Measurement of Proton Translocation in Thylakoids Under Flashing Light Using a Spin-Labelled Amine

The acidification of the internal compartment of thylakoids induced by single turnover flashes was monitored using the electron spin resonance (ESR) probe tempamine. The internal concentration of the probe, which reflects the internal pH, is measured using a non-penetrating agent (chromium oxalate) which quenches the contribution from the externally located tempamine. The method is used to estimate internal volume, internal buffering capacity, and the number of translocated protons per photochemical turnover. Analysis of the results obtained when submitting dark-adapted chloroplasts to a sequence of flashes seems to support the (1, 0, 1, 2) scheme for proton liberation by the oxygen-evolving system, and suggests that more than one proton is translocated inward by the intersystem electron transfer chain.