Disulfide bonds play a key function in determining the structure of proteins, and are the most strongly conserved compositional feature across proteomes. They are particularly common in extracellular environments, such as the extracellular matrix and plasma, and in proteins that have structural (e.g. matrix) or binding functions (e.g. receptors). Recent data indicate that disulfides vary markedly with regard to their rate of reaction with two-electron oxidants (e.g. HOCl, ONOOH), with some species being rapidly and readily oxidized. These reactions yielding thiosulfinates that can react further with a thiol to give thiolated products (e.g. glutathionylated proteins with glutathione, GSH). Here we show that these ‘oxidant-mediated thiol-disulfide exchange reactions’ also occur during photo-oxidation reactions involving singlet oxygen ( 1 O 2 ). Reaction of protein disulfides with 1 O 2 (generated by multiple sensitizers in the presence of visible light and O 2 ), yields reactive intermediates, probably zwitterionic peroxyl adducts or thiosulfinates. Subsequent exposure to GSH, at concentrations down to 2 μM, yields thiolated adducts which have been characterized by both immunoblotting and mass spectrometry. The yield of GSH adducts is enhanced in D 2 O buffers, and requires the presence of the disulfide bond. This glutathionylation can be diminished by non-enzymatic (e.g. tris-(2-carboxyethyl)phosphine) and enzymatic (glutaredoxin) reducing systems. Photo-oxidation of human plasma and subsequent incubation with GSH yields similar glutathionylated products with these formed primarily on serum albumin and immunoglobulin chains, demonstrating potential in vivo relevance. These reactions provide a novel pathway to the formation of glutathionylated proteins, which are widely recognized as key signaling molecules, via photo-oxidation reactions.

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Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen ( 1 O 2 )-mediated oxidation of multiple proteins (α-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDS-PAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the 1 O 2 concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with 18 O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta-2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide ‘proof-of-concept’ for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies.