The enhancement of the fluorescence quantum yield of 1,8- anilinonaphthalenesulfonic acid (ANS) upon binding to intestinal fatty acid protein (I-FABP) was exploited to devise an assay for free I-FABP. With this assay, we monitored the competition for free I-FABP between ANS and fatty acids and thereby extracted values for the dissociation constants (K(FA)) of fatty acids for I-FABP. We obtained these constants for the I-FABP ligands oleic acid, arachidonic acid, and palmitic acid. In addition, we measured the dependence of K(FA) for oleic acid upon temperature and at two pH values. From these data, we calculate the van't Hoff enthalpy of oleic acid binding. This enthalpy is compared with the enthalpies of binding obtained directly from titration calorimetry. Our experiments with the fluorescence-based assay generate values of K(FA) which disagree with older values obtained from calorimetry and other methods. Our own calorimetric data were analyzed with a view to improving the technique involved in subtraction of a 'reference' dilution of the ligand into solution in the absence of the protein. By this maneuver, we obtained 'corrected' titrations which could be fitted to values of K(FA) more in agreement with the values we determined via the fluorescence-based assay than were the older literature values. Our new values for K(FA) also agree substantially with values derived using a complementary assay technique, one measuring the concentration of free fatty acid, that has recently been developed by Richieri et al. [Richieri et al. (1995) J. Biol. Chem. 270, 15076-15084]. We compare the values of ΔH°, ΔS°, and ΔC(p)° for fatty acid binding we have obtained in this work with those we found in earlier work with ANS binding to I-FABP [Kirk et al. (1996) Biophys. J. 70, 69-83]. Our interpretation of the origin of the thermodynamic changes for ANS binding in our earlier work is here substantiated and extended to include an evaluation in physical terms of the interaction of I- FABP with fatty acids.
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