The effect of F190L to peroxidase activity and sensitivity to hyperoxidation.

<p><b>a.</b> Time course of PrxIII dependent NADPH oxidation coupled to H₂O₂ reduction for PrxIII wild-type and F190L. NADPH oxidation was monitored at 340 nm in a 1 ml reaction mixture containing with 5 μM of PrxIII, 2.1 μM Trx, 1.5 μM TrxR2 and 0.5 mM NADPH in 50 mM NaH₂PO4 buffer, pH 7.4 containing 150 mM NaCl, 1 mM EDTA. A reaction mix without PrxIII was used as the negative control. This is a representative result from three independent experiments with similar results. <b>b.</b> Dose-dependent increase in PrxIII wild-type and F190L mutant hyperoxidation after exposure to hydrogen peroxide at the indicated concentrations. PrxIII hyper-oxidation was monitored by western blotting using an antibody that recognizes primarily the sulfinic or sulfonic acid forms of Prxs (α-SO_2-3). The assay was carried out in the presence of 1 mM DTT as reductant. Western blots were used for quantification of hyperoxidized PrxIII by densitometry. Intensity of hyper-oxidized PrxIII was normalized against loading control blotted with an anti-His antibody. Error bars represent the mean ±S.D. from three independent experiments.