Our experiments were performed at 170 mM Na þ, which may facilitate solubility and reduce possible self-association of the antibiotic, while the concentrations of MgCl 2 varied depending on the technique used (see Materials and Methods). The effect of multivalent cations on the DNA–ligand interactions is greater than expected on the basis of their contribution to the ionic strength (50,51). The presence of Mg 2 þ in the binding buffer merits further consideration not only because of the effects on the ionic strength, but also because of the direct binding (condensation) of the dication to the deoxyribonucleic acids (50). The Mg 2 þ requirement for the binding of mithramycin and related antibiotics to DNA has long been known and the presence of a dication is essential for the formation of the dimers that bind to DNA (12). For MTA, correcting the Á H b obtained by DSC, using the calculated Á C p, renders an enthalpy, at 25 8 C, higher than its value determined directly by ITC, which points to intrinsic difficulties in measuring the DNA binding constants for Mg 2 þ -coordinated antibiotics by this technique. These changes indicate that Á H estimates for MTA binding to DNA are temperature dependent, a situation sometimes neglected in van’t Hoff’s analyses of drug–DNA interactions obtained from spectroscopic data (49). Besides, the comparison of these Á H reveals the presence of heat capacity changes ( Á C p ), which we determined from the solvent-accessible areas in drug–DNA complexes (Table 2). Our observation that the binding enthalpies determined by van’t Hoff’s analysis were higher than those reported using direct ITC measurements is consistent with previous reports. The differences in the calculated Á H may be attributed to an ionic dependence of the enthalpy of binding (48).
These enthalpy values were measured under quite different ionic strength conditions-higher in our experiments-(cf. This value is about 2-fold higher than the enthalpy we obtained directly using ITC at 25 8 C ( Á H 1⁄4 þ 2.6 kcal mol À 1 ). In those experiments, the enthalpy of binding ( Á H 1⁄4 þ 5.1 kcal mol À 1 ) was estimated by application of the van’t Hoff relationship to the spectroscopic data (12,17).
In spectroscopic studies, positive enthalpy was also observed under conditions consistent with the formation of Mg 2 þ -coordinated MTA dimers.
It is interesting to compare the thermodynamic profile of the MTA binding to DNA described here with previous determinations of the enthalpy of binding by spectroscopic titration and fluorescence (12,17). Binding of MTA and MSK to DNA is clearly entropically driven (Table 1), and the positive sign of both Á H and Á S can be considered hallmarks of a predominantly hydrophobic binding reaction (23,47). Although the Á H values obtained by DSC or ITC differ from those obtained by spectroscopic techniques (12) they are consistent with the view that positive enthalpy is involved in the binding of Mg 2 þ -coordinated MTA dimers in the minor groove of DNA. Binding to C/G-rich DNA has been addressed for intercalating agents (23,45), yet some of these agents also bind along the minor groove in the vicinity of the intercalating site in DNA tracts of diverse sequences (40,41,46).
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