Oxidation of disulfide bonds: a novel pathway to protein glutathionylation

Disulfide bonds, formed from two cysteine (Cys) residues, play a key role in defining and stabilizing protein structures. Whilst the reactions of Cys are well characterized, little is known about oxidation of disulfides, despite their high abundance in some proteins. Recent data indicate that some disulfides are rapidly oxidized by a range of oxidants, with rate constants, k, 105–107 M-1 s-1. Here we show that these reactions yield intermediates, potentially thiosulfinates [RSS(=O)R•], that can undergo further reaction, including disulfide bond cleavage and reaction with another thiol. Thus, oxidation of alpha-lactalbumin, which contains no free Cys residues, by HOCl or ONOOH gives long-lived reactive thiosulfinates that can react with GSH to give a glutathionylated protein, which can be detected by both anti-GSH antibodies and mass spectrometry. The thiosulfinate has a lifetime of many hours allowing glutathionylation to occur hours after initial oxidation. Disulfide oxidation can therefore give rise to long-lived oxidants on proteins that can undergo further reaction with thiols, including GSH and other proteins, to give mixed disulfides and protein dimers. These reactions may play a key role in GSH-dependent cell signaling and tissue damage.