van Roosmalen Dick, Lazzara Matthew J, van den Broeke Leo J P, Keurentjes Jos T F, Blankschtein Daniel
Department of Chemical Engineering, Room 66-444, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge 02139, USA.
Biotechnol Bioeng. 2004 Sep 20;87(6):695-703. doi: 10.1002/bit.20172.
The separation and purification of biomolecules in aqueous media driven by excluded-volume interactions is a well-established concept. In this article we propose a new separations method, based on excluded-volume principles, consisting of an aqueous micellar-gel system (AMGS). Specifically, an outer aqueous phase containing cylindrically shaped micelles of the nonionic surfactant n-decyl tetra (ethylene oxide) (C10E4) is physically separated from an inner aqueous phase defined by the interior volume of gel beads, from which the micelles are completely excluded because of their shape and size. In the AMGS, the concentration of the micelles outside the gel beads is sufficiently high that the volume excluded to a biomolecule in the solution external to the gel beads is much larger than that within the gel beads. Accordingly, when biomolecules are introduced into the AMGS, they partition preferentially into the gel-bead phase, according to their sizes, as a result of the greater effect of the excluded-volume interactions with the C10E4 micelles present in the aqueous phase outside the gel beads. The new AMGS is more versatile and adaptable than the conventional two-phase aqueous C10E4 micellar system because the micelle volume fraction is independent of the temperature and because the effects of entrainment are eliminated. After demonstrating the experimental feasibility of creating the new AMGS, the three proteins myoglobin, ovalbumin, and BSA-FITC, and the enzyme G6PD, were partitioned in the AMGS and their partitioning behavior was found to follow the experimental excluded-volume trends dictated by the interactions of the biomolecules with the C10E4 micelles. Specifically, the measured partition coefficients of the four biomolecules into the micellar phase were found to be less than unity and to decrease with increasing biomolecule size. A theoretical description of the partitioning behavior of the biomolecules in the new AMGS was formulated, based on excluded-volume considerations, and the predicted biomolecule partition coefficients were found to compare favorably with those measured for the four biomolecules studied.
由体积排阻相互作用驱动的水相介质中生物分子的分离和纯化是一个已确立的概念。在本文中，我们基于体积排阻原理提出了一种新的分离方法，该方法由水相胶束 - 凝胶系统（AMGS）组成。具体而言，含有非离子表面活性剂正癸基四（环氧乙烷）（C10E4）圆柱形胶束的外部水相与由凝胶珠内部体积定义的内部水相物理分离，由于其形状和大小，胶束被完全排除在凝胶珠内部水相之外。在AMGS中，凝胶珠外部的胶束浓度足够高，以至于溶液中生物分子在凝胶珠外部被排除的体积远大于在凝胶珠内部的体积。因此，当生物分子被引入AMGS时，由于与凝胶珠外部水相中存在的C10E4胶束的体积排阻相互作用的影响更大，它们会根据大小优先分配到凝胶珠相中。新的AMGS比传统的两相水相C10E4胶束系统更通用且适应性更强，因为胶束体积分数与温度无关，并且消除了夹带效应。在证明创建新的AMGS的实验可行性之后，将肌红蛋白、卵清蛋白和BSA - FITC这三种蛋白质以及酶G6PD在AMGS中进行分配，发现它们的分配行为遵循由生物分子与C10E4胶束相互作用所决定的实验体积排阻趋势。具体而言，发现这四种生物分子进入胶束相的实测分配系数小于1，并且随着生物分子大小的增加而降低。基于体积排阻考虑，对生物分子在新的AMGS中的分配行为进行了理论描述，发现预测的生物分子分配系数与所研究的四种生物分子的实测值相比具有良好的一致性。
