Shen Ching-Wei, Yu Tiing
Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30050, Taiwan.
J Chromatogr A. 2007 Jun 1;1151(1-2):164-8. doi: 10.1016/j.chroma.2007.01.079. Epub 2007 Jan 27.
A protein mixture consisting of myoglobin, cytochrome c, and lysozyme was separated by high-speed counter-current chromatography using a two-phase aqueous/reverse micelle-containing organic solvent system. About 50% stationary phase retention ratio was obtained in most chromatographic experiments. Separations were manipulated mainly by pH gradients that controlled the electrostatic interactions between the protein molecules and reverse micelles. Separations were further improved by incorporating an ionic strength gradient along with the pH gradient. Control of ionic strength in the aqueous solution helped fine-tune protein partitioning between the stationary and mobile phases. Although non-specific protein interactions affected baseline resolution, recovery of cytochrome c and lysozyme reached 90% and 82%. Furthermore, concentration or enrichment of these two proteins was achieved from a large-volume sample load. This technique can potentially be employed in the recovery and enrichment of proteins from large-volume aqueous solutions.
使用含反胶束的水相/有机溶剂两相体系，通过高速逆流色谱法分离了由肌红蛋白、细胞色素c和溶菌酶组成的蛋白质混合物。在大多数色谱实验中，获得了约50%的固定相保留率。分离主要通过控制蛋白质分子与反胶束之间静电相互作用的pH梯度来进行。通过引入离子强度梯度以及pH梯度，进一步改善了分离效果。控制水溶液中的离子强度有助于微调蛋白质在固定相和流动相之间的分配。尽管非特异性蛋白质相互作用影响基线分辨率，但细胞色素c和溶菌酶的回收率分别达到了90%和82%。此外，通过大量进样实现了这两种蛋白质的浓缩或富集。该技术有望用于从大量水溶液中回收和富集蛋白质。
