Magic-angle spinning and solution 13C nuclear magnetic resonance studies of medium- and long-chain cholesteryl esters in model bilayers.
Magic-angle spinning and solution 13C nuclear magnetic resonance studies of medium- and long-chain cholesteryl esters in model bilayers.
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模型双层中中链和长链胆固醇酯的魔角旋转和溶液 13C 核磁共振研究。
DOI：
10.1021/bi00049a021
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Hamilton,JA
中科院分区：
文献类型：
--
作者：
Salmon,A;Hamilton,JA
材料和方法化学品。富集13 C的CE可从先前的合成中获得（Sripada，1988），或使用相同的程序从相应的富集1 - 13 C的脂肪酸合成（剑桥同位素实验室，Andover，MA）。硬脂酸和棕榈酸是90原子%的13 C;油酸和辛酸是99原子%的13 C。用二环己基碳二亚胺（Aldrich，密尔沃基，WI）将脂肪酸转化成其酸酐，并在4-吡咯烷基吡啶（Sigma，St. Louis，MO）存在下，在无水氯仿中将酸酐与胆固醇（NuChek Prep，Elysian，MN）缩合。通过在1/1（v/v）乙醚/己烷中展开的薄层色谱法（TLC），随后用含有10%硫酸铜的8%磷酸水溶液炭化，并通过13 C NMR光谱与真实的未标记CE（NuCheck Prep，Elysian，MN）的比较来检查纯度。囊泡和多层膜。通过将氯仿或环己烷中的CE与氯仿中的40-200 mg卵PC（Avanti Polar Lipids，雪花石膏，AL）混合，并在具有氮气流的大Pyrex离心管中将混合物干燥成均匀的薄膜来制备NMR样品。在高真空（< 50毫托）下使干燥混合物不含残余溶剂，并在氩气下封盖。将密封的样品在高于CE熔点的油浴中加热1-2 min，并用0.5 mL pH 7.4磷酸盐缓冲液进行超声处理，或用0.25 mL氧化氘（Wilmad，Buena，NJ）或双蒸水进行MAS样品水合。
MATERIALS AND METHODSChemicals. 13C-enriched CE were available from previous syntheses (Sripada, 1988) or were synthesized using the same procedure from the corresponding l-13C-enrichedfatty acid (Cambridge Isotope Laboratories, Andover, MA). Stearic and palmitic acids were 90 atom% 13C; and oleic and octanoic acids were 99 atom% 13C. The fatty acid was converted to its anhydride with dicyclohexylcarbodiimide (Aldrich, Milwaukee, WI) and the anhydride was condensed with cholesterol (NuChek Prep, Elysian, MN) indry chloroform in the presence of 4-pyrrolidinopyridine (Sigma, St. Louis, MO). Purity was checked by thin-layer chromatography (TLC) developed in 1/1 (v/v) diethyl ether/hexane followed by charring with 8% aqueous phosphoric acid containing 10% copper sulfate and by comparison of l3C NMR spectra with authentic unlabeled CE (NuCheck Prep, Elysian, MN). Vesicles and Multilayers. NMR samples were prepared by mixing the CE in chloroform or cyclohexane with 40—200 mg of egg PC (Avanti Polar Lipids, Alabaster, AL) in chloroform and drying the mixture to an even thin film in a large Pyrex centrifuge tube with a stream of nitrogen. The dry mixture was freed from residual solvent under high vacuum(< 50 mTorr) and capped under argon. The sealed sample was heated in an oil bath above the melting point of the CE for 1—2 min and hydrated either with 0.5 mL of pH 7.4 phosphate buffer for sonication or with 0.25 mL of deuterium oxide (Wilmad, Buena, NJ) or double-distilled water for MAS samples.