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

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 ki netic 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-hash photolysis technique and their reaction ki netics was examined by time-resolved transient absorption or fluorescence s pectroscopy, respectively. The reaction between triplet benzophenone and DH I produced with unit efficiency the corresponding 6O-centered semiquinone r adical, 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 DHI CA (3.3-5.5 x 10(9) M-1 s(-1)) were near the diffusion-controlled limit, in dicating excellent antioxidant properties, Kinetic solvent effects were obs erved, The reactivity of synthetic melanin, assessed through the quenching rate constant of Fluorazophore-P and normalized to the number of monomer un its, was more than one order of magnitude lower (2.7 x 10(8) M-1 s(-1)) tha n that of its precursors. The trend of the quenching rate constants, i.e, D HI > DHICA approximate to alpha-tocopherol > melanin, along nith the prefer ential solubility of DHICA in aqueous environments, serves to account for s everal experimental results from biochemical studies on the inhibition of L ipid peroxidation by these natural antioxidants.