[12] Fluorescence anisotropy applied to biomolecular interactions

This chapter focuses on the applications of polarization or anisotropy data. The appeal of fluorescence spectroscopy in the study of biomolecular systems lies in the characteristic time scale of the emission process, the sensitivity of the technique, and its ability to accommodate rapid and facile changes in the solvent milieu under conditions corresponding to thermodynamic equilibrium. On excitation of a fluorescent solution and observation of the emission at right angles both to the direction of propagation of the exciting light and to the direction of the electric vector, the polarization of the emission is defined in this chapter. The optical densities of the solution may be unavoidably high (even allowing for smaller cuvette or front-face geometries), and the requisite inner filter corrections may lower confidence in the binding data. Polarization/anisotropy measurements—such as lifetime measurements—are intensive quantities and as such are not subject to first-order inner filter corrections.