Thermodynamic Affinity and Nature of Forces Defining Glycosaminoglycan-Protein Systems Using Fluorescence...

Among the biophysical techniques used to study glycosaminoglycan (GAG)-protein interactions, fluorescence spectroscopy is a quantitative tool that has been extensively used to provide structural and dynamical information. Its advantages include high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. A large majority of protein-GAG systems have been studied using either intrinsic (e.g., Trp) or extrinsic (e.g., a noncovalent fluorophore) probes. It forms the basis for measurement of dissociation constant and stoichiometry of GAG-protein complexes. We describe step-by-step procedures to measure the affinity of GAG-protein complexes, parse the ionic and non-ionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments.