Kinetics of the reaction of bleomycin-Fe(II)-O2 complex with DNA

The kinetics of the reaction of BLM-Fe(II)-O2 with DNA in the absence or presence of 2-mercaptoethanol (2-ME) were studied. The total number of bases released by BLM-Fe(II)-O2 in the presence of 2-ME increased about 6.5 times more than that in the absence of 2-ME in the reaction of 6 hours at 37 degrees C. The molar ratios of the released bases ware little affected by the reaction time, temperature or 2-ME. Among the four bases, thymine was preferentially released. On the basis of a reaction scheme of BLM-Fe(II)-O2 with DNA, the equations were derived by the steady-state method. In the absence of 2-ME, the release of bases from DNA was dependent on the concentration of BLM-Fe(II)-O2, but independent of the concentration of DNA. In the presence of 2-ME, a biphasic reaction was observed; the first one is due to BLM-Fe(II) which originally existed and the second one is due to BLM-Fe(II) produced by the reduction of BLM-Fe(III) with 2-ME. In the second reaction, the rate of the release of bases from DNA was proportional to the concentration of BLM-Fe(II) and 2-ME, but inversely proportional to the concentration of DNA. The rate-determining step in the reaction of BLM-Fe(II)-O2 with DNA in the presence of 2-ME was found to be the reduction of BLM-Fe(III) to BLM-Fe(II). By these kinetic studies, the reaction of BLM-Fe(II)-O2 with DNA in the presence of 2-ME was elucidated to proceed in a catalytic fashion. Furthermore, the maximum number of bases released by BLM from DNA was one base per twelve to thirteen bases.