Photocurrents of dark-adapted bacteriorhodopsin on black lipid membranes

Abstract

The main constituent of purple membranes from Halobacterium halobium is bacteriorhodopsin, the light-driven proton pump [I]. The addition of purple membranes (i.e., bacteriorhodopsin) to one side of a lipid bilayer induces on illumination photocurrents [2--61. The purple membranes are not integrated in the lipid bilayer, but they form, with the supporting black lipid membrane, a sandwich-like structure. This membrane system has been used for measurements under stationary light conditions [2-51. Photocurrent kinetics induced by a laser flash have been studied: Single steps of the photocurrent could be assigned to the photocycle of bacteriorhodopsin [6]. Bacteriorhodopsin exists in a dark-adapted form, the chromophore, retinal, is in -1: 1 equilibrium between the 134s and the all-trans configuration [7-91, whereas in the light-adapted form almost all of the chromophore exists in the all-tt-ans form [ 10,l I]. Here, the change of the photocurrent amplitude during the dark-adaptation process of purple membranes attached to a black lipid membrane is reported. absorbance was 0.04. The membrane was excited by laser pulses of -10 ns duration, 575 mm (Dye-laser, Molectron DL lO/UV 12). The energy flash was monitored via a semi-transparent mirror by which half of the light intensity was deflected to a Joulemeter J 3 (Molectron). Therefore, photocurrent and laser flash could be measured simultaneously. This was necessary because the light intensity of the laser flash was fluctuating within a factor of 2. The low light intensity was adjusted to -60 pJ/cm’ which corresponds to 1.7 X 1 014 quanta/cm’. Since the absorption crosssection of bacteriorhodopsin is -u = 2.1 X lo-l6 corresponding to an extinction coefficient e = 54 000 M-r. cm-’ [12] means that u . n = 3.6 X lo-*, so that -3% of the present bacteriorhodopsin molecules can be excited by a single flash.