Characterization of disulfide (cystine) oxidation by HOCl in a model peptide: Evidence for oxygen addition, disulfide bond cleavage and adduct formation with thiols

Disulfide bonds play a key role in stabilizing proteins by cross-linking secondary structures. Whilst many disulfides are effectively unreactive, it is increasingly clear that some disulfides are redox active, participate in enzymatic reactions and/or regulate protein function by allosteric mechanisms. Previously (Karimi et al., Sci. Rep. 2016, 6, 38752) we have shown that some disulfides react rapidly with biological oxidants due to favourable interactions with available lone-pairs of electrons. Here we present data from kinetic, mechanistic and product studies for HOCl-mediated oxidation of a protected nine-amino acid model peptide containing a N- to C-terminal disulfide bond. This peptide reacts with HOCl with k₂ 1.8 × 10⁶ M⁻¹ s⁻¹, similar to other highly-reactive disulfide-containing compounds. With low oxidant excesses, oxidation yields multiple oxidation products from the disulfide, with reaction predominating at the N-terminal Cys to give sulfenic, sulfinic and sulfonic acids, and disulfide bond cleavage. Limited oxidation occurs, with higher oxidant excesses, at Trp and His residues to give mono- and di- (for Trp) oxygenated products. Site-specific backbone cleavage also occurs between Arg and Trp, probably via initial side-chain modification. Treatment of the previously-oxidised peptide with thiols (GSH, N-Ac-Cys), results in adduction of the thiol to the oxidised peptide, with this occurring at the original disulfide bond. This gives an open-chain peptide, and a new mixed disulfide containing GSH or N-Ac-Cys as determined by mass spectrometry. Disulfide bond oxidation may therefore markedly alter the structure, activity and function of disulfide-containing proteins, and provides a potential mechanism for protein glutathionylation.