Abstract
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)
Highlights
Using the "ghost method" previously applied to study the binding of palmitate to bovine serum albumin (BSA)
The binding capacities and high affinities of fatty acids (FA) to BSA and to the membrane ensure that FA bound to albimin or ghost membranes (B-FA) per ml ghost and [water-phase FA in equilibrium with B-FA (W-FA)] of the ghost suspension remain essentially unchanged after the complete removal of BSA outside the ghosts by the described washing procedure and by the final four different dilutions of the washed ghost suspension
The data conform with the expected dilution effect presented in Fig. lA, and the intercept is the equilibrium [W-FA] corresponding to B-FA of the washed ghosts. [W-FA] is the same in the chargebuffer, provided the ghosts are not depleted significantly by washings and by the final dilutions
Summary
Using the "ghost method" previously applied to study the binding of palmitate to bovine serum albumin (BSA) A large turnover in the body of FA is normally ensured by the large transport capacity of serum albumin and the strong binding of FA to this protein This strong binding minimizes undesirable detergent effects that depend strongly on the water-phase concentrations below the critical micelle concentrations [3]. T h e W-FA concentration is usually, but erroneously, considered as a direct determinant of the transport between serum and cells, it must be more or less offset by a great flow of the FA, dependent on rate constants of dissociation [4,5,6]. Diffusion to pure buffer of protonated palmitic acid (PA) has been observed through polyethylene membranes [11]
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