Affinity of fatty acid for (r)rat intestinal fatty acid binding protein:further examination.
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 wer 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 Richiere et al [Richiere et al. (1995) J. Biol. Chem. 270, 15076-15084]. We compare the values of delta H degrees, delta S degrees, and delta C(p)degrees 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.
Kurian
E
E
Department of Biochemistry and Molecular Biology, Mayo Foundation and Clinic, Rochester, Minnesota 55905, USA.
Kirk
W R
WR
Prendergast
F G
FG
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001667:Binding, Competitive; D002151:Calorimetry; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism; D018996:Myelin P2 Protein / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D017186:Titrimetry
D000817:Anilino Naphthalenesulfonates; D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids; D010169:Palmitic Acids; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D019301:Oleic Acid; D019308:Palmitic Acid; C027132:1-anilino-8-naphthalenesulfonate