Fluorescence spectra of Hoechst 33258 bound to chromatin

Fluorescence spectra of Hoechst 33258 bound to rat thymocytes were measured by flow cytometry. At low dye concentrations (less than or equal to 2 micrograms/ml) the fluorescence maximum was situated at 460 nm irrespective of solvent composition. With higher dye concentrations the fluorescence maximum was shifted upwards, the intensity decreased and the width of the fluorescence peak increased. Linear combinations of a spectrum obtained at a low dye concentration (0.5 microgram/ml, type 1 binding) and one obtained at a high dye concentration (42.4 micrograms/ml, type 2 binding) failed to reproduce spectra measured at intermediate dye concentrations (0.15 M NaCl). Hence, Hoechst 33258 forms at least three different fluorescing complexes with DNA in chromatin. The shift in the fluorescence maximum of the Hoechst 33258/chromatin complex towards higher wavelengths decreased with ionic strength. 25% ethanol in the 0.15 M NaCl staining buffer reduced the wavelength shift at high dye concentrations, indicating that the strength of type 2 binding depends on DNA conformation in addition to ionic strength. The fluorescence spectrum was independent of whether DNA in chromatin was complexed with histones or not. However, histone-depleted thymocytes fluoresced more intensely than cells in which DNA was complexed with histones, the difference being greater at low concentrations of Hoechst 33258. Hence, type 2 binding to DNA in chromatin appears to be less restricted by histones than type 1 binding.