Temperature dependence of the volumetric parameters of drug binding to poly[d(A-T)].Poly[d(A-T)] and Poly(dA).Poly(dT)

Abstract

We report the temperature and salt dependence of the volume change (DeltaVb) associated with the binding of ethidium bromide and netropsin with poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]. The DeltaV(b) of binding of ethidium with poly(dA).poly(dT) was much more negative at temperatures approximately 70 degrees C than at 25 degrees C, whereas the difference is much smaller in the case of binding with poly[d(A-T)].poly[d(A-T)]. We also determined the volume change of DNA-drug interaction by comparing the volume change of melting of DNA duplex and DNA-drug complex. The DNA-drug complexes display helix-coil transition temperatures (Tm several degrees above those of the unbound polymers, e.g., the Tm of the netropsin complex with poly(dA)poly(dT) is 106 degrees C. The results for the binding of ethidium with poly[d(A-T)].poly[d(A-T)] were accurately described by scaled particle theory. However, this analysis did not yield results consistent with our data for ethidium binding with poly(dA).poly(dT). We hypothesize that heat-induced changes in conformation and hydration of this polymer are responsible for this behavior. The volumetric properties of poly(dA).poly(dT) become similar to those of poly[d(A-T)].poly[d(A-T)] at higher temperatures.

FIGURE 1

Temperature dependence of the molar volume change of DNA denaturation. The volume change is per mole of basepairs. The open circles are data for poly(dA)·poly(dT) obtained in this work and the solid circles are from the literature (20). The open triangles are data for poly[d(A-T)]·poly[d(A-T)] obtained in this work and the solid triangles are from the literature (21). The lines fit to the data are given by ΔV (cm³ mol bp⁻¹) = −1.82 (± 1.04) + (0.088 ± 0.017) × T_m (°C) for poly(dA)·poly(dT) and ΔV (cm³ mol bp⁻¹) = −6.64 (± 0.72) + (0.145 ± 0.012) × T_m (°C) for poly[d(A-T)]·poly[d(A-T)].

FIGURE 2

Pressure dependence of the helix-coil transition temperature of DNA with or without ethidium bromide at two sodium chloride concentrations. Poly(dA)·poly(dT), circles; poly[d(A-T)]·poly[d(A-T)], triangles; 25 mM NaCl, open symbols; 75 mM NaCl, solid symbols; DNA only, solid line; basepair/drug ratio of ∼5:1 (dotted line); and basepair/drug ratio of 2:1 (dashed line). Please refer to the text for details.

FIGURE 3

Pressure dependence of the helix-coil transition temperature of DNA with or without netropsin at various salt concentrations. Poly(dA)·poly(dT), circles; poly[d(A-T)]·poly[d(A-T)], triangles; 25 mM salt, open symbols; 75 mM salt, solid symbols; DNA only, solid line; basepair/drug ratio of 2:1, dotted line).

FIGURE 4

Temperature dependence of volume change of ethidium binding with poly(dA)·poly(dT) and poly[d(A-T)]·poly[d(A-T)] in 20 mM Tris-HCl, 50 mM NaCl, pH 7.2.

FIGURE 5

Temperature dependence of equilibrium constant of ethidium binding with poly(dA)·poly(dT) and poly[d(A-T)]·poly[d(A-T)] in 20 mM Tris-HCl, 50 mM NaCl, pH 7.2. A typical plot of lnK_a versus pressure (poly[d(A-T)]·poly[d(A-T)], 26.2°C) is shown in the inset.