Binding of arachidonate and oleate to bovine serum albumin.
Using the "ghost method" previously applied to study the binding of palmitate to bovine serum albumin (BSA) (Bojesen, I. N., and E. Bojesen. 1992. J. Lipid Res. 33: 1327-1334) we have measured the water-phase concentrations of arachidonate (AR) and oleate (OL) in 165 mM KCl and in 165 mM NaCl, 2 mM phosphate buffer at pH 7.3 in equilibrium with AR and OL bound to BSA (about 30 microM) inside resealed human red cell ghosts at low molar ratio (v). Data were obtained at 0 degree C, 10 degrees C, 23 degrees C, and 38 degrees C for v between 0.012 and 1.5. Regression analyses of the data suggest that BSA has three equivalent binding sites for AR and OL at the four temperatures. The equilibrium dissociation constants (Kd) were the same in potassium and in sodium buffers. They were calculated for AR and OL on basis of three equivalent binding sites per mol BSA. Kd values increase with temperature and the AR values are, on average, approximately 5-fold greater than those of OL within the investigated temperature range. At 23 degrees C, Kd values for three equivalent sites are 15.60 +/- 0.73 nM and 2.89 +/- 0.14 nM corresponding to -44.2 +/- 0.15 kJ/mol and -48.3 +/- 0.1 kJ/mol in free energies of binding AR and OL, respectively. The difference, 4.1 kJ/mol, fits fairly well with the theoretical difference in hydrophobic effects of the two aliphatic chains, 3.7 kJ/mol.(ABSTRACT TRUNCATED AT 250 WORDS)
Bojesen
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Department of Medical Biochemistry and Genetics, University of Copenhagen, Panum Institute, Copenhagen, Denmark.
Bojesen
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D016718:Arachidonic Acid / Q000097:blood*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D019301:Oleic Acid; D009829:Oleic Acids / Q000097:blood*; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000378:metabolism*
D009829:Oleic Acids; D012710:Serum Albumin, Bovine; D016718:Arachidonic Acid; D019301:Oleic Acid