Aggregation of DNA due to ethidium bromide binding
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The effects of ethidium bromide (EB) binding to DNA were investigated over a range of DNA concentrations by equilibrium dialysis and the 1H NMR spectroscopy. Scatchard plots showed varying degrees of curvature, depending on the concentrations of DNA used. The empirical data (apparent slopes, intercepts, k and n values) were also affected, suggesting the possibility of DNA-DNA interaction when EB binds to DNA. From the nmr measurements, plots of relative intensity loss versus R values for the titrations of the 50 mM, 20 mM, 10 mM and 5 mM base pair concentration with EB showed a shifting of the break point in the EB titrations where the maximum effect of EB on the intensity loss in the nmr spectra is seen. The break points did come out to be exactly 0.5 (or 1 EB bound per 2 base pairs), the ratio expected with an intercalation process. The nmr titrations and equilibrium dialysis experiments implied aggregation to be a possible mode of interaction besides intercalation; the extent of DNA-DNA interaction was subjected to mass action, making any changes in DNA concentration to be a sensitive factor in determining the intensity loss in the nmr spectra and the binding curves of the r/c versus r plots.