H-1-NMR INVESTIGATION OF THE INTERACTION OF ETHIDIUM-BROMIDE WITH TETRADEOXYRIBONUCLEOSIDE TRIPHOSPHATE 5'-D(GCGC)

Complex formation between ethidium bromide and self-complementary tetr adeoxyribonucleoside triphosphate 5'-d(GCGC) in water-salt solution wa s studied by one-dimensional and two-dimensional 500-MHz H-NMR spectro scopy. Concentration dependences of proton chemical shifts were measur ed at a constant temperature of 308 degrees K. Different schemes of co mplex formation between the dye and tetranucleotide were considered. T he equilibrium constants of reactions were determined, as well as the limit values of the chemical shifts of protons of ethidium bromide in the complex. The peculiarities of the dynamic equilibrium between diff erent types of complexes depending on relative concentrations of dye a nd tetranucleotide were revealed. The conclusion is made that ethidium bromide intercalates preferentially into the pyrimidine-purine fragme nt of the tetranucleotide duplex (CG site). Ethidium bromide intercala tes at the minor groove of the duplex, in contrast to proflavine, whic h binds at the major groove of the double helix. The most plausible st ructure of the 1:2 complex of the dye with tetranucleotide is suggeste d, which agrees well with the results of X-ray investigations of Sobel l et al.