Taylor M E, Bogomolni R A, Weber H J
Proc Natl Acad Sci U S A. 1983 Oct;80(20):6172-6. doi: 10.1073/pnas.80.20.6172.
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.
我们已经开发出一种用于纯化处于光化学活性状态的嗜盐视紫红质的方法。用辛基葡糖苷溶解来自一株无细菌视紫红质的嗜盐菌菌株的膜，然后在羟基磷灰石和辛基琼脂糖凝胶上进行色谱分离。所有步骤均在高离子强度溶液中进行。该方法使富集倍数达到270倍，产率为35%。洗脱的色素在575nm处有最大吸收峰，A280/A575比值为2。通过透析去除去污剂后，纯化的嗜盐视紫红质经化学漂白、用[3H]视黄醛再生并用氰基硼氢化钠还原。在对十二烷基硫酸钠/聚丙烯酰胺凝胶进行染色或荧光显影后，此类样品显示出一条主带和一条卫星带。主带的表观分子量为25,000。纯化的嗜盐视紫红质在脉冲激光激发后经历光循环，并且在去除氯离子后（在中性pH下）显示出9nm的蓝移。该色素在碱性pH下也会发生光可逆转变，形成在410nm处有最大吸收的形式。所有这三个反应都与膜结合的嗜盐视紫红质的反应非常相似。
