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.