Formation of peroxynitrite at high pH and the generation of S-nitrosothiols from thiols and peroxynitrous acid in acid solution

Abstract

The formation of peroxynitrite from hydrogen peroxide and S-nitrosothiols at high pH is shown to involve reaction of the hydroperoxide anion. Kinetic measurements over a pH range 10–13 yield a value close to the literature value for the pK_a of hydrogen peroxide and give a quantitative measure of the reactivity of the hydroperoxide ion. S-Nitrosothiols are formed in high yield when peroxynitrous acid reacts with thiols when the thiol is in very large excess, within the pH range 2.7–3.7. Kinetic measurements have shown that reaction occurs by rapid oxidation of the thiol to the disulfide, with release of nitrite ion, which at these pH values is sufficiently protonated to effect nitrosation of the thiol, which is in large excess. Quantitative yields of RSNO can be obtained when there is a very large excess [thiol], when the spontaneous decomposition of peroxynitrous acid cannot compete. The individual kinetic results are fully first order and analysis of the results yields values for the third-order rate constants k (in rate = k [RSH][HNO₂][H⁺]) which are in acceptable agreement with the values in the literature for the direct nitrosation of thiols with nitrous acid. Results were obtained for three thiols [glutathione (GSH), cysteine (Cys) and cysteine ethyl ester], confirming the generality of the reaction. At higher pH values the yield of RSNO drops off sharply as nitrous acid becomes deprotonated. A separate experiment with stoichiometric concentration ratios of thiol and peroxynitrous acid in acid solution gave high yields of nitrous acid. At higher acidities (>0.3 mol dm⁻³) S-nitrosothiols are still formed, but now there are clearly two sequential first-order processes which can be fitted to a two exponential model. One reaction is believed to be nitrous acid nitrosation, following oxidation of the thiol as before, whilst the other reaction, which is also acid catalysed, is interpreted as nitrosation via a protonated form of peroxynitrous acid as recently reported in the literature, which can compete with the isomerisation of peroxynitrous acid to nitrate anion, when the thiol is at high concentration.