IDENTIFICATION OF CU-OXIDE FORMATION FROM S-NITROSOTHIOLS (RSNO)( AS THE EFFECTIVE REAGENT IN NITRIC)

Decomposition of S-nitrosothiols (RSNO) in aqueous solution at pH 7.4 is brought about by copper ions, either present as an impurity or spec ifically added. The primary products are nitric oxide and the disulfid e. In the presence of the specific Cu+ chelator, neocuproine, reaction is progressively inhibited as the [neocuproine] is increased, the rea ction eventually stopping completely. The characteristic UV-VIS spectr um of the Cu+ adduct can be obtained from the reaction solutions. This shows clearly that Cu+ and not Cu2+ is the effective catalyst. Two li miting kinetic conditions can be identified for a range of S-nitrosoth iols at specific copper ion concentrations (a) a first-order dependenc e and (b) a zero-order dependence upon [RSNO]. Normally both situation s also have a short induction period. This induction period can be rem oved by the addition of the corresponding thiol RSH. A mechanism is pr oposed in which Cu+ is formed by reduction of Cu2+ by thiolate anion v ia an intermediate, possibly RSCu(+). Loss of nitric oxide from RSNO i s then brought about by Cu+, probably via another intermediate in whic h Cu+ is bound to the nitrogen atom of the NO group and another electr on-rich atom (such as nitrogen from an amino group, or oxygen from a c arboxylate group) involving a six-membered ring. As well as NO this pr oduces both RS(-) and Cu2+ which then are part of the cycle regenerati ng Cu+. Thiolate ion is oxidised to RS(.) which dimerizes to give the disulfide. Depending on the structure (and hence reactivity) of RSNO e ither Cu+ formation or its reaction with RSNO can be rate-limiting. Co mputer modelling of the reaction scheme allows the generation of absor bance time plots of the same forms as those generated experimentally, i.e. first- or zero-order, both with or without induction periods. We suggest that the thiolate ion necessary to bring about Cu2+ reduction is either present as a thiol impurity or is generated in small quantit ies by partial hydrolysis of the nitrosothiol, which results in an ind uction period. Addition of small quantities of thiol removes the induc tion period and leads to catalysis but larger quantities bring about a rate reduction by, it is suggested, complexation of the Cu2+. For two very unreactive substrates, S-nitrosoglutathione and S-nitroso-N-acet ylcysteine very large induction periods were observed, typically three hours. This results, we suggest, from competitive re-oxidation of Cu to Cu2+ by the dissolved oxygen. Experiments carried out anaerobicall y confirm this, since there is then no induction period. Addition of h ydrogen peroxide extends the induction period ever further. The result s are discussed in terms of the biological properties of S-nitrosothio ls which are related to nitric oxide release.