Use of the Hummel and Dreyer method for the study of nucleotide binding on chloroplast ATPase CF1

The study of the binding of the nucleotides ADP and ATP on the exchangeable sites of chloroplast ATPase CF1 has been carried out by the Hummel and Dreyer method applied to HPLC. It has been shown that this method was well fitted to the problem: rapidity of exchange, absence of noticeable modification after binding, presence of a constant concentration of ligand during the chromatography, which stabilizes these low affinity complexes. The dissociation constants of binding of ADP, ATP and of their magnesium salt complexes have been determined. In order to measure the simultaneous binding of ADP and ATP when present in mixture, we have modified the method by using an anion-exchange column in place of the gel filtration column: the two nucleotides were easily separated, while the binding on the protein was unchanged. The extension of this method to the reversed-phase chromatography could also be considered for the binding of hydrophobic ligands.