Oxidation of 5-thio-2-nitrobenzoic acid, by the biologically relevant oxidants peroxynitrite anion, hydrogen peroxide and hypochlorous acid

The modification of protein and non-protein thiols by oxidants including hydrogen peroxide (H2O2), peroxynitrite anion (ONOO−) and hypochlorous acid (HOCl) is well documented. Using an aromatic thiol, 5-thio-2-nitrobenzoic acid, and biologically relevant oxidants, we have identified higher oxidation states of sulfur including the sulfonic acid derivative and the disulfide S-oxide, a thiosulfinate, by HPLC and mass spectrometry. The initial reaction of ONOO− with 5-thio-2-nitrobenzoic acid yielded a transient red intermediate, the sulfenate anion. The red intermediate was observed when ONOO− and H2O2 were used to oxidize 5-thio-2-nitrobenzoic acid and it persisted for several seconds at pH 7. HOCl oxidized the disulfide, 5,5′dithiobis(2-nitrobenzoic acid) to the corresponding sulfonic acid and no additional products were detected. Using this system, we can directly compare the thiol-oxidizing abilities of several oxidants. Because 5-thio-2-nitrobenzoic acid is the product of the reaction of Ellman’s reagent with protein thiols, a detailed study of its stability in biological matrices where oxidants may be generated is warranted.