Study of protein-ligand binding by fluorescence

A practical class experiment is described to show the students an application of fluorescence spectroscopy in the study of protein-ligand binding. This class is part of an undergraduate physical biochemistry course for life science students. The major aim is to introduce the students to the basic concepts of fluorescence emission (intrinsic and extrinsic), the effect of environment on fluorescence parameters, and how this could be applied to the study of ligand binding to proteins. In particular, binding of the fluorescent probe 1-anilinonaphthalene-8-sulfonate to albumin (native and oxidized) is described. From the obtained results, the stoichiometry of complex formation, the binding equilibrium constants, and alterations of the environment of the binding sites are determined and discussed with the students.