Molecular Pharmacology, Vol 14, 337-346, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Competitive Binding of Long-Chain Free Fatty Acids, Octanoate, and Chlorophenoxyisobutyrate to Albumin

¹ Department of Pharmacology and Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

The mechanism of binding of certain fatty acids and the drug chlorophenoxyisobutyrate (CPIB) to defatted bovine serum albumin has been studied by analysis of the mutual displacement of these ligands. The displacement of one ligand by another was measured at pH 7.45 by equilibrium partitioning between hexane and an aqueous phase, and by ultrafiltration. The ligand-inhibitor pairs were [¹⁴C]palmitate vs. CPIB, octanoate, or stearate, [¹⁴C]CPIB vs. octanoate or palmitate, and [¹⁴C]octanoate vs. CPIB. Association constants (K_a) were measured for ligands in separate experiments, and inhibition constants (K_i) for competitors were estimated from linear double-reciprocal plots of the low ligand concentration region. In all cases a competitive type of displacement of ligand from a high-affinity site(s) by the inhibitor was observed. When K_a inhibitor was equal to or greater than K_a ligand, the inhibition constant (K_i) equaled K_a inhibitor, indicative of classical competitive inhibition. An example of this type is [¹⁴C]CPIB (K_a 2 x 10⁵ M^-1) displacement by palmitate (K_i = K_a = 5 x 10⁶ M^-1). However, when K_a inhibitor was less than K_a ligand, as in the case of [¹⁴C]palmitate displacement by CPIB, the calculated K_i was less than K_a inhibitor. Furthermore, K_i. decreased as the inhibitor concentration rose. Although data can be interpreted by a model in which common primary binding sites exist on albumin for medium- or long-chain free fatty acids and CPIB, a more complex interpretation than simple competitive inhibition may be required to explain the unequal K_i and K_a values.

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ACKNOWLEDGMENTS We thank Mrs. Rosario Bowen for her excellent technical assistance, and Dr. Robert Ainslie for helpful discussions during the preparation of the manuscript.