Absolute rate constants for the quenching of reactive excited states by melanin and related 5,6-dihydroxyindole metabolites: implications for their antioxidant activity

Abstract

The triplet-excited state of benzophenone and the singlet-excited state of 2,3-diazabicyclo[2.2.2]oct-2-ene (Fluorazophore-P) have been employed as kinetic probes to obtain information on the antioxidant activity of the skin and eye pigment melanin and its biogenetic precursors 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The excited states were generated by the laser-flash photolysis technique and their reaction kinetics was examined by time-resolved transient absorption or fluorescence spectroscopy, respectively. The reaction between triplet benzophenone and DHI produced with unit efficiency the corresponding 6O-centered semiquinone radical, which was characterized by its characteristic transient absorption. The quenching rate constants for DHI (3.1-8.4 x 10(9) M-1 s-1) and DHICA (3.3-5.5 x 10(9) M-1 s-1) were near the diffusion-controlled limit, indicating excellent antioxidant properties. Kinetic solvent effects were observed. The reactivity of synthetic melanin, assessed through the quenching rate constant of Fluorazophore-P and normalized to the number of monomer units, was more than one order of magnitude lower (2.7 x 10(8) M-1 s-1) than that of its precursors. The trend of the quenching rate constants, i.e. DHI > DHICA approximately alpha-tocopherol > melanin, along with the preferential solubility of DHICA in aqueous environments, serves to account for several experimental results from biochemical studies on the inhibition of lipid peroxidation by these natural antioxidants.