COMPONENT ANALYSIS OF THE FAST PHOTOELECTRIC SIGNAL FROM MODEL BACTERIORHODOPSIN MEMBRANES .4. A METHOD FOR ISOLATING THE B2 COMPONENT AND THE EVIDENCE FOR ITS POLARITY REVERSAL AT LOW PH

Thin films formed from oriented purple membranes of Halobacterium halo bium exhibit fast photoelectric signals upon pulsed light stimulation. These signals are generated by light-induced rapid charge separation and recombination. The fast photosignal consists of several components . We have previously developed methodology to separate these component s on the basis of their pH dependence. We used the Trissl-Montal metho d of membrane reconstitution to generate a photosignal which contains the pH-independent B1 component and the pH-dependent B2 component, whe reas we used a modified multilayered thin film method to obtain a pure B1 signal. The B2 component is prominent in the medium to high pH ran ge (pH 7-11) and has a polarity opposite to that of B1. As the pH decr eases, the B2 amplitude declines progressively. However, the B2 amplit ude does not just eventually become zero but rather undergoes a polari ty reversal when the pH declines below 2.7, i.e. the B2 component acqu ires the same polarity as the B1 component below pH 2.7. Its amplitude grows as pH decreases further. Experimentally, the polarity reversal is demonstrated by a pH titration, in which successive signals were re corded as the pH is reduced in small decrements (about 0.2-0.3 pH unit s). The null point of titration, where the B2 component is completely absent (pH 2.7), is determined by the superposition of the signal (fro m a Triss-Montal film) and the standard pure B1 signal from a multilay ered thin film after normalization. The justification of this titratio n procedure is based on an equivalent circuit analysis previously esta blished.