Merck KGaA, Frankfurter Str. 250, D-64293 Darmstadt, Germany.
J Chromatogr A. 2013 Apr 12;1285:48-56. doi: 10.1016/j.chroma.2013.01.104. Epub 2013 Feb 1.
In protein chromatography, the size of the protein determines which fraction of pores it can access within a resin and at which rate of diffusion. Moreover, in the presence of grafted polymers like in advanced materials, adsorbed proteins and electrolytes complicate the interaction pore-protein. In this study, we evaluated in a comparative way the behavior of Fractogel EMD SO3 (M) and (S), "tentacle"-type, strong cation exchangers, as well as a reference material without tentacles, all of which are commonly used for protein purification. ISEC experiments were carried out with a set of Dextran tracers of largely different molecular size covering the typical range of protein sizes. Experimental values of porosity (internal and external to the particles) as well as of pore diffusion coefficients have been measured at different NaCl concentrations and under protein loading. These results provide useful insights into the complex interplay among mentioned factors: first, the presence of tentacles induces size exclusion selectivity in the materials; second, the salt induces conformational changes of the tentacles, leading to porosities larger than expected in tentacle materials; third, protein adsorption mainly leads to a reduction of porosity due to pore space occupied by the protein and to a decrease of pore diffusion coefficient.
在蛋白质层析中，蛋白质的大小决定了它可以进入树脂中哪个部分的孔径以及扩散的速度。此外，在存在接枝聚合物的情况下，如在先进材料中，吸附的蛋白质和电解质会使孔-蛋白质相互作用变得复杂。在这项研究中，我们以比较的方式评估了 Fractogel EMD SO3（M）和（S）这两种“触手”型强阳离子交换剂以及一种无触手的参考材料的行为，它们都是常用于蛋白质纯化的。使用一组大小差异很大的葡聚糖示踪剂进行 ISEC 实验，这些示踪剂涵盖了典型的蛋白质大小范围。在不同的 NaCl 浓度和蛋白质负载下，测量了内部和外部颗粒的孔隙率以及孔扩散系数的实验值。这些结果为理解上述因素之间的复杂相互作用提供了有用的见解：首先，触手的存在会在材料中诱导尺寸排除选择性；其次，盐会引起触手的构象变化，导致触手材料的孔隙率大于预期；第三，蛋白质吸附主要会导致孔隙率降低，这是由于蛋白质占据了孔隙空间，以及孔扩散系数的降低。
