A fluorimetric method for the detection of copper-mediated hydroxyl free radicals in the immediate proximity of DNA.

ABSTRACT

An optical method to detect copper-mediated hydroxyl free radicals generated close to DNA and other biomolecules has been developed. Low-molecular-weight polylysines were labeled with SECCA, a derivative of coumarin that generates the fluorescent 7-OH-SECCA following its interaction 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 mumol 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 corresponding to the formation of the fluorescent 7-OH-SECCA in the polylysine-nucleic acid complex. This fluorescence was not decreased significantly by addition of high concentrations of hydroxyl free-radical scavengers (DMSO, methanol, ethanol and tert-butanol), but was diminished by addition of relatively low concentrations of EDTA (0.1 mmol dm-3), histidine (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 the absence of DNA, the induced fluorescence was diminished by all hydroxyl free-radical scavengers. The efficiency by which the scavengers reduce the fluorescence increases as their hydroxyl rate constant increases. The data indicate that the detector molecule SECCA can be used to detect copper-mediated hydroxyl free radicals generated close to DNA.