Thermodynamic Aspects of Fatty Acids Binding to Human Serum Albumin : A Microcalorimetric Investigation

Abstract

Thermodynamic parameters have been evaluated for the binding interaction between human serum albumin (HSA) and unbranched fatty acids (FFA) on the basis of a flow microcalorimetric measurement at pH 7.4 and 37°C by computer-fitting to single- and two-class binding models. The heat of binding increased exothermically with increasing alkyl chain length. FFA with nine or less carbons bound to only one class of binding sites (n=2) with a binding constant (K) of 10⁴M^-1. FFA with ten or more carbons bound to the first class of binding sites with high affinity K in the order of 10⁵ to 10⁶M^-1, and to the second class with a lower affinity and high capacity. The free energy change of first class of binding sites (ΔG₁) became more negative as the chain length of FFA was increased. The enthalpy change per mol of FFA (ΔH) decreased at the rate of -7.47kJ·mol^-1·CH₂^-1 to a minimum at C9 and then increased due to the hydrophobicity of alkyl chains. Compensation analysis for the i th class of HSA molecule by plotting molar changes of enthalpy (ΔH_mi) against entropy (ΔS_mi) and free energy (ΔG_mi) indicates two distinct binding sites. The first class (i=1) of the long-chain FFA on HSA is an entropy-driven reaction associated with nearly constant values of ΔH_m1 (-43.0±4.8kJ·mol^-1), slightly negative values of ΔS_m1 (-47.4≤ΔS_m1≤-8.1J·mol^-1·K^-1) and -ΔG_m1 values, increasing with increasing alkyl chain length. The second class (i=2) of the long-chain FFA may lie in the same region as the binding sites of the short- and medium-chain FFA with a linear relationship between ΔH_mi-ΔS_mi.