STUDIES OF THE BINDING OF ETHIDIUM-BROMIDE TO CELLS BEFORE AND AFTER ENZYME TREATMENT

Binding of ethidium bromide (EB) to [mouse Ehrlich ascites tumor] cells before and after HCl, [bovine pancreas] pepsin and RNase treatment was investigated by spectrophotometry and fluorimetry. Binding isotherms, calculated with the McGhee-von Hippel equation, taking EB as a non-interacting ligand, revealed the influence of these treatments on the fluorescence characteristics of the cells measured by flow-through cytofluorimetry. Pepsin- and RNase-treated cells have a reduced intercalation capacity due to the loss of cytoplasmic RNA and RNA hydrolysis, respectively. HCl alone or with pepsin considerably increased the equilibrium constant K. At low free EB concentrations the enhanced EB affinity of acid-pretreated cells generates a high fluorescence intensity, compared to treatments at neutral pH. The view that high EB binding to acid pretreated cells is due to hydrolytic histone removal from potential intercalation sites is not supported. With increasing free EB concentrations the fluorescence intensities of RNase- and pepsin-treated cells culminate at the same level due to their almost identical intercalation capacities. Quantitative DNA analysis of pretreated cell suspensions with EB can only be performed if the alteration, induced by the pretreatment was previously studied.