Salt Effects on Fluorescence Spectral Shifts of DNA-Bound Hoechst 33258 and Reaction Volumes of the Minor Groove Binding

Abstract

Abstract In this study, salt effects on the interaction of Hoechst 33258 (2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride) with several synthetic polynucleotide duplexes were studied by fluorescence spectral shifts and reaction volumes derived from the pressure dependence of binding equilibria. Both the excitation and emission spectra of Hoechst 33258 saturated by polyd(A)·polyd(T) or poly[d(A-T)]·poly[d(A-T)] shifted to shorter wavelengths as NaCl concentrations increased. The peak intensities first increased ([NaCl] < 100 mM) then started to decrease ( > 100 mM NaCl). Salt dependent blue shifts were also observed in their absorption spectra. These results were in contrast to what occurred to the drug complexed with poly[d(G-C)]·poly[d(G-C)] or poly[d(A-G)]·poly[d(C-T)]. The pressure dependence of the association equilibrium constants (Kp) of the drug and two A·T polymers was examined at several salt concentrations; the results helped to derive reaction volumes. In the case of the A-T copolymer, negative volume changes were found at all ionic concentrations studied. In contrast, in the case of the homopolymer, negative volume changes were found at lower salt concentrations (10 and 50 mM) while positive ones were found at higher concentrations (100 and 200 mM NaCl). These results are discussed in terms of the local structural deformation and hydration rearrangement of DNA.