Choe J, VanderNoot V A, Linhardt R J, Dordick J S
Department of Chemical and Biochemical Engineering, College of Pharmacy, University of Iowa, Iowa City 52242, USA.
Biotechnol Prog. 1997 Jul-Aug;13(4):440-5. doi: 10.1021/bp970049h.
Affinity-based reversed micellar extraction and separation (ARMES) is an effective method for purifying both low and high molecular weight glycoproteins via liquid-liquid extraction. A range of extraction conditions were examined to gain insight into the mechanism of ARMES. Concanavalin A (Con A) was used as the model affinity ligand to bind soybean peroxidase (SBP) and beta-galactosidase as model glycoproteins. Factorial design was used to investigate the effect of various system variables on the extraction of SBP via ARMES. A quadratic model described the systems well, resulting in a standard deviation of 7% between calculated and experimental extraction efficiencies. Sensitivity analysis suggested that the key criteria in ARMES were the NaCl concentration and pH of the aqueous feed phase. Extraction of both glycoproteins decreased above pH 7 but fell to zero only at pH values significantly above the pI of the model glycoproteins and the Con A affinity ligand. It is proposed that the complex of the affinity lectin with the glycoprotein results in a sufficiently hydrophobic species that can be extracted into a reversed micellar organic phase even at pH's far above the pI's of the individual proteins that comprise the complex. This finding has practical considerations for the use of ARMES in the resolution and purification of protein glycoforms.
基于亲和作用的反胶束萃取与分离（ARMES）是一种通过液-液萃取纯化低分子量和高分子量糖蛋白的有效方法。研究了一系列萃取条件以深入了解ARMES的机制。以伴刀豆球蛋白A（Con A）作为模型亲和配体，结合大豆过氧化物酶（SBP）和β-半乳糖苷酶作为模型糖蛋白。采用析因设计研究各种系统变量对通过ARMES萃取SBP的影响。二次模型能很好地描述这些系统，计算得到的萃取效率与实验萃取效率之间的标准差为7%。敏感性分析表明，ARMES中的关键标准是水相进料的NaCl浓度和pH值。两种糖蛋白在pH 7以上的萃取率均下降，但仅在显著高于模型糖蛋白和Con A亲和配体pI值的pH值下才降至零。有人提出，亲和凝集素与糖蛋白的复合物会形成一种疏水性足够强的物质，即使在远高于构成该复合物的单个蛋白质pI值的pH值下，也能被萃取到反胶束有机相中。这一发现对于在蛋白质糖型的分离和纯化中使用ARMES具有实际意义。
