Fig. 2.

Peroxidase activity of Prx1. Mean values of triplicate experiments are shown. (A) DTT-dependent removal of H₂O₂ (left panel) and t-BOOH (right panel) by Prx1. Peroxidase reaction was carried out at room temperature in 200 μl of reaction mixture containing 50 mM HEPES (pH 7.0), 5 mM DTT, 100 μM H₂O₂ or 100 μM t-BOOH, and in the absence (closed square) or presence of Prx1 (closed circle, 2 μM; closed triangle, 4 μM). At the indicated times, the remaining peroxides were measured using FOX1 reagent as described in Materials and methods. (B) Peroxidase reaction was carried as in (A) with 100 μM H₂O₂ (left panel) or 100 μM t-BOOH (right panel) in the absence (closed square) or presence of 4 μM Prx1 (wild type, closed circle; C69S, closed triangle). (C) Peroxidase reaction was performed using 4 μM Prx1 as in (A), except the presence of GSH instead of DTT as a reducing equivalent: closed square, without Prx1; closed triangle, with Prx1. (D) Peroxidase activity linked to NADPH oxidation with S. cerevisiae Trx system was monitored by decreases of A₃₄₀ in a 200-μl reaction mixture containing 50 mM HEPES (pH 7.0), 0.2 mM NADPH, 2 μM ScTrr1, 5 μM ScTrx1, 4 μM Prx1, and either 0.1 mM H₂O₂ (left panel) or 0.1 mM t-BOOH (right panel): closed square, without Prx1; closed triangle, with Prx1.