FIGURE 8.

Oxidation of Cys-107 of RidA by different oxidative and nitrosative stressors and reversibility by ascorbate of these oxidations. RidA (70 μm) was incubated with nitrosative (peroxynitrite, GSNO) and oxidative stressors (hydrogen peroxide, diamide) at a molar ratio of 1:10 in potassium phosphate buffer at pH 7.3 for different lengths of times (peroxynitrite, 5 min; GSNO, 30 min; hydrogen peroxide and diamide, 20 min). Untreated RidA served as a control. A, concentration of free thiol groups in the samples was quantified spectroscopically by monitoring the release of thio-nitrobenzoic acid from 5,5′-dithiobis-2-nitrobenzoic acid (Ellman reagent) at 412 nm and 20 °C. B, reversibility of the thiol modifications of Cys-107 upon treatment with GSNO and peroxynitrite stress was analyzed by subsequent ascorbate reduction. Nonstressed and stressed RidA samples were incubated with 1 mm ascorbate for 2 h.