AFFINITY OF FATTY-ACID FOR RRAT INTESTINAL FATTY-ACID-BINDING PROTEIN- FURTHER EXAMINATION

The enhancement of the fluorescence quantum yield of 1,8-anilinonaphth alenesulfonic 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 fa tty acids and thereby extracted values for the dissociation constants (K-FA) Of fatty acids for I-FABP. We obtained these constants for the I-FAB ligands oleic acid, arachidonic acid, and palmitic acid. In addi tion, we measured the dependence of K-FA for oleic acid upon temperatu re 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 ent halpies of binding obtained directly from titration calorimetry. Our e xperiments with the fluorescence-based assay generate values of K-FA w hich disagree with older values obtained from calorimetry and other me thods. Our own calorimetric data were analyzed with a view to improvin g the technique involved in subtraction of a ''reference'' dilution of the ligand into solution in the absence of the protein. By this maneu ver, we obtained ''corrected'' titrations which could be fitted to val ues of K-FA more in agreement with the values we determined via the fl uorescence-based assay than were the older literature values. Our new values for K-FA also agree substantially with values derived using a c omplementary assay technique, one measuring the concentration of free fatty acid, that has recently been developed by Richieri et al. [Richi eri et al. (1995) J. Biol. Chem. 270, 15076-15084]. We compare the val ues of Delta H-o, Delta S-o, and Delta C-p(o) for fatty acid binding w e 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 bind ing in our earlier work is here substantiated and extended to include an evaluation in physical terms of the interaction of I-FABP with fatt y acids.