Isolation and properties of the native chromoprotein halorhodopsin.

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Abstract
The native chromoprotein of the light‐driven chloride pump halorhodopsin (HR) was isolated from Halobacterium halobium strain L‐33 which lacks bacteriorhodopsin but contains ‘slow cycling rhodopsin‐like pigment’ (SR). A membrane fraction was prepared in low salt and dissolved in a high salt medium by the detergents Lubrol PX or octylglucoside. These conditions destroyed the chromophore of SR but not the HR pigment. Chromatography on phenyl‐Sepharose and hydroxylapatite produced, in 60% yield, a 230‐fold enriched monomeric chromoprotein with an apparent mol. wt. of 20,000. The chromoprotein was stable in 1 M NaCl and 1% octylglucoside and remained stable upon removal of detergent. It reacted with borohydride in the dark and with hydroxylamine in the light. The absorption maximum of the light‐adapted state is at 580 + 2 nm and its molar extinction approximately 50,000/M/cm. Upon illumination in the presence of detergent it was converted into a 410 nm absorbing species with concomitant release of protons. A thermal reconversion to the 580 nm species occurred with a half time of 76 s at ‐6 degrees C. Blue light absorbed by the photoproduct accelerated the re‐conversion as well as the re‐uptake of protons. Removal of the detergent prevented the light‐induced formation of the 410 nm species. Under these conditions a photochemical behaviour similar to that in intact cells and cell vesicles, i.e., a photocycle in the 10‐20 ms range was observed. These findings form the basis for functional reconstitution of HR.