Biophysical studies on the base specificity and energetics of the DNA interaction of photoactive dye thionine: Spectroscopic and calorimetric approach

In this study absorbance, fluorescence, circular dichroic spectroscopy, viscosity, thermal melting and calorimetric techniques were employed to understand the binding of the phenothiazinium dye, thionine, with deoxyribonucleic acids of varying base composition. Strong hypochromic and bathochromic effects and quenching of fluorescence were observed that showed strong binding of thionine to the DNAs. The binding parameters evaluated from Scatchard analysis through McGhee-von Hippel analysis showed that the binding was non-cooperative and affinities of the order of 105 M−1. The results of ferrocyanide fluorescence quenching studies and viscosity experiments, taken together suggested the intercalation of thionine while thermal melting, differential scanning calorimetry and circular dichroic studies provided evidence for the thermal stabilization and conformational perturbations associated with the binding. The thermodynamic parameters elucidated through sensitive isothermal titration calorimetric studies suggested that the binding was exothermic, characterized by negative enthalpy and large positive entropy changes and that the non-electrostatic contributions play a significant role for thionine association to DNA. The heat capacity changes obtained from the temperature dependence of enthalpy changes gave negative values suggesting substantial hydrophobic contribution in the DNA binding process of thionine. Further, an observation of enthalpy-entropy compensation in the DNA binding also suggested the involvement of multiplicity of non covalent interactions in the binding process. The base specificity of the complexation and energetics of the interaction of thionine to DNA are obtained for the first time from this study.