10–30 µM PRX-IIE in 100 mM Tris-HCl, pH 8, was reduced for 30 min at room temperature (RT) with 4 mM DTT. Desalting was achieved by passing the solution through PD10 columns. S-glutathionylation was carried out by disulfide exchange with oxidized glutathione (GSSG) for 1 h at RT. Excess GSSG was removed via acetone precipitation. Afterward, S-glutathionylation was detected by Western blot using a monoclonal anti-GSH antibody (Thermo Scientific, Schwerte, Germany). In addition, molecular masses of modified and unmodified proteins were assessed by ESI-MS as mentioned before. For deglutathionylation, 10 µM glutathionylated PRX-IIE was incubated with 10 µM of GRX-S12, GRX-C5, or SRX and 0.5 mM GSH at 25 °C. The decrease of glutathionylated PRX-IIE was determined using Western Blot with anti-GSH antibodies. The spot intensities were analyzed using ImageJ.

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