ALKYL GLYCOSIDE DETERGENTS - A SIMPLER SYNTHESIS AND THEIR EFFECTS ON KINETIC AND PHYSICAL-PROPERTIES OF CYTOCHROME-C OXIDASE
ALKYL GLYCOSIDE DETERGENTS - A SIMPLER SYNTHESIS AND THEIR EFFECTS ON KINETIC AND PHYSICAL-PROPERTIES OF CYTOCHROME-C OXIDASE
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DOI：
10.1021/bi00558a032
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发表时间：
1980-01-01
期刊：
影响因子：
2.9
通讯作者：
FERGUSONMILLER, S
中科院分区：
文献类型：
--
作者：
ROSEVEAR, P;VANAKEN, T;FERGUSONMILLER, S
辛基葡糖苷是一种有效的非离子膜蛋白增溶剂，其优点是易于通过透析去除。为了研究细胞色素c氧化酶的去污剂敏感活性，选择这种去污剂是因为它的结构简单，并且可以合成类似物来测试去污剂特异性的结构依赖性。开发了一种有利于大规模制备辛基葡糖苷和相关烷基葡糖苷的方法，通过消除结晶步骤并在Dowex 1上采用最终产物的一步纯化来改进先前的方法。这种新的纯化方法对于达到获得可溶形式的二糖、较长烷基链去污剂所需的纯度水平特别重要。在所制备的烷基糖苷中（辛基β- D-吡喃葡糖苷，辛基β- D-乳吡喃糖苷，十二烷基β- D-乳吡喃糖苷，十二烷基β- D-纤维双吡喃糖苷和十二烷基β- D-麦芽吡喃糖苷），月桂基（十二烷基）麦芽糖苷是最成功的纯化牛肉和脉孢菌细胞色素氧化酶的激活剂，比辛基葡糖苷和其他市售洗涤剂，吐温-20和Triton X-100的活性高2至10倍。使用2种不同稳态测定系统的动力学研究表明，活性变化不是底物结合改变的结果，而是反映了对酶（作为单体、二聚体聚合物）缔合状态及其内在活性的去污剂效应。通过凝胶过滤程序，月桂基麦芽糖苷和辛基葡糖苷分别以50，000和8000道尔顿的胶束的单分散群体存在。月桂基麦芽苷和辛基葡萄糖苷的小而均匀的胶束和化学上明确的结构使它们在一般膜蛋白和特别是细胞色素氧化酶的研究中上级其他非离子洗涤剂，因为其在月桂基麦芽苷中的活性最接近生理状态。
Octyl glucoside is an effective, nonionic, solubilizing agent for membrane proteins with the advantage of ease of removal by dialysis. In order to study the detergent-sensitive activity of cytochrome c oxidase, this detergent was chosen because of its simple structure and the possibility of synthesizing analogues to test the structural dependence of the detergent specificity. A procedure was developed that facilitates large-scale preparation of octyl glucoside and related alkyl glycosides, improving on previous methods by eliminating crystallization steps and employing a 1-step purification of the final product on Dowex 1. This new purification procedure is particularly important for achieving the level of purity required to obtain the disaccharide, longer alkyl chain detergents in soluble form. Of the alkyl glycosides prepared (octyl .beta.-D-glucopyranoside, octyl .beta.-D-lactopyranoside, dodecyl .beta.-D-lactopyranoside, dodecyl .beta.-D-cellobiopyranoside and dodecyl .beta.-D-maltopyranoside), lauryl (dodecyl) maltoside was the most successful as an activator of purified beef and Neurospora cytochrome oxidases, giving 2- to 10-fold higher activities than octyl glucoside and other commercially available detergents, Tween-20 and Triton X-100 . Kinetic studies using 2 different steady-state assay systems indicate that the activity changes are not the result of altered binding of the substrate, but rather reflect a detergent effect on the state of association of the enzyme (as a monomer, dimer polymer) as well as on its intrinsic activity. By gel filtration procedures, lauryl maltoside and octyl glucoside existed as monodisperse populations of micelles of 50,000 and 8000 daltons, respectively. The small uniform micelles and chemically well-defined structures of lauryl maltoside and octyl glucoside make them superior to other nonionic detergents for the study of membrane proteins in general and cytochrome oxidase in particular, since its activity in lauryl maltoside most closely approaches that of the physiological state.