Carbon Dioxide Stimulates the Production of Thiyl, Sulfinyl, and
Disulfide Radical Anion from Thiol Oxidation by Peroxynitrite*
Marcelo G.
Bonini and
Ohara
Augusto
From the Departamento de Bioquímica, Instituto de
Química, Universidade de São Paulo, CP 26077, CEP
05513-970, São Paulo, SP, Brazil
Received for publication, September 15, 2000, and in revised form, December 20, 2000
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ABSTRACT |
Reaction of peroxynitrite with the
biological ubiquitous CO2 produces about 35% yields
of two relatively strong one-electron oxidants, CO
3
and ·NO2, but the remaining of peroxynitrite
is isomerized to the innocuous nitrate. Partial oxidant deactivation
may confound interpretation of the effects of
HCO
/CO2 on the oxidation of targets that react with peroxynitrite by both one- and two-electron mechanisms. Thiols are example of such targets, and previous studies have reported that
HCO
/CO2 partially inhibits GSH oxidation by peroxynitrite at pH 7.4. To differentiate the
effects of HCO
/CO2 on
two- and one-electron thiol oxidation, we monitored GSH, cysteine, and albumin oxidation by peroxynitrite at pH 5.4 and 7.4 by thiol disappearance, oxygen consumption, fast flow EPR, and EPR spin trapping. Our results demonstrate that
HCO
/CO2 diverts thiol
oxidation by peroxynitrite from two- to one-electron mechanisms
particularly at neutral pH. At acid pH values, thiol oxidation to free
radicals predominates even in the absence of HCO
/CO2. In addition to
the previously characterized thiyl radicals (RS·), we also
characterized radicals derived from them such as the corresponding
sulfinyl (RSO·) and disulfide anion radical
(RSSR·
) of both GSH and cysteine. Thiyl,
RSO· and RSSR·
are reactive radicals that
may contribute to the biodamaging and bioregulatory actions of
peroxynitrite.
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INTRODUCTION |
Peroxynitrite1
(ONOO
+ ONOOH), which is formed by the fast reaction
between ·NO and O
2, has been receiving increasing
attention as a mediator of human diseases and as a toxin against
invading microorganisms (1-5). The compound is a potent oxidant that
is able to oxidize and nitrate a variety of biological targets by
mechanisms that are presently being elucidated (6-16). In general
terms, peroxynitrite-mediated oxidations are either bimolecular, first
order on peroxynitrite and biomolecule concentration, or unimolecular,
first order on peroxynitrite and independent of biomolecule
concentration. Bimolecular processes can result in product yield either
around stoichiometry and over, as is the case for thiol oxidation (17),
or around 35% , as is the case for carbon dioxide (CO2)
oxidation (10, 14-16). Presently, most investigators accept that
product yields around 30% are characteristic of peroxynitrite-mediated
free radical processes. Indeed, it has been established that
ONOO
protonation (pKa = 6.6) leads to
its fast decomposition (k = 0.17 s
1 at pH 7.4) to yield approximately 70%
nitrate and 30% hydroxyl radical (·OH) and nitrogen dioxide
(·NO2) (Fig. 1, path
1) (7-9, 11-14). These radicals are the
species responsible for peroxynitrite-mediated unimolecular oxidations. In the presence of HCO
,
ONOO
decomposes much faster because of its
reaction with CO2 (k = 2.6 × 104 M
1
s
1 at pH 7.4, 25 °C) (18, 19) to produce
approximately 65% nitrate and 35% carbonate radical anion
(CO
3) and ·NO2 (Fig. 1, path 2) (10,
14-16). The fast rate constant of this reaction and the ubiquity of
the HCO
3/CO2 pair in biological environments indicate
that CO
3 and ·NO2 are likely to play
relevant roles in peroxynitrite-mediated oxidations in
vivo.

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Fig. 1.
Schematic representation of the possible
competing paths for thiol oxidation by peroxynitrite in the presence
and in the absence of CO2. The shown rate constant
values are those at 25 °C.
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Recent detailed studies have established the role of CO
3
in the potentiating effects of
HCO
/CO2 on
peroxynitrite-mediated tyrosine nitration (20-23). This process has a
zero order dependence on the target, being exclusively dependent on
free radicals produced from peroxynitrite decay (20). The higher fluxes
of free radicals produced in the presence of
HCO
/CO2 and the more
specific reactivity of the CO
3 compared with the ·OH radical toward tyrosine result in higher yields of the
tyrosyl radical and its recombination products with
·NO2 (nitrotyrosine) and itself (dityrosine)
(21-23). The effects of
HCO
/CO2 on the oxidation
of targets that compete with CO2 for the oxidant by
reacting with peroxynitrite through second order processes have yet to
be elucidated.
Among these targets, thiols are particularly relevant because they are
present in high concentrations in biological environments and react
relatively fast with peroxynitrite (k = 6.6 × 102 M
1
s
1 for GSH at pH 7.4, 25 °C) (Fig. 1, path
3) (17, 24-26). Additionally, GSH is an important antioxidant, and
thiol groups play a major role in maintaining the native conformation
of proteins and in regulating enzyme activity. Previous studies have
shown that HCO
/CO2 partially inhibited GSH and albumin thiol oxidation at pH 7.4, as
monitored by thiol disappearance (19, 26). However, this parameter does
not differentiate between two- and one-electron mechanisms, and thiyl
radicals (RS·) have been shown to be produced during the
oxidation of thiols by peroxynitrite (6, 25, 27-29). In this context,
it can be hypothesized that
HCO
/CO2 is likely to
inhibit thiol oxidation by two-electron mechanisms while stimulating their oxidation to free radicals (Fig. 1). To test this hypothesis, we
studied the effects of
HCO
/CO2 on thiol
disappearance and free radical production during the oxidation of GSH,
cysteine, and albumin by peroxynitrite.
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EXPERIMENTAL PROCEDURES |
Materials--
All reagents were purchased from Sigma, Merck, or
Fisher and were analytical grade or better. Peroxynitrite was
synthesized from 0.6 M sodium nitrite and 0.65 M hydrogen peroxide in a quenched flow reactor; excess
hydrogen peroxide was used to minimize nitrite contamination. To
eliminate excess hydrogen peroxide, the peroxynitrite solution was
treated with manganese dioxide. Synthesized peroxynitrite contained low
levels of contaminating hydrogen peroxide (<1%) and nitrite
(10-30%) which were determined as described previously (29) by the
titanyl method and by absorbance measurements at 354 nm (
= 24.6 M
1.
cm
1), respectively. The concentration of
peroxynitrite stock solutions was determined spectrophotometrically at
302 nm using an extinction coefficient of 1.670 M
1. cm
1
(30). The thiol group of bovine serum albumin (fraction V) was blocked
by reaction with N-ethylmaleimide as described previously (27). Concentrations of CO2 were calculated from the added
HCO
concentrations by using
pKa = 6.4 (15). Buffers were pretreated with
Chelex-100 to remove contaminant metal ions. All solutions were
prepared with distilled water purified with a Millipore Milli-Q system.
Thiol Oxidation--
Total thiol oxidation was measured after a
20-min incubation at 25 ± 2 °C by spectrophotometric
quantitation of sulfhydryls using the 5,5'-dithiobis-2-nitrobenzoic
acid assay as described previously (25, 26).
Oxygen Consumption--
Oxygen uptake studies were performed
using an oxygen monitor (Gilson 5/6 oxygraphy) at 25 °C ± 1 °C. The saturation oxygen concentration at this temperature was
taken as 250 µM.
EPR Experiments--
The EPR fast flow spectra were recorded at
room temperature (25 ± 2 °C) on a Bruker EMX spectrometer
operating at 9.65 GHz and 100 KHz field modulation equipped with a
Bruker ER4117 D-MTV dielectric mixing resonator with a 9-mm distance
between the mixing cell and the resonator center. Thiol solutions were
pre-prepared in appropriated buffers to which sodium bicarbonate was
added or not; in the former case, the solutions were left undisturbed for 5 min to permit
HCO
/CO2 equilibration. Peroxynitrite solutions were prepared with water. Thiol and
peroxynitrite solutions were transferred to 60-ml plastic syringes
mounted on a syringe infusion pump (Harvard apparatus pump 22). Spectra
were recorded 3.5 and 12 ms after mixing at continuous flow of 20 ml/min and 6 ml/min, respectively. The dispensed mixtures were
collected for pH measurement at the end of the experiments to detect
changes caused by mixing with alkaline solutions of peroxynitrite. The magnetic field was calibrated with
4-hydroxy-2,2,6,6,-tetramethyl-1-piperidinyloxy (g = 2.0056) (15). Computer simulation of spectra was performed using a
program written by Duling (31). In the EPR spin trapping experiments,
the incubation mixtures were transferred to a flat cell and the spectra
recorded at room temperature 1 min after the addition of
peroxynitrite.
Calculations--
The concentrations of expected products during
the oxidation of GSH and cysteine by peroxynitrite were estimated by
calculating the percentage of the oxidant that decomposes to
NO
, ·OH, and
·NO2 (Fig. 1, path 1; k), reacts with
thiols (Fig. 1, path 3; kRSH), and reacts with
CO2 producing CO2,
NO
, CO
3, and
·NO2 (Fig. 1, path 2;
kCO2) under our experimental conditions
(1 mM RSH, 1 mm CO2, and 0.5 mM peroxynitrite at 25 °C) by Equation 1 (32).
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(Eq. 1)
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The rate constants were k = 1.1 and 0.17 s
1 (11, 21), kGSH = 1.4 102 and 6.6 × 102
M
1 s
1,
kCys = 6.4 × 102 and 2.0 × 103 M
1
s
1 (24, 25), and
kCO2 = 1.8 × 103 and 2.6 × 104
M
1 s
1
(18) at pH 5.4 and 7.4, respectively. The calculated ONOO
concentration that decays through path 3 (Fig. 1) was taken as the
expected RSOH concentration. Because paths 1 and 2 (Fig. 1) produce
radical and nonradical products, the calculated ONOO
concentrations were multiplied by 0.3 and 0.35 to estimate the produced
·OH and ·NO2 and CO
3 and
·NO2, respectively (Table
I).
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Table I
Comparison of the experimental yields of depleted thiol with calculated
yields of products formed during the oxidation of 1 mM GSH
or 1 mM cysteine by 0.5 mM peroxynitrite in the
presence and in the absence of 1 mM carbon dioxide at
25 °C
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RESULTS |
Oxidation of GSH and Cysteine--
To evaluate the effects of
HCO
/CO2 on
peroxynitrite-mediated oxidation of thiols, we monitored cysteine and
GSH oxidation at pH 5.4 and 7.4 by both thiol disappearance (Table I)
and oxygen consumption (Fig. 2). Oxygen
uptake provides a nonspecific measurement of radicals that react fast
with oxygen such as RS· (33, 34). The results show that
HCO
/CO2 inhibited GSH and
cysteine thiol disappearance at pH 7.4 (Table I) but in parallel
increased the amount of consumed oxygen (Fig. 2). At pH 5.4, HCO
/CO2 had marginal effects on both thiol depletion (Table I) and oxygen consumption (Fig.
2). These results indicate that
HCO
/CO2 diverts
peroxynitrite-mediated thiol oxidation from two- to one-electron mechanisms as initially hypothesized (Fig. 1). At pH 5.4 the
CO2 effects were marginal because at this pH most of the
thiol oxidation occurs by free radical mechanisms even in the absence
of HCO
/CO2 . These
conclusions are well supported by comparison of the experimental yields
of depleted thiol with the yield of products, radicals (·OH/·NO2;
CO
3/·NO2), and thiol oxidized by two
electrons (RSOH), which can be estimated from the known rate constants
of the main competing reactions occurring under the experimental
conditions used (Fig. 1, paths 1-3) (see "Experimental Procedures"
and Table I). The data presented in Table I show that GSH oxidation at
pH 5.4 occurs through the radicals produced from peroxynitrite in the
presence or absence of
HCO
/CO2 . The produced radicals are different, but they are expected to be formed in similar
yields (Table I), and all of them
(·OH/·NO2/CO
3) react quickly
with GSH to produce GS· (Table
II). Relevantly, the experimental values
of depleted thiol (0.30 and 0.26 mM) were the same as the
calculated total radical yields in the presence (0.10 + 0.20 mM) and absence (0.26 mM) of
HCO
/CO2 , respectively
(Table I). Compared with GSH, a larger fraction of cysteine is oxidized
by two-electron mechanisms at pH 5.4 in the absence of
HCO
/CO2 because of the
higher second order rate constant of its reaction with peroxynitrite
(24, 25). In this case,
HCO
/CO2 inhibits total
thiol disappearance to some extent, but the oxidized fraction should
result from the produced radicals whose expected total yield is, again,
similar to the measured depleted thiol (Table I). At pH 7.4 both thiols
are oxidized mainly by two-electron mechanisms in the absence of
HCO
/CO2 . In this case,
the experimental value of depleted thiol is considerably higher than
the calculated one, confirming that the direct reaction between
peroxynitrite and RSH consumes more than one thiol (Fig. 1, path 3)
(17). At pH 7.4 in the presence of
HCO
/CO2 , total depleted
thiol decreased, but most of it should result from free radical
mechanisms because of the similar concentration values of produced
radicals and depleted thiols (Table I). In agreement, consumed oxygen
increased in the presence of
HCO
/CO2 , particularly in
the case of GSH (Fig. 2).

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Fig. 2.
Effects of
HCO /CO2 on oxygen
consumption during the oxidation of 1 mM GSH
(A) and 1 mM cysteine (B)
by 0.5 mM peroxynitrite at 25 °C at pH 7.4 (full
bars) and pH 5.4 (open bars). The
reactions were started by the addition of peroxynitrite, which
triggered an extremely fast consumption of oxygen. Concentrations of
CO2 were calculated from the added
HCO by using pKa = 6.4.
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Table II
Reactions shown in the text for a general thiol (RSH) are listed with
their corresponding available rate constants for GSH and cysteine
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Although the yield of depleted thiol by free radical mechanisms was
roughly similar to total radical yields (Table I), it is not possible
to infer that RS· are decaying mainly by reaction with
themselves (Reaction 1) because a fast consumption of oxygen was
associated with thiol oxidation by peroxynitrite under all experimental
conditions tested (Fig. 2) (see also Ref. 25). Because oxygen
consumption was lower than oxidized thiol (Fig. 2 and Table I), it is
likely that RS· is decaying by at least three competing routes,
i.e. dimerization, reaction with oxygen, and reaction with
excess thiolate (RS
) (Reactions 1-R3).
Other decay routes also occur as indicated by the detection of low
levels of GSNO2 (41) and GSNO (42) in incubations of
GSH with peroxynitrite. The importance of Reactions 2 and 3 arises from
the production of sulfonyl (RSOO·) and disulfide radical anions
(RSSR·
), respectively (Table II). In previous
studies of thiol oxidation by peroxynitrite, we detected RS·
radicals by EPR spin trapping and obtained indirect evidence for their
conversion to both RSOO· and RSSR·
(25).
Here, we present direct EPR evidence for the formation of these species
and for the stimulatory effects of
HCO
/CO2 on their yields.
EPR Detection of Thiyl, Sulfinyl, and Disulfide Radical
Anion--
We have demonstrated previously that fast flow EPR of
concentrated solutions of peroxynitrite and CO2 produces
detectable concentrations of CO
3 (15). In the presence of
5 mM cysteine (Fig. 3) or 5 mM GSH (Fig. 4), the one-line
EPR signal of CO
3 produced from 5 mM
peroxynitrite and 5 mM CO2 became barely
detectable (indicated by
in Figs. 3 and 4) and new EPR signals
appeared, particularly at pH 7.4. Cysteine oxidation produced a
three-line signal (a2H = 9.3 G; line width = 3.5 G; g = 2.0107) (Fig. 3) which has been
characterized previously as the corresponding sulfinyl radical
(CysSO·) by fast flow EPR studies of cysteine oxidation by
Ti(III)-H2O2 (43). GSH oxidation produced a
four-line signal (aH = 7.1 G and
aH = 10.7 G; line width = 2.6 G;
g = 2.0109) whose EPR parameters are also consistent
with the corresponding sulfinyl radical (GSO·) (Fig. 4). Both
CysSO· and GSO· have two
-methylene hydrogens, but in
the latter, a hindered rotation of the methylene group that is adjacent
to a chiral center is likely to result in the magnetic nonequivalence
(44, 45) reflected in the different hyperfine splitting constants
(aH) obtained for its two hydrogens (Fig.
4B).

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Fig. 3.
EPR continuous flow spectra of
cysteine-derived radicals produced from mixing 5 mM
peroxynitrite and 5 mM cysteine with or without 5 mM CO2. A, in 0.3 M
phosphate buffer, pH 7.4; B, in 0.3 M phosphate
buffer, pH 7.4, equilibrated with 5 mM CO2;
C, in 0.3 M acetate buffer, pH 5.4;
D, in 0.3 M acetate buffer, pH 5.4, equilibrated
with 5 mM CO2; E, same as
B but in the presence of 20 mM DMPO. Detectable
traces of CO 3 are indicated by in B. The
spectrum in E was displaced to the left because
of the marked differences in the g values of CysSO·
and DMPO/·SCys. The specified concentrations are those in the
final reaction mixture. The flow rate was 20 ml/min. Instrumental
conditions: microwave power, 2 mW; time constant, 81.9 ms; scan rate,
0.6 G/s; modulation amplitude, 2 G; gain, 8.93 × 105
except for E, where 1.78 × 105 was
used.
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Fig. 4.
EPR continuous flow spectra of GSH-derived
radicals produced from mixing 5 mM peroxynitrite and 5 mM GSH with or without 5 mM
CO2. A, in 0.3 M
phosphate buffer, pH 7.4; B, in 0.3 M phosphate
buffer, pH 7.4, equilibrated with 5 mM CO2;
C, in 0.3 M acetate buffer, pH 5.4;
D, in 0.3 M acetate buffer, pH 5.4, equilibrated
with 5 mM CO2; E, same as
B but in the presence of 20 mM DMPO. Detectable
traces of CO 3 are indicated by in B. The
spectrum in E was displaced to the left because
of the marked differences in the g values of GSO· and
DMPO/·SG. The specified concentrations are those in the final
reaction mixture. The flow rate was 20 ml/min. Instrumental conditions:
microwave power, 2 mW; time constant, 81.9 ms; scan rate, 0.6 G/s;
modulation amplitude, 2 G; gain, 1.78 × 106 except
for E, where 3.56 × 105 was used.
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Direct EPR detection of RSO· during peroxynitrite-mediated
oxidation of thiols is consistent with the initial formation of RS· that react with oxygen to produce RSOO· which, in
turn, as metastable intermediates, yield RSO· (Reaction 2) (46).
Thiyl radicals cannot be detected by direct EPR in aqueous solutions at
room temperature because of the large anisotropy in their g
tensors which broadens the EPR signal beyond detection (43). They are
detectable in aqueous solutions by EPR spin trapping. Indeed, addition
of the spin trap DMPO to the fast flow mixtures containing the thiols,
HCO
/CO2 and peroxynitrite led to the
substitution of the RSO· spectra by those characteristic of
DMPO/·SCys (aN = 15.2 G;
aH = 17.4 G) (Fig. 3E) and
DMPO/·SG (aN = 14.9 G;
aH = 15.6 G) (Fig. 4E) radical
adducts (47), confirming that RS· are the RSO· precursors
(Reaction 2).
The effects of HCO
/CO2 in
increasing the concentrations of RSO· radicals detected from
cysteine and GSH oxidation by peroxynitrite are shown in Figs. 3 and 4.
In agreement with the conclusion that thiols are oxidized mainly by
two-electron mechanisms at pH 7.4 in the absence of
HCO
/CO2 (Fig. 2 and Table
I), EPR signals were not detectable under these conditions. Although
the calculated total yields of peroxynitrite-derived radicals at pH 7.4 in the presence of
HCO
/CO2 were similar to
those at pH 5.4 in any condition (Table I), the detectable radical
concentrations varied considerably (Figs. 3, B-D, and 4,
B-D). This is because EPR flow experiments detect instantaneous radical concentrations whose values depend not only on
radical yields but also on the observation time and rates of radical
formation and decay. Rates of radical formation become more important
at the short observation time used to detect RSO· (3.5 ms) (48).
Consequently, the higher RSO· concentrations detected in the
presence of HCO
/CO2 at pH
7.4 than at pH 5.4 (Figs. 3, B and D, and 4,
B and D) are likely to be due to the usually
higher rate constants of thiol oxidation at alkaline pH values (25, 36,
49).
Detection of RSO· was possible in incubations containing
equimolar concentrations of peroxynitrite and RSH (Figs. 3 and 4). Increases in thiol molar ratios led to composite EPR spectra whose partial characterization was possible with a 5 molar excess thiol. Such
excess, low oxygen tensions, and the presence of
HCO
/CO2 led to the
detection of EPR spectra dominated by the spectra of GSSG·
(Fig.
5B) or
CysSSCys·
(Fig. 5D). These spectra have
been characterized previously in flow mixtures of horseradish
peroxidase/H2O2/acetaminophen and the
corresponding thiols (50). The spectra shown in Fig. 5 were obtained
with four times lower concentrations of thiols and consequently were
less resolved than those published previously (50). Computer simulation
(noiseless lines in Fig. 5) of the experimental spectra obtained in mixtures of excess thiol, peroxynitrite, and
HCO
/CO2 confirmed that
GSSG·
(a2H = 6.9 G;
a2H = 7.0 G) and CysSSCys·
(a2H = 6.6 G; a2H = 7.7 G) are the predominant species produced under these conditions. Again,
RSSR·
attained detectable concentrations only in
the presence of HCO
/CO2 (Fig. 5).

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Fig. 5.
EPR continuous flow spectra of
GSSG· and
CysSSCys· radical anions produced from mixing 5 mM peroxynitrite and 25 mM GSH or cysteine with
or without 5 mM CO2 at pH 7.4. A, GSH; B, GSH in the presence of
CO2; C, cysteine; D, cysteine in the
presence of CO2. The specified concentrations are those in
the final incubation mixture. The flow rate was 6 ml/min. Instrumental
conditions: microwave power, 2 mW, time constant, 81.9 ms; scan rate,
0.6 G/s; modulation amplitude, 2 G; gain, 1.78 × 106.
The noiseless lines correspond to computer simulation of the
GSSG· (a2H = 6.9 G;
a2H = 7.0 G) and CysSSCys·
(a2H = 6.6 G; a2H = 7.7 G) spectra.
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Oxidation of Albumin Thiol--
The main target of peroxynitrite
in albumin is its free thiol group (17, 51). It has been reported that
HCO
/CO2 partially
inhibits albumin thiol oxidation by peroxynitrite (19). This result was
confirmed under our experimental conditions where 0.5 mM
peroxynitrite at pH 7.4 depleted 0.68 ± 0.02 mM and
0.56 ± 0.02 mM albumin thiols in the absence and in
the presence of 1 mM CO2, respectively.
Although it inhibited total thiol depletion, 1 mM
CO2 increased about two times the yield of albumin thiyl radical that was trapped by POBN (Fig. 6,
A and B). All POBN/·protein radical
adducts have similar EPR spectra, a broad triplet characteristic of
high molecular weight nitroxides (52). Thus, to prove that
albumin-thiyl is the main radical produced under the experimental
conditions employed, parallel experiments were performed with albumin
pretreated with N-ethylmaleimide. EPR signals were barely
detectable from albumin whose thiol group was previously blocked with
N-ethylmaleimide (Fig. 6, C and D).
Under conditions of high molar excess of peroxynitrite over albumin
thiol, other protein-derived radicals are produced and detected (53),
as expected from the reactivity of peroxynitrite-derived radicals.

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Fig. 6.
EPR spectra of the POBN/·S-albumin
radical adduct obtained from a 1-min incubation of 1 mM
albumin with 25 mM POBN and 0.5 mM
peroxynitrite at pH 7.4. A, in the absence of added
CO2; B, in the presence of 1 mM
CO2; C, the same as A but with
albumin whose thiol group was blocked; D, the same as
B but with albumin whose thiol group was blocked.
Instrumental conditions: microwave power, 20 mW, time constant, 81.9 ms; scan rate, 0.6 G/s; modulation amplitude, 1G; gain, 8.93 × 105.
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DISCUSSION |
Presently, most investigators agree that CO2 is likely
to be the major sink of peroxynitrite in most physiological
environments (2, 5, 10). Carbon dioxide deactivates about 65% of
peroxynitrite because it catalyzes isomerization of the oxidant to the
innocuous nitrate, although the remaining products (about 35%) are
CO
3 and ·NO2 (Fig. 1, path 2). The
high rate constant of the reaction between ONOO
and
CO2 and the resulting deactivation of 65% of the oxidant may lead to the conclusion that
HCO
/CO2 inhibits the
oxidation of targets that react with peroxynitrite directly, competing
with CO2 for the oxidant. Thiols are examples of such
targets (Fig. 1, path 3) (17, 24-26). Here we demonstrate that
although HCO
/CO2 inhibits
the total yield of GSH and cysteine oxidized by peroxynitrite at pH 7.4 (19, 26), the oxidized fraction produces free radicals (Figs. 1-6 and
Table I). Carbon dioxide inhibits thiol oxidation because it
outcompetes the thiols for the direct reaction with peroxynitrite. As a
consequence, thiols are oxidized to RS· by CO
3 and
·NO2 that escape solvent cage (Fig. 1 and Table I).
Subsequent reactions of RS· (Reactions 1-R3) produce the
detectable RSO· (Figs. 3 and 4) and RSSR·
(Fig. 5). Undoubtedly, the effects of
HCO
/CO2 in diverting
peroxynitrite reactivity from two- to one-electron mechanisms may apply
to other important biological targets that react directly with the
oxidant such as hemoproteins (54, 55). The demonstration that at acid
pH values peroxynitrite acts as an one-electron thiol oxidant in the
presence and in the absence of
HCO
/CO2 (Figs. 2-4) was
also relevant. Acid pH values are important for phagocyte function and
ischemic tissue pathology but have been overlooked in studies
addressing peroxynitrite reactivity in biological environments. Because
the three radicals that can be produced from peroxynitrite are strong (·OH, E = 2.3 V (24); and CO
3,
E = 1.8 V (15,
56-58)2or moderately strong
(·NO2, E = 0.99 V) (24) one-electron
oxidants, they are likely to mediate some of the biological damage
attributed to peroxynitrite such as nitration of protein tyrosine
residues (2, 5, 20-23) and protein thiol oxidation (27, 59, 60).
Formation of RS· radicals during the oxidation of low and high
molecular weight thiols by peroxynitrite has been reported in the
literature by our group and others (6, 25, 27-29). However, this
report provides the first direct EPR detection of RSO· (Figs. 3
and 4) and RSSR·
(Fig. 5) formed from the
initially produced RS· (Reactions 1-R3). All low molecular
weight radicals, GSO·, GSSG·
,
CysSO·, and CysSSCys·
were unambiguously
characterized by the parameters of their corresponding EPR spectra (see
"Results"). Moreover, it was shown that the presence of
HCO
/CO2 stimulated the
production of RS· and of all the radicals derived from them
(Figs. 3-6). Among the identified radicals those that may have greater
physiological consequences are GSSG·
(Fig.
5B) and protein-S· (Fig. 6). Formation of
protein-S· and protein-SO· may alter protein structure
and function (61, 62), and relevantly, protein-thiol oxidation is a
recurrent event in cell signaling cascades (63). On the other hand,
because GSSG·
is produced under conditions of
excess GSH over peroxynitrite, it is likely to be the prevalent radical
produced from peroxynitrite attack on thiols in aqueous intracellular
environments where oxidant concentration is expected to be much lower
than the GSH and HCO
/CO2 concentration. The GSSG·
radical reacts fast with
oxygen, producing superoxide anion (Table II), which can further react
with thiols, although not particularly fast, to produce RS·
(64). Consequently, intracellular GSH oxidation by
peroxynitrite-derived radicals may trigger an
oxygen-dependent free radical chain reaction, as was shown
to occur in vitro (25). This possibility argues against GSH
being particularly effective in directly counteracting the oxidant
properties of peroxynitrite-derived radicals. In contrast, an
antioxidant such as ascorbate which is oxidized to a radical that
reacts very slowly with oxygen is expected to be more effective. Moreover, ascorbate reacts faster with CO
3 than GSH
(65).Consequently, in aqueous environments, ascorbate may be more
important than GSH in counteracting the oxidant action of free
radicals. This has been shown to occur in the case of the
acetaminophen-derived radical (50) and more recently, in the case of
peroxynitrite-derived radicals (65).
In conclusion, our results demonstrate that
HCO
/CO2 diverts low and
high molecular weight thiol oxidation by peroxynitrite from two- to
one-electron mechanisms particularly at neutral pH. At acid pH values,
thiol oxidation to free radicals predominates in the presence and
absence of HCO
/CO2 . The
produced thiol-derived radicals were identified as RS·,
RSO·, and RSSR·
. These are reactive
radicals that may contribute to the biodamaging and bioregulatory
actions of peroxynitrite.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Ronald P. Mason, Rafael Radi,
Sergei V. Lymar, and Roger Bisby for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by grants from Fundação
de Amparo à Pesquisa do Estado de São Paulo, Conselho
Nacional de Desenvolvimento Científico e Tecnológico, and
Financiadora de Estudos e Projetos.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Depto. de
Bioquímica, Instituto de Química, Universidade de
São Paulo, Caixa Postal 26077, 05513 970, São Paulo, SP,
Brazil. Tel.: 55 11 3818 3873; Fax: 55 11 3818 2186 and
55 11 3815 5579; E-mail: oaugusto@iq.usp.br.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M008456200
2
The reduction potential of CO
3 has
usually been assumed to be 1.6 V. The determined value was 1.58 ± 0.02 V(56, 58) and corresponds to the pair
CO
3/CO
. At pH 7.0, the relevant pair is
CO
3C,H+/HCO
(pKa of HCO
= 10.32) whose reduction potential can be calculated as 1.78 V (ECO
3,H+/HCO
=
ECO
3/HCO
+ 0.059 × (pKa
pH).
 |
ABBREVIATIONS |
The abbreviations used are:
peroxynitrite, the
sum of peroxynitrite anion (ONOO
, oxoperoxonitrate
(
1)), and peroxynitrous acid (ONOOH, hydrogen oxoperoxonitrate)
unless specified;
HCO
/CO2, the sum of
HCO
and CO2 unless
specified;
DMPO, 5,5-dimethyl-1-pyrroline-N-oxide;
POBN,
-(4-pyridyl-1-oxide)-N-t-butylnitrone;
RS·, thiyl radical(s);
RSO·, sulfinyl radical(s);
RSOH, sulfenic acid(s);
RSOO·, sulfonyl radical(s);
RSSR·
, disulfide radical anion.
 |
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