[Study of complex formation of ethidium bromide with the self-complementary deoxytetranucleotide 5'-d(ApGpCpT) by...

Complex formation of ethidium bromide (3,8-diamino-6-phenyl-5-ethyl-phenanthridine) with the self-complementary desoxytetraribonucleosidetriphosphate d(ApGpCpT) was studied in aqueous solution by uni- and two-dimensional 1H NMR spectroscopy (500 and 600 MHz). The concentration dependence of the chemical shifts of protons of the complex at a fixed temperature (T = 308 K) were measured. The schemes of complexing of the dye with the tetranucleotide which take into account eventual molecular associates in solution are discussed. Equilibrium constants of the reactions and the limiting values for the chemical shifts of the protons of ethidium bromide incorporating into the complex were calculated. Relative contents of different complex types were analyzed. The relationship between the dynamic equilibrium and dye/tetranucleotide concentration ratio was examined. The conclusion is made that, as with the complex of proflavine with the d(AGCT) examined earlier, ethidium bromide is intercalated essentially into the G-C site of the tetranucleotide being in the duplex form. The difference in intercalation between ethidium bromide and proflavine lies in the fact that ethidium bromide incorporate on the side of the narrow groove, whereas proflavine is built in on the side of the large groove. Based on the calculated values of the induced chemical shift of ethidium bromide protons and 2-d NOE spectal data, an adequate model (1:2) of the complex of the dye with tetranucleotide in solution was constructed.