Reaction of peroxynitrite with the melanin precursor, 5,6-dihydroxyindole-2-carboxylic acid

The reaction of peroxynitrite with the melanin precursor, 5,6-dihydroxyindo le-2-carboxylic acid (DHICA), was investigated by the stopped-flow techniqu e under deaerated conditions. The reaction is fast and proceeds through the intermediacy of unstable intermediates with absorption in the UV and visib le regions which finally gave brownish-black polymeric products. The appare nt second-order rate constant for the oxidation of DHICA by peroxynitrite i s (6.3 +/- 0.1) x 10(3) M(-1)s(-1) at pH 7.4 and 25 degrees C. LC-MS analys is of the reaction mixture of DHICA and peroxynitrite did not show the pres ence of nitrated products but dimers and trimers of DHICA as reaction produ cts. Thus, peroxynitrite, which nitrates phenolic compounds, did not produc e nitro-derivatives of DHICA, but oxidized ones. The oxidation of DHICA by peroxynitrite produced indole-5,6-quinone-2-carboxylic acid (DHICA-Q) as ev idenced by its characteristic absorption in the 380-400 nm range followed b y its transformation to a melanin like polymer as the final stable product. The rate of build-up of the absorption at 380 nm increased linearly with t he concentration of DHICA. Overall second-order kinetics is observed for th e peroxynitrite-mediated oxidation of DHICA suggesting that both DHICA and peroxynitrite be involved in the rate-determining step. The dependence of t he apparent second-order rate constant on pH displayed a bell-shaped curve with a maximum near pH 7.0. The observed pH dependence clearly indicates th e involvement of two acid-base equilibria in this reaction, those of peroxy nitrite and DHICA respectively. This novel reaction may have physiological relevance in the biosynthesis of melanin and in the understanding of the mo lecular mechanisms of UVB- induced melanogenesis. Since the precursors of p eroxynitrite, nitric oxide and superoxide, are produced by melanocytes in r esponse to UVB radiation it is possible that peroxynitrite could play a rol e in UV induced pigmentation.