應用親和性染料剛果紅於離子液體中萃取蛋白質

Abstract

室溫離子液體(Room Temperature Ionic Liquids, ILs) 因具有不揮發性、低毒性、高熱穩定性、良好導電度、可回收與重複使用等特殊的物理與化學性質，近年來已引起廣泛的研究，並用於取代傳統會揮發的有機溶劑，以達到安全與環保的目的。 本實驗嘗試以具親和性的染料剛果紅(Congo Red)將其應用於疏水性離子液體[Bmim][PF6]中，以液相/液相萃取的方式來萃取牛血清蛋白，並藉由銀離子交換法合成[Bmim]2[Congo Red] ，並將其成功溶於[Bmim][PF6]中，並以此系統對牛血清蛋白作萃取，在pH為4.0時正向萃取率可達近100%，並藉由調控pH值與鹽類濃度，得知靜電力也為親和性染料能吸引蛋白質的其中一項因素，選用較為溫和的pH值，在不破壞系統的穩定性之下，以pH9.0 並添加1.0M的NaSCN其蛋白質總回收率約為50%。 另外本實驗也嘗試萃取其他蛋白質，以近一步探討本系統對蛋白質間的作用力，在pH為7.0時，加入萃取劑[Bmim]2[Congo Red]對牛血清蛋白萃取則無萃取率，而在pH為4.0或7.0萃取核醣核酸□A時，萃取劑的加入與否對萃取率的影響並不顯著，最後在pH為4.0或7.0萃取溶菌□，結果顯示萃取劑的加入對蛋白質萃取率有顯著提升，綜合以上得知本系統與蛋白質間必須要同時有靜電力與親和力，才會有較好的萃取率。 由於染料配體數量眾多，本實驗期望為離子液體液相/液相萃取蛋白質技術提供一個新的選擇與發展。

Owing to the special physical and chemical properties, such as non-volatility, minimal toxicity, high thermal stability, good conductivity and recyclability, room temperature ionic liquids (RTILs) have attracted wide research interests. Replacement of traditional volatile organic solvents with RTILs may potentially achieve environmental safety and protection goals. In this study, we try to dissolve the affinity dye Congo Red in hydrophobic ionic liquid [Bmim][PF6] and apply this solution for liquid/liquid extractions of Bovine Serum Albumin (BSA) from aqueous solutions. During the extraction, Congo Red is easily dissolved into the aqueous solution. Thus this solution is inadequate for protein extraction. We then apply a simple ion exchange method to synthesis [Bmim]2[Congo Red] and dissolve it in [Bmim][PF6] to form aa extraction soluteion. The forward extraction efficiency (from the aqueous solution to the RTIL phase) reached almost 100 % while the aqueous solution was at pH 4.0. Protein molecules can be extracted back to another aqueous solution by manipulating the pH and the ionic strength. Under pH 9.0 and 1.0 M NaSCN, the back extraction reached 50%. In addition, other proteins are extracted using this solution in order to examine the selectivity of this affinity compound. Under pH 7.0, negligible extraction of BSA is found. Addition with [Bmim]2[Congo Red] to the IL does not significantly improve extraction of Ribonuclese A at either pH 4.0 or 7.0. As for lysozyme, addition of extractant [Bmim]2[Congo Red] helps promote extraction under pH 4.0 and 7.0. In addition to the affinity of the dye to proteins, the electrostatic force between the dye and the protein is found to influence the extraction.