Effect of GSH, Cys-SH, or NAC on CSE-induced Cys34 oxidation.

<p>(<b>A</b>) HSA-SH solutions (60 µM) were incubated with vehicle (control; filled circles) or 0.3 µM (triangles), 3 µM (squares) or 30 µM (open circles) GSH (molar ratio [GSH]/[HSA] = 0.005–0.05–0.5, respectively), for 30 min, before exposing protein solutions to 0%, 1%, 4% and 16% (v/v) CSE, for 60 min. (<b>B</b>) HSA-SH solutions (60 µM) were incubated with vehicle (control; filled circles) or 1 µM (triangles), 10 µM (squares) or 100 µM (open circles) Cys-SH (molar ratio [Cys-SH]/[HSA] = 0.016–0.16–1.6, respectively), for 30 min, before exposing protein solutions to 0%, 1%, 4% and 16% (v/v) CSE, for 60 min. (<b>C</b>) HSA-SH solutions (60 µM) were incubated with vehicle (control; filled circles) or 1 µM (triangles), 10 µM (squares) or 100 µM (open circles) NAC (molar ratio [NAC]/[HSA] = 0.016–0.16–1.6, respectively), for 30 min, before exposing protein solutions to 0%, 1%, 4% and 16% (v/v) CSE, for 60 min. (<b>D</b>) Experiment was performed as in (<b>A</b>) except that HSA solutions were exposed to CSE immediately after GSH addition. (<b>E</b>) Experiment was performed as in (<b>B</b>) except that HSA solutions were exposed to CSE immediately after Cys-SH addition. (<b>F</b>) Experiment was performed as in (<b>C</b>) except that HSA solutions were exposed to CSE immediately after NAC addition. Conditions of incubation and estimation of HSA Cys34 free sulfhydryl group by biotin–HPDP binding and Western blot analysis are described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029930#s2" target="_blank">Materials and methods</a>. Densitometric analyses of biotin-HPDP incorporation are shown. Data are presented as the mean ± SD of three independent determinations.