Phase-lifetime spectrophotometry of deoxycholate-purified bacteriorhodopsin reconstituted into asolectin vesicles

A rapid reconstitution procedure has been developed to insert deoxycholate-purified bacteriorhodopsin (bR) into asolectin vesicles. The procedure relies on the ability of the hydrophobic resin Bio-Beads SM-2 to remove octyl glucoside from a mixture of deoxycholate-purified bR, asolectin, and the detergent. Light-dependent acidification of the vesicle interior is observed with the reconstituted preparations as judged by the fluorescence quenching of an entrapped pH indicator, pyranine. Inhibition of proton pumping by the addition of LaCl3 to the external medium indicates that approximately 90% of the bR is oriented such that it pumps protons into the vesicles. Phase-lifetime spectrophotometry was used to study the relaxation processes associated with the intermediate in the photocycle of the reconstituted bR which absorbs at 410 nm. Amplitude spectra indicate that these absorbance changes are associated with the M intermediate in the bR photocycle. Two relaxation processes are observed. One is characterized by a relaxation time of approximately 4 ms and is independent of pH over the range 4.4-9.4. The longer relaxation time varies from 4 to 200 ms in the same pH range. By digitization of transients, which are observable when the actinic source is modulated at a low frequency, information about the dependence of the slower process on the light intensity and carbonyl cyanide m-chlorophenylhydrazone was obtained. The results can be interpreted in terms of two different forms of the M intermediate that decay on parallel kinetic paths. To explain the pH dependence of the decay rate, the slower decaying form must have three coupled protonation states, each with a different decay rate.