Protein binding chiral discrimination of HPLC stationary phases made with whole, fragmented, and third domain turkey ovomucoid.
Protein binding chiral discrimination of HPLC stationary phases made with whole, fragmented, and third domain turkey ovomucoid.
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用完整、片段和第三域火鸡卵类粘蛋白制成的 HPLC 固定相的蛋白质结合手性辨别。
DOI：
10.1021/ac00110a006
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发表时间：
1995
影响因子：
7.4
通讯作者：
Markley,JL
中科院分区：
文献类型：
--
作者：
Pinkerton,TC;Howe,WJ;Ulrich,EL;Comiskey,JP;Haginaka,J;Murashima,T;Walkenhorst,WF;Westler,WM;Markley,JL
火鸡卵类粘蛋白经酶解和化学裂解后，用分子排阻色谱和离子交换色谱分离纯化，得到单个蛋白结构域和两个组合结构域。硅胶键合相HPLC柱由火鸡卵类粘蛋白的完整或分离的结构域制成。蛋白质柱通过其在宽范围的外消旋体中拆分对映体的能力来测试手性识别。从整个火鸡卵粘蛋白制成的列显示对许多外消旋体的手性活性，其中作为第一和第二结构域的组合，仅分辨选定数量的芳香族弱碱。第一和第二结构域独立地没有明显的手性活性。火鸡卵类粘蛋白第三结构域对稠环芳香族弱酸表现出对映选择性蛋白质结合。第三个结构域的糖基化不影响手性识别。用模型化合物滴定第三结构域，结合NMR测量，能够鉴定负责结合的氨基酸。配体-蛋白质复合物的分子模拟提供了对蛋白质表面基于多种分子间相互作用区分对映体的能力的深入了解。在过去的十年中，许多HPLC手性固定相已经被开发出来，每个相采用其独特的分离策略来分离对映体。12由于手性识别特性是高度特异性的，已经设计了大量的对映选择性相来分离广泛的手性化合物。这些相通过配体交换络合作用产生手性拆分。T-供体/
Individual protein domainsand two domains in combina-tion were prepared by enzymatic and chemical cleavage of turkey ovomucoid followed by isolation and purification by size-exclusion and ion-exchange chromatography. Silica bonded-phase HPLC columns were made from either whole or isolated domains of turkey ovomucoid. The protein columns were tested for chiral recognition by their abilities to resolve enantiomers among a wide range of racemates. The columns made from whole turkey ovo-mucoid displayed chiral activity toward many racemates, where as a combination of the first and second domain resolved only a selected number of aromatic weak bases. The first and second domains independently gave no appreciable chiral activity. The turkey ovomucoid third domain exhibited enantioselective protein binding for fused-ring aromatic weak acids. Gtycosylation of the third domain did not affect chiral recognition. Titration of the third domain with model compounds in conjunction with NMR measurements enabled the identification of the amino acids responsible for binding. Molecular modeling of the ligand-protein complexation provided insights into the ability of a protein surfaceto discriminate enantiomers on the basis of multiple intermolecular interactions.Over the past decade, many HPLC chiral stationary phases have been developed to separate enantiomers with each phase employing its own unique separation strategy. 12 Since chiral recognition properties are highly specific, a large number of enantioselective phases have been designed to separate an extensive array of chiral compounds. These phases generate chiral resolution by ligand-exchange complexation,. T-donor/