Lack of tyrosine nitration by peroxynitrite generated at physiological pH

Nitration of tyrosine residues of proteins has been suggested as a marker of peroxynitrite-mediated tissue injury in inflammatory conditions. The nitration reaction has been extensively studied in vitro by bolus addition of authentic peroxynitrite, an experimental approach hardly reflecting in vivo situations in which the occurrence of peroxynitrite is thought to result from continuous generation of (NO)-N-. and O-2(radical anion) at physiological pH, In the present study, we measured the nitration of free tyrosine by (NO)-N-. and O-2(radical anion) generated at web defined rates from the donor compound (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]-diazen-1-ium-1,2-diolate] (spermine NONOate) and the xanthine oxidase reaction, respectively. The results were compared with the established nitration reaction triggered by authentic peroxynitrite, Bolus addition of peroxynitrite (1 mM) to tyrosine (1 mM) at pH 7.4 yielded 36.77 +/- 1.67 mu M 3-nitrotyrosine, corresponding to a recovery of about 4%. However, peroxynitrite formed from (NO)-N-. and O-2(radical anion), which were generated at equal rates (similar to 5 mu M X min(-1)) from 1 mM spermine NONOate, 28 milliunits/ml xanthine oxidase, and 1 mM hypoxanthine was much less efficient (0.67 +/- 0.01 mu M; similar to 0.07% of total product flow). At O-2(radical anion) fluxes exceeding the (NO)-N-. release rates, 3-nitrotyrosine formation was below the detection limit of the high performance Liquid chromatography method (<0.06 mu M) Nitration was most efficient (similar to 0.3%) with the (NO)-N-. donor alone, i.e. without concomitant generation of O-2(radical anion). Nitration by (NO)-N-. had a pH optimum of 8.2, increased progressively with increasing tyrosine concentrations (0.1-2 mM), and was not enhanced by NaHCO3 (up to 20 mar), indicating that it was mediated by (NO2)-N-. rather than peroxynitrite, Our results argue against peroxynitrite produced from (NO)-N-. and O-2(radical anion) as a mediator of tyrosine nitration in vivo.