Kinetics of reaction of DNA-bound Fe(III)bleomycin with ascorbate: interplay of specific and non-specific binding.
The aerobic redox reaction of Fe(III)bleomycin (Blm) and ascorbate was examined in the absence of DNA and in the presence of 7.5 and 25 calf thymus DNA base pairs per-drug molecule, in order to investigate the effect of DNA binding on the properties of FeBlm activation and DNA strand cleavage. Under these successive conditions, the rate of initial reduction of Fe(III)Blm became progressively slower and biphasic. Using 7.5 base pairs per-molecule of FeBlm, 2-3 times as much drug reacted in the faster step as with the larger DNA to drug ratio. In each case, the more rapid process was identified with the reaction of high spin Fe(III)Blm-DNA. With the smaller ratio, dioxygen consumption, formation of HO(2)-Fe(III)Blm-DNA, and production of DNA strand breaks as measured by the formation of base propenal were largely rate limited by the initial reaction of ascorbate with Fe(III)Blm-DNA. After a burst of reaction with the larger ratio of base pairs to Fe(III)Blm, a small fraction of the total Fe(III)Blm, representing high spin Fe(III)Blm, entered a steady state as HO(2)-Fe(III)Blm-DNA. Thereafter, reaction of dioxygen and base propenal formation occurred slowly with similar first-order rate kinetics. In order to explain these results, it is hypothesized that the metal domain-linker of Fe(III)Blm adopts two conformations with respect to DNA. One, at specific binding sites, is relatively unreactive with ascorbate. The other, present at non-specific sites as HPO(4)-Fe(III)Blm, is readily reactive with ascorbate to generate HO(2)-Fe(III)Blm-DNA. At the larger base pair to drug ratio, movement of Fe(III)Blm between specific and non-specific sites to generate HO(2)-Fe(III)Blm is a necessary part of the mechanism of strand scission.
Li
Wenbao
W
Department of Chemistry, University of Wisconsin-Milwaukee, P.O. Box 413, Milwaukee WI 53201, USA.
Antholine
William E
WE
Petering
David H
DH
D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001761:Bleomycin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism
D005290:Ferric Compounds; D008024:Ligands; C023503:iron bleomycin; D001761:Bleomycin; D004247:DNA; D001205:Ascorbic Acid; D010100:Oxygen