Measurement of ligand binding to proteins by fluorescence spectroscopy.

Publisher Summary This chapter illustrates that fluorescence spectroscopy has developed into a routine experimental procedure for the study of protein-ligand interactions. To study ligand binding by this method, one only requires that a change in quantum yield be consequent upon ligand binding, whether one is observing ligand fluorescence, intrinsic protein fluorescence, or fluorescence of a covalently or noncovalently bound fluorescent probe which is sensitive to ligand binding. Because of the indirect observation of ligand binding by such changes in fluorescent intensity, methods of data analysis differ from those which are used routinely to analyze binding data obtained using methods such as equilibrium dialysis, where the free ligand concentration is measured directly. This chapter describes methods that can be used to calculate the binding constants and stoichiometry of particular protein-ligand interactions from the dependence of the observed fluorescence of a protein ligand mixture upon total ligand concentration. The relevant theory, approach, and possible pitfalls associated with the fluorescent measurement of interactions is presented with respect to both perturbation of ligand fluorescence on binding, along with the perturbation of the intrinsic fluorescence of acceptor on interacting with ligand.