Use of Nile Red as a long-wavelength fluorophore in dual-probe studies of ligand-protein interactions

The long-wavelength fluorescence probe, Nile Red, has polarity-dependent fluorescence intensity and wavelength properties that can be used to monitor the binding of drugs and other ligands to plasma proteins such as albumin and α-1 acid glycoprotein. This paper shows that it can be used in tandem with another fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, to study two or more types of ligand binding sites simultaneously. Some ligands displace one or the other probe from the protein/dual-probe complex, other ligands displace both probes. In each case the resulting decrease in fluorescence can be used to estimate the numbers of binding sites and their association constants.