Chemical synthesis of radiolabeled bleomycin a2 and Its Binding to DNA

Samar N. Roy, George A. Orr, C. Fred Brewer, Susan Band Horwitz

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44 Scopus citations


A method for the preparation of biologically active [3H]- and [13C]bleomycin A2 is described. Demethyl Cu(ll):bleomycin A2, isolated after pyrolysis of Cu(ll):bleomycin A2( was methylated with either [3H]- or [13C]methyl iodide, which resulted in Cu(ll): bleomycin A2 labeled in the dimethylsulfonium moiety. Copper was removed by treatment with dithizone in chloroform, and structures were verified by thin-layer chromatography and 1H and 13C nuclear magnetic resonance spectroscopy. Copper-free [3H] and [13C]bleomycin A2 are active in the degradation of DNA in vitro. Gel exclusion chromatography and equilibrium dialysis were used to determine the apparent equilibrium constants for binding of [3H]bleomycin A2 and Cu(ll):[3H]bleomycin A2 to calf thymus DNA, noncovalently associated polydeoxyguanylate: polydeoxycytidylate, and noncovalently associated polydeoxyadenylate:polydeoxythymidylate. In 2.5 mM sodium phosphate buffer, pH 7.0, binding data obtained by gel filtration with calf thymus DNA reveal an apparent equilibrium constant for [3H]bleomycin A2 of 5.7*105/mol and for Cu(ll):[3H]bleomycin A2 of 3.9*105/mol. One molecule of [3H]bleomycin A2 binds for every 3.7 base pairs in DNA, and one molecule of Cu(ll):[3H]bleomycin A2 binds for every 2.8 base pairs in DNA. Analysis of binding data with calf thymus DNA, noncovalently associated polydeoxyguanylate:polydeoxycytidylate, and non-covalently associated polydeoxyadenylate:polydeoxythymidy-late obtained by equilibrium dialysis reveals, in each instance, 2 types of binding sites for both the copper and metal-free form of the antibiotic. For those sites in calf thymus DNA with tighter binding affinity, the apparent equilibrium constant for [3H]bleomycin A2 was 6.8 * 106/mol and for the Cu(ll):[3H]bleomycin A2 complex, 4.4 * 105/mol. As seen with calf thymus DNA, the affinity of [3H]bleomycin A2 is slightly greater than that of Cu(ll):[3H]bleomycin A2 for the synthetic DNAs, although more of the copper form of the drug binds to these polymers.

Original languageEnglish (US)
Pages (from-to)4471-4477
Number of pages7
JournalCancer research
StatePublished - Nov 1 1981

ASJC Scopus subject areas

  • Oncology
  • Cancer Research


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