Direct and simultaneous ultraviolet second-derivative spectrophotometric determination of nitrite and nitrate in preparations of peroxynitrite.

We have determined the initial concentrations of nitrite and nitrate for three different methods of synthesizing peroxynitrite using an ultraviolet second-derivative spectroscopy method (Fig. 3). As expected, the net nitrogen balance in these preparations (Fig. 4) and the yields of nitrite and nitrate (Table II) indicate that, at pH 6.0, peroxynitrite decomposes to give essentially NO3-. Stock solutions of peroxynitrite prepared using method I (ozonation of azide) consistently contain more NO2- and NO3- than method II (isoamyl nitrite with hydrogen peroxide) and method III (hydrogen peroxide with nitrous acid). Method II gives the least amount of NO2- contaminants, and NO3- impurities are the lowest in method III (Table I).

Nitrosation by peroxynitrite: use of phenol as a probe.

Nitrosation is an important pathway in the metabolism of nitric oxide, producing S-nitrosothiols that may be critical signal transduction species. The reaction of peroxynitrite with aromatic compounds in the pH range of 5 to 8 has long been known to produce hydroxylated and nitrated products. However, we here present evidence that peroxynitrite also can promote the nitrosation of nucleophiles. We chose phenol as a substrate because the nitrosation reaction was first recognized during a study of the CO2-modulation of the patterns of hydroxylation and nitration of phenol by peroxynitrite (Lemercier et al., Arch. Biochem. Biophys. 345, 160-170, 1997). 4-Nitrosophenol, the principal nitrosation product, is detected at pH 7.0, along with 2- and 4-nitrophenols; 4-nitrosophenol becomes the dominant product at pH >/= 8.0. The yield of 4-nitrosophenol continues to increase even after pH 11.1, 1. 2 units above the pKa of phenol, suggesting that the phenolate ion, and not phenol, is involved in the reaction. Hydrogen peroxide is not formed as a by-product. The nitrosation reaction is zero-order in phenol and first-order in peroxynitrite, suggesting the phenolate ion reacts with an activated nitrosating species derived from peroxynitrite, and not with peroxynitrite itself. Under optimal conditions, the yields of 4-nitrosophenol are comparable to those of 2- and 4-nitrophenols, indicating that the nitrosation reaction is as significant as the nitration of phenolic compounds by peroxynitrite. Low concentrations of CO2 facilitate the nitrosation reaction, but excess CO2 dramatically reduces the yield of 4-nitrosophenol. The dual effects of CO2 can be rationalized if O=N-OO- reacts with the peroxynitrite anion-CO2 adduct (O=N-OOCO-2) or secondary intermediates derived from it, including the nitrocarbonate anion (O2N-OCO-2), the carbonate radical (CO*-3), and *NO2. The product resulting from these reactions can be envisioned as an activated intermediate X-N=O (where X is -OONO2, -NO2, or -CO-3) that could transfer a nitrosyl cation (NO+) to the phenolate ion. An alternative mechanism for the nitrosation of phenol involves the one-electron oxidation of the phenolate ion by CO*-3 to give the phenoxyl radical and the oxidation of O=N-OO- by CO*-3 to give a nitrosyldioxyl radical (O=N-OO*), which decomposes to give *NO and O2; the *NO then reacts with the phenoxyl radical giving nitrosophenol. Both mechanisms are consistent with the high yields of NO-2 and O2 during the alkaline decomposition of peroxynitrite and the potent inhibitory effect of N-3 on the nitrosation of phenol by peroxynitrite and peroxynitrite/CO2 adducts. The biological significance of the peroxynitrite-mediated nitrosations is discussed.