Steady-State Fluorescence Anisotropy To Investigate Flavonoids Binding to Proteins

Abstract

A laboratory experiment using steady-state fluorescence polarization or anisotropy to determine the binding constant for a flavonoid–protein interaction is described. Using the intrinsic fluorescence of quercetin (a model flavonoid), the fluorescence anisotropy is measured as a function of human serum albumin (HSA) concentration. From the anisotropy data the fraction of bound quercetin at each protein concentration is calculated. Then, employing both linear (double reciprocal) and nonlinear regression analyses, the binding constant and Gibbs free energy change for the quercetin–HSA interaction are determined. This interdisciplinary exercise investigates a fluorescence technique not typically covered in the undergraduate curriculum and explores many aspects of chemistry such as equilibrium, thermodynamics, biochemical interactions, and data-analysis methods. The experiment can be completed in one, three-hour laboratory period and is appropriate for analytical or instrumental analysis, physical chemistry, and biochemistry courses.