[47] A centrifuged-column procedure for the measurement of ligand binding by beef heart F1

This chapter describes the method suitable for determination of the binding of most of these ligands by the enzyme, utilizes very small amounts of protein, and is highly sensitive because the ligand which was bound to protein is measured in the absence of free ligand.

Abstract

This chapter discusses that beef heart mitochondrial ATPase (F1) contains a variety of binding sites for small ligands. These include five sites for adenine nucleotides, two sites for aurovertin, and one or more sites for oxyanions such as 2,4-dinitrophenol and at least one site for Pi. It describes the method suitable for determination of the binding of most of these ligands by the enzyme, utilizes very small amounts of protein (as little as 50/μg), and is highly sensitive because the ligand which was bound to protein is measured in the absence of free ligand. However, the method is not an equilibrium binding procedure, and while it is suited for quantitating the occupancy of binding sites under differing experimental conditions, calculation of dissociation constants from the binding data obtained with F1 or other proteins should be made with caution. The chapter also discusses binding of inorganic phosphate by F1.