Osmolyte changes the binding affinity and mode of interaction of minor groove binder hoechst 33258 with calf thymus...

The aim of this work was to investigate the effect of altered water activity on Hoechst 33258-calf thymus DNA (CtDNA) interaction by using osmotic stress approach. Water activity was changed by using osmolytes viz., sucrose and triethylene glycol (TEG). We have reported the results of thermal denaturation, absorption and fluorescence spectroscopy and binding affinity measurements as a function of osmolytes concentration. TEG dramatically lowered the thermal stability of CtDNA, ΔT(m)=-16 °C whereas sucrose induced very little decrease. Hoechst 33258 increases the stability of CtDNA, but in the presence of TEG, the ΔT(m) was -37 °C and a marginal decrease was observed with sucrose. Binding affinity of Hoechst 33258 with CtDNA was found to be reduced from 4.75×10⁷ to 0.16×10⁷ M⁻¹ in TEG and this was accompanied with the increased uptake of 74±2 water molecules. In the presence of sucrose this uptake of water molecules was found to be 30±1. Method of continuous variation suggests that the osmolytes lowered the stoichiometry of Hoechst 33258-CtDNA complex. On the contrary, van't Hoff plot revealed the hydrophobic interaction (ΔS=130.66 J mol⁻¹ K⁻¹) between the Hoechst 33258 and CtDNA. The detailed absorption and fluorescence spectral measurements including the fluorescence lifetime and anisotropy indicated bound state of Hoechst 33258 in osmotic stress condition. Fluorescence lifetime measurement revealed that the contribution from the planar conformer of Hoechst 33258 dominated the binding interaction with CtDNA in presence of TEG. These results can be useful for understanding of interaction of Hoechst 33258 with genomic DNA in a complex environment having altered water activity.