Herve F, Millot M C, Eap C B, Duche J C, Tillement J P
Service Hospitalo-Universitaire de Pharmcologie of Paris XII, Cedex, France.
J Chromatogr B Biomed Appl. 1996 Mar 29;678(1):1-14. doi: 10.1016/0378-4347(95)00366-5.
Alpha1-Acid glycoprotein (AAG) or orosomucoid was purified to homogeneity from human plasma by a separate two-step method using chromatography on immobilized Cibacron Blue F3G-A to cross-linked agarose and chromatography on hydroxyapatite. The conditions for the pre-purification of AAG by chromatography on immobilized Cibacron Blue F3G-A were first optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 40 ml sample of human plasma. This method was applied to the purification of AAG samples corresponding to the three main phenotypes of the protein (FIS/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampholytes showed that the microheterogeneity of the purified F1S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding plasma, thus suggesting that no apparent desialylation of the glycoprotein occurred during the purification steps. This method was also applied to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/AAD, S/AX0 and F1/A*C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded by the second gene of AAG (i.e. the A, AD, X0 and C1 variants) strongly bound to immobilized Cu(II) ions. These results suggested that chromatography on an immobilized affinity Cu(II) adsorbent could be helpful to distinguish between the respective products of the two highly polymorphic genes which code for human AAG.
通过一种单独的两步法从人血浆中纯化出了均一的α1-酸性糖蛋白（AAG）或血清类粘蛋白，该方法包括使用固定化的汽巴蓝F3G-A交联琼脂糖进行色谱分离以及羟基磷灰石色谱分离。首先使用不同pH值的不同缓冲系统优化了通过固定化汽巴蓝F3G-A色谱对AAG进行预纯化的条件。这些组合技术的总产率为80%，并且从约40ml人血浆样品中最初约15mg的总量中纯化出了约12mg的AAG。该方法应用于从先前已对AAG进行表型分析的个体人血浆中纯化与该蛋白的三种主要表型（FIS/A、F1/A和S/A）相对应的AAG样品。一项使用载体两性电解质进行等电聚焦的研究表明，纯化后的F1S/A、F1/A和S/A AAG样品的微不均一性与相应血浆中AAG的微不均一性相似，因此表明在纯化步骤中该糖蛋白没有明显的去唾液酸化现象。该方法还应用于纯化与该蛋白的罕见表型（F1/AAD、S/AX0和F1/A*C1）相对应的AAG样品，然后通过在亚氨基二乙酸琼脂糖-Cu(II)凝胶上进行色谱分析，在pH 7条件下研究了这些变体与固定化铜(II)离子的相互作用。结果发现，人类中编码AAG的两个基因中的第一个基因所编码的不同变体（即F1和S变体）与固定化配体非特异性相互作用，而AAG第二个基因所编码的那些变体（即A、AD、X0和C1变体）则与固定化Cu(II)离子强烈结合。这些结果表明，在固定化亲和Cu(II)吸附剂上进行色谱分析有助于区分编码人AAG的两个高度多态性基因的各自产物。
