FIGURE 2.
Analysis of Prx2 nitration after peroxynitrite treatment. A, QTOF-ESI MS analysis. Disulfide-oxidized Prx2 was treated with a 5-fold excess peroxynitrite, then reduced with 5 mm DTT for 15 min at room temperature, followed by addition of 20 mm NEM to block the reduced thiols. The remaining DTT and NEM were removed by gel filtration, and samples in 30 mm ammonium bicarbonate buffer were analyzed by QTOF-ESI MS. Control Prx2 is shown in black (shown as 100%), and the gray dotted line shows the peroxynitrite-treated enzyme. B, Western blot analysis. Disulfide Prx2 was treated with a 5-fold excess peroxynitrite (ONOO−) or ROA. 2.4 μg of control or peroxynitrite-treated Prx2 was resolved on 15% SDS-PAGE under reducing conditions and analyzed by Western blot using α-NO2Y antibodies.