9-Amino-acridine as a probe of the electrical double layer associated with the chloroplast thylakoid membranes

Chloroplasts washed with monovalent cations are found to quench 9-amino-acridine fluorescence after resuspension in a cation-free medium. This quenching occurs in the absence of a high energy state and can be reversed by the addition of salts. The effectiveness of these salts is related to the charge carried by the cations and appears to be essentially independent of the associated anions. The order of effectiveness is polyvalent > divalent > monovalent, and virtually no variation is found within the groups of monovalent cations and divalent cations tested. Furthermore, choline and lysine are as effective as alkali metal cations, and lysyl-lysine is almost as effective as alkaline earth metal cations. These results are consistent with an effect mediated by the electrical double layer at the membrane surface rather than chemical bonding, and can be qualitatively explained in terms of the Gouy-Chapman theory.It appears that 9-amino-acridine acts as a diffusible monovalent cation which increases its fluorescence when displaced from the diffuse layer adjacent to the negatively charged membrane surface. The 9-amino-acridine fluorescence changes have been experimentally correlated with the cation-induced chlorophyll a fluorescence changes also observed with isolated chloroplasts.