Abstract
Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization (FP) provides a nondisruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP(3)) to N-terminal fragments of IP(3) receptors can be characterized at different temperatures and in competition with other ligands. The assay allows the standard Gibbs free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) changes of ligand binding to be determined. The method is applicable to any purified ligand-binding site for which an appropriate fluorescent ligand is available. FP can be used to measure low-affinity interactions in real time without the use of radioactive materials, it is nondestructive and, with appropriate care, it can resolve ΔH° and ΔS°. The first part of the protocol, protein preparation, may take several weeks, whereas the FP measurements, once they have been optimized, would normally take 1-6 h.