Intrinsic and artifactual pH buffering in chloroplast thylakoids

After an increase in pH of the suspension medium, a pH gradient across the membrane of chloroplast thylakoids stored at pH greater than or equal to 6.5 is often maintained for several minutes. The intrinsic hydrogen ion buffering capacity of the thylakoid membranes between pH 6.5 and 8.5 is about 40 neq/mg chlorophyll, but can be artificially inflated by penetration of the external buffer into the thylakoid vesicle. A delta pH imposed across the thylakoid membrane by an acid/base transition cannot be estimated accurately by the fluorescent probe 9-aminoacridine, especially with osmotically shrunken thylakoids in which 9-aminoacridine appears to become bound or adsorbed to the membrane. This interaction may be related to the existence of the previously demonstrated special pool of slowly equilibrating, "sequestered" protons.