Time-resolved, flash-induced difference absorbance spectra (300-700 am) at pH 10.5 and 5C for the bacteriorhodopsin photocycle fast and slow decaying forms of the M intermediate (M' and MS, respectively) MATERIALS AND METHODS Purple membranes were isolated from Halobacterium halobium, S9 strain, and suspended in 30 mM piperazine/30 mM glycylglycine, pH 10.5. All samples contained 40%o (vol/vol) glycerol except those used for the flash-induced absorbance changes in Fig. 4. All measurements were carried out on light-adapted BR.Two spectrophotometers were used to obtain the lightinduced difference spectra. For the faster measurements, the data were taken and analyzed as described (10,11). For the slower measurements a Hewlett-Packard model 8452A diode array spectrophotometer was used to take complete lightinduced difference spectra from 300 to 700 nm with 2-nm spectral resolution. (The absorption spectrum of the lightadapted sample was used as the baseline.) The actinic source was a commercial photoflash with Coming long-pass filter (CS 3-67, wavelength >550 nm). The half-duration time was 150 tUs. Since the measuring beam of the diode array spectrophotometer was relatively strong white light, special care was taken to check and avoid a possible actinic effect of the measuring beam. Sample OD570 was between 1.3 and 1.5.
RESULTSSeparation of the Difference Spectra of Intermediates M', ME, and R. From the time-dependent series of difference spectra, we can determine the difference spectra of the late intermediates involved in the BR recovery at high pH (Mf, MS, and R). We have chosen conditions for which the differences in the lifetimes and yields of these three components are maximal. We assume that the intermediates before M can be neglected because of their much shorter lifetimes and that no significant amount of the late intermediate 0 is formed because of the low temperatures and high pH values (refs. 5, 6, and 10 and unpublished data). Fig. 1
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