Interactions of oleic acid with liver fatty acid binding protein: a carbon-13 NMR study

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Abstract
13C NMR spectroscopy as used to probe the structural interactions between carboxyl-13C-enriched oleic acid (18:1) and rat liver fatty acid binding protein (FABP) and the partitioning of 18:1 between FABP and unilamellar phosphatidylcholine (PC) vesicles. Spectra of systems containing 2-8 mol of 18:1/mol of FABP (but no PC) exhibited one carboxyl resonance (182.2 ppm) corresponding to FABP-bound 18:1. At pH values <8.0, an additional carboxyl resonance, corresponding to unbound 18:1 in a lamellar phase, was observed. Both resonances exhibited ionization shifts with estimated apparent pKa values of 7 (unbound 18:1). The intensity of the resonance corresponding to FABP-bound 18:1 increased with increasing 18:1/FABP mole ratio and at 8/1 mole ratio indicated that at least 2 and 6 mol of 18:1/mol of FABP were FABP-bound at pH 7.4 and 8.6, respectively. NMR spectra of systems containing equal concentrations (w/v) of FAPB and PC and from 1 to 4 mol of total fatty acid (FA)/mol of FABP exhibited two 18:1 carboxyl resonances (182.2 and 178.5 ppm, pH 7.4). The downfield resonsance corresponded to FABP-bound 18:1 and the upfield resonance to PC vesicle bound 18:1. At 1/1 mole ratio (FA/FABP), the intensities of both resonances were approximately equal, but at 4/1 mole ratio the resonance for PC vesicle bound 18:1 as 3-fold more intense than that for FABP-bound 18:1. The following conclusions are reached: (i) The carboxyl groups of 18:1 bound to liver FABP experience only one type of binding environment (the aqueous milieu adjacent to the protein surface). (ii) 18:1 bound to FABP is fully ionized at neutral pH, and the anionic 18:1 carboxyl groups are not involved in electrostatic interactions ith cationic residues on FAPB. (iii) Each mole of FABP bound at least 2 mol of 18:1 at physiological pH. However, in the presence of phospholipid vesicles, FABP bound up to 1 mol of 18:1/mol of FABP.