Pergamon Free Radical Biology & Medicine,Vol. 18, No. 4, pp. 669-678, 1995 Copyright © 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0891-5849/95 $9.50 + .00 0891-5849(94)00184-7 Original Contribution A FLUORIMETRIC METHOD FOR THE DETECTION OF COPPER- MEDIATED HYDROXYL FREE RADICALS IN THE IMMEDIATE PROXIMITY OF DNA G. MIKE MAKRIGIORGOS,* EDWARD BUMP,* CHRISTINE HUANG,* JANINA BARANOWSKA-KORTYLEWICZ, ¢ and AMIN I. KASSIS* *Joint Center for Radiation Therapy, Department of Radiation Oncology, and *Department of Radiology, Harvard Medical School, Boston, MA, USA; and *University of Nebraska Medical Center, Omaha, NE, USA (Received 2 May 1994; Revised 15 August 1994; Accepted 18 August 1994) 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 #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 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 × 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. Keywords--Fluorescence, Free radicals, DNA damage, Transition metals, Copper INTRODUCTION Oxygen-derived free radicals have been implicated in the etiology of a variety of biological phenomena such as aging, mutagenesis, carcinogenesis, inflammation, and other pathological conditions. 1-6 Reactive oxygen species such as superoxide anion (02"-) and hydrogen peroxide (H202) are generated during cellular metabo- lism or by external factors such as environmental muta- gens, chemicals, radiation, or certain chemotherapeutic drngs. 6'7 It has been suggested that copper and other transition metals have a high affinity to some cellular biomolecules including DNA, and that traces of metal can catalyze the formation of hydroxyl free radicals (HO') causing damage to cellular biomolecules. 8-~° Be- cause the average diffusion distance of hydroxyl radi- cals in cells is very short, the deleterious effects are Address correspondence to: G. Mike Makrigiorgos, Joint Center for Radiation Therapy, 50 Binney Street, Boston, MA 02115, USA. 669 thought to be site-specific, in close proximity to the metal-binding site. 1~ Damage caused by biomolecule-bound metals is a subject of current interest and is often studied in cell- free chromatin and DNA model systems. 11'12 In most studies, the effects of the damage caused by metal- induced free radicals rather than the radicals them- selves are measured. Methods for studying radicals di- rectly in biomolecule-containing samples have also been applied; 5"13 however, these lack information on the site-specificity of free radical formation, which is a central element in understanding the processes in- volved. In this article, a fluorimetric method for the site- specific detection on biomolecules of copper-catalyzed hydroxyl radicals is presented. A molecular probe (SECCA, the succinimidyl ester of coumarin-3-car- boxylic acid) has been recently described, which, in neutral aqueous solutions, is nonfluorescent, but which is converted upon interaction with ionizing-radiation- induced hydroxyl radicals to hydroxylated derivatives