RESUMEN
Ascorbate is as a potent antioxidant in vivo protecting the organism against oxidative stress. In this process, ascorbate is oxidized in two steps to dehydroascorbate (DHA), which if not efficiently reduced back to ascorbate decomposes irreversibly to a complex mixture of products. We demonstrate that a component of this mixture specifically reacts with the thiol group of cysteine residues at physiological pH to give a protein adduct involving the addition of a 5-carbon fragment of DHA (+112 Da). Incubations of glutaredoxin-1 expressed in Escherichia coli and dehydroascorbate revealed abundant adducts of +112, +224 and +336 Da due to the addition of one, two and three conjugation products of DHA, respectively. ESI-MS of carbamidomethylated glutaredoxin-1 before incubation with DHA, deuterium exchange together with tandem mass spectrometry analysis and LC-ESIMS/MS of modified peptides confirmed structure and sites of modification in the protein. Modification of protein thiols by a DHA-derived product can be involved in oxidative stress-mediated cellular toxicity.
Asunto(s)
Ácido Deshidroascórbico/química , Glutarredoxinas/química , Proteínas Recombinantes/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Liquida , Medición de Intercambio de Deuterio , Escherichia coli/genética , Glutarredoxinas/genética , HumanosRESUMEN
A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the oxidized bases including 8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiquitous marker of almost every type of oxidative stress in cells. Efforts to standardize the nomenclature and abbreviations of the main DNA degradation products that arise from oxidative pathways are reported. Information is also provided on the main oxidative radicals, non-radical oxygen species, one-electron agents and enzymes involved in DNA degradation pathways as well in their targets and reactivity. A brief classification of oxidatively generated damage to DNA that may involve single modifications, tandem base modifications, intrastrand and interstrand cross-links together with DNA-protein cross-links and base adducts arising from the addition of lipid peroxides breakdown products is also included.