Figure 3.

Effect of varying the concentration of TrxS₂ on the formation of the Cys⁴⁹⁷/SeCys⁴⁹⁸ thioselenide and the Cys⁵⁹/Cys⁶⁴ disulfide. NADPH-reduced TrxR1 (20 μg) was incubated for 10 min at room temperature with the indicated amount of TrxS₂ in oxygen-free PBS buffer (pH 7.2) containing 1 mM EDTA. The samples were then reacted with 1 mM IAM at pH 8.8 in 6 M guanidine-HCl as described in the legend to Fig. 2C. The reaction mixtures after dialysis were digested with Lys-C; peptides were purified by HPLC; and the selenium contents of the S-Se peptide and the CAM-Cys/CAM-SeCys peptide were determined by atomic absorption spectroscopy. The amounts of Cys/Cys peptide and CAM-Cys/CAM-Cys peptide were estimated from the HPLC peak areas. (Left) The formation of the oxidized, thioselenide-containing peptide, expressed as a percentage of the sum of the oxidized (S-Se) and alkylated (CAM-Cys/CAM-SeCys) derivatives of reduced Cys⁴⁹⁷/SeCys⁴⁹⁸. (Right) The formation of the disulfide-containing peptide, expressed as a percentage of the sum of the oxidized (S-S) and alkylated (CAM-Cys/CAM-Cys) derivatives of the reduced Cys⁵⁹/Cys⁶⁴ pair.