Abstract
Bacteriorhodopsin (BR) essentially free of native lipids has been prepared in a highly stable state. Purple membrane was solubilized in Triton X-100 and BR was purified by size exclusion chromatography using 3-[cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonic acid (CHAPSO) detergent at pH 5. Molar ratios of phospholipid/BR ranged from 0.4 to 0.05 corresponding to 94-98% phospholipid removal. Purified BR has an absorbance ratio (A280nm/A548nm) of 1.5-1.6 in the dark-adapted state which is the highest purified BR/protein ratio reported to date. The purified BR in CHAPSO shows maximum stability in the pH range 5.0-5.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of native purple membrane and solubilized BR from most Halobacterium halobium JW-3 cultures show 3 higher molecular weight bands in addition to BR. Immunological staining and amino acid sequencing indicates that these additional proteins are partially processed forms of the BR precursor protein. The BR preprotein contains 13 additional amino acids on the NH2 terminus which are removed by post-translational processing in at least four steps. Isoelectric focusing separated most delipidated and non-delipidated BR samples into 8 bands. Incomplete BR post-translational processing BR is thought to be largely responsible for the multiplicity of isoelectric BR species. The principal components have pI values of 5.20 and 5.24 and both have absorption maxima at 550 nm, characteristic of detergent-solubilized BR. BR in Triton X-100 or nonylglucoside, delipidated BR in CHAPSO, and BR in intact purple membrane all have a dark-adapted ratio of 13-cis to all-trans-retinal of 1.9:1.
Highlights
1.5-1.6 in the dark-adapted statwe hich is the highest We required highly stable, delipidated BR in an purified BR/protein ratio reported to date
BR in TritonX-100 or nonylglucoside, delipidated BR in CHAPSO, and BR in intact purple membrane all have a dark-adapted ratio of 13-cis to all-trans-retinal of 1.9:l
This method was not utilized since lauryl sucrose is notapureproduct, is not commercially available, and presumably has a very low CMC (CMC of lauryl maltoside is 0.2 mM)
Summary
The results for BR solubilization, pH dependence of stabilitiyn solubilization detergents, column purification in CHAPSO detergent, absorption spectrumof purified BR, and thepH stability of the purified BR are described inthe Miniprint Section. No significant improvement of phospholipid removal was obtained by a second passage through the size exclusion column (first pass 95% phospholipid free; second pass 96% phospholipid free). This suggests thatthe remaining phosphorous may be covalently bound. Multiple BR Polypeptides-BR isolated from most but not all of the halobium JW-3 cultures show two higher molecular weight species in additionto theprincipal band when analyzed by SDS-PAGE visualized with Coomassie Blue or silver stain (Fig. 5). The relative amounts of SDS-PAGE bands a, b, c, and d on the Western blot in Fig. 6 was approximately 63, 21, 12, and 4% of the total,respectively, as determined by quantitative scanning of the gel. Gel contained 15%acrylamide and was run at 10 "Cand silver stained
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