Abstract
Purpose: The aim of this work was to adapt a recently developed fluorometric method for use in the detection of hydroxyl radical (HO) generated in the immediate vicinity of chromatin core particles reconstituted from pUC19 plasmid DNA and isolated core histones. Materials and methods: The procedure followed involves labelling nucleosomal histones with SECCA, a non-fluorescent coumarin derivative that generates the fluorescent 7-hydroxy-SECCA after reaction with HO. Core particles are formed using histones and pUC19 DNA in a salt-dialysis procedure. Results: Electron microscopy and micrococcal nuclease digestion are consistent with successful formation of core particles. No significant differences between core particle formation in the unlabelled and SECCA-labelled samples were detected. Exposure to HO generated by radiation or copper-- ascorbic acid-hydrogen peroxide results in a gradual induction of fluorescence. Studies using dimethyl sulphoxide (DMSO) demonstrate that, unlike HO produced by radiation, the majority of HO generated by copper-- ascorbic acid--hydrogen peroxide occurs primarily in the immediate vicinity of core particles and DNA and cannot be scavenged. Conclusions: The present procedure demonstrates the feasibility to quantitate HO generated by several agents in the immediate vicinity of nucleosomes (chromatin-associated HO) or associated with specific regions within the genome.
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