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 (^{\bullet}{\rm OH}$) in aqueous solution. The ^{\bullet}{\rm OH}$ was generated by γ irradiation or chemically by the ${\rm Cu}^{2+}\text{-}\text{mediated}$ oxidation of ascorbic acid (ASC). The excitation and emission spectra of 3-CCA, hydroxylated either chemically or by γ 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 γ 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 (∼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 ^{\bullet}{\rm OH}$ generation. The kinetics of 3-CCA hydroxylation by γ radiation is linear, although the kinetics of 3-CCA hydroxylation by the ${\rm Cu}^{2+}\text{-}{\rm ASC}$ 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 $5.0\pm 1.0\times 10^{9}\ M^{-1}\ {\rm s}^{-1}$. The scavenging of ^{\bullet}{\rm OH}$ by 3-CCA was ∼14% for chemical generation with ${\rm Cu}^{2+}\text{-}{\rm ASC}$ and ∼50% for γ-radiation-produced ^{\bullet}{\rm OH}$. The yield of 7-OHCCA under the same radiation conditions was ∼4.4% and increased linearly with radiation dose. The 3-CCA method of detection of ^{\bullet}{\rm OH}$ is quantitative, sensitive, specific and therefore accurate. It has an excellent potential for use in biological systems.