This proposal requests funds to support research investigating the complex interactions between nitric oxide (.NO) and biological thiols, Such interactions have been postulated to have importance in physiology and pathology. The long-term objective of this proposal is to delineate, at the molecular level, the chemical mechanisms of the reactions of .NO and peroxynitrite with biologically relevant low- and high-molecular weight thiols. S-Nitrosothiols, formed from .NO and thiols have been implicated in the maintenance of vascular tone and in cardiovascular protection. More recently, S-nitrosohemoglobin has been detected in vivo and may play an important role in oxygen delivery. It is hypothesized that: (i) the mechanism of reaction between .NO and thiols is affected by the concentration of oxygen, (ii) the biological effects of S-nitrosothiols are dictated by transnitrosation equillbria between thiols and S-nitrosothiols and the reduction of S-nitrosothiols to form the nitroxyl anion and (iii) S-nitrosation of cysteinyl residues of nitric oxide synthase (NOS) regulates NOS activity. These ideas will be explored by pursuing the following specific aims: l) Determine the reaction mechanism between .NO and biologically relevant thiols, 2) Determine the thermodynamic and kinetic parameters of the reactions between thiols and S-nitrosothiols, 3) Investigate the reaction between peroxynitrite and thiols, 4) Investigate the effect of endogenous and exogenous thiols on the fate of .NO generated from NOS and 5) Determine the effect of S-nitrosothiols and .NO on the activity of NOS. The information gained from the accomplishment of these goals will advance our understanding of the fate of .NO in biological systems and the pathological consequences of altered .NO generation.
