Title: Superoxide Reacts with Nitric Oxide to Nitrate Tyrosine at Physiological pH via Peroxynitrite
Abstract: Tyrosine nitration is a widely used marker of peroxynitrite (ONOO−) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280–27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkalinecis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo. Tyrosine nitration is a widely used marker of peroxynitrite (ONOO−) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280–27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkalinecis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo. In the decade since its discovery, nitric oxide has been shown to have multiple physiological actions and is implicated in the pathology of a wide range of diseases. However, nitric oxide itself is neither highly reactive nor particularly toxic, but rather forms secondary oxidants responsible for tissue injury. A major pathway that enhances the toxicity of nitric oxide is the near diffusion-limited reaction with superoxide to form peroxynitrite (ONOO−). One of the most easily identified products of peroxynitrite attack on proteins is 3-nitrotyrosine (2Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (912) Google Scholar). Nitrotyrosine has been identified in human atherosclerosis, pulmonary and heart disease, acute and chronic kidney rejection, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (3Beckman J.S. Ye Y.Z. Anderson P. Chen J. Accavetti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1069) Google Scholar, 4Kooy N.W. Lewis S.J. Royall J.A. Ye Y.Z. Kelly D.R. Beckman J.S. Crit. Care Med. 1997; 25: 812-819Crossref PubMed Scopus (215) Google Scholar, 5Kooy N.W. Royall J.A. Ye Y.Z. Kelly D.R. Beckman J.S. Am. J. Respir. Crit. 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Ishida H. Ichimori K. Nakazawa H. Free Radic. Biol. Med. 1997; 22: 771-774Crossref PubMed Scopus (164) Google Scholar). From 1 to 3% of tyrosine in spinal cords of amyotrophic lateral sclerosis patients is nitrated (22Ferrante R.J. Shinobu L.A. Schulz J.B. Matthews R.T. Thomas C.E. Kowall N.W. Gurney M.E. Beal M.F. Ann. Neurol. 1997; 42: 326-334Crossref PubMed Scopus (219) Google Scholar, 23Ferrante R.J. Browne S.E. Shinobu L.A. Bowling A.C. Baik M.J. MacGarvey U. Kowall N.W. Brown Jr., R.H. Beal M.F. J. Neurochem. 1997; 69: 2064-2074Crossref PubMed Scopus (657) Google Scholar). The mechanisms by which tyrosine is nitrated in vivo remain an area of active investigation and controversy (2Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (912) Google Scholar, 24Eiserich J.P. Cross C.E. Jones A.D. Halliwell B. van der Vliet A. J. Biol. Chem. 1996; 271: 19199-19208Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 25Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1357) Google Scholar). Most experiments have used preformed alkaline peroxynitrite, which can be commercially purchased or easily synthesized by a variety of methods (26Koppenol W.H. Kissner R. Beckman J.S. Methods Enzymol. 1995; 269: 296-302Crossref Google Scholar, 27Pryor W.A. Cueto R. Jin X. Koppenol W.H. Ngu-Schwemlein M. Squadrito G.L. Uppu P.L. Uppu R.M. Free Radic. Biol. Med. 1995; 18: 75-83Crossref PubMed Scopus (233) Google Scholar). Peroxynitrite in alkaline solution is present only in thecis conformation (28Tsai J.-H.M. Harrison J.G. Martin J.C. Hamilton T.P. van der Woerd M. Jablonsky M.J. Beckman J.S. J. Am. Chem. Soc. 1994; 116: 4115-4116Crossref Scopus (119) Google Scholar, 29Tsai H.-H. Hamilton T.P. Tsai J.-H. van der Woerd M. Harrison J.G. Jablonsky M.J. Beckman J.S. Koppenol W.H. J. Phys. Chem. 1996; 100: 15087-15095Crossref Scopus (82) Google Scholar), which contributes to the unusual stability of peroxynitrite by preventing the rearrangement of the terminal oxygen to form nitrate (NO3−). Curiously, peroxynitrite in the solid state is entirely in thecis conformation (28Tsai J.-H.M. Harrison J.G. Martin J.C. Hamilton T.P. van der Woerd M. Jablonsky M.J. Beckman J.S. J. Am. Chem. Soc. 1994; 116: 4115-4116Crossref Scopus (119) Google Scholar, 30Lo W.J. Lee Y.P. Tsai J.H. Tsai H.H. Hamilton T.P. Harrison J.G. Beckman J.S. J. Chem. Phys. 1995; 103: 4026-4034Crossref Scopus (34) Google Scholar). The x-ray structure of the tetramethylammonium salt of peroxynitrite has recently been determined and was also in the cis conformation (31Worle M. Latal P. Kissner R. Nesper R. Koppenol W.H. Chem. Res. Toxicol. 1999; 12: 305-307Crossref PubMed Scopus (23) Google Scholar). However, it remains possible that nitric oxide plus superoxide reacting at neutral pH might form peroxynitrite in a different conformation or in an excited electronic state that could be substantially less reactive than preformed cis-peroxynitrite (1Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Co-generation of superoxide and nitric oxide is experimentally difficult to control. The products and reactants used to generate superoxide and nitric oxide can competitively inhibit tyrosine nitration (32Goldstein S. Czapski G. Lind J. Merényi G. J. Biol. Chem. 2000; 275: 3031-3036Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). For example, urate produced by xanthine oxidase is a strong antioxidant and decreases both dihydrorhodamine oxidation and phenolic nitration by peroxynitrite (33Kooy N.W. Royall J.A. Ischiropoulos H. Beckman J.S. Free Radic. Biol. Med. 1994; 16: 149-156Crossref PubMed Scopus (670) Google Scholar, 34Skinner K.A. Crow J.P. Skinner H.B. Chandler R.T. Thompson J.A. Parks D.A. Arch. Biochem. Biophys. 1997; 342: 282-288Crossref PubMed Scopus (59) Google Scholar). A second issue is the limited availability of dissolved oxygen in buffer, which is generally in the range of 200–250 μm depending upon ionic strength and temperature. Both xanthine oxidase and SIN-1 can quickly make a solution anaerobic, thereby stopping superoxide production unless special precautions are taken to refresh oxygen supplies (35Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Crossref PubMed Scopus (264) Google Scholar). However, increased nitration of tyrosine in cellular proteins by co-generation of nitric oxide plus superoxide has been demonstrated by a number of laboratories (7Ara J. Przedborski S. Naini A.B. Jackson-Lewis V. Trifiletti R.R. Horwitz J. Ischiropoulos H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7659-7663Crossref PubMed Scopus (381) Google Scholar, 18Zou M.-H. Ullrich V. FEBS Lett. 1996; 382: 101-104Crossref PubMed Scopus (202) Google Scholar, 36Haddad I.Y. Crow J.P. Hu P. Ye Y.Z. Beckman J.S. Matalon S. Am. J. Physiol. 1994; 267: L242-L249PubMed Google Scholar). Co-generation of nitric oxide and superoxide has been shown to be equally toxic or slightly more toxic than the bolus addition of peroxynitrite to bacteria and cultured cells (35Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Crossref PubMed Scopus (264) Google Scholar,37Shin J.T. Barbeito L. MacMillan-Crow L.A. Beckman J.S. Thompson J.A. Arch. Biochem. Biophys. 1996; 335: 32-41Crossref PubMed Scopus (39) Google Scholar). Recently, Pfeiffer and Mayer (1Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) reported that the co-generation of superoxide from xanthine oxidase and nitric oxide from spermine NONOate yielded far less nitrotyrosine than produced by preformed alkaline peroxynitrite. They confirmed the rate of peroxynitrite formation by monitoring the oxidation of dihydrorhodamine. To explain the apparent lack of nitration, Pfeiffer and Mayer (1Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) proposed that superoxide and nitric oxide react at neutral pH following a pathway whose intermediate products decay without nitrating tyrosine. To address whether peroxynitrite was formed from nitric oxide reacting with superoxide at neutral pH, we developed a stopped-flow method to mix potassium superoxide with nitric oxide in phosphate buffer. We report that this resulted in the stoichiometric formation of peroxynitrite, which gave the same yields of nitrotyrosine as with preformed alkaline peroxynitrite. Xanthine oxidase was purchased from CalBiochem (La Jolla, Ca). All other reagents were purchased from Sigma-Aldrich. All water was deionized and had a conductivity greater than 18 megaohms. Dimethyl sulfoxide (Me2SO) was dried over 3-Å molecular sieves and used to dissolve potassium superoxide (KO2). Alkaline peroxynitrite was synthesized from acidified nitrite and hydrogen peroxide as described previously (38Beckman J.S. Wink D.A. Crow J.P. Feelisch M. Stamler J.S. Methods in Nitric Oxide Research. John Wiley & Sons Ltd., Chichester1996: 61-70Google Scholar). Generally, hydrogen peroxide was not removed by treatment with manganese dioxide since it did not interfere in the present assays. To investigate whether peroxynitrite was generated at neutral pH, we utilized an SX.18MV stopped-flow spectrophotometer (Applied Photophysics, Leatherhead, United Kingdom) to rapidly mix superoxide dissolved in anhydrous Me2SO with nitric oxide dissolved in anaerobic phosphate buffer. The stopped-flow spectrophotometer was fitted with two different sized drive syringes to minimize the final concentration of Me2SO. A 100-μl drive syringe holding Me2SO/KO2 was placed in the left most mixing port of the stopped-flow, while a second 2.5-ml syringe contained nitric oxide in anaerobic buffer (Fig.1). Because of the 25-fold dilution of Me2SO/KO2 with the asymmetric mixing in the stopped-flow cuvette, the final Me2SO concentration was 0.5m. A stock solution of KO2 was prepared by adding an excess of KO2 (5 g) to 5 ml of Me2SO, strongly mixed with a vortex mixer for 1 min at room temperature, and centrifuged at 1,000 × g for 5 min to remove excess solid KO2. This yielded a stock concentration of approximately 3.6 mm KO2 in Me2SO. Solid KO2 is caustic and potentially combustible, requiring appropriate caution in handling and waste disposal. The final concentration of superoxide in the stopped-flow cell was determined by monitoring the reduction of cytochrome c substituted for nitric oxide in drive syringe B (Fig. 1). A light-path of 2 mm was used due to the strong absorption of cytochrome c. The smaller drive syringe A was filled with Me2SO/KO2 and the larger syringe was filled with 100 mm sodium phosphate, 100 μm diethylenetriaminepentaacetic acid, and 200 μm cytochrome c and the reaction was followed at 550 nm (Δε550 nm = 21 mm−1cm−1). An apparent rate of 1 × 106m−1 s−1 was obtained from the converted reduction traces by non-linear fit of a second order equation and was consistent with that previously determined by pulse radiolysis (39Butler J. Koppenol W.H. Margoliash E. J. Biol. Chem. 1982; 257: 10747-10750Abstract Full Text PDF PubMed Google Scholar). To work with nitric oxide in the stopped-flow spectrophotometer, solutions in the 2.5-ml syringe were carefully maintained under anaerobic conditions following the manufacturer's instructions. A tonometer designed for anaerobic work was fitted to the loading port of drive syringe B. Both the bottom of the drive syringes and the nitric oxide tonometer junction were shrouded with a continuous flow of nitrogen to minimize oxygen leakage into the drive syringes (Fig. 1). In addition, the water bath was continuously bubbled with nitrogen. Sodium dithionite (500 mg) was also directly added to the water in the bath to further scavenge residual oxygen. Buffer in the tonometer was bubbled with helium for 10 min to remove dissolved oxygen and carbon dioxide before addition of nitric oxide. Residual carbon dioxide dissolved in non-sparged buffers increased tyrosine nitration to a variable extent by 1–2-fold. The stock buffer was 104 mmsodium phosphate plus 104 μmdiethylenetriaminepentaacetic acid at pH 7.37. Nitric oxide was added from a saturated stock solution to sparged buffer in the tonometer and mixed by repeatedly drawing the NO solution in and out of drive syringe B. The nitric oxide stock solution was prepared by bubbling 100 ml of helium-sparged 104 mm sodium phosphate, pH 7.37, at room temperature in a 300-ml gas sampling tube (Fisher Scientific Inc., Pittsburgh, PA) with 100% nitric oxide gas for 5 min. The nitric oxide solution was determined to be 1.32 mm by measuring the oxidation of 6 μm oxy-hemoglobin tetramer to met-hemoglobin (40Kelm M. Dahmann R. Wink D. Feelisch M. J. Biol. Chem. 1997; 272: 9922-9932Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The differential extinction coefficient at 401 nm of 52.1 mm−1cm−1 was used to calculate the amount ofmet-hemoglobin formed (40Kelm M. Dahmann R. Wink D. Feelisch M. J. Biol. Chem. 1997; 272: 9922-9932Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The dissolved nitric oxide concentration was less than in distilled water due to the increased ionic strength of the buffer. The concentration of nitric oxide after dilution in the stopped-flow tonometer was confirmed by removing a small amount of the solution via a gas-tight Hamilton syringe fitted with a cannula for assay with oxy-hemoglobin. To study the nitration of tyrosine, we added 1.04 mmtyrosine to the buffer in the tonometer before adding nitric oxide. There was no detectable nitrotyrosine formation in this solution for as long as 4 h. To study the effects of dissolved CO2, we added a final concentration of 26 mm sodium bicarbonate to sparged buffer before addition of nitric oxide. The bicarbonate-containing buffer was then sparged briefly with helium to remove any oxygen introduced with the bicarbonate. The bicarbonate was then allowed to re-establish equilibrium with free CO2 for 2 min before addition of nitric oxide. The final pH of all buffers was 7.43, which was measured in the stopped-flow effluent. All stopped-flow reactions were performed at 37 °C. Alkaline stock peroxynitrite was used as a comparison to the nitric oxide/superoxide system by placing alkaline peroxynitrite in the 100-μl drive syringe in place of superoxide. Because peroxynitrite stock solutions are unstable in concentrated Me2SO, we controlled for the effects of Me2SO by adding 0.5m Me2SO into the 2.5-ml drive syringe B with the buffer. Because the mixing chamber in the stopped-flow is not symmetric with respect to the drive syringes, greater mixing artifacts were apparent during the initial mixing but did not interfere with the last 90% of the reaction course. Decay of peroxynitrite anion was followed at 302 nm (1.70 mm−1 cm−1) (41Bohle D.S. Hansert B. Paulson S.C. Smith B.D. J. Am. Chem. Soc. 1994; 116: 7423-7424Crossref Scopus (120) Google Scholar). Formation of nitrotyrosine was observed at 430 nm (3.87 mm−1 cm−1 as determined using nitrotyrosine standards in the stopped-flow instrument under identical reaction conditions). Reaction rates were determined using Applied Photophysics stopped-flow software. The spectrum of the intermediate product from the reaction of superoxide and nitric oxide was constructed from consecutive stopped-flow traces between 250 and 450 nm taken in 5-nm steps. These data plus the initial concentrations of superoxide and nitric oxide were used by a single value decomposition algorithm in the kinetic software Pro-K supplied with the Applied Photophysics stopped-flow spectrophotometer to estimate the spectrum and extinction coefficient of the intermediate product generated from superoxide and nitric oxide as a function of wavelength. The spectrum of preformed peroxynitrite decreases at more acidic pH because peroxynitrous acid does not absorb significantly in the near UV. The pK a of peroxynitrite in phosphate buffer is 6.8 (42Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1277) Google Scholar), which was used to calculate the apparent absorption spectrum at pH 7.4 via the Henderson-Hasselbalch equation. The pH-adjusted extinction coefficient is thus εpH = εONOO− × 10pH/(10pH+106.8), where εONOO− is the extinction coefficient of peroxynitrite at alkaline pH. At 302 nm, the apparent extinction coefficient for peroxynitrite decreases from 1.7 mm−1 cm−1 (41Bohle D.S. Hansert B. Paulson S.C. Smith B.D. J. Am. Chem. Soc. 1994; 116: 7423-7424Crossref Scopus (120) Google Scholar) at alkaline pH to 1.36 mm−1 cm−1. We repeated the xanthine oxidase/spermine NONOate co-generation experiments of Pfeiffer and Mayer (1Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) at 22 °C using 10 milliunits of xanthine oxidase, 1 mm hypoxanthine, 1 mm tyrosine, and 1 mm spermine NONOate in 50 mm potassium phosphate, pH 7.4. The activity of xanthine oxidase was measured using xanthine and 1 unit was defined as the amount of enzyme needed to produce 1 μmol of urate min−1at 25 °C in 50 mm potassium phosphate, pH 7.4. Urate formation was measured at 292 nm (ε = 1.1 × 104m−1 cm−1) (43Fridovich I. J. Biol. Chem. 1970; 245: 4053-4057Abstract Full Text PDF PubMed Google Scholar). Superoxide was measured by the reduction of cytochrome c at 550 nm as described above. Dihydrorhodamine oxidation was followed by withdrawing 1-ml aliquots of the xanthine oxidase reaction at timed intervals, adding dihydrorhodamine, and then measuring the rate of increase in absorbance at 550 nm. The accumulation of an inhibitor of tyrosine nitration was monitored by adding 0.5 mmperoxynitrite to aliquots taken at progressively longer times following the addition of xanthine oxidase. Superoxide reacted with nitric oxide at neutral pH to produce an intermediate product with the same spectrum as preformed alkaline peroxynitrite (Fig. 2). The solid line shown in Fig. 2 is not a fitted curve to the experimental data, but rather the spectrum of cis-peroxynitrite at alkaline pH adjusted to account for the 20% peroxynitrous acid present at pH 7.4. Utilizing all of the experimental data in Fig. 2 and the starting concentration of superoxide, a simple irreversible bimolecular reaction was fitted by a single value decomposition algorithm and yielded an extinction coefficient of 1.34 mm−1s−1 at 302 nm for the intermediate product at pH 7.4. As described under “Materials and Methods,” the expected apparent extinction coefficient for peroxynitrite at pH 7.4 is 1.36 mm−1 s−1 when taking into account the 20% peroxynitrous acid present. The formation of the intermediate product was complete within the dead time of the stopped-flow spectrophotometer, consistent with the near diffusion-limited rate reported for the reaction of superoxide with nitric oxide (44Huie R.E. Padmaja S. Free Rad. Res. Commun. 1993; 18: 195-199Crossref PubMed Scopus (2015) Google Scholar). The intermediate product then decomposed at the same rate as preformed peroxynitrite (Fig. 3). The yield of peroxynitrite was 105 ± 9% of the starting superoxide and 101 ± 10% of the nitric oxide concentration (mean ± S.D. from seven separate experiments). These data indicated that peroxynitrite was quantitatively produced at neutral pH in the stopped-flow system. When tyrosine was added to the syringe buffer plus nitric oxide, equimolar nitric oxide plus superoxide produced the same amount of nitrotyrosine and at the same rate as produced by preformed peroxynitrite (Fig. 2). Carbon dioxide present in the buffer accelerated the decomposition of the intermediate product from superoxide and nitric oxide (Fig. 4) and increased the yield of nitrotyrosine by 3–4-fold to the same extent as preformed peroxynitrite (Table I). Reducing the initial concentration of nitric oxide resulted in a proportional decrease in the formation of peroxynitrite and nitrotyrosine. In the absence of superoxide, even saturated solutions of nitric oxide mixed with 100% oxygen-saturated phosphate buffer containing 1 mm tyrosine did not yield detectable nitrotyrosine after as long as 2000 s. Me2SO, necessary to dissolve superoxide, did not interfere with nitration by peroxynitrite in the presence or absence of carbon dioxide as previously reported (45Beckman J.S. Ischiropoulos H. Zhu L. van der Woerd M. Smith C. Chen J. Harrison J. Martin J.C. Tsai M. Arch. Biochem. Biophys. 1992; 298: 438-445Crossref PubMed Scopus (733) Google Scholar, 46Van der Vliet A. Eiserich J.P. O'Neill C.A. Halliwell B. Cross C.E. Arch. Biochem. Biophys. 1995; 319: 341-349Crossref PubMed Scopus (374) Google Scholar).Table IObserved yields and rates of the reactions producing peroxynitrite and nitrotyrosinePeroxynitrite decayRate of tyrosine nitrationYield nitrotyrosines −1%No CO2O⨪2/NO1.8 ± 0.13 (10)1.6 ± 0.425.3 ± 0.4ONOO−1.9 ± 0.05 (5)1.7 ± 0.155.1 ± 0.4With CO2O⨪2/NO76 ± 9.4 (9)77 ± 1114 ± 1.1ONOO−75 ± 3.5 (10)83 ± 5.815 ± 1.0Rates were calculated by fitting the equation y =y 0 + A 0 e −k t to the stopped-flow reaction traces. Reactions were conducted with 100 μm peroxynitrite or 100 μm nitric oxide plus 100 μm superoxide. Nitrotyrosine yield is calculated as the final nitrotyrosine concentration divided by the initial concentration of peroxynitrite and expressed as a percentage. Open table in a new tab Rates were calculated by fitting the equation y =y 0 + A 0 e −k t to the stopped-flow reaction traces. Reactions were conducted with 100 μm peroxynitrite or 100 μm nitric oxide plus 100 μm superoxide. Nitrotyrosine yield is calculated as the final nitrotyrosine concentration divided by the initial concentration of peroxynitrite and expressed as a percentage. Consistent with the results of Pfeiffer and Mayer (1Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), we have observed little nitration of free tyrosine using hypoxanthine plus xanthine oxidase with spermine NONOate to generate superoxide and nitric oxide. Hypoxanthine, xanthine oxidase, and spermine NONOate tested separately did not interfere with nitration by bolus additions of peroxynitrite. However, hypoxanthine is oxidized to urate by xanthine oxidase and urate is known to inhibit peroxynitrite-mediated nitration (47Skinner K.A. White C.R. Patel R. Tan S. Barnes S. Kirk M. Darley-Usmar V. Parks D.A. J. Biol. Chem. 1998; 273: 24491-24497Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Under our assay conditions using 10 milliunits of xanthine oxidase, urate was formed from hypoxanthine at a rate of 5.2 μmmin−1 and superoxide was produced at 6.7 μmmin−1. Hypoxanthine donates four electrons to xanthine oxidase, which under our experimental conditions resulted in 32% univalent reduction of oxygen to superoxide. The remaining 68% was consumed by direct divalent reduction of oxygen to form hydrogen peroxide as previously demonstrated by Fridovich (43Fridovich I. J. Biol. Chem. 1970; 245: 4053-4057Abstract Full Text PDF PubMed Google Scholar). When bolus additions of preformed peroxynitrite were made to the xanthine oxidase reaction, tyrosine nitration was progressively inhibited as urate accumulated in the assay system (Fig. 5). Addition of 50–100 μm urate into the stopped-flow system also strongly inhibited tyrosine nitration to an equal extent with either preformed peroxynitrite or the combination of superoxide and nitric oxide (not shown). Pfeiffer and Mayer (1Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) quantified peroxynitrite formation from xanthine oxidase plus spermine NONOate by measuring the rate of dihydrorhodamine oxidation only at the start of the assay. The oxidation of dihydrorhodamine is known to be strongly inhibited by urate (33Kooy N.W. Royall J.A. Ischiropoulos H. Beckman J.S. Free Radic. Biol. Med. 1994; 16: 149-156Crossref PubMed Scopus (670) Google Scholar). Immediately after the addition of xanthine oxidase, the rate of dihydrorhodamine oxidation was 1.4 μm/min−1, which corresponds to 4.8 μm/min−1peroxynitrite after correction for