The inhibition of horse liver alcohol dehydrogenase by acridine orange was studied as a function of the concentration of the two coenzyme and substrate forms, the inhibitor concentration, pH, and in the presence of other inhibitors of the enzyme. The changes in optical properties, of the dye occurring during its binding to the enzyme (especially the absorption spectra and the fluorescence polarization) were also studied. The existence of an efficient resonance energy transfer from the excited NADH molecule to the acridine orange molecule in the corresponding ternary complex with the enzyme has also been demonstrated. The results obtained provide evidence showing that the binding site of alcohol dehydrogenase for acridine orange differs from the binding sites of this enzyme for both the coenzyme and the substrate. This binding site most likely is localized in a large substrate pocket of the enzyme near to the binding sites for o-phenanthroline and berberine and very close to the binding site for tricyclic psychochemicals.
