Carbonate Radical Anion Research Articles

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite.

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.

Oxidative generation of guanine radicals by carbonate radicals and their reactions with nitrogen dioxide to form site specific 5-guanidino-4-nitroimidazole lesions in oligodeoxynucleotides.

A simple photochemical approach is described for synthesizing site specific, stable 5-guanidino-4-nitroimidazole (NIm) adducts in single- and double-stranded oligodeoxynucleotides containing single and multiple guanine residues. The DNA sequences employed, 5'-d(ACC CG(1)C G(2)TC CG(3)C G(4)CC) and 5'-d(ACC CG(1)C G(2)TC C), were a portion of exon 5 of the p53 tumor suppressor gene, including the codons 157 (G(2)) and 158 (G(3)) mutation hot spots in the former sequence with four Gs and the codon 157 (G(2)) mutation hot spot in the latter sequence with two Gs. The nitration of oligodeoxynucleotides was initiated by the selective photodissociation of persulfate anions to sulfate radicals induced by UV laser pulses (308 nm). In aqueous solutions, of bicarbonate and nitrite anions, the sulfate radicals generate carbonate anion radicals and nitrogen dioxide radicals by one electron oxidation of the respective anions. The guanine residue in the oligodeoxynucleotide is oxidized by the carbonate anion radical to form the neutral guanine radical. While the nitrogen dioxide radicals do not react with any of the intact DNA bases, they readily combine with the guanine radicals at either the C8 or the C5 positions. The C8 addition generates the well-known 8-nitroguanine (8-nitro-G) lesions, whereas the C5 attack produces unstable adducts, which rapidly decompose to NIm lesions. The maximum yields of the nitro products (NIm + 8-nitro-G) were typically in the range of 20-40%, depending on the number of guanine residues in the sequence. The ratio of the NIm to 8-nitro-G lesions gradually decreases from 3.4 in the model compound, 2',3',5'-tri-O-acetylguanosine, to 2.1-2.6 in the single-stranded oligodeoxynucleotides and to 0.8-1.1 in the duplexes. The adduct of the 5'-d(ACC CG(1)C G(2)TC C) oligodeoxynucleotide containing the NIm lesion in codon 157 (G(2)) was isolated in HPLC-pure form. The integrity of this adduct was established by a detailed analysis of exonuclease digestion ladders by matrix-assisted laser desorption ionization with time-of-flight detection MS techniques.

Reactions of Manganese Porphyrins with Peroxynitrite and Carbonate Radical Anion

We have studied the reaction kinetics of ten manganese porphyrins, differing in their meso substituents, with peroxynitrite (ONOO-) and carbonate radical anion (CO3.) using stopped-flow and pulse radiolysis, respectively. Rate constants for the reactions of Mn(III) porphyrins with ONOO- ranged from 1 x 10(5) to 3.4 x 10(7) m(-1) s(-1) and correlated well with previously reported kinetic and thermodynamic data that reflect the resonance and inductive effects of the substituents on the porphyrin ring. Rate constants for the reactions of Mn(III) porphyrins with CO3. ranged from 2 x 10(8) to 1.2 x 10(9) m(-1)s(-1) at pH <or= 8.5 and increased with pH as a consequence of the ionization of the complexes. Mn(II) porphyrins reacted with CO3. with rate constants ranging from 1 x 10(9) to 5 x 10(9) m(-1)s(-1) at pH 10.4. Hence, fast scavenging of ONOO- and CO3. by manganese porphyrins could occur in vivo because of the catalytic reduction at the expense of a number of cellular reductants. Additionally, we determined the pKa of the axial water molecules of the Mn(III) complexes at pH 7.5-13.2 by spectrophotometric titration. Results were consistent with two acid-base equilibria for most of the complexes studied. The pKa values also correlated with the resonance and inductive effects of the substituents. The correlations of E(1/2) with the rate constants with ONOO- and with the pKa values display a deviation from linearity when N-alkylpyridinium substituents included N-alkyl moieties longer than ethyl, which is interpreted in terms of a decrease in the local dielectric constant.

Bicarbonate-dependent Peroxidase Activity of Human Cu,Zn-Superoxide Dismutase Induces Covalent Aggregation of Protein: INTERMEDIACY OF TRYPTOPHAN-DERIVED OXIDATION PRODUCTS

This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1WT) induced by bicarbonate (HCO3-)-mediated peroxidase activity. Higher molecular weight species (apparent dimers and trimers) of hSOD1WT were formed from incubation mixtures containing hSOD1WT, H2O2, and HCO3-. HCO3--dependent peroxidase activity and covalent aggregation of hSOD1WT were mimicked by UV photolysis of hSOD1-WT in the presence of a [Co(NH3)5CO3]+ complex that generates the carbonate radical anion (CO3.). Human SOD1WT has but one aromatic residue, a tryptophan residue (Trp-32) on the surface of the protein. Substitution of Trp-32 with phenylalanine produced a mutant (hSOD1W32F) that exhibits HCO3--dependent peroxidase activity similar to wild-type enzyme. However, unlike hSOD1WT, incubations containing hSOD1W32F,H2O2, and HCO3-did not result in covalent aggregation of SOD1. These findings indicate that Trp-32 is crucial for CO3.-induced covalent aggregation of hSOD1WT. Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1WT, but not from hSOD1W32F. Spin traps also inhibited the covalent aggregation of hSOD1WT. Fluorescence experiments revealed that Trp-32 was further oxidized by CO3., forming kynurenine-type products in the presence of oxygen. Molecular oxygen was needed for HCO3-/H2O2-dependent aggregation of hSOD1WT, implicating a role for a Trp-32-dependent peroxidative reaction in the covalent aggregation of hSOD1WT. Taken together, these results indicate that Trp-32 oxidation is crucial for covalent aggregation of hSOD1. Implications of HCO3--dependent SOD1 peroxidase activity in amyotrophic lateral sclerosis disease are discussed.

Reactivity of the Carbonate Radical Anion Towards Carbohydrate and Lignin Model Compounds

The kinetic selectivity for the reaction of the carbonate radical anion towards lignin and carbohydrate model compounds was determined by pulse radiolysis in the pH-span 8–13. For the cellulose model compound methyl-β-d-glucopyranoside a pKA-value of 14.4 was estimated.

Ionic Species in Pulse Radiolysis of Supercritical Carbon Dioxide. 2. Ab Initio Studies on the Structure and Optical Properties of (CO2)n+, (CO2)2-, and CO3- Ions

Gas-phase (CO2)+2-4 cations and (CO2)2-, CO3-, CO3-·H2O and CO3-·CO2 anions have been examined using density functional theory calculations. It is shown that the lowest energy (CO2)n+ cations are C2h symmetric “ladder” structures with complete delocalization of charge and spin between the monomer units. These cations absorb in the visible due to a charge resonance band that involves their lowest 2Bu and 2Ag states. The “ladder” structure accounts for several trends observed in the photodestruction spectra of the (CO2)n+ cations. For the (CO2)2+ dimer cation, the energy and the oscillator strength of the charge resonance band compare favorably with these parameters for the solvent radical cation in supercritical (sc) CO2. A close similarity between the VIS spectra of the (N2O)2+ dimer cation (in sc CO2) and the solvent cation suggests that the latter has the (CO2)2+ dimer cation as the chromophore core. It is demonstrated that optical, Raman, and magnetic resonance spectra of the carbonate radical anion, CO3-, can be consistently accounted for by a C2v symmetric structure with the unique O−C−O angle of ≈113° (in water) to ≈100° (in carbonate minerals). The oscillator strength of the A 2A1 ← X 2B2 transition in the visible correlates with this O−C−O angle. In aqueous CO3-, the trigonal distortion is due to hydrogen bonding to a single water molecule. In sc CO2 (and other nonpolar liquids), the trigonal distortion is weak, and CO3- should be a poor light absorber. This may be the reason no dissociative electron capture in sc CO2 has been observed by optical spectroscopy.

Copper, Zinc Superoxide Dismutase and H2O2: EFFECTS OF BICARBONATE ON INACTIVATION AND OXIDATIONS OF NADPH AND URATE, AND ON CONSUMPTION of H2O2

Copper,zinc superoxide dismutase (Cu,Zn-SOD) catalyzes the HCO(3)(-)-dependent oxidation of diverse substrates. The mechanism of these oxidations involves the generation of a strong oxidant, derived from H(2)O(2), at the active site copper. This bound oxidant then oxidizes HCO(3)(-) to a strong and diffusible oxidant, presumably the carbonate anion radical that leaves the active site and then oxidizes the diverse substrates. Cu,Zn-SOD is also subject to inactivation by H(2)O(2). It is now demonstrated that the rates of HCO(3)(-)-dependent oxidations of NADPH and urate exceed the rate of inactivation of the enzyme by approximately 100-fold. Cu,Zn-SOD is also seen to catalyze a HCO(3)(-)-dependent consumption of the H(2)O(2) and that HCO(3)(-) does not protect Cu,Zn-SOD against inactivation by H(2)O(2). A scheme of reactions is offered in explanation of these observations.

The Mechanism by which 4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (tempol) diverts peroxynitrite decomposition from nitrating to nitrosating species.

Tempol is a stable nitroxide radical that has been shown to protect laboratory animals from the injury associated with conditions of oxidative and nitrosoactive stress. Tempol's protective mechanisms against reactive oxygen species have been extensively studied, but its interactions with reactive nitrogen species remain little explored. Recently, it has been shown that tempol is a potent inhibitor of peroxynitrite-mediated phenol nitration while it increases phenol nitrosation by a complex mechanism [Carrol et al. (2000) Chem. Res. Toxicol. 13, 294]. To obtain further mechanistic insights, we reexamined the interaction of peroxynitrite with tempol in the absence and presence of carbon dioxide. Stopped-flow kinetic studies confirmed that tempol does not react directly with peroxynitrite but levels off the amount of oxygen (monitored with an oxygen electrode) and nitrite (monitored by chemiluminescence) produced from peroxynitrite in the presence and absence of carbon dioxide to about 30% and 70% of the initial oxidant concentration at pH 5.4, 6.4, and 7.4. Tempol inhibited phenol nitration while increasing the amounts of 4-nitrosophenol, that attained yields close to 30% of the peroxynitrite in the presence of carbon dioxide at pH 7.4. Fast-flow EPR experiments showed detectable changes in the instantaneous tempol concentration (maximum of 15%) only in the presence of carbon dioxide. Under these conditions, the instantaneous concentration of the carbonate radical anion was reduced by tempol in a concentration-dependent manner. The results indicate that tempol is oxidized by peroxynitrite-derived radicals (*OH and CO(3)(*-), in the absence and presence of carbon dioxide, respectively) to the oxoammonium cation which, in turn, is reduced back to tempol while oxidizing peroxynitrite to oxygen and nitric oxide. The latter reacts rapidly with peroxynitrite-derived nitrogen dioxide to produce the nitrosating species, dinitrogen trioxide. Overall, the results support a role for peroxynitrite and its derived radicals in the tissue pathology associated with inflammatory conditions.

Bicarbonate Enhances Peroxidase Activity of Cu,Zn-Superoxide Dismutase: ROLE OF CARBONATE ANION RADICAL AND SCAVENGING OF CARBONATE ANION RADICAL BY METALLOPORPHYRIN ANTIOXIDANT ENZYME MIMETICS

Much evidence exists for the increased peroxidase activity of copper, zinc superoxide dismutase (SOD1) in oxidant-induced diseases. In this study, we measured the peroxidase activity of SOD1 by monitoring the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF). Bicarbonate dramatically enhanced DCFH oxidation to DCF in a SOD1/H(2)O(2)/DCFH system. Peroxidase activity could be measured at a lower H(2)O(2) concentration ( approximately 1 microm). We propose that DCFH oxidation to DCF is a sensitive index for measuring the peroxidase activity of SOD1 and familial amyotrophic lateral sclerosis SOD1 mutants and that the carbonate radical anion (CO(3)) is responsible for oxidation of DCFH to DCF in the SOD1/H(2)O(2)/bicarbonate system. Bicarbonate enhanced H(2)O(2)-dependent oxidation of DCFH to DCF by spinal cord extracts of transgenic mice expressing SOD1(G93A). The SOD1/H(2)O(2)/HCO(3)(-)-dependent oxidation was mimicked by photolysis of an inorganic cobalt carbonato complex that generates CO(3). Metalloporphyrin antioxidants that are usually considered as SOD1 mimetic or peroxynitrite dismutase effectively scavenged the CO(3) radical. Implications of this reaction as a plausible protective mechanism in inflammatory cellular damage induced by peroxynitrite are discussed.

Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation

The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.

The Carbonate Radical Is a Site-selective Oxidizing Agent of Guanine in Double-stranded Oligonucleotides

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.

Bicarbonate Enhances the Hydroxylation, Nitration, and Peroxidation Reactions Catalyzed by Copper, Zinc Superoxide Dismutase: INTERMEDIACY OF CARBONATE ANION RADICAL

The effect of bicarbonate anion (HCO(3)(-)) on the peroxidase activity of copper, zinc superoxide dismutase (SOD1) was investigated using three structurally different probes: 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2, 2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO(3)(-) enhanced SOD/H(2)O(2)-dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UV-visible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H(2)O and not from H(2)O(2). This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO(3)(-) enhances SOD1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO(3)(-)-mediated oxidative reactions of SOD1 may help us understand the free radical mechanism of SOD1 and related mutants linked to amyotrophic lateral sclerosis.

Role of the Carbonate Radical Anion in Tyrosine Nitration and Hydroxylation by Peroxynitrite

Peroxynitrite has been receiving increasing attention as the pathogenic mediator of nitric oxide cytotoxicity. In most cases, the contribution of peroxynitrite to diseases has been inferred from detection of 3-nitrotyrosine in injured tissues. However, presently it is known that other nitric oxide-derived species can also promote protein nitration. Mechanistic details of protein nitration remain under discussion even in the case of peroxynitrite, although recent literature data strongly suggest a free radical mechanism. Here, we confirm the free radical mechanism of tyrosine modification by peroxynitrite in the presence and in the absence of the bicarbonate–carbon dioxide pair by analyzing the stable tyrosine products and the formation of the tyrosyl radical at pH 5.4 and 7.4. Stable products, 3-nitrotyrosine, 3-hydroxytyrosine, and 3,3-dityrosine, were identified by high performance liquid chromatography and UV spectroscopy. The tyrosyl radical was detected by continuous-flow and spin-trapping electron paramagnetic resonance (EPR). 3-Hydroxytyrosine was detected at pH 5.4 and its yield decreased in the presence of the bicarbonate–carbon dioxide pair. In contrast, the yields of the tyrosyl radical increased in the presence of the bicarbonate–carbon dioxide pair and correlated with the yields of 3-nitrotyrosine under all tested experimental conditions. Taken together, the results demonstrate that the promoting effects of carbon dioxide on peroxynitrite-mediated tyrosine nitration is due to the selective reactivity of the carbonate radical anion as compared with that of the hydroxyl radical. Colocalization of 3-hydroxytyrosine and 3-nitrotyrosine residues in proteins may be useful to discriminate between peroxynitrite and other nitrating species.

Theoretical studies of the reduction of ethylene carbonate

A mechanism for the reduction of ethylene carbonate proposed by D. Aurbach, M.D. Levi, E. Levi and A. Schechter [J. Phys. Chem. B 101 (1997) 2195] is analyzed using quantum ab initio and classical transition state theory methods. The reduction reaction leads to open-chain anion products. The two-electron transfer reduction mechanism forming carbonate and ethylene di-carbonate radical anions is thermodynamically feasible. The first electron transfer is the rate-determining step. Further reaction of the carbonate ion with lithium ion or with another ethylene carbonate molecule yields Li 2CO 3 as the most probable product, with lithium ethylene di-carbonate most likely to be present at high solvent concentrations.

Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase: ROLE OF CARBONATE ANION RADICAL

We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro-gamma-tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H(2)O(2)-mediated nitration of gamma-tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H(2)O(2)-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H(2)O(2)-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H(2)O(2), suggest that SOD1/H(2)O(2) oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H(2)O(2) by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226-1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H(2)O(2). However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.

The Carbonate Radical (HCO3·/CO3-·) as a Reactive Intermediate in Water Chemistry: Kinetics and Modelling

Results from kinetic laboratory studies of reactions of the carbonate radical anion (CO - 3 .) with aromatic compounds in aqueous solution at T = 298 K are presented. Data were obtained in using a laser photolysis laser long-path absorption (LP-LPLA) apparatus which was designed for direct time-resolved studies of radical reactions. For the reactions of CO - 3 . with hydroquinone dimethyl ether (2), methyl anisole (3), benzene (4), p-xylene (5), toluene (6), chlorobenzene (7), nitrobenzene (8), and benzonitrile (9), rate coefficients of k 2 = (3.0 ± 0.6). 10 7 M-' s -1 , k 3 = (9.7 ± 1.7). 10 5 M -1 s -1 , k 4 = (3.2 ± 0.7). 10 5 M -1 s -1 , k 5 = (3.8 ± 0.9). 10 4 M -1 s -1 , k 6 = (6.8 ± 2.3). 10 4 M -1 s -1 , k 7 = (2.7 ± 0.6). 10 5 M -1 s -1 , k 8 = (1.4 ± 0.5). 10 4 M -1 s -1 , and k 9 < 1.3. 10 2 M -1 s -1 were obtained. In further studies the effect of temperature on the reactions (2), (4), and (5) has been studied. The kinetic data obtained for the reaction of the carbonate radical anion with aromatic compounds were compared to the corresponding reactions of the hydroxyl radical. Finally, these kinetic data were used within a simple model system to investigate the implications of carbonate radical anion kinetics within water treatment processes. It is shown that the degradation of organic pollutants in. OH-radical based water treatment may proceed via the CO - 3 ./HCO 3 . radical under certain conditions.

Direct EPR Detection of the Carbonate Radical Anion Produced from Peroxynitrite and Carbon Dioxide

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-) 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 (CO-3). 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.

Formation of nitrous oxide from the reaction of peroxynitrite with sodium azide

The reaction of peroxynitrite (oxoperoxonitrate) with azide ion (N3–), the residual starting material in the peroxynitrite preparation by the ozonolysis of azide, was investigated in phosphate and carbonate buffers. The observed rate constants (kobs) for the decay of peroxynitrite at pH 4.5 in the presence of azide up to 30 mM are within experimental error identical to the self decomposition rate of peroxynitrite indicating that the reaction is zero order in azide concentration. However, when the concentration of azide is increased beyond 40 mM a slight increase in kobs is noticed. From the dependence of kobs on the concentration of azide the second-order rate constants for the reaction of peroxynitrite with azide are determined to be 3.5 and 1.2 M–1 s–1 at pH 4.5 and 7.4 respectively. In the presence of added bicarbonate the reaction between peroxynitrite and azide is zero order in azide concentration. The reaction of peroxynitrite with azide led to the production of nitrous oxide (N2O) in the absence and presence of bicarbonate as identified by GC-MS analysis of the reaction mixture. At a given pH and peroxynitrite concentration, the yield of N2O increased linearly with an increase in the concentration of azide up to 100 mM and attained saturation beyond that. Under identical conditions, the yield of N2O obtained in the absence of bicarbonate is 50% more compared to that obtained in the presence of bicarbonate. Based on kinetics and product studies, it is proposed that the reaction of peroxynitrite with azide involves a one-electron oxidation of N3– to azide radical (N3˙) by the activated form of HOONO (HOONO*). Also that the combination between N3˙ and nitrogen dioxide (NO2˙) is rapid and leads to nitryl azide (N3NO2) as a transient intermediate and precursor for nitrous oxide (N2O). In the presence of bicarbonate the peroxynitrite–carbon dioxide adduct (ONOOCO2–) or the carbonate radical anion (CO3˙–) is proposed as a one-electron oxidant towards N3– forming N3˙. The proposed mechanism for N2O formation from nitryl azide is supported by high level ab initio calculations.

Formation of Peroxynitrate from the Reaction of Peroxynitrite with CO2: Evidence for Carbonate Radical Production

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-.

Electron transfer reaction rates and equilibria of the carbonate and sulfate radical anions

Electron transfer reactions of the carbonate radical anion with azide, bromide, and hypochlorite ions have been studied by pulse radiolysis. From the equilibrium Br .- 2 + CO 2- 3 ⇆ 2Br - + CO .- 3 and the known redox potential for Br .- 2/2Br - = 1.62V vs NHE we derive E = 1.59V for CO .- 3/CO 2- 3. From this value and the equilibrium CO .- 3 + ClO - ⇆ CO 2- 3 + ClO . we derive E = 1.41V for ClO ./ClO -. The equilibrium reaction SO .- 4 + Cl - ⇆ SO 2- 4 + Cl . was studied by laser photolysis, by using 248 nm excitation of S 2O 2- 8 to produce SO .- 4 and examining the rate of reaction of this radical with Cl - in the presence of varying concentrations of SO 2- 4. By modeling the reactions taking place in this system, we derive the rate constant for the reaction of Cl . with SO 2- 4 (2.5 × 10 8dm 3mol -1s -1 at 0.1 mol dm -3 ionic strength), and from the known rate constant for reaction of SO .- 4 with Cl - (4.7 × 10 8dm 3mol -1s -1 at the same ionic strength) we derive the equilibrium constant. From the known redox potential for Clf .- 2/Cl - = 2.41V we derive E = 2.43V for SO .- 4/SO 2- 4.