STUDY OF COMPLEX-FORMATION BETWEEN ETHIDI UM-BROMIDE AND THE SELF-COMPLEMENTARY DESOXYTETRANUCLEOTIDE 5'-D(APGPCPT) BY UNI-DIMENSIONAL AND 2-DIMENSIONAL H-1-NMR SPECTROSCOPY

Complex formation of ethidium bromide (3,8-diamino-6-phenyl-5-ethyl-ph enanthridine) with the self-complementary desoxytetraribonucleosidetri phosphate d(ApGpCpT) was studied in aqueous solution by uni- and two-d imensional H-1 NMR spectroscopy (500 and 600 MHz). The concentration d ependence 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 molecula r associates in solution are discussed. Equilibrium constants of the r eactions and the limiting values for the chemical shifts of the proton s of ethidium bromide incorporating into the complex were calculated. Relative contents of different complex types were analyzed. The relati onship between the dynamic equilibrium and dye/tetranucleotide concent ration ratio was examined. The conclusion is made that, as with the co mplex of proflavine with the d(AGCT) examined earlier, ethidium bromid e is intercalated essentially into the G-C site of the tetranucleotide being in the duplex form. The difference in intercalation between eth idium bromide and proflavine lies in the fact that ethidium bromide in corporate on the side of the narrow groove, whereas proflavine is buil t in on the side of the large groove. Based on the calculated values o f the induced chemical shift of ethidium bromide protons and 2-d NOE s pectal data, an adequate model (1:2) of the complex of the dye with te tranucleotide in solution was constructed.