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Figure 3
Intermolecular disulfide-bond formation in VvPrx3 (C73S) on treatment with peroxide. Proteins were treated with H2O2 (a) or tert-butyl hydrogen peroxide (t-BOOH) (b) at the concentrations shown for 30 min and the reactions were stopped by adding iodoacetamide. Samples were boiled in the presence (+) or absence (−) of 10 mM DTT for SDS–PAGE analysis. (c) Determination of the second-order reaction rate constant for the reaction of VvPrx3 and H2O2 using competition kinetics with HRP. The fitting line was calculated by the least-squares method and the error bars reflect the standard deviation from three independent experiments. (d) Purified VvPrx3 (C73S) (70 µM) was reacted with 50 µM H2O2 for the given times and analyzed by SDS–PAGE. At a time of 30 s, ∼30 µM protein was linked by a disulfide bond as estimated using the density-measuring program ImageJ. The protein band for disulfide bond-containing VvPrx3 (D) and bands that lack a disulfide bond (M) are indicated.

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