Figure 1
From: Light-independent phospholipid scramblase activity of bacteriorhodopsin from Halobacterium salinarum
Biochemical characterization of BR purified from purple membrane. (a) Absorbance spectrum of purified BR. The ratio of peak heights, A560/A280 = 0.6/1.22, indicates that the sample is 98% pure. (b) Size exclusion chromatography (SEC) purification of BR using a Sephadex 200 column. The void volume Vo (determined using Blue Dextran 2,000 kDa) and the elution positions of alcohol dehydrogenase (150 kDa) and bovine serum albumin (66 kDa) are indicated. (c) Coomassie-stained SDS-PAGE of purified BR. (d) Fluorescence of pyranine as a function of pH (calibration plot). (e) Schematic representation of the proton pumping assay. (f) pH inside protein-free liposomes (‘no protein’, pH20 = 7.57 ± 0.2, mean ± s.d. n = 6) and BR-proteoliposomes (‘BR’, pH20 = 6.90 ± 0.14, mean ± s.d. n = 3) upon illumination at time = 0 min (indicated by *). Samples that were either not illuminated or supplemented with valinomycin (final concentration 50 nM, pH20 = 6.45 ± 0.16, mean ± range. n = 2) are indicated; pH20 corresponds to the pH value 20 min after the start of the experiment.
