Robert G. Lowery

CHAPTER 18 – Measurement of Peptide Binding Affinities Using Fluorescence Polarization

This chapter discusses measurement of peptide binding affinities using fluorescence polarization. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding and enzyme catalysis. When a fluorescently labeled molecule is excited with plane polarized light, it emits light that has a degree of polarization that is inversely proportional to its molecular rotation. Large fluorescently labeled molecules remain relatively stationary during the excited state and the polarization of the light remains relatively constant between excitation and emission. Equilibrium binding experiments using fluoresceinated peptides are set up over a 2-day period. Peptide labeling and purification is done on the first day and the equilibrium binding measurements are done on the second day. A single labeling reaction will generate enough labeled peptide for several hundred binding experiments. Three important considerations in labeling peptide tracers for fluorescence polarization include the peptide molecular weight and composition, the labeling method, and the purity of the labeled product. Thin layer chromatography is a convenient way to remove unwanted hydrolysis products and to resolve the multiple fluoresceinated products that result when peptides that contain amino acids with amine side chains are labeled. The common pitfall in fluorescence polarization equilibrium binding experiments is also elaborated.