Hervé F, Duché J C, Barré J, Millot M C, Tillement J P
Laboratoire Hospitalo-Universitaire de Pharmacologie de Paris XII, Centre Hospitalier Intercommunal de Créteil, France.
J Chromatogr. 1992 May 20;577(1):43-59. doi: 10.1016/0378-4347(92)80597-j.
Using a two-dimensional isoelectrofocusing (IEF)-electrophoresis technique, the pH titration curves of the three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG) were studied to assist in the development of a fractionation method for the AAG variants. For this purpose, different AAG samples, each corresponding to one of the three main phenotypes of the protein (F1S/A, F1/A and S/A), were first purified by chromatographic separation of individual human plasma samples on immobilized Cibacron Blue F3G-A. The purified AAG samples were then disialylated and their heterogeneity was checked by analytical IEF. The pH-mobility curves of the desialylated AAG samples were displayed in polyacrylamide gel slabs, under a constant set of experimental conditions, by carrying out electrophoresis of the protein samples perpendicularly to two stationary pH gradients: a large gradient (pH 3.5-9.5) and a narrow gradient (pH 5-8). The curves showed that all the desialylated variants of AAG exhibited small charge differences and moved closely together between about pH 3.5-5.5 and pH 7.5-9.5. However, the variants were found to show microheterogeneity in their total charge between about pH 5.5 and 7.5 due to the titrated ionizable groups involved along this pH zone. This microheterogeneity was assumed to be accounted for by the existence of differences between the titratable histidyl residues of the AAG variants. Consequently, the interactions of the variants with immobilized transition metal ions were studied at pH 7, using affinity chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the A variant was strongly bound by immobilized Cu(II) ions, whereas the F1 and S variants interacted non-specifically with the immobilized ligand. This finding allowed the development of a rapid and effective fractionation method for desialylated AAG into its A and F1 or S variants, depending on the AAG phenotype, by chromatography on an immobilized affinity Cu(II) adsorbent. The quantitative relationships between immobilized Cu(II) ions and desialylated AAG (the apparent association constant and gel protein-binding capacity) were also determined using a non-chromatographic equilibrium binding technique.
采用二维等电聚焦（IEF）-电泳技术，研究了人α1-酸性糖蛋白（AAG）的三种主要去唾液酸化变体（F1、S和A）的pH滴定曲线，以辅助开发一种针对AAG变体的分离方法。为此，首先通过在固定化的汽巴蓝F3G-A上对个体人血浆样品进行色谱分离，纯化了不同的AAG样品，每个样品对应于该蛋白的三种主要表型（F1S/A、F1/A和S/A）之一。然后将纯化的AAG样品去唾液酸化，并通过分析IEF检查其异质性。在一组恒定的实验条件下，通过在垂直于两个固定pH梯度（一个大梯度（pH 3.5 - 9.5）和一个窄梯度（pH 5 - 8））的方向上对蛋白质样品进行电泳，在聚丙烯酰胺凝胶板中显示去唾液酸化AAG样品的pH-迁移率曲线。曲线表明，AAG的所有去唾液酸化变体都表现出较小的电荷差异，并且在约pH 3.5 - 5.5和pH 7.5 - 9.5之间紧密移动。然而，由于在该pH区域涉及滴定的可电离基团，发现这些变体在约pH 5.5和7.5之间的总电荷上表现出微异质性。这种微异质性被认为是由于AAG变体的可滴定组氨酸残基之间存在差异所致。因此，在pH 7下，使用亚氨基二乙酸琼脂糖-Cu（II）凝胶上的亲和色谱法研究了变体与固定化过渡金属离子的相互作用。结果发现，A变体被固定化的Cu（II）离子强烈结合，而F1和S变体与固定化配体非特异性相互作用。这一发现使得开发出一种快速有效的方法，可根据AAG表型，通过在固定化亲和Cu（II）吸附剂上进行色谱分离，将去唾液酸化的AAG分离为其A和F1或S变体。还使用非色谱平衡结合技术确定了固定化Cu（II）离子与去唾液酸化AAG之间的定量关系（表观缔合常数和凝胶蛋白结合容量）。
