CONTINUOUS DETECTION OF RADIATION OR METAL GENERATED HYDROXYL RADICALS WITHIN CORE CHROMATIN PARTICLES

Purpose: The aim of this work was to adapt a recently developed fluoro metric method for use in the detection of hydroxyl radical (HO.) gener ated in the immediate vicinity of chromatin core particles reconstitut ed from pUC19 plasmid DNA and isolated core histones. Materials and me thods: The procedure followed involves labelling nucleosomal histones with SECCA, a non-fluorescent coumarin derivative that generates the f luorescent 7-hydroxy-SECCA after reaction with HO.. Core particles are formed using histones and pUC19 DNA in a salt-dialysis procedure. Res ults: Electron microscopy and micrococcal nuclease digestion are consi stent with successful formation of core particles. No significant diff erences between core particle formation in the unlabelled and SECCA-la belled samples were detected. Exposure to HO . generated by radiation or copper(--)ascorbic acid(--)hydrogen peroxide results in a gradual i nduction of fluorescence. Studies using dimethyl sulphoxide (DMSO) dem onstrate that, unlike HO. produced by radiation, the majority of HO. g enerated by copper(--)ascorbic acid(--)hydrogen peroxide occurs primar ily in the immediate vicinity of core particles and DNA and cannot be scavenged. Conclusions: The present procedure demonstrates the feasibi lity to quantitate HO. generated by several agents in the immediate vi cinity of nucleosomes (chromatin-associated HO.) or associated with sp ecific regions within the genome.