OXIDATION OF 3-HYDROXYANTHRANILIC ACID TO THE PHENOXAZINONE CINNABARINIC ACID BY PEROXYL RADICALS AND BY COMPOUND-I OF PEROXIDASES OR CATALASE

Since 3-hydroxyanthranilic acid (3HAA), an oxidation product of tryptophan metabolism, is a powerful radical scavenger [Christen, S., Peterhans, E., & Stocker, R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2506], its reaction with peroxyl radicals was investigated further. Exposure to aqueous peroxyl radicals generated at constant rate under air from the thermolabile radical initiator 2,2'-azobis[2-amidinopropane] hydrochloride (AAPH) resulted in rapid consumption of 3HAA with initial accumulation of its cyclic dimer, cinnabarinic acid (CA). The initial rate of formation of the phenoxazinone CA accounted for almost-equal-to 75% of the initial rate of oxidation of 3HAA, taking into account that 2 mol of 3HAA are required to form 1 mol of CA. Consumption of 3HAA under anaerobic conditions (where alkyl radicals are produced from AAPH) was considerably slower and did not result in detectable formation of CA. Addition of superoxide dismutase enhanced autoxidation of 3HAA as well as the initial rates of peroxyl radical-induced oxidation of 3HAA and formation of CA by almost-equal-to almost-equal-to 40-50%, whereas inclusion of xanthine/xanthine oxidase decreased the rate of oxidation of 3HAA by almost-equal-to 50% and inhibited formation of CA almost completely, suggesting that superoxide anion radical (O2.-) was formed and reacted with reaction intermediate(s) to curtail formation of CA. Formation of CA was also observed when 3HAA was added to preformed compound I of horseradish peroxidase (HRPO) or catalytic amounts of either HRPO, myeloperoxidase, or bovine liver catalase together with glucose/glucose oxidase. In the case of preformed HRPO compound I, oxidation of 3HAA into CA proceeded to the same extent under both aerobic and anaerobic conditions, indicating that it did not require molecular oxygen per se. The results suggest that oxidative dimerization of 3HAA into CA can be obtained by initial and successive one-electron oxidation reactions mediated by either free radicals or compound I of peroxidases or catalase.