LINAC/LASER determination of the absolute rate constant for thiyl and hydroxyl radical reaction with sulfhydryls in aqueous solution: Mercaptoethanol, cysteamine, and N-acetyl-L-cysteine

Article abstract of DOI:10.1021/jp953067v

The technique of combined pulse radiolysis/laser photolysis has been used to investigate the disulfide radical anion formation reactions for mercaptoethanol, cysteamine, and N-acetyl-L-cysteine over the pH range 7.0-13.0. The photolysis of the transient radical anions, formed by the one electron oxidation of the sulfhydryl, perturbs the disulfide radical anion/thiyl radical equilibrium, allowing rate constants for thiyl radical reaction with the parent sulfhydryl to be uniquely determined from the absorption bleach and subsequent recovery. These pH-dependent values were combined with measured disulfide radical anion equilibrium constants to calculate first-order disulfide radical anion dissociation rate constants. By computer modeling of established mechanisms for the disulfide radical anion growths, rate constants for the reaction of hydroxyl radicals with individual sulfhydryl species were calculated. These values are contrasted with previously reported values determined by using competition kinetics.

Full text of DOI:10.1021/jp953067v

J. Phys. Chem. 1996, 100, 8295-8301
8295
LINAC/LASER Determination of the Absolute Rate Constant for Thiyl and Hydroxyl
Radical Reaction with Sulfhydryls in Aqueous Solution: Mercaptoethanol, Cysteamine, and
†
N-Acetyl-L-cysteine
Stephen P. Mezyk*
Radiation Laboratory, UniVersity of Notre Dame, Notre Dame, Indiana 46556
X
ReceiVed: October 16, 1995; In Final Form: January 2, 1996
The technique of combined pulse radiolysis/laser photolysis has been used to investigate the disulfide radical
anion formation reactions for mercaptoethanol, cysteamine, and N-acetyl-L-cysteine over the pH range 7.0-
13.0. The photolysis of the transient radical anions, formed by the one electron oxidation of the sulfhydryl,
perturbs the disulfide radical anion/thiyl radical equilibrium, allowing rate constants for thiyl radical reaction
with the parent sulfhydryl to be uniquely determined from the absorption bleach and subsequent recovery.
These pH-dependent values were combined with measured disulfide radical anion equilibrium constants to
calculate first-order disulfide radical anion dissociation rate constants. By computer modeling of established
mechanisms for the disulfide radical anion growths, rate constants for the reaction of hydroxyl radicals with
individual sulfhydryl species were calculated. These values are contrasted with previously reported values
determined by using competition kinetics.
Introduction
the formation rate constants are of the same order of magni-
1
3
tude, and the observed growths represent an approach to
equilibrium. This difficulty is reflected in the scatter in the
The important role of sulfides (RSH) and disulfides (RSSR)
1
,2
in radiation protection has been known for many years. The
redox properties of these organic sulfur compounds, and
especially their radicals, are of considerable interest for the
understanding of many processes in biological and related model
systems. The discovery of in-situ thiyl (RS ) radicals in
biological material and the establishment of their versatile
chemistry as intermediates in the repair reaction of carbon
1
3
available literature data.
In several recent investigations
1
4-16
the technique of com-
bined LINAC radiolysis/LASER photolysis was used with
computer modeling to determine the individual rate constants
for these reactions for the sulfhydryls cysteine and glutathione.
These experiments involved using electron pulse radiolysis to
generate the radical disulfide, and then photolyzing this anion
•
3
,4
5
centered radicals have prompted much attention being paid to
the reactions of such sulfur-centered radicals. These RS radicals
1
4,16
•
using the laser pulse
are stronger oxidizing species than the simple aliphatic peroxyl
RSSR•- + hν f RS + RS-
•
(-4)
•
6-8
radicals, ROO , and have been demonstrated to be of similar
•
reactivity to their oxyanalogues, RO , toward many biologically
9
The photolysis of this transient causes a decrease in the RSSR•-
absorption, which subsequently recovers by first-order kinetics.
The bleach and recovery of the transient absorption is due only
available electron donors. Thiyl radicals have even been found
to initiate lipid peroxidation.1
0,11
These findings indicate that
sulfur-centered radicals play a very important role in free-
radical-mediated tissue damage.
•
•-
to the perturbation of the RS /RSSR equilibrium and thus the
exponential recovery gives the unique (pseudo-first-order) rate
constant for the disulfide anion formation reaction. By repeating
these experiments at different pHs and sulfhydryl concentrations,
specific rate constants for the disulfide formation by reactions
The reaction of the hydroxyl radical with free sulfhydryls,
in neutral or slightly basic solution, results in the formation of
•-
the reducing radical disulfide anion, (RSSR ), via the general
1
2
mechanism
4
and 5 were obtained.
These rate constants were then combined with pH-dependent
-
-
RSH + OH h H O + RS
(1)
(2)
2
disulfide radical anion equilibrium constants, independently
determined from concentration-dependent integrated RSSR
•
•
•-
OH + RSH f H O + RS
2
1
5
yield measurements, to give rate constants for the disulfide
dissociation reaction. Knowing both component rate constants
in these two equilibria, the observed pH-dependent growth
profiles of the RSSR•- transient could then be fitted to the
general reaction mechanism to determine individual rate con-
stants for reactions 2 and 3.
•
-
-
•
OH + RS f OH + RS
(3)
(4)
(5)
•
-
•-
RS + RS h RSSR
•
•-
+
RS + RSH h RSSR + H
The determination of specific rate constants for these reactions
from only the disulfide transient absorption is difficult, as all
The hydroxyl radical reaction rate constants obtained in this
1
5
16
manner for both cysteine and glutathione were much lower
than previously reported literature values, which had been
determined by using competition kinetics. Most of these
1
3
*
Present address: Research Chemistry Branch, AECL-Whiteshell
Laboratories, Pinawa, Manitoba, R0E 1L0, Canada.
The research described herein was supported by the Office of Basic
Energy Sciences of the Department of Energy. This is contribution No.
NDRL 3883 from the Notre Dame Radiation Laboratory.
-
competition studies used SCN as a reference reaction, which
†
complicates the elucidation of individual rate constants as the
two intermediates formed, RSSR•- and (SCN)2 , have over-
lapping spectra. This means that accurate absorption coefficients
•-
X
Abstract published in AdVance ACS Abstracts, March 15, 1996.
S0022-3654(95)03067-X CCC: $12.00 © 1996 American Chemical Society

8
296 J. Phys. Chem., Vol. 100, No. 20, 1996
Mezyk
are required to calculate the hydroxyl radical rate constant with
the sulfhydryls. Furthermore, it has been reported that an
-
interfering reaction occurs in the irradiated glutathione/SCN
17
system, to give another product that absorbs in the same region.
This latter study also used tetramethyl urea as a competitor and
obtained a much lower rate constant for hydroxyl radical
reaction. Based on these potential complications, the competi-
tion-kinetics literature values must be treated with some caution.
Another interesting aspect observed for hydroxyl radical
reaction with cysteine and glutathione in the LINAC/LASER
studies was that the overall rate constant decreased at higher
pHs. This behavior was opposite to that determined in previous
measurements for glutathione,18,19 although only sporadic pH-
dependent investigations had been performed, and inconsistent
with analogous studies of hydroxyl radical reaction with simple
amines, which showed an order of magnitude increase in rate
constant upon deprotonation of the amino group.20
In order to further investigate the generality of the behavior
observed for cysteine and glutathione, this study utilized the
LINAC/LASER technique to determine rate constants for the
reactions of hydroxyl radicals with other simple thiols in aqueous
solution. The sulfhydryls used were mercaptoethanol, cyste-
amine (2-aminoethanethiol), and N-acetyl-L-cysteine, due to their
relatively intense disulfide anion transient absorption across a
large pH range and because the photolysis of this transient
recovered fully, implying that there was no additional chemistry
Figure 1. (a) Transient absorption change at 410 nm observed in the
electron pulse radiolysis (9) of N
2
O-saturated mercaptoethanol (9.61
-4
-3
×
10 mol dm ) solution at pH 10.0. Second trace (o) shows this
-
such as photoionization of RS occurring on the experimental
transient which has subsequently been photolyzed at 308 nm (130 mJ),
resulting in a reduction of absorption followed by recovery. (b)
Difference between the above two absorption profiles, inverted only
for display purposes. Solid line is an exponential decay fit to the
difference data, corresponding to a pseudo-first-order rate constant of
time scales. The determined hydroxyl radical rate constants
have been compared to the results of previous determinations,
and by utilizing known ionization constants, the constituent rate
constants for hydroxyl radical reaction with individual sulfhydryl
species have also been calculated.
6
-1
(4.76 ( 0.28) × 10 s
.
solutions were flowed through the irradiation cell at a sufficient
rate to ensure that a fresh sample was irradiated each time.
During the irradiation process, the solution vessels were bubbled
Experimental Section
The experimental setup used for these measurements has been
detailed in several previous publications,
with only the minimum amount of N O required to prevent air
2
21-23
hence only a brief
ingress, to prevent loss of the volatile sulfhydryl. Typically
description shall be given here. The pulse radiolysis system
consists of a 7-MeV LINAC, which delivered 10-20-ns electron
pulses to the sample cell. An excimer laser (Lumonics Hyper
EX-400), which could be used to pump a dye laser (Lumonics,
Hyperdye-300), had its excitation pulse synchronized to pho-
tolyze the irradiated solution after a fixed time period. The
pulse radiolysis traces in all cases were normalized for the
radiolysis dose; the laser intensity did not vary significantly
during an experiment so its normalization was not required. Most
data in this study were collected by using the output of the
excimer laser at 308 nm, of typical power ca. 140 mJ/pulse.
Some experiments used the dye laser output over the range 400-
50-80 pulses were averaged to obtain a single trace. -
Dosimetry was performed using N2O saturated SCN solu-
-2
-3
4 24
tions, (10 mol dm , λ ) 472 nm, Gꢀ ) 4.92 × 10 ). All
experiments were performed at room temperature (22 ( 2 °C)
using initial hydroxyl radical concentrations of 2-5 × 10 mol
-6
-3
dm .
Results and Discussion
Mercaptoethanol. This compound was chosen for initial
study, as mercaptoethanol, HSCH CH OH, is one of the simplest
2
2
25
sulfhydryls with only a single pK of 9.50. In neutral or basic
a
•-
4
30 nm; however, the significantly lower energy (ca. 7 mJ/
solution, the mercaptoethanol radical disulfide anion, MSSM ,
pulse) meant that the signals obtained were much smaller and
correspondingly more noisy. The few rate constants obtained
using the dye laser photolysis were found to be in good
agreement with those measured with the 308-nm excimer
excitation.
has a characteristic absorbance with a maximum at 410-420
2
6
nm; at lower pHs this transient is replaced by a very weak
•
absorbance due to the thiyl radical MS . Under the experimental
conditions of this study, the best signals were obtained at 410
•
nm; at this wavelength the interference from the MS radical
The sulfhydryls used in this study (Aldrich) were of the
highest purity available and used as received. Care was taken
to avoid oxidation of these compounds; all solutions were
prepared immediately before irradiation by dissolving known
amounts of the sulfhydryl into N2O saturated Millipore Milli-Q
filtered water which had been buffered to the appropriate pH
by using Baker Analyzed monobasic phosphate or borax at a
concentration of 2.0 × 10 mol dm . Exact pH values were
obtained by additions of small amounts of NaOH (Fisher, ACS)
or HClO4 (Aldrich, ACS 70%) and using a pH meter. All
absorption was negligibly small.
The optical transient obtained from only the pulse radiolysis
-4
-3
of a mercaptoethanol solution (9.61 × 10 mol dm at pH
10.0) is shown in Figure 1a. The additional photolysis of this
transient causes a bleach in the absorbance, and the subtraction
of these two traces gives the recovery trace shown in Figure 1b
(data inverted only for presentation purposes). This recovery
trace shows first-order (decay) kinetics. By performing this
-
2
-3
-
experiment at different total (MS ) MSH + MS ) mercapto-
ethanol concentrations the scavenging curve shown in Figure

Determination of Absolute Rate Constant
J. Phys. Chem., Vol. 100, No. 20, 1996 8297
Figure 2. (a) Total mercaptoethanol concentration dependence of
measured rate constants for radical disulfide anion formation in N
2
O-
saturated solution at pH 10.0. Solid line corresponds to fitted second
9
3
-1
order rate constant, with value k
s
6
) (3.80 ( 0.27) × 10 dm mol
-1
. (b) pH dependence of calculated radical disulfide anion formation
rate constants of this study. Error bars are one standard deviation
obtained from the linear fits in (a). Individual values are given in Table
1
. Solid line corresponds to predicted values using eq 10 and limiting
thiyl radical reaction rate constants as given in the text.
2
a was obtained. The straight line fit to this data gives the rate
constant for mercaptoethanol disulfide anion formation,
•
•-
MS + MS f MSSM
(6)
at pH 10.0 as k6 ) (3.80 ( 0.27) × 10 dm mol s-1. The
9
3
-1
rate constants obtained in this manner over the pH range 8.0-
2.5 are shown in Figure 2b, the error bars shown for these
1
points correspond to one standard deviation obtained from the
straight line fits. These individual values are also listed in Table
1
. No comparison values could be found in the literature.
Over the pH range of this study, the charge on this sulfhydryl
changes from 0 to -1, therefore at intermediate pH values, the
overall reaction is by a combination of the two limiting reactions
•
•-
+
MS + MSH f MSSM + H
(7)
(8)
•
-
•-
MS + MS f MSSM
where these two species exist in equilibrium
+
-
HSCH CH OH h H + SCH CH OH
(9)
2
2
2
2
On the basis of these reactions, a general expression for the
overall measured rate constant, k6, can be derived as
+
k [H ] + k K
7
8
9
k6 )
(10)
+
[
H ] + K
9
By fitting eq 10 to the data of Figure 2b, with the value of K9
fixed at its pKa, the limiting rate constants k7 ) (1.22 ( 0.08)
9
9
3
-1 -1
×
10 and k8 ) (4.85 ( 0.19) × 10 dm mol
s
were
obtained. The predicted rate constant values using this model

8
298 J. Phys. Chem., Vol. 100, No. 20, 1996
Mezyk
Figure 4. pH dependence of calculated rate constants for the first order
dissociation of mercaptoethanol disulfide radical anion, reaction 13,
from measured equilibrium values of Figure 3b and measured (9) or
calculated (0) scavenging rate constants of Figure 2b. Error bars
correspond to one standard deviation as calculated from the uncertainty
in these two measured values. Individual values are given in Table 1.
the mercaptoethanol radical disulfide anion,
Figure 3. (a) Equilibrium constant determination from integrated
transient yields for mercaptoethanol radical disulfide anion formation
MSSM•- f MS + MS-
•
(13)
in N
2
O-saturated solution at pH 10.0. From fitted linear slope and
intercept values (see text), an equilibrium constant of K11 ) 1099.0 (
can readily be calculated. These values are shown in Figure 4
and also listed in Table 1. The obtained pH behavior is
complex; showing a minimum value around pH 10.5 and rising
with increasing pH to a plateau. A slower rise at lower pHs is
also observed. This behavior is consistent with that observed
3
-1
2
6.3 dm mol is calculated. (b) pH dependence of the calculated
equilibrium constants for mercaptoethanol radical disulfide anion
formation. Individual values are given in Table 1.
are shown as the solid line in Figure 2b and are seen to be in
very good agreement with the experimental data.
The overall equilibrium constants for mercaptoethanol di-
sulfide radical anion formation
1
5
16
for cysteine and glutathione previously and also in accord
29
with previous measurements for cystamine and cystine, where
the pH-dependence of the decay of the disulfide radical anion
was directly measured by following the reaction of the hydrated
electron with the disulfide.
•
•-
MS + MS h MSSM
(11)
Knowing the individual, pH-dependent rate constants for the
component reactions in equilibrium (11), the individual rate
constants for the reactions of hydroxyl radicals with mercap-
toethanol
were determined by using integrated yield measurements. These
measurements were performed at low initial radical concentra-
tions in order to minimize the disulfide radical decay. The
dependence of total mercaptoethanol concentration, [MS], on
2
7,28
•
•
the total yield (Gꢀ) of the formed radical anion is given by
OH + MS f MS
(14)
Gꢀ∞
1
can then be determined. By use of the established general
reaction mechanism, each mercaptoethanol disulfide radical
anion growth was modeled by using the differential equation
)
1 +
(12)
Gꢀ
K [MS]
11
-1
30
where Gꢀ∞ is the limiting absorbance. Thus a plot of (Gꢀ)
solving code FACSIMILE to optimize the best pseudo-first-
against [MS]- should give a straight line of slope (Gꢀ∞K11) ,
1
-1
order rate constant for reaction 14. Typical fits to three
mercaptoethanol concentrations at pH 10.0 are shown in Figure
5.
-
1
intercept (Gꢀ∞) , and ratio K11 ) intercept/slope. Typical data
obtained at pH 10.0 are shown in Figure 3a, where from the
-
8
3
-1
calculated slope of (2.31 ( 0.03) × 10 dm mol and
These fitted rate constants were then plotted against total
mercaptoethanol concentration to obtain linear scavenging
curves, at pH 10.0 the overall rate constant was k14 ) (4.37 (
-
5
intercept (2.54 ( 0.02) × 10 values, K11 ) 1099.0 ( 26.3
3
-1
dm mol is obtained.
9
3
-1 -1
These measurements were repeated over the pH range 7.0-
3.0, with the individual equilibrium constants obtained sum-
marized in Table 1 and shown in Figure 3b. The pH dependence
of these values is similar to that observed for cysteine and
0.47) × 10 dm mol s . By repeating this analysis over
the pH range 8.0-12.0, the values shown in Figure 6a and listed
in Table 1 were obtained. This plot again exhibits the
1
1
5
characteristic sigmoidal shape obtained previously for cysteine
1
6
15,16
glutathione previously, increasing from pH 7.0 to 11.0 (ca.
and glutathione,
pHs.
with the higher rate constants being at lower
3
-1
1
50 to >1600 dm mol ) and then decreasing to a limiting
value at higher pHs. Although in very good agreement with
the values determined for cysteine and glutathione, these
equilibrium constants are much lower than measured for other,
similar, sulfhydryls.
From the measured, pH-dependent k6 rate and K11 equilibrium
Over the pH range 10.5-12.0, the calculated hydroxyl radical
reaction rate constants are equivalent within error, which implies
that the rate constant for reaction of hydroxyl and oxide radical
with mercaptoethanol is essentially the same. Thus, the
composite values shown in Figure 6a are well described by only
the reactions
1
2
constants, the rate constants for the first-order dissociation of

Determination of Absolute Rate Constant
J. Phys. Chem., Vol. 100, No. 20, 1996 8299
•
•
OH + HSCH CH OH f H O + SCH CH OH (15)
2
2
2
2
2
•
-
-
•
OH + SCH CH OH f OH + SCH CH OH (16)
2
2
2
2
and, by analogy to eq 10, limiting rate constants of k15 ) (8.62
9
9
3
-1 -1
(
0.49) × 10 and k16 ) (2.67 ( 0.18) × 10 dm mol
s
are calculated. The predicted rate constants by using these
values in eq 10 are shown as the solid line in Figure 6a and are
seen to be in very good agreement with the experimental data.
There have been several previous determinations of the rate
constant for hydroxyl radical reaction with mercaptoethanol. By
•-
monitoring the formation of MSSM at 420 nm, a value of
9
3
-1
s-1 at pH 11 has been
(
4.0 ( 0.6) × 10 dm mol
2
6
measured; this rate constant is in reasonable agreement with
the values of this study. However, attempts to measure the rate
constant for hydroxyl radical reaction with the fully protonated
-
Figure 5. Typical fits obtained in the determination of pseudo-first-
order rate constants for hydroxyl radical reaction with mercaptoethanol
at pH 9.0. Data shown corresponds to mercaptoethanol concentrations
form of mercaptoethanol, using SCN competition kinetics (k
1
0
3
-1 -1 13
10
)
1.1 × 10 dm mol s ), gave values of 3.2 × 10 (pH
2
6 10 3 -1 -1 31
7
)
and 1.9 × 10 dm mol
s
(pH 6.5). These values
-
3
-3
-4
-3
of 2.00 × 10 (0), 1.26 × 10 (4), and 7.44 × 10 (]) mol dm
.
are much greater than those of this study and also higher than
another competition kinetics determination, using ferrocyanide
Fitted lines correspond to calculated values, using determined pH-
dependent rate constants for reactions 6 and 13 (Table 1) and hydroxyl
radical rate constants of 8.28 × 10 , 6.81 × 10 , and 4.48 × 10 s
9
3
-1 -1 31
6
6
6
-1
as a reference, which gave (6.5 ( 0.7) × 10 dm mol s .
Based on the conclusion that thiocyanate was not suitable as a
reference for mercaptoethanol, the only comparison rate constant
shown in Figure 6a at lower pHs is the ferrocyanide value, which
is seen to be slightly lower than the rate constants calculated in
this study.
,
respectively.
Cysteamine. The rate constants for cysteamine thiyl radical,
•
CyS , reaction with cysteamine (CyS), to give the cysteamine
disulfide radical anion
•
•-
CyS + CyS f CySSCy
(17)
have been previously measured by using the LINAC/LASER
1
4
technique.
reactions
From the determined limiting values for the
•
-
•-
CyS + CyS f CySSCy
(18)
•
•-
+
CyS + CySH f CySSCy + H
(19)
9
8
of k18 ) (1.21 ( 0.04) × 10 and k19 ) (3.39 ( 0.31) × 10
3
-1 -1
25
dm mol s , and the known pKa value of 8.3, individual
rate constants for the formation of this transient could be
calculated for the pH range of this study (see Table 1).
Equilibrium constants for cysteamine disulfide radical anion
formation
•
•-
CyS + Cys h CySSCy
(20)
Figure 6. (a) pH-dependent rate constants of this study, for the reaction
of hydroxyl and oxide radicals with mercaptoethanol in aqueous
solution, in comparison to previous literature data: ref 26 (4) and ref
were determined over the pH range 6.0-13.0 as for mercap-
toethanol. The concentration-dependent yields at the transient
maximum of 410 nm were used for these measurements, where
3
1 (0). Individual values are given in Table 1. Error bars are one
standard deviation obtained from second-order rate constant determina-
tions (not shown). Solid line corresponds to best fit of eq 10 with
limiting rate constants as given in text. (b) pH dependence of hydroxyl
and oxide radical reaction with cysteamine in aqueous solution (values
in Table 1). Fitted solid line corresponds to eq 28, with limiting rate
constants as given in text. (c) pH dependence for hydroxyl and oxide
radical reaction with N-acetyl-L-cysteine (see also Table 1). Fitted line
corresponds to eq 10 with values as given in text.
•
the absorbance due to the CyS radical was again seen to be
negligible. These equilibrium and rate constants were again
used to calculate the pH dependence of the equivalent first-
order disulfide anion dissociation rate constants,
•
CySSCy•- f CyS + CyS-
(21)
All these individual values are listed in Table 1. The observed
pH dependence again displays the characteristic shape seen for
mercaptoethanol and also is in qualitative agreement with a
with the disulfide. The lower pH rate constant is in very good
5
-1
agreement with the value of this study, (8.5 ( 0.7) × 10 s ;
however, at the basic pH, the value of this study, (3.23 ( 0.20)
1
2
6
-1
previous pH-dependent study performed for this sulfhydryl.
There have been several previous measurements for the k21
× 10 s , is considerably higher. Although this discrepancy
cannot be explained at this time, it is important to note that the
calculated values of this study correspond to the total rate of
loss of the disulfide radical anion under the exact experimental
5
32
rate constant, with values of ca. 8 × 10 (pH 8) and 1.3 ×
6
-1
12
1
0 s (pH 11.8), obtained from the hydrated electron reaction

8
300 J. Phys. Chem., Vol. 100, No. 20, 1996
Mezyk
conditions used to determine hydroxyl radical reaction rate
constants, and moreover, it has previously been demonstrated
that the hydroxyl radical rate constants are not very sensitive
values gave a calculated pKa of 10.27 ( 0.06, higher than the
2
5
literature value of 9.52. Using the calculated pKa value of
this study, the rate constants for the two limiting reactions
16
to the dissociation rate constants used in the fitting procedure.
•
-
•-
On the basis of the K20 equilibrium and k17/k21 rate constant
values of this study, individual cysteamine disulfide radical
anion transient absorptions were fitted as for mercaptoethanol,
to obtain the pseudo-first-order hydroxyl radical reaction rate
constants for the reaction
nCysS + nCysS f nCysSSnCys
(30)
(31)
•
•-
+
nCysS + nCysSH f nCysSSnCys + H
9
were determined as k30 ) (1.10 ( 0.03) × 10 and k31 ) (1.31
8
3
-1 -1
(
0.19) × 10 dm mol s , respectively. The k30 value of
•
•
OH + CyS f CyS
(22)
this study is in excellent agreement with a previous determi-
9
3
-1 -1 12
nation at pH 11.2, of k ) 1.1 × 10 dm mol s .
Equilibrium constants for N-acetylcysteine disulfide radical
anion formation
From the variation in cysteamine concentration, scavenging rate
constants over the pH range 7.0-12.0 were obtained, these
values are shown in Figure 6b and listed in Table 1. This plot
again shows the characteristic pH-dependence obtained for
mercaptoethanol, with decreasing rate constants at higher pHs.
For this sulfhydryl there are three species present in the pH
range of study, formed by the two ionization equilibria
•
•-
nCysS + nCysS h nCysSSnCys
(32)
were determined over the pH range 7.0-12.0 as before. These
equilibrium and rate constants were again used to calculate the
pH dependence of the equivalent first-order disulfide anion
dissociation rate constants,
+
+
-
+
HSCH CH NH h H + SCH CH NH
3
(23)
(24)
2
2
3
2
2
-
+
+
-
SCH CH NH h H + SCH CH NH
2
2
2
3
2
2
nCysSSnCys•- f nCysS + nCysS-
•
(33)
with known pKa values of 8.6 and 10.75.25 The calculated total
pH-dependent hydroxyl radical rate constants are therefore
described by the three reactions
All these individual values are listed in Table 1. The observed
pH dependence again displays the characteristic shape seen for
the other two sulfhydryls of this study.
Based on the K32 equilibrium and k29/k33 rate constant values
of this study, individual N-acetylcysteine disulfide radical anion
transient absorptions were again fitted to the general formation
mechanism, to obtain the pseudo-first-order hydroxyl radical
reaction rate constants for the reaction
•
+
•
+
3
OH + HSCH CH NH f H O + SCH CH NH
(25)
2
2
3
2
2
2
•
-
+
-
•
+
3
OH + SCH CH NH f OH + SCH CH NH (26)
2
2
3
2
2
•
-
-
•
OH + SCH CH NH f OH + SCH CH NH
2
(27)
2
2
2
2
2
with oxide radical reaction being treated as equivalent to
hydroxyl, as for mercaptoethanol. By analogy with eq 10, the
general expression for the total rate constant for this sulfhydryl
is
•
•
OH + nCysS f nCysS
(34)
The N-acetylcysteine concentration dependence of these values
gave the scavenging rate constants shown in Figure 6c and listed
in Table 1. This plot also shows the characteristic pH-
dependence obtained previously, with decreasing rate constants
at higher pHs.
As this sulfhydryl has only a single pKa value, the calculated
total pH-dependent hydroxyl radical rate constants are described
by the two limiting reactions
+
2
+
k [H ] + k K [H ] + k K K
25
26 23
27 23 24
krxn
)
(28)
+
+ 2
K K + K [H ] + [H ]
2
3
24
23
Fitting this equation to the data shown in Figure 6b, fixing K23
and K24 at their known pKa values, gives the limiting rate
9
constants k25 ) (3.66 ( 0.31) × 10 , k26 ) (3.14 ( 0.16) ×
9
9
3
-1 -1
•
-
1
0 , and k27 ) (1.29 ( 0.08) × 10 dm mol s . The
OH + HSCH CH(NHCOCH )CO f
2
2
3
predicted rate constant values using these values in eq 28 are
shown as the solid line in Figure 6b; again excellent agreement
with the experimental data is observed.
•
-
H O + SCH CH(NHCOCH )CO (35)
2
2
3
2
•
-
-
The hydroxyl radical rate constants of this study are signifi-
cantly lower than the only previous measurements for this
OH + SCH
2
CH(NHCOCH
3
)CO f
2
-
•
-
2
OH + SCH CH(NHCOCH )CO (36)
2
3
3
2
sulfhydryl, where over the pH range 6.5-9, a value of 8.5 ×
9
3
-1 -1
-
1
0 dm mol
s
was obtained by the SCN competition
again equating the hydroxyl and oxide radical reaction rate
constants under basic conditions. By fitting eq 10 to this data,
using the fitted pKa value of this study, the rate constants k35 )
kinetics method.
N-Acetyl-L-cysteine. The rate constants for N-acetylcysteine
•
thiyl radical, nCysS , reaction with N-acetylcysteine (nCysS),
9
9
3
-1
(
s
3.66 ( 0.31) × 10 and k36 ) (3.14 ( 0.16) × 10 dm mol
to give the disulfide radical anion
-1
are obtained. The predicted rate constant values by using
•
•-
these values in eq 10 are shown as the solid line in Figure 6c,
again excellent agreement with the experimental data is
observed.
The hydroxyl radical rate constants of this study are signifi-
cantly lower than the previous measurements for this sulfhydryl;
nCysS + nCysS f nCysSSnCys
(29)
were determined in this study as for mercaptoethanol. The
individual pH-dependent rate constants, calculated from the total
N-acetylcysteine, [nCysS], concentration dependence on the laser
bleach recovery, are given in Table 1. The pH-dependence of
these values follows the expected sigmoidal curve, consistent
with its single pKa value. However, the fitting of eq 10 to these
1
0
3
-1 -1
values of 1.0 × 10 dm mol
s
at pH 5.2 and 8.4 were
3
3
obtained by using pulse radiolysis techniques (unfortunately
no further experimental details were given), and a rate constant

Determination of Absolute Rate Constant
J. Phys. Chem., Vol. 100, No. 20, 1996 8301
of 1.36 × 10 dm mol s-1 at pH 7.034 was obtained by using
1
0
3
-1
•
-
-
OH + SCH CH(NHCOCH )CO
f
2
3
2
-
the SCN competition method.
-
•
-
2
OH + SCH CH(NHCOCH )CO (36)
2
3
9
Summary
being determined as k ) (1.10 ( 0.03) × 10 , k ) (1.31 (
3
0
31
8
9
0
.19) × 10 , k35 ) (3.66 ( 0.31) × 10 , and k36 ) (3.14 (
The techniques of electron pulse radiolysis and laser pho-
tolysis have been used to determine individual rate constants
for the mercaptoethanol disulfide radical anion formation
reactions
9 3 -1 -1
0
.16) × 10 dm mol s , respectively.
The calculated hydroxyl radical reaction rate constants for
these three sulfhydryls are considerably lower than previously
reported competition kinetics measurements using SCN , and
-
all exhibit the same pH dependence, with faster values at lower
pHs, in agreement with the behavior seen for cysteine and
glutathione previously.
•
HOCH CH S + HOCH CH SH f
2
2
2
2
-
+
HOCH CH S∴ SCH CH OH + H (7)
2
2
2
2
References and Notes
•
-
HOCH CH S + HOCH CH S f
2
2
2
2
(
1) Bacq, Z. M. In Chemical Protection against Ionizing Radiation;
Thomas, C. C., Ed.; Springfield, IL, 1965.
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-Centered
-
HOCH CH S∴ SCH CH OH (8)
2
2
2
2
(
9
9
3
as k7 ) (1.22 ( 0.08) × 10 and k8 ) (4.85 ( 0.19) × 10 dm
(
-1 -1
mol s , respectively. The values have been combined with
pH-dependent equilibrium constant measurements, determined
from mercaptoethanol disulfide radical anion transient intensi-
ties, to allow computation of rate constants for the hydroxyl
radical reactions
ReactiVe Intermediates in Chemistry and Biology; Chatgilialoglu, C., Asmus,
K.-D., Eds.; NATO-ASI Series A; Life Sciences; Plenum-Press: New York,
1
990; Vol. 197, p 401.
(
4) Mason, R. P.; Maples, K. R. In Sulphur-Centered ReactiVe
Intermediates in Chemistry and Biology; Chatgilialoglu, C., Asmus, K.-D.,
Eds.; NATO-ASI Series A; Life Sciences; Plenum-Press: New York, 1990;
Vol. 197, p 429.
(
5) Von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor
and Francis: London, 1987; p 353.
6) Forni, L. G.; M o¨ nig, J.; Mora-Arellano, V. O.; Willson, R. L. J.
•
•
OH + HOCH CH SH f H O + HOCH CH S (15)
2
2
2
2
2
(
•
-
-
•
Chem. Soc., Perkin Trans. 2 1983, 961.
OH + HOCH CH S f OH + HOCH CH S (16)
2
2
2
2
(7) Forni, L. G.; Willson, R. L. Biochem. J. 1986, 240, 897.
(8) Forni, L. G.; Willson, R. L. Biochem. J. 1986, 240, 905.
9
9
(9) Erben-Russ, M.; Michel, C.; Bors, W.; Saran, M. J. Phys. Chem.
987, 91, 2362.
as k15 ) (8.62 ( 0.49) × 10 and k16 ) (2.67 ( 0.18) × 10
1
3
-1 -1
dm mol s .
(
10) Tieu, M.; Bucher, J. R.; Aust, S. D. Biochem. Biophys. Res.
Similar measurements were also performed for cysteamine,
where from literature rate constants for the disulfide radical
anion formation and measured equilibrium constants, limiting
hydroxyl radical reaction rate constants for the three separate
species,
Commun. 1982, 107, 279.
(11) Searle, A. J. F.; Willson, R. L. Biochem. J. 1983, 212, 549.
(12) Hoffman, M. Z.; Hayon, E. J. Phys. Chem. 1973, 77, 990.
(13) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J.
Phys. Chem. Ref. Data 1988, 17, 513 and references therein.
(14) Mezyk, S. P. Chem. Phys. Lett. 1995, 235, 89.
(
(
15) Mezyk, S. P. Radiat. Res. 1996, 145, 102.
16) Mezyk, S. P. J. Phys. Chem., in press.
•
+
•
+
3
OH + HSCH CH NH f H O + SCH CH NH
(25)
2
2
3
2
2
2
(17) Liphard, M.; Bothe, E.; Schulte-Frohlinde, D. Int. J. Radiat. Biol.
990, 58, 589.
1
(
18) Eriksen, T. E.; Fransson, G. J. Chem. Soc., Perkin Trans. 2 1988,
•
-
+
-
•
+
3
OH + SCH CH NH f OH + SCH CH NH (26)
1117.
2
2
3
2
2
(19) Quintiliani, M.; Badiello, R.; Tamba, M. Int. J. Radiat. Biol. 1977,
3
2, 195.
•
-
-
•
OH + SCH CH NH f OH + SCH CH NH
2
(27)
(20) Simic, M.; Neta, P.; Hayon, E. Int. J. Radiat. Phys. Chem. 1971,
, 309.
2
2
2
2
2
3
(
21) Janata, E.; Schuler, R. H. J. Phys. Chem. 1982, 86, 2078.
9
were calculated as k25 ) (3.66 ( 0.31) × 10 , k26 ) (3.14 (
(22) Ebbesen, T. W. Radiat. Phys. Chem. 1989, 34, 619.
(23) Natarajan, P.; Fessenden, R. W. J. Phys. Chem. 1989, 93, 6095.
9
9
3
-1 -1
0
.16) × 10 , and k27 ) (1.29 ( 0.08) × 10 dm mol s .
(
24) Buxton, G. V.; Stuart, C. R. J. Chem. Soc., Faraday Trans. 1995,
1, 279.
25) Fasman, G. D., Ed. Handbook of Biochemistry and Molecular
These measurements were also repeated for N-acetyl-L-
9
cysteine, with limiting rate constants for the reactions
(
Biology, Physical and Chemical Data, 3rd ed.; CRC Press Inc.: Boston,
1976; Vol. 1.
•
•
•
-
SCH CH(NHCOCH )CO +
2
2
3
(
26) Karmann, W.; Granzow, A.; Meissner, G.; Henglein, A. Int. J.
Radiat. Phys. Chem. 1969, 1, 395.
27) Baxendale, J. H.; Bevan, P. L. T.; Stott, D. A. Trans. Faraday
Soc. 1968, 64, 2389.
-
-
SCH CH(NHCOCH )CO f
2
2
3
(
-
-
O C(CH CONH)CHCH S∴ SCH CH(NHCOCH )CO
2
3
2
2
3
2
(28) Baxendale, J. H.; Bevan, P. L. T. J. Chem. Soc. A 1969, 2240.
(29) Hoffman, M. Z.; Hayon, E. J. Am. Chem. Soc. 1972, 94, 7950.
(30) Curtis, A. R.; Sweetenham, W. P. FACSIMILE/CHECKMAT Users
(
30)
-
Manual; Harwell Research Report AERE-R 12805; 1988.
31) Jayson, G. G.; Stirling, D. A.; Swallow, A. J. Int. J. Radiat. Biol.
1971, 19, 143.
SCH CH(NHCOCH )CO
+
2
3
2
(
-
+
HSCH CH(NHCOCH )CO f H +
2
3
2
(32) Adams, G. E.; McNaughton, G. S.; Michael, B. D. In Chemistry
-
-
2
O C(CH CONH)CHCH S∴ SCH CH(NHCOCH )CO
of Ionization and Excitation; Johnson, G. R. A., Scholes, G., Eds.; Taylor
and Francis: London, 1967; p 281.
2
3
2
2
3
(
31)
(33) Rougee, M.; Bensasson, R. V.; Land, E. J.; Pariente, R. Photochem.
Photobiol. 1988, 47, 485.
(34) Aruoma, O. I.; Halliwell, B.; Hoey, B. M.; Butler, J. Free Rad.
Biol. Med. 1989, 6, 593.
-
OH + HSCH CH(NHCOCH )CO f
2
2
3
•
-
2
H O + SCH CH(NHCOCH )CO (35)
2
2
3
JP953067V