Measurement of copper-dependent oxidative DNA damage by HOCl and H2O2 with the ethidium-binding assay.
The ETB-binding assay provides a rapid means to investigate time profiles for oxidative degradation of double-stranded DNA, by detecting the loss of ability of DNA to form a fluorescent intercalation complex with ETB. The assay was applied to demonstrate copper-dependent damage to DNA by HOCl using ascorbate as reductant. DNA degradation in this system proceeds with a rate comparable to that of reaction of HOCl with the DNA-Cu(I) complex, as monitored by the loss of DNA-Cu(I) absorption. The reaction of HOCl with DNA-Cu(I) is more than two orders of magnitude faster than the reaction of H2O2 with DNA-Cu(I). HOCl presumably reacts like H2O2 with DNA-Cu(I) to generate strongly oxidizing OH radicals immediately at the Cu(I) binding site. Quenching of the DNA/ETB fluorescence by DNA-bound copper was investigated because this may interfere with detection of copper-dependent damage to DNA with the ETB-binding assay, if not appropriate chelators are applied to remove the copper from the DNA.
Prütz
W A
WA
Universität Freiburg, Institut für Biophysik und Strahlenbiologie, Germany.
D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects* / Q000378:metabolism*; D004249:DNA Damage; D004996:Ethidium / Q000378:metabolism*; D005453:Fluorescence; D006861:Hydrogen Peroxide / Q000633:toxicity*; D006997:Hypochlorous Acid / Q000633:toxicity*; D007364:Intercalating Agents / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction
D007364:Intercalating Agents; D006997:Hypochlorous Acid; D003300:Copper; D004247:DNA; D006861:Hydrogen Peroxide; D004996:Ethidium