Purification of photochemically active halorhodopsin.

We have developed a procedure for the purification of halorhodopsin in a photochemically active state. Solubilization of membranes from a bacteriorhodopsin-negative Halobacterium strain with octyl glucoside was followed by chromatography on hydroxylapatite and octyl-Sepharose gels. All steps were carried out in high-ionic-strength solutions. The procedure resulted in 270-fold enrichment with a 35% yield. The eluted pigment had an absorption maximum at 575 nm and an A280/A575 ratio of 2. On removal of the detergent by dialysis, the purified halorhodopsin was chemically bleached, regenerated with [3H]retinal, and reduced with cyanoborohydride. Such samples showed one main and one satellite band after staining or fluorography of NaDodSO4/polyacrylamide gels. The apparent molecular weight of the main band was 25,000. Purified halorhodopsin underwent a photocycle after excitation with pulsed laser light and showed a 9-nm blue shift (at neutral pH) on removal of chloride ion. The pigment also underwent a photoreversible shift at alkaline pH to a form absorbing maximally at 410 nm. All three reactions closely resembled those of membrane-bound halorhodopsin.