Interaction kinetics of liposome-incorporated unsaturated fatty acids with fatty acid-binding protein 3 by surface plasmon resonance |
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| Institution: | 1. JST ERATO, Lipid Active Structure Project, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan;2. Department of Chemistry, De La Salle University, 2401 Taft Avenue, Malate, Manila 1004, Philippines;3. Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan;4. Project Research Center for Fundamental Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan;1. INRIA, 655 Avenue de l’Europe, 38334 Saint Ismier cedex, France;2. Grenoble Institute of Technology, France;3. Institut Universitaire de France, France;1. Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki, Japan;2. Research Institute for Science and Engineering, Waseda University, Tokyo, Japan;3. Chalmers University of Technology, SE-412 96 Goteborg, Sweden;4. University of Houston, Houston, TX 77204-5005, USA;5. Texas A & M University, College Station, TX 77843-3133, USA;6. East Carolina University, Greenville, NC 27858, USA;7. Royal Military College, Kingston, Ontario K7K 7B4, Canada;8. Medical College of Soochow University, 215123 Suzhou, Jiangsu Province, China;9. Roanoke College, Salem, VA 24153, USA;10. National Institute of Radiological Sciences, Chiba, Japan;3. Cell Signaling Laboratory, Department of Biomedical Science (DIBINEM), University of Bologna, 40126 Bologna, Italy;;4. Institute of Molecular Genetics, National Research Council of Italy (IGM-CNR), 40126 Bologna, Italy;;5. SC Laboratory of Musculoskeletal Cell Biology, Rizzoli Orthopedic Institute, 40126 Bologna, Italy;;6. Laboratory RAMSES, Rizzoli Orthopedic Institute, 40126 Bologna, Italy |
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| Abstract: | The role of heart-type fatty acid-binding protein (FABP3) in human physiology as an intracellular carrier of fatty acids (FAs) has been well-documented. In this study, we aimed to develop an analytical method to study real-time interaction kinetics between FABP3 immobilized on the sensor surface and unsaturated C18 FAs using surface plasmon resonance (SPR). To establish the conditions for SPR experiments, we used an FABP3-selective inhibitor 4-(2-(1-(4-bromophenyl)-5-phenyl-1H-pyrazol-3-yl)-phenoxy)-butyric acid. The affinity index thus obtained was comparable to that reported previously, further supporting the usefulness of the SPR-based approach for evaluating interactions between FABPs and hydrophobic ligands. A pseudo-first-order affinity of FABP3 to K+ petroselinate (C18:1 Δ6 cis), K+ elaidate (C18:1 Δ9 trans), and K+ oleate (C18:1 Δ9 cis) was characterized by the dissociation constant (Kd) near micromolar ranges, whereas K+ linoleate (C18:2 Δ9,12 cis/cis) and K+ α-linolenate (C18:3 Δ9,12,15 cis/cis/cis) showed a higher affinity to FABP3 with Kd around 1 × 10?6 M. Interactions between FAPB3 and C18 FAs incorporated in large unilamellar vesicles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and FAs (5:1 molar ratio) were also analysed. Control DMPC liposomes without FA showed only marginal binding to FABP3 immobilized on a sensor chip while liposome-incorporated FA revealed significant responses in sensorgrams, demonstrating that the affinity of FAs to FABP3 could be evaluated by using the liposome-incorporated analytes. Significant affinity to FABP3 was observed for monounsaturated fatty acids (Kd in the range of 1 × 10?7 M). These experiments demonstrated that highly hydrophobic compounds in a liposome-incorporated form could be subjected to SPR experiments for kinetic analysis. |
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| Keywords: | Fatty acid binding proteins Large unilamellar vesicles Surface plasmon resonance |
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