The ferrous-dependent xylenol orange assay (FOX) was used to analyze the activity of PRX-IIE and its phosphomimic variants with DTT as electron donor. The reaction mixture contained 2 µM PRX-IIE and 4 mM dithiothreitol (DTT) in 40 mM K-Pi, pH 7.2. The measurement was started by the addition of peroxides (400 µM H2O2, 200 µM t-BOOH, or 200 µM CuOOH) in a time course of 90 s at 15 s intervals. The remaining peroxides were detected by ferrous-dependent oxidation of xylenol orange as reported previously [20].

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