Fung B K, Hubbell W L
Biochemistry. 1978 Oct 17;17(21):4396-402. doi: 10.1021/bi00614a007.
Papain and thermolysin are shown to cleave bovine rhodopsin in native membranes in two temporally distinct steps at room temperature. The final product of the proteolysis consists of two membrane-bound fragments of molecular weights 27 000 (Rh27) and 12 500 (Rh12). The molecular weights are not changed by reduction with dithiothreitol. The two fragments remain closely associated in both the membrane and nondenaturing detergents before and after bleaching and can be selectively cross-linked with carbodiimides. The sulfhydryl chemistry of the cleaved protein in nearly indistinguishable from native rhodopsin, and of the total of six sulfhydryl groups, two are located on Rh12 and four on Rh27. In the membrane-bound protein, two sulfhydryl groups are accessible for modification, one on Rh12 and the other on Rh27. The sulfhydryl on Rh12 is particularly reactive and may be selectively labeled with maleimides. Continuous irradiation with white light induces additional sulfhydryl reactivity on Rh27.
木瓜蛋白酶和嗜热菌蛋白酶在室温下能在天然膜中以两个时间上不同的步骤切割牛视紫红质。蛋白水解的最终产物由分子量分别为27000（Rh27）和12500（Rh12）的两个膜结合片段组成。用二硫苏糖醇还原后分子量不变。这两个片段在漂白前后在膜和非变性去污剂中都紧密相连，并且可以用碳二亚胺进行选择性交联。切割后的蛋白质的巯基化学性质与天然视紫红质几乎无法区分，在总共六个巯基中，两个位于Rh12上，四个位于Rh27上。在膜结合蛋白中，有两个巯基可用于修饰，一个在Rh12上，另一个在Rh27上。Rh12上的巯基特别活泼，可以用马来酰亚胺进行选择性标记。用白光持续照射会使Rh27上产生额外的巯基反应活性。
