title: Titration calorimetry as a binding assay for lipid-binding proteins.
abstract: Titration calorimetry has been evaluated as a method for obtaining binding constants and thermodynamic parameters for the cytosolic fatty acid- and lipid-binding proteins. An important feature of this method was its ability to accurately determine binding constants in a non-perturbing manner. The equilibrium was not perturbed, since there was no requirement to separate bound and free ligand in order to obtain binding parameters. Also, the structure of the lipid-protein complex was not perturbed, since native ligands were used rather than non-native analogues. As illustrated for liver fatty acid-binding protein, the method distinguished affinity classes whose dissociation constants differed by an order of magnitude or less. It also distinguished endothermic from exothermic binding reactions, as illustrated for the binding of two closely related bile salts to ileal lipid-binding protein. The main limitations of the method were its relatively low sensitivity and the difficulty working with highly insoluble ligands, such as cholesterol or saturated long-chain fatty acids. However, the signal-to-noise ratio was improved by manipulating the buffer conditions, as illustrated for oleate binding to rat intestinal fatty acid binding protein. Binding parameters are reported for oleate interactions with several wild-type and mutant lipid-binding proteins from intestine. Where possible, the binding parameters obtained from calorimetry were compared with results obtained from fluorescence and Lipidex binding assays of comparable systems.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
authors.forename: K R
authors.initials: KR
authors.lastname: Miller
authors.forename: D P
authors.initials: DP
authors.lastname: Cistola
mesh_terms: D000818:Animals; D002151:Calorimetry / Q000379:methods*; D002352:Carrier Proteins / Q000378:metabolism*; D007422:Intestines / Q000737:chemistry; D050356:Lipid Metabolism; D051381:Rats; D013816:Thermodynamics
chemical_list: D002352:Carrier Proteins