Buffer effects on electric signals of light-excited bacteriorhodopsin

Buffers change the electric signals of light-excited bacteriorhodopsin mole cules in purple membrane if their concentration and the pH of the low-salt solution are properly selected. "Positive" buffers produce a positive compo nent, and "negative" buffers a negative component in addition to the signal s due to proton pumping. Measurement of the buffer effects in the presence of glycyl-glycine or bis-tris propane revealed an increase of similar to 2 and a change of sign and a decrease to similar to 0.5 in the translocated c harge in these cases, respectively. These factors do not depend on temperat ure. The Arrhenius parameters established from the evaluation of the kineti cs indicate activation enthalpies of 35-40 kJ/mol and negative activation e ntropies for the additional signals. These values agree with those found by surface-bound pH-sensitive probes in the search of the timing of proton re lease and uptake. The electric signals were also measured in the case of D2 O solutions with similar results, except for the increased lifetimes. We of fer a unified explanation for the data obtained with surface-bound probes a nd electric signals based on the clusters at extracellular and cytoplasmic sites of bacteriorhodopsin participating in proton release and uptake.