Nonspecific ligand-DNA equilibrium binding parameters determined by fluorescence methods

This chapter focuses on the use of steady-state fluorescence techniques to monitor changes in the ligand (protein) that accompany binding, with examples drawn from studies of ligands and proteins that bind nonspecifically to nucleic acids. Spectroscopic methods—such as fluorescence, UV absorbance, and circular dichroism—offer many advantages for the study of ligand–nucleic acid as well as other macromolecular interactions. To use a change in a spectroscopic signal that is induced on formation of a ligand–nucleic acid complex to obtain a true equilibrium binding isotherm, either the relationship between the signal change and the degree of binding must be known or a method of analysis must be used that does not require knowledge of this relationship. If some relationship between the signal change and the degree of binding is assumed (e.g., linear), then the resulting binding isotherm and thermodynamic parameters determined from that binding isotherm are only as valid as the assumed relationship. The chapter also presents ligand binding density function (LBDF) analysis in which it reviews the case in which binding is accompanied by a change in the signal (fluorescence) of the ligand.