A METHOD FOR DETECTION OF HYDROXYL RADICALS IN THE VICINITY OF BIOMOLECULES USING RADIATION-INDUCED FLUORESCENCE OF COUMARIN

A novel method is described to quantitate radiation-induced hydroxyl r adicals in the vicinity of biomolecules in aqueous solutions. Coumarin -3-carboxylic acid (CCA) is a non-fluorescent molecule that, upon inte raction with radiation in aqueous solution, produces fluorescent produ cts. CCA was derivatized to its succinimidyl ester (SECCA) and coupled to free primary amines of albumin, avidin, histone-H1, polylysine, an d an oligonucleotide. When SECCA-biomolecule conjugates were irradiate d, the relationship between induced fluorescence and dose was linear i n the dose range examined (0.01-10 Gy). The fluorescence excitation sp ectrum of irradiated SECCA-biomolecule conjugates was very similar to that of 7-hydroxy-SECCA-biomolecule conjugates, indicating the convers ion of SECCA to 7-hydroxy-SECCA following irradiation. Control studies in environments that excluded certain radiation-induced water radical s for both the conjugated and unconjugated forms of irradiated SECCA d emonstrated that: (1) the induction of fluorescence is mediated by the hydroxyl radical; (2) the presence of oxygen enhances induced fluores cence by a factor of about 1.4, and (3) other primary water radicals a nd secondary radicals caused by interaction of primary water radicals with biomolecules do not significantly influence the induced fluoresce nce. The data indicate that the induction of fluorescence on SECCA-bio molecule conjugates records specifically the presence of the hydroxyl radical in the immediate vicinity of the irradiated biomolecule. The m ethod is rapid and sensitive, uses standard instrumentation, and the s ample remains available for further studies.