Fig. 7.

Hyperoxidation sensitivity of 2-CysPrx WT and variants. Representive data are shown for 2-CysPrx F84R. Similar assay conditions were applied as used for the peroxidase activity. A fixed concentration of 15 μM Ec-Trx was used, and the reaction was started with various concentrations (0.5, 1, 2, 5, and 10mM) of cumene hydroperoxide. (A) The NADPH consumption was monitored over 5min. (B) The number of turnovers per 2-CysPrx molecule was calculated and plotted against time. The data were fitted to an equation describing a saturation curve. (C) The calculated limits of turnover were plotted against cumene hydroperoxide concentrations. Data were fitted to an equation describing an exponential curve with offset. The calculated parameters are displayed in Table 5. (D) Western blot analysis of hyperoxidized 2-CysPrx using antiserum against sulfinic acid 2-CysPrx (2-CysPrx-SO₃ ^2–). WT, C176S and His₆-WT were reduced with 10mM DTT and treated three times with 2mM H₂O₂ for 15min at 37 °C. Untreated C176S was loaded in lane 2. One microgram of each 2-CysPrx variant was loaded per lane.