Oxidant-induced glutathionylation at protein disulfide bonds

Disulfide bonds are a key determinant of protein structure and function, and highly conserved across proteomes. They are particularly abundant in extracellular proteins, including those with critical structural, ligand binding or receptor function. We demonstrate that oxidation of protein disulfides induces polymerization, and results in oxygen incorporation into the former disulfide via thiosulfinate generation. These intermediates, which have half-lives of several hours in vitro, undergo secondary reactions that cleave the disulfide bond, by irreversible hydrolysis to sulfinic and sulfonic acids, or reaction with thiols in a process that yields thiolated proteins (e.g. glutathionylated species in the case of reaction with glutathione). The adducts have been characterized by mass spectrometry (as ions corresponding to the addition of 306 and 712 Da for addition of one and two glutathione molecules, respectively) and immunoblotting. These modifications can be induced by multiple biologically-important oxidants, including HOCl, ONOOH, and H₂O₂, and on multiple proteins, demonstrating that this is a common disulfide modification pathway. Addition of glutathione to give glutathionylated proteins, can be reversed by reducing systems (e.g. tris(2-carboxyethyl)phosphine), but this does not repair the original disulfide bond. Exposure of human plasma to these modifying agents increases protein glutathionylation, demonstrating potential in vivo relevance. Overall these data provide evidence for a novel and facile route to glutathionylated proteins involving initial oxidation of a disulfide to a thiosulfinate followed by rapid reaction with GSH ('oxidant-mediated thiol-disulfide exchange'). These data elucidate a novel pathway for protein glutathionylation that may have significant implications for redox biology and cell signaling.