The interaction of bisbenzimide with DNA

The interaction of bisbenzimide (Hoechst 33258) with DNA has been studied by microcalorimetry, fluorimetry and fluorescence polarization measurements. The binding does not involve intercalation, takes place in at least two modes and occurs in the major groove at low dye/DNA molar ratio (first mode of binding), in agreement with previous proposals by other Authors. Furthermore it has been shown that i) at high dye/DNA molar ratio the binding (second mode) occurs in both grooves; ii) at pH 7.0 the protonation of a dye nitrogen accompanies the binding in both modes; iii) the quantum yield of the dye-DNA complex is sensitive to the DNA conformation change caused by an increase in ionic strength. Basically the same type of binding takes place on chromatin, as detected by calorimetry. However, at high dye/DNA molar ratio about 30% of DNA is not accessible to the dye. The decreased accessibility appears to be mainly related to the nucleosomal structure of chromatin.