Chiral recognition based on a protein-domain-bonded column

用2-芳基丙酸衍生物U-80,413滴定火鸡和鸡类卵粘蛋白的第三结构域(分别为OMTKY3和OMCHI3)，结合核磁共振测量，能够识别负责结合的氨基酸。配体-蛋白质络合的分子模拟提供了对蛋白质表面基于多个分子间相互作用区分U-80,413对映体的能力的见解。我们发现U-80,413的R-和S-对映体在取向和分子间相互作用方面有异同之处。每个对映体的羧基与Arg 21上的正电荷发生静电相互作用。U-80,413‘S中心环上的羰基与赖氨酸34的NH_3~+基团形成氢键。不同对映体的区别在于R-对映体的苯基与Phe 53的接近。用阴离子交换层析和阳离子交换层析对市售鸡卵粘蛋白(OMCHI)和用有机溶剂沉淀从粗蛋清中分离的OMCHI进行了分离。用反相色谱、N-末端测序、基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱仪、糖含量测定和胰酶抑制活性等方法对得到的四个组分进行了表征。其中三个组分是糖类组成不同的OMCHI变种，特别是唾液酸含量不同，另一个组分暂定为卵糖蛋白(OGCHI)。将这些OMCHI变异体和OGCHI与氨基丙基硅胶结合，以评价手性识别能力。然而，两种OMCHI变体都没有表现出明显的手性识别能力，而OGCHI具有比先前报道的OMCHI更优异的手性识别性能。这表明以前报道的OMCHI的手性识别能力来自于OGCHI，OGCHI以杂质的形式存在于OMCHI粗品中。

Titration of the third domain of turkey and chicken ovomucoid (OMTKY3 and OMCHI3, respectively) with U-80,413, a 2-arylpropionic acid derivative, 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 ability of a protein surface to discriminate the U-80,413 enantiomers on the basis of multiple intermolecular interactions. We found that similarities and differences in orientation and intermolecular interactions between the R- and S-enantiomers of U-80,413. The carboxyl groups of each enantiomer engage in electrostatic interactions with positive charge on Arg 21. The carbonyl group on U-80,413's central ring shares a hydrogen bond with NH_3^+ group of Lys 34. The distinguishing difference between enantiomers is the proximity of the phenylgroup of R-enantiomer and Phe 53.Commercial chicken ovomucoid (OMCHI) and isolated OMCHI by precipitation with organic solvents from egg whites, which are crude, were fractionated by anion- and cation-exchange chromatography. The obtained four fractions were characterized by reversed-phase chromatography, N-terminal sequencing, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, determination of sugar contents and trypsin-inhibitory activities. Three fractions were OMCHI variants differing in carbohydrate composition, especially in sialic acid content, and the other fraction was tentatively termed ovoglycoprotein (OGCHI). These OMCHI variants and OGCHI were bound to aminopropyl-silica gels to evaluate chiral recognition ability. However, neither OMCHI variant gave appreciable chiral recognition ability, while the OGCHI gave much more excellent chiral recognition properties than those of the OMCHI reported previously. This reveals that chiral recognition ability of the OMCHI reported previoulsy comes from the OGCHI,which is present in crude OMCHI as an impurity.