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

The formation of peroxynitrite from hydrogen peroxide and S-nitrosothiols a t 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 va lue for the pK(a) of hydrogen peroxide and give a quantitative measure of t he 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, wit h release of nitrite ion, which at these pH. values is sufficiently protona ted to effect nitrosation of the thiol, which is in large excess. Quantitat ive 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 r esults 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 liter ature for the direct - nitrosation of thiols with nitrous acid. Results wer e obtained for three thiols [glutathione (GSH), cysteine (Cys) and cysteine ethyl ester], confirming the generality of the reaction. At higher pH valu es the yield of RSNO drops off sharply as nitrous acid becomes deprotonated . A separate experiment with stoichiometric-concentration ratios of thiol a nd 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 n ow there are clearly two sequential first-order processes which can be fitt ed to a two exponential model. One reaction is believed to be nitrous acid nitrosation, following oxidation of the thiol as before, whilst the other r eaction, which is also acid catalysed, is interpreted as nitrosation via:a protonated form of peroxynitrous acid as recently reported in the literatur e; which can compete with the isomerisation of peroxynitrous acid to nitrat e anion, when the thiol is at high concentration.