Light-dependent and biochemical properties of two different bands of bacteriorhodopsin isolated on phenyl-sepharose CL-4B

We report a detailed description of the light-dependent and biochemical properties of two different bands of isolated and nearly delipidated bacteriorhodopsin obtained from chromatography on phenyl-Sepharose CL-4B. The two bands (BR I and BR II) showed a number of markedly different spectroscopic and biochemical characteristics: different absorption maximums in the dark, different light/dark adaptations, different M decay kinetics, different stabilities, different responses to titration with alkali in the dark and different circular dichroism (CD) spectra. Organic phosphate contents of BR I and BR II were measured; we found that more than 90% of purple membrane organic phosphate was removed in the course of chromatography and that the phospholipid/protein molar ratio was always higher in BR I than in BR II. In many functional aspects (high stability, response to light adaptation, spectral changes in the dark by alkali addition and bilobate CD spectrum) the first band appeared to be similar to the purple membrane. We suggest that the functional differences between the two bands depend on the fact that the first band (BR I) contains mostly bacteriorhodopsin aggregates corresponding to purple membrane trimers, while the second band (BR II) contains only bacteriorhodopsin monomers.