Fatty-acid binding proteins (FABPs) belong to a small family of a cytosolic transport proteins whose main function is related to binding and relocation of highly hydrophobic long-chain fatty acids (FA). Currently, eight tissue-specific types of FABP are known: liver, intestinal, heart, adipocyte, epidermal, ileal, brain, and testis. It is proposed that FABPs also protects tissues from the high toxicity of FA and their metabolic intermediates.
In this work, we focused on binding characterization of FA and FA-carnitine esters to human heart-type-FABP (H-FABP or FABP3) by means of isothermal titration calorimetry (ITC) and NMR. Nevertheless, FA binding studies in vitro are limited to FA solubility and micellization. Some attempts were performed to solubilize FA by formation of heterogeneous micelles with DMPC. It allowed to reach the desired concentration range but completely changed binding thermodynamics. Here we present alternative approach of FA solubilization by addition of Trition-X that allowed to solubilize even long-chain FA and determine binding affinity and thermodynamic parameters in the ITC assay. 2D ¹⁵N-HSQC NMR spectroscopy was used for validation of the assay and for characterization of the binding specificity. Additionally, ITC experiments varying temperature were performed on mid-length FA to evaluate additive effect on the protein-ligand binding and to determine its impact on heat capacity.