Measurement of ligand-protein binding interactions in a biphasic aqueous polymer system

Abstract

In this paper we introduce a new technique, which we term the method of equilibrium partition, for studies of the interaction of small ligands with macromolecules. The method utilizes a biphasic aqueous solution of the polymers dextran and polyethylene glycol; in this system, proteins tend to partition chiefly into one phase while small ligands are distributed among both phases. The quantity of ligand bound to the protein in the protein-containing phase can be calculated using measurements of ligand concentration in the other phase, if the other phase contains only negligible quantities of protein. Compared with other methods commonly in use for ligand binding studies, the equilibrium partition method has the advantages that it is very rapid (equilibrium is reached within a few minutes) and can easily be scaled down to microliter volumes in order to perform binding studies with the consumption of minimal quantities of protein. However, the method is applicable only to those proteins which exhibit the required partitioning behavior. The equilibrium partition method has been used successfully in studies of nucleotide binding to the enzymes aspartate transcarbamylase and formyltetrahydrofolate synthetase. In the case of ATCase (but not FTHF synthetase) ligand binding affinities measured in the presence of the highly concentrated dextran-polyethylene glycol solutions appear to differ from the corresponding affinities observed in dilute aqueous solutions.