Instrumentation and procedures for real time measurements of proton motive force, membrane potential, ΔpH, proton extrusion, and oxygen uptake in respiring cells and vesicles

Abstract

The construction and use of two polyvinyl chloride, solid-state membrane ion-selective electrodes, one selective to tetraphenylphosphonium (TPP+) [to measure membrane potential (ΔΨ)] and the other selective to salicylate ion [to measure the pH gradient (ΔpH)], are described. A reaction vessel is fitted with these two electrodes plus a pH and an oxygen electrode. The signals from all four electrodes pass through an electronic interface to a microcomputer which filters out noise, corrects for relaxation time delay of electrodes as needed, converts the raw readings to real time kinetic values for rate of oxygen uptake, rate of proton extrusion, ratio of protons extruded per atom of oxygen consumed, membrane potential, the pH gradient across the membrane and the proton motive force. These values are continuously updated every 90 ms and passed to a multipen recorder for observation.