Summary
Binding of ethidium bromide (EB) to cells before and after HCl, pepsin and RNase treatment was investigated by spectrophotometric and fluorimetric methods. Binding isotherms, calculated with the McGheevon Hippel equation, taking EB as a non-interacting ligand, revealed the influence of these treatments on the fluorescence characteristics of the cells which were measured by flow-through cytofluorimetry. Thus pepsin- and RNase-treated cells have a reduced intercalation capacity due to the loss of cytoplasmic RNA and RNA hydrolysis, respectively. HCl alone, or in association with pepsin, increased the equilibrium constant K considerably. Thus at low free EB concentrations the enhanced EB affinity of acid-pretreated cells generates a high fluorescence intensity, by comparison with treatments at neutral pH. This result contradicts the interpretation of high EB binding to acid pretreated cells which is commonly believed to be due to hydrolytic histone removal from potential intercalation sites.
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. Consequently, quantitative DNA analysis of pretreated cell suspensions with EB can only be performed if the alteration, induced by the pretreatment, has previously been studied.
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Eisenhut, M., Choné, B. Studies of the binding of ethidium bromide to cells before and after enzyme treatment. Histochemistry 62, 207–220 (1979). https://doi.org/10.1007/BF00493321
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DOI: https://doi.org/10.1007/BF00493321