Interactions of iron bleomycin, phosphate or cyanide, and DNA: sequence-dependent conformations and reactions

The hypothesis was investigated that axial ligands bound to Fe(III)-bleomyc in [Fe(III)Blm] are destabilized at specific 5'-guanine-pyrimidine-3' bindi ng sites but are stable at nonselective dinucleotides. DNA oligomers and ca lf-thymus DNA were used in reactions with L-Fe(III)Blm, where phosphate and cyanide served as examples of large and small ligands (L). Both ligands un derwent dissociation when L-Fe(III)BLm was bound to d(GGAAGCTTCC)(2) (I) bu t not d(GGAAATTTCCC)(2) (II) and at large ratios of calf-thymus DNA to drug . Fe(III)Blm is high spin in 20 mM phosphate buffer, signifying the presenc e of phosphate adduct. In the titration of HPO4-Fe(III)Blm with calf-thymus DNA, a large excess of DNA was needed to reach the low-spin state, consist ent with an equilibrium competition between phosphate and DNA for Fe(III)Bl m. Equilibrium constants for binding Fe(III)Blm and CN-Fe(III)BLm to calf-t hymus DNA (6.8x10(5)M(-1) and 5.9x10(4)M(-1), respectively, in HEPES buffer at 25 degreesC and pH 7.4) showed that the CN- ligand also reduced the aff inity of DNA for the drug. The kinetics of dissociation of CN- from CN-Fe(I II)Blm-DNA were slow and first order in bound drug. The reversible nature o f these dissociation reactions was shown using H-1 NMR spectroscopy of Fe(I II)Blm-I in the absence and presence of large excesses of CN- or phosphate. The results are discussed in terms of a two-state hypothesis for the bindi ng of L-Fe(III)Blm to specific and nonspecific dinucleotides. It is propose d that steric restrictions at specific sites inhibit binding of these ligan ds.