Reactions of Peroxynitrite with Aldehydes as ... - ACS Publications

We have examined the reactions of peroxynitrite with short-chain aliphatic aldehydes to model the reaction of the peroxynitrite anion (ONOO-) with CO2...
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Chem. Res. Toxicol. 1997, 10, 1331-1337

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Reactions of Peroxynitrite with Aldehydes as Probes for the Reactive Intermediates Responsible for Biological Nitration Rao M. Uppu, Gary W. Winston,† and William A. Pryor* The Biodynamics Institute, Louisiana State University, Baton Rouge, Louisiana 70803-1800 Received April 15, 1997X

We have examined the reactions of peroxynitrite with short-chain aliphatic aldehydes to model the reaction of the peroxynitrite anion (ONOO-) with CO2. Aldehydes, like CO2, react rapidly with peroxynitrite and catalyze its decomposition. The pH dependence of the reaction is consistent with the addition of ONOO- (not ONOOH) to the carbonyl carbon atom of the free aldehyde forming a 1-hydroxyalkylperoxynitrite anion adduct (5), which structurally resembles the nitrosoperoxycarbonate adduct (1) formed from the reaction of ONOO- with CO2. Intermediate 5, or the secondary products derived from it, decays to give NO3- and regenerated aldehyde, with small but significant yields of H2O2, organic acids, and organic nitrates. In analogy with the peroxynitrite/CO2 system, it is suggested that 5 undergoes homolytic or heterolytic cleavage at the O-O bond, giving a caged radical pair [RCH(OH)O•/ •NO ] (7) or intimate ion pair [RCH(OH)O-/+NO ] (8). The radicals and ions in intermediates 2 2 7 and 8 can recombine within the solvent cage to form 1-hydroxyalkylnitrate [RCH(OH)ONO2] (6), which can then dissociate to give nitrate and regenerate the aldehyde. The aldehyde/ peroxynitrite adducts 5-8 mediate the oxidation of 2,2′-azinobis(3-ethylbenzthiazoline-6sulfonate) but not the nitration of 4-hydroxyphenylacetate. The significance of these findings is discussed in relation to the mechanism(s) of the CO2-catalyzed isomerization of peroxynitrite to nitrate and biological nitrations involving peroxynitrite/CO2 adducts. Peroxynitrite1 can be formed in biological systems from the reaction of nitric oxide (•NO), a potent biological mediator (1-3), and superoxide (O2•-).2 Recent studies suggest that in physiological fluids devoid of peroxidases (4), ONOO- reacts almost exclusively with CO2 giving ONOOCO2- (1) (5-11). Adduct 1 is believed to undergo homolytic or heterolytic cleavage at the weak O-O bond, giving a caged radical pair [•NO2/CO3•-] (3) or an intimate ion pair [+NO2/CO32-] (4), which can then recombine within the solvent cage to form the metastable O2NOCO2(2) (Chart 1) (6, 10-13). One or more of the peroxynitrite/CO2 adducts 1-4 mediate(s) the electrophilic nitration of phenolic residues in proteins and, possibly, nucleic

Chart 1. Possible Intermediates from Peroxynitrite/CO2 (1-4) and Aldehyde/ Peroxynitrite (5-8) Reactions

* To whom correspondence should be addressed. Fax: (504) 3884936. E-mail: [email protected]. † Present address: Department of Toxicology, North Carolina State University, Raleigh, NC. X Abstract published in Advance ACS Abstracts, November 15, 1997. 1 Official IUPAC name is oxoperoxonitrate(1-) for ONOO- and hydrogen oxoperoxonitrate for ONOOH. We use the term “peroxynitrite” to indicate total concentration of ONOO- + ONOOH. The individual concentrations of ONOO- and ONOOH are governed by the pKa of 6.8 for this conjugate acid/base system. The term carbonate is used to indicate the sum of all carbonated species (CO2, H2CO3, HCO3-, and CO32-). If a particular species is referred to, it is represented by its chemical formula. The term aldehyde refers to the sum of both the free aldehyde and its hydrate, the gem-diol. For discussion specifically limited to either the free aldehyde or the gem-diol, either its name or structure is given. 2 Abbreviations: ABTS, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid); ABTS•+, ABTS radical cation; ADP, adenosine-5′-diphosphate; ATP, adenosine-5′-triphosphate; CO3•-, carbonate radical; DTPA, diethylenetriaminepentaacetic acid; FAD, flavin adenine dinucleotide; H2O2, hydrogen peroxide; 4-HPA, 4-hydroxyphenylacetic acid; MAH, molecule-assisted homolysis; •NO, nitric oxide; +NO2, nitronium ion; •NO2, nitrogen dioxide; 3-NO2-4-HPA, 3-nitro-4-hydroxyphenylacetic acid; O2•-, superoxide; ONOO-, peroxynitrite anion; ONOOH, peroxynitrous acid; ONOOCO2-, nitrosoperoxycarbonate anion adduct; O2NOCO2-, nitrocarbonate anion; RCHO, aldehyde; RCH(OH)2, aldehyde gem-diol; RCH(OH)OONO, 1-hydroxyalkylperoxynitrite; RCH(OH)ONO2, 1-hydroxyalkylnitrate.

acids, a hallmark of peroxynitrite formation in vivo (1417). We reasoned that ONOO- adds to the carbonyl carbon atom of aldehydes, forming adducts similar to those produced in the peroxynitrite/CO2 reaction; therefore, we examined the reactions of peroxynitrite with some shortchain aliphatic aldehydes to better understand the mechanism(s) of nitration by peroxynitrite/CO2 adducts.

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Experimental Procedures Chemicals. Acetaldehyde (99.7%), ATP disodium salt (99%), 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid ammonium salt) (ABTS) (∼98%), diethylenetriaminepentaacetic acid (DTPA), FAD disodium salt (94%), NADH disodium salt (98%), NADPH tetrasodium salt (98%), N-(1-naphthyl)ethylenediamine dihydrochloride (98%), phospho(enol)pyruvate monosodium salt (98%), acetate kinase from Escherichia coli (EC 2.7.2.1, 790 units/mg of protein), catalase from bovine liver (EC 1.11.1.6, 11 000 units/mg of solid), lactic dehydrogenase from rabbit muscle (EC 1.1.1.27, 880 units/mg of protein), nitrate reductase from Aspergillus species (EC 1.6.6.2, 0.45 unit/mg of solid),

© 1997 American Chemical Society

1332 Chem. Res. Toxicol., Vol. 10, No. 12, 1997 peroxidase from horseradish (EC 1.11.1.7, 290 purpurogallin units/mg of solid), and pyruvate kinase (EC 2.7.1.40, 535 units/ mg of protein) were purchased from Sigma (St. Louis, MO). 4-Hydroxyphenylacetic acid (4-HPA) (98%), propionaldehyde (97%), and sulfanilamide (98%) were obtained from Aldrich (Milwaukee, WI); sodium azide was from EM Science (Cherry Hill, NJ); triethanolamine (99.9%) was from Fisher Scientific (Fair Lawn, NJ). All other chemicals were of the highest grade available. Caution: Ozone is a strong oxidizing agent. It is highly reactive and cytotoxic, and therefore, any direct contact or exposure should be avoided. The reactions involving ozone must be carried out behind a safety shield in a fume hood, and excess unreacted ozone must be absorbed and destroyed in a solution of 10% (w/v) potassium iodide in 0.07 M phosphate buffer, pH ∼7.0. Similarly, care should be taken while handling and disposing of azide solutions. There have been some reports of poisoning and explosions associated with the use of azide solutions (18). Synthesis of Peroxynitrite. Peroxynitrite was synthesized by the ozonation of 0.1 M sodium azide in water at pH 12, as described earlier (19). To reduce the contamination by unreacted azide, ozonation was prolonged even after a maximal yield of peroxynitrite was obtained (20). This procedure helps to reduce the amount of unreacted azide to e5 µM with a loss of peroxynitrite yield of about 20-30% (20). The concentration of peroxynitrite in the final preparations was estimated (after a 50- or 100-fold dilution with 0.1 N NaOH) based on the absorbance at 302 nm ( ) 1670 M-1 s-1) (21). Stopped-Flow Kinetics. Peroxynitrite (0.5 mM) was allowed to react with 2-20 mM acetaldehyde or propionaldehyde at 25 ( 1 °C in 0.1 M phosphate buffer, pH 5.3-7.5, containing 0.1 mM DTPA. Stock solutions of aldehydes in water were mixed with the phosphate buffer at least 5 min before the addition of peroxynitrite to allow aldehydes to reach equilibrium with regard to hydration. A stopped-flow spectrophotometer (On-Line Instrument Systems, Jefferson, GA) equipped with an OLIS-RSM rapid scanning monochromator was used to collect data on the decay of peroxynitrite, which was routinely measured over a wavelength range of 260-390 nm (22, 23). The mixing time in the spectrophotometer was less than 2 ms, and the monochromator used had the capability of collecting up to 1000 scans/s. The data were analyzed for pseudo-first-order rates (kobs) using OLIS-RSM software. Apparent second-order rate constants (kapp) for the reaction of peroxynitrite with aldehydes were calculated from the slopes of plots of kobs versus the concentration of aldehyde. Oxidation of ABTS. Peroxynitrite-mediated oxidation of ABTS to the ABTS radical cation (ABTS•+) was studied at pH 7.0, as described earlier (6, 24). Stock solutions of peroxynitrite were diluted to 2 mM using a freshly prepared solution of 0.005 N NaOH. An aliquot (100 µL) of the diluted peroxynitrite solution was mixed over a period of 10 s with 1.9 mL of 0.1 M phosphate buffer, pH 7.0, containing 0.1 mM DTPA, 2 mM ABTS, and 0-40 mM propionaldehyde. Throughout the course of addition of peroxynitrite, the contents of the reaction mixture were continuously stirred using a vortex mixer, and stirring was continued for an additional 10 s at room temperature. The ABTS radical cation formed was measured within 2 min after the addition of peroxynitrite (660nm ) 12 000 M-1 cm-1) (25). Control experiments were performed using decomposed solutions of peroxynitrite as described earlier (19, 24). Nitration of 4-Hydroxyphenylacetate. Peroxynitrite (2 µmol) was allowed to react with 8 µmol of 4-HPA in 2 mL of 0.1 M phosphate buffer, pH 7.0, containing 0.1 mM DTPA and 0-40 mM propionaldehyde. As described above, peroxynitrite was added to a rapidly stirred solution of reactants over a period of 10 s, and stirring was continued for an additional 10 s at room temperature. 3-Nitro-4-hydroxyphenylacetate (3-NO2-4-HPA) was estimated spectrophotometrically at 430 nm following the addition of 0.5 mL of 0.5 N NaOH ( ) 4400 M-1 cm-1) (26). Analysis of Products. Known amounts of peroxynitrite (0.2-2.2 µmol) were decomposed by incubation with 2 mL of

Uppu et al. 0.1 M phosphate buffer, pH 7.0, containing 0.1 mM DTPA and 40 mM acetaldehyde or propionaldehyde for 2-5 min at room temperature. In the first set of experiments, the aldehyde was added 2 min before the addition of peroxynitrite; in the second, the aldehyde was added 2 min after the peroxynitrite, so the peroxynitrite had already decomposed. Nitrate and H2O2 in the decomposed peroxynitrite solutions were estimated by coupled enzyme assays using NADPH/nitrate reductase (6, 27) and ABTS/peroxidase (24, 28) systems, respectively. Nitrite was determined through measurement of an azo complex that results from the diazotization and subsequent coupling of sulfanilamide to N-(1-naphthyl)ethylenediamine in 0.4 N HCl (19, 29). Acetate was estimated using a coupled enzyme assay that involves phosphorylation of acetate by ATP/acetate kinase (30) followed by a second phosphorylation reaction of the product ADP by phospho(enol)pyruvate/pyruvate kinase (31). The product pyruvate formed in the second reaction was then coupled to the oxidation of NADH to NAD+ through use of lactic dehydrogenase (31), which was monitored spectrophotometrically at 340 nm ( ) 6220 M-1 cm-1).

Results and Discussion Aldehydes are close analogues of CO2. We reasoned that ONOO- forms addition products with RCHO in much the same way it reacts with CO2, and that the aldehyde/peroxynitrite adducts might unveil mechanistic details of the peroxynitrite/CO2 reaction. Chart 1 shows the structures of the primary adducts nitrosoperoxycarbonate (1) and 1-hydroxyalkylperoxynitrite (5) that can be formed from the reactions of ONOO- with CO2 and RCHO, respectively. The structures 2-4 and 6-8 represent the possible secondary products that can be formed from 1 and 5 (respectively) through a homolytic or heterolytic cleavage at the O-O bond and the subsequent recombination of the radical or ion pairs within the solvent cage. Except for protonation differences and the presence of electron-donating alkyl substituents, there are striking similarities between 1 and 5, 2 and 6, 3 and 7, and 4 and 8, respectively. We find that aldehydes do react rapidly with peroxynitrite and catalyze its decomposition to a species that does not absorb significantly between 260 and 390 nm. Figure 1A shows the pseudo-first-order rates for the decay of peroxynitrite (0.5 mM) at various initial concentrations of propionaldehyde (2-20 mM) at pH 7.0 and 25 ( 1 °C. The apparent second-order rate constant for the propionaldehyde/peroxynitrite reaction (kapp) is 530 ( 4 M-1 s-1. We have measured the apparent secondorder rate constants for reactions of peroxynitrite with several short-chain aliphatic aldehydes and some hydroxy and keto aldehydes. At pH 7.0 and 25 ( 1 °C, typical values of kapp for the reaction of most aldehydes are in the range of 102-103 M-1 s-1 (data not shown). Identification of the Reactant Pair. Aldehydes in aqueous solutions exist in an equilibrium mixture of free (RCHO) and hydrated [RCH(OH)2] forms (32, 33). Both RCHO and RCH(OH)2 have pKa values above 13 (34). Therefore, at and around pH 7.0, aldehydes and the corresponding hydrates exist predominantly in their protonated forms. Considering the protonation equilibrium of ONOO- (pKa ) 6.8) (35) and the forms in which aldehydes exist, the reactant pair in the aldehyde/ peroxynitrite reaction can be ONOO-/RCHO, ONOO-/ RCH(OH)2, ONOOH/RCHO, or ONOOH/RCH(OH)2. As part of the understanding of the nature of the reactants, we studied the pH dependence of peroxynitrite decay in the presence of excess propionaldehyde (Figure 1B). The values of kobs shown in Figure 1B represent the pseudo-

Reactions of Peroxynitrite with Aldehydes

Figure 1. (A) Pseudo-first-order rate constant (kobs) for the decomposition of peroxynitrite (0.5 mM) at 25 ( 1 °C in 0.1 M phosphate buffer, pH 7.0, containing 0.1 mM DTPA and 0-20 mM propionaldehyde. The values of kobs were corrected for the pH-induced self-decomposition of peroxynitrite. Each data point represents the mean of 6-10 measurements on the sample. (B) pH dependence of the observed pseudo-first-order rate constant (kobs) for the reaction of peroxynitrite with propionaldehyde. Peroxynitrite (0.5 mM) was allowed to react at 25 °C with 10 mM propionaldehyde in 0.1 M phosphate buffer, pH 5.3-7.5, containing 0.1 mM DTPA. The values of kobs have been corrected for the pH-induced self-decomposition of peroxynitrite. Each data point represents the mean of 6-10 measurements on the sample.

first-order rates for the propionaldehyde-catalyzed decomposition of peroxynitrite (kobs,ald). In these assays, the pseudo-first-order rates for the spontaneous decomposition of peroxynitrite (kobs,spont) varied between 1.06 and 0.18 s-1, depending on the pH. The values of kobs,spont were subtracted from the respective rates (kobs,total) to obtain the aldehyde-dependent rates of peroxynitrite decomposition. To test the influence of pH on the equilibrium ratio of [RCHO]/[RCH(OH)2], solutions of propionaldehyde (20 mM) in 0.1 M phosphate buffer at pH 5.2-7.8 were incubated for 30 min at 25 ( 1 °C and the concentration of free aldehyde was measured spectrophotometrically at 280 nm (33). We do not find significant changes in concentration of free aldehyde with the change in pH from 5.2 to 7.8. This shows that the hydration equilibrium also is not dependent on the pH under the conditions employed in the assay. As shown in Figure 1B, a plot of kobs versus the pH exhibits a sigmoidal profile with an inflection point (pH 6.2) close to the pKa of peroxynitrite (35). The pH dependence is consistent with the reactant pair being either ONOO-/RCHO or ONOO-/RCH(OH)2. We choose ONOO-/RCHO as the most probable reactant pair, consistent with a strong electrophile-nucleophile interaction between the electron-deficient carbonyl carbon atom of the aldehyde and the nucleophilic terminal oxygen atom of the peroxo group of ONOO- (eqs 1-3).

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Figure 2. Levels of NO3- (0, 1) and NO2- (b, O) in peroxynitrite solutions (0.1-1 mM) decomposed by incubation with 0.1 M phosphate buffer, pH 7.0, containing 0.1 mM DTPA and 0 or 40 mM propionaldehyde at 25 °C: open symbols, peroxynitrite decomposed in the absence of propionaldehyde; closed symbols, peroxynitrite decomposed via the reaction with propionaldehyde. Each data point represents the mean ((SD) of two measurements carried out in either duplicate or triplicate.

The reaction of ONOO- with RCHO gives the 1-hydroxyalkylperoxynitrite anion (5a) (eq 4) which, presumably, is in equilibrium with its protonated form, 1-hydroxyalkylperoxynitrite (5b) (eq 5).

Products of Peroxynitrite Reaction with Aldehydes. (A) Nitrite Is Not Formed. As shown in Figure 2, at pH 7.0, the yields of NO2- do not differ whether peroxynitrite was decomposed in the absence (curve B) or presence (curve A) of 40 mM propionaldehyde. From the apparent second-order rate constant of 530 M-1 s-1 for the propionaldehyde/peroxynitrite reaction, it can be calculated that in assays that contained 40 mM propionaldehyde, more than 98% of peroxynitrite decomposed by reaction with the aldehyde. In the absence of propionaldehyde, at pH 7.0, the decomposition of peroxynitrite proceeds primarily through the isomerization of ONOOH, resulting in the formation of NO3(36, 37). Therefore, the NO2- in these solutions represents nitrite contamination in the original peroxynitrite preparation and not NO2- formed during the decomposition of peroxynitrite. (B) Nitrate Is a Major Product. In the absence of additives that react with peroxynitrite, the concentration of NO3- in solutions of peroxynitrite decomposed at pH 7.0 (Figure 2, curve C) represents the contamination of nitrate in the original peroxynitrite preparation plus nitrate formed from the isomerization of peroxynitrite. Assuming that the isomerization of peroxynitrite gives stoichiometric yields of NO3- (36, 37), the NO3- contamination in the original peroxynitrite preparation was calculated to be 1.06 mol/mol of peroxynitrite. When

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Uppu et al.

Scheme 1. Proposed Mechanism of Decay of 1-Hydroxyalkylperoxynitrite Formed from the Reaction of Peroxynitrite Anion and Free Aldehyde

corrected for this NO3-, the yield of nitrate was 0.65 ( 0.12 mol/mol of peroxynitrite decomposed in the presence of 40 mM propionaldehyde at pH 7.0 (Figure 2, curve D). This shows that the aldehyde-assisted decomposition of peroxynitrite gives NO3- as a major product, but the yields are substantially lower than that observed during the spontaneous decay of peroxynitrite (Figure 2, curve C). As shown in Figure 2 (curve B), we do not find any indication of increased formation of NO2- that, in combination with the lowered yields of NO3-, could explain the nitrogen balance in the aldehyde/peroxynitrite system. This lack of nitrogen balance suggests that organic nitrates are formed as minor products in the aldehyde/ peroxynitrite system. Such a proposition is supported by the fact that the enzyme nitrate reductase used in these assays for the determination of NO3- does not reduce organic nitrates (27). (C) Organic Acids Are Formed in Low Yields. We measured the yield of acetate in reaction mixtures in which a low concentration of peroxynitrite (1 mM) was allowed to react with excess acetaldehyde (40 mM) at pH 7.0. (We chose to study the reaction of acetaldehyde because it was convenient to estimate the product acetate using a coupled-enzyme assay.) Calculations based on the concentration of acetaldehyde and the apparent second-order rate constant for the acetaldehyde/peroxynitrite reaction (kapp ) 830 ( 5 M-1 s-1) suggest that more than 99% of peroxynitrite in these reactions reacted with acetaldehyde. The yield of acetate was found to be 0.19 ( 1 mol/mol of peroxynitrite employed in the reaction. This yield is negligibly small in the control assays in which decomposed solutions of peroxynitrite were used. (D) H2O2 Is Formed in Small Yields. Hydrogen peroxide is produced in yields of about 0.04 mol/mol of peroxynitrite decomposed via the reaction with propionaldehyde. In these reactions, a low concentration of peroxynitrite (1.1 mM) was allowed to react with excess propionaldehyde (40 mM) at pH 7.0. The assay of H2O2 was based on the horseradish peroxidase coupled oxidation of ABTS to ABTS•+ (28). Preincubation of peroxynitrite-treated aldehyde solutions with catalase nearly abolished the oxidation of ABTS, confirming that most

of the peroxidic material formed is H2O2. The yield of H2O2 is negligibly small (0.2 mol %) in the control assays in which decomposed solutions of peroxynitrite were used. Possible Mechanisms for the Aldehyde-Assisted Isomerization of Peroxynitrite. A mechanism for the aldehyde-catalyzed decay of peroxynitrite must be consistent with these observations: (i) Free aldehyde and ONOO- are the reactants. (ii) NO3- and regenerated aldehyde3 are the major products. (iii) Small but significant yields of H2O2, organic acids, and organic nitrates are produced. A mechanism that fits these data is given in Scheme 1. According to this scheme, the adduct 1-hydroxyalkylperoxynitrite undergoes a homolytic or heterolytic cleavage at the weak O-O bond giving a caged radical pair [RCH(OH)O•/•NO2] (7) or an intimate ion pair [RCH(OH)O-/+NO2] (8). The radical 7 or ion 8 pairs can recombine within the respective solvent cage to form 1-hydroxyalkylnitrate [RCH(OH)ONO2] (6), which then dissociates to give RCHO and NO3- (eq 6).

The product 6 is the addition product of aldehyde and nitric acid. Carbonyl compounds have been reported to form 1:1 adducts with nitric acid (in 99.5% nitric acid solvent) at -40 to -15 °C (38). These adducts exist in the form of oxonium ion/nitrate salts (39). The pKa of 3 In assays pertaining to the stoichiometric analysis of products, the concentration of aldehyde was at least 40-fold higher than the concentration of peroxynitrite (see curves C and D in Figure 2). A high concentration of aldehyde was required to compete with the spontaneous decomposition of peroxynitrite and allow a quantitative reaction of peroxynitrite with the aldehyde. Therefore, it was difficult to establish the regeneration of aldehyde by direct measurement of the changes in aldehyde concentration before and after the reaction with peroxynitrite. Circumstantial evidence for the regeneration of aldehyde comes from the observation that there were no substantial (stoichiometric) yields of organic acids, alcohols, and nitrite that are characteristic of the oxidation-reduction reactions of aldehydes and peroxynitrite. As shown in eq 6, the regeneration of aldehyde is consistent with the formation of nitrate as a major product.

Reactions of Peroxynitrite with Aldehydes

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Scheme 2. Schematic Representation of the Formation of Various Minor Products in the Aldehyde/Peroxynitrite System Involving (A) an Alkoxyl [RCH(OH)O•] and/or (B) an Acyl Radical [R(CdO)•]

Figure 3. Oxidation of ABTS (2 mM) by peroxynitrite (0. 1 mM) in the presence of propionaldehyde (0-40 mM) in 2 mL of 0.1 M phosphate buffer, pH 7.0, containing 0.1 mM DTPA at 25 °C. The ABTS radical cation formed was measured spectrophotometrically at 660 nm ( ) 12 000 M-1 cm-1). Each data point represents the mean ((SD) of two measurements carried out in either duplicate or triplicate. Inset is a double-reciprocal plot of fractional inhibition of ABTS oxidation against the concentration of propionaldehyde.

the aldehyde oxonium ion is -8 (40). Therefore, we presume that, at pH 7.0, the equilibrium for the reaction shown in eq 6 lies far to the right, and this precludes the existence of an appreciable concentration of 6 under steady-state conditions. We suggest that the homolytic cleavage of 5 and the subsequent escape of RCH(OH)O• and •NO2 radicals from the solvent cage are responsible for small yields of H2O2, organic acids, and organic nitrates. Scheme 2 outlines reactions that can explain the formation of these minor products based on the known chemistry of acyl, alkoxyl, and/or nitrogen dioxide radicals (41, 42). Oxidative Chemistry of Aldehyde/Peroxynitrite Adducts. To understand the oxidative chemistry of 1-hydoxyalkylperoxynitrite (5) and the secondary products derived from it, we have studied two known reactions of peroxynitrite: the oxidation of ABTS (Figure 3) and the nitration of 4-hydroxyphenylacetate at pH 7.0 (Figure 4). Propionaldehyde, which was used as a representative aldehyde, was included in the assay at a final concentration of 0-40 mM, allowing the isomerization of peroxynitrite to form NO3- to go from predominantly H+-catalyzed (37, 43) to more than 97% aldehydeassisted. In the absence of propionaldehyde, the extent of oxidation of ABTS is 0.36 mol/mol of peroxynitrite (Figure 3). Addition of propionaldehyde (0-40 mM) results in a progressive decrease in the ABTS oxidation to about 0.15 mol/mol of peroxynitrite (Figure 3). The maximum possible inhibition of ABTS oxidation, calculated using a Scatchard-like plot (44), is 57%, and the yield of ABTS•+ under saturating conditions is 0.15 mol/mol of peroxynitrite (Figure 3, inset). The yield of ABTS•+ in the peroxynitrite/CO2/ABTS system is 0.40 mol/mol peroxynitrite (6),4 150% higher than in the aldehyde/peroxynitrite system. This shows that aldehyde/peroxynitrite adducts (5-8) (Scheme 1), or the secondary intermediates 4 In the peroxynitrite/CO /ABTS system, the yields of ABTS•+ were 2 calculated using 404nm ) 36 800 M-1 s-1 based on a report by Everse et al. [Everse, J., Johnson, M. C., and Marini, M. A. (1994) Peroxidase activities of hemoglobin and hemoglobin derivatives. Methods Enzymol. 231, 547-561]. This molar absorption coefficient value is in error, and

Figure 4. Nitration of 4-HPA (4 mM) by peroxynitrite (1 mM) in the presence of propionaldehyde (0-40 mM) in 2 mL of 0.1 M phosphate buffer, pH 7.0, containing 0.1 mM DTPA at 25 °C. The 3-nitro-4-hydroxyphenylacetate formed was measured spectrophotometrically at 430 nm following the addition of 0.5 mL of 0.5 N NaOH ( ) 4400 M-1 s-1). Each data point represents the mean ((SD) of two measurements carried out in either duplicate or triplicate. Inset is a double-reciprocal plot of fractional inhibition of 4-HPA nitration against the concentration of propionaldehyde.

derived from them (Scheme 2), mediate the oxidation of ABTS, but to a lesser extent than do peroxynitrite/CO2 adducts (1-4). Although the list of potential oxidants in the aldehyde/peroxynitrite system includes 5, 6, •NO2, +NO , and RCH(OH)O•, we eliminate 6 and +NO for the 2 2 following reasons: We eliminate 6 because this intermediate may not exist at an appreciable concentration under steady-state conditions (discussed above). Regarding the involvement of +NO2 in the oxidation of ABTS in the aldehyde/peroxynitrite system, +NO2 reacts rapidly with water to give NO3- (k ) 5 × 108 s-1) (45), and therefore, the correct value is 25 000 M-1 s-1 (S. Goldstein, personal communication). Once corrected for this difference in molar absorption coefficient, the maximum yield of ABTS•+ in the peroxynitrite/CO2/ABTS system is 0.40 mol/mol of peroxynitrite.

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the reaction of ABTS (2 mM) with +NO2 occurs in competition with the reaction of water. The rate constant for the +NO2 reaction with ABTS is not known; however, even if it is assumed that the +NO2/ABTS reaction proceeds at a rate close to the diffusion limit (1 × 1010 M-1 s-1), the maximum yield of ABTS•+ in the aldehyde/ peroxynitrite reaction should have been less than 0.04 mol/mol of peroxynitrite, a value smaller than the observed yield of 0.15 mol of ABTS•+/mol of peroxynitrite (Figure 3, inset). Figure 4 shows the formation of 3-nitro-4-hydroxyphenylacetate (3-NO2-4-HPA) when a low concentration of peroxynitrite (1 mM) was allowed to react with excess 4-HPA (4 mM) in the presence of 0-40 mM propionaldehyde at pH 7.0 and 25 °C. In the absence of propionaldehyde, peroxynitrite reacts with 4-HPA giving a 8.2 mol % yield of 3-NO2-4-HPA (Figure 4) (the yield of 3-NO2-4-HPA was expressed based on the concentration of peroxynitrite employed in the reaction). The yield of 3-NO2-4-HPA progressively decreases to a value of about 1.5 mol % as the concentration of propionaldehyde in the reaction mixture increases to 40 mM (Figure 4). The maximum possible inhibition of 4-HPA nitration by the aldehyde, calculated using a Scatchard-like plot (44), is 90% (Figure 4, inset). Thus, under saturating conditions of the aldehyde, the yield of 4-HPA nitration in the aldehyde/peroxynitrite system is e1 mol % (based on the concentration of peroxynitrite). The yield of 4-HPA nitration by the peroxynitrite/CO2 adducts (1-4) is 20 mol % (6). This large difference (g20-fold) in the yield of 3-NO2-4-HPA in the two systems suggests that adducts 5, 6, RCH(OH)O•/•NO2, and RCH(OH)O-/+NO2 are poor mediators of phenolic nitration.

General Implications Aldehydes are ubiquitous in biological systems. Several biogenic aldehydes (such as glyceraldehyde-3phosphate, pyridoxal-5-phosphate, and retinal) serve as key intermediates in metabolism (46). Aldehydes also are products of lipid peroxidation and are formed in substantially larger amounts as a result of natural and xenobiotic-induced oxidative stress (47, 48). Nevertheless, we do not believe that aldehydes compete with CO2 for the reaction of peroxynitrite in vivo. This conclusion stems from the fact that carbonated species are abundant (g25 mM) in all physiological fluids and the values of kapp for the aldehyde/peroxynitrite reaction are at least 40-300 times smaller than the rate constant for the CO2/ peroxynitrite reaction.5 Nevertheless, aldehyde/peroxynitrite reactions are an instructive variation on the chemistry of peroxynitrite/ CO2 adducts. Both the nature of the reactants and the product profiles are similar in these two systems, but a striking difference lies in their ability to bring about phenolic nitration. The peroxynitrite-mediated nitrations are facilitated by CO2 by 3-4-fold (6-8, 10-12, 49). Aldehydes, on the other hand, bring about a near total inhibition of phenolic nitration by peroxynitrite (Figure 4). Chart 1 shows the comparable intermediates in the two systems. The list of potential oxidants responsible for phenolic nitration in the peroxynitrite/CO2 system 5 It is possible that aldehydes react with 1 or the secondary products 2-4 derived from 1. These reactions may have some physiological significance in terms of producing organic acids and organic nitrates. We are currently investigating the reactions of peroxynitrite with aldehydes in the presence of excess HCO3-/CO2.

Uppu et al.

includes 1, 2, CO3•-/•NO2, and +NO2/CO32-. We eliminate 1 and +NO2/CO32- from the list for the following reasons. If +NO2/CO32- performs nitrations, one also would expect nitration by the ion pair consisting of the RCH(OH)O-/ + NO2, but no nitration is observed in the aldehyde/ peroxynitrite system. We considered the possibility that 1 brings about nitration by a molecule-assisted homolytic (MAH) type of reaction (50), as suggested by Hurst and his colleagues (49). Here again, if 1 performs nitration, one would expect nitration by 1-hydroxyalkylperoxynitrite formed in the aldehyde/peroxynitrite system, which was not found to be the case (Figure 4). Therefore, by comparison of these intermediates, we suggest that 2 and/or CO3•-/•NO2 are responsible for nitration of phenolic substrates by peroxynitrite in vivo. Why does the radical pair RCH(OH)O•/•NO2 not give nitration in the way the pair CO3•-/•NO2 is proposed to do in the peroxynitrite/CO2 system (10-12)? The carbonate radical is a strong one-electron oxidant [E°(HCO3•/ HCO3-) ) 1.5 V] (51), and the nitration of phenol [E°(phenoxyl/phenol) ) 0.8 V] (52) by CO3•-/•NO2 presumably proceeds via the intermediate formation of phenoxyl radical which then reacts with •NO2 to yield the nitro product(s). The reason for low yields of nitration in the aldehyde/peroxynitrite system is that RCH(OH)O• is not a good one-electron oxidant and that •NO2 itself is a poor nitrating species (53).

Acknowledgment. This publication was made possible by Grant ES-06754 from the National Institute of Environmental Health Sciences, NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIEHS, NIH. We thank Prasanna L.Uppu for her technical help with stopped-flow kinetic measurements.

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