa
Br/nstedrelationin pK"s of the conjugatethiols(RSH):pnu"- 0.5.This valueis similarto that for reductionof Ellman'sreagent:0nu"= 0.4-0.5.Analysisof a numberof rate and equilibriumdata,takenboth from this work and from the literature.
indicatesthat rate constants,
interchange
reactions
k, for a rangeof thiolate-disulfide
are correlatedwell by equationsof the
formlogk=C*0nu.pKunu"*0"pK""*0repKule(nuc=nucleophile.c=central,andlg=leavinggroupsulfur):eq36-38
giverepresentative
valuesof the Br/nstedcoefficients.
The valuesof theseBr/nstedcoefficients
are not sharplydefinedby the
availableexperimental
data.althougheq 36-38 provideusefulkineticmodelsfor ratesof thiolate-disulfide
interchange
reactions.The uncertaintyin theseparametersis suchthat their detailedmechanistic
interpretation
is not worthwhile.but their
qualitativeinterpretation-thatall threesulfuratomsexperience
a significanteffectivenegativechargein the transitionstate,
but that the chargeis concentrated
on the terminalsulfurs-is justified.Equilibriumconstantsfor reductionoiCSSG using
a,cr-dithiolshavebeenmeasured.
The reducingpotentialof the dithiol is stronglyinfluencedby the sizeof the cyclicdisulfide
formedon its oxidation:the moststronglyreducingdithiolsare thosewhichcan form six-membered
cyclicdisulfides.
Separate
e q u i l i b r i u mc o n s t a n tfso r t h i o l a t e a n i o n - d i s u l f iidnet e r c h a n g(eK s - ) a n d f o r t h i o l - d i s u l f i dien t e r c h a n g(eK s H ) h a v eb e e ne s t i matedfrom literaturedata:Ks- is roughlyproportionalto2ApK. is the differencebetweenthe pKusof the two thiolsinvolved
in the interchange.
The contributions
of thiol pKu valuesto the observedequilibriumconstantsfor reductionof GSSG with
a,co-dithiols
appearto be much smallerthan thoseascribableto the influenceof structureon intramolecularring formation. Theseequilibriumand rate constantsare helpfulin choosingdithiolsfor useas antioxidants
in solutionscontainingpro(DMP), and 2-mercaptoethanol
teins:dithiothreitol(DTT), 1.3-dimercapto-2-propanol
usefulproperties.
haveespecially
: 0.34).and B rockl ehurst
of
et al . haveexami n edr educt ions
= 0.23).r2'Thes
(pnu"
di
sul
fi
de
e
2,2-di
pyri
dyl
st
udies
indicat
e
rl
O x idat iono f c a ta l y ti c a l l o
y r s rru c tu ra l ley ssenti al
cystei ne
i
s
that
thi
ol
ate-di
sul
fi
de
i
nterchange
a
mech
anist
icaily
sim
ple
t hiol gr oupsc a n b e a n i m p o rta n tm e c h a ni smfor enzyme
but.suggest
that the valuesof t he
reacti on,
deac t iv at ion .l
It i s i mp ra c ti c ato
i e x c l u d eo x ygencompl etel y S x 2 di spl acement
characteristic
Brpnsted
sensitiveto the parcoefficients
are
f r om s olut io n o
s f p ro te i n sd u ri n gth e i rm a n i pul ati on,
and the
ti
cul
ar
The
extensi
o of
n t hesem odel
set
of
reactants
chosen.
s t r at egynor m a l l yfo l l o w e dto m i n i m i z eth e i nfl uenceof autFir st .t he
studi esto GS S G w ascarri edout for tw o re asons.
ox idat ionon e n z y m a ti ca c ti v i tyi s b a s e do n th e protecti on
afwit
h
l
'
or
di
aryl
di
sul
fi
des
t
ypical
aliequi
l
i
bri
a
reducti
on
of
t or dedby or g a n i cth i o l s(e s p e c i a l l2y-m e rc aptoethanol
and
phati
c
l
i
e
thi
ol
that
e
quilibr
ium
conthi
ol
s
so
far
tow
ard
aryl
dithiothreitola's)
presentin excess.
Theseaddedthiolsprobably
conveni entl y.
I t was t hus not
stantscoul d not be measured
inhibit t he ir re v e rs i b l o
e x i d a ti v ed e a c ti v a ti on
of protei nsby
possible
to
explore
the
influence
of
the
structure
of the reducing
s ev er alm ec h a n i s msT.h e y re v e rs eth e i n i ti ai sragesof the
equilibria.
Second,aryl
thioi
on
the
thiol-disulfide
interchange
proteinautoxidation
protein
reactions
by reduction
of
disulfides
poor
protei
n
cyst
inegr oup.
for
a
thi
ol
s
are
arguabl
y
model
s
and s ulf enica c i d s 6to th i o l s .T h e y m a y , b y coordi nati on,
m odif y t he c a ta l y ti ca c ti v i tyo f tra n s i ti o n -metal
i onsi n thi ol
aut ox idat ion .l T
' 7h e i r o w n o x i d a ti o nc o n s u mes
R esul ts
di oxvsenand
hy dr ogenper o x i d eps re s e nitn s o l u ti o n
Ratesof Reductionof OxidizedGlutathioneby Mono- and
As part of a projectaimedat thedevelopment
of techniques Dithiols.The rate of reieaseof GSH from GSSG wasdeterto permit the useof enzymesas practicalcatalystsin the synmi ned usi nga fast enzymati cfol l ow i ngreact ionat pH 7. 0
thesisof organicand biochemicals,e
we havebegunto study
(0.06 M phosphate
buffer)and 30.0* 0.5 ' C underar gon.
the relationbetweenthe structuresof organicthiolsand their
CSH wasconvertedto S-lactoylglutathione(GS-lac)by reability to preventor reversethe autoxidationof proteins.The
o f glyoxalase- l
acti on w i th methyl gl yoxali n the presence
work describedin this paperis focusedon one aspectof this
(GX -l ), and the concentrati on
of GS -l ac was m onit or ed
pr oblem ,v iz ., ra te - a n d e q u i l i b ri u m -s tru c t ure
rel ati onsfor
spectrophotometri calatl y 240 nm, es 3.37 m M - l cm - i. l'1
the reductionof a modelcystine-containing
pepride(oxidized
EquationsI -6 list the reactionspertinentto this assay.In these
glut at hione,G S SG, 7 -g l u ta my l c y s te i n y l gl yci ne)
by base- equations,RSH is a monothiolreducingagent.Reaction6, the
catalyzedthiol-disulfideinterchange.
Oxidizedglurathione enzymaticconversion
to GS-lac,is essentially
of hemithioacetal
wasselectedas substratein this investigation
for threereasons: i rrevei si bl e.l a
it is readilyavailablein pureform:its presence,
as wellasthat
KrRsH
of reducedglutathione(GSH), can be monitoredin the pres(I)
RSH_
RS- + H+
enc eof ot hert h i o l sa n d d i s u l fi d e u
s s i n ge n z ymari cassays;10
and, becauseit occursin biologicalsystems,l.llinformation
kt
( 2)
GS S R + GSR S - + GS S G_
about its reactionswith variousthiolsshouldprovegenerally
k-r
us ef ul.W el2a n d H u p e e t a l .l 3h a v ea l re a d yreportedratesof
k't
t hiol- dis ulf id ei n te rc h a n g e
re a c ti o n si n v o l v i n gE Il man' sre(3)
R S - + G S S RR S S R+ G S agent(5.5'-dithiobis(2-nitrobenzoic
acid))and foundthemto
k-z
foilowa Brlnstedcorrelation(pnu"= 0.5).Clelandet al. have
p K " C S H= g . l l t S
(4)
carriedout relatedstudieswith 4,4'-dipyridyldisulfide(pnu"
GS- + H+
GSH
Introduction
(1980).1
zjt2
Journal of the Anterican Chemical Society / 102:6 / March 12, lgB0
o
( G S S R ) > ( R S S R ) : s i n c et h e r a t e c o n s t a n t sk r , k : , k - 1 ,a n d
k -2 areall of the samemagnitude,the second.third. and fourth
t e r m s o f e q 8 c a n b e n e g l e c t e d ,a n d t h i s e x p r e s s i o nm a y b e
combined with eq 7c and rewritten as eq 9a. For convenience,
this equation was used in a form (eq 9b) involving an observed
r a t e c o n s t a n t .k l o b ' d b
, a s e do n t h e t o t a l c o n c e n t r a t i o no f t h i o l
and thiolate anion in solution.The rate constantsll I and k robsd
a r e r e l a t e db y e q 1 0 .
(
F4
9.
x
1
I
(n
9-?
d ( G S - l a cf )d t - k r ( R S - ) ( G S S G )
= f t , o b s d t ( R s+- ) ( R S H ) l ( G S S G )
k t = k , o b s1dl + l 6 p x " n s x - p H )
ot23456789tO
(9a)
(eb)
(l0)
To ensurethe accuracy of the assumptionsand approximations
m a d e t o g e n e r a t ee q 9 , i t i s n e c e s s a r yt o a d j u s t t h e r e l a t i v e
Figure l. Formationof S-lactoyl glutarhione(CS-tac, M) with time at 30.0
c o n c e n t r a t i o n so f t h e v a r i o u ss p e c i e sp r e s e n ti n s o l u t i o n . R e o
C
,
* 0.5
u n d e ra r g o n i n a p H 7 . 0 p h o s p h a t eb u f f e r ( 0 . 0 6 6 M ) c o n t a i n i n g
actionswere carried out using the following initial conceno x i d i z e dg l u t a t h i o n e( G S S C . 0 . 3 5 m M ) . d i t h i o t h r e i t o l( 5 . 0 7 m M ) , a n d
t r a t i o n s :( R S H ) 0 = 0 . 7 7 m M , ( G S S G ) = 0 . 3 5 m M , a n d
m e t h y l g l y o x a(lC H : C O C H O , 0 . 7 7 m M ) . a n d u s i n g( A ) 2 4 , ( B ) 2 . 4 .( C )
( G X - l ) - 2 . 5 X l 0 - 4 m M ( 2 . 4 U / m L ) . 1 6 B e c a u s et h e s r a r t i n g
1 . 8 .( D ) 1 . 2 ,( E ) 0 . 3 U / m L . r e s p e c t i v e l yo.f g l y o x a l a s e - (l G X - l ) . T h e
c o n c e n t r a t i o no f G S - l a c w a s d e t e r m i n e ds p e c t r o p h o t o m e t r i c a l l ay t 2 4 0
c o n c e n t r a t i o no f t h i o l i s m u c h g r e a t e r t h a n a n y o f t h e o t h e r
n m u s i n ge o3 . 3 7 m M - l c m - 1 . T h e r e i s n o s i g n i f i c a n td i f f e r e n c eb e r w e e n
s o l u t i o n c o m p o n e n t s ,t h i s c o n c e n t r a t i o n i s n o t s i g n i f i c a n t l y
d ( G S - l a c ) / d / a t e n z y m ec o n c e n r r a r i o n o
sf 24 and 1.8 U/mL: the conperturbedby hemithioacetalformation and remainsessentially
c e n t r a t i o no f e n z y m eu s e dr o u t i n e l y i n t h e a s s a yw a s 2 . a U l m L .
c o n s t a n tt h r o u g h o u t t h e c o u r s eo f a k i n e t i c r u n . T h e c o n c e n t r a t i o n o f C H I C O C H O u n d e r t h e s ec o n d i t i o n si s , o f c o u r s e .
u
nknown: although a significant fraction oi this material is
K5=J6fYfla
p r o b a b l . vc o n v e r t e dt o h e m i t h i o a c e t a l ,t h e v e l o c i t y o f e q u i l i GSC HO HCO CHT
GSH + CH3COCHO (5)
l i t h a l d e h y d ea n d h e m i h y d r a t e
b r a t i o n o f t h e h e m i t h i o a c e t aw
GSCHOHCOCHT
+ GX-I9 G S C O C H O H C H+3c x - l
i s f a s t c o m p a r e d t o t h e e n z y m a t i c r e a c t i o n sw h i c h f o l l o w . r a
Regardlesso[ the equilibrium concentrationof hemithioacetal.
(GS-lac)
( C H I C O C H O ) o > ( G S H ) i n t h e i n i t i a l s t a g e so f t h e r e a c t i o n .
(6)
F o r s m a l l e x t e n t so f r e a c t i o n( i n t h e s ee x p e r i m e n t st y p i c a l l v
l0-20Vo) the concentrationof CHICOCHO can be considered
T o d e r i v e r a t e e x p r e s s i o n sf o r k I f r o m t h e s ee q u a t i o n s ,w e
c o n s t a n t .T h u s . w i t h t h e a s s u m p t i o n si n d i c a t e d ,t h e r a t e o f
r e q u i r e t h a t t h e o v e r a l l r a t e - l i m i t i n g s t e p i n c o n v e r s i o no f
a p p e a r a n c eo f G S - l a c w i l l b e d i r e c t l y p r o p o r t i o n a l t o t h e
G S S G t o G S - l a c b e t h e i n i t i a l t h i o l a t e - d i s u l f i d ei n t e r c h a n g e
c o n c e n t r a t i o no f G S H . F o r t h e s t e a d y - s t a t ea p p r o x i m a t i o nt o
r e a c t i o n( e q 2 ) . W e w i l l a s s u m et h a t t h i o l a t ea n i o n i s t h e o n l y
h o l d f o r G S H , i t i s n e c e s s a r yt h a t i t b e c o n v e r t e dt o G S - l a c
n u c l e o p h i l ep a r t i c i p a t i n g i n t h e t h i o l - d i s u l f i d e i n t e r c h a n g e
much more rapidly than it is formed from GSSG. The rate of
r e a c t i o n , l Tt h a t r e d u c e dg l u t a t h i o n e( G S H ) i s p r e s e n to n l y a t
c o n v e r s i o no i G S H t o G S - l a c c a n b e a d j u s t e d o v e r a w i d e
a low, steady-stateconcentration.and that glyoxalase-lfollows
m a r g i n b y s i m p l y c h a n g i n gt h e c o n c e n t r a t i o no f g l y o x a l a s e |typical Michaelis-Menten kineticswith the hemithioacetal
o r C H 3 C O C H O . T h e c o n c e n t r a t i o no f e n z y m e r e q u i r e d t o
C S C H O H C O C H I a s i t s o n l y s u b s t r a t e( e q 7 a ) . t e . r 6 ' r 8 ' r e
a c h i e v e s t e a d y - s t a t ec o n d i t i o n s i n G S H , a t a c o n v e n i e n t
d(GS-lac)/dr
C H 3 C O C H O c o n c e n t r a t i o nw
, a s e s t a b l i s h e de x p e r i m e n t a l l y .
The concentration(activity) of enzyme in solution was varied,
Krn GSCHOHCOCHT
(ia)
a n d t h e v a l u e o f ( G S - l a c ) m e a s u r e d ,h o l d i n g a l l o t h e r p a ( G S C H O H C O C H 3] )rameters constant.At valuesof enzymatic activity greater than
t
s K m c s c H o H C o c H i l -I
2 UlmL, increasingthe activity of enzyme in solution
= k : ( G X - l ) ( G S H ) l ( c S+H K
)
shortened the duration of a non-steady-stateinterval at the
t
b e g i n n i n go f t h e r e a c t i o n ,b u t d i d n o t i n f l u e n c e t h e v a l u e o f
(7b)
d(GS-lac)/dt once the steady-stateregion had been reached
= [ k 3 ( G X - l ) ( C H 3 C O C H O ) / 3 m M 2 ] ( G S H )( 7 c )
( F i g u r e I ) . M o r e g e n e r a l l y ,t h e v a l i d i t y o f e q 9 r e q u i r e st h a t
d
( G S - l a c )l d t b e z e r o o r d e r i n G X - l a n d C H 3 C O C H O a n d
Substitutionof the equilibriumexpression
for K5 (eq 5) into
f
i
r s t o r d e r i n R S H a n d G S S G . T a b l e I , w h i c h l i s t s k ' o b s dr n 6
eq 7a followed by rearrangementgives eq 7b. At a low
velocities [d(GS-lac) ldt] for formation of GSH from GSSG
steady-state
7c
7b
concentrationof GSH, eq reducesto eq for
K s - 3 m M and K . c s c H o H c o c H :- I m M .2 0T h e b ra c k eted using 2-mercaptoethanolas reducing agent, establishesthat
theseconditions are fulfilled. The data in the first line of this
portionof eq 7c may,in principle,bemadearbitrarilylargeand
table were obtained using the standard conditions employed
constantby adjustingthe concentrations
of either GX-l or
routinely in this work; the other runs were performed on soCH3 C O CHO ; in pr ac t i c eth
, e q u a n ti tyo f G X -l u s e di s the
lutions in which these conditions were systematically varied.
moreeasilyvaried.At the pH usedfor the assay(pH 7.0),the
Analysis of the velocity data and extraction of the rate constant
majorityof the reducedglutathioneis presentas GSH rarher
than GS-. Assumingthese conditionsand a steady-srare k,ob'd from these data are discussedlater in the text. Under
the conditions used, the assumptionsmade in obtaining eq 9
concentrationfor GSH, we'relatethe rate of productionof
seem to be justified, since large deviations from the standard
CS-lacto k 1 by the equation
conditions have no influence on krobsd.
0 : d ( C S H )/ d t = k r ( R S - ) ( c S S c ) + k 2 ( R S - ) ( G S S R )
Equation 9 applies to monothiol reducing agents. In this
- k_r(cssR)(GS-) - k_z (RSSR)(GS-)
work, we were particularly interested in dithiols. In treating
reducing agents, HSRSH, containing two identical thiol
- f t 3 [ ( c x - l ) ( C H 3 C O C H O ) / 3m M 2 ] ( G S H ) ( 8 )
groups, we used an entirely analogous procedure, with two
We ensurethat kineticsobservations
extendonly over the
additional assumptions.First, we assumed that the nucleo-SRS-,
in i ti a l stagesof t he r ea c ti o n ,i n w h i c h re g i m e (G S SG) >
p h i l i c i t i e so f t h e m o n o - a n d d i a n i o n s ,H S R S - a n d
Timc (min)
= k :( G X - l l+I r
@
2013
Szajewski, Whitesides/ Thiol-Disulfide InterchangeReactions
(CSSG)by 2-MercaPtoethanol4
(k,ou'a,
Glutathione
of Oxidized
M-l min-l) for Reduction
TableI. RateConstants
k , obsd
d(GS-lac)/drD IHOCH2CH25HI'
8.8
9.5
8.2
9.1
7. 3
9. 3
8. 9
8.0
8.8
9.2
0 . 76
0.77
0.77
0.77
0.76
0.75
0.31
1.50
0.76
0.76
5 .0 7
5 .l 0
5 .r 0
5.09
14.5
0.49
5.r2
4.99
5 .0 5
5 .0 7
t4.4
r5.6
13.2
t4.9
39.4
1.5
t4.7
12.6
28.3
2.5
l c H r c o c H o l . lcsscl.
0.32
0.32
0.32
0.32
0.32
0.3r
0.33
0.32
0.64
0.054
IGS-rld
1 . t
:.1
a a
1
0.48
1.2
1A
L-.+
2.1
1A
'tA
L.1
1A
L.a
comments€
standard
XIOGX-I
X O . 2G X - I
x0.5GX-l
X3 RSH
X O . IR S H
xo.5 cHTCOCHO
x2 CH3COCHO
X2 GSSG
X O . I 7G S S G
2.4
oC,
at 240nmusing€o3.37mM-l cm-t,
underargon,wcrcfollowed
buffer,pH 7.0,30.0* 0.5
" All kincticslrercrun in 0.056M phosphate
unitsare mM. d In enzyme
ii rnt. , Unir; of d(Cs-tac)/drarc M-r min:r x 106.. Concertration
and werecoftectedfor blankreactions,
'
mnditions.
standard
fiom
the
units(prnol/min)pcr mL. Thiscolumnindicctesthe majorchangc
Table II. Rate Constants(k'ou'a M-l min-r) for Reductionof OxidizedGlutathioneby Dithiothreitolo
k , obsa
14.0
14.6
8.4
t3.l
r3.0
t4.2
9.8
d(GS-lac)/dt'
47.0
26.9
41.3
81.6
4.5
31.9
l 2 .r
J t,l
12.7
14.0
8 0 .l
5.8
IDTTI.
lcHscocHol.
IGSSGl.
0.76
0.77
0.77
0.77
0 . 76
0 . 76
0.39
L50
0.76
0.'t6
0.32
0.32
0.32
0.32
0.32
0.32
0 . 33
0 . 32
0.64
0.042
4.94
4.97
4.97
4.94
9.94
0.49
5.01
4.86
4.93
1.94
AA'I
IGS-rld
1^
'tA
1
0.48
1.8
1A
1A
.\^
1,1
1A
11
commentse
standard
X I OG X - I
X O . 2G X . I
X 0 . 7 5G X . I
X2 RSH
X O . IR S H
xo.5cH3COCHO
x2 CHTCOCHO
X2 GSSG
X O .I 3 G S S G
d All kinetics were run in 0.05 M phospharebuffer, pH 7.0, 30.0 * 0.5 'C, under argon. ' Velocity offormation ofGS-lac as determined
a! 240 nm u.;ng io :.:l mM-i cm-l and correctedfor blank riactions.if any. Unitsof d(Cslac)/d, are M min-l
spectrophoromerrically
'
i t Oo.I Con""ntr",ionunirsrre mM. ; ln unirsof enzymeacriviryper mL. This columnindicates(he majordeviationfrom stlndard condi!ions.
w e r e i n d i s t i n g u i s h a b l e .T h i s a s s u m p t i o n n e g l e c t se l e c t r o n i c
effects and a statistical factor of 2, both of which would make
the dianion more reactive than the correspondingmonoanion.
T h e c o n c e n t r a t i o no f d i a n i o n w i l l , h o w e v e r .b e s m a l l r e l a t i v e
to monoanion at pH 7 for the dithiols we have examined.
M o r e o v e r ,t h e o b s e r v a t i o n( s e eb e l o w ) t h a t m o n o - a n d d i t h i o l s
f a l l o n t h e s a m e B r / n s t e d c o r r e l a t i o n l i n e s u g g e s t st h a t t h e
c u m u l a t i v e e r r o r i n t r o d u c e d i n t o e s t i m a t e so f r a t e c o n s t a n t s
b y t h i s a s s u m p t i o ni s s m a l l . S e c o n d ,w e a s s u m et h a t , f o r t h e
c o n c e n t r a t i o no f d i t h i o l u s e d , t h e r a t e o f t h e i n i t i a l i n t e r m o lecular thiol-disulfide interchangeis lower than that of a
s u b s e q u e n ti n t r a m o l e c u l a r r e a c t i o n w h i c h r e l e a s e sa s e c o n d
e q u i v a l e n to f C S H ( e q I l ) . A s i m i l a r a s s u m p t i o nr.n a d e i n o u r
HSRSH + CSSG
g
HSRSSG + GSH
'-";
fast
sn3 + GSH
HSRSSG
(I IA)
( I lb)
could
previoustreatmentof the reductionof EIlman'sreagent.
esti mates
of
bejus t if iedex p e ri m e n ta l lbye c a u s ien d e p e n d ent
fo r re l e a s o
e f a ry l th i o l sfro m di aryldi sul t he r at e c ons ta n ts
fidesand aryl alkyl disullidesindicateda sufficientdifference
for
t o be det ec t ed| 2. '1 34 n o l ts i s o f th e e q u i l i b ri u mconstants
,
r educ t ionof CS SG b y a l l o f th e d i th i o l se x a mi nedbel ow exa significant
established
cept for glycol dimercaptoacetate,
l f w e assume
s n o th i o ls.
enhanc em enr e
t l a ti v eto a n a l o g o umo
reactionof mono-anddithiols
that the ratesof intermolecular
hav ings im ilarth i o l p K " v a l u e so n GS SGw i l l a l sobesi mi l ar.
that
suggests
differencein equilibriumconstants
the observed
involving
thiol-disulfide
interchange
inlramolecular
the rateof
is probablylast relative
the secondthiol groupof an cv,c.,.r-dithiol
e e eb e l ow ).N onethet o t he f ir s t int e rm o l e c u l ai nr te rc h a n g(s
for
Iess,sincewe havenot beenableto measurerateconstants
r eleas oi
e CS H i ro m GS SGa n d GS SR SHb y thi ol -di sul fi de
ex p e ri m e n ta l l ya,n d s i n c eth e s ev a l uesare probint er c hange
a b l y s i m i l a r f o r m o s t o f t h e t h i o l se x a m i n e dh e r e . l l ' 1 2w e r e l v
o n a n a l o g y w i t h t h i s p r e v i o u sw o r k t o j u s t i i y o u r a s s u m p t i o n .
N o t e , h o w e v e r ,t h a t . i f t h i s a s s u m p t i o ni s i n c o r r e c t .i t i n t r o d u c e s a n e r r o r o f o n l y a f a c t o r o i 2 i n t h e r a t e c o n s t a n t sf o r
dithiols.
W i t h t h e s e a s s u m p t i o n sa. t r e a t m e n ts i m i l a r t o t h a t d e s c r i b e dp r e v i o u s l yl e a d st o t h e e x p r e s s i o nisn e q l 2 r e l a t i n gt h e
o b s e r v e dr a t e o f p r o d u c t i o no f G S - l a c t o t h e r a t e c o n s t a n to f
i n t e r e s t ,k 1 . A g a i n k 1 a n d k l o b s oa r e r e l a t e db y e q 1 0 .
d ( G S - l a c )f d r = 2 k T U H S R S - )
+ (-SRS-)l(GSSG)
(l2a)
= 2krob'd[(HSRSH)
+ (HSRS-) + (-SRS-)I(GSSG)
(l2b)
T a b l e I I l i s t sv a l u e so f k , o u s aa n d v e l o c i t i e s[ d ( G S - l a c ) / d r ] f o r
formation oi GSH from GSSG usingdithiothreitol as reducing
a g e n t . T h e f i r s t l i n e i n t h e t a b l e r e p o r t sd a t a o b t a i n e d u s i n g
o u r s t a n d a r d c o n d i t i o n s :t h e o t h e r r u n s w e r e p e r f o r m e d o n
s o l u t i o n s i n w h i c h t h e s e s t a n d a r d p a r a m e t e r sw e r e v a r i e d .
A g a i n . u n d e r t h e c o n d i t i o n su s e d . t h e a s s u m p t i o n sm a d e i n
o b t a i n i n g e q l 2 s e e mt o b e j u s t i f i e d .
S t r a i g h t f o r w a r d c o n s i d e r a t i o no f m a t e r i a l b a l a n c ea n d i n t e g r a t i o n o f e q 9 ( o r e q l 2 ) b y s t a n d a r d m e t h o d sg i v e s r z ' : :
, (GSSG)o
In-())o
( S ) o- ( G S S G ) o
( GS
S - l a cc))//nn
* [ t s l o - -t c( C S + * ) / r lI ( l 3 )
[(GS S G)o
are definedas follows:for moThe terms in the expression
n o t h i o l sS o = [ ( R S - ) + ( R S H ) ) , n = l ; f o r d i t h i o l sS o =
= 2 . F i g u r e2 r e p r o [ ( - S R S - ) + ( - S R S H ) + ( H S R S H ) ] ,r
ducestypi calki neti cdata for reducti onoi C SSC by sever al
mono-and di thi ol sas deri vedusi ngthe i nte gr at edr at e exdatau sedt o gener at e
(eq l 3). The crudeabsorbance
pressi on
f t , o b s d 1=
20t4
Journal of the American chemical society
/ t 02:6 / March I 2, I gg0
(GMA)
ct
\l
c
\
o l
o
-
E
.o
!19
s1
6l
(,1(J
-l
i
t
ctr
o
'ld
/,1
3\(DTT)
, ,7 (ME)
!{
_q6
cnl.r,
-g
ro
8 l v-) e
i^l
tl
Y I
s
l^o
l(9
Itn
|(t
-1(9
*-
t ^
l ^ v
('r
t9
Figure
2.Rate-constanr
prots
,",-,*1...1"1
o
r.t""*,l,on*tunoot,,n,o,,
w i r h o x i d i z c dg l u t a t h i o n e( G S S G ) i n p H 7 . 0"r
p h o s p h a t eb u f f e r ( 0 . 0 6 6 M )
at 30-0 + 0.5 "c under argon.The termq in the expression
on the axis are
d e f i n e da s f o l l o w s : f o r m o n o t h i o l sS
, e= [(RS-) +'(RSHt] , n = t; for di_
r h i o t s .s 6 = [ ( - S R S - ) + ( H S R S - ) + ( A S R S H ) J , l r =
z'(ir. eq r3 or the
t e x t ) . R e d u c i n ga g e n r s :r , g r y c o rd i m e r c a p t o a c e r a t ev;,
dithiothreitor:
o. 2-mercaptoethanol:
r. thiogl.vcolic
acid.Kineticsare sho*n .espectivery
t o 6 0 , 5 0 , 2 0 . a n d 2 0 v oo f c o m p r e t i o nT. h e k i n e t i c p o i n t s
sho*n herewere
o b t a i n e df r o m t h e e x p e r i m e n t Ia A z c o a t t r i b u t a b r teo
GS-lacand are corrected for the relativel-vslow blank reactionobservedin
thesesysrems*hen
u s i n g p h o s p h a t eb u f f e r ( s e er e f 2 4 ) .
2 (DMP)
pKrs*
F i g u r e 3 .P l o t s o f( A ) l o g , t l t u a n d ( B ) I o g & r ( M - r m i n - r ) v s .t h i o r p K "
f o r t h e r e d u c t i o no f o x i d i z e dg r u t a r h i o n ei n p H ? . 0 p h o s p h a t eb u f f e r
at
3 0 . 0 + 0 . 5 o c u n d e ra r g o nb y r h e m o n o -o n a ' a i t n i o t t i s t e di n T a b l e
III.
D T T i s d i t h i o t h r e i t o l . v l E i s 2 - m e r c a p r o e t h a n oD
l.Mp is 1.3-dimercap
topropanol, and G M A
t-vc^ol mgcaproacetate. The least_sq
uares slope
.is.e
!
!
f o r ( B ) i s 6 n , " = 0 . 5 0 1 r 2 = 0 . 8 9 ) .T h e e q u a t i o nu s e d, o g . n . r . , . t h e
rine
in (A) is log k 1oH = -_1,29* 0-50pK. - Iog ( lgpKe-7.0+" t ti,. equation
);
i n ( B ) i s l o gk r = - 1 . 2 9 * 0 . 5 0 p K " .
Ta b l e sI - lll wer ec or r e c te d
fo r th e s l o wp h o s p h a te
i o n cara_
lyzedreactionbetweenmethylglyoxal
and thiolsbeforeanalysi s to obt ain v aluesof k l o b ' du s i n gth i s e q u a ti o n .2 a
T a b l eI I I . R a t ec o n s r a n r (st . M - r m i n - r ) f o r R e d u c r i o o
R ate
nf
constantsfor reductionof GSSG by a seri'es
O x i d i z e dG l u t a t h i o n (eG S S G )b y M o n o -a n d D i t h i o l s "
of rnono-and dith i o l su nders t andar dc o n d i ti o n a
s re l i s te di n T a b te 1 1 y .zr.::
thiol
&"obsd
Figure3 summarizes
k1
PK^
thesedataasplotsof log ft 1and log k ,obsd
vs. th e p K " of t he r educi n gth i o l .T h e a ro rn a ti cth i o l J w hi ch
: M A ) 7. 1 1 9 . 9 1 t 90
I ( H S C H T C O : C H : _()G
4.9X 102
were usedto obtaindata for valuesof pK"RSH( g with
2 HSCH2CHOHCHTSH
9 . 0( 1 0 . 3 ) b 9 . 3 9 . 3x 1 0 2
Eil_
(DMP)
ma n 'sre agent ll' 13
c ouldn o t b e u s e di n i tu d y i n gth e re d u cti on
of GSSG, becausethey absorbedstrongiyat ine waverength 3 d i t h i o t h r e i t o( lD T T )
9 . 2( 1 0 . 1 ) 6 r 4 . l 2 . 2x 1 0 3
4 DTT/DTE mixture.
9 . 2( r 0 . 1 ) b 8 . 4 l . l x t 0 3
use dto m onit ort he c on c e n rra ti ooni G S I-i a c(2 a 0 n m). For
g.5b
5 HOCH2CHOHCH2SH
7 . 9 2 . 5x 1 0 3
comparison,
Figure4 reproduces
analogous
datafor reduction 6 H S C H 2 C H ( N H C O C H 3 ) _ g . 5 d
5 . 2 1 . 8x 1 0 3
o f El l man' sr eagent t; hi s p l o t i n c l u d .i d u ru b o th fro m our
COzH
w o rkl 2and f r om t hes t ud yo f H u p e e t a l .r3
7 H O C H : C H 2 S H( M E )
9.6,
8 . 7 3 . 4x 1 0 3
The Br/nsted coefficient(pnu"= 0.50,coefficientof de_
8 HSCH:COzH
9.8r
t.J
4 . 6x l 0 l
termination= 12= 0.89)obtainedby least-squares
9 HSCH2CHzCOzH
analysisof
10.68
3 . 2 1 . 2x 1 0 4
the rate constantsfor GSSG is similar to tiat obtainld for
a All rateswerederermined
in 0.066M phosphate
buffer,pH 7.0,
reductionof EIlman'sreagentby thiols.This lattersystemhas
30.0+ 0.5 oc, underargon.Data typicailyrepresent
an averageof
nowbeenthesubjectof two independent
r:.
r:
studies( Figure4).
threedeterminations.
The rateconstantsarenot correctedstatistically
our study^analyzed
a groupof at[yt and aryr thiorsrogether,
of two equivalent
sulfuratomsin GSSG.Reproduilo11hepresence
an d g a ve6 nu.= 0. 30,12- 0 .g 5 .2 H
5 u p ee t a l . a n a yl :z e dd,, ata ibility
in theseexperimenrs
was* l5zo.Arremptsto measureratesfor
for a similar set of thiors,but suggested
severalotherrepresentative
thar alkyi and aryl
thiolswereunsuccessful.
The rateof rethiols
duction
separare
correlation-fines,
GSSG by CHrcoSH wasnot significantlyfasterthan the
pnu.irkyt
=
with
6.49
l.ll_:n
9f
(r.l = 0.98) and Bnu"a'vl 0..48(r2 = 0.96).nnatysis
rate of the blank reaction:k,obsdis smallerthan lri-r v-' min-r.
of our
^=
Amino thiols (cysteine,Et2NcH2cH2sH) react quickrywith the
alkyl dataaloneyieldedpnu"ulkrl
= 0.4| (r2'= 0.67;.Our data
methylglyoxalin the assaysolutionsto form speciiswhich absorb
for aryl thiolscovera smallrangeof pK" valuesunJar" l.r,
stronglyat 240 nm, presumabrys-ractoyr thioesters(see ref 24 for
accuratethan thoseof Hupe.26The value of the Brlnsted
a discussion
of the mechanism
of theseblank reactions).GX-l was
coefficientselectedfrom theseseveralanalysesdependsin purt
inactivatedby HocH2cHSHcH2sH. , Sourcesof thesepK" valuis
judgmenrsconcerningthe ihoi". oi dutu to be
are listedin ref 12.Thesevaluesfor DTT havealsobeenreportedby
9n 9uljgctive
incl u d e d w
. e agr ee. wit hH u p e e t a l .-th a tth e a rk y ra n d a ryr
E. L. Loechlerand T. C. Hollocher.-/. Am. Chem..Soc.,'g7,323i
thiolsshouldbe analyzedseparately,2T
q9]5i c Preparedas described
and not" th"r there is
in ref 12. d From M. Friedman,J.
no independent
F. Cavins,andJ. S. Wall, J. Am. Chem.Soc.,g7, 3672(1965).' This
evidence
whichcan be usedto selectbetween
valuesof pnu""lkrl
pK" is taken from ref 13./Reference 15. c From R. J. Irving, L.
lying between-0.4and 0.5.For simpricity,
w e w i l l ta ke6 - 0. 5;ar gu m e n ts
Nelander,and I. Wadso,Acta Chem.Scand.,18, 769 ( I964).
o u tl i n e dl a te rs u g g e sthat
t
thesep valuesmay not, in any event,translatedirectly
into
fractionalcharges,and the ty,n.rtry
and chargedistribution
-demanda
s h o u l d n o t b e c o n s i d e r e d ' h i g h l yp r e c i s e .Q u a r i t a t i v e r y m o r e
of the transitionstatedo not
specificialue of B.zs.n
important is the observation that reductions of GSSG and
The valueof pnu"alkrl
= 0.5 for reductionof GSSG determined
Ellman's reagent by thiol-disulfide interchange appear to be
in the presen^t
s.tydythusseemsphysicailyreasonabre,
but, since
closely related reactions. Each is characterized by a Brf,nsted
the rangeof thiol pKrs covet.ais reraiiverysmail,
sincethe
coefficient of similar size, and neither shows any evidJnce of
scatterin the experimentar
pointsis significant,and sincethe
curvature in the Brfnsted plots suggestinga change in mechassaysystemusedto followthe reactions
is complex,thisvalue
anism or an intermediate.Since Ellman's reagentand oxidized
2015
Szajewski, Whitesides/ Thiol-Disulfide InterchangeReactions
*.
I
MMA
5
-\
.-
b
a-t--
O
aa
o
94
a
--
./
.o
.|
zVEEs
Wilron, Boyrr.Hugr
O O
Whit.lid.!,Lilburn,S:oienliat
A
z
3
4
7
6
5
8
9
ll
lO
pKo
F i g u r e 4 . C o l l e c t e dr a t e c o n s t a n t s( M - r s - r ) f o r r e d u c t i o no i E l l m a n ' s
r e a g e n tb y t h i o l s . D a t a f r o m r e f l ? a r e r e p r e s e n t e d
b y s o l i d p o i n t s ;d a t a
f r o m r e f l 3 a r e r e p r e s e n t e db y o p e n p o i n t s . D a t a f o r a l k y l t h i o l s a r e
s u m m a r i z e db y o a n d O : d a t a f o r a r y l t h i o l s b y I a n d E ; d a t a f l o rt h i o l
a c i d s b y r . M E i s m e r c a p t o e t h a n o lG
; MA is glycoldimercaptoacetate;
MMA is methyl mercaptoacetate.
Other pointsare identifiedin ref l2 and
13.The linesrepresentleast-squares
fits to thesesetsof data: A (-), alkyls
( o ) . r r 0 n u "= 0 . 4 3 ,r 2 = 0 . 9 1 ;B ( - - - - ) , a l k y l s( o ) r 3 e x c l u d i n gm e t h y l
m c r c a p t o a c e t a t1e3. n u=. 0 . 4 9 ,r l = 0 . 9 7 . C ( ' - ' - ) , a l k y l s( O ) , 1 2g n u "=
0 . - 1 1r,l = 0 . 6 7 :D ( . . - - - ) , a r y l s ( u ) , r l d n u "= 0 . 4 1 .r 2 = 0 . 9 ' 1 .
g l u a t h i o n e r e p r e s e n tv e r y d i f f e r e n t t y p e s o i d i s u l f i d e s ,t h e i r
k i n e t i c s i m i l a r i t i e se n c o u r a g et h e c o n c l u s i o nt h a t t h i o l - d i s u l f i d e i n t e r c h a n g ei s , i n m o s t i n s t a n c e s ,a m e c h a n i s t i c a l l y
u n c o m p li c a t e d r e a c t i o n .
Although we have not explicitly determined Brpnsted
c o e f f i c i e n t sl o r t h e l e a v i n g( p r e )a n d c e n t r a l ( d . ) t h i o l g r o u p s
i n t h e t h i o l - d i s u l f i d ei n t e r c h a n g ee, x a m i n a t i o no f o u r d a t a , a n d
t h o s eo f C r e i g h t o n , r ly i e l d sa v a l u e f o r t h e s u m o f p . * 1 3 1 W
n.e
h a v ed e t e r m i n e dt h e r a t e so f r e d u c t i o no i G S S G a n d E l l m a n ' s
the rate
r e a g e n tw i t h a n u m b e r o f t h i o l s :C r e i g h t o ne s t a b l i s h e d
o f o x i d a t i o n o i D T T i n t h e p r e s e n c eo f s e v e r a ls y m m e t r i c d i s u l f i d e s .R e p r e s e n t a t i v ed a t a a r e s u m m a r i z e d i n F i g u r e 5 . I t
i s e v i d e n tt h a t , f o r t h i s h e t e r o g e n e o u g
s r o u p o f d i s u l f i d e s ,0 "
+ r j r e = - 1 . 0 .C r e i g h t o n h a s r e a c h e dt h e s a m e c o n c l u s i o n .
Equilibrium Constants for Thiol-Disulfide Interchange
R e a c t i o n s .T h e m e a s u r e m e n ta n d i n t e r p r e t a t i o n o f e x p e r i m e n t a l v a l u e s f o r e q u i l i b r i u m c o n s t a n t sf o r t h i o l - d i s u l f i d e
i n t e r c h a n g er e a c t i o n si n a q u e o u ss o l u t i o n sa r e c o m p l i c a t e db y
t w o c o n s i d e r a t i o n sF. i r s t . t h e s i m p l e s tt y p e o f e q u i l i b r i u m r e a c t i o n t o i n t e r p r e t - c o m p l e t e r e d u c t i o no f a s y m m e t r i c a l d i sulfide ESSE to a thiol ESH with concomitantoxidation of the
r e d u c i n gt h i o i R S H t o a d i s u l f i d e R S S R ( e q l . l ) - i s a c h i e v e d
o n l y i n t w o s t e p s b y w a y o f a n i n t e r m e d i a t eu n s y m m e t r i c a l
d i s u l f i d e ( e q 1 5 , l 6 ) . S e c o n d ,b o t h t h i o l a n d t h i o l a t e s p e c i e s
2 ( R S H+ R S - ) + E S S E s
R S S R+ 2 ( E S H+ E S - )
(l4)
( R S H+ R S - ) + E S S E$
* S S E+ ( E S H+ E S - )
(l5)
( R S H+ R S - ) + R S S Es
R S S R+ ( E S H+ E S - )
(l6)
6 ' o b s d K l o b s a 6' r! o b s d :
(I7)
\"'
(ESSE)[(RSH)+(RS-)]2
m ay be pr es e nitn a p p re c i a b lceo n c e n tra ti oinn sol uti on,and
m eas ur ed
o rd i n a ri l yc o ntai ntermsi n
equ i l i b ri u mc o n s ta n ts
(eq
t hes umof t heco n c e n tra ti o nosf th i o la n dth i o l atespeci es
4
5
6
7
8
9
lO
ll
pKoESH
F i g u r e 5 . R a t e s o f r e d u c t i o n, t ( l v 1 - t m i n - r ) o f s y m m e t r i c a l d i s u l f i d e s
E S S E b y t h i o l a t ea n i o n . u s i n gd i t h i o t h r e i t o l( D T T ) o r g l y c o l d i m e r c a p t o a c e t a t e( C M A ) a s r e d u c i n ga g e n t .T h e l i n e sh a v es l o p e- I . 0 . D a t a f o r
r e d u c t i o nu s i n g D T T a r e i n d i c a t e db y f i l l e d s y m b o l s .a n d a r e t a k e n f r o m
t h i s p a p e ro r r e f l 2 ( r ) o r f r o m C r e i g h t o n( o ) ; 2 2d a t a f o r r e d u c t i o n su s i n g
G V I A a r e i n d i c a t e db y a . T h e d i s u l f l d e sa r e i d e n t i f i e db y a b b r e v i a t i o n s
f o r t h e c o r r e s p o n d i n gt h i o l s : E , E l l m a n ' s a n i o n : M A , 2 - m e r c a p t o e t h y l : E . 2 - m e r c a p t o e t h a n oAl :, m e r : S H . g l u t a t h i o n eM
a m i n e :C . c y s t e i n eG
c a p t o a c e t i ca c i d .
l 4 - 1 7 ) . T h e s ee q u i l i b r i u mc o n s t a n t s h o u l dt h u s . i n p r i n c i p l e .
d e p e n db o t h o n t h i o l a n d d i s u l f i d es t r u c t u r e sa n d o n s o l u t i o n
p H . I n t h e s e e q u a t i o n s ,a n d s u b s e q u e n t l ya, n e q u i l i b r i u m
c o n s t a n tr e f e r r i n gt o a d i s u l f i d ei n t e r c h a n g ei n v o l v i n ga m i x t u r e o f t h i o l a n d t h i o l a t es p e c i e sw i l l b e d e n o t e db y t h e s u p e r s c r i p t " o b s d " . T h e i n f l u e n c eo f t h e o r g a n i cg r o u p s R a n d E o n
t h e p o s i t i o no f e q u i l i b r i u m i n i n t e r c h a n g er e a c t i o n sb e t w e e n
d i s u l f i d e sa n d t h i o l a t e i o n s w o u l d n o t n e c e s s a r i l yb e e x p e c t e d
t o b e p a r a l l e l :r e a c t i o ni n v o l v i n go n l y t h i o l a t ea n i o n s ( K s - . e q
l 8 ) s h o u l d b e s t r o n g l y i n f l u e n c e db y t h e p K " s o f R S H a n d
2RS-
r"nsnlf
65+ ESSE -i-+
KSH
2 R S H + E S S E =-
RSSR + 2ES-
'Jf
*""r" (1s)
RSSR + 2 E S H
E S H . w h i l e r e a c t i o nw i t h t h i o l s ( K s H ) s h o u l d b e m u c h l e s s
d e p e n d e n to n t h e s ea c i d i t i e s .Q u a l i t a t i v ea n a l y s e so i e q u i i i b r i u m d a t a o b t a i n e d i n n o n a q u e o u sm e d i a i n w h i c h t h e e q u i l i b r i u n r c o n c e n t r a t i o no f t h i o l a t e a n i o n i s v e r y s m a l l h a v e
s u g g e s t e dt h a t t h i o l - d i s u l f i d e i n t e r c h a n g ei s r e l a t i v e l y i n s e n s i t i v e t o o r g a n i c s t r u c t u r e . l 0 ' l t l n 6 . O e n d e n te x a m i n a t i o n so f
KsH and Ks- in aqueous media have not been reported.
Our primary concern in the work reportedhere is the inf l u e n c e o f r i n g s i z e o n e q u i l i b r i u m c o n s t a n t sf o r i n t e r c h a n g e
reactionswhich involve cyclic disulfides.We have approached
this problem by measuring equilibrium constants for reduction
o f a s t a n d a r d d i s u l f i d e ( o x i d i z e dl i p o a m i d e ) b y a n u m b e r o f
a.c,l-dithiols having different separations between the thiol
groups. Since thesethiols have different pKrs, it is important.
i n t r y i n g t o u n d e r s t a n dt h e i n f l u e n c eo f r i n g s i z e i n t h e c y c l i c
d i s u l f i d e so n t h e m e a s u r e de q u i l i b r i u m c o n s t a n t s ,t o i d e n t i f y
the contributions to theseequilibrium constantswhich reflect
the relative acidities of the thiols.
Separationof a vaiue for KoM (eq l7) measuredat a known
pH into valuesof KsH and Ks- requiresonly knowledgeof the
pK,,s for the two thiols ESH and RSH. The fact that oniy one
e x p e r i m e n t a le q u i l i b r i u m m e a s u r e m e n ti s r e q u i r e d t o o b t a i n
valuesof KsH and Ks- reflectsthe fact that theseequilibrium
c o n s t a n t sa r e n o t i n d e p e n d e n t :t h e r e l a t i v ec o n c e n t r a t i o n so f
J o u rnalof the A meri canC henti calS oci ety/ 102:6I March 12, 19 80
20l6
?
a^
ko
or
-9
'Yz
CI
I
+l
ESSE+zRS-= RSSR+zES-
a,
7o
Y
!o
,t 6,/
t,
0
"a %V,
)<
or
A
o
Y
g-2
,y't
'
tto
9a"
pH -trzlpKo*,
p"ott")
F i g u r e6 . s c h e m a r i c r e p r e s e n r a t i oonf t h e r e s p o n s eo f l o g .K o b ' d( d e f i n e d
- pKusH to
b f e q l 7 ) f o r s e l e c t e dv a l u e so f t h e P a r a m e t e rp K " * s |
. h " n g . r i n t h e s o l u t i o np H l t h e s ec u r v e sw e r eg e n e r a t e df r o m e q 2 5 . F o r
c o n v J n i e n c ei n p l o t t i n g , t h e z e r o o i t h e l o g 6 o u ' a a x i s i s t a k e n t o b e t h e
l/z(log
m e a no f t h e l o g a r i t h * o i t i , . e q u i l i b r i u mf o r t h i o l a n d t h i o l a t ei o n ,
K s H + l o g K s = ) . a n d t h e z e r o o f t h e p H s c a l ei s t a k e n t o b e m e a n o f t h e
* p K r s H ) . E a c h c u r v e i s t a b e l e dw i t h a v a l u eo f
pK, valuei. l/z(pKuRSH
=
p r " n s H - p r " ' e s H T h e b o l d f a c ec u r v e i s f o r p K " R S H p K " E S . H 2 ' 0 :
n o t e t h a t t h e o b s e r v e de q u i l i b r i u mc o n s t a n tX ' o b sfde l t h i s c u r v e i n c r e a s e s
b y ,l 0 a a s r h e p H i s c h a n g e df r o m v a l u e ss u f f i c i e n t l l , a c i d i ct h a t b o t h R S H
rna eSU are essentiallyiompletelyprotonatedto valuessufficientlybasic
t h a t b o t h c o m p o n e n t sa r e e s s e n t i a l l yc o m p l e t e l yi o n i z e d '
-b-4-262468
z (ptrf$-proEsH)
F i g u r e7 . P l o t so f ( A ) l o g , ( s H a n d ( B ) l o g K s - v s ' 2 ( p K " R S " : ! K " t t " ) '
= 2.12'
T h " en u m b e r s r e f e r t o f a b l e I V . ( B ) i n c l u d e st h e l i n e l o g 5 s t
hrough
l
i
n
e
l
e
a
s
t
s
q
u
a
r
e
s
b
e
s
t
t
h
e
w
h
i
c
h
r
e
p
r
e
s
e
n
t
s
(pK"RsH pK"sH).
(0.0)' The
i i . i o i n t r I - S l i n v o l v i n g a l k y l t h i o l s ) w h i c h p a s s - et.hl r o u g h
=
(B) log KS- =
d a s h e dl i n e sa r e i n c l u d e df o r r e f e r e n c e (: A ) l o g K s H 0 ;
p
K
"
E
s
H
)
.
2(pK"RsH
only on the differences in the characteristics (pK,. KsH' Ks-)
of the thiols involved. rather than on their absolute values. The
thiols with which we are concerned in the following have pr("
values between 7.5 and I 1.0.and experimental measurements
w e r e m a d ea t p H 7 . T h u s ,t y p i c a l l yp, H - l / 2 ( p K , R S H+
- p6" ESH
pK ,E S Hhad
) val uesfrom -3.0 to -0.5, andpK " R S H
ci
rcumsta
nces,
these
t h i o la n dt hiolat eanionsa refi x e db y th e k n o w nv a l u e so f pH '
U
nder
2.5.
and
0
had val uesbetw een
T o d e ri v eKS H a n d K S- fro m 7 1 ' obsd, Fi gure6 i ndi cates
p KrRSHa. nd pK oE S H.
that 6obsdcoul dcontai nsi gni fi cantc ong e ra ti o o f th i o l ate rri buti onsfrom boththi ol - andthi ol ate-di sul fi de
. p re s s i nth
ar e us e fu l Ex
i nterchange
s e ve rael quat ions
intoeq I7, oneobtains e q u i l i b r i a .
to thiolby eq l9 and 20,andsubstituting
of t he
andconsi stency
reasonabl eness
To checkthephysi cal
(le)
( E S - ) / ( E S H ) - l o P H - P ^ u * "= 6
wc have
of 1'trbsd,
valuesof KsH andKs- obtainedby dissection
Figure
constants.
( 2 0 ) examineda numberof literatureequilibriun'l
( R S - ) i ( R S H ; = l g n H - R K ' R t "= 6
pl otsof l og K sH and l og K S - vs. 2(pK uRSH
7 show sseparate
- pK ,E s11)
deri vedfrom the data summari zedi n Tabl e I V.
( 2 1)
KSH= !] i :l; 6obsd
i n the upperpl otshow sno obvious
The
equi
l
i
bri
umconstants
(l + 0)'
pK,s.
are inlluencedby steric effects:
but
with
correlation
- 1' * :-iJ; x'obsd
(22)
76ssteri cal l yhi nderedthi ol sgi ve l ow val uesof K sl ' 1.W e n ot e,
(l * d-';'
how ever,that the data i n thi s fi gurehavebeendraw n from
several
sources,and differencesin experimentalconditions
(23)
Ks- = { ^tn
in individualdata,may
betweensetsof data,and inaccuracies
ApK"' fl':.lpKsH-on
log
of
weak
dependence
disguise
a
(24)
log 6s- = 2(pKrRSH - pKaESH)+ log KsH
pro"ximate
linearityol the plot of log KS- vs' 2!Ptr,*t"
by the acithat K s- i s strongl yi nfl uenced
pK " E S Hi)ndi cates
( l + 6 ) ( l+ 6 - r )
conj ugatethi ol s.and that the d at a
di ti esof the parti ci pati ng
log6ou'o- | lz.loeKsH+ logtrs-1= log ( l + e ) ( l * e - r )
are adequatelrati
y onal i i edby the schemeof eq 18.In pr inshould.be(/3nr"
ciple,the slopeof a plot of log KsH vs-^2ApK"
= (pKuESH - pKoRSH)
-' gr, = I ) and that of a plot of log KsH vs' 2ApKoshouldbe
I I * I gpH-r /2(pK"nsH+pKaEsH)-l/21px^esu-pr"RsH)]
e de(B ^,,:- 6,,) (seebel ow );i t shoul dthereforebe possi blto
r 2log
experimentallyfrom theseplots.Figure7B
i!iri". P"";"- ,619
line drawn throughthe
(2s) includesfor referencethe least-squares
that
suggests
and
thiols.
6nu.- fl,r- l '2' We
setof aliphatic
unKSH,and KS- summarized rei teratethat the qual i tyof the data makesthi s esti mate
between6'obsd,
the relationships
*.20Vo.
least
bY
at
certain
and
eq
23
of
sides
both
of
logarithm
by eq 21-23.Taking the
w e can i nterpretequi l i bri umc onW i th thi s background,
exp a n d i n gy ieldseq24.M u l ti p l y i n go f e q 2 l a n d 2 2 ' ta k i ng
lipoamideby a seriesof
oxidized
of
reduction
for
stants
expression,
the squareroot and the logarithmof the resulting
both by differentseparationsbecharacterized
a.<r:-dithiols
eq 25. This lastequation,although
and expandinggenerates
tw eenthe thi olgroupsand by di fferentval uesof thi ol pK" in
the functionalrelationsbetweenlog
cumbeisome.describes
the influenceof ring sizein the product
waythat separates
a
plotted
is
easily
that
pH
form
pKusH, and
in a
Kohd,pKoRSH,
that
of
the aci di tyof the starti ngdi thi ol .The
from
( Figure6). The particularfunctionalform of eq25 waschosen di sul hde
for reductionof oxidizedliK1;*obsd
constants
equilibrium
so that the shapeof the curvesgeneratedfrom it woulddepend
20t7
Szajewski, Whitesides/ Thiol-Disulfide InterchangeReactions
2l and22
Vaiues
fe15obsd
intoKs- andKsHUsingEquations
of Literature
TabfetV. Separation
I
2
J
4
5
6
7
8
9
l0
lt
t2
IJ
t4
r5
l6
t7
l8
r9
20
disulfide
(ESSE)
t h i o l( R S H )
no.
(cH3)3csH
dithiothreitol
cysteine
glutathione-SH
N HzCHzCHzSH
NHzCHzC(CHr)zSH
NH2CH(CH3)CH(CrH7)SH
NHZCHZCH(CH3)SH
NH2CH(CH3)C(CHr)25H
t.)
7.4
6.5
6.0
6.0
6.0
T
T
.f
'7.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6'SH a
ref
2.4
3.6
0.75
2.t
1.4
3.0
3.3
1.7
b
b
b
b
c
d
e
e
0 .t 6 )
0.28)
0.e8)
0.54)
s
s
s
s
8 . 0x t O l
3.6
1 . 9x 1 0 2
3.1
3.6
0.80
1.7
1.4
3.0
3.3
1.7
0.16
0.28
0.98
0.54
31.
0.98
l.l
2.1
0.33
0.14
0.36
0.09
7.4
7.4
cystine
cystine
glutathione
glutathione
glutathione
cystine
cystine
glutathione
(cHr(cH2)5s)2
(CHr(CHz)sS)z
(cHr(cH2)2s)2
(cH3(cH2)3s)2
lipoamide
BSSBi
BSSB
BSSB
BSSB
BSSB
BSSB
BSSB
cHCONHCHzCHzSH
NH2CH2CH:SH
CHTCONHCHuCHzSH
NH2CH2CHzSH
NH2CH2CHzSH
glutathione-SH
HSCH2CO2H
HSCH2CO2H
CoHsSH
HOCH2CH2SH'
CHTCHzCH(CH3)SH
KS- O
gobsd a
pH
0.r8
0.15
28.
5.6X 104
2.7X t02
(4.4x t0-s
( 1 . 4x l 0 - 3
(le.
(4.0
3.0
1 . 2x 1 0 4
1 . 3x 1 0 5
2.6X t04
2.2X t03
1 . 1x 1 0 3
2.3X 103
6.2x t02
h
31.
0.r2
0.14
0. 26
0.04
0. 02
0. 05
0.0r
j
i
i
i
i
i
i
o Equilibrium constanr5arc dimensionless.t L. Eldjarn and A. Pihl. J. Am. Chem. Soc.,79, 4589 ( 1957).' L. Eldjarn and A. Pihl, "/. aior'
Chen.: 2?5,4gg(t957 ). I C. corin and c. Doughty.Arch. Bioihem. B{ophys.,126, 547(1968). ' t. M. Kolthoff.W. Strick, and R. C. Kapoor.
J. Am. Chem. Soc.,77, 4733 ( 1955)./ Thesecquilibrationswere perfo.med in hyd.ocarbonsolventsand are includedfor rcference.For each.
ir wasassumedthat KsH in water would equalthe listedvalueof,(ot"d. s Rcference31. ' Reference4. i RSSBis 4.4'-dithiobis(b€nzenesulfonic
acid).i G. Gorin,G. Doughty,and R. Gideon../.Chem.Soc-B,729 (1961).
p o a m i d e b y s e v e r a lm o n o - a n d d i t h i o l s w e r e d e t e r m i n e d e x p e r i m e n t a l l yu s i n g a p r o c e d u r ed e v e l o p e db y C l e l a n d : ad e t a i l s
of this procedureare summarized in the ExperimentalSection.
I n t h e s i m p l e s tc a s e .r e D r e s e n t e d
b v e q 2 6 a n d e x e m p l i f i e db v
HS-R-SH
s
Iff;a'j;"*"
.Aqp,
\
.I
^,,
^\11!/
-
)--SH
-
p .H- _7
tl
(cH,,).co\H
\
\
o
b
(cHr).,coNHr
+ NADH
+
+ NAD' :
HS-R-SH
(cH,).coNHr
-K
' 7=7
Kupoobtd
i-
s-R-s
5H
+
SH
S-R-S
+ H+ ('/lat
+ NADH + H'
(lipoox)(NADH) =
0.0858
(28a)
(27b)
(lipored)(NAD')
( 26al
(cH:)..coNH.,
KNeDobd
=
(s-R -S X N A D H )
(H S -R -S H X N A D ' )
KtipoobdK.,,
(28b)
f o r K 1 ; o , r u bf' rdo m a p a r t i c u l a r s e t o f s t a r t i n g a n d o b s e r v e d
concentrationd
s e p e n d so n t h e c h o i c eo f e q u i l i b r i u m e q u a t i o n .
(HS-R-SH
)(liPoox;
E q u a t i o n 2 6 a s s u m e st h a t t h e p r o d u c t o f o . x i d a t i o n o f a n
c r . c o - d i t h i oH
l S - R - S H i s t h e c o r r e s p o n d i n gc y c l i c d i s u l f i d e .
HS - R- S H = D T T (s e eb e l o w ),re a c ti o no f I equi vof d,ord d ucedl i poami de In principle.this reactionmight alsogeneratedimeric or higher
dit hiolwit h o x i d i z e dl i p o a mi d eg e n e ra tere
oxi dati ve o l i g o m e r i c c y c l i c p o l y ( d i s u l f i d e s ) o, r l i n e a r p o l y m e r i c d i s u l and t he c y c l i cd i s u l fi d efo rm e db y i n tra mo l ecul ar
fides. The experimental procedureused here measuresdirectll
. h i s e q ui l i brati onw as
r i o l g ro u p sT
c ouplingof t h e a a n d c .'th
c ar r iedout in th e p re s e n coef N A D + a n d l i p o ami dedehydro- o n l y t h e c o n c e n t r a t i o no f N A D H ( a n d b y i n f e r e n c e t h a t o f
e q 2 7 ) .T h e c o n c e n t r a t i oonf N A D H a t e q u i l i b r i u m r e d u c e d l i p o a m i d e ) ; i t d o e s n o t i d e n t i f y t h e s t r u c t u r e s o r
- q e n a s( e
concentrationsof the disulfidespresent.We have not attempted
was m eas ur e da, n d s tra i g h tfo rw a rdc a l c u l a ti onsbasedon
thiol,oxidizedlipoamide, t o r e s o l v et h e a m b i g u i t y c o n c e r n i n gt h e s t r u c t u r e so f t h e d i of a,r,.r-di
known initialconcentrations
s q u iredto cal cul ate sulfidesproducedin thesethiol-disulfideinterchangeequilibria
and NA D+ y i e l d e dth e c o n c e n tra ti o nre
b y t h e i r i s o l a t i o n :s i n c e a l l o f t h e p l a u s i b l et y p e s o f p r o d u c t s
K , , *oo' o( eq2 6 b ).M u l ti p l i c a ti o no f K" * o l o b y K zt gi vesthe
(e
q
c
a n i n t e r c o n v e r t ,t h e r e i s n o g u a r a n t e et h a t a n y s p e c i e si d e n bioc hem ic a l lre
y l e v a nqt u a n ti tyKr,rD o b ' d 27,28). In eq
l y e concentrati ons t i f i e d i n c o n c e n t r a t e d ,i s o l a t e df o r m w o u l d b e t h a t p r e s e n ta t
27b,( lipou^)a n d (l i p o " d )a re re s p e c ti v eth
e q u i l i b r i u m u n d e r t h e c o n d i t i o n su s e d t o m e a s u r . K , , * o o ' d .
K n and
. c o n v enti on,
ol ox idiz edan d re d u c e dl i p o a mi d eBy
I n s t e a d .w e h a v ee x a m i n e dt h e c o n c e n t r a t i o nd e p e n d e n c e( o r
pH
value
particular
of
solution
Kr,rDub'dare definedfor a
i n d e p e n d e n c e )o f e q u i l i b r i u m c o n s t a n t sc a l c u l a t e d f r o m e x ( her e,pH 7) a n d a re d i me n s i o n l e s(e
s q 2 7 b and 28b).The
p e r i m e n t a i d a t a m e a s u r e da t d i f f e r e n t c o n c e n t r a t i o n so i d i balanc edequ a ti o n to
s w h i c hth e y re fe r(e q 2 7 a and 28a) i neq 26 (for formation
thiols. for three equilibrium expressions:
v olv epr ot onsth
: u s K2 7a n d K 5 l n p o b s;n
y
d ' tp 1 l 6i tli ncorporate
and dependon solution o f a c y c l i c m o n o m e r i c d i s u l f i d e ) ,2 9 ( i o r r e a c t i o n g e n e r a t i n g
a term in hydrogenion concentration,
a c y c l i c o l i g i m e r o f n m e m b e r s ) .a n d 3 0 ( f o r [ i n e a r p o l y m e r pH. Kripuob'd,K7j, KNn Dobtd,and K655cobtd(see below) are
i z a t i o n t o a h i g h e r m o l e c u l a rw e i g h t a , o r - d i t h i o l ) .T h e s e c o n d
all basedon calculationswhich utilize the totalconcentrations
pq{ il_gg_2c
is derived from eq 29b by assuming that
o f t h i o l a n d t h i o l a t es p e c i e sd e r i v e d f r o m e a c h S H - c o n t a i n i n g
(
S
R
I
S
S
R
]
n
c o m p o u n d .W e t a k e t h e v a l u e o f K z t t o b e 0 . 0 8 5 8 . 3 2
r S ) = | I n ( l i p o r c o )( t h a t i s , i n a s ! ' s t e mo r i g i n a l l y
c o n t a i n i n g o n l y a . c , . r - d i t h i oal n d o x i d i z e d l i p o a m i d e . t h e
T h e n u m e r i c a iv a l u e o f t h e e q u i l i b r i u m c o n s t a n tc a l c u l a t e d
Kliooobtd
=
(S-R-SXliPorea)
(%b)
2018
Journal of rhe American chemical society / 102:6 / March 12, rg80
TableV. EquilibriumConstantsfor Thiol-DisulfideInterchangeReactionand DerivedConstantsa
chain
length
compd
thiol
l4
HOCH2CHSHCH25H (BAL)
25
HSCH2CHOHCH2SH (DMP)
HSCH2CH2CH25H
35
45
lipoamidc (lipo'd)
56
dithiothrcitol (DTT)
66
dithioerythreitol ( DTE)
76
HS(CH2)4SH
(HSCH2CH2)20
87
(HSCH2CHOH)2CH2
97
(HSCH2CONH)2'
l0
8
(HSCH2CHOHCH2)2CH2
n8
( H S C H 2 C O 2 C H z ) z( G M A )
t2
t0
t3
monothiolH S C H 2 C H 2 O H ( M E )
14 monothiolglutathione (GSH)
PK,
8 . 6( I 0 . 5 ) .
9 . 0( 1 0 . 3 ) "
9 . 8( r 0 . 9 /
r 0 . 7( r 0 . 7 ) r
9 . 2( 1 0 . 1 ) "
9 . 2( 1 0 . 1 ) a
1 0 . 0( 1 0 . 7 ) "
e.2(e.e),
9.I (r0.6)/
8.0(9.6/
e . 3( r l . l y
7 . 7( 8 . 9 ) .
9.U
8 . 7k
o
K,,*o*
t
5.8
0.51
0.12
I
l5
ll
22
0.68
0.88
0.50
9 . 2x l 0 - l
2 . 0X l 0 - l
1 . 8x l 0 - l
disulfidc
form
KxeDoH
6
cyclic dimer
cyclic monomer
cyclic monomcr
cyclic monomer
cyclic monomcr
cyclic monomcr
cyclic monomcr
cyclic monomer
cyclic monomcr
cyclic monomer
cyclic monomer
polymer
dimcr
dimcr
4.2X l0-2
4 . 4X l 0 - 2
1 . 6x l 0 - 2
8 . 6x , t 0 - 2 i
1.3
0.95
1.9
0.64
5 . 8x t 0 - 2
? . 6X l 0 - 2
4 . 3x l 0 - 2
7 . 9x l 0 - 4
1 . 7x l 0 - .
1 . 5x l 0 - 4
cyclic monomer
6.4
Kcsscs- t
KcsscoM'
2 . 9x
3 . 1x
l.l x
5 . 9x
8 . 8x
6 . 6x
1 . 3x
4 . 4x
4 . 0x
5 . 2x
3 . 0x
5.4
t.2
I
102 -0.298
1 0 2 -0.279
l 0 l -0.296
102 -0.288i
t03 -0.323
l0l -0.319
104 -0.328
103 -0.3t4
102 -0.283
102 -0.286
102 -0.279
-0.227
-0.207
-0.205
-0.344
1 . 0X 1 0 3
3 . 8X I 0 r
7.8 X 105
l.l x 107
6 . 0X t 0 5
5 . 2X t 0 5
9 . 6X 1 0 6
8 . 0x t 0 ' 1
4 . 6X l 0 r
4 . 4X t O r
8 . 4X l 0 r
4 . 0X 1 0 2
7 . 1x l 0 r
I
KcsscsH "
5 . 8x t 0 2
6.2x t02
2.0 X l0l
1 . 3x t 0 3
1 . 8x t 0 4
1 . 4x I 0 4
2 . 6X 1 0 4
8 . 9x t o l
7 . 8x t 0 2
1 . 2x t 0 3
6 . 0x 1 0 2
3.9
t.2
I
8 . 6x 1 0 4/
" All equilibrationswerc carricd out in 0.05-0.02 M phosphatebuffer at pH 7.0, 30.0 + 0.5 oC, undcr argon usingthc lipoamide-lipoamide
delydrogenasccouple.'i(1pooH and Kp4Dos havcunits oi M-l for entriesl, 12,
2, 13,and 14;
the other
l4; for all of
ofthc
oher thiols,
thi;k. they
the; are
arc dimensionless.
dimensiontcss.
' (csscobd, .Kcsscs-, and K6556ss for
entries l, 12, 13, and 14 are dimensionless;for all the orher thiols they havc units of M. Rcference
36 dcsc.ibesthe calculation of /(qss6s-.and-,(6gs6sHfrom Kq556oH. d.Eg (V) vaiuesvs. srandardhydrogenilectrode at pH i.0 and 30.0
oC (seefef 32). ' Thesep,(. values
arc takcn,fromrif tZ. / Octcrminedby the methodsdescribedin ref li. c IlM. Gascoigneand G. K. Radda,
&ioc.him.BioPhvs.Acta, l3l,498 (1967). ' Standa.d valuc; seeref 32. i Synthesisof this material is describedin ref-41. I This pK" value
is takcn from rcf 13, r Refercncc15. r O*idiz€d dithiodiketopiperazinewas cquilibratedagainstHS(CHr)dH in CHCl3, and an equilibrium
-'ofK = 3.3 determinedby lH NMR spectroscopy.
This value corrcspondsto kqss6sH =-A.e x IOoM.
aHSRSH+ nlipo* ,- f,p]ffirt;,J
* rlipo."d (29a)
,#.--ll3
tflioo..at,
K".-,,-.b'd= I:Il!!5Lr
lution,thedcrivedequilibrium
constanr
shouldbe invariant
withchanges
in thestartingconcenrration
of dirhiol.Exami-
"c,c'c- (HSRSH).*;;.)"'(2eb)
ffi[:"*hiTT|*'J:H]:,,1T;::]".,**:,";o#*l
r../,
( K.."ri"uo.d)
'
fSTf SSn i-l];sl r/n{rin^,cd\
(HSRSH)(lipoo\)
/ '(lipo*d\t+ |/ "
_ [l I n]t
( HER-SE!1-1*-|
,.^^,
tzvct
!/" is alsoinvariant.Equation
with concentration.
( '(":,.ri.obd)
l9c differs from that for K1,pooM
(eq 26b) only in rhe exponent
of the rerm (lipo'"d)and
ru.ior, ior K1;*ofu rhis
"ion*uni
exponent is_-2:
for formation
of a cyclicd imer (eq 26i, n = 2),
it is 3/2. Thus, equilibriumexpiessions
for glneration oi
: R,ssRR,sH
+R-sH
+ripoox
(3oa)fliil:ffT:li1',1"Jf,1'.:ilh:jlX.,j"J_::1$:f|'""$.
+ripo,d
r
i ouru
=
( R,SSR-)(lioo.d)
( R'SH)(R"SH)(lip
,- - (E:SSRj')(liPo'd)
60b)
- '
[2(HSR" SH)]r(lipoo')
perimenrallydifficutrro disringuish.
K*5;,* u, u tuncrronot rhe concenrrarion
ofsraning d.&rdiihiot for rhreedithiolsspanning rhe rangeof inrrathiolseparationsexamined:
(BAL), DTT, ana
2,3-dimercaptopropanol
t;iT
number
ofdisurnde
bonds
formcd
from
theformcr
must
equa, fl:illjjT,1T,Xl"T::1H?#,tl
fi:::riil'j1)'i;:
the number of lipoamidedisulfidebondsreduced).Taking the
n th root ofeq.29bandconve.tingthe numeratorby substitution
to an exprcssioncontainingonly thc concentrationof reduced
Iipoamidegeneratcsthe final expression
in eq 29c.
Equation
30
assumcs
groups
that
presentin the soall
SH
.
Iution arc equally rcactivein eachthiol-disulfide interchange;
that is' it.assumesthat the thiol groupsof an a,<.r-dithiolrcact
completely-indcpeldently,
and that intramolecularinterchange
is neither favored nor disfavored rclative to intermoleculir
intercharg-e.In this equation, R'SH and R"SH arc-thusarbitrary- SH groups in the reaction mixture, either from the
original dithiol or from someoligomer,and (HSR"'SH) refers
to the total conccntrationof all the a,ar-dithiolspresentin the
systcm. If intramolecular reactionsarc excludedand only intermolecularreactionsoccur.the factorof4 in the denominator
of eq 30b disapp€a$.Thc concentrations
of the sprecies
derived
from the c,<o-dithiol in cq 29 and 30 arc casily estimatedin
thescsystemsby balancingrcdox equations,following proceduresanalogousto thoseoutlined in the ExperimentalSection
for calculation of-K1;*obd.
l.hc purposeol this analysisis to distinguish betweenthe
equilibriarepresented
by eq 26,29,and 30.1f the equilibrium
exprcssioncorrectly representsthe reaction occurring in so-
As expected,this figure contlritt iial DTi forms a cyclic
monomer,and not aiincar polymer. Thc data for GMA indicatc that this material forms a linear polymer. The range of
usablethiol concentrations
in this lattercaseis limited on the
low end by thc limited reducingability of C MA and on the
high end by its low solubility. Tie data for BAL are not com_
patiUtewith formationof a linearpolymer(eq 30). They do not,
however,clearly distinguishbetrieenequilibration roa cyclic
monomer,dimcr, or pirluty higtt"t
oligomer,although
the slight slopeobscrvcafoi f1i.M "y"iic
suggeststhat a dimer;r
oligomermight be the preferred'species.-Efforts
to resolvethis
ambiguity using othir techniques indicate that a cyclic
monomeris not iormed and suggestthat a cyclic dimer is the
probableoxidation product una;r thescconiitions.s3-1s
Thc studiessummarizedin Figure8 are compatiblewith the
hypothesesthat oxidation of DiT yietJs a cyitic monomeric
disulfidc,thar oxidationof BAL yieidsa cyclic dimer, and that
oxidation of CMA yields a linear polymer. Table V lists
K1;rob.dand Kpapod for thesereactions,and corrcsponding
daia for other mono-and dithiols.Allofrhe other a,r,r_dithiols
were assumedto form cyclic monomericdisulfideson oxidation; thejusrificationfoi this assumptionis discussed
below.
Table V ilso containsseveralother usiful conslants:,(csscohd
Szajewski, Whitesides/ Thiol-Disulfide InterchangeReactions
2019
si-:
o.g
Kil:.
s(e
o3
3
,r
r
\z
's
::e
:lo
rl
I
oi
-9
I
t2l
t
ftr
LS
t
Ko!..
''lcal
stS
s87
"6
a
^^l
a
|
| (-"-.'\
F.-/\^
AE
)-)
r
o o.l
t.
I
'
*#
143 er
I
o3
2:
'z
08
os 3l
- t to
gz
^t
'29
)*il.
t
678
Choin Lenglh
I
x4
. . F
'lD-':'
aa
a
o
o
For comparison, Table V includes a value for a conformat i o n a l l y r i g i d d i t h i o d i k e t o p i p e r a z i n e ' , v h i c hf o r m s a s i x - m e m -
aa
.*l
|-fI
:<
or
IL
-l
I
-2
o
oo
ooo
-5
otz
roefxsnsn] (mM)
F i g u r e8 . P l o t so f l o g K a s a f u n c t i o no f t h e c o n c e n t r a r i o o
n f r e d u c i n gt h i o l
t h a r o x i d a t i o no f t h e d i t h i o l sy i e l d s
I H S R S H J c a l c u l a t e do n r h ea s s u m p t i o n
( o ) c y c l i c m o n o m e r i cd i s u l f i d e s( K r i p o o M , e q2 6 ) ; ( r ) c y c l i cd i m e r i cd i sulfides (K"y.ti.obd,eq 29): or (O) polymericdisulf'ides(K*i"oM, eq 30).
K"y"ri"obtdand Kpu"obd have units of M: K1;*obtdis dimeniionless. AII
e q u i l i b r a t i o n sw e r e c a r r i e d o u r i n 0 . 0 5 - 0 . 2 M p h o s p h a t eb u f f e r , p H 7 . 0
at i0.0 + 0.5 oC under argon. Dara are shown ior (A) dithiothreitol( DTf.
5 , T a b l e V ) ; ( B ) 2 , 3 - d i m e r c a p t o - t - p r o p a n(oBl A L . l . T a b t e V ) ; ( C ) g t y c o l
d i m e r c a p t o a c e t a t e( C M A , 1 2 , T a b l e V ) .
is t he equilibr iu mc o n s ta n fo
t r e q 3 1 , a n d i s o b t ai nedby di vidingKNRoobtd
by I .5 X l0-4 tr- r (thevalueof Kx.roobdfor
glutathione);
Eo for eachof the thiolsis relatedto the half-cell
p ot ent ialf or t he N 4 D + /N AD H c o u p l e(e q 3 2) by eq 33;
KcsscS- and KcsscSHare calculatedfrom K6556;ubJ
and the
thiol pK. valuesusingeq 2l and 22.36
HS . R. S H + C SS G # E" Y
2e- * NAD+ + H+
S.
pH7
E ' N A D= 0 ' 3 2 0v r 2
+ 2 GS H
NADH
(3I)
(32)
vs. SCE. pH 7
f oRsn - E o N n o:
#log
K ;* 1 a p u b * d
Figure 9. Plotsof log K6556obsd
on6 log K6556sH vs. chain length for the
r e d u c t i o no f o x i d i z e dg l u t a t h i o n e( G S S G ) b y a s e r i e so f d i t h i o l s a t 3 0 . 0
o
C
+ 0.5
i n p H 7 . 0 p h o s p h a t eb u f f e r u n d e ra r g o n .T h e n u m b e r sr e f e r r o
T a b l e V . A l l d i t h i o l s a r e b e l i e v e dt o f o r m c y c l i c d i s u l f i d e so n o x i d a t i o n .
w i t h t h e e x c e p r i o no f 2 , 3 - d i m e r c a p t o p r o p a n(ool, r , l ) , w h i c h m a y f o r m
a c y c l i c d i m e r ( s e et h e t e x t ) , a n d g l y c o ld i m e r c a p r o a c e r a (t e
0. O, l2),
w h i c h f o r m s a l i n e a rp o l y m e r .T h e p o i n r f o r t h e d i t h i o k e t o p i p e r a z i n(ev .
l 5 ) w a s o b t a i n e d i n a n o r g a n i c s o l v e n t .T h e u n i t s o f K 6 5 5 6 o b d a n d
K c s s c s H a r e M . T h e e q u i l i b r i u m c o n s t a n t sf o r t h e s ed i t h i o l s c a n n o r b e
c o n t r a s t e dd i r e c t l y f o r t h a t w i t h m o n o t h i o l ss, i n c et h e u n i r s a r e d i f f e r e n t .
F o r c o m p a r i s o nh. o w e v e ra. d d i t i o no f a s m a l l q u a n r i t yo f G S S G t o a I M
s o l u t i o n o l 2 - m e r c a p t o e t h a n o la, 0 . 2 2 M s o l u r i o n o f g l y c o l d i m e r c a p t o a c e t a t ea. n d a 1 . 4X l 0 - ' t M s o l u t i o no f D T T w o u l d p r o d u c er h e s a m e
r a t i oo f ( C S H ) / ( C S S G ) .
(JJl
b e r e d c y c l i c d i s u l f i d eo n o x i d a t i o n .T h i s c o m p o u n d i s n o r s o l 'uble
i n w a t e r . a n d i t s e q u i l i b r i u mc o n s t a n tw a s m e a s u r e di n
c h l o r o f o r m b y e q u i l i b r a t i o no f t h e d i k e t o p i p e r a z i n ed i s u l f i d e
a g a i n s t b u t a n e - 1 , 4 - d i t h i o lT. h e d e r i v e d c o n s r a n r K c s s c s H
s h o u l d . h o w e v e r .b e c o m p a r a b l et o t h o s eo b r a i n e d i n a q u e o u s
s o l u t i o n s .s i n c et h e s ec o n s t a n t sa r e p r o b a b l y r e l a t i v e l v i n s e n s i t i v et o m e d i u m . r l
Figure 9 illustratesthe variation in Kcsscob'dand K6556sH
( f r o m T a b l e V ) w i t h i n t r a t h i o l c h a i n l e n g t h .T h e f o r m e r d a t a
( p H 7 . 0 ) a r e u s e f u l i n t h e p r a c t i c a l p r o b l e m o f c h o o s i n ga r e d u c i n g a g e n t f o r p H 7 a q u e o u ss o l u t i o n .T h e p l o t g i v e s o n l y
upper limits for a,c,.r-dithiols
connectedby chainsof two (BAL)
a n d e i g h t ( G M A ) a t o m s .S i n c e t h e s ec o m p o u n d sa p p e a r e dt o
form respectivelya cyclic dimer and polymer, it is nor possible
to establishequilibrium constantsfor intramolecular disulfide
f o r m a t i o n . I t i s , h o w e v e r , p o s s i b l et o e s r i m a t e w h a t t h e s e
e q u i l i b r i u m c o n s t a n t sw o u l d h a v e h a d t o h a v e b e e n f o r t h e
p r o d u c t o f i n t r a m o l e c u l a rc o u p l i n gt o h a v e b e e n p r e d o m i n a n t
u n d e r t h e r e a c t i o nc o n d i t i o n s .T h e s ec o n s t a n r sa r e i n d i c a t e d
on the figure as upper limits. The plot of log KcsscSH vs. chain
l e n g t h i s u s e f u l i n e s t a b l i s h i n gt h e s t r u c t u r a l b a s i s o f t h e r e d u c i n g a b i l i t y o f t h e d i t h i o l s . T h e s ed a t a c o n t a i n n o c o n t r i bution from the relativeaciditiesof the thiol groups. The close
q u a l i t a t i v e s i m i l a r i t y b e t w e e nt h e p l o t s o f l o g X ' o b s d
and log
K S H v s . c h a i n l e n g t h s u g g e s t st h a t d i f f e r e n c e si n t h e a c i d i t i e s
o f t h e t h i o l g r o u p sa r e r e l a t i v e l yu n i m p o r t a n t i n d e t e r m i n i n g
t h e r e d u c i n g a b i l i t y o f t h e s ed i t h i o l s .
Several featuresof the data summarized in Figure 9 warrant
discussion.First. the equilibrium constant K6556SH = 3.9 for
g l y c o l d i m e r c a p t o a c e t a t e( G M A ) i s a p p r o x i m a t e l y t h a t f o r
m o n o t h i o l s ( f o r e x a m p l e ,K c s s c S H = I f o r - e l u t a t h i o n ea. n d
KcsscsH = 1.2 for mercaptoethanol).The fact that a solution
oi GMA is not significantlymore reducing than a solution
c o n t a i n i n g a n e q u a l c o n c e n t r a t i o no f m o n o t h i o l S H g r o u p s i s
2020
Journal of the American Chenticar society
/ 102.6 / March
8.O
7.O
6.O
5,O
4 .O
3 .O
2.O
t,
C)
l.o
o
()
*
o
= l 6 M - r ; ( l l ) = l 5 M - r . T h e s e c o n s t a n r s a r le0caah. i g h e r
than thosefor otherthi ol sw hi chform i ntramol ecul adrim er s
(GSH, ME) or polymer(GMA). Sinceall of theevidence
from
the w ork suggests
that thi s constanti s not very sensit ive
to
structuralvari ati onsi n the starti ngthi ol ,w e concl ud e
eit her
that di thi ol shavi ngsevenor ei ghtbondsseparati ng
the t hiol
groupsshowanomalously
high reactivityin polymerformation
or that they form cyclicdisulfides.
The latter alternativeseems
the more probable.It is more difficult to distinguishintramoleculardisulfideformationfrom formationof a ivclic dimer
for reasons
discussed
above.Third, the observation
that a solutionof BAL is muchmorestronglyreducingthana solution
of a monothi olcontai ni ngan equalconcentrati on
of t hiol
groupssupportsthe hypothesis
that BAL formsa cyclic di_
mericdisulflde:an initiallyformed.half-oxidized
dimer from
B A L enj oysthe sameadvantage
i n cl osi ngto an ei ght - m em beredri ng asdoesan a,or-di thi ol
of si mi l arstructure,b ut t he
i ni ti al formati onof the hal f-oxi di zed
di mer requi resa bimol ecul arstep.
OH
Ctt
8 .O
t( ^ . _. J n
Y|
\
7.O
12,Igg0
-sH
RSsR
-- 2 R S H
"'-{::v'"
RssR
6.O
-z,.r'n
.--
HO
\
/-
s-s-\
-
oH
/
#
\r-r/
5,O
Discussion
4.O
T h e r a r eo f r e d u c r i o o
n f o x i d i z e dg r u t a t h i o n eG,S S G ,b y
t h i o l - d i s u l f i d ei n t e r c h a n gien a q u e o u ss o l u t i o nf o l l o w s
a
B rpnsted
rel ati on.
3.O
The anal yti cal
methodusedto fol l owth ese
rateswaslessgeneralthanthat employed
in our previous
study
of the ratesof reducti on
of E Il man' ri .og.nt, and fe* er thiols
2 .0
w ere exami nedas reduci ngagents.N onethel ess,
the close
p a r a l l ebl e t w e etnh er e s u r tosf t h e s et w os t u d i e sa.n dt h es i m _
l.o
i l ari ty of the B nu.val uesderi vedfrom them suggesrs
tha t a
t.o ?.o 3.O 4.O 5.O 6.0 7.0 g.o
correl ati onbetw een
the protonbasi ci tyof the tn]i tatenu cleophi l eandtherateof i ts attackon a steri cal l unemcumb
y
er ed
log k (expr)
sul fur-sul furbond i s characteri stiof
c thi oi -di sul fi dei nt er c h a n g e .A p i c t u r e o f t h i o r - d i s u r i i d e
Figure10.(A) PIorof rareconstants
i n t e r c h a n g ea s a
for thior-disurfide
interchange
(k)
mechani sti cal luncompl
y
calculated
i cated
usingeq 36 againstcorresponding
reacri oni s i n acci rd wit h
experimental
rareconsranrs:
unitsfor rateconstants
theoreti calresul ts,w hi chsuggesta S r,.r2
areM-r min-1.poinisr-g (o t ur. t"t.n fromrhis
transi ti onstatein_
work:9-13(v) fromref 22:l4-27and4g_50(r)
volvinglittled-orbitalparticipation
fromref l2: 2g_35(O)
by
the
central
sulfur,rT
and
from Tablelv, footnote
j of G. Gorin,c. Doughty.unJ n.'cioeon.-/.
w i th X -raysrudi es,
w hi chi ndi catethatthepreferred
di
rec
r ion
Soc.
j
B,
729
(t96j):36-4:
jirorn
(v) fromref r3:"5r_S i"
9!"^.
w"U"r.
for-approach
of nucreophi rcs
to a di surfi debondi s arongthe
Hartter,and Flohe(ref 2r). points42-s2referto un.y,n,n.,ricar
disur_
s u l f u r * s u l f uar x i s . 3 8
fides;all othersaresymmetricar.
The least-squares
correra[ion
lineshown
=.9'?8'slope= 0.98.^and
two,B,r/nstedcoefficients
determinedexperimentally
=
inrercept
0.l r. (B) simirarprotfor eq
l:r It
,_
38. Detailedidentification
In Il:
thtsw ork (pnu"-. 0.5: 13.^
of thepointsii givenin ,uppr.r.niuri material
* 0E ! - 1.0)di fi ei i n thei ra c_
in the microfilmedition(seeparagraph
curacy.The fi rsti s deri vedfromi rargenumberof data,
a"tendof paper).
and
seemsrel i abl e.The secondi s basedon a smal l erset,and dec o m p a t i b l e w i t h o u r c o n t e n t i o nt h a t t h e t h i o l s
of GMA act
pendsheavi l yon exampres
i nvorvi ngE i l man' sreagent:the
i n d e p e n d e n t l ya n d t h a t c M A f o r m s p o r y m e r s
on oxidation.
general i tyof thi scoeffi ci ent
i s not yer defi ned.w e-havenor
I f o x i d a t i o n o f G M A g e n e r a t e da c y c r i c d i s u r f i d e .
we wourd
explicitlydetermined
separare
valuei forB"and B1_*.
we have,
e x p e c t i t t o b e a s t r o n g e rr e d u c i n g a g e n t t h a n
a monothiol.
however,estimatedvaruesfor theseparameters,usingrate
second, a,c.r-dithiolscapabreof foriring five-, six-.
seuen-.and
constantsfrom this work and frorn literaturesourcesfor reeight-membered rings are stronger redicing agenrs
than moactiorsinvolving
unsymmetrical
disulfides
havingbothRnr"SH
nothiols. The stability of many cyclic five- a-ndiix-membered
and R " S H al i phati c.A ssumi ngthat theseratesaredescri b ed
d i s u l f i d e si s w e l l k n o w n . n t t n o u g i , w e h a v e n o t
e x p l i c i t r ye s by an equati onof the form of eq 35,i mposi ngthe condi ti o ns
tablishedthe form of.tlrgequilibrium reactions
inuotuingseventhat B nu.= * 0.5 andB " = - 1.0- 0,* ,andsol -vi ng
for the un a n d e i g h t - m e mb e r e dd i s ur f id e - c o n t a i n i n gr i n g s ,. ^ u r ]
n ut i o n
uld 0,, by least-squares
anaTysis
*e esririar. ti,"r Bj,
o f t h e m a g n i t u d e s , o fe q u i l i b r i u m c o n s t a n t sc a l c u l a t e d
lno*lj
using
= -0.72, 0, = -0.27,andC = 7.0.Theabilityof the l-esultin!
the various equilibrium expressions(eq 26, 29, and30)
suggesrs
e q u a t i o n( e q3 6 ,k = M - r m i n - r ) t o f i t t h ea v a i l a b l de a t ai i
t h a t i n t r a m o l e c u l a r d i s u r h d ef o r m a t i o n i s
occurring.in p"rs,u.mmari zed
by Fi gurel 0A as a pl ot of l og k(exptl )vs. l o g
ticular, equilibrium constantscalculated
for compo"undg, 9,
k(calcd).
1 0 , a n d l I ( T a b l e v ) a s s u m i n gt h a t o x i d a t i o n
y i e r d sa p o r v m e r i c d i s u l f i d ea r e K p r y - o h (d8 ) = 2 6 M - r .
19) ='2g trtj,, (lO)
ft,nuc5-+ R .S S R trl
pnu" 5S R .+ -S R re
(3a )
2021
S:ajewski, Whitesides/ Thiol-Disulfide InterchangeReactions
log k + C + g n u " p K" n u* t p .p K" c* d p* pK ,l s (35)
l o g k = 7 . 0 + 0 . 5 O p K , , n u ' - 0 . 2 1 p-K0,. 7 3 p K . , r s ( 3 6 )
l o gk :
5 . 5 * 0 . 6 3 p K . n u-c 0 . 3 0 p K , -c 0 . 6 3 p K , r B( 3 7 )
l o g k = 6 . 3 + 0 . 5 9 p K , n u-" 0 . 4 0 p K " ' - 0 . 5 9 p K " r s ( 3 8 )
log /gou'o= log k
-
(l + l0PKrnuc-PH)
(3e)
l f t h e a t t a c k i n gt h i o l a t ea n i o n a n d t h e l e a v i n gt h i o l a t ea n i o n
are both derivedfrom aromatic thiols, the only changein eq
3 6 i s t h a t t h e c o n s t a n tC h a s t h e v a l u e 7 . 9 .
Figure l0A establishesthat eq 36 providesa good model for
a l a r g e n u m b e r o f r a t e s .I n f a c t , t h e q u a l i t y o f t h e c o r r e l a t i o n
i s p r o b a b l y h i g h e r t h a n i n d i c a t e d b y t h e c o e f f i c i e n to f d e t e r mination (rl = 0.98), becausemost of the points rvhich fall far
off the tine (points 13. 26, 40, 46, and 50) involveeither
t h i o g l y c o l i ca c i d ( o r i t s a n i o n ) o r m e t h y l t h i o g l y c o l a t e( R O C O C H z S H , R = H , C H 3 ) . H u p e e t a l . h a v es u m m a r i z e de v idence that thesecompounds behaveanomalouslyin other
B r p n s t e dc o r r e l a t i o n sI.l
A l t h o u g h e q 3 6 f i t s t h e e x p e r i m e n t a ld a t a w e l l , t h e r e r e mains some ambiguity concerningthe mechanisticsignificance
o i t h e v a l u e o f d r eu s e di n i t . T h i s e q u a t i o ni m p l i e st h a t B n u ., J , * : 0 . 5 0 + 0 . 7 3 - 1 . 2 ,a n d b o t h t h e i n e q u a l i t yi n t h e m a g n i t u d e s o f t h e s e B r l n s t e d c o e f f i c i e n t sa n d t h e f a c t t h a t t h e i r
d i f f e r e n c ei s g r e a t e r t h a n 1 . 0 a r e r e a s o n st o c o n s i d e rt h e p o s s i b i l i t y t h a t t h e y m i g h t r e f l e c ts o m ee l e m e n to f e x p e r i m e n t a l
o r a n a l y t i c a l a r t i f a c t . I n p r i n c i p l e ,i t s h o u l d b e p o s s i b l et o e s timate the parameter /3nr.- d', independentlyfrom the slopes
o f F i g u r e 7 , r e b u t . f o r r e a s o n sd i s c u s s e dp r e v i o u s l y ,e x p e r i mental estimatesbasedon theseslopesare too uncertain to be
o f g r e a t v a l u e . F u r t h e r ,i t i s p o s s i b l et o f i n d o t h e r c o m b i n a t i o n s
o f B r / n s t e d i 3 ' sr v h i c h g i v e g o o d c o r r e l a t i o n s .F o r e x a m p l e .i f
*. ro'lu.for the set of i'r ruii.h bestfit the data summarized
i n F i g u r e l 0 u n d e r t h e i m p o s e dc o n d i t i o n st h a t d . = - 0 . 3 0 a n d
d n u . = - d ' * , w e o b t a i n t h e p a r a m e t e r ss u m m a r i z - e di n e q 3 7
( r l = 0 . 9 6 ) . W e h a v e a l s oe x p l o r e ds o l u t i o n st o e q 3 5 i n w h i c h
- p t g a n d i n w h i c h d . i s a l l o w e dt o a s s u m ev a l u e so t h e r
, d n u .=
t h a n 0 . 3 . T h e b e s ts o l u t i o n a 0w i t h t h e s ec o n s t r a i n t si s g i v e n b y
e q 3 8 ( r 2 = 0 . 9 6 ) :t h e a g r e e m e n tb e t w e e ne x p e r i m e n t a rl a t e s
a n d t h o s ec a l c u l a t e du s i n g t h i s e q u a t i o ni s a l s o s u m m a r i z e d
i n F i g u r e1 0 .
T h e o b s e r v a t i o nt h a t a l l t h r e e o f t h e s e c o r r e l a t i o n s g i v e
similar coefficients of determination indicates that the
B r p n s t e dc o e f f i c i e n t sa r e n o t s h a r p l y d e f i n e d b y t h e a v a i l a b l e
d a t a . F u r t h e r , t h e p o p u l a t i o n o f t h i o l s a n d d i s u l f i d e sr e p r e sented by the experimental rates is sufficiently heterogeneous
t h a t g r e a t p r e c i s i o ni s n o t e x p e c t e d .F o r a m o r e l i m i t e d s e t o f
t h i o l s a n d d i s u l f i d e s ,i t w o u l d p r o b a b l y b e p o s s i b l et o d e v e l o p
a Brpnstedequation which would correlate experimental rates
m o r e c l o s e l yt h a n e q 3 6 - 3 8 , b u t f o r s e m i q u a n t i t a t i v ee s t i m a t i o n o f r a t e c o n s t a n t s f o r t h i o l - d i s u l f i d e i n t e r c h a n g eg i v e n
known valuesfor the pK.s of the participating thiols,or for
kinetic measurementsof thiol pKos using rate constants for
thiol-disulfide interchange,any one of theseequationsshould
p r o v e e q u a l l y u s e f u l ( a n d e q u a l l y a c c u r a t e ) . 1 rW e r o u t i n e l y
use eq 36 and 38. and find little difference between the two. We
emphasize,however, that theseequations should be considered
primarily as kinetic models for thiol-disulfide interchange,
a n d t h a t d e t a i l e d m e c h a n i s t i ci n t e r p r e t a t i o n o f t h e 0 c o e f f i c i e n t s s h o u l d w a i t o n s i m u l t a n e o u s ,d i r e c t , e x p e r i m e n t a ld e t e r m i n a t i o n o f t h e s ec o e f f i c i e n t su s i n g a c a r e f u l l y l i m i t e d s e t
of thiols and disulfides.
Although quantitative discussionof the charge distribution
in the transition state for thiol-disulfide interchangebasedon
t h e c o e f f i c i e n t si n e q 3 6 - 3 8 i s n o t j u s t i f i e d , q u a l i t a t i v e d i s c u s s i o nc e r t a i n l y i s . H y d r o g e n a n d s u l f u r h a v e s i m i l a r e l e c tronegatives(Xn = 2.20,XS = 2.44),12and there is no evidence
o f c u r v a t u r e i n t h e B r / n s t e d p l o t s s u g g e s t i n ga n i n t e r m e d i a t e
i n r a t e - l i m i t i n gs t e p .T h u s t h e t r a n s i t i o ns t a t e a p or chanree
p e a r s t o b e s v m m e t r i c a l .r v i t h s i g n i f i c a n t e f f e c t i v e n e g a t i v e
c h a r g eo n a l l t h r e es u l f u r s .b u t c o n c e n t r a t e do n t h e t e r m i n a l
p o s i t i o n s . rH
l u p e e t a l . h a v e r e a c h e ds i m i l a r c o n c l u s i o n s . l l
A n i m p o r t a n t o b j e c t i v eo f t h i s w o r k . u n r e l a t e dt o q u e s t i o n s
o f t r a n s i t i o n - s t a t es t r u c t u r e ,w a s t h a t o f t r y i n g t o u n d e r s t a n d
t h e r e l a t i o n s h i p sb e t w e e nt h e s t r u c t u r e so f t h e t h i o l s a n d t h e i r
r e d u c i n g p o t e n t i a l si n t h i o l - d i s u l f i d e i n t e r c h a n g e r e a c t i o n s :
e s p e c i a l l yw
, h a t s t r u c t u r a l f e a t u r e sc h a r a c t e r i z es t r o n g i y r e ducing thiols? Four useful conclusionsemerge from this work.
F i r s t . a s a n t i c i p a t e d ,m a n y a , r o - d i t h i o l sc a p a b l e o f f o r m i n g
c y c l i c d i s u l i i d e sa r e m o r e s t r o n g l y r e d u c i n g t h a n t h e c o r r e sponding monothiols.This effect is attributable in major part
H S . R - S H+ E S S E $
k-1
"S.N.SSE
+ HSE
-
kz
t-*-S + 2HSE (40)
2RSH+ESSE$NSH+RSSE
k'-t
+ H S E5
n s s R+ 2 H S E ( 4 1)
k'-2
to a much higher forward rate for the secondstep of eq 40 than
f o r t h a t o i e q 4 l . r e f l e c t i n gt h e h i g h e f f e c t i v ec o n c e n t r a t i o no f
t h i o l g r o u p si n t h e i n t r a m o l e c u l a rt h i o l - d i s u l f i d ei n t e r c h a n g e
s t e p . r 5W e h a v e n o t b e e n a b l e t o c o m p a r e t h e r a t e c o n s t a n t s
k l a n d t ' 1 d i r e c t l y ,s o t h a t w e c a n n o tp r e s e n t l yj u d g e r v h e t h e r
o t h e r s t r u c t u r a l f e a t u r e so f t h e i n t e r m e d i a t em i . r e d d i s u l f i d e
o i e q . l I m i g h t c o n t r i b u t e t o i t s r a p i d c o n v e r s i o nt o p r o d u c t s .
S e c o n d .t h e r ei s a c l e a r c o r r e l a t i o nb e t w e e nt h e r e d u c i n gp o t e n t i a l o f a n a . c , : - d i t h i oal n d t h e s i z e o f t h e c y c l i c d i s u l f i d e
f o r m e d o n i t s o x i d a t i o n .T h e m a x i m u m e q u i l i b r i u ma d v a n t a g e
is observedior dithiols capableof forming six-memberedrings.
d n d f i v e - m e m b e r e dr i n g s c a n a l s o
a l t h o u g h s e v e n - m e m b e r ea
h a v e g o o d s t a b i l i t y . A l l - c a r b o n s y s t e m ss e e m t o f a v o r s i x We were surprised
memberedrings in a wide variety of cases.a6
to iind that the sameapparently holdstrue for cyclic disulfides,
sincea CSSC dihedral angle of 90" is tavored both experim e n t a l l y ' * 6a n d t h e o r e t i c a l l y , aaTn d e l e m e n t a l s u l f u r i t s e l f
forms eight-memberedrings. The relativestabilitiesof six- and
cyclic disulfidesseemsto representa delicate
seven-membered
b a l a n c e b e t w e e nC S S C a n g l e s t r a i n a n d e n t r o p y o f r i n g c l o s u r e . 4 7T h i r d . a l a r g e c o n t r i b u t i o n t o t h i o l - d i s u l f i d e i n t e r c h a n g ee q u i l i b r i u mc o n s t a n t sc a n , i n p r i n c i p l e ,a r i s e f r o m t h e
r e l a t i v e p K , s o f t h e r e d u c i n gt h i o l a n d t h e t h i o l d e r i v e df r o m
the disulfide.This contribution is proportionalto the difference
b e t w e e n t h e t h i o l p K " S , a n d i s l a r g e s tw h e n b o t h t h i o l s a r e
p r e s e n te n t i r e l y a s t h i o l a t e a n i o n s ( i . e . ,w h e n t h e e q u i i i b r i u m
consideredis a thiolate-disulfideinterchange).For the systems
c o n s i d e r e dh e r e - a q u e o u s s o l u t i o n . p H 7 , t h i o l s h a v i n g p K "
v a l u e s b e t w e e n8 a n d l 0 - c o n t r i b u t i o n s 1 s ( o b s d f r o m t h i s
s o u r c e a r e l e s si m p o r t a n t t h a n t h o s e d u e t o i n t r a m o l e c u l a r
disuliide bond formation. Fourth. the rare of reduction of a
d i s u l f i d e b y a t h i o l d o e s n o t n e c e s s a r i l yc o r r e l a t e w i t h t h e
equilibrium constant for the reduction. The former is determined by the relationof thiol pK" to solutionpH, with the most
rapidly reactingthiols normally being thosehaving pK, values
c l o s et o t h e s o l u t i o np H ; 1 2 t h e l a t t e r i s d e t e r m i n e db y f a c t o r s
unrelatedto thiol pK", exceptin solutionssufficiently basicthat
the interconvertingthiols are significantlyionized. Dithiols are
ordinarily more rapidly reducing than monothiols of the same
p K , , e s p e c i a l l yi n d i l u t e s o l u t i o n :a l t h o u g h t h e i n i t i a l c o n v e r s i o n o f s t a r t i n g d i s u l f i d e t o a n i n t e r m e d i a t em i x e d d i s u l f i d e
p r o c e e d se q u a l l y r a p i d l y w i t h e i t h e r , t h e c o n v e r s i o no f t h e
mixed disulfide to products is more rapid rvith the dithiol.
W i t h i n t h e g r o u p o f d i t h i o l s , t h e s e l e c t i o no f r a t e a n d e q u i l i b r i u m c o n s t a n tc a n u s u a l l y b e m a d e o n t h e b a s i so f t h e t h i o l
2022
Journal of the AmericanChemicalsociery
/
SS
tl
SS
il
r o oJ
to-'
ro-'
:
.o
ro-.
F
Q|r
.c
c.)
ro{,
q)
ro-c.
0a
t3579ttt3
/,)
El \
g=:RE
,iit
EgE
Compound
F i g u r e l l . C o n c e n r r a r i o n so f t h i o r sr e q u i r e d
t o r e d u c eb y h a r f t h e c o n centrarionof the indicateddisurfidegroup in
sorurions
containing
o x i d i z e dI i p o a m i d e( I 0 - 3 M , r o x - i d i z e g
d l u t a t h i o n"i,gi#iL"
et j O : i p f . O ) . a n d
)
o x i d i z e dl i p o a m i d e( l 0 - 6 M , o ) . C o m p o u n d
n u m b e r sr e f e r r o T a b l e V .
T h e s c h e m a r i cs r r u c r u r e sa t t h e t o p o f t h e f i g u r e
r e p r e s e ntrh e t y p e o f d i sulfide believedto be formed from ihe thior
re-ducing'ug.ni(..u.ricdimeric,
cycl ic monomcric, a nd i n tcrmolecular. respect
iveiy).*
102:6/ March t2. rgg0
acrerisrics
of thior-disurfide
interchange
in sufficientdetairto
be able to rarionalizeraresand equiliiria of ,.Jo*
reactions
i nvol vi ngprotei ncystei neand cysti n. groupr.-Thi s
ar ea is
rel evantnotonryto mechani stibi
c och.#.tty, uut arsot o t he
practi calprobremof stabi ri zi ngthe sensi ti ve
cystei ne- containingproteinsimportantin enzyme-cataryzedorganic
synthesisagainstautoxidation.e
studlesof raresand equilibrium
constantsfor thiol-disulfideinterchangeinvolving
structurally
simple thiols
work for
.and disurfidesprovide-backlrou?-,a
studiesof simirarreacrionswith proteins.bit ii is
important
lot to try ro infer the behaviorof the latter from thai of the
former.Our work with proteinsis_inco,'pt.t.,Uu,
,*o qualitativeconclusions
are evidentwhichur. ,"i.uunt-toti,. subject
9f thif paper.Firsr,the ratesof reductionoi.lriain protein
disulfidegroupsby thiorsarsofoilow BrpnsteJcorrerations.
Second.the fact that a thiol is a strongredlcing agent
doesnot
guaranteethat it is an effectiveantioxiaant.piotein
autoxi dati oni s a comprexprocess,
and the anti oxi dantacti vit yof
addedthi ol si s onrypartryduero thi or-di sutfi ae
i nterchange.
we have,for exampre,
encountered
oneinstance
in whichDTT
is a lesseffectiveantioxidant(arthougnu n'u.n stronger
re2-mercaproethanj,pr.ru,,'ulry because
9rylg agent)th.a.n
DTT is morerapidryautoxidized
than z-r.r.upioeirranot.Tire
H :oz producedi n theseautoxi dati ons
i s i tsel tan effi cient
enzymeoxidant.E
Experimental
Section
Methodsand Materiars.pH wasdererminedusinga
Radiometer
a e r em e a s u r euds i n ga G i r f o r d
l o d e l 2 8 p H m e r e ra. n d U V s p e c r r w
Model 240 specrromerer
pK,1 and of the ring size that would
equippedwith a sampre.h;;;,
rhermobe formed on oxidation
statedat 30.0+ 0.5'c anda stripchartrecorder.Distiiledwater
of th e d i thiolt o a dis ulf i d eT. h u s ,D T T (p ,< ,,,_ _g .Z l
was
re acrs p a s s e tdh r o u g ha n i o n - e x c h a n gc eo r u m nr,e d i s r i i l e a
wi th c.sSc appr ox im ate ry
u s i n fa C o r n i n g
a fa c to ro f 6 ' mo re s ro w ryth an A G - l B s t i l l ,a n d p u r g e dw i t h a r g o n
p r i o r t o u s e .A r r r e l c t i o n sa n d
gl yco ld i m er c apt oac er a(p
teK" r = 7 .7 )i n p H 7 a g u l o u ss ol u_ m e a s u r e m e n w
t se r ec a r r i e do u t u n d e r a n a r g o n a t m o s p h e r e .
Dit io n . b u t DT T is a m uc hs i ro n g .,re d u c i n gu g " n i
fo ,.G S SG t h i o t h r e i t o tN. A D + . N A D H . G S S G .G S H , m . t i , y r l r l o ^ J rs o r u r i o n ,
t h a ni s G M A , e s p e c i a l li yn l o wc o n c e n t r a t i o n s .
l i p o a m i d el,i p o a m i d ed e h - v d r o g e n a( ys e a s r )( E . a r . 6 . q . 3 ) g, r u t a t h i o n er e d u c r a s(eE . C . 6 , 4 . 2 ) . a ngdl . v o x a l a s e( 8- l. C . 4 . 4 .
. A .majorpracticarobjectiveoi this work is to providedata
I .5)were
^
l
rvh i chw o u ldm ak eit pos si b re
to s e re cat th i o rre d u c i n ga g e nr o b t a i n e df r o mS i g m aC h e m i c a r c oi.h i o t a c e t i ca c i d( 9 7 % ) , t h i o g r y coll
(98vo);
ic
havi n ga re duc t ionpot enti a r-a p p ro p rifo
acid
2.3'd.i
mercaptoa te
r -propanor(g5Eo).2-mercaptor p u rti .u t" , u ppli ca ti o n It
. r equir ess om ee ffo i t to c o mp a re
ror (97To), i.3-di-...oiio-z-propunot
"a i l o f th e th i ol s ethanol (98vo), r -thiogryce
(technical),3-mercaptopropionic
exa mi n e don t he bas isof t h e i r e q u i ri b ri u mc o n s ta n ts ,
acid (ggEo),Elrlian;s reagent
s i n ce (99+%), 2,2'-oxydiethanethiol
(technical),t,5_hexadiene
theseconstantshavedifferentunits.To assistin the
(9g7o),
ur. or these z-chloroperoxybenzoic
acid ( g5zo),l .3-propanedi
thior liluoy, ana
t hio l s,we h av ec ar c ur at efdro m th e d a tai n T a b rev
th e c o n1,4-butanedithiol
(99vo)were.btainedfiom Aldrich chemical
co.
c e n tra ri o ns
of t hiolsr equi re dto re d u c eb y h a l f th e c o n c en- l ' 4 - P e n t a d i e nwea so b t a i n e d
f r o mC h e m i c a r s a m p r e
- os. c t y c o t o i trationof the indicateddisulfidegroupsprisentin
wasa gift of Evanschemetics.barien, conn.
threesolu_ mercaptoacerate
All
tions:(lipoo*)o
= 0; (lipoo^)e= t o-, M,
(lipo-'"I)'
other chemicalsusedwereAR grade.organic ttriots*ere
M,
_1
distiiled
( l. ip o 'd )s-- Q ;(:-19
G S S G ) o= i c i -, M , (C S ri l , = o i p i g u re I l ) .
underargonbeforeuse.Enzymaiicactivity-isdefinedin
units having
dimensions
Thesedisulfidesand concentrations
(prmotproductproduced
werechoiento ruig.st the
) (mln- I ).
N'N'-Dimercaptoacetyrhydrazide
cond i ti o nw
s hic hm ight be re q u i re dto re d u c ea s ta b i e ,
was preparedfoilowinga riteri n tra atureprocedure,
mp I
156oC (lit. mpaaiSO_f 5g oC). The pK"s
mo l e cu l a rcy
, s t inelink ageo f i p ro te i ni n d i ru tes o ru ti o n((ri:1of the thiol groupsin this materiaiwere measuredas previously
der i p o o * )o
= l0 j
P9l')o :.1 0 - uM ) , ands t abl ei n tra m o l e c u l a((l
scribed:12
pK^r = 8.0, pK^2= 9.6.
M) a n d l e sss t ableint er m o l e c u l a((G
r S SG);' : tO :j V ) A il,GDimercapto-2.5-hexanediol.
To 4.1 g (50 mmot) of 1,5_hexa_
..
sulfideIinkagesat concentrations
representative
d i e n ei n 4 0 0 m L o f C H 2 C I 2a t 0 o C w a ss l o w l ya d d e d
of thoseen2 0 . 0g ( 1 0 0
countered
in solutions
of low molecularweightdisulfides.
m.mol)of.finely dividedo'-chroroperoxybenzoic
This
acid with ,tiriing.
that, for generalwork in .nryrnoiofy, rhree The reactionmixturewasailowedio
!qu.r. suggests
ro room temperatureand
thiols haveusefulcharacieristics.
"or.
after l4 h gaveonly a weak positive
2-Mercap*.,nuior is inreactionto starch-iodidetest
paper.Peroxideswere removedby addition of
expensive,
but is a weakreducingagent.Ditiriothreitor,crer5 mL of water and I
g of Na2s2o3andstirringuntirthereactionrni*tur. *",
land's.reagent,is the strongestr.iu"ing agentamong
n"!utlu. to
the thiors s.tarch-iodide
resrpaper(about r0 min). The reactionmixiure *as
examined.It is, however,expensive
(atihougha i'i*tur. or
drlutedwith 20 mL^ofwaterand srowryaddedwith stirring
diastereomeric
to I00 g
1,4-dithiobutane-2,3-diols
hJvin! .luiu.r.nt
of Na2co3. Afrer frothing had subsid'ed,
the reactionmiiture *as
propertiesis readilyavailableby synthesis).rz
ionifouna Z
filteredthroughfritted glassand the cHzClz removeduy Jistiltation
(l'3-dimercaptopropanor,
DMp)-isa compromise:
it is armost at room temperatureand_reduced
pressureto give the crudediepoxide
asstronga reducingagentas DTT, but it is ress
asa clearoil: IR (neat)3050,2990,2920,28?0,t 260.830, and 740
expensive.
Ail
threehavegood*atei sorubirityand torerabre
) 3 . 0 - 2 . 8( m . 2 ) , 2 . s - 2 . 3( m , 2 ) . r . 7 _ r . 4( m .
odor. we now
9r-l; NMR (CDCt3d
useDMP in placeof DTT for muchof our *o.k in
4). caution. This diepoxide and that used in ih" p'rrporation
.nryiof ogy,
of
as a.general-purpose
1.5-dimercapto-2,4-pentanediolare probably mutagenic. Thev
antioxidant,it so far appearcio U. interch a n g e a ble
should be handledonly with caution in a good hood. oid ,or" tokrn
wit h DT T .
A l o n g -range
c onc er nof t h i sa n d re l a te dw o rk i n o u r Ia b o - to auoid contacr with tiquid or uapor. fh. di"po*ide was treated
withoutfurther purificationunderargonwith r r.6 g ( r I0 mmol)
ratory is to understandthe mechanistic
of
and structurarcharthiolaceticacid.After 48 h ail volatiliswereremovedat room
tem-
Szajewski, Whitesides/ Thiol-Disulfide InterchangeReactions
peratureunder wateraspiratorvacuum.Argon-degassed
methanol
( 3 0 m L ) a n d 2 m L o f a I N s o l u t i o no f H C I i n d i e t h v le t h e rw e r e
added,the mixturewasrefluxedunderargonfor 5 h andcooled.and
erher,methylacetate,and methanolwereremovedby disrillationat
pressure.
The residualoil wasdistilledbulb to bulb at
atmospheric
1 3 0 - 1 5 0" C ( 0 . 0 5 - 0 . 1T o r r ) t o g i v e 1 . 8 6g ( 1 0 . 2m m o l )o i 1 , 6 - d i mercapto-2,5-hexanediol
as a clear,foul:smellingliquid: IR (neat)
3 4 5 0 , 2 9 2 02,8 6 0 ,1 0 5 0c m - r ; N M R ( C C l 4 )6 4 . 1 - 3 . 9( m . 2 ) , 3 . 5 - 3 . 3
( m , 2 ) , 2 . 8 - 2 . 4( m , 4 ) , 2 . 4 - 1 . 8( m , 4 ) , 1 . 6( t , 2 . J = 9 H z ) . T h e e p imericcomposition
of thismaterialwasnot determined.
The pK"sof
the thiol groupsin this materialwere measuredas previouslydes c r i b e d : l 2 P K=a 9
r . 3 , p K ^ 2 =l l . l
A n a l .C a l c df o r C 6 H l c O z S zC:, 3 9 . 5 6 H
; , 7 . 7 5 .F o u n dC
: ,39.75:
H. 7.63.
1,5-Dimercapto-2,4-pentanediol.
Application oi the epoxidation,
thioacetylation,
and transesterification
sequence
as described
above
t o 3 . 4g ( 5 0 m m o l ) o f 1 , 4 - p e n t a d i egnaev e3 . 1 6g ( 1 9 m m o l )o f 1 , 5 dimercapto-2,4-pentanediol
as a clear, odiferousoil: IR (neat)
3 4 0 0 - 3 2 0 0 , 2 9 2 0 , 2 5 5t 04 .2 0 ,1 0 5 0c m - { ; N M R ( C C l a )d 3 . 8 - i . 2
( m . 2 ) ,2 . 8 - 2 . 2( m , 6 ) , 2 . 2 - 1 .(8m . 2 ) ,1 . 4( b r t , 2 , J = 9 H z ) ;p K u r=
9 . 1 ,p K u ,= 1 0 . 6 .
A n a l .C a l c df o r C 5 H 1 2 O 2 SC2,:3 5 . 7 2 H
: , 7 . 1 9 .F o u n dC
: ,35.39;
H.6.91.
Kineticsof Reductionof OxidizedGlutathione(GSSG)by Thiols.
one representative
kineticrun usingthe coupledenzymaticindicator
reactionwill be describedin detlil as will the corresponding
control
experiments.
other runswereperformedin an analogousrnanner.To
t 000pL of 0.2 M phosphate
buffer,pH 7.0,containing0.t%oeggalbumin,underargonar 30.0* 0.5 oC in cuvetteI wasaddedZe00pL
of a 7.6 mM solution(as determinedby Ellman'sassayae)
of dithiothreitolpreparedby dissolvingI I 7 mg of that thiol in t 00 mL of
water.To this solutionwasadded50pL of a freshlysteamdistilled
solutionof methylglyoxal(47 mM) in water.The concentration
of the
stocksolutionof methylglyoxalwasdeterminedby enzymaricconversionto s-lactoylglurathioneusingan excess
of rey'uced
glutathione
. sb0l a n ks o l u t i o ni.n c u v e t t e2 , w a sp r e p a r esdi m a n dg l y o x a l a s e - 1A
i l a r l y .A s l o wb l a n kr e a c t i o nb e r w e e m
n e t h y l g l y o x a ln da d d e dt h i o t
wasnotedundertheseconditions.
To cuvetteI wasadded20 gL of
a g l y o x a l a s e s- lo l u t i o ne x h i b i t i n g0 . 3 7 U l l t L a c t i v i t y .T h e a c t i v i t y
of stockglyoxalase[ wasdeterminedin a separate
experimentutilizing
reducedglutathioneand merhylglyoxal
followinga literatureprocedure.So
Comparisonof the blankreacrions
in cuvettesI and 2 overa
5-min periodshowedno difference.thusestablishing
thar,underrhese
c o n d i t i o n sg,l y o x a l a s e -dli d n o t m e d i a t et h e r e d o xc o u p l i n go f t h e
addedthiol with methylglyoxai.
To borhcuvettesweresimultaneously
addedl0-pL aliquotsof a 99 mM stocksolutionof GSSG prepared
by dissolving
68.3mg of GSSGin 10001tLof 0.2 M phosphate
buffer.
pH 7.0.The endpoinrof the kineticrun. aftercorrecrionfor theslow
blankreaction,indicateda concenrration
of S-lactoytglutathioneof
0.69 mM in the assay.This valuecorresponds
to a concentration
of
106mM GSSG in thestocksolutionpreparedasabove.The concentrationcouldbe furthercheckedby oxidationof NADPH by GSSG
utilizingtheenzymeglutathione
reductase.t0
The absorbance
in both
cuvetteswas followedsimultaneously
by appropriaterepositioning
o[ thecuvetteholder(at 240nm. thetr-u* for GS-lacla)until theslopes
of both curveswereagainparallel.As a further control,the above
assaywasrepeated,
the onlydifferencebeingin the useof 100pL of
the glyoxalase-l
solution.The absorbance
curvesproducedwereessentiallyidentical.Figurel2 illustratesthisexperiment.
The quantities
addedcorrespond
to the followinginitial assayconditions:66 mM
phosphate.5.07 mM dithiothreitol.0.'11mM methylglyoxal,2.4
U lmL glyoxalase-1,
0.35 mM GSSG.The final pH of the assaysolutionwas7.02.The raw data wererefinedby subtractionof the blank
from the reactioncurve,determination
of the CS-lacconcentration
u s i n ge e 2 4 0 n=- 3 . 3 7m M - l c m - l , l a a n d s u b s t i t u t i oinn t o t h e i n t e gratedform of the appropriare
rateequation(eq I 3). Thesedataare
illustratedin Figure2. Alternatively,subtractionof the blankfrom
the reactioncurve,extrapolation
ro time 0 (pastthe shortinitial rog
phase),determination
of the initial slope,and divisionby e6gavean
approximationof the initial velocity,d(GS-lac)/dr,of rhe reaction
i n u n i t so f m M m i n - r . D i v i s i o no f t h i sv e l o c i t yb y t h e i n i t i a lG S S G
and RSH concentrations
givesa valueof k,obsaindistinguishable
from
that obtainedusingeq I 3.
Determinationof RelativeReducingAbility of Mono- and Dithiols
towardNAD+ Utilizing a CoupledEnzymaticAssay.One representativeequilibrationusingthe coupledenzymaticindicatorreaction
2023
r.o
o.8
o.6
o.4
o.2
o.o
o 2 4 6I
lo
tor,,'."
3o
(,r,in)
40
F i g u r e 1 2 . P l o t o f A 2 a 6v s . t i m e i n 0 . 0 6 6 M p h o s p h a t eb u f f e r , p H 7 . 0 , a t
3 0 . 0 o C u n d e r a r g o n w i t h 5 . 0 7 m M d i t h i o t h r e i t o la s r e d u c i n ga g e n t a n d
0 . 7 7 m M m e t h y l g l y o x a la s e n z y r n ec o s u b s t r a t eC
. urve I is the reacrion
mixture;curve 2 is a blank.The arrows indicate(a)addition of glyoxalase-l
Q.a U lnL) to cuvettel; (b) simultaneousadditionof oxidizedglutathione
( G S S G , 0 . 3 2 m M ) t o c u v e t t e sI a n d 2 : ( c ) a t t a i n m e n t o f s t e a d y - s t a r e
c o n c e n t r a t i o nos l r e d u c e dg l u t a t h i o n ei n c u v e t t el . U s e o f l a r g e r a m o u n t s
o f g l y o x a l a s e -(l 1 2 U / m L ) g a v ec u r v e 3 . T h e r a n g e n ru s e dt o c a l c u l a t e
d ( G S - l a c ) / d t i s i n d i c a t e db y t h e b r o k e nl i n e ( . - . - ) .
with DTT as reducing thiol will be describedas will the corresponding
control experiments.Other runs were performed in an analogous
m a n n e r . T o 2 9 0 0 p L o f 0 . 2 M p h o s p h a t eb u f f e r . p H 7 . 0 . c o n t a i n i n g
O . l V oe g g a l b u m i n . a t 3 0 . 0 + 0 . 5 o C u n d e r a r g o n , w a s a d d e d l 0 p L
o f a 4 8 m l v l N A D + s t o c k s o l u t i o n p r e p a r e d b y d i s s o l v i ng 3 2 . 4 m g o f
N A D + i n i 0 0 0 p L o f 0 . 2 M p h o s p h a t eb u f f e r , p H 7 . 0 . T o t h e r e s u l t i n g
s o l u t i o n w e r e a d d e d l 0 p L o i 9 . 0 m M l i p o a m i d es o l u t i o n ( p r e p a r e d
b y d i s s o l v i n g3 . 6 8 m g o f o x i d i z e d l i p o a m i d e i n 2 0 0 0 p L o f d e g a s s e d
m e t h a n o l )a n d 5 0 p L o f a 1 0 5 m M d i r h i o t h r e i t o sl t o c k s o l u t i o n i n 0 . 2
M p h o s p h a t e b u t ' f e r .p H 7 . 0 . T h e i n i t i a l c o n c e n r r a r i o no f t h e d i thiothreitol stock solution was checked by Ellman's merhod,l8 and
t h e i n i t i a l c o n c e n t r a t i o no f N A D + c o u l d b e c h e c k e db y t o t a l c o n v e r s i o n o f N A D H u s i n g a l a r g e e x c e s so f t h i o l . T h e a b s o r b a n c ea r 3 4 0
n m w a s n o t e d a n d 3 0 p L o f s o l u t i o nc o n t a i n i n ga p p r o x i m a t e l y I U l p L
o f l i p o a m i d e d e h y d r o g e n a s ew a s a d d e d . T h e a b s o r b a n c e a r 3 4 0 n m
w a s f o l l o w e d u n t i l i t b e c a m e c o n s t a n r .C o n t r o l s i n d i c a t e d t h a t n o
f o r m a t i o n o f N A D H o c c u r r e d w h e n l i p o a m i d e .l i p o a m i d e d e h y d r o g e n a s e .o r o r g a n i c t h i o l w a s a b s e n t . F r o m t h e a b s o r b a n c ea r 3 4 0 n m ,
t h e c o n c e n t r a t i o no f N A D H w a s o b t a i n e d . T h e c o n c e n r r a t i o n o f
NAD+ is given by (NAD+; = (NAD+)o - (NADH), where
(NAD+)o is the concentration of NAD+ presentbefore adding the
l i p o a m i d e d e h y d r o g e n a s eT. h e r a t i o o f o x i d i z e d t o r e d u c e d l i p o a m i d e
g i v e n b y i n s er t i n g v a l u e s i o r ( N A D + ) a n d ( N A D H ) i n t o e q
irr:n""
(lipoo*_
) ( N A D + ) o- ( N A D H ) Kzt = ^YKz'r
(N=-=( l i p s ' . 4 )= [ l + 7 K n ] - r ( l i p o ) s
(42b)
(lipoox)= 7i(27(lipo'ed)
G2c)
(lipo,d)
Q2a)
T h i se q u a t i o ny i e l d sv a l u e sf o r ( l i p o o ^a) n d ( l i p o ' " d )s, i n c et h e i rs u m
is (lipoo*)e,the knownconcentration
of oxidizedlipoamidebefore
a d d i t i o no f l i p o a m i d d
e e h y d r o g e n a sTeh.e s u mo i ( N A D H ) a n d ( l i pot"d)equals(DTTox),the concentration
of oxidizedDTT presentat
equilibrium.Sincethe originalconcenrration
of DTT betoreaddition
of lipoamidedehydrogenase
of
is known.(DTT)0,the concentration
( D T T ) a t e q u i l i b r i u mi,s e a s i l yc a l c u l a t e dC. o m b i n a t i o no f t h e s e
concentrations
yieldseq 43 and 44 whichdirectlyrelateK1;*oBdrnd
K N e D H o b tdo k n o w nq u a n t i t i e s .
K t i p oo x a -
( N A D H ) ( | t : y K z t )* ( l i p o ) o
I
7 K 2 7[ ( D T T ) 6- ( N A D H ) l ( l * T K z t ) - ( l i p o ) o
( 4 3)
KNeDHobtd' DTT = KzrKri*obtd
(44)
I n t h e p a r t i c u l a re x p e r i m e nut s e dh e r ef o r i l l u s t r a t i o nt,h e i n i t i a l
of materials
were0.2 M phosphate.
pH 7.0:0.166mM
concentrations
2024
Journal of the Anterican ChenricalSociery'/ t02;6 / March t2. tgg0
N A D + : 0 . 0 3 0m M l i p o a m i d e|;. 6 gm M d i t h i o t h r e i t o ;
l0 U/mL liT a b l eV I . I n i t i a la n d E q u i l i b r i u mC o n c e n t r a r i o n( m
s M) in Thiot_
poamidedehydrogenase.
The concentrarion,
of ;p.;;", presenrat
Disulfide lnterchange
Reactions
equilibrium
w e r e0 . 0 1 4 6m M N A D + ; 0 . 1 5 3m M f r i e O f f
; 1 . 4 9m M
DTT'd: 0.183mM DTTo*;0.OZg71nt,t
tipo."o.-3 X l0--3mM lipoox.
In otherruns.higherconcentrations
expt
initial
of oigunictr,iotr-*erenecessary
ro achievecomparabrereductionsof NRb+.
ln tt," reastfavorabre
la
G S S GI . O I
case.540 mM 2-mercaptoethanor
0 . 5l 3
0.5r4
was required.For severarruns it
DTTrd 0.497
provedmoreconvenientto start with known
0.000
0.004
mixturesoioxidizedand
GSH
reducedthiol.Thus,a stocksorutionprepared
0.994
0.9 86
by dissorvin
g 23.7mg
(-0.132 mmol) of a mixtureof oxid'ized
DTTOX
0.497
0.4 93
and ,.ou..J dimercapto_
2b
lipoox1.04
acetvlhvdrazide
( r0. Tabrev) in 2.0 mL of 0.2 M
0.497
0.231
buffer,
;;;;phate
BAL'"d 7.73
pH 7.0'showeda dithiorconcenrrarion
7.22
6.92
of 44.5n.,M*hJn assayed
by
liPot"a
Ellman'smethod.ae
0.507
0.809
The composition
of the mixedstocksorutionis
BALO'
then 44.5mM in dimercaptoicetyrhydrazicilercd
0.254
0.405
mM in di3a
lipoor1.04
mercaptoacetylhydrazideox.
0.540
"nazl.:
0.5
38
The remainderof the
equilibration*as
DTT.cd0.531
carriedout asdescribed
0.031
0.028
above.Initial concentrations
of materialswere
lipo'd
0 . 2 M p h o s p h a t ep,H 7 . 0 ;0 . 1 6 2m M N A D + ;
0.500
0.s 03
l+.g mia dimercapDTTOX
toacetylhydrazide'd;
0.500
0.5 03
6.9gmM dimercaptoacetylhydrazideor;
0.00367
lipoox1.04
mM lipoamideo*;I O Lt
0.538
0.497
lmL tipoamided.hyd;;;;;r".
ir... concen_
yErcd 592.
5
0
r
of
species.pr..r:1t
500.
at
equitibriu,
*..."0.139
l.1tl?lr
mM NAD+;
lipet"o
0.0223mM NADH: 14.7mM dimercaptoacetylhydrazider"d;
0.502
0.5 45
7.01
MEO'
mM dimercaptoacetylhydrazideox;
0.502
0.5 45
0.0012g 111M
li;;;iA-."-, O.OOZ:q
mM liPoaml6.rcd.
" Conductedin aqueousbuffer sorution.6 conducted in r:r
Equilibrationof l.,4-Dimercaptobutane
and Dithiodiketopiperazine. methanol-aqueous
buffersolution.
Easily monitored
propertiesof r,+-oimercap,olur"n"
.physicar
l'2-dithianeare^their'H NM-R spectrawhich
"no
are. for r,4-dimerc a p t o b u t a n(eC D ^ C t : ) , 62 . 6 _ 2 . 4
beforethe additionof lipoamideand after equilibriumhad beenobi m , q , C C H 2 5 ) ,f . S _ r . 6( m , 4 ,
tained.Extinctioncoefficients
=290 M-r cm-r for 1.2-dithianes
€2es
9 C H r C ) , 1 . 3( t , 2 . J^ = 8 _ H z -. S H ) , a n d f o r t . 2 - d i t h i a n (eC D C l 3 ) ,
( D T T o * ;a n d e 3 3 e= 1 4 7 M - l c m - l f o r 1 , 2 - d i t h o l a n (eosx i d i z e dl i q 3 . 0 - 2 8 ( m , 4 , C C H 2 S ) ,z . A : s ( m , 4 , C C H ' 6 ; ! i
Oxidized
d . i t h i o d i k e t o p i p e r a zsihnoew s( C D C t 3 id 4 . 1_ t . t
poamide)wereusedin the work.3a
iur;, q, 3.1_2.6
( b r m , 2 ) , 2 . s - 2 - 0( b r m , 6 ) . T h i s r a t t e r
N M R s p e c t r u mi s r i t t r e
Acknowledgmento
. u r c o l l e a g u e sJ o h n D e u t c h a n d J a m e s
c h a n g e do n r e d u c t i o nt o t h ed i s u r f i d eA. m i x t u r eo i q l . s
m g ( 0 .r g 5
Barber
provided
with
us
most usefur discussionsof the sepammol) of oxidized.
dithiodikeropiperazine
and 29.0mg (0.23gmmor)
r a t i o n o p6 ' o b s d
into KsH and Ks-, and of the compatibilitvof
of-I.4-dimercaptobutane
in r.0 mL of cDCr3 under,;g';". at 30.0*
t h e k i n e t i c a n d t h e r m o d y n a m i c e q u a t i o n s .w e a l s o t h a n k
0.5 oc, showedinitiailya I H N M R spectrumwhichw-as
just the su_
perimposition
william Rastetter and Robert wiiliams for the gift of the
of the specrraof theconsriruenrs.
The,p..*u, of this
mixturewasmonirored.over
d i t h i o d i k e t o p i p e r a z i n eu s e d i n t h e s e s t u d i e s . a n I E d w a r d
a periodof 7 daysouring*trich signars,
6 3 - 0 - 2 . 8 .c h a r a c r e r i s t o
i cf 1 , 2 - d i t h i a naep p e a r e d
S o l o m o n a n d A I H . p p f o r d e t e r m i n a t i o no f R a m a n s p e c r r a .
w h i l es i g n a r sd.
I .8- I .6. characteristic
of.r ,4.-dimercaptobutane
ze'ev Shaked provideddara concerningraresof thiol-disulfide
disappeared
in equar
amountsas determinedby integration.After 5 days,the
spectrum
i n t e r c h a n g er e a c t i o n si n v o l v i n gp r o t e i n su s e d t o t e s t s o m e o f
b e c a m es t a t i c T
. h e i n t e g r a t erda l i oo f s i g n ailn t e n s i i a
v t 6 r .g- r .6to
the Brfnsted equationssummarizedhere. we thank especially
t h a t a t d 3 . 0 - 2 . 8i s e q u i v a t e nt or ( H S ( C i t 2 ) o S H ) / ( { - H 2 ) i S ) .
i;;
Professorw. w. cleland for detailed readings of severalverrario of oxidizedro reduceddiketopiperazine
ma.vbe cariuratedbv
sionsof this paper and for exceptionailyuseful suggestionsand
the law of massbarancesincethe ln'itiarquantities a;;-it_ii
criticisms. Professorsw. Jencksand D. Hupe alsoiontributed
mercaproburane
and dith.iodiketopiperazine
"iMultiplication
are known.
s u b s t a n t i a l l yi n c o r r e c t i n ge r r o r s i n t h e s ee a r l y v e r s i o n s .
o f t h e s er a t i o sg i v e sa v a l u eo f 3 . 3a st h ee q u i l i b r i u m
c o n s t a nfto r r e d u c t i o no f 1 , 2 - d i r h i a nbey d i t h i o d i k e t o p i p e r a z itnhei s; v a l u e
corresupplementary
Material Available:Identificationand sourcesof
spondsto KNeooM = 7.1X l0-l andKcsscsH= 4.6X 105mM.
The
thedata in Figurel0 (4 pages).
Orderinginformarionis givenon any
definirionsand interconversions
of ttreseconstanrs
in Table
currentmastheadpage.
V andin thetext.
"pp""i
Experimentalchecks
of Carcurated
Datain Figurer r. Fourequiribrationswerecarriedout to verifyrepresentative
calculations
included
i n F i g u r eI l . I n e a c hi n s t a n c ea, q u a n r i t yo f t h i o rc a r c u r a r e d
to be
sufficientto reduce50voof GSSGio GSH (or lipoo'ioiipo-ay *ui
addedto a solutioncontainingGSSC (or lipoox)and the'final
concentrationsof thiolsand disulfidesweremeasured.
Theseexperiments
w er e s e m i q u a n t i r a t i vt e
h :ed i f f i c u l t i eisn m e a s u r i nsgm a l lq u a n t i t i e s
of air-sensitive
thiols.and the low solubilityof oxidlzeJiipJamiaein
water.precludedhigh precision.They nonetheless
servedio establish
that the data in Figurer r werenot grossryin.error.Representative
experimenraldetailsfollow,and reiults are summarized
in Table
vt.
The absorbance
of a solutionof 0.003g4 g of DTT in 50.0mL of
q.0i M phosphate
at pH 7-0.30 + 0.5 oc, wis monitoredar 290 nm,
the tr,n",for 1,2-dithianes,sa
beforeand after the additionof 0.030g
g.of oxidizedglutathione.After equilibration,
0.0304g of oxidized
glutathionewasaddedand theabsoibance
againmeasur.?to establish
a b l a n kc o r r e c t i o nf o r t h a r q u a n r i t yo f g l u t a t h i o n e .
I n i t i a lc o n c c n trarionsusedin thisexperim.nt*eri o.+gzmtr4Drr
unJ i.007 mM
cssc. The absorbance
changeat 290 nm indicateatn.
olilr::9 DTT presentat equiribrium.From thisvarueconcenrrations
"o,ount-6i
o f G S H ' G S S G .a n d D T T a t e q u i r i b r i u m
c o u r db e c a r c u r a t e d
T.h e
concentrarion
a sc h i e v e ad t e q u i r i b r i u m
a r e l i s t e di n T a b l eV I .
Equilibrations
againstripoamide
wereperformed
by.Jai"g known
q u a n r i t i t i eosf o x i d i z e dl i p o a m i d e
t o s o l u r i o nosf B A L . D T i : o r M E
i n I : I M e o H - H 2 0 b u f f e ra n d m e a s u r i ntgh ea b s o r b a n c e
a t 3 3 0n m
Referencesand Notes
( 1) Partsof thisresearchweresupported
byNrH(GM26543,24o2s,andcA
09112CT).
(2) NIHPostdoctorat
Feltow,1976-1978(FeilowshipA j05337).
(3) M. Friedman,"The chemistryand Biochemistryof the
SurfhydryrGroup",
press,Elmsford,N.y., rgzSi p.
C. .tocetyn,:igiochem_
.1sted.,,Pergamon
istry of the SH Group", Academic press, New york, 1972: yu. M.
Torchinskii,"surfhydrylandDisulfldeGroupsof proteins",plenumpiess, New
York' 1974;A. L. Flahartyin "The chemistry of the Thiorcr-up';, s. patai,
Ed.,Wiley,New york, 1974,p 589.
(4) W. W. Ctetand.Biochemistry,3,480 (1964).
(ll y Konigsberg,MethodsEnzyrnot.,25,185 (19721.
(6) W. S. Ailison, Acc. Chem.Bes.,9, ZSg(rSZb).
(7) E. S. G. Barron,Adv. Enzymot.,,fi,2O1(rgSr); A. K. Ahmed,
S. W.
Sciraffer,and D. B. Weflaufer,J. Biol. Ctrcm, ZSO,gqZt (1975);t. G. D"nce,
R. C. Conrad, and J. E. Cline, J. Chem. Soc., Chem. iommun., 13
(1 s 7 4 ) .
(8) Hyeogen peroxide_is
generatedduing rnostoxidationsof thiorsby diorygen:
P. P. Trotto,L. M. pinkus_and A- MeiJrer,J. Biot. Clpm., Zcg, tgts
t$iql:
M. Costa, L. Pecci. B. pensa, and C. Cannella,Biochem. biopnys.
Resi.
Commun.,78, 596 (1977\.
(9) A. Pollak,R. L. Ba,ghn,.andG. M. Whiresides,
J. Am.CEm. fu.,99, 2366
andG. M. Whilesioes,iora, rOo,
ll9l1l,-1.L.Baughn, 0. Aroarstetnsson,
3 0 a ( 1 9 7 8 ) ;Y . - S .S h i h a n d G . M . W h i l e s i d e s , , tO
. r g .C h e m . , 4 2 , 4 1 6 5
(19771.
(10) E. Berntand H. U._Bergmeyer
"Methods
in
of EnzymaticAnalysis",Vor.
4,Znd ed., H. U. Bergmeyer,Ed.,Academicpress, New yori<,1974,p
16 4 3 .
(11) W. E. Knox in "The Enzymes",Vol.2,2nd ed.,p. Boyer,Ed.,Academic
Press,New york, 1960,p 253; E. Boylandand L. F. Cirasseaud,
Adv. En_
z y m o l . , 3 2 , 1 7 3( 1 9 6 9 )A
; . M e i s r eirn . . T h eE n z y m e s , V
' , o l . 1 0 .3 r de d . ,p .
Boyer,Ed.,Academicpress,New york. 1974,9 67 1; N. S. Kosoweranct
'
l:L
S:ajewski, Whitesides/ Thiol-Disulfide InterchangeReactions
E. M. Kosower in "Free Radicals in Biology", Vol. ll' W. A. Pryor' Ed.' Academic Press, New York, 1976, Chapter ll; l. M. Arias and W- B. Jakoby'
"Glutathione: Metabolism and Function", Raven Press, New York,
1976.
(12) G. M. Whitesides, J. Lilburn, and R- P. Szalewski , J. Org- Chem., 42, 332
(1e77).
(13) J. tvt. Witson, R. J. Bayer, and D. J. Hupe, J. Am- Chem. Soc-, 99, 7922
(1977): C. E. Grimshaw, R. L- Whistler, and W. W. Cleland, ibid','1O1,1521
171,389 (1978): K.
irgz9); M. Shipton and K. Brocklehurst, Biochem' J-,
- 0'45
Brocklehr.nst ano C. Uttle, rbrd., 128, 471(1972). The vah.p of 0.,c
has been reported for the reaction of amines with disulfides: H. Al-Rawi,
K. A. Stacy, R. H. Weatherhead, and A. William, J- Chem. Soc', Perkin
Trans.2,663 (1978).
(14) The substrate for glyoxalase I is the hemithioacetal formed between gl|Jtathione-SH and an-a-keto aldehyde. See E. E- Cliffe and S. C' Waley'
Biochem. J..79,475 (1961); M. V. Kester and S. J. Norton, Biochim' Biophys. Acta, 3g'1.,212 (1975): L.-P. B. Han' L- M. Davison, and D' L' Vander
|lagt, ioia-,445, 486 (1976); D. L- Vander Jagt, E Daub..J' A' Krohn' and
f-.-F. A. Han, Biochemistry, 14, 3669 (1975). For a discussion of the
magnih.des of equilibrium constants between GSH and aldehydes, see M.
S. (anchuger and L D- Byers, J. Am. Chem. Soc., 101, 3005 (1979)'
(15) O, M. E. Reuben and T. C. Bruice, J. Am. Chem- Soc', 98, 114.(1976)'
Robenstein [D. L Rob€nstein , J. Am. Chem. fuc.,95'2797 (1973)] reports
microscopiC pK.s of 8.93 (amino group protonated) and 9'08 (amino group
unprotonated) blsed on NMR chemical shifts. and Jung [G. Jung, Eur. J.
Biochem., 24,438 (1972)] reports pKr= 9.2. M. S. Kanchuger and L. D.
Byers, J. Am. Chem. Soc., 101, 3005 (1979), Indicated pK" = 9.1 by alkylation. The origin of the differences between these values is not entirely
clear. In any event, the estimate of Bruice (pK' = 8.7) is compatible with
our data, and is used throughout this paper.
(16) The maximum specific activity reported for this enzyme is -103 pmol of
product mg-t 61n-t. Using a molecular weight of 48 000t4 for GX-|, this
value gives a turnover number of -5 X 10s mol of product min-r (mol of
enzyme)-! as the maximum value of k"., (k3). Typically, kinetic runs were
performed using enzyme with specific activity ol -200 U mg-t.
(17) N. Kharasch, "Organic Sulphur Compounds", Vol. l, Pergamon Press,
'1961:
A. Fava. l. llliceto, and E. Camera. J. Am. CEm. Soc.,
Efmsford. N.Y.,
79, 833 (1957); L. Eldiarn and A. Pihl, J. Biol. Chem., 225, 499 (1957).
(18) H. U. Bergmeyer in ref 10, Vol. 1, p 95 ff.
(19) H. R. Mahler and E. H. Cordes. "Biological Chemistry", Harper and Row,
New York, 1966, p 237 tl.
(20) R. H. Weaver and H. A. Lardy, J. 9iol. Chem.,236, 313 (1961)'
iZrt ff'e values of klob$ in this work are comparable to rates (1.9-33 M-1
min-11 observed for the formation ot GSSG from GSH and GSSR at pH 7'0:
U. Weber, P. Hartter, and L. Flohe, Hoppe-Seyler's Z. Physiol. Chem.,351,
1389 (1970). fhe values of k1 in this work are also comparable to those
found by' SJ.-R.
Garel, FEBS Lett.,79, 135 (1977), tor the reduction ot a
-S
cystine
O o n Oi n r i b o n u c l e a s e A o f 3 . 0 X 1 0 2 M - r m i n - r a t p H 8 . 5 .
(221 T E. Creighton, J. Mo|.9iot.,96,767 (1975). Several corrections were
applied to Creighton's data before they were incorporated into Figure 5.
First. the pK" values are those used consistently throughout this paper'
and do not always match those used by Creighton. Second, Creighton's
rate constants are calculated on the basis of the appearance of oxidized
DTT, and ours on the appearance of reduced thiol by reaction of a disulfide
with a monothiol. Creighton's rate constants were multiplied by a factor
of 4 to make them compatible with ours: a factor of 2 to account for the
dilterence in the rates of appearance of oxidized DTT and reduced thiol
(2dIDTTo']/dt = d[Rs-]/dt), and a second factor of 2 to account for the
presence of two symmetry-equivalent thiol groups in DTT.
(23) A. A. Frost and R. G. Pearson, "Kinetics and Mechanisms", 2nd ed., Wiley,
New York, 1961.
(24) S. S. Hall, A. M. Doweyko. and F. Jordan, J. Am. Chem. Soc., 100, 5934
(1978).
(25) The value of pnr" given in ret 12 was calculated from the rate constants
incorrectly, and is in error.
(26) The data ot Hupe et al. also show some small sensitivity to the particular
data inch.rded in the analysis- Two sels of experiments were carried out
in obtaining rate constants for reductions with aryl thiols: one used the
reducing agent in excess, one had Ellman's r^eagent in excess. Leastsquares analysis of the first set of data yielded Fn*nv' = 0.48 (l = 0.96);
anatysis of thb second se!of data yielded in a = 0.41 1F = 1.0); analysis
of both together yielded d*-ry' = 0.44 1ts = 0.97). Inclusion. o1 a point for
methyl mercaptoacetate in Hupe's analysis changes Fn,-"'*v' to 0.43 (ts
= 0.91).
(27) Although the data for alkyl thiols in these studies are in good agreement,
there is a small but real difference in the data for the aryl thiols. The similarity in the alkyl thiol rate constants suggests that no consistent experimental difference accounts for this difference. All ot orr data were obtained
using the same procedure, although their experimental precision was lower
for the more rapidly reacting thiols. Hupe et al. used slightly different procedures for alkyl and aryl thiols. Either this difference in procedure, or
differences in the compositions of solutions (especially ionic strengith and
buffer composition), might confibute to fie consistent tactor ot ca. 2 which
separates these sets of rate constants.
(28) We note that the values of 0^* derived from these two-independent in= 0.4112
vestigations are. in fact, experimentally indistingnristraole (Fo.ry
vs. 0.4313) if the datum for methyl mercaptoacetate is included in the calculation of the latter point- Hupe et al. point out frat this compound shows
deviations in analyses of rate constants derived from both thioesters and
disulfides, and exclude it on that basis.
(29) C. O. Johnson, Chem. Rev., 75,755 (1975).
(30) H. A. Smith, G. Doughty, and G. Gorin, J. Org. Chem.,29,1484 (1964).
1480
(31) G. Dafman, J. McDermed, and G. Gorin, J. Org. Chem.,29,
(1964).
(32) O. R. Sanadi, M. Langley, and R. L. Searls, J. Biol. Chem., 234, 178 (195-9)'
oC,
report Eq' tor the reduciion of lipoamide to be -0.294 V at pH 7.1' 22
(33)
(34)
(35)
(36)
(37)
(38)
(39)
2025
vs. the standard hydrogen electrode. This correspords to a vah.re of -o.2gg
V at pH 7.0,30 "C. V. Ma,ssey, Biochim. Eiophys- Acta,3Z,314 (1960),
reports a value ol -0.288 V vs. standard hydrogen electrode at pH 7.0.
oc. we have repeated
30
the equilibrations described by Dandi et at. .r,o
obtained the same value for this potential. These potentials were all ou
tained by equilibration against an NAO+/NADH couple. We have used a
vah.p of E9' = -0.30 V at pH 7.0, 30.0 oC, vs. standard hyctogen electooe
for this couple: K. Brton and T. H. Wilson, BirJpm. J., 54, 86 (1953). Aq,
values and equilibrium constants were interconverted using eq 33; Kr, =
(lipoo'[NADH)/(lipddXNADH+) = 0.08_58at.pH 7.0, 3O-"C. rc)ri;t
Kn^od'i are thus pH dependent but K27[H+]-' (and Kxeo@ [H+]--riare
pH independent.
UV spectra of solutions obtained by mixing BAL and Ellrnan's reagent
showed only those features attributable to Ellman's anion and to acyclic
S-S bonds (Ellman's reagent). However, consideration of the trerds observed in the UV of various disulfidesJ'(acyclic, €ru 480 at tr 250 nm;
1,2{ithiepan€. €mar 444 al tr,,la, 258 nm; 1,2{ithiane. €m.r 290 at tr,*-,
290 nm; 1,2{ithiolane , enex 147 at tr,.", 330) suggest that the e,r,., torfour-membered cyclic disulfide might be vanishingly srnall. Rarnan specta
of an identical mixture of BAL and Ellrnan's reagent show, in addition to
the expected absorptions, a new band at 510 cm-r. This position is consistent with there being no special st'ain in ft€ S-S bord and is substantially
diflerent from the y 500 cm-r predicted for a l,2dithietane.$ Thus. these
spectral data are compatible with formation of either a cryclic dimer or
higher oligomer o( a polymer on oxidation of BAL. The isolation of a dithietane has not been reported, but stable disulfide-containing rings containing seven or more members are well established: A. E. Asato and R.
E. Moore, Tetrahdron Lett., 4841(1973); G. H. Wahl. J. Bordner, D. N.
Harpp, and J. G. Gleason. J. Chem. Soc. Chem. Commun'985 (1972); T.
C. Owen, J. M. Fayach, and J. S. Chen, J. Org. Chem.,38, 937 (1973).
J. A. Barltrap, P. M. Hayes, and M. Calvin, J. Am. Chem. Soc., 76, 4348
(1954).
H. E. Van Wart and H. A. Scheraga, J. Phys. Chem.,80, 1812, 1823
(1976).
We assume in doing these calculations that the pf. ol HSR-SSR' eqtnls
that of HSRSH (see Discussion section in text). The calculated values of
Kosscs and K6556sH include a statistical factor of 2 whenever a dithiol
reducing agent is involved.
J. P. Snyder and L. Carlsen, J. Am. Chem. Soc., 99, 2931 (1977).
R. E. Rosenfield, Jr., R. Partfiasarathy, and J. D. Dunitz, J. Am. Chem. Soc.,
99, 4860 (1977).
In principle. it should be possible to estimate 0n,. * grs from a plot of the
sort shown in Figure 7, assuming that 36 (with constant parameters)
characterized all the thiol-disulfide reactions in an equilibrating mixture
of RSH and ESSE (eq 18). At equilibrium, defining the equilibrium constant
in terms of rate constants for forward and back reactions:
xs- = 2(dn,- - p'gXpK""sH - pKaESH)
The data in Figure 7 cover both alkyl and aryl thiols and disulfides' and tying
tofitthema||toasing|eequation'naynot|eadtousefu|parameters.||we
to thiolanalyze. however, oity tfte group oi points 1-8 corresponding
OisuitiOeinterchange involving aliphatic thiols. and force the le.ast-squares
-O'7'l'
0,n.= 1'?7. n tJ's=
line to go hrough (d,O),we estirrnie trat dogrc = -0'73 genof
vilue
with
the
quaiitative
agreement
is
in
This va-lue
Hupe.
erated by analyzrng rates. R. Freter, E. R. Pohl, J. M. Wilson, and,,D.J. -o'3
=
J- Org. Chem.', 44: fl70(1979), have independently estirnated /J"
form
for dlsplacement of Ellman's anion from mixed disulfides of the
RSS-Ellman's.
: -0.30 (0n* = =g,g= 0.63,C = 5'5.1)'
0"-= -0'35 (0'"*
t+ol
' ' - ' viies';ilj:
_ : d ; = b . o z .C = S . S i r ' j l n d P : : 0 . 4 0 ( t , =
. , 8- d ' n = 0 . ! 1 -C = 6 . 2 9 )
arlnale ts = 0.96for plotsof thetypeshownin Figure10' Thevalues
to d. = -O'40 werechosenas "best"becausatheslope
-orresponolng
- a
K4itcd
a of the correlatlonline and the intercept b in the eguation-log
=
for
=
log A-'ot * bwereclosestto the valuesof a 1'0, b 0'0=expected
-0'35 (a =
=
a oerlectconetatioi:-1i.: :o'50 (a = 0'93' a 0'34);0'
i c = -0.40 (a = 0'98' b = 0'09)'
o . ' g Oo, = 0 . 2 0 ) d
of the
(41) In practice,in appticationsof these equalionsto the determination
we find
pK's of proteinttiolg-toupi u"ing thiol-disulfide intercfrange-rates'
is less than
that fre difference-be-t*# G p"r"s estirnatedusingeq.3F3.8
*9fr'^^"
the uncertaintyfrom other aspectl of the experimental
269 (1958);
(42) A. L. Aflred ano E. G- Rocnow' J. lnorg' Nuci'Chem'' 5' 26.4'
a' J' Am' Chem' Soc" 99'
E. Clementi,F. Cavallone,and R. Scordamagli
5 5 3 1( 1 9 7 7 ) .
in eq 36 can'
(43) The relative magniMes of the Bronstedcoefficientsused
state in which
in principte,o" |'"tloi-"ii.Jt ,rig itnodrl for the transition
on ona
RSre and RS'chave eqtral charge, providedthat the substituent
to acconrsulfur significanttyintGnces thi ability of an adiacent.sulfur
distributionbasedon this
a
modate charge.An;;;Gi;;iti."tt
"Ltg"
-groundof p.is lircarly
modef, and on u.,eaoaitionafassumptionthat fre rnagniMe.
and transition
the
in
sulfur
on
charga
in
related to the difference
results'
unreasonable
physically
to
however,
lead.
state,
UniversityPress'
(44) R. P. Bell, "fne p;ln l[ Crremijtry" , 2nd ed" Cornell
y anclEnzynptogy"'
Itfraca.N.y.. 1973;Ui. F..l*"f", "Cit"rV"S in Ci=mist
New York' 1969'
McGraw-Hill,
( 4 5 ) T h e e f | e c t i v e m o t a r i t y o t t h i o t i n i n t r a m o | e c u l a r a t t a c k o n t h e cisa r1'
b4o n y |
moiety of fnitrophenyl M(2<nercaptopnenyllMm€hylcarbarnate
A'm' Clem'
J'
Wing'
S'
Hutchin"'
EC.
JFile'
x 1os M: cf. T. H.
"'io'u' intramolecularetfects
Soc., 97, 5s78 (1975).For a generaldiscuision of
tnt'Ed' Enst'' 16' 449 (19771'
see M. l. Page, eigi;. Cnii,
6308
B:'c; a.i" e^" ctnm' soc" ee'
(46) G. iltuminari.r. #,iooirii";;
(1977),and referencescited therein'
plenum Press' New York' 1977'
(47)
' ' ' S. Oae, "organ," bitrnrttw ot Surfw"'
(1976)'
Cnapter7; Bi Meyer,Chem.Rev- 76' 367
J' tuld' AEn'
(48) E. R. Atkinson,G-R. l{at'tdrii' R. J' Ekuni'and i' '' eanch€lli'
2026
Journal of the American Chemicarsocietl,
8,29(1e6s).
(49) c. L. Eilnran.Arch; Bjo_chgm.Biophys.,gZ,ZO(1959);
A. F. S, A' l'{abeeb'
Methods Enzymot., 25, 457 (19i2f.
(50) K. Crawehn
and H. U. Bergmeyerin ref 10, Vol. 3, p 1496.
/ r02.6 / March r2, tgg0
(S1) H. U. Bergmeyer, K. Gawehn,
and M. Grassl in ref 10, Vol. 1, p 469.
(52) N-r.
-'rilJin"irti""t
crrairoeilain J.l. n. Reed;;
chemisryor Sutrur
"noPart lll, "Nr.rclear tutagnetic
and lts Compounds",
nesonance
Compounds',, J. H. Karchmer, Ed., Wit"V]frr-.i york. j971. Data of Sulfur