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Abstract
A recently developed method has been utilized to demonstrate the generation of hydroxyl radicals (HObullet) in the immediate proximity of DNA by cop-per(II)/iron(HI)–adriamycin in the presence of ascorbate and hydrogen peroxide. SECCA, a succinylated derivative of coumarin, generates the fluorescent 7-hydroxy-SECCA following reaction with HObullet. SECCA was coupled to polylysine or to histone HI and then complexed to DNA. When HO' was generated in the proximity of DNA by polylysine-coupled iodine-125, which emits short range Auger electrons, 7-hydroxy-SECCA was produced. DMSO was only moderately efficient in reducing the fluorescence induction, demonstrating the “local” generation of HObullet in this system. Copper(II)/iron(III)–adriamycin in the presence of ascorbate and hydrogen peroxide generated the fluorescent 7-hydroxy-SECCA both when SECCA was free in solution and when SECCA was DNA-conjugated. With SECCA free in solution, the fluorescence induction was almost eliminated in the presence of HObullet scavengers (ethanol, tertbutanol or DMSO) and the relative efficiency of the scavengers in reducing the fluorescence followed their rate constant with HObullet. Furthermore, SECCA incubated with a singlet oxygen-generating compound demonstrated no fluorescence induction. When SECCA was positioned in close proximity to DNA as a SECCA-histone-H1–DNA complex, the relative efficiency of the scavengers in reducing the fluorescence still followed their rate constant with HO'; overall however the scavengers were much less effective in reducing the fluorescence, due presumably to the formation of HObullet radical in the immediate vicinity of DNA. These data suggest that copper(II)/iron(III)–adriamycin produces HO' in the presence of ascorbate and hydrogen peroxide whether unbound or bound to DNA and suggest that in the latter case scavengers would not prevent HObullet from attacking chromatin. In addition, the ability of DMSO to trap HObullet was shown to decrease as the conformation of the HI–DNA complex becomes more compact indicating the strong dependence of the trapping ability on chromatin conformation.