Phenyl-Sepharose-mediated detergent-exchange chromatography: its application to exchange of detergents bound to membrane proteins.
Phenyl-Sepharose-mediated detergent-exchange chromatography: its application to exchange of detergents bound to membrane proteins.
复制标题
苯基琼脂糖介导的去垢剂交换层析：其在交换与膜蛋白结合的去垢剂方面的应用。
DOI：
10.1021/bi00320a034
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
Talbert,L
中科院分区：
文献类型：
--
作者：
Robinson,NC;Wiginton,D;Talbert,L
Neal C. Robinson、* Diane Wiginton 和 Linda Talbert 摘要：去污剂饱和的苯基琼脂糖用于在膜蛋白存在的情况下相互交换去污剂。烷基去污剂月桂基麦芽糖苷、辛基葡糖苷和十二烷基硫酸盐分别成功地替换为细胞色素 c 氧化酶、线粒体内膜蛋白混合物或红细胞膜蛋白混合物溶液中存在的 Triton X-100、Triton N-101 或 Nonidet P-40。该方法包括 (1) 在 pH 为 8 或 9、离子强度为 0.01 的情况下，用一种烷基去污剂饱和一小支苯基琼脂糖柱 (1-2 mL)，(2) 使用去污剂溶解的 mem-。内在膜蛋白研究过程中面临的主要问题是用更适合下一步实验的去污剂替换项目某一阶段用于溶解蛋白质的去污剂。例如，对于细胞色素 c 氧化酶，通常需要相互交换胆酸盐、脱氧胆酸盐、Triton X-100、Nonidet P-40、月桂基麦芽糖苷、1 个 Tween 20、Tween 80 或 Brij 96。由于 Tri-ton 型去垢剂、胆酸盐和/或脱氧胆酸盐经常用于纯化复合物，因此需要更换这些去垢剂（Hartzell 等，1978）； Triton X-100、Nonidet P-40 或胆酸盐非常适合复合物的脱脂（Robinson 等人，1980；Yu 等人，1975）；月桂基麦芽糖苷、吐温 20、吐温 80 或 Brij 96 对于最大电子传输活性最有用[例如，Thompson & Ferguson-Miller (1983)、Robinson & Capaldi (1976)、Yu 等人 (1975) 和 Vanneste 等人 (1974)]；胆酸盐是将复合物重建为磷脂囊泡的最佳选择（Carroll & Racker，1977）。去垢剂交换的必要性并不是细胞色素c氧化酶特有的问题。不幸的是，对于大多数内在膜蛋白，通常没有一种去污剂能够很好地适合项目的每个部分。有许多去污剂交换方法可用，但其中大多数方法相对较慢，并且不能与多种去污剂或内在膜蛋白一起使用。例如，在存在过量脱氧胆酸盐的情况下，可以使用 Triton X-100 溶解的蛋白质进行凝胶过滤，以将脱氧胆酸盐交换为 Triton X-100（Robinson & Capaldi，1976），但只有当蛋白质-去污剂复合物的尺寸足够大于去污剂混合胶束的尺寸以通过凝胶过滤色谱分离时，才能使用该方法。对于大多数非离子去垢剂和膜蛋白来说，情况并非如此。因此，我们开发了一种使用苯基琼脂糖介导的去污剂交换的方法，这似乎是一种非常有用的交换方法
Neal C. Robinson,* Diane Wiginton, and Linda Talbert abstract: Detergent-saturated phenyl-Sepharose was used to exchange detergents for one another in thepresence of membrane proteins. The alkyl detergents lauryl maltoside, octyl glucoside, and dodecyl sulfate were each successfully exchanged for Triton X-100, Triton N-101, or Nonidet P-40 present in a solution of either cytochrome c oxidase, a mixture of inner mitochondrial membrane proteins, or a mixture of erythrocyte membrane proteins. The method involves (1) saturating a small column of phenyl-Sepharose (1-2 mL) with one of the alkyl detergents at a pH of 8 or 9 and an ionic strength of 0.01,(2) applying a detergent-solubilized mem-. major problem that is faced during the study of intrinsic membrane proteins is the replacement of the detergent that is used to solubilize the protein at one stage of a project with a detergent that is better suited for the next experiments. For example, with cytochrome c oxidase, it is often necessary to exchange cholate, deoxycholate, Triton X-100, Nonidet P-40, lauryl maltoside, 1 Tween 20, Tween 80, or Brij 96 for each other. Exchange of these detergents is required since Tri-ton-type detergents, cholate, and/or deoxycholate is often used to purify the complex (Hartzell et al., 1978); Triton X-100, Nonidet P-40, or cholate is excellent for delipidation of the complex (Robinson et al., 1980; Yu et al., 1975); lauryl maltoside, Tween 20, Tween 80, or Brij 96 is the most useful for maximum electron-transport activity [eg, Thompson & Ferguson-Miller (1983), Robinson & Capaldi (1976), Yu et al.,(1975), and Vanneste et al.(1974)]; cholate is the best for reconstitution of the complex into phospholipid vesicles (Carroll & Racker, 1977). The necessity of detergent ex-change is not a problem that is unique for cytochrome c ox-idase. Unfortunately, with most intrinsic membraneproteins, no single detergent is usually well suited for each part of a project.A number of detergent-exchange methods are available, but most of these are relatively slow and cannot be used with a wide variety of detergents or intrinsic membraneproteins. For example, gel filtration of a Triton X-100solubilized protein in the presence of excess deoxycholate can be used to exchange deoxycholate for Triton X-100 (Robinson & Capaldi, 1976), but this method can only be used if the size of the protein-detergent complex is sufficiently larger than the size of the detergent mixed micelles to separate by gel filtration chromatography. This is not true with most nonionic detergents and membrane proteins. We, therefore, have developed a method using phenyl-Sepharose-mediated detergent exchange that appears to be an extremely useful approach for exchanging