51] Measurement of membrane ΔpH

Abstract

Publisher Summary This chapter describes three procedures which estimate the magnitude of the proton concentration gradient (∆pH) across thylakoid membranes. In silicone fluid centrifugation method, thylakoids are illuminated in the presence of radioactive amines within a microcentrifuge. The thylakoids are then rapidly centrifuged through a layer of silicone fluid to separate them from the suspending medium. Internal volumes may be estimated from the uptake of 3 H 2 O. In the fluorescent amines method, a suspension of thylakoids including a fluorescent amine (9-aminoacridine or N -l-naphthylethylenediamine) contained in a glass cuvette is placed in a fluorimeter and the changes in the fluorescence of the amine are continuously followed. Illumination of the thylakoid suspension results in the uptake of amine by the thylakoid which will be manifested as a reversible decrease of the fluorescence emitted from the cuvette. In the third method of stimulation of proton uptake by amines, the pH increase in the medium of an illuminated thylakoid suspension is measured. Following addition of an amine having a p K lower than the external pH, an increase in the extent of proton uptake is observed. Increment in proton uptake resulting from internal buffering of protons by the amine is used to calculate the internal pH.