Characterisation of the major autoxidation products of 3-hydroxykynurenine under physiological conditions

Abstract

3-Hydroxykynurenine (3-OHKyn) is a tryptophan metabolite that is readily autoxidised to products that may be involved in protein modification and cytotoxicity. The oxidation of 3-OHKyn has been studied here with a view to characterising the major products as well as determining their relative rates of formation and the role that H2O2 and hydroxyl radical (HO*) may play in modifying the autoxidation process. Oxidation of 3-OHKyn generated several compounds. Xanthommatin (Xan), formed by the oxidative dimerisation of 3-OHKyn, was the major product formed initially. It was, however, found to be unstable, particularly in the presence of H2O2, and degraded to other products including the p-quinone, 4,6-dihydroxyquinolinequinonecarboxylic acid (DHQCA). A compound that has a structure consistent with that of hydroxyxanthommatin (OHXan) was also formed in addition to at least two minor species that we were unable to identify. Hydrogen peroxide was formed rapidly upon oxidation of 3-OHKyn, and significantly influenced the relative abundance of the different autoxidation species. Increasing either pH (from pH 6 to 8) or temperature (from 25 degrees C to 35 degrees C) accelerated the rate of autoxidation but had little impact on the relative abundance of the autoxidation species. Using electron paramagnetic resonance (EPR) spectroscopy, a clear phenoxyl radical signal was observed during 3-OHKyn autoxidation and this was attributed to xanthommatin radical (Xan*). Hydroxyl radicals were also produced during 3-OHKyn autoxidation. The HO* EPR signal disappeared and the Xan* EPR signal increased when catalase was added to the autoxidation mixture. The HO* did not appear to play a role in the formation of the autoxidation products as evidenced using HO* traps/scavengers. We propose that the cytotoxicity of 3-OHKyn may be explained by both the generation of H2O2 and by the formation of reactive 3-OHKyn autoxidation products such as the Xan* and DHQCA.

A1M, an extravascular tissue cleaning and housekeeping protein

“…Thus, Lys69 [47], 92, 118 and 130 [3] are covalently modified in human A1M isolated from urine. Furthermore, the tryptophan metabolite 3-OH-kynurenin, which has a propensity to form free radicals [48], was identified as a lysyl modification on urinary A1M from hemodialysis patients [49]. The side-chains of Lys92, 118 and 130 may therefore serve a dual role both in activation of the Cys34 thiol by lowering its pK a [38] and as electron-donors in the radical-trapping mechanism.…”

Novel Protein Modification by Kynurenine in Human Lenses

“…The consequences of 3-OHKyn binding to human lens protein will be even more deleterious. When 3-OHKyn binds to protein through the amino acid side chain, as described here, the reactive o-aminophenol functionality is still able to undergo oxidation reactions that, in turn, may lead to localized formation of superoxide and hydrogen peroxide (42). Oxidation of bound 3-OHKyn could also produce protein cross-links (43) and may also result in protein coloration, precipitation, and cataract formation.…”

3-Hydroxykynurenine-mediated Modification of Human Lens Proteins

“…Kynurenines are unstable at physiological pH; they undergo side chain deamination to produce ␣, ␤ unsaturated ketoalkenes (15). Kynurenine levels decrease in the lens with age (16) and cataract formation (17), which may be due to their reaction with lens proteins.…”