Binding of ethidium bromide to self-complementary deoxydinucleotides

Optical spectra titrations were performed with ethidium bromide and the self-complementary deoxydinucleotides pdCpdG, pdGpdC, pdTpdA, and pdApdT. The titrations were performed in 7.5 mM phosphate buffer, pH 7.0, at 7 degrees C, and with varying dinucleotide concentrations always in large excess of the dye concentration. Well-defined isosbestic points were present in each titration after correction for dinucleotide light absorption. The binding curves were evaluated in terms of simple bimolecular or termolecular reaction models. The bimolecular reaction model gave a significantly better fit to the experimental data, judging from a computerized nonlinear least-squares fitting procedure. The following equilibrium constants were obtained: KC-G = 2000 M-1; KG-C = 950 M-1; KT-A = 370 M-1; KA-T = 350 M-1. From these data the absorption spectra of the completely bound dye were evaluated. These spectra showed bathchromic shifts of their maxima, increasing with the magnitude of K. Fluorescence spectra of ethidium bromide/dinucleotide mixtures were recorded under conditions similar to those for absorption spectra. From the known equilibrium constants the contributions of the bound dye could be estimated. The following fluorescence enhancements Ib/If were found: IC-Gb/If = 6.5; IG-Cb/If = 3.0; IT-Ab/If = 2.0; IA-bT/If = 2.0. From our results, in relation to other theoretical and experimental studies, we conclude that electrostatic phosphate-dye interactions give rise to a major part of the binding energy, which varies with dinucleotide geometry. The more strongly bound complexes exhibit less exposure of the dye to the solvent.