Kinetic studies of anthracycline-DNA interaction by fluorescence stopped flow confirm a complex association mechanism

The kinetics of association and dissociation between calf thymus DNA and five anthracyclines, including doxorubicin, daunorubicin, and three synthetic analogues, were investigated with stopped flow using fluorescence detection. The sensitivity of this technique allowed us to work with submicromolar drug concentrations, thus excluding formation of aggregates, and with ratios of DNA base pairs to drug in the range 10-250, where site exclusion effects could be taken into account with a simple correction of DNA concentration and pseudo-first-order conditions were nearly fulfilled. In all cases, both association and dissociation reactions required a sum of three exponential terms to be fitted. However, satisfactory interpretation of reciprocal relaxation times as functions of DNA concentration was only achieved with kinetic models comprising a total of five steps. One of the extra steps was tentatively assigned to formation of a weakly bound, probably nonintercalated species. Another step was deduced from a comparison between results of association and dissociation experiments. The five steps are arranged, for convenience, in an association mechanism with two branches, though other mechanisms cannot be definitely ruled out. Correlation of cytotoxicity data with both association and dissociation rates is not found to be significant. This suggests that other factors must be involved in modulating the different biological properties of the investigated anthracyclines.