Hydrogen--deuterium exchange analysis of ligand--macromolecule interactions: ethidium--deoxyribonucleic acid system

The interaction between DNA and the intercalating dye, ethidium bromide, was studied by use of a novel approach in which hydrogen--deuterium (H--D) exchange between ethidium amino groups and solvent was measured spectrophotometrically in a stopped-flow mode. The method depends on the fact that ethidium H exchange is greatly slowed on complexation with DNA and that H--D exchange kinetics of the chromophore can be monitored via an accompanying change in its spectral absorbance. The H--D exchange dependent spectral character of ethidium was characterized, and the catalyzed exchange behavior of the free dye and the dye--DNA complex was studied. From such measurements, one can obtain rate and equilibrium constants for the interaction and possibly also some stereochemical information. The constants obtained were checked in more conventional mixing experiments. At 20 degrees C in high salt, the equilibrium binding constant is approximately 5 x 10(4) M-1, and on and off rate constants are 1.6 x 10(6) M-1 s-1 and 30 s-1, respectively. The results independently confirm that one dye molecule is bound for each 2--2.5 base pairs. The method should be applicable to a range of binding interactions. Among other advantages, this approach can allow tight binding interactions to be studied at concentrations of the reactants far above the characteristic Kdiss value.