DISSECTION OF THE FREE-ENERGY OF ANTHRACYCLINE ANTIBIOTIC BINDING TO DNA - ELECTROSTATIC CONTRIBUTIONS

Fluorescence titration methods and equilibrium dialysis were used to s tudy the thermodynamics of the interaction of doxorubicin, the beta an omer of doxorubicin, daunorubicin, and hydroxyrubicin with DNA. All of these except hydroxyrubicin carry a net charge of +1 at neutral pH, a rising from the protonation of the daunosamine moiety. Hydroxyrubicin is a synthetic anthracycline antibiotic in which the amine moiety has been replaced by a hydroxyl group, which is uncharged but polar at neu tral pH. The comparative binding studies we describe offer a unique op portunity to evaluate the electrostatic contributions to the DNA bindi ng free energy of these anthracycline antibiotics and to test specific predictions arising from current polyelectrolyte theory as applied to ligand-DNA interactions. We find that the quantity (delta log K/delta log [M+]) changes from a value of -0.95 for doxorubicin to -0.18 for hydroxyrubicin. The latter value is in excellent agreement with the va lue of -0.24 predicted by the theory of Friedman and Manning for the i nteraction of an uncharged intercalating ligand with DNA. The DNA bind ing free energy decreases from -8.8 kcal mol-1 for doxorubicin to -7.2 kcal mol-1 for hydroxyrubicin under solution conditions of 200 mM Na, pH 7.0, 20-degrees-C. The results we obtain allow us to dissect the DNA binding free energy into its electrostatic and nonelectrostatic co mponents. Knowledge of these values helps to clarify the precise role of the amine group in doxorubicin binding to DNA. In addition, compari son of the DNA binding of doxorubicin with the beta anomer of doxorubi cin provides a striking example of stereoselective antibiotic binding to DNA.