FRACTIONATION OF THE GENETIC-VARIANTS OF HUMAN ALPHA(1)-ACID GLYCOPROTEIN IN THE NATIVE FORM BY CHROMATOGRAPHY ON AN IMMOBILIZED COPPER(II) AFFINITY ADSORBENT - HETEROGENEITY OF THE SEPARATE VARIANTS BY ISOELECTROFOCUSING AND BY CONCANAVALIN-A AFFINITY-CHROMATOGRAPHY
FRACTIONATION OF THE GENETIC-VARIANTS OF HUMAN ALPHA(1)-ACID GLYCOPROTEIN IN THE NATIVE FORM BY CHROMATOGRAPHY ON AN IMMOBILIZED COPPER(II) AFFINITY ADSORBENT - HETEROGENEITY OF THE SEPARATE VARIANTS BY ISOELECTROFOCUSING AND BY CONCANAVALIN-A AFFINITY-CHROMATOGRAPHY
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DOI：
10.1016/0378-4347(93)80289-g
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发表时间：
1993-05-19
期刊：
影响因子：
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通讯作者：
TILLEMENT, JP
中科院分区：
文献类型：
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作者：
HERVE, F;GOMAS, E;TILLEMENT, JP
研究了通过在固定化铜（II）亲和吸附剂上进行色谱法分离人α 1-酸性糖蛋白（AAG）的三种主要遗传变体（F1、S和A）的天然（唾液酸化）形式。该色谱法先前已开发用于分离去唾液酸化蛋白变体。为此，使用了三种主要的AAG表型样品（F1 S/A、F1/A和S/A）（先前已从个体人血浆样品中分离）和一种来自商业来源的AAG样品（表型混合物）。这些不同的样品在亚氨基二乙酸琼脂糖-铜（II）凝胶在pH 7的亲和色谱解析两个蛋白质峰，无论使用的天然AAG样品的来源。发现未结合峰1由F1、S或两种变体组成，具体取决于色谱法中使用的AAG样品的表型。发现结合峰2由纯形式的A变体组成。发现用天然AAG获得的分级分离结果与最初由去唾液酸化蛋白质产生的分级分离结果相同。然而，本机和去唾液酸化AAG与固定化铜（II）离子的相互作用的比较，分别使用亲和色谱法和非色谱平衡结合技术，表明去唾液酸化增加了与固定化铜（II）离子的蛋白质的非特异性相互作用。当使用固定化的锌、镍或钴（II）离子代替铜进行亲和层析时，AAG变体未被分级分离。在纯化唾液酸化形式的每种变体（F1、S和A）后，在pH 2.5-4.5范围内，通过使用载体两性电解质的分析等电聚焦研究它们各自的异质性。此外，凝集素结合行为的单独的唾液酸化AAG变体进行了研究，通过亲和色谱固定刀豆球蛋白A。
Fractionation of the three main genetic variants (F1, S and A) of human alpha1-acid glycoprotein (AAG), in their native (sialylated) form, by chromatography on immobilized copper(II) affinity adsorbent was investigated. This chromatographic method had been previously developed to fractionate the desialylated protein variants. For that purpose, the three main AAG phenotypes samples (F1S/A, F1/A and S/A), which had been previously isolated from individual human plasma samples, and an AAG sample from commercial source (a mixture of the phenotypes) were used in the native form. Affinity chromatography of these different samples on an iminodiacetate Sepharose-copper(II) gel at pH 7 resolved two protein peaks, irrespective of the origin of the native AAG sample used. The unbound peak 1 was found to consist of the F1, the S or both variants, depending on the phenotype of the AAG sample used in the chromatography. The bound peak 2 was found to consist of the A variant in a pure form. The fractionation results obtained with native AAG were found to be the same as those originally yielded by the desialylated protein. However, comparison of the interactions of native and desialylated AAG with immobilized copper(II) ions, using an affinity chromatographic method and a non-chromatographic equilibrium binding technique, respectively, showed that desialylation increased the non-specific interactions of the protein with immobilized copper(II) ions. The AAG variants were not fractionated when affinity chromatography was performed using immobilized zinc, nickel or cobalt(II) ions, instead of copper. After purification of each variant in the sialylated form (F1, S and A), their respective heterogeneity was studied by analytical isoelectrofocusing with carrier ampholytes in the pH range 2.5-4.5. In addition, the lectin-binding behaviour of the separate sialylated AAG variants was investigated by affinity chromatography on immobilized concanavalin A.