Formation of peroxynitrate from the reaction of peroxynitrite with CO2: Evidence for carbonate radical production

Article abstract of DOI:10.1021/ja9733043

The decomposition of peroxynitrite in the presence of bicarbonate and various substrates was studied using the stopped-flow technique. We have shown that peroxynitrite (ONOOH/ONOO^-) reacts with excess of CO₂ to form peroxynitrate (O<

Full text of DOI:10.1021/ja9733043

3458
J. Am. Chem. Soc. 1998, 120, 3458-3463
Formation of Peroxynitrate from the Reaction of Peroxynitrite with
CO2: Evidence for Carbonate Radical Production
Sara Goldstein* and Gidon Czapski
Contribution from the Department of Physical Chemistry, The Hebrew UniVersity of Jerusalem,
Jerusalem 91904, Israel
ReceiVed September 19, 1997
Abstract: The decomposition of peroxynitrite in the presence of bicarbonate and various substrates was studied
using the stopped-flow technique. We have shown that peroxynitrite (ONOOH/ONOO-) reacts with excess
of CO2 to form peroxynitrate (O2NOOH/O2NOO-) in aerated solutions containing HCO2-, H2O2, CH3OH, or
(CH3)2CHOH. The yield of peroxynitrate increased with the increase in these substrate concentrations
approaching 30-33% of added peroxynitrite. Competition kinetics study yields rate constants which are similar
to those determined directly for the reactions of these substrates with the carbonate radical anion. We therefore
suggest that the formation of peroxynitrate takes place via the following steps: (i) the rapid reaction of
-
peroxynitrite with CO2 to form ONOOCO2-; (ii) the homolytic cleavage of 30-33% of ONOOCO2 into
•-
•NO2 and CO3•-; (iii) the reaction of CO3 with the various substrates, yielding ultimately superoxide in
•
aerated solutions; and (iv) the fast reaction of superoxide with NO2 to form O2NOO-.
-
ONOO- + CO2 f ONOOCO2
(1)
Introduction
Peroxynitrite (ONOOH/ONOO-) is formed from the very fast
•
reaction of superoxide with NO (k ) (4.3-6.7) × 109 M-1
The concentrations of CO2 in vivo are relatively high due to
high levels of bicarbonate in intracellular (12 mM)16 and
interstitial fluids (30 mM).16 This suggests that the reaction of
peroxynitrite with CO2 must be the predominant pathway for
peroxynitrite disappearance in biological systems.9-15,17 There-
fore, the chemical properties of this adduct are of great
importance.
s-1).1,2 It is a powerful oxidant,3-5 that can be formed by
activated macrophages and neutrophils, and may be the major
damaging species produced after cerebral and myocardial
ischemia, inflammation, sepsis, and many other pathological
conditions.3
Peroxynitrite is unstable in the presence of carbonate,6,7 and
low concentrations of bicarbonate were shown to protect
Escherichia coli from the toxic effect of peroxynitrite.8 Lymar
and Hurst9 were the first to show that ONOO- reacts rapidly
Recent studies have demonstrated that bicarbonate enhances
peroxynitrite-mediated nitration of aromatics,10-12,18,19 DNA,20
and ethylacetoacetate.13 It partially or completely inhibited the
oxidation of various compounds by peroxynitrite, e.g., dimethyl
sulfoxide,18 benzoate,18 glutathione,15,18 Ni(II)cyclam,21 but had
hardly any effect on the oxidation yield of ferrocyanide and
ABTS (2,2′-azinobis(3-ethyl-1,2-dihydrobenzothiazoline 6-sul-
fonate).21
with CO2 (k ) 3 × 104 M-1 s-1), apparently forming an adduct
- 9-15
whose composition is ONOOCO2
.
* To whom all correspondence should be addressed: Tel. 972-2-6586478;
Fax: 972-2-6586925; E-mail: SARAG@HUJI.VMS.AC.IL.
(1) Huie, R. E.; Padmaja, S. Free Rad. Res. Comms. 1993, 18, 195.
(2) Goldstein, S.; Czapski, G. Free Rad. Biol. Med. 1995, 19, 505.
(3) Beckman, J. S. In: The Biological and Pathological Chemistry of
Nitric Oxide; Lancaster, J., Ed.; Academic Press: New York, 1995; pp
140-206, and references therein.
(4) Pryor, W. A.; Squadrito, G. L. Am. J. Physiol. (Lung Cell. Mol.
Physiol.) 1995, 268, L699, and references therein.
(5) Goldstein, S.; Squadrito, G. L.; Pryor, W. A.; Czapski, G. Free Radic.
Biol. Med. 1996, 21, 965, and references therein.
(6) Keith, W. G.; Powell, R. E. J. Chem. Soc. A 1969, 90.
(7) Radi, R.; Cosgrove, T. P.; Beckman, J. S.; Freeman, B. A. Biochem.
J. 1993, 290, 51.
(8) Zhu, L.; Gunn, C.; Beckman, J. S. Arch. Biochem. Biophys. 1992,
298, 452.
(9) Lymar, S. V.; Hurst, J. K. J. Am. Chem. Soc. 1995, 117, 8867.
(10) Uppu, R. M.; Squadrito, G. L.; Pryor, W. A. Arch. Biochem. Biophys.
1996, 327, 335.
(11) Lymar, S. V.; Jiang, Q.; Hurst, J. K. Biochemistry 1996, 35, 7855.
(12) Gow, A.; Duran, D.; Thom, S. R.; Ischiropoulos, H. Arch. Biochem.
Biophys. 1996, 333, 42.
(13) Uppu, R. M.; Pryor, W. A. Biochem. Biophys. Res. Commun. 1996,
229, 1996.
(14) Pryor, W. A.; Lemercier, J.-N.; Zhang, H.; Uppu, R. M. Free Radic.
Biol. Med. 1997, 23, 331.
-
It has been suggested that ONOOCO2 can be a source for
several reactive intermediates including the carbonate radical/
nitrogen dioxide radical pair, which may be formed via the
homolytic cleavage of the peroxo O-O bond of ONOOC-
O2 ,
- 9-15,17-19 the nitronium ion/carbonate ion pair, which may
be formed via the heterolytic cleavage of the peroxo O-O bond
- 10,12-15,18,19
of ONOOCO2
,
or the nitrocarbonate anion (O2-
NOCO2-), which may be formed via the isomerization of
- 10,13-15,19
ONOOCO2
.
We have recently shown that the amount
of peroxynitrite available for the oxidation of various substrates
in the presence of excess CO2 over peroxynitrite does not exceed
(16) Carola, R.; Harely, J. P.; Noback, C. R. Human Anatomy &
Physiology; McGraw-Hill: New York, 1990.
(17) Lymar, S. V.; Hurst, J. K. Chem. Res. Toxicol. 1996, 9, 845.
(18) Denicola, A.; Freeman, B. A.; Truijillo, M.; Radi, R. Arch. Biochem.
Biophys. 1996, 333, 49.
(19) Lemercier, J.-N.; Padmaja, S.; Cueto, R.; Squadrito, G. L.; Uppu,
R. M.; Pryor, W. A. Arch. Biochem. Biophys. 1997, 345, 160.
(20) Yermilov, V.; Yoshie, Y.; Rubio, J.; Ohshima, H. FEBS Lett. 1996,
399, 67.
(15) Zhang, H.; Squadrito, G. L.; Uppu, R. M.; Lemercier, J.-N.; Cueto,
R.; Pryor, W. A. Arch. Biochem. Biophys. 1997, 339, 183.
(21) Goldstein, S.; Czapski, G. Inorg. Chem. 1997, 36, 5113.
S0002-7863(97)03304-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/26/1998

Formation of Peroxynitrate
J. Am. Chem. Soc., Vol. 120, No. 14, 1998 3459
33%.21 Therefore, we have suggested that 30-33% of
ONOOCO2- decomposes into an intermediate, X, which either
forms nitrate and bicarbonate or oxidizes/nitrates the substrate.
Our data was not sufficient for the identification of X. We
Table 1. kobs of the Decay of Peroxynitrite in the Absence and
Presence of Formate Ionsa
pH
[PN], M
[HCO2-], M
λ, nm
∆OD
kobs, s-1
4.1
4.1
4.5
4.5
5.0
5.0
6.3
6.3
7.0
7.0
7.0
8.3
8.3
8.3
2.2 × 10-4
2.2 × 10-4
2.1 × 10-4
2.1 × 10-4
2.0 × 10-4
2.0 × 10-4
3.6 × 10-4
3.4 × 10-4
3.4 × 10-4
3.2 × 10-4
1.3 × 10-4
2.8 × 10-4
1.2 × 10-4
1.2 × 10-4
280
280
280
280
280
280
302
302
302
302
302
302
302
302
0.059
0.061
0.056
0.058
0.057
0.054
0.21
0.20
0.39
0.39
0.14
0.79
0.83
0.82
0.86
0.81
0.81
0.76
0.73
0.46
0.53
0.63
0.046
0.86
0.94
•
suggested that X may be the couple NO2 and CO3•-, as both
0.48
0.42
0.25
0.25
radicals are capable of oxidizing ferrocyanide, ABTS and Ni-
2-
(II)cyclam.21,22 The identity of X as the couple NO2+ and CO3
was ruled out because the half-life of NO2+ in water is 1.4 ns,23
which is far too short to be scavenged by the various
substrates.11-15,18-21
In this study, the reaction of peroxynitrite with excess of CO2
was investigated in the presence of HCO2-, H2O2, methanol,
2-propanol, and DTPA (diethylenetriaminepentaacetic acid). We
will demonstrate that in the presence of these substrates up to
30-33% of added peroxynitrite is converted into peroxynitrate,
0.25
0.40
0.41
0.19
0.20
0.50
1.0
•
•-
and will show that X is the couple NO2 and CO3
.
a All solutions were air saturated and contained 0.1 M acetate or
phosphate buffer.
Experimental Section
Chemicals. Peroxynitrite was synthesized by the reaction of nitrite
with acidified H2O2 at room temperature. Syringe pump (Model SP
230IW) was used to inject 0.606 M nitrite and 0.60 M H2O2 in 0.7 M
HClO4 through four tangential inlets of the first mixing chamber, and
the combined solutions flowed through a short connector to the second
mixing chamber where 3.6 M NaOH was pushed at the same flow rate
(45 mL/min) through four inlets to quench the reaction.24 The yield
of peroxynitrite was 85% (0.2 M) as determined by measuring the
absorption at 302 nm using ꢀ ) 1670 M-1 cm-1 25
contained about 2.5% nitrite and 1.5% H2O2.24
, and the stock solution
Kinetic Measurements. Kinetic measurements were carried out
using the Bio SX-17MV Sequential Stopped-Flow from Applied
Photophysics with a 1-cm long mixing cell. The reaction of peroxy-
nitrite with CO2 was followed at 290 or 302 nm by mixing equal
volumes of alkaline peroxynitrite with 0.2 M phosphate buffer
containing bicarbonate and other reactants. Bicarbonate was added as
a sodium salt to the buffer solutions 2-5 min before mixing to allow
the equilibration of the various carbonated species. The concentration
of CO2 in the buffer solutions was determined by measuring the pH of
these solutions and using pK ) 6.2 (I ) 0.25 M) for the equilibrium
CO2 +H2O h HCO3- + H+.26 Under all our experimental conditions,
the reaction of peroxynitrite with CO2 was faster than the hydration of
CO2.26,27 Therefore, the concentration of CO2, immediately after
mixing, was half of that at equilibrium before mixing. Peroxynitrite
concentration was measured before mixing by transferring a portion
from the syringe into a cuvette and measuring the absorption at 302
nm. The pH of the mixture was measured at the outlet of the flow
system. Each run was repeated for at least five times. The kinetic
measurements were carried out at 21.7 ( 0.1 °C.
Figure 1. The decay of peroxynitrite in the presence of 1.2 mM CO2
in solutions containing 0.1 M phosphate at pH 7.7. Lower curve: 0.26
mM peroxynitrite and no formate; middle curve: 0.28 mM peroxynitrite
and 5 mM formate; upper curve: 0.29 mM peroxynitrite and 0.2 M
formate.
The Formate System. It has been previously demonstrated,3
and confirmed in this study (Table 1), that formate enhances
the rate of the self-decomposition of peroxynitrite at pH > 7.3
The mechanism of the reaction is complex and is strongly
affected by the pH and by the presence of oxygen (data not
shown). Peroxynitrite reacts relatively slowly with formate
(Table 1), and, therefore, the contribution of this reaction to
the decomposition of peroxynitrite in the presence of bicarbonate
can be ignored as reaction 1 predominates.
The effect of up to 0.5 M formate on the decomposition of
peroxynitrite in the presence of excess of CO2 over peroxynitrite
was studied at pH 4.8-9.5, where peroxynitrite decayed via
two sequential first-order reactions. Typical kinetic traces at
two concentrations of formate are given in Figure 1.
The observed rate constant of the first fast decay was linearly
dependent upon [CO2], independent of [HCO2-] and [phos-
phate], and decreased with the decrease in pH, suggesting that
ONOO- is the reactive form. As ONOOH does not react with
CO2,9,10 we determined the rate constant of the reaction of
Results and Discussion
The reaction of peroxynitrite with CO2 was studied under
limiting concentrations of peroxynitrite at pH 4.7-9.3 in the
presence of 0.1 M acetate or phosphate buffers. The decay of
peroxynitrite in the presence of excess of CO2 was first order,
and kobs was linearly dependent upon [CO2] and highly
dependent upon pH. We determined k1 ) (2.3 ( 0.2) × 104
M-1 s-1 using pKa ) 6.8 for ONOOH3,4 and pK ) 6.2 for the
26
hydration of CO2 (data not shown). Our value is somewhat
lower than that determined by Lymar and Hurst (k1 ) 3 × 104
ONOO- with CO2 in the presence of formate by plotting kobs
-
M-1 s-1)9 due to different methods of calculations.28
(Ka + [H+])/Ka as a function of [CO2] at several pH’s using
the value of 6.8 for the pKa of ONOOH4 (Figure 2). From the
slope of the line we determined k1 ) (2.2 ( 0.3) × 104 M-1
s-1, which is in excellent agreement with that determined in
(22) Ross, A. B.; Mallard, W. G.; Helman, W. P.; Buxton, J. V.; Huie,
R. E.; Neta, P. NIST Standard References Database 40, Version 2.0 1994.
(23) Moodie, R. B.; Schofield, K. Taylor, P. G.; J. Chem. Soc., Perkin
Trans. 2 1979, 133.
(24) Saha, A.; Goldstein, S., Cabelli, D.; Czapski, G. Free Radic. Biol.
Med. In press.
(25) Hughes, M. N.; Nicklin, H. G. J. Chem. Soc. (A) 1968, 450.
(26) Alberty, R. A. J. Phys. Chem. 1995, 99, 11028.
(27) Kern, D. M. J. Chem. Edu. 1960, 37, 14.
(28) Lymar and Hurst9 used a pH profile to fit three parameters of k1,
pKa of ONOOH, and pK of the hydration of CO2 and got a good fit for 3
× 104 M-1 s-1, 6.6 and 5.96, respectively. We used a pH profile to fit only
one parameter, k1, using the literature values of 6.8 for the pKa of ONOOH3,4
and 6.2 for the pK of the hydration of CO2.26

3460 J. Am. Chem. Soc., Vol. 120, No. 14, 1998
Goldstein and Czapski
Figure 2. kobs(Ka + [H+])/Ka of the first decay as a function of [CO2].
(+) pH 6.27, 0.33 mM peroxynitrite, 0.25 M formate; (9) pH 7.13,
0.37 mM peroxynitrite, 0.01-0.25 M formate; (b) pH 8.35, 0.28 mM
peroxynitrite, 0.2 M formate. All solutions contained 0.1 M phosphate
buffer.
Figure 4. ∆ꢀ290 of the second decay as a function of pH. Solutions
contained 0.3 mM peroxynitrite, 1 mM CO2, 0.2-0.25 M formate, and
0.1 M phosphate. The solid line is the best fit calculated using eq 2
(∆ꢀ replaces k) with ∆ꢀa ) 0, ∆ꢀb ) 440 M-1 cm-1, and pKa ) 6.15.
Figure 5. The spectrum obtained immediately and 0.1 s after mixing.
The final solution contained 0.36 mM peroxynitrite, 1.6 mM CO2, 0.5
M formate, and 0.1 M phosphate at pH 8.44.
Figure 3. kobs of the second decay as a function of pH. Solutions
contained 0.3 mM peroxynitrite, 1 mM CO2, 0.2-0.25 M formate and
0.1 M phosphate. The solid line is the best fit calculated using eq 2
with ka ) 0, kb ) 0.97 s-1, and pKa ) 6.15.
of added peroxynitrite is available for the oxidation of ferro-
cyanide, Ni(II)cyclam, and ABTS in the presence of excess of
CO2 over peroxynitrite.21 Therefore, we assume that 30-33%
the absence of formate, suggesting that the pKa of ONOOH is
unaffected by the presence of formate.
-
of ONOOCO2 is also available for the oxidation of formate,
The observed first-order rate constant of the second slow
decay was independent of [peroxynitrite], [CO2], [HCO2-] and
[phosphate] but decreased with the decrease in pH (Figure 3).
The observed rate constant of the second decay is given by eq
2, where ka and kb are the rate constants of the decay of the
acid and anion forms, respectively:
and the extinction coefficient of the species formed at the end
of the first decay should be 3-fold the value determined in alkali
pH’s (440 ( 30 M-1 cm-1, Figure 4), i.e., ꢀ290 ) 1400 ( 150
M-1 cm-1
. All these features strongly suggest that in the
presence of sufficient concentrations of formate, 30-33% of
peroxynitrite is converted to peroxynitrate as the properties of
the latter species are the following: (i) The pKa of O2NOOH
has been determined to be 5.9 ( 0.1.29,30 (ii) The rate constants
of the decay of O2NOOH and O2NOO- have been determined
kobs ) (ka[H+] + kbKa)([H+] + Ka)
(2)
The best fit of eq 2 to the experimental results given in Figure
3 was obtained with ka ) 0, kb ) 0.97 ( 0.05 s-1, and pKa )
6.15 ( 0.05. The contribution of the second decay to the whole
process increased with the increase in [HCO2-] and leveled off
at all pH’s at [HCO2-] > 0.15 M. In the presence of 0.2-0.25
M formate, the contribution of the second decay was highly
dependent upon pH (Figure 4). The best fit of eq 2 to the
experimental results given in Figure 4, where ∆ꢀ290 replaces k
(∆ꢀ290 ) ∆OD290/[peroxynitrite]o), was obtained with ∆ꢀa )
0, ∆ꢀb ) 440 ( 30 M-1cm-1, and pKa ) 6.15 ( 0.05. The
spectrum of ONOO-, as measured immediately after the mixing,
differs than that measured at the end of the first decay (Figure
5).
We conclude that a new species with a pKa ) 6.15 ( 0.05 is
formed at the end of the reaction of ONOO- with CO2 in the
presence of formate. The acid form of this species is relatively
stable and does not absorb at 290 nm, whereas the anion form
has a maximum absorption at 290 nm, and it decays with kb )
0.97 ( 0.05 s-1. We have recently shown that only 30-33%
to be 7 × 10-4 s-1 28 and 1.0 s-1 29,30
,
respectively. (iii) O2-
NOOH does not have any appreciable absorption above 280
nm,29,31 whereas the maximum absorption of O2NOO- is at
285-290 nm (ꢀ ) 1500-1650 M-1 cm-1).29,30
The H2O2 System. The decomposition of 0.26 mM peroxy-
nitrite in the presence of 1.2 mM CO2 and 0.047-66 mM H2O2
was studied at pH 7.7 (0.1 M phosphate). Under these
conditions, peroxynitrite decayed via two sequential first-order
reactions as in the presence of formate. The rate of the first
decay was independent of [H2O2] with kobs ) 27 ( 0.2 s-1
which correlates well with k1) (2.3 ( 0.2) × 104 M-1 s-1
,
.
The rate constant of the second decay was determined to be
0.9 ( 0.03, independent of [H2O2], in agreement with the value
determined in the presence of formate at pH 7.7 (Figure 3).
The contribution of the second decay increased with increasing
(29) Løgager, T.; Sehested, K. J. Phys. Chem. 1993, 97, 10047.
(30) Goldstein, S.; Czapski, G. Inorg. Chem. 1997, 36, 4156.
(31) Appelman, E. H.; Gosztola, D. J. Inorg. Chem. 1995, 34, 787.

Formation of Peroxynitrate
J. Am. Chem. Soc., Vol. 120, No. 14, 1998 3461
CO3•- + ROH f R• + HCO3
k9(MeOH) ) 5.0 × 103 M-1 s-1
(9)
-
[H2O2] and leveled off at [H2O2] > 70 mM, where ∆ꢀ290
)
460 ( 10 M-1cm-1, which corresponds to 30-33% yield of
peroxynitrate, similarly to the case of formate.
k9(2-PrOH) ) (3.9-5.0) × 104 M-1 s-1 22
The Alcohol System. The decay of peroxynitrite was
followed under identical conditions to those described for the
H2O2 system but in the presence of up to 5 M methanol or
2-propanol. As in the former cases, the decay of peroxynitrite
followed two sequential first-order reactions with the same rate
constants as in the presence of H2O2. We found that the yield
of peroxynitrate in the presence of high concentrations of these
alcohols approached 30-33% of added peroxynitrite. Under
anaerobic conditions, only the first decay was observed.
R‚ + O2 f RO2
(10)
(11)
•
k10 ) (3.5-4.9) × 109 M-1 s1 22
•
•-
RO2 f R+ + O2
k11(MeOH) < 3 s-1
k11(2-PrOH) ) 700-960 s-1 22
The DTPA System. DTPA is usually added in most
peroxynitrite studies to eliminate the possible catalysis of the
decomposition of peroxynitrite by traces of metal impurities.4
We avoided the use of DTPA because it can be oxidized
by the intermediates formed during the decomposition of
ONOOCO2-. Indeed, the decay of 0.26 mM peroxynitrite in
the presence of 1.2 mM CO2 and various concentrations of
DTPA at pH 7.7 (0.1 M phosphate) followed two sequential
first-order reactions as in the presence of HCO2-, H2O2,
methanol, and 2-propanol. The contribution of the second
process in the presence of 0.1 and 2 mM DTPA was 3% and
15% of added peroxynitrite, respectively.
In the presence of DTPA, reactions 12 and 13 most probably
take place, though the yield of superoxide may be lower than
100%:33
CO3•- + DTPA f CO32- + DTPA•
k12 ) 1.7 × 107 M-1 s-1 at pH 1122
(12)
DTPA• + O2 f O2•- + products
(13)
The reaction sequences 5-6, 7-8, 9-11, or 12-13 are followed
by reactions 14-17:
Mechanism and Competition Kinetics. We have shown
that the reaction of peroxynitrite with excess of CO2 in aerated
solutions containing formate, H2O2, methanol, and 2-propanol
yields peroxynitrate. We have previously demonstrated that
O2•- + •NO2 f O2NOO-
k14 ) 4.5 × 109 M-1 s-1 29
(14)
(15)
(16)
(17)
•NO2 + •NO2 h N2O4
•-
only 30-33% of ONOOCO2 is capable of oxidizing the
-
k15 ) 4.5 × 108 M-1 s-1
k-15 ) 6.9 × 103 s-1 34
substrates, and we will now show that 30-33% of ONOOCO2
•
is converted into NO2 and CO3•-. We suggest the following
N2O4 + H2O f NO3- + NO2- + 2H+
mechanism:
k16 ) 1.0 × 103 s-1 34
–
NO3– + CO2/HCO3
2k3
–
O2NOO- f NO2- + O2
k17 ) 1.0 s-1 29,30
ONOOCO2
(3)
k3
CO3•– + •NO2
The rate constants of the reactions of •NO2 with formate and
H2O2 have not been determined. However, since the rate
CO3•- + •NO2 f CO2 + NO3
(4)
-
•
-
constants of the reactions of ClO2 with HCO2 and H2O2 are
k4 ) 1.0 × 109 M-1 s-1 22
lower than 0.01 and 4 M-1 s-1, respectively,22 it is expected
•
-
that the rate constants of the reactions of NO2 with HCO2
and H2O2 will not exceed these values,35 and, therefore, these
reactions will not compete with superoxide for •NO2. It is also
In the presence of formate reactions 5 and 6 take place:
2-
important to note that reaction 11 is catalyzed by HPO4 and
CO3•- + HCO2- f CO2•- + HCO3
(5)
(6)
-
OH-,22 and, therefore, superoxide is formed rapidly in the
presence of 0.1 M phosphate at pH 7.7. The results in the
k5 ) (1.1-1.6) × 105 M-1 s-1 (I f 0) 22
presence of DTPA indicates that reaction 13 is also catalyzed
CO2•- + O2 f CO2 + O2
by HPO4 .
•-
2-
According to our suggested mechanism, if HCO2-, H2O2, or
alcohol (RH) compete with •NO2 for CO3•-, peroxynitrate will
subsequently be formed. We found that the yield of peroxyni-
trate as measured at 290 nm increased with increasing [RH]. If
k6 ) (2.0-4.2) × 109 M-1 s-1 22
In the presence of H2O2 reactions 7 and 8 take place:
(32) Bielski, B. H. J.; Cabelli, D. E.; Arudi, R. L.; Ross, A. B. J. Phys.
Chem. Ref. Data 1985, 14, 1041.
(33) von Sonntag, C. In Free Radicals, Metal Ions and Polymers,
Beaumont, P. C., Deeble, D. J., Rice-Evans, C., Eds.; Richelieu Press:
London, 1989; p 73.
(34) Graetzel, M.; Henglein, A.; Lilie, J.; Beck, G. Ber. Bunsen-Ges.
Phys. Chem. 1969, 73, 646.
•
H2O2 + CO3•- f HCO3- + HO2
(7)
(8)
k7 ) 8.0 × 105 M-1 s-1 22
HO2 h H+ + O2
•
•-
pK ) 4.8 32
(35) Neta, P.; Huie, R. E.; Ross, A. B. J. Phys. Chem. Ref. Data 1988,
17, 1027. The redox potential of ClO2. (0.938 V) is somewhat lower than
•
that of •NO2 (1.03 V), but the self-exchange rate for ClO2 /ClO2- is higher
In the presence of methanol and 2-propanol (ROH) reactions
than that for •NO2/NO2. Therefore for most cases, the rate constants of the
•
•
9-11 take place:
one-electron oxidation by ClO2 exceeds that by NO2.

3462 J. Am. Chem. Soc., Vol. 120, No. 14, 1998
Goldstein and Czapski
Table 2. Comparison of the Rate Constants Determined Directly with Those Determined from the Slope of the Lines in Figure 6 for the
•-
Reaction of CO3 with the Various Substrates
k(CO3•- + RH),
slope ) k4[•NO2]/k, M-1b
k × slope,
RH
M-1 s-1a
(Figure 6)
s-1
H2O2
HCO2
k12 ) 8.0 × 105
1.9 × 10-3
1.5 × 103
k4 ) (1.1-1.6) × 105 (I f 0)
(2.2-3.2) × 105 (I ∼ 0.28 M)c
k15(2-PrOH) ) (3.9-5.0) × 104
k15(MeOH) ) 5.0 × 103
5.7 × 10-3
(1.3-1.8) × 103
-
2-PrOH
CH3OH
2.2 × 10-2
(0.9-1.1) × 103
0.47
2.3 × 103
a These rate constants were determined directly and were taken from ref 21. b All solutions contained 0.29-0.31 mM peroxynitrite, 1.2 mM CO2
and 0.1 M phosphate at pH 7.7. c Under our experimental conditions the ionic strength exceeds 0.28 M.
Figure 6. ODmax/OD - 1 as measured at 290 nm versus 1/[RH] for
Figure 7. The dependence of 1/∆OD270 of the second decay on
RH ) H2O2, HCO2-, 2-PrOH, and CH3OH. All solutions contained
ferricyanide concentration. Solutions contained 0.28 mM peroxynitrite,
0.29-0.31 mM peroxynitrite, 1.2 mM CO2, and 0.1 M phosphate at
1.2 mM CO2, 0.2 M HCO2-, and 0.1 M phosphate at pH 8.1.
pH 7.7.
∆OD ) ∆ODok6[O2](k6[O2] + k19[Fe(CN)63-])
(20)
RH competes with NO2 for CO3•-, eq 18 will be obtained,
•
where k(RH + CO3•-)[RH] ) k5[HCO2-] or k7[H2O2] or k9-
A plot of 1/∆OD270 as a function of [Fe(CN)63-] yields a
straight line with intercept/slope ) k6[O2]/k19 ) (2.8 ( 0.4) ×
10-4 M (Figure 7). As [O2] ) 0.12 mM, it is calculated that
k6/k19 ) 2.3 ( 0.3, which is in agreement with the literature
values.22 The same effect is expected in the case of methanol
and 2-propanol as the radicals derived from these substrates
react rapidly with ferricyanide.22
[ROH]:
OD
max ) 1 +
k4[•NO2]
k(RH + CO3•-)[RH]
(18)
OD
Plots of ODmax/OD - 1 as a function of 1/[RH] under identical
experimental conditions yielded straight lines with intercepts
close to zero (Figure 6). The various slopes obtained for
formate, H2O2, methanol, and 2-propanol from Figure 6, and
We conclude that the competition kinetics study shows that
30-33% of ONOOCO2- decomposes via the homolytic cleav-
•-
•
age of the peroxo O-O bond into CO3 and NO2.
•-
the literature values for the rate constants of CO3 with these
Lymar and Hurst9 synthesized peroxynitrite through the
reaction of nitrite with acidified hydrogen peroxide. They
destroyed the excess of H2O2 with MnO2 and did not add DTPA.
Therefore, it is expected that in their system peroxynitrite in
the presence of excess of CO2 would decay via a single
exponent. Denicola et al.18 synthesized peroxynitrite by the
same method and used 0.1 mM DTPA in their kinetic measure-
ments. However, they reported only on a single first-order
decay of peroxynitrite. It is possible that they did not look at
a longer time scale of seconds or that their preparation contained
substrates are summarized in Table 2. As the slope of these
lines equals k4[•NO2]/k(RH + CO3•-), the last entry in Table 2
(slope × k(RH + CO3•-)) is similar for the various RH,
suggesting that the formation of peroxynitrate in all four systems
•-
is initiated by the reaction of RH with CO3
If the conversion of peroxynitrite to peroxynitrate takes place
.
-
•
•-
through the homolysis of ONOOCO2 into NO2 and CO3
,
the addition of ferricyanide should lower the yield of O2NOO-
in the case of the formate, as it would compete with oxygen
•-
for CO2 (reaction 19).
relatively high concentrations of nitrite, which might compete
•- 22
with DTPA for CO3
.
Uppu et al.10 synthesized peroxynitrite
Fe(CN)63- + CO2•- f Fe(CN)64- + CO2
k19 ) 1.1 × 109 M-1 s-1 (I ) 0.3 M)22
(19)
by the ozonation of 0.1 M azide at pH 12 and reported only on
a single first-order decay of peroxynitrite in the presence of
0.1 mM DTPA and excess of CO2. Therefore, we have to
assume that either they did not look at a longer time scale of
seconds or that their solutions are contaminated with azide. The
latter may compete with DTPA for the carbonate radical, and
superoxide will not be formed. To test this hypothesis, we
added azide to the mixture containing 0.26 mM peroxynitrite,
1.2 mM CO2, and 0.1 mM DTPA at pH 7.7 (0.1 M phosphate
buffer). Under these conditions, the contribution of the second
decay in the absence of azide was ca. 3% of added peroxynitrite,
and it diminished completely in the presence of [N3-] > 0.1
When a deaerated solution of 0.56 mM peroxynitrite at pH 12
was mixed with an aerated solution containing 2.4 mM CO2,
3-
0.4 M HCO2-, 0.2 M phosphate, and 0.105-1 mM Fe(CN)6
(the final pH was 8.1), the yield of O2NOO- decreased with
the increase in ferricyanide concentration. The yield of per-
oxynitrate was measured at 270 nm, where the absorption of
ferricyanide is relatively low, and is given by eq 20, where
∆ODo is the absorption measured in the absence of ferricy-
anide:

Formation of Peroxynitrate
J. Am. Chem. Soc., Vol. 120, No. 14, 1998 3463
mM. We conclude that the use of relatively high [DTPA] as
chelator for traces of metal impurities should be avoided in these
kind of experiments.
0.2 M formate, or 2 M 2-PrOH, about 30% of peroxynitrite
will be converted into peroxynitrate, and the latter can be
efficiently quenched with strong acid.
It has been previously suggested that the reaction of peroxy-
nitrite with CO2 must be the predominant pathway for peroxy-
nitrite disappearance in many biological systems.9,17 Therefore,
we suggest that the reaction of peroxynitrite with CO2 is a
plausible pathway for the formation of peroxynitrate in biologi-
cal systems, which contain high concentrations of various
solutes, which scavenge CO3•- and yield ultimately superoxide
(e.g., DNA, glucose). This reaction can take place if peroxy-
nitrite is formed in the absence of relatively high concentrations
of •NO and SOD as theses compounds will compete with •NO2
for superoxide. Thus, bicarbonate will be protective or enhance
the toxicity of peroxynitrite depending on the chemical proper-
ties and reactivity of peroxynitrate.
Conclusions
We have demonstrated that the decomposition of peroxynitrite
in the presence of excess of CO2 and various organic and
inorganic solutes yields up to 30-33% peroxynitrate. The
competition kinetics study shows that the formation of perox-
ynitrate is initiated by the reaction of CO3•- with these solutes.
-
Therefore we conclude that 30-33% of ONOOCO2 decom-
•-
•
poses into CO3 and NO2. The chemical properties and
reactivity of peroxynitrate is not well understood as it is difficult
to prepare it in aqueous solutions.29-31 This paper describes a
new method for the preparation of the relatively stable O2NOOH
(pKa ) 5.9, τ1/2 ∼ 30 min)29-31 via the reaction of peroxynitrite
with CO2 in the presence of H2O2, formate, or 2-PrOH, which
may enable a further study on this interesting compound. The
optimal conditions are around pH 6, where the concentration
of CO2 is relatively high (pK ) 6.2), and the half-life of
peroxynitrate is longer than 1 s. In the presence of 0.1 M H2O2,
Acknowledgment. This research was supported by grant
4129 from The Council For Tobacco Research and by The Israel
Science Foundation.
JA9733043