Equilibrium binding of daunomycin and adriamycin to calf thymus DNA. Temperature and ionic strength dependence of thermodynamic parameters

Abstract

Absorbance and fluorescence quenching monitoring of the binding of the anthracyclines adriamycin (ADM) and daunomycin (DNM) to calf thymus DNA, provides reproducible binding data only when moderate drug/DNA molar ratios are used in the assays. Under these conditions, the fraction of DNA-bound drug, in equilibrium with free anthracycline, which can be reliably detected, ranged from 40-60% to 80-95% of the total added drug, depending upon ionic strength and temperature. Use of the neighbour exclusion model adequately fits such data and predicts that (i) the affinity of ADM for binding to the DNA is always higher than that corresponding to DNM, under similar experimental conditions, (ii) the binding constant for both drugs exhibits a strong salt and temperature dependence, and (iii) the exclusion parameter, indicative of the size of the anthracycline binding sites on the DNA, equals 3.1 +/- 0.4 and 3.3 +/- 0.4 base pairs for ADM and DNM, respectively, and is independent of salt concentration. The salt and temperature dependence of the binding constant is used to estimate the thermodynamic parameters involved in the interaction of the drugs with the DNA. Binding of the drugs is an exothermic process and the binding free energy arises primarily from a large negative enthalpy which, as the entropy, strongly depends upon ionic strength, and is much larger than predicted by polyelectrolyte theory. The enthalpy and entropy changes observed, appear to compensate each other over the entire range of salt concentrations used, and may arise from a complex variety of contributions, including salt-induced changes in secondary structure of the DNA, as indicated by circular dichroism techniques.