Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.

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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.Three retinal-containing pigments are synthesized by Halobacterium halobium. These membrane-bound pigments are bacteriorhodopsin (bR), halorhodopsin (hR), and the slow rhodopsin-like pigment (s-rhodopsin; sR); they have similar UV and visible absorption spectra, with maxima at 568 nm (bR), 578 nm (hR), and 587 nm (sR), and all three undergo characteristic photochemical cycles after excitation by actinic light (1-4). Whereas bR, the light-driven proton pump, converts light energy into a protonmotive force that can be harnessed by bacterial cells for ATP synthesis (for review see ref. 1), hR is probably involved in electrogenic transport of Cl-into the cells (5). The physiological significance of this light-driven process is not known with certainty. Some evidence suggests that sR participates in the phototactic responses of halobacterial cells (3).Many structural and functional studies require purification of the photoactive chromoproteins. This task is relatively simple for bR, because it is the only protein of the purple membrane that can be fractionated on sucrose gradients (6). In our available halobacteria strains, hR is not so organized and has to be purified by detergent solubilization and subsequent fractionation.Using a strain deficient in bR [determined by flash spectroscopy as described (4, 7) were suspended in 150 ml of 4 M NaCl and lysed by dialysis in the presence of 10 mg of DNase at 4°C for 18 hr against 6 liters of 50 mM Hepes (pH 7.0). The lysate was cleared of large debris (containing little or no hR) by centrifugation (6,000 x g, 15 min) and the membranes were then sedimented (260,000 x g, 1 hr). The bluish pellet was suspended in 50 mM Hepes buffer (pH 7.0). The sedimentation and resuspension of membranes was repeated twice in Hepes buffer and then once in 4 M NaCl. Finally, the membranes were stored in 4 M NaCl at 4°C at 10-20 mg/ml.Pro...

Asolectin lipid vesicles containing halorhodopsin show light-induced acidification in the presence of proton ionophores. This effect is abolished by triphenyltin chloride, a chloride/hydroxyl antiporter, and is greatly diminished by valinomycin in the presence of potassium ions, which collapse the membrane potential. This indicates that halorhodopsin orients in the lipid vesicles preferentially inside out, pumping chloride into the extravesicular compartment. The absorption maximum of halorhodopsin in asolectin vesicles in 3 M NaCl is at 567 nm, and the action spectrum for the lightinduced pH changes followed closely the absorption spectrum. Replacement of chloride by acetate or sulfate causes a shift in the absorption maximum to =559 nm and renders the pump inactive. The different photocycles of the two forms were used to show that 80% of the molecules have the extracellular side exposed to the vesicle interior and that the halide-binding site(s) associated with the spectral transition is accessible from the extracellular side of the molecule. The data presented demonstrate that the purified chromoprotein is the light-driven chloride pump in Halobacterium halobium.The membranes of Halobacterium halobium contain at least three retinyl-proteins. Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-driven proton (outward) and chloride (inward) pumps, respectively (1,2). A third pigment, slow rhodopsin (sR), does not mediate light-driven electrogenic ion translocation and it may be the receptor in phototaxis (3). The visible absorption maxima of bR, hR, and sR are at 568 nm, 578 nm, and 587 nm, respectively (4). Both bR and hR undergo cyclic photoreactions with half-times of a few milliseconds (5,6 The photochemical properties of hR are chloride dependent. At physiological chloride concentrations (-3 M), the photocycle half-time is -10 msec and intermediates are observed as maxima in the difference spectrum at 500 nm (P500) and 680 nm (P680) (8).In the light and in the presence of chloride or bromide, hR generates a negative transmembrane electrical potential and protons are driven into the cell. These proton fluxes are greatly enhanced in the presence of proton ionophores and have been used to monitor the chloride transport by hR in intact cells (6, 9) and in cell envelope vesicles (10, 11).In chloride-free medium, the hR absorption maximum shifts to 565 nm (12, 13), and in the photocycle only a redshifted intermediate (P640) has been observed. It shows a maximum of 640 nm in the difference spectrum and decays into the 565 nm ground state with a half-time of -2 msec (14). No light-driven ion transport has been observed under these conditions (2).From the membranes of a bacteriorhodopsin-deficient H. halobium strain, we have recently purified a retinyl-polypeptide of -25 kDa, with the spectroscopic and photochemical properties of hR (15), and similar preparations have been obtained in other laboratories (16,17). However, it remains to be shown that this isolated retinyl-protein is the ion pump and that ...

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