BACTERIORHODOPSIN INTERMEDIATE SPECTRA DETERMINED OVER A WIDE PH RANGE

The pH dependence of the absorption spectra of bacteriorhodopsin and i ts photocycle intermediates was studied in the pH range 4.5-9. The spe ctra of the intermediates were determined from difference spectra take n during the photocycle with an optical multichannel analyzer. The dat a analysis was based on various criteria concerning the shape of the s pectra, but no assumption was made about the kinetic model that descri bes the photocycle. The strategy for calculation of the spectra was on e described earlier, as well as a newly introduced algorithm based on the Monte Carlo method. The search methods used gave very similar resu lts. Like the absorption spectrum of bacteriorhodopsin in the above-me ntioned pH range, the spectra of all the intermediates were found to b e almost unchanged. The spectrum of intermediate M displayed a 2 nm, a nd that of intermediate L a 1 nm, red shift with rising pH, but this l atter shift was within the overall error of the measurements. The simi larity of all the intermediate spectra calculated with different proce dures at all pH values points to the reliability of the method and the validity of the spectra. Averaging of the calculated spectra over the whole pH range furnished a well-determined set of intermediate spectr a, suitable for further kinetic and spectroscopic studies of the photo cycle.