Abstract
For studies on the intracellular structure of DNA, the binding of ethidium bromide (EB) to isolated DNA and chromatin was studied by examining their fluorescence spectra in the absence and presence of sodium chloride (NaCl) or ethylenediaminetetraacetic acid (EDTA). Rat liver chromatin of a natural type showing an A240/A260 ratio of 80% and a F450/F330 ratio of about 20% was prepared with a modified version of Marushige and Bonner's method. Both DNA-EB and chromatin-EB showed two excitation peaks at wavelengths of 300 and 330nm and emissions at 585 and 600nm. The relative fluorescence at 585nm (RF585) and the ratio of the RF585 values on excitations at 300 and 330nm (F-ratio) were measured.
With the addition of 0.6M NaCl or 25mM EDTA the RF585 of both DNA-EB and chronation-EB decreased. The decrease caused by NaCl was recovered by removal of NaCl. NaCl and EDTA had a specific effect on the fluorescence of chromatin-EB excited at 300nm but not on that of DNA-EB. With increase in the NaCl concentration, the F-ratio of chromatin-EB increased to a maximum at 0.15M NaCl and then decreased at higher salt concentrations. The optimal conditions for fluorometry of the chromatin were found to be in the presence of 0.15M NaCl and low concentrations of EDTA (less than 3mM) and EB (3-10mM).
