KINETICS OF THE DAUNOMYCIN DNA INTERACTION

The kinetics of the interaction of daunomycin with calf thymus DNA are described. Stopped-flow and temperature-jump relaxation methods, using absorption detection, were used to study the binding reaction. Three relaxation times were observed, all of which are concentration dependent, although the 2 slower relaxations approach constant values at high reactant concentrations. Relaxation times over a wide range of concentrations were gathered, and the data were fit by a minimal mechanism in which a rapid bimolecular association step is followed by 2 sequential isomerization steps. The 6 rate constants for this mechanism were extracted from data by relaxation analysis. The values determined for the 6 rate constants may be combined to calculate an overall equilibrium constant that is in excellent agreement with that obtained by independent equilibrium measurements. Additional stopped-flow experiments, using 1st sodium dodecyl sulfate to dissociate bound drug and 2nd pseudo-1st-order conditions to study the fast bimolecular step, provide independent verification of 3 of the 6 rate constants. The temperature dependence of 4 of the 6 rate constants was measured, allowing estimates of the activation energy of some of the steps to be made. The 3 steps in the proposed mechanism may correspond to a rapid outside binding of daunomycin to DNA, followed by intercalation of the drug, followed by either conformational adjustment of the drug or DNA binding site or redistribution of bound drug to preferred sites.