Rate constant determination for the reaction of hydroxyl and glutathione thiyl radicals with glutathione in aqueous solution

Article abstract of DOI:10.1021/jp9535553

The techniques of pulse radiolysis, laser photolysis, and absorption spectroscopy have been used to investigate the glutathione disulfide radical anion formation over the pH range 7.0-13.0 in aqueous solution. Photolysis of the disulfide anion formed from the one-electron oxidation of reduced glutathione perturbs the disulfide anion/thiyl radical equilibrium, allowing the rate constant for. thiyl radical reaction with glutathione to be uniquely determined from the transient absorption bleach and subsequent pseudo-first-order recovery. These pH-dependent values were combined with measured disulfide equilibrium constants to calculate glutathione radical anion dissociation rate constants. From computer modeling of established mechanisms of the observed disulfide radical anion growths, pH-dependent rate constants for the reaction of hydroxyl radicals with glutathione to produce the thiyl radical were obtained. Utilizing literature ionization constants, values for hydroxyl and thiyl radical reactions with individual glutathione species were determined. The similarity of the measured values over the pH range 10-13 suggests that the rate constants for both the hydroxyl and oxide radical reaction with glutathione are essentially the same. These hydroxyl radical rate constants are contrasted with previously reported values determined using competition kinetics.

Full text of DOI:10.1021/jp9535553

J. Phys. Chem. 1996, 100, 8861-8866
8861
Rate Constant Determination for the Reaction of Hydroxyl and Glutathione Thiyl Radicals
with Glutathione in Aqueous Solution†
Stephen P. Mezyk*
Radiation Laboratory, UniVersity of Notre Dame, Notre Dame, Indiana 46556
ReceiVed: NoVember 28, 1995; In Final Form: February 15, 1996X
The techniques of pulse radiolysis, laser photolysis, and absorption spectroscopy have been used to investigate
the glutathione disulfide radical anion formation over the pH range 7.0-13.0 in aqueous solution. Photolysis
of the disulfide anion formed from the one-electron oxidation of reduced glutathione perturbs the disulfide
anion/thiyl radical equilibrium, allowing the rate constant for thiyl radical reaction with glutathione to be
uniquely determined from the transient absorption bleach and subsequent pseudo-first-order recovery. These
pH-dependent values were combined with measured disulfide equilibrium constants to calculate glutathione
radical anion dissociation rate constants. From computer modeling of established mechanisms of the observed
disulfide radical anion growths, pH-dependent rate constants for the reaction of hydroxyl radicals with
glutathione to produce the thiyl radical were obtained. Utilizing literature ionization constants, values for
hydroxyl and thiyl radical reactions with individual glutathione species were determined. The similarity of
the measured values over the pH range 10-13 suggests that the rate constants for both the hydroxyl and
oxide radical reaction with glutathione are essentially the same. These hydroxyl radical rate constants are
contrasted with previously reported values determined using competition kinetics.
Introduction
previous kinetic investigations8 have shown that the rate
constants for reactions 1, 3, and 4 are all similar in aqueous
solution and also because the observed growths represent an
approach to an equilibrium. This difficulty is reflected in the
scatter of the available literature data for sulfhydryls.8
The role of sulfhydryl (RSH) and disulfide (RSSR) com-
pounds in radiation protection has been known for many years.1,2
The in-situ discovery of thiyl (RS•) radicals in biological
material3,4 and the establishment of their versatile chemistry as
intermediates in the repair reactions of carbon-centered radicals5
have resulted in increased attention being paid to the reactions
of such sulfur-centered radical species. Within cells glutathione
is the most abundant non-protein-bound sulfhydryl that can act
in this manner;6,7 consequently, the investigation of its thiyl
radical formation by primary water radiolysis radicals has been
intensively pursued.8 Although a general scheme for the one-
electron oxidation of glutathione by the hydroxyl radical in
neutral and alkaline solution has been established,9 there is little,
and inconsistent, rate constant data available for this reaction.8
In neutral or basic aqueous solutions, the production of the
RS• radicals from free sulfhydryls results in the formation of
the reducing radical disulfide anion, RSSR•-. A large number
of these radical anions have been shown to be formed by the
mechanism10
In a recent investigation,11 absolute rate constants for reaction
4 were determined for the sulfhydryls cysteine and cysteamine,
using a combined LINAC radiolysis-LASER photolysis tech-
nique. These experiments used electron pulse radiolysis to
generate the radical disulfide anion via reactions 1-4 and then
photolyzed the anion using the laser pulse. The photolysis of
this transient causes a decrease in the RSSR•- absorption, which
subsequently recovers via first-order kinetics. As the bleach
and subsequent recovery of the RSSR•- absorption were
demonstrated to be due only to the perturbation of the RS•/
RSSR•- equilibrium of reaction 4, the exponential recovery
gives a direct and unique measurement of the (pseudo-first-
order) rate constant for the disulfide anion formation reaction.
In a subsequent study12 these rate constants were combined
with measured cysteine disulfide radical anion equilibrium
constants to allow calculation of rate constants for the disulfide
dissociation reaction over a wide range of pH’s. Knowing both
component rate constants in reaction 4, the observed growth
profiles of the cysteine RSSR•- transient could then be used to
accurately determine rate constants for hydroxyl and oxide
radical reaction with this sulfhydryl.
•OH + RSH f H2O + RS•
RSH + -OH h H2O + RS-
(1)
(2)
This study utilizes these techniques in the determination of
rate constants for the reaction of hydroxyl radicals with
glutathione in aqueous solution, over the pH range 7.0-13.0 at
room temperature. Using literature ionization constants, the
constituent rate constants for hydroxyl radical reaction with
individual glutathione species have also been calculated. These
values are compared to the results of previous determinations.
•OH + RS- f OH- + RS•
(3)
RS• + RS- (RSH) h RSSR•- (+H+)
(4,-4)
The elucidation of individual rate constants from only the
temporal behavior of the disulfide transient is complicated, as
* Present address: Research Chemistry Branch, AECL-Whiteshell
Laboratories, Pinawa, Manitoba, R0E 1L0 Canada.
Experimental Section
† The research described herein was supported by the Office of Basic
Energy Sciences of the Department of Energy. This is contribution No.
NDRL 3864 from the Notre Dame Radiation Laboratory.
The setup used for these experiments has been described in
detail in several previous publications;13-15 hence only a brief
description shall be given here. The pulse radiolysis system
X Abstract published in AdVance ACS Abstracts, April 15, 1996.
S0022-3654(95)03555-6 CCC: $12.00 © 1996 American Chemical Society

8862 J. Phys. Chem., Vol. 100, No. 21, 1996
Mezyk
consists of an 8 MeV LINAC, which delivered 10 ns electron
pulses to the sample cell. An XeCl* excimer laser (Lumonics
Hyper EX-400) was synchronized to photolyze the irradiated
solution after a fixed time period. The pulse radiolysis traces
were normalized for small fluctuations in radiolysis dose; the
laser intensity did not vary significantly in these experiments.
Glutathione (Aldrich, 98% reduced) was used as received.
Care was taken to minimize air oxidation of the glutathione as
well as decomposition at higher pH. All solutions were prepared
immediately before irradiation, by dissolving weighed amounts
of glutathione in N2O-saturated Millipore Milli-Q filtered water,
which had been buffered to the appropriate pH using Baker
Analyzed monobasic phosphate or borax at a concentration of
2.0 × 10-2 mol dm-3. Exact pH values were obtained by
addition of small amounts of NaOH (Fisher, ACS) or HClO4
(Aldrich, ACS 70%) and using a pH meter. In N2O-saturated
solution, quantitative conversion of hydrated electrons to
hydroxyl radicals occurs via
eaq- + N2O + H2O f •OH + -OH + N2
(5)
Pulse radiolysis experiments were performed at room temper-
ature (22 ( 1 °C) using initial hydroxyl radical concentrations
of (2-4) × 10-6 mol dm-3. Solutions were flowed through
the irradiation cell at a rate sufficient to ensure that a fresh
sample was irradiated each time, with typically about 80 pulses
being averaged to obtain a single trace. Absolute dosimetry
was carried out using N2O-saturated SCN- solutions16 (10-2
mol dm-3, λ ) 472 nm, Gꢀ ) 4.92 × 104).
Figure 1. (a) Transient absorption change at 410 nm observed in the
pulse radiolysis (9) of N2O-saturated glutathione (2.57 × 10-3 mol
dm-3) solution at pH 9.0. Second trace (O) shows this system which
has also been photolyzed by a laser pulse at 308 nm (135 mJ), resulting
in a reduction of absorption (bleach) followed by recovery. (b)
Difference between these two absorption profiles. Solid line is the
predicted values of eq 8 in text, using the rate parameters k7 ) 8.1 ×
Results and Discussion
Photolysis Measurements. The glutathione radical disulfide
anion, GSSG•-, has a characteristic absorbance with a maximum
at 420-430 nm.17 At lower pH’s, this transient is replaced by
another weak absorbance due to GS•, which has a peak at 330
nm and extends to 450 nm. Under the experimental conditions
of this study, the best signals were obtained at 410 nm; at this
wavelength the interference from the GS• radical absorption was
negligibly small (<2%).
103 s-1 and k6[GS] ) (9.94 ( 0.83) × 105 s-1
.
The optical transient obtained from only the pulse radiolysis
of a solution of glutathione, 2.57 × 10-3 mol dm-3 at pH 9.0,
is shown in Figure 1a. The additional photolysis of this transient
causes a bleach in this absorbance, as observed for cysteine
previously.11 By subtracting this bleach from only the pulse
radiolysis absorption, the recovery trace was obtained, as shown
in Figure 1b. The subtracted trace exhibited first-order kinetics
and corresponds to the forward “averaged” rate constant, k6,
for glutathione thiyl radical reaction with both protonated and
deprotonated glutathione to form the disulfide anion,
GS• + GS h glutathione disulfide radical anion (6,-6)
Figure 2. Measured pH and glutathione concentration dependence of
the rate constants for glutathione thiyl radical intramolecular rearrange-
ment to form the carbon-centered R-amino radical (reaction 7 in text),
as taken from ref 19.
However, it has previously been demonstrated9,18-21 that an
additional decomposition pathway exists, where the glutathione
thiyl radical undergoes an intramolecular rearrangement to form
the carbon-centered R-amino radical, written as the first-order
reaction
concentration and pH dependence observed for these values,
as shown in Figure 2, with an upper rate constant for reaction
7 of ca. 1.5 × 105 s-1 determined at all glutathione concentra-
tions and pH’s. This limiting rate constant is in reasonable
agreement with a subsequent determination at pH 10.5,18 which
GS• f GC•
(7)
Individual rate constants for reaction 7 have been previously
determined over the pH range 8-11;19 these values are shown
in Figure 2. These rate constants were determined from
extrapolated zero-dose, half-life measurements of the glutathione
disulfide radical anion decay as well as from the growth kinetics
of the carbon-centered radical. There was a marked glutathione
calculated k7 to be 2.2 × 105 s-1
.
There was also a significant dose dependence seen for reaction
7, with the observed rate constant doubling for a ca. 5 Gy
increase in dose. However, the magnitude of the experimental
photolytic-induced bleach measured in this study (typically 10-
15% of the glutathione disulfide anion intensity) corresponds

Reaction of Hydroxyl and Glutathione Thiyl Radicals
J. Phys. Chem., Vol. 100, No. 21, 1996 8863
TABLE 1: Summary of the Kinetic Parameters Used in the
Determination of Hydroxyl Radical Reaction Rate Constants
with Glutathione in Aqueous Solution
10-8 k6
K6
10-5 k-6
10-9 k18
pH
(dm3 mol-1 s-1
)
(dm3 mol-1
)
(s-1
)
(dm3 mol-1 s-1
)
7.0
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
13.0
0.75 ( 0.12
1.38 ( 0.07
2.15 ( 0.09
3.48 ( 0.23
4.90 ( 0.14
5.79 ( 0.25
6.04 ( 0.19
5.85 ( 0.31
6.09 ( 0.30
5.98 ( 0.27
6.25 ( 0.16
338 ( 242 2.22 ( 1.62
478 ( 265 2.70 ( 1.50
1595 ( 167 1.35 ( 0.15
2798 ( 242 1.24 ( 0.14
3063 ( 390 1.60 ( 0.21
2205 ( 269 2.59 ( 0.34
2226 ( 109 2.71 ( 0.16
2079 ( 465 2.81 ( 0.64
2016 ( 134 3.02 ( 0.25
2023 ( 337 3.06 ( 0.53
1990 ( 144 3.14 ( 0.24
3.34 ( 0.14
2.87 ( 0.18
2.34 ( 0.09
1.41 ( 0.05
1.06 ( 0.06
0.79 ( 0.04
0.79 ( 0.03
0.80 ( 0.06
0.77 ( 0.02
0.75 ( 0.03
0.81 ( 0.03
the second-order rate constant for reaction 6 as k6 ) (3.48 (
0.23) × 108 dm3 mol-1 s-1
.
Analogous experiments were performed over the pH range
7.0-13.0 to obtain the rate constants shown in Figure 3b and
individually listed in Table 1. Values for k7 at these pH’s, over
the experimental glutathione concentration range (1-5) × 10-3
mol dm-3, were estimated by linear extrapolation/interpolation
of the pH 8-11 rate constants given in Figure 2. Also shown
are several previous determinations of this rate constant;10,18,22,23
the results of this study are seen to be in excellent agreement
with a “direct” measurement from the pseudo-first-order forma-
tion of GSSG•- of 6.2 × 108 dm3 mol-1 s-1 at pH 11.7,10 but
to differ significantly from values obtained at lower pH’s.
Over the pH range of this study, there are three different
glutathione species, designated GSH, GS-, and GS2-, all in
equilibrium,24
Figure 3. (a) Total glutathione concentration dependence of measured
rate constants for radical disulfide anion formation in N2O-saturated
solutions at pH 9.0. Error bars are one standard deviation obtained from
the fitting of eq 8 in text. Solid line corresponds to fitted second-order
rate constant, with value k6 ) (3.48 ( 0.23) × 109 dm3 mol-1 s-1. (b)
pH dependence of measured radical disulfide anion formation rate
constants of this study, in comparison to previous literature determina-
tions: (4) ref 10, (]) ref 18, (3) ref 22, and (4) ref 23. Solid line
corresponds to predicted values using eq 16 given in text with limiting
thiyl radical reaction rate constants as given in Table 2. Individual values
are summarized in Table 1.
GSH h H+ + GS-
GS- h H+ + GS2-
pK11 ) 8.75
pK12 ) 9.65
(11)
(12)
and therefore the measured rate constants in Figure 3b are
combinations of the limiting reactions
to the production of an initial glutathione thiyl radical concen-
tration equivalent to a dose of ca. 1 Gy, which is low enough
that the values shown in Figure 219 can be used without
significant error.
Therefore, the observed recovery of the photolyzed transient
incorporates both pathways for the formed GS• radical, reactions
6 and 7, as given by the competition-kinetics expression
GS• + GSH f GSSG•- + H+
GS• + GS- f GSSG•-
(13)
(14)
(15)
GS• + GS2- f GSSG•2-
[GS•]0k6[GS]
It was assumed that the effects of the GS• radical deprotonation
at higher pH’s were small. Transient absorption spectra of the
disulfide radical anion measured at pH 9.0 (mostly GSSG•-)
and pH 11.0 (mostly GSSG•2-) were the same within experi-
mental error; thus, on the basis of these three reactions, a general
expression for the overall measured rate constant is given by25
[GSSG•-]t )
{1 - e-(k [GS]+k )t
}
(8)
6
7
k6[GS] + k7
Fitting the recovery of Figure 1b to this equation, with a fixed
value of k7 ) 8.1 × 103 s-1, a pseudo-first-order rate constant
(k6[GS]) of (9.94 ( 0.83) × 105 s-1 was obtained (see Figure
1b).
k13[H+]2 + k14K11[H+] + k15K11K12
k6 )
(16)
There are other potential pathways for GS• radical reaction,
notably the radical combination reactions
K11K12 + K11[H+] + [H+]2
Fitting this equation to the data shown in Figure 3b, with K11
and K12 fixed at their known pKa values, gives the limiting rate
constants k13 ) (6.62 ( 0.76) × 107, k14 ) (4.64 ( 0.19) ×
108, and k15 ) (6.21 ( 0.10) × 108 dm3 mol-1 s-1 (Table 2).
The predicted rate constants using these calculated values in
eq 16 are shown as the solid line in Figure 3b and are seen to
be in excellent agreement with the experimental data.
Equilibrium Constant Determinations. The overall equi-
librium constants for glutathione radical disulfide anion forma-
tion, K6, were calculated from only the radiolysis transient
growth kinetics at 420 nm, as performed for cysteine previ-
GS• + GS• f GSSG
(9)
GS• + GSSG•- f products
(10)
Although the extent of these two reactions is believed to be
small under the experimental conditions employed, their possible
interferences were further minimized by performing the radi-
olysis-photolysis experiments over a range of total glutathione,
GS, concentrations, to give scavenging plots as shown in Figure
3a. The weighted straight line fit to this data at pH 9.0 gives

8864 J. Phys. Chem., Vol. 100, No. 21, 1996
Mezyk
TABLE 2: Calculated Rate Constants Obtained in This
Study for Glutathione Thiyl and Hydroxyl Radical Reaction
with Glutathione Species in Aqueous Solution
reaction
rate constant (dm3 mol-1 s-1
)
GS• + GSH f GSSG•- + H+
(6.62 ( 0.76) × 107
(4.64 ( 0.19) × 108
(6.21 ( 0.10) × 108
(3.48 ( 0.11) × 109
(9.00 ( 0.74) × 108
(7.68 ( 0.13) × 108
GS• + GS- f GSSG•-
GS• + GS2- f GSSG•2-
•OH + GSH f H2O + GS•
•OH + GS- f OH- + GS•
•OH + GS2- f OH- + GS•-
ously.12 The temporal behavior of the disulfide anion absorption
was fitted to a combined first-order growth and decay expo-
nential model, the second term being used to approximate the
small amount of decay that occurred for some of these transients
on the experimental time scales. By plotting the total glu-
tathione concentration dependence on the fitted growth rate
constants, a linear relationship was obtained, with the equilib-
rium constant given by K6 ) slope/intercept. The obtained data
for pH 9.0 is shown in Figure 4a, with a calculated slope value
of (3.47 ( 0.03) × 108 dm3 mol-1 s-1 and an intercept of (1.16
( 0.10) × 105 s-1, corresponding to an equilibrium constant
of K6 ) 2798 ( 242 dm3 mol-1. This value is in very good
agreement with, and more precise than, the equilibrium constant
determined from the slope and intercept values of the second-
order plots obtained from the radiolysis-photolysis experiments
(Figure 3a), K6 ) 2892 ( 738 dm3 mol-1
.
The 420 nm measurements were repeated over the pH range
7.0-13.0, with the individual equilibrium constants obtained
summarized in Table 1 and shown in Figure 4b. The pH
dependence of these values is similar to that determined for
cysteine previously;12 increasing from ca. 300 to >3000 dm3
mol-1 from pH 7.0 to 9.5 and then decreasing to a limiting
value at higher pH’s.
Figure 4. (a) Equilibrium constant determination for glutathione radical
disulfide anion formation in N2O-saturated solutions at pH 9.0 from
420 nm growth kinetics. From fitted linear slope and intercept values
(see text), an equilibrium constant of K6 ) 2798 ( 242 dm3 mol-1 is
calculated. (b) pH dependence of the calculated equilibrium constants
for glutathione radical disulfide anion formation. Individual values are
given in Table 1.
From the pH-dependent k6 rate and K6 equilibrium constants,
the rate constants for the dissociation of the glutathione radical
disulfide anion can readily be calculated; these values are shown
in Figure 5 and listed in Table 1. It is important to note that
this calculation only gives the k-6 value; however these rate
constants are in excellent agreement with the total GSSG•-
decay rate constants obtained from the intercept values of the
photolysis plots (Figure 3a). This means that the dissociation
of GSSG•- to again form the glutathione thiyl radical is the
dominant pathway under these conditions, which implies that
the direct conversion of the disulfide anion to the carbon-
centered radical
GSSG•- f GC•
(17)
previously proposed19 does not occur to any significant extent
under the present conditions.
Figure 5. pH dependence of calculated rate constants for reaction -6,
determined using measured disulfide radical equilibrium constants
(Figure 4b) and scavenging rate constants (Figure 3b), in comparison
to previous literature determinations: (]) ref 10, (4) ref 18, (3) ref
21, (0) ref 22, and (O) ref 23. Error bars correspond to one standard
deviation as calculated from the uncertainty in these two measured
values. Individual values are given in Table 1.
Despite the large errors associated with these dissociation rate
constants at lower pH’s, there is a discernible trend in the
obtained pH behavior, consisting of a minimum value around
pH 9.0 and an indication of a rise with increasing pH. An
increase to a limiting value at higher pH’s is also seen. This
pH dependence is consistent with previous measurements for
cystamine and cystine,26 where the decay of the disulfide radical
anion was directly observed following the reaction of the
hydrated electron with the disulfide. For these two compounds,
the increase at lower pH was attributed to protonation of the
disulfide anion to give the short-lived sulfenium (RSSRH•)
radical, which would dissociate to give RS• and RSH, and the
rise at higher pH was shown to exactly follow the pKa for
deprotonation of the disulfide amino group. Although in
qualitative agreement, ascertaining whether glutathione also
exhibits this behavior at higher pH’s is not possible in this study,
due to the large errors and because there are two pKa values in
this pH regime (8.75 and 9.65).24
There have been several previous direct measurements for
the GSSG•- decay rate constant,17,18,22,23,26 and these values are
also shown in Figure 5. Although no pH dependence has been
performed in any other single study, these values are seen to
be in good agreement with the calculated rate constants of this
study.

Reaction of Hydroxyl and Glutathione Thiyl Radicals
J. Phys. Chem., Vol. 100, No. 21, 1996 8865
Figure 6. Typical fits obtained in the determination of pseudo-first-
order rate constants for hydroxyl radical reaction with glutathione at
pH 10.0. Data shown corresponds to glutathione concentrations of 3.51
× 10-3 (0), 2.89 × 10-3 (4), and 1.64 × 10-3 (]) mol dm-3. Fitted
lines correspond to calculated values, using determined rate constants
for reactions 6 and -6 (Table 1) and hydroxyl radical reaction rate
constants of k18[GS] ) 2.59 × 106, 2.17 × 106, and 1.27 × 106 s-1
,
respectively.
Hydroxyl Radical Reactions. Knowing the pH-dependent
rate constants for reactions 6 and -6, individual rate constants
for the reaction of hydroxyl radicals with glutathione,
Figure 7. (a) Total glutathione concentration dependence of calculated
rate constants for hydroxyl radical reaction in N2O-saturated solutions
at pH 10.0. Solid line corresponds to fitted second-order rate constant,
with value k18 ) (7.90 ( 0.42) × 108 dm3 mol-1 s-1. (b) pH dependence
of rate constants for the reaction of hydroxyl and oxide radicals with
glutathione in aqueous solution in comparison to previous literature
data: (O) ref 9, (4) ref 22, and (0) ref 28. Individual values are given
in Table 1. Error bars correspond to one standard deviation obtained
from second-order rate constant determinations. Fitted solid line
corresponds to the best fit of eq 16 in text, with limiting hydroxyl radical
reaction rate constants as given in Table 2.
•OH + GS f GS•
(18)
can then be determined. Using the three-reaction mechanism,
consisting of reactions 6, -6, and 18, each transient glutathione
disulfide radical anion growth was modeled using the numerical
differential equation solving code FACSIMILE27 to optimize
the best pseudo-first-order rate constant for reaction 18. Typical
fits obtained for three glutathione concentrations at pH 10.0 are
shown in Figure 6.
The calculated first-order rate constants were then plotted
against total glutathione concentration to give scavenging curves,
as shown in Figure 7a. At pH 10.0, the overall rate constant is
k18 ) (7.90 ( 0.40) × 108 dm3 mol-1 s-1. These determinations
were repeated over the pH range 7.0-13.0, with the values
obtained listed in Table 1 and shown in Figure 7b.
tions. Based on these potential complications, the previously
reported competition-kinetics reaction rate constants must be
treated with some caution.
At higher pH’s, deprotonation of the hydroxyl radical also
occurs,
•OH h H+ + •O-
(19)
There have been several previous measurements of hydroxyl
radical reaction rate constants with glutathione,9,21,22 and these
values are also shown in Figure 7b. The rate constants obtained
in this study are significantly different from all the previous
competition-kinetics determinations, being lower in magnitude
and showing the opposite pH behavior of decreasing with
increasing pH. The observed pH behavior in this study is
consistent with previous measurements of hydroxyl and oxide
radical reaction with cysteine.12
with a measured pKa value of 11.9.29 In principle this means
that there are six separate reactions that can occur, with both
the hydroxyl and oxide radicals reacting with GSH, GS-, and
GS2-
. However, as the overall measured rate constant is
essentially the same over the pH range 10.0-13.0, this implies
that the rate constants for oxide and hydroxide reaction with
GS- and GS2- are effectively the same. Given that oxide
radical reaction with GSH will only be a very minor reaction,
the rate constants shown in Figure 7b are thus well described
by the three limiting reactions
Most of the competition-kinetics determinations have used
hydroxyl radical reaction with SCN- as a reference reaction,9,22
which introduces the additional complication that the two
intermediates formed, GSSG•- and (SCN)2•-, have overlapping
spectra. To accurately determine the hydroxyl radical reaction
rate constant under these conditions requires accurate absorption
coefficients for the glutathione radical species; however, the
anion peak values have been reported over the range 6300-
•OH + GSH f GS• + H2O
(20)
•OH + GS- f GS• + OH-
•OH + GS2- f GS•- + OH-
(21)
(22)
8300 dm3 mol-1 cm-1 9,17
Moreover, it has been proposed that
.
there may be an interfering reaction of the formed GS• radicals
with SCN-, to give a product that absorbs in the same region.28
There has only been one other reference compound used,
tetramethylurea,28 which gave a glutathione reaction rate
constant that was much lower than all of the SCN- determina-
By analogy to reactions 13-15, eq 16 can again be used to
determine individual rate constants for these reactions. By
fixing the two pKa values of glutathione at their known values,24
individual rate constants of k20 ) (3.48 ( 0.11) × 109, k21
)
(9.00 ( 0.74) × 108, and k22 ) (7.68 ( 0.13) × 108 dm3 mol-1

8866 J. Phys. Chem., Vol. 100, No. 21, 1996
Mezyk
s-1 were obtained (Table 2). The predicted rate constants using
these values in eq 16 are shown as the solid line in Figure 7b
and are seen to be in excellent agreement with the experimental
data.
(2) Hollaender, A.; Doherty, D. G. Radiation Damage and Sulfhydryl
Compounds; International Atomic Energy Agency: Vienna, 1969.
(3) Mason, R. P.; Ramakrishna Rao, D. N. In Sulphur-Centred ReactiVe
Intermediates in Chemistry and Biology; Chatgilialoglu, C.; Asmus, K.-D.,
Eds.; NATO-ASI Series A, Life Sciences, Vol. 197, Plenum-Press: New
York, 1990; p 401.
(4) Mason, R. P.; Maples, K. R. In Sulphur-Centred ReactiVe Inter-
mediates in Chemistry and Biology; Chatgilialoglu, C.; Asmus, K.-D., Eds.;
NATO-ASI Series A, Life Sciences, Vol. 197; Plenum-Press: New York,
1990; p 429.
(5) Von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor
& Francis: London, 1987; p 353.
Conclusion
By combining the techniques of electron pulse radiolysis and
laser photolysis, individual rate constants for the reactions
GS• + GSH f GSSG•- + H+
GS• + GS- f GSSG•-
(13)
(14)
(15)
(6) Sa´ez, G. T.; Bannister, W. H.; Bannister, J. V. In Glutathione:
Metabolism and Physiological Functions; Vina, J., Ed.; CRC Press: Boca
Raton, FL, 1990; p 237.
(7) Wardman, P. In Glutathione Conjugation: Mechanism and Biologi-
cal Significance; Sies, H., Ketterer, B., Eds.; Academic Press: London,
1988; p 43.
(8) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J.
Phys. Chem. Ref. Data 1988, 17, 513 and references therein.
(9) Eriksen, T. E.; Fransson, G. J. Chem. Soc., Perkin Trans. 2 1988,
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(10) Hoffman, M. Z.; Hayon, E. J. Phys. Chem. 1973, 77, 990.
(11) Mezyk, S. P. Chem. Phys. Lett. 1995, 235, 89.
(12) Mezyk, S. P. Radiat. Res. 1996, 145, 102.
GS• + GS2- f GSSG•2-
have been elucidated from pH-dependent rate constants of
radical disulfide anion formation as k13 ) (6.62 ( 0.76) × 107,
k14 ) (4.64 ( 0.19) × 108, and k15 ) (6.21 ( 0.10) × 108 dm3
mol-1 s-1. These values were combined with measured, pH-
dependent, glutathione disulfide radical anion equilibrium
constants to allow computation of rate constants for the hydroxyl
radical reactions
(13) Janata, E.; Schuler, R. H. J. Phys. Chem. 1982, 86, 2078.
(14) Ebbesen, T. W. Radiat. Phys. Chem. 1989, 34, 619.
(15) Natarajan, P.; Fessenden, R. W. J. Phys. Chem. 1989, 93, 6095.
(16) Buxton, G. V.; Stuart, C. R. J. Chem. Soc., Faraday Trans. 1995,
91, 279.
(17) Shafferman, A. Isr. J. Chem. 1972, 10, 725.
(18) Zhao, R.; Lind, J.; Mere´nyi, G.; Eriksen, T. E. J. Am. Chem. Soc.
1994, 116, 12010.
•OH + GSH f GS• + H2O
(20)
(19) Grierson, L.; Hildenbrand, K.; Bothe, E. Int. J. Radiat. Biol. 1992,
62, 265.
(20) Eriksen, T. E.; Fransson, G. Radiat. Phys. Chem. 1988, 32, 163.
(21) Sjo¨berg, L.; Eriksen, T. E.; Re´ve´sz, L. Radiat. Res. 1982, 89, 255.
(22) Quintiliani, M.; Badiello, R.; Tamba, M. Int. J. Radiat. Biol. 1977,
32, 195.
(23) Baker, M. Z.; Badiello, R.; Tamba, M.; Quintiliani, M.; Gorin, G.
Int. J. Radiat. Biol. 1982, 41, 595.
(24) Fasman, G. D. Ed.; Handbook of Biochemistry and Molecular
Biology, Physical and Chemical Data, 3rd ed.; CRC Press: Boston, 1976;
Vol. 1, p C-703.
•OH + GS- f GS• + OH-
•OH + GS2- f GS•- + OH-
(21)
(22)
as k20 ) (3.48 ( 0.11) × 109, k21 ) (9.00 ( 0.74) × 108, and
k22 ) (7.68 ( 0.13) × 108 dm3 mol-1 s-1. These rate constants
are much lower and show the opposite pH dependence of
previously reported competition kinetics values.
(25) Mezyk, S. P. J. Phys. Chem. 1995, 99, 13970.
(26) Hoffman, M. Z.; Hayon, E. J. Am. Chem. Soc. 1972, 94, 7950.
(27) Curtis, A. R.; Sweetenham, W. P. FACSIMILE/CHECKMAT Users
Manual. Harwell Research Report AERE-R 12805, 1988.
(28) Liphard, M.; Bothe, E.; Schulte-Frohlinde, D. Int. J. Radiat. Biol.
1990, 58, 589.
Acknowledgment. I wish to thank Dr. David Armstrong for
his insightful comments on an earlier version of the manuscript.
References and Notes
(29) Rabani, J.; Matheson, M. S. J. Am. Chem. Soc. 1964, 86, 3175.
(1) Bacq, Z. M. Chemical Protection against Ionizing Radiation;
Thomas, C. C.: Springfield, IL, 1965.
JP9535553