THERMODYNAMICS OF FATTY-ACID-BINDING TO FATTY-ACID-BINDING PROTEINS AND FATTY-ACID PARTITION BETWEEN WATER AND MEMBRANES MEASURED USING THEFLUORESCENT-PROBE ADIFAB

Using the fluorescent probe ADIFAB (acrylodan-derivatized intestinal f atty acid-binding protein) to determine the equilibrium concentration of the free (unbound) fatty acid (FFA), dissociation constants were me asured between 10 and 50 degrees C for the interaction of five differe nt long chain fatty acids (FA) with fatty acid-binding proteins (FABP) from adipocyte, intestine, and heart. Gibbs free energies (Delta G) d etermined from the dissociation constants were between about -9 and -1 1 kcal/mol at 25 degrees C. Thermodynamic parameters for binding were determined using van't Hoff plots of the dissociation constants, which range, over the entire temperature region, between 2 and 3000 nM. For all the unlabeled FABPs, free energies of binding were dominated by l arge negative enthalpies that ranged from -7 to -12 kcal/mol, and the enthalpies tended to decrease with increasing FA unsaturation, The ent ropic contributions (-T Delta S) at 25 degrees C ranged between -4 and +2 kcal/ mol and tended to increase with increasing FA unsaturation. To assess the role of FA aqueous solubility in FABP binding, measureme nts of the partition of FA between unilamellar lipid vesicles and wate r were also done using ADIFAB; the lipid/water partition coefficients (K-p) determined from these measurements were found to be independent of temperature. The binding of FA to FABP is governed by the sum of co ntributions of various interactions between FA, water, and FABP. An an alysis of the individual contributions suggests that the net free ener gy of binding results from the canceling in part of a number of separa te quite large contributions. The entropic contributions sum almost to zero for most FA and FABPs as a result of the canceling of a large in crease in bulk solvent entropy by decreases in configurational entropy upon FA binding to FABP. The net, approximately -10 kcal/mol enthalpy of binding, probably results from an increase in FA configurational e nthalpy upon binding to FABP plus a large negative enthalpy from the i nteraction between the FA and the FABP. This large enthalpy of the FA- FABP interaction suggests that in addition to previously identified sp ecific interactions between the carboxylate portion of the FA and char ged amino acids within the binding cavity, other significantly larger enthalpic interactions, presumably involving the hydrocarbon portion o f the FA, must contribute to the binding energy.