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 ot 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 longchain 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.
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Abbreviations
- I-FABP:
-
recombinant (E. coli-derived) rat Intestinal Fatty Acid-Binding Protein
- I-FABP (R106 Q):
-
a mutant form of I-FABP in which arginine-106 has been replaced by glutamine
- CRBP-II:
-
recombinant rat Cellular Retinol-Binding Protein
- CRBP-II (Q109R):
-
a mutant form of CRBP-II in which glutamine-109 has been replaced by arginine
- L-FABP:
-
recombinant rat Liver Fatty Acid-Binding Protein
- I-LBP:
-
recombinant rat Ileal Lipid-Binding Protein, n, lipid: protein molar binding stoichiometry
- Kd and Ka:
-
dissociation and association constants for ligand binding, respectively
- ΔH:
-
enthalpy of binding
- ΔG:
-
Gibbs free energy of binding
- ΔS:
-
enthalpy of binding
- T:
-
Temperature in °K
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Miller, K.R., Cistola, D.P. Titration calorimetry as a binding assay for lipid-binding proteins. Mol Cell Biochem 123, 29–37 (1993). https://doi.org/10.1007/BF01076472
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DOI: https://doi.org/10.1007/BF01076472