Coumarin-3-carboxylic acid as a detector for hydroxyl radicals generated chemically and by gamma radiation

Coumarin-3-carboxylic acid (3-CCA) was used as a detector for hydroxyl radicals ((OH)-O-.) in aqueous solution. The (OH)-O-. was generated by gamma irradiation or chemically by the Cu2+-mediated oxidation of ascorbic acid (ASC). The excitation and emission spectra of 3-CCA, hydroxylated either chemically or by gamma irradiation, were nearly identical to those of an authentic 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). The pH-titration curves for the fluorescence at 450 nm (excitation at 395 nm) of 3-CCA, hydroxylated either chemically or by gamma radiation, were also identical to those of authentic 7-OHCCA (pK = 7.4). Time-resolved measurements of the fluorescence decays of radiation- or chemically hydroxylated 3-CCA, as well as those of 7-OHCCA, indicate a monoexponential fit. The fluorescence lifetime for the product of 3-CCA hydroxylation was identical to that of 7-OHCCA (similar to 4 ns). These data, together with analysis of end products by high-performance liquid chromatography, show that the major fluorescent product formed by radiation-induced or chemical hydroxylation of 3-CCA is 7-OHCCA. Fluorescence detection of 3-CCA hydroxylation allows real-time measurement of the kinetics of (OH)-O-. generation. The kinetics of 3-CCA hydroxylation by gamma radiation is linear, although the kinetics of 3-CCA hydroxylation by the Cu2+-ASC reaction shows a sigmoid shape. The initial (slow) step of 3-CCA hydroxylation is sensitive to Cu2+, but the steeper (fast) step is sensitive to ASC. Analysis of the kinetics of 3-CCA hydroxylation shows a diffusion-controlled reaction with a rate constant 5.0 +/- 1.0 X 10(9) M-1 s(-1). The scavenging of (OH)-O-. by 3-CCA was similar to 14% for chemical generation with Cu2+-ASC and similar to 50% for gamma-radiation-produced . OH. The yield of 7-OHCCA under the same radiation conditions was similar to 4.4% and increased linearly with radiation dose. The 3-CCA method of detection of (OH)-O-. is quantitative, sensitive, specific and therefore accurate. It has an excellent potential for use in biological systems. (C) 1997 by Radiation Research Society.