Buffers change the electric signals of light-excited bacteriorhodopsin molecules in purple membrane if their concentration and the pH of the low- salt solution are properly selected. 'Positive' buffers produce a positive component, and 'negative' buffers a negative component in addition to the signals due to proton pumping. Measurement of the buffer effects in the presence of glycyl-glycine or bis-tris propane revealed an increase of ~ 2 and a change of sign and a decrease to ~ -0.5 in the translocated charge in these cases, respectively. These factors do not depend on temperature. The Arrhenius parameters established from the evaluation of the kinetics indicate activation enthalpies of 35-40 kJ/mol and negative activation entropies for the additional signals. These values agree with those found by surface-bound pH-sensitive probes in the search of the timing of proton release and uptake. The electric signals were also measured in the case of D2O solutions with similar results, except for the increased lifetimes. We offer a unified explanation for the data obtained with surface-bound probes and electric signals based on the clusters at extracellular and cytoplasmic sites of bacteriorhodopsin participating in proton release and uptake.
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