Quantification of sulfenic acid on model protein

Aldolase contains eight free cysteine residues without disulfide formation in its native state, which makes it a suitable model to manipulate the oxidative modifications on cysteine. H2O2 was chosen to treat aldolase since the principle product between which is sulfenic acid.To assess the reduction of the newly formed sulfenic acids on aldolase by arsenite, a differential alkylation strategy was adapted.To further investigate the formation of other reversible cysteine oxidations such as disulfide on aldolase, arsenite was replaced by DTT in the differential alkylation method to reduce all the reversible cysteine oxidations.To further validate the selectivity of the reducing ability of arsenite on sulfenic acid but not other oxidative modifications especially disulfide, lysozyme was employed in the differential alkylation method. Lysozyme has 8 cysteine residues all bound into disulfide bonds, thereby was not treated with H2O2 to avoid further oxidation.