Quantification of the percentage of oxidized cysteine thiols in GrxS12 under normal and H2O2 treatments.

To evaluate the percentage of oxidized cysteine thiols, the GrxS12 proteins underwent either with or without H2O2 treatment were alkylated by iodoacetamide (IAM), reduced with DTT, and subsequently the newly generated free Cys were alkylated with N-ethylmaleimide (NEM). The spectrometric analysis of the GrxS12 sample indicated that less than 30% of monomers formed an intramolecular disulfide linkage. This confirmed the formation of intramolecular disulfide bonds between Cys34 and Cys92 and demonstrated that the percentage of intramolecular disulfide linkage remained unaltered under normal and H₂O₂- oxidized conditions.