FIGURE 3.

Differential overoxidation of control and peroxynitrite-treated Prx2. After treatment with the ROA control or a 5-fold excess of peroxynitrite (ONOO⁻), Prx2 was treated with DTT for reduction of its thiols, and residual DTT was removed. 5 μm Prx2 was incubated with 0, 0.01, 0.02, 0.06, 0.12, 0.24, or 5 mm H₂O₂ in 50 mm phosphate buffer, pH 7.4. After 5 min, 30 mm NEM was added to alkylate the remaining thiols. Samples were prepared for SDS-PAGE under nonreducing conditions, analyzed by Western blotting using α-Prx2 antibody and α-C_P-SO_2/3H after mild stripping. One μg of protein was loaded on each lane (D_ss, dimer with one C_P disulfide-oxidized; D_ss/ssSS/SS, dimer with both C_P disulfide-oxidized; M, monomer).