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Mass spectrometry and NMR analysis of ligand binding by human liver fatty acid binding protein
Author(s) -
Santambrogio C.,
Favretto F.,
D'Onofrio M.,
Assfalg M.,
Grandori R.,
Molinari H.
Publication year - 2013
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.3237
Subject(s) - chemistry , ligand (biochemistry) , fatty acid binding protein , electrospray ionization , cytosol , binding site , mass spectrometry , protein ligand , carboxylate , binding protein , stereochemistry , biochemistry , chromatography , receptor , enzyme , gene
Human liver fatty acid binding protein (hL‐FABP) is the most abundant cytosolic protein in the liver. This protein plays important roles associated to partitioning of fatty acids (FAs) to specific metabolic pathways, nuclear signaling and protection against oxidative damage. The protein displays promiscuous binding properties and can bind two internal ligands, unlike FABPs from other tissues. Different topologies for the ligand located in the more accessible site have been reported, with either a ‘head‐in’ or ‘head‐out’ orientation of the carboxylate end. Electrospray‐ionization mass spectrometry and nuclear magnetic resonance titrations are employed here in order to investigate in further detail the binding properties of this system, the equilibria established in solution and the pH dependence of the complexes. The results are consistent with two binding sites with different affinity and a unique head‐out topology for the second molecule of either ligand. Competition experiments indicate a higher affinity for oleic acid relative to palmitic acid at each binding site. Copyright © 2013 John Wiley & Sons, Ltd.