Study of the Simultaneous Binding of ADP and ATP on Coupling Factor CF-1 by a Modification of the Hummel and Dreyer Method

A modification of the Hummel and Dreyer method(1), based on anion exchange separation is used here for the study of the simultaneous binding of ADP and ATP on spinach coupling factor CF1. This method gives the same results as gel filtration (dissociation constant and number of sites) when ADP alone is present. The extent of binding of ADP and ATP is approximately the same when mixed in equimolecular ratio, but since endogenous ADP is irreversible bound, this nucleotide is predominant on CF1. The binding of one nucleotide is partly prevented by prealable mixing of CF1 with the other nucleotide. This phenomenon occurs likely at the level of high affinity sites, where binding would not be entirely reversible, contrarily to low affinity sites. This method is of potential application for other ligands separable by anion exchange chromatography and for other types of chromatography (reversed phase).