Method for studying kinetics of light-induced transport across membranes

A technique based on phase spectrophotometry is described for studying the rates of elementary processes associated with the light-driven transport of ions and molecules across membranes. The light-induced pumping of protons by bacteriorhodopsin reconstituted into phospholipid vesicles and by chloroplast thylakoids has been studied to illustrate the potential of this technique. The exciting light is modulated by a mechanical chopper over the frequency range 5 Hz to 2 kHz. The internal pH of the membrane vesicles is modulated at the same frequency as the exciting light but differs in phase because of the finite rate of proton pumping. Measurement of this phase difference or of the frequency dispersion of the amplitude of the internal pH modulation is accomplished by use of a lock-in amplifier. The results can be interpreted in terms of relaxation times characterizing the chemical steps in proton pumping. The shortest relaxation time that can be measured is about 50 microseconds, although the time resolution could be easily extended by use of faster light chopping techniques. At pH 8.0, two relaxation processes are associated with proton pumping by bacteriorhodopsin reconstituted into phospholipid vesicles; the relaxation times are 2 and 28 msec. Two relaxation processes also were found with chloroplast thylakoids at pH 7.8, with relaxation times of 2 and 16 msec. The former can be associated with photosystem II and the latter, with photosystem I.