利用反微胞相在高速逆向流層析儀上萃取蛋白質

Abstract

當界面活性劑加入非極性的有機溶劑中，會以親水基朝內，親油基朝外的方式聚集，形成反微胞的形式，反微胞的功能是可將極性分子藉由反微胞中的水核而溶於非極性的溶液之中。反微胞萃取法的過程分為兩個步驟：正向萃取和反向萃取，正向萃取是將水溶液中之蛋白質分子萃取至含有界面活性劑的有機溶液中，反向萃取則是將溶於有機溶液中的蛋白質分子再萃取至另一水溶液中。 本實驗是將含有陰離子型界面活性劑sodium bis(2-ethylhexyl) sulfosuccinate的正己烷溶液(或異辛烷溶液)與含有KCl的水溶液混合，以上層有機相為靜相，下層水相為動相；以高速逆向流層析儀(high speed countercurrent chtomatography)當作一高效率之萃取裝置。當含有cytochrome c蛋白質分子的水溶液流經管柱中的靜相時，蛋白質分子會被萃取至反微胞相中而滯留在分離管柱中；待正向萃取完成後，注入高pH值的鹼液，藉由動相的帶動改變反微胞相的環境，使蛋白質分子轉移至此一水溶液中，完成反向萃取，並在過程中達成濃縮蛋白質分子。例如，含有6.05 mg cytochrome c 700 mL之水溶液樣品可被濃縮成含有4.97 mg 40 mL之水溶液，回收率為82%。

As surfactants are added into non-polar organic solvent, they will aggregate to form reverse micelles. Reverse micelles provide micro water pools that enable solubilization of polar molecules in non-polar solutions. Extraction using reverse micelles includes two steps, i.e. forward and backward extractions. Forward extraction is a process that protein molecules in aqueous solution are extracted to surfactant-containing organic solution, and backward extraction is a process that protein molecules in reverse micelle phase are extracted into another aqueous solution. The extraction solvent was prepared by thouroughly mixing sodium bis(2-ethylhexyl)sulfosuccinate in n-hexane (or isooctane) solution with potassium chloride aqueous solution. The upper organic phase thus obtained was used as the stationary phase in a countercurrent chromatography apparatus; while the lower aqueous phase as the mobile phase. Sample solutions were prepared by dissolving cytochrome c in the mobile phase. The forward extraction was operated by delivering the sample solution through the CCC apparatus. The protein molecules were dissolved in the surfactant-containing organic phase, i.e. the stationary phase. A small volume of base was then injected into the column to extract the proteins back to the aqueous mobile phase and collected at the outlet. During the forward and backward extractions, the dilute protein solution was effectively concentrated. For example, a 40 mL of 124.23 mg/L cytochrome c solution could be obtained from a sample of 700 mL of 8.64 mg/L cytochrome c solution.