Abstract
Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. Nitrosation reactions are quenched by O-2, while the oxidation chemistry mediated by peroxynitrite is quenched by excess flux of either NO or O-2. A solution of glutathione (GSH), a model thiol-containing tripeptide, exclusively yielded S-nitrosoglutathione when exposed to the NO donor, Et2NN(O)NONa. However, when xanthine oxidase was added to the same mixture, the yield of S-nitrosoglutathione dramatically decreased as the activity of xanthine oxidase increased, such that there was a 95% reduction in nitrosation when the fluxes of NO and O-2 were nearly equivalent. The presence of superoxide dismutase reversed O-2-mediated inhibition, while catalase had no effect. Increasing the flux of O-2 yielded oxidized glutathione (GSSG), peaking when the flux of NO and O-2 were approximately equivalent. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance.
Highlights
Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation
We have explored the oxidative and nitrosative resulting from the interaction of NO and O2. with thiols and demonstrate that there is a balance between fluxes of NO and O2. with respect to oxidative and nitrosative products of glutathione
The lack of effect of catalase suggests that O2. and not hydrogen peroxide attenuates nitrosation. Such results lessen the concerns of oxygen depletion by xanthine oxidase, since catalase will adequately reoxygenate the solution by converting hydrogen peroxide to oxygen
Summary
Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance. The endogenously formed radicals, NO1 and superoxide (O2.), are thought to play key roles in the regulation of a number of physiological and pathophysiological mechanisms. This interaction has been suggested to be pivotal in cytotoxic mechanisms involving the modulation of oxidative stress by NO. In biological systems, perhaps explaining why proteins and peptides containing thiols that undergo nitrosation as well as oxidation reactions might modulate cellular function. Reactions of chemical species derived from NO can result in the nitrosation of thiols to form S-nitrosothiol complexes [1, 2]. We have shown that oxidative and nitrosative chemistry resulting from a continuous chemical generator of NO, SPER/NO (a NONOate complex that spontaneously releases NO at physiological pH), in the presence of a source of
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