A FLUOROMETRIC METHOD FOR THE DETECTION OF COPPER-MEDIATED HYDROXYL FREE-RADICALS IN THE IMMEDIATE PROXIMITY OF DNA

An optical method to detect copper-mediated hydroxyl free radicals gen erated close to DNA and other biomolecules has been developed. Low-mol ecular-weight polylysines were labeled with SECCA, a derivative of cou marin that generates the fluorescent 7-OH-SECCA following its interact ion with hydroxyl free radicals in aqueous solution. These polylysines were then complexed with DNA to place the detector molecule SECCA in the vicinity of the nucleic acid. Following addition of copper sulfate (0-10 mu mol dm(-3)), free radicals were generated by incubation with ascorbic acid (0-1 mmol dm(-3)) and hydrogen peroxide (0-1 mmol dm(-3 )). A rapid increase in the induced fluorescence was observed correspo nding to the formation of the fluorescent 7-OH-SECCA in the polylysine -nucleic acid complex. This fluorescence was not decreased significant ly by addition of high concentrations of hydroxyl free-radical scaveng ers (DMSO, methanol, ethanol and tert-butanol), but was diminished by addition of relatively low concentrations of EDTA (0.1 mmol dm(-3)), h istidine (0.1 mmol dm(-3)) or catalase (8.3 x 10(-5) mmol dm(-3)). On the other hand, when such reaction mixtures were incubated with SECCA molecules that were free in solution or SECCA-labeled polylysine in th e absence of DNA, the induced fluorescence was diminished by all hydro xyl free-radical scavengers. The efficiency by which the scavengers re duce the fluorescence increases as their hydroxyl rate constant increa ses. The data indicate that the detector molecule SECCA can be used to detect copper-mediated hydroxyl free radicals generated close to DNA.