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J Biol Chem, Vol. 274, Issue 16, 10802-10806, April 16, 1999
,
, and**
From the The biological effects of peroxynitrite have been
recently considered to be largely dependent on its reaction with carbon dioxide, which is present in high concentrations in intra- and extracellular compartments. Peroxynitrite anion
(ONOO Peroxynitrite1 is formed
from the very fast reaction between nitric oxide and superoxide anion
(k = (6.7 The pathway shown in Scheme 1A is similar to the one initially proposed
to explain the oxidations mediated by peroxynitrite in the absence of
carbon dioxide (Scheme 1B). It has been proposed that peroxynitrous
acid (ONOOH) promotes one-electron oxidations following a rate-limiting
unimolecular activation to a species whose chemical identity, an
activated form of peroxynitrous acid (ONOOH*) or the hydroxyl radical,
remained under debate for a long time (3, 14-18). It was only recently
that clear experimental evidence was obtained demonstrating that a
significant portion of the oxidative activity of peroxynitrous acid is
because of the hydroxyl radical (19,20). The controversy was difficult
to resolve because spin trapping of the hydroxyl radical with
DMPO,2 the most
unambiguous method for detecting the hydroxyl radical by EPR (21), was
compromised by the reactivity of peroxynitrite with spin traps and spin
adducts (15, 19). A direct demonstration of the participation of the
carbonate radical in oxidations mediated by peroxynitrite in the
presence of carbon dioxide (Scheme 1A) could be anticipated to be
similarly difficult.
There is only one report of spin trapping of the carbonate radical with
nitromethane in irradiated aqueous solutions at pH 12 (22). At
physiological pHs, the DMPO-carbon dioxide radical anion produced
during irradiation of bicarbonate and DMPO solutions was incorrectly
attributed to the carbonate radical adduct (23). We have tried to trap
the carbonate radical in systems containing peroxynitrite and
bicarbonate under different experimental conditions but did not
succeed. Consequently, we considered it worth trying to detect the
carbonate radical directly by continuous fast flow EPR of peroxynitrite
and bicarbonate solutions. Our results provided unambiguous evidence
for the formation of the carbonate radical in these mixtures.
Materials--
All reagents were purchased from Merck, Aldrich,
or Sigma and were analytical grade or better. Sodium bicarbonate
labeled with 13C (98%) was obtained from Isotec
(Miamisburg, OH). Peroxynitrite was synthesized from sodium nitrite
(0.6 M) and hydrogen peroxide (0.65 M) in a
quenched-flow reactor (3, 9); excess hydrogen peroxide was used to
minimize nitrite contamination (24). To eliminate excess hydrogen
peroxide, peroxynitrite was treated with manganese dioxide. Synthesized
peroxynitrite contained low levels of contaminating hydrogen
peroxide (< 1%) and nitrite (10-30%) that were determined as
described previously (2) by the titanyl method and by absorbance
measurements at 354 nm ( EPR Experiments--
The EPR fast-flow spectra were recorded at
room temperature (25 ± 2 °C) on a Bruker EMX spectrometer
operating at 9.68 GHz and 100 KHz field modulation equipped with a
Bruker ER 4117 D-MTV dielectric mixing resonator with a 9-mm distance
between the mixing cell and the resonator center. Sodium bicarbonate
solutions were prepared in 0.1-1 M phosphate buffer of
various pHs and left undisturbed for 15 min to permit
bicarbonate-carbon dioxide equilibrium (7); the pH of each solution was
determined after bicarbonate addition and equilibration. Peroxynitrite
solutions were prepared with water or 0.2 M dipotassium
hydrogen phosphate to a final pH of 12. These solutions were
transferred to 2 20-ml syringes mounted on a syringe infusion pump
(Harvard apparatus pump 22). Most spectra were recorded 15 ms after
mixing under a continuous flow of 7 ml/min. The dispensed mixtures were
collected for pH measurements at the end of the experiments to detect
changes caused by mixing with the alkaline stock solutions of
peroxynitrite. In some experiments, stock solutions of bicarbonate and
peroxynitrite were prepared in buffers bubbled with nitrogen for 20 min. The concentrations of the carbonate radical were estimated by
double integration of their EPR spectra and comparison with a doubly
integrated signal obtained under the same experimental conditions from
10 µM 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy radical (g = 2.0056) (25), which was also used to calibrate the
magnetic field.
Computer Simulations--
Simulations were carried out using the
Gepasi software, version 3.2 (26, 27).
Fast flow mixing of concentrated solutions of peroxynitrite (10 mM final concentration) and bicarbonate (50 mM
final concentration) in 0.5 M phosphate buffer at pH 6.9 led to the detection of a one-line EPR spectrum (Fig.
1A) that was not detectable
after stopping the flow (Fig. 1B). Also, no signal was
detected in fast flow mixtures of the buffer with either bicarbonate
(Fig. 1C) or peroxynitrite alone (Fig. 1D),
indicating that the radical is produced from the reaction between
peroxynitrite and carbon dioxide. Accordingly, the EPR signal varied
significantly with the pH of the mixtures, and the intensities were
maximum around pH 6.4 (Fig. 2). This
value is similar to those obtained for maximum decomposition rates of
peroxynitrite in the presence of carbon dioxide, which was in the pH
range of 6.2-6.5 (7, 9). These values are consistent with a
rate-limiting reaction between the peroxynitrite anion
(pKa = 6.8) (3) and carbon dioxide (pKa apparent = 6.4) (28) to produce the
nitrosoperoxocarboxylate adduct whose homolysis would generate the
carbonate radical (Scheme 1A).
Departamento de Bioquímica and
Departamento de Química, Instituto de Química,
Universidade de São Paulo, São Paulo, Brazil 05599-970 and
§ Departamento de Bioquimica,
ABSTRACT
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) reacts rapidly with carbon dioxide, forming
an adduct, nitrosoperoxocarboxylate (ONOOCO2), whose
decomposition has been proposed to produce reactive intermediates such
as the carbonate radical (CO
3). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we
directly detected the carbonate radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of
6-9. The radical was unambiguously identified by its EPR parameters (g = 2.0113; line width = 5.5 G) and by experiments with
bicarbonate labeled with 13C. In this case, the singlet EPR
signal obtained with 12C bicarbonate splits into the
expected doublet because of 13C (a(13C)= 11.7 G). The singlet spectrum of the unlabeled radical was invariant between
pH 6 and 9, confirming that in this pH range the detected radical is
the carbonate radical anion (CO3). Importantly, in addition to
contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct EPR spectroscopy.
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19) × 109
M1 s1) (see Reaction 1) (1, 2).
The compound is a potent oxidant that has been receiving increasing
attention as a potential pathogenic mediator in human diseases and as a
cellular toxin in host defense mechanisms against invading
microorganisms (3-6). At present, a significant part of the biological
reactivity of peroxynitrite is ascribed to the adduct produced by its
reaction with carbon dioxide (7-13). The peroxynitrite anion
(ONOO), which is the predominant form at physiological
pHs (pKa = 6.8) (see reaction 2, Table II) (2, 3),
reacts fast with carbon dioxide (pH-independent k = 5.8 × 104 M1 · s1 at 37 °C) (11), producing an adduct
whose structure is proposed to be
([ONOOCO2], nitrosoperoxocarboxylate) (see
reaction 3, Table II) (7). Taking into account the concentrations of
carbon dioxide in equilibrium with bicarbonate present in physiological
fluids, model calculations have suggested that most of the
peroxynitrite that might be formed in these fluids will produce the
carbon dioxide adduct before reacting with other biological targets (5,
13).
Carbon dioxide modulates the reactivity of peroxynitrite by
altering reaction rates, product yields, and product distribution (7-13). In these reactions, formation of the adduct
nitrosoperoxocarboxylate is rate-limiting, as first proposed by Lymar
and Hurst (7). This suggestion was confirmed by other authors (8-13),
and the current proposal is that in the absence of substrates, the
carbon dioxide adduct decomposes to nitrate and carbon dioxide, but in their presence, a fraction of the adduct (~35%) can engage in one-electron oxidations (and aromatic nitrations), probably through homolysis to nitrogen dioxide and carbonate radical anion (Scheme 1A). The participation of the carbonate
radical in the oxidation of substrates was inferred from the reactivity
of peroxynitrite with inorganic ions in the presence of carbon dioxide
(11,12).
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Scheme 1.
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= 24.6 M1
cm1), respectively. The concentration of peroxynitrite
stock solutions was determined spectrophotometrically at 302 nm
using an extinction coefficient of 1670 M1 cm1 (3, 9). All solutions
were prepared with distilled water treated in a Millipore Milli-Q system.
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Fig. 1.
EPR continuous flow spectra of the carbonate
radical produced from mixing peroxynitrite and bicarbonate solutions at
room temperature. A, peroxynitrite (20 mM)
and bicarbonate (100 mM) solutions in 1 M
phosphate buffer, pH 6.9; B, same as A but with
the spectrum scanned after stopping the flow; C,
peroxynitrite (20 mM) mixed with 1 M phosphate
buffer, pH 7.1; D, bicarbonate (100 mM) mixed
with water, pH 12. The spectra were recorded 15 ms after mixing (flow
of 7 ml/min) as described under "Experimental Procedures" except
for the spectrum (B) that was scanned immediately after
stopping the flow. The final concentrations of peroxynitrite,
bicarbonate, and phosphate buffer were 10, 50, and 500 mM;
the final pH was 6.9. Instrumental conditions: microwave power, 2 mW;
modulation amplitude, 5G; time constant 0.16 s; scan rate 0.84 G/s. Each spectrum is the accumulation of two scans.
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Fig. 2.
Representative plot of the effects of pH on
the yield of the carbonate radical anion produced in fast flow mixtures
of peroxynitrite and bicarbonate. The yields of the radical were
expressed as the peak height of the EPR signal in arbitrary units
(a.u.). Final concentrations of peroxynitrite, bicarbonate
and phosphate buffer were 10, 50, and 500 mM, respectively.
Bicarbonate solutions were prepared in 1 M phosphate buffer
of various pHs, and the pH was checked for each mixture before and
after mixing with peroxynitrite solutions (pH = 12). The changes
in pH before and after mixing were 
0.2 pH units, and the average pH
values were used in the plot. The spectra were recorded 15 ms after
mixing (flow of 7 ml/min) as described under "Experimental
Procedures" with the same instrumental conditions as shown in Fig. 1.
The data represent a series of experiments obtained on the same day
with the same peroxynitrite preparation. The same profile was obtained
in a different set of experiments; the data were not presented as the
average ±S.D. because of the difficulties in obtaining exactly the
same final pH values in different sets of experiments.
To better characterize the radical detected during fast flow mixing of
the peroxynitrite and bicarbonate solutions, we tried to increase the
detectable radical concentrations under our instrumental conditions. As
expected, the EPR signal shown in Fig. 1 increased with the flow in the
4-8 ml/min range, but the available instrumentation only permitted
flows of up to 8 ml/min. The EPR signal did not change significantly
when the buffer solutions were purged free of oxygen but grew when the
concentrations of both peroxynitrite or bicarbonate were increased.
Consequently, to obtain higher radical concentrations, we took
advantage of the relatively slow carbon dioxide hydration-dehydration
equilibrium as compared with peroxynitrite protonation and decay to
nitrate (Scheme 1B) (7). For this purpose, solutions of 100 mM bicarbonate in 0.1 M phosphate buffer, pH
6.4, were fast mixed with alkaline solutions of 40 mM
peroxynitrite to give solutions of pH 8.4 whose transient carbon dioxide concentrations (~25 mM) were much higher than
equilibrium values (~1.23 mM). Under these conditions,
the EPR signal obtained (Fig.
3A) was more intense than that
obtained under conditions of equilibrium (Fig. 1A).
Qualitatively, the signal was the same and showed EPR parameters, a
line width of 5.5 G, and a g value of 2.0113, which were in good
agreement with those previously reported for the carbonate radical in
solid matrices if allowance is made for the very different media used,
i.e. solutions versus solid matrix (29, 30).
Indeed, the matrix itself changes the EPR parameters of the carbonate
radical, and some of the values reported in the literature are listed
in Table I and compared with those
determined here in aqueous solutions (Fig. 3A).
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Unequivocal proof that the radical detected directly in fast flow mixtures of peroxynitrite with bicarbonate is the carbonate radical was obtained in experiments with bicarbonate labeled with 13C (Fig. 3B). In this case, the singlet signal of the unlabeled carbonate radical (Fig. 3A) split into a half-height doublet signal (Fig. 3B) because of the contribution of the 13C atom (I = 1/2). The determined hyperfine splitting constant of 13C is 11.7 G (Fig. 1B), a value in excellent agreement with those previously reported as experimental (Table I) (29, 30) and calculated values (11 G) (31) for the labeled carbonate radical.
The concentrations of the carbonate radical were estimated to be about
3 × 106 M under equilibrium conditions
at pH 6.9 (Fig. 1A) and 5 × 106
M under nonequilibrium conditions at pH 8.4 (Fig.
3A). These values should be taken as rough estimates,
because the experiments were run close to the detection limits of the
instrument, and EPR itself presents inherent quantification problems
(32). Still, the obtained values are of the same order of magnitude as
those calculated by computer simulation considering reactions 2-10
(Table II) in which the decomposition of
nitrosoperoxocarboxylate produces the carbonate radical anion with 35%
yield (reaction 5a, Table II) and with k4
assumed to be 1 × 106 s1 (7, 17). In
this scheme (Table II), the carbonate radical anion was assumed to
decay via three major pathways, including O transfer to
nitrogen dioxide (reaction 7), dismutation (reaction 8), and oxidation
of residual nitrite (reaction 9) (33-35). Under the same conditions as
shown in Fig. 1A, the simulation showed that the transient
concentration of the carbonate radical anion is 6 µM at
15 ms (Fig. 4).
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It is important to note that the one-line signal shown in Fig. 3A did not visibly split when the pH was changed over the 6-9 range or by the use of modulation amplitudes as low as 1 G (data not shown). This indicates that either the proton hyperfine splitting constant of the carbonate radical is lower than 1 G or that the species is not protonated (see Reaction 11) over the studied pH range of 6-9. Although the literature contains conflicting evidence regarding the protonated state of the carbonate radical over the 6-12 pH range (36, 37), recent studies by time resolved RAMAN spectroscopy have found no evidence for its protonation at pHs between 7.5 and 12.3 (38). These studies are in agreement with our EPR data indicating that the radical is not protonated at physiological pHs.
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DISCUSSION |
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A role for the carbonate radical in peroxynitrite-mediated
processes was first suggested by Radi and co-workers (39), who demonstrated that bicarbonate greatly increased luminol
chemiluminescence triggered by peroxynitrite. The direct reaction
between peroxynitrite and carbon dioxide was then characterized by
Lymar and Hurst (7). That this reaction should be a significant
fraction of the biological reactivity of peroxynitrite and that it
produces reactive oxidizing and nitrating species is currently
supported by the work of many investigators (8-13). The chemical
nature of these reactive intermediates has also been proposed, but this
is the first report presenting direct spectroscopic evidence for one of
these intermediates, the carbonate radical anion (Figs 1 and 3, Table
I). The proposed mechanisms of carbonate radical formation (reactions 3 and 5a) and consumption (reactions 7-9) (Fig. 4; Table II) are
consistent with the carbonate radical levels (~3-5 × 106 M) quantified experimentally under
equilibrium and nonequilibrium conditions.
Our results confirmed that the carbonate radical is not protonated at physiological pHs (Figs. 1-3) (38). As discussed by Bisby et al. (38), these results indicate that the carbonate radical anion is slightly more oxidizing in neutral solutions (Em,i ~ 1.80 V) than previously assumed considering a pKa of 9.6 (Em,i ~ 1.64 V) (38). Although local concentrations of carbon dioxide and biological targets will determine the decomposition pathway of peroxynitrite that might be formed in a particular physiological compartment, a significant fraction of peroxynitrite (35%) will produce reactive free radicals such as nitrogen dioxide and carbonate radical anion (Scheme 1A). Formation of hydroxyl radical is also possible (Scheme 1B) (19, 20) but unlikely to be significant under physiological conditions, because the rate of peroxynitrite homolysis is too slow to efficiently compete with peroxynitrite attacking the many available biological targets (5, 13). Both the hydroxyl radical and the carbonate radical anion can oxidize substrates by hydrogen atom abstraction, but in the case of the hydroxyl radical, the faster addition reactions would be likely to predominate in most conditions (40). This can explain why peroxynitrite-mediated nitration of aromatic amino acids is increased in the presence of carbon dioxide (5, 13).
Direct EPR detection of the carbonate radical anion is important to unravel mechanistic details of oxidative damage inflicted not only by peroxynitrite but also by other oxidizing species such as hydrogen peroxide and the hydroxyl radical. On the one hand, normal plasma bicarbonate and carbon dioxide concentrations are 25 and 1.3 mM, but higher levels could be achieved during pathologic events such as respiratory distress syndrome or ischemia reperfusion (41). On the other hand, the carbonate radical has been proposed to be responsible for the effects of bicarbonate increasing photodamage to erythrocytes (42) and the bactericidal activity of the hydroxyl radical (5, 23). The carbonate radical, however, has never been characterized in these systems. In a recent paper published by Hurst and co-workers (23), a role for the carbonate radical in the bactericidal effects of the hydroxyl radical is evidenced by the overall data. The claimed detection of the carbonate radical, however, was not substantiated by the presented spin trap results. The authors show that irradiated solutions of DMPO and bicarbonate presented a composite EPR spectrum whose components were the DMPO-hydroxyl radical adduct, the DMPO-hydrogen radical adduct, and a third adduct whose EPR parameters coincided with those of the DMPO-carbon dioxide radical adduct. This coincidence led the authors to conclude that they trapped the carbonate radical. However, in the carbon dioxide radical, most of the electron density is on the carbon atom, whereas in the carbonate radical, most of the electron density is on the oxygen atoms (30, 38), and adducts of these radicals with DMPO are unlikely to have similar EPR parameters. In addition, to the best of our knowledge, nobody has been able to trap the carbonate radical with DMPO. There is a report of trapping of the carbonate radical with nitromethane by irradiation at alkaline pHs, and as expected, the nitromethane-carbonate radical adduct has EPR parameters similar to those of the hydroxyl radical (22), an oxygen-centered radical. The radical detected by Hurst and co-workers should, indeed, be the carbon dioxide radical anion produced from reduction of carbon dioxide by hydrogen atoms not completely quenched in their system; the presence of hydrogen atoms is proved by their detection of the DMPO-hydrogen radical adduct (Reactions 12-R14). In our experience, it is common to detect DMPO-carbon dioxide radical adduct from air-equilibrated buffer solutions in the presence of reducing agents such as hydrazine (43).
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FOOTNOTES |
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* This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP) (to O. A.), and Swedish Agency for Research Cooperation, Consejo Nacional de Investigaciones Científicas y Técnicas, and Consejo Superior de Investigaciones Científicas, Universidad de la República (to R. R.).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: Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, 05599-970, São Paulo, SP. Brazil. Tel.: 55-11-818-3873; Fax: 55-11-8182186; E-mail: oaugusto{at}quim.iq.usp.br.
1
Unless otherwise specified, the terms
peroxynitrite and bicarbonate are used to refer to the sum of
peroxynitrite anion (ONOO) and peroxynitrous acid (ONOOH)
and to the sum of CO2, H2CO3, HCO3, and
CO32, respectively. IUPAC-recommended
names for peroxynitrite anion, peroxynitrous acid,
nitrosoperoxocarboxylate (ONOOCO2),
and nitric oxide are oxoperoxynitrate(-1), hydrogen oxoperoxynitrate, 1-carboxylato-2-nitrosodioxidane, and nitrogen monoxide, respectively.
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ABBREVIATIONS |
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The abbreviation used is: DMPO, 5,5-dimethyl-1-pyrroline-N-oxide.
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DISCUSSION
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M. G. Bonini and O. Augusto Carbon Dioxide Stimulates the Production of Thiyl, Sulfinyl, and Disulfide Radical Anion from Thiol Oxidation by Peroxynitrite J. Biol. Chem., March 23, 2001; 276(13): 9749 - 9754. [Abstract] |
