Mechanistic studies of S-nitrosothiol reactions with reference to potential physiological activity

Abstract

A study of the reactions of various S-nitrosothiols, particularly S-nitroso-N- acetylpenicillamine (SNAP), was undertaken. These compounds were known to produce nitric oxide (NO) when decomposing, which has important and diverse biological roles. An example of their use in physiological research was demonstrated. The Griess method was used to determine the stoichiometry of nitrite production from S-nitrosothiol decomposition in various buffer solutions. In all cases the production was found to be almost quantitative. The kinetic measurement of SNAP decomposition in a variety of buffers and pH was undertaken. The results were complex and often erratic, conforming to first order but also half order kinetics in many cases. There was some indication that decomposition products and light could affect the reaction. The presence of disulphide (dimer) as a major reaction product was confirmed in the case of SNAP. Free-radical traps were used to probe the decomposition mechanism, as were hemin and haemoglobin as NO detectors to determine decomposition kinetics. The true agent of S-nitrosothiol decomposition was found to be intrinsic copper in the water supply and buffer salts. S-nitrosothiols were found to be stoichiometrically decomposed by Hg(^2+) ions, but catalytically decomposed by Cu(^2+) ions. Kinetic measurement confirmed the complex nature of the catalysis. The importance of SNO and NH(_2), and SNO and COO- as binding sites was demonstrated. Some explanation was found for the differing structure/reactivity relationships observed. It was shown that transnitrosation of a thiol could occur, involving thiolate anion attack upon the S-nitrosothiol. However, the reaction appeared to be very slow at physiological pH. The nitrosation of N-methylaniline by S-nitrosothiols was found to occur only in the presence of oxygen - direct transfer of NO did not occur, nitrosation being mediated after SNAP decomposition.