ETHIDIUM BROMIDE FLUORESCENCE OF THE CHROMATIN STRUCTURE IN RELATION TO SODIUM CHLORIDE AND EDTA CONCENTRATIONS

Publisher Website
Google Scholar
Abstract
The intracellular structure of DNA was studied by binding ethidium bromide (EB) to isolated DNA and chromatin and by examining their fluorescence spectra in the absence and presence of NaCl or EDTA. Rat liver chromatin of a natural type showing an A [absorption] 240/A260 ratio of 80% and a F [fluorescence] 450/F330 ratio of about 20% was used. Both DNA-EB and chromatin-EB showed 2 excitation peaks at wavelengths of 300 and 330 nm and emissions at 585 and 600 nm. The relative fluorescence at 585 nm (RF585) and the ratio of the RF585 values on excitations at 300 and 330 nm (F-ratio) were measured. With the addition of 0.6 M NaCl or 25 mM EDTA the RF585 of both DNA-EB and chromatin-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 300 nm but not on that of DNA-EB. With increase in the NaCl concentration, the F-ratio of chromatin-EB increased to a maximum at 0.15 M NaCl and then decreased at higher salt concentrations. The optimal conditions for fluorometry of the chromatin were found in the presence of 0.15 M NaCl and low concentrations of EDTA (less than 3 mM) and EB (3-10 mM).