Glycosaminoglycan–Protein Interaction Studies Using Fluorescence Spectroscopy
Fluorescence spectroscopy is a quantitative analytical tool that has been extensively used to provide structural and dynamical information on GAG–protein complexes. It possesses major advantages including high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. It has been applied to practically every protein–GAG system through the use of either intrinsic (e.g., Trp) or extrinsic (e.g., a non-covalent fluorophore) probe. For studies involving GAGs, it forms the basis for measurement of dissociation constant of complexes and the stoichiometry of binding, which helps elucidate many other thermodynamic and/or mechanistic parameters. We describe the step-by-step procedure to measure the affinity of GAG–protein complexes, parse the ionic and nonionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments.
Key wordsActivation Binding affinity Competitive binding Fluorescence spectroscopy GAG–protein interactions Inhibition Serpins
This work was supported by the grants HL090586 and HL107152 from the National Institutes of Health.
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