Relations between high-affinity binding sites for l-tryptophan, diazepam, salicylate and Phenol Red on human serum albumin

Abstract

Binding of L-tryptophan, diazepam, salicylate and Phenol Red to defatted human serum albumin was studied by ultrafiltration at pH 7.0. All ligands bind to 1 high-affinity binding site with association constants of the order of 104-105 M-1. The number of secondary binding sites varied from zero to five with association constants about 103 M-1. Competitive binding studies with different pairs of the ligands were performed. Binding of both ligands was determined simultaneously. L-Tryptophan and diazepam competed for a common high-affinity binding site on albumin. The following combinations of ligands do not bind competitively to albumin: L-tryptophan-Phenol Red, L-tryptophan-salicylate and Phenol Red-salicylate. On the other hand, high-affinity bindings of the 3 ligands do not take place independently but in such a way that binding of one of the ligands results in a decrease in binding of the other ligands. The decreases in binding are reciprocal and can be accounted for by introducing a coupling constant. The magnitude of the constant is dependent on the ligands being bound. The mutual decrease in binding was more pronounced with L-tryptophan-salicylate and Phenol Red-salicylate than with L-tryptophan-Phenol Red.