Molecular Pharmacology, Vol 18, 230-236, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Quantitative Assessment of the Competitive Binding of Anionic Ligands to Albumin

¹ Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106
² Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106

A generalized model has been developed to test quantitatively the equilibrium displacement of ligands that bind to successive sites on bovine serum albumin with decreasing affinity. The equations, which are based on the site binding model of Scatchard, assume that competition exists at individual sites, that the affinity constants for ligand and competitor pertain to individual sites, and that the affinity constants for ligand or competitor at any given site are independent of ligand or competitor bound at other sites. The equations are unique in that they account for (a) variation in free competitor concentration as a function of ligand concentration and (b) the partitioning of a competitor into the organic phase. For the ligand-competitor pairs ¹⁴C-octanoate-chlorophenoxyisobutyrate and ¹⁴C-palmitate-stearate, the site binding model gives an excellent fit to the data, and apparent competitor constants (k_i') are generated that approximate the independently determined association constants (k_i) for the competitor, indicative of classical site-site competition. In the case of ¹⁴C-stearate-chlorophenoxyisobutyrate and ³H-anilinonapthalenesulfonate (ANS)-chlorophenoxyiosbutyrate, while the computer simulations for the site binding model fit the data well, the apparent k_i' of chlorophenoxyisobutyrate for the first site is 10- to 100-fold lower than the apparent k₁' value for the same site. In this instance, therefore, the equations assuming site-site competition do not account entirely for the data. A comparison was made of association constants of monomer ANS determined by fluorescence or radiolabeled ³H-ANS binding. A single high-affinity site of 6.8 x 10⁵ M^-1 existed when ³H-ANS binding was measured, compared to an apparent k₁ of 3.2 x 10⁴ M^-1 by fluorescence. Chlorophenoxyisobutyrate competitively displaced ANS with a k₁' of 1.4 x 10⁴ M^-1 measured by ³H-ANS, but only 48 M^-1 when fluorescence was determined. It is concluded that fluorescence is not a quantitative indicator of ANS binding to albumin and is not suitable for determination of competitive interactions.

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ACKNOWLEDGMENTS We wish to thank Dr. Robert Ainslie, Mr. Val Schurowliew, and Ms. Avo Bowen for assistance in various aspects of the project.