KINETICS OF NITROSATION OF THIOLS BY NITRIC-OXIDE IN THE PRESENCE OF OXYGEN

Nitrosothiols are powerful vasodilators, They act by releasing nitric oxide, which activates the heme protein guanylate cyclase, We have stu died the kinetics of nitrosothiol formation of glutathione, cysteine, N-acetylcysteine, human serum albumin, and bovine serum albumin upon r eaction with nitric oxide (NO) in the presence of oxygen, These studie s have been made at low pH as well as at physiological pH, At pH 7.0, contrary to published reports, nitric oxide by itself does not react w ith thiols to yield nitrosothiol. However, formation of nitrosothiols is observed in the presence of oxygen, For all thiols studied, the rat es of nitrosothiol formation were first order in O-2 concentration and second order in NO concentration and at lower concentrations (<5 mM t hiol) also depended on thiol concentrations, analysis of the kinetic d ata indicated that the rate-limiting step was the reaction of NO with oxygen, Analysis of the reaction products suggest that the main nitros ating species is N2O3: RSH + N2O3 --> RSNO + NO2- + H+. Rate constants for this reaction for glutathione and several other low molecular wei ght thiols are in the range of 3-1.5 x 10(5) M(-1) s(-1), and for huma n and bovine serum albumins 0.3 x 10(5) M(-1) s(-1) and 0.06 x 10(5) M (-1) s(-1), respectively, The data further indicate that the reaction rate of the nitrosating species N2O3 with thiols is competitive with i ts rate of hydrolysis. At physiological concentrations nitrosoglutathi one formation represents a significant metabolic fate of N2O3, and at glutathione concentrations of 5 mM or higher almost all of N2O3 formed is consumed in nitrosation of glutathione. Implications of these resu lts for in vivo nitrosation of thiols are discussed.