Banerjee P, Joo J B, Buse J T, Dawson G
Department of Chemistry, City University of New York, College of Staten Island 10314, USA.
Chem Phys Lipids. 1995 Aug 1;77(1):65-78. doi: 10.1016/0009-3084(95)02455-r.
Membrane proteins are typically extracted by detergent concentrations of 0.5-2.0%, using detergent/protein ratios of 1:1 to 3:1. We have compared the ability of 14 different detergents from seven different structural and ionic classes, at a concentration of 2.0% and a detergent/protein ratio of 2:1, to extract an integral membrane protein (the serotonin 5-HT1A receptor) in active form and have observed profound differences in both lipids and proteins. All extracts were freed from detergents and dialyzed to form vesicles containing 95-100% of the extracted lipids, prior to [3H]8-hydroxy-2-(N,N-di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding. The most efficient detergents in extracting active 5-HT1A receptor protein were the zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 3-[(cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), followed by the neutral n-dodecyl-beta-D-maltoside. Zwitterionic detergents also produced the highest solubilized lipid/protein ratio (3.0 and 2.5, respectively) and in general the relative amounts of extracted lipids and proteins followed inverse profiles. Thus, hydrophobic detergents such as Tritons (with critical micelle concentrations similar to CHAPS) and Thesit (structurally similar to Lubrol) extracted the most protein, but relatively little lipid (ratios of less than 0.2) and very little active 5-HT receptor. Dramatic differences were also observed in the ratios of individual lipids extracted by the same concentrations of different detergents and resolved by high-performance thin-layer chromatography. For example, galactosylceramide (GalCer) content ranged from 2.7% (CHAPSO) to 13.4% (sodium cholate) of the total lipid extract and cholesterol ranged from 0% (digitonin) to 17.9% (Triton X-100). The detergent-extractability profile for phosphatidylethanolamine (PE) (range 15-40% of total lipid) paralleled that of phosphatidylinositol (PI) (range 4-10%), but was inverse to that for GalCer and cholesterol. Detergent-extractability profiles for phosphatidylcholine (PC) and phosphatidylserine (PS) also followed inverse profiles, with zwitterionic detergents giving high PS/PC and high PE/PC ratios (approximately 2:1), whereas the Tritons and digitonin gave ratios of 1:2. We believe that differential solubilization of lipids, as well as proteins, by detergents is important for the biological activity of the extracted proteins, and lipid extractability should be taken into account when purifying membrane proteins.
膜蛋白通常用浓度为0.5 - 2.0%的去污剂提取，去污剂与蛋白质的比例为1:1至3:1。我们比较了来自七个不同结构和离子类别的14种不同去污剂在浓度为2.0%且去污剂与蛋白质比例为2:1时，提取活性形式的整合膜蛋白（血清素5 - HT1A受体）的能力，并观察到脂质和蛋白质方面都存在显著差异。在进行[3H]8 - 羟基 - 2 -（N，N - 二正丙基氨基）四氢萘（[3H]8 - OH - DPAT）结合之前，所有提取物都去除了去污剂并进行透析，以形成含有95 - 100%提取脂质的囊泡。提取活性5 - HT1A受体蛋白最有效的去污剂是两性离子型的3 - [(3 - 胆酰胺丙基)二甲基铵基]-1 - 丙烷磺酸盐（CHAPS）和3 - [(胆酰胺丙基)二甲基铵基]-2 - 羟基 - 1 - 丙烷磺酸盐（CHAPSO），其次是中性的正十二烷基 - β - D - 麦芽糖苷。两性离子去污剂还产生了最高的溶解脂质/蛋白质比例（分别为3.0和2.5），并且一般来说，提取的脂质和蛋白质的相对量呈现相反的分布。因此，诸如Tritons（临界胶束浓度与CHAPS相似）和Thesit（结构与Lubrol相似）等疏水性去污剂提取的蛋白质最多，但脂质相对较少（比例小于0.2），且活性5 - HT受体极少。在相同浓度的不同去污剂提取并通过高效薄层色谱分离的单个脂质比例方面也观察到了显著差异。例如，半乳糖神经酰胺（GalCer）含量占总脂质提取物的比例从2.7%（CHAPSO）到13.4%（胆酸钠）不等，胆固醇含量从0%（洋地黄皂苷）到17.9%（Triton X - 100）不等。磷脂酰乙醇胺（PE）（占总脂质的15 - 40%）的去污剂提取谱与磷脂酰肌醇（PI）（占总脂质的4 - 10%）相似，但与GalCer和胆固醇的提取谱相反。磷脂酰胆碱（PC）和磷脂酰丝氨酸（PS）的去污剂提取谱也呈现相反的分布，两性离子去污剂给出高的PS/PC和高的PE/PC比例（约为2:1），而Tritons和洋地黄皂苷给出的比例为1:2。我们认为，去污剂对脂质以及蛋白质的差异增溶对于提取蛋白质的生物活性很重要，并且在纯化膜蛋白时应考虑脂质的提取能力。
