Isolation, purification, and partial characterization of stable forms of monomeric bacteriorhodopsin in lauryl sucrose

Abstract

The purple membrane of Halobacterium halobium was solubilized by treatment with lauryl sucrose and bacteriorhodopsin was found to be more stable in that detergent than in any other. While bacteriorhodopsin could not be completely delipidated by chromatography in lauryl sucrose alone, the purified bacteriorhodopsin monomers previously delipidated by treatment with Triton X-100 could be stabilized for a long time once Triton was exchanged for lauryl sucrose. In that state, an acid–base equilibrium exists between two spectral forms of the pigment absorbing, respectively, at 540 and 480 nm. The pKa of the equilibrium is 6.8. Consideration of the pKa value together with the amplitude of the spectral shift and the fact that both bacteriorhodopsin species, upon illumination, form different bathointermediates but share the same metaintermediate suggests that the acid–base equilibrium corresponds to the titration of the retinal Schiff base of the pigment. Since this equilibrium is only observable in completely delipidated bacteriorhodopsin, we propose that some specific lipid–protein interaction is responsible for the prevention of its occurrence in the membrane and for the maintenance of structural and functional integrity of bacteriorhodopsin.