title: Thermodynamics of fatty acid binding to fatty acid-binding proteins and fatty acid partition between water and membranes measured using the fluorescent probe ADIFAB.
abstract: Using the fluorescent probe ADIFAB (acrylodan-derivatized intestinal fatty acid-binding protein) to determine the equilibrium concentration of the free (unbound) fatty acid (FFA), dissociation constants were measured between 10 and 50 degrees C for the interaction of five different long chain fatty acids (FA) with fatty acid-binding proteins (FABP) from adipocyte, intestine, and heart. Gibbs free energies (delta G) determined from the dissociation constants were between about -9 and -11 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 large negative enthalpies that ranged from -7 to -12 kcal/mol, and the enthalpies tended to decrease with increasing FA unsaturation. The entropic contributions (-T detla S) at 25 degrees C 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, measurements of the partition of FA between unilamellar lipid vesicles and water were also done using ADIFAB; the lipid/water partition coefficients (Kp) determined from these measurements were found to be independent of temperature. The binding of FA to FABP is governed by the sum of contributions of various interactions between FA, water, and FABP. An analysis of the individual contributions suggests that the net free energy of binding results from the canceling in part of a number of separate quite large contributions. The entropic contributions sum almost to zero for most FA and FABPs as a result of the canceling of a large increase 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 enthalpy upon binding to FABP plus a large negative enthalpy from the interaction between the FA and the FABP. This large enthalpy of the FA-FABP interaction suggests that in addition to previously identified specific interactions between the carboxylate portion of the FA and charged amino acids within the binding cavity, other significantly larger enthalpic interactions, presumably involving the hydrocarbon portion of the FA, must contribute to the binding energy.
authors.affiliation: Medical Biology Institute, La Jolla, California 92037, USA.
authors.forename: G V
authors.initials: GV
authors.lastname: Richieri
authors.forename: R T
authors.initials: RT
authors.lastname: Ogata
authors.forename: A M
authors.initials: AM
authors.lastname: Kleinfeld
mesh_terms: D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010713:Phosphatidylcholines; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D025521:Tumor Suppressor Proteins; D014867:Water
chemical_list: D002352:Carrier Proteins; C107608:FABP7 protein, human; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005456:Fluorescent Dyes; D008081:Liposomes; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C079339:acrylodated intestinal fatty acid binding protein, recombinant; D014867:Water