56] Determination of the net proton-hydroxide ion permeability across vesicular lipid bilayers and membrane proteins by optical probes

Abstract

Publisher Summary This chapter describes a methodology for the determination of proton-hydroxide ion permeability across lipid bilayers of vesicles and through the membrane protein, bacteriorhodopsin, which is also applicable to native biological membrane systems. The optical probe pyranine used in this approach allows the continuous measurement of pH changes in the vesicle interior with high sensitivity and high time resolution. An optical probe suitable for the measurement of internal pH changes in vesicles, organelles, and cells have a fast response time, high sensitivity, small permeability, high solubility in the aqueous medium, a well-understood reaction mechanism, and spectroscopic properties that are easy to monitor with commercially available equipment. To apply flux equations for the evaluation of the net H+–OH– permeability coefficient, Pnet, the experiments are performed close to pH 7.0 and with small pH differences. The fluorescent 9-aminoacridine (9-AA) is the most often used optical probe for the determination of the magnitude of proton gradients across biological and artificial membranes. The driving force for the creation of a proton-hydroxide ion flux across a membrane can either be a pH gradient (∆pH) or a membrane potential (∆ψ).