The binding of eosin‐labeled subunit δ to the isolated chloroplast ATPase, CF₁, as revealed by rotational diffusion in solution

We investigated the binding of subunit δ to solubilized chloroplast ATPase. Purified δ was covalently labeled with eosin 5‐isothiocyanate and its rotational correlation time was determined by a photoselection technique as a function of added CF 1 (containing δ) and of CF 1 (−δ) (lacking δ). In aqueous buffer the rotational correlation time of labeled δ was 33 ns. This is compatible with a rather elongated shape with the dimensions 2 b = 100 Å/2 a = 28 Å. Binding of δ to CF 1 decreased the rotational correlation time about 10‐fold. The result was a biphasic decay of the laser flash‐induced absorption anisotropy which was analyzed to yield the proportion of δ (bound to CF 1 ) relative to δ (free). CF 1 (−δ), which completely lacked the δ‐subunit, bound one δ (mol/mol) with high affinity ( K d ≈ 100 nM) and at least another δ with about 20‐fold lower affinity. The δ‐containing CF 1 , revealed only the low‐affinity site(s) for δ. This was compatible with a 1:1 stoichiometry of δ in isolated CF 1 .