Determination of ligand to DNA binding parameters from two-dimensional DSC curves

Abstract

The application of the theory of DNA denaturation linked to ligand binding to differential scanning calorimetry (DSC) is a useful tool to simultaneously characterize the energetics of denaturation and binding. Although the general theory is well known, the current DSC-based approaches to study the DNA–ligand interaction do not utilize the full potential of this method. In this paper, we propose the analytical approach for detailed analysis of DNA–ligand interaction from DSC data. The DNA macromolecule is represented as an assembly of cooperative units which melt by two-state model. The explicit account of ligand distribution on polymeric DNA and the temperature dependences of melting and binding constants, as well as of enthalpies, are considered. Such approach enables to extract the binding constant, stoichiometry, enthalpy, entropy, and heat capacity changes from multiple excess heat capacity profiles obtained at varying concentrations of the ligand (i.e. two-dimensional DSC curves). The applicability of the developed approach was demonstrated using an example salmon testes DNA–proflavine DSC experiment. The full set of DNA melting and proflavine binding thermodynamic parameters was obtained. Comparison of the proflavine binding parameters obtained from DSC with those determined from alternative experimental methods has proved the usefulness of the DSC method for evaluation of the binding thermodynamics in DNA–ligand system. In addition, the approach developed in the present study, allows to evaluate the concentration dependences of all species in solution as a function of temperature. Analysis of these dependences has enabled to interpret fine effects on the DSC curves of DNA–ligand complexes.