MEASUREMENT OF HYDROXYL RADICALS CATALYZED IN THE IMMEDIATE VICINITY OF DNA BY METAL-BLEOMYCIN COMPLEXES

A recently developed sensitive fluorimetric assay has been used to exa mine whether free hydroxyl radicals (HO.) are generated in the immedia te vicinity of DNA by Fe(II)-bleomycin. When aqueous solutions of SECC A (the succinimidyl ester of coumarin-3-carboxylic acid) are irradiate d with gamma rays or incubated with Fe(II)-bleomycin or Fe(II) or Fe(I I)-EDTA in the presence of ascorbate and H2O2, 7-hydroxy-SECCA, a fluo rescent product of the interaction of HO. with SECCA, is generated. St udies with catalase and several HO. scavengers indicate that the fluor escence induction is mediated by HO.. On the contrary, Cu(II)-bleomyci n complexes under similar conditions fail to induce 7-hydroxy-SECCA fl uorescence. When SECCA is conjugated to DNA via SECCA-polylysine-DNA c omplexes and incubated in the same iron-containing systems, the relati ve ability of the scavengers to reduce the fluorescence again demonstr ates the generation of 7-hydroxy-SECCA by HO.. However, while the fluo rescence is practically eliminated by high concentrations of DMSO (100 mu mol dm(-3)) in the systems with Fe(Il) or Fe(II)-EDTA, it is not p ossible to reduce it similarly in the case of Fe(II)-bleomycin. These data demonstrate the generation of HO. by Fe(II)-bleomycin in the imme diate vicinity of DNA. Because the experiments simulate the lifetime o f HO. expected in cells, these data suggest that, if such DNA-associat ed HO. radicals are also produced in vivo by bleomycin, these would no t be scavengable by intracellular scavengers and they could interact w ith chromatin.