SEARCH

SEARCH BY CITATION

Abstract

An electrochemical cell was previously reported in which bacteriorhodopsin (BR, purple membrane) was adsorbed on the surface of a transparent SnO2 electrode, and illumination resulted in potential or current changes (Koyama et al., Science 265:762–765, 1994; Robertson and Lukashev, Biophys. J. 68:1507–1517, 1995; Koyama et al., Photochem. Photobiol. 68:400–406, 1998). In this paper, we concluded that pH changes caused by proton transfer by the deposited BR or proteorhodopsin (PR) films lead to the flash-induced potential change in the SnO2 electrode. Thus, the signals originate from BR and PR acting as light-driven proton pumps. This conclusion was drawn from the following observations. (1) The relation between the potential of a bare electrode and pH is linear for a wide pH range. (2) The flash-induced potential changes decrease with an increase in the buffer concentration. (3) The action spectrum of PR agrees well with the absorption spectrum. (4) The present electrode can monitor the pH change in the time range from 10 ms to several hundred milliseconds, as deduced by comparing the SnO2 signal with the signals of pH-sensitive dyes. Using this electrode system, flash-induced proton transfer by BR was measured for a wide pH range from 2 to 10. From these data, we reconfirmed various pKa values reported previously, indicating that the present method can give the correct pKa values. This is the first report to estimate these pKa values directly from the proton transfer. We then applied this method to flash-induced proton transfer of PR. We observed proton uptake followed by release for the pH range from 4 to 9.5, and in other pH ranges, proton release followed by uptake was observed.