Kinetics of S-nitrosation of thiols in nitric oxide solutions

The S-nitroso adducts of nitric oxide(NO) may serve as carriers of NO and play a role in cell signaling and/or cytotoxicity. A quantitative understanding of the kinetics of S-nitrosothiol formation in solutions containing NO and O-2 is important for understanding these roles of S-nitroso compounds in vivo. Rates of S-nitrosation in aqueous solutions were investigated for three thiols: glutathione, N-acetylcysteine, and N-acetylpenicillamine. Nitrous anhydride (N2O3), an intermediate in the formation of nitrite from NO and O-2, is the most likely NO donor for N-nitrosation of amines as well as for S-nitrosation of thiols, at physiological pH. This motivated the use of a competitive kinetics approach, in which the rates of thiol nitrosation were compared with that of a secondary amine, morpholine, The kinetic studies were carried out with known amounts of NO and O-2 in solutions containing one thiol (400 mu M) and morpholine (200-5700 mu M) in 0.01 M phosphate buffer at pH 7.4 and 23 degrees C. It was found that disulfide formation, transnitrosation reactions, and decomposition of the S-nitrosothiol products were all negligible under these conditions. The rate of formation of S-nitrosothiols was expressed as k(7)[N2O3][RSH], where RSH represents the thiol. The rate constant for S-nitrosation relative to that for N2O3 hydrolysis (k(4)) was found to be k(7)/k(4) = (4.15 +/- 0.28) x 10(4), (2.11 +/- 0.11) x 10(4), and (0.48 +/- 0.04) x 10(4) M(-1) for glutathione, N-acetylcysteine, and N-acetylpenicillamine, respectively. The overall (observed) rates of nitrosothiol formation reflect the fact that [N2O3] OC [NO](2)[O-2] and that [N2O3] also depends on [RSH] and the concentration of phosphate. Using a detailed kinetic model to account for these effects, the present results could be reconciled with apparently dissimilar findings reported previously by others.