Ionic Liquid-Based Extraction And Purification Of Proteins

A novel liquid phase extraction technique based on ionic liquid is established for separation and purification of proteins. In order to avoid the waste of ionic liquid, solid phase extraction is developed by using immobilized ionic liquid as new adsorbent material. After the solid phase extraction of proteins with the immobilized ionic liquids, the adsorption of hemoglobin causes significant quenching of the fluorescence intensity. A solid surface fluorimetry was established for the direct determination of hemoglobin. Finally, the interaction of immidazolium ionic liquid with bovine serum albumin is investigated.A water-in-ionic liquid reverse microemulsion is prepared with water, sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and ionic liquid1-butyl-3-methylimidazolium hexafluorophosphate (BmimPFg). The microemulsion system has been proved to entail selective extraction of hemoglobin.96%of the hemoglobin in a500μL of aqueous solution (100μg ml/1, pH6.3) could be readily extracted by an equal volume of microemulsion (with an AOT concentration of50mmol L"1, and the molar ratio of6/50/5for water/AOT/BmimPF6).73%of the hemoglobin in the microemulsion system could afterwards be rapidly back extracted into an aqueous phase with a6mol L-1urea as stripping reagent. The hemoglobin in the microemulsion system might exist either in the bulk of the ionic liquid phase or in the "water pools" of the microemulsion. Coordination between imidazolium cation and heme group of hemoglobin facilitates the transfer of hemoglobin from aqueous phase into the ionic liquid continuous phase. In addition, electrostatic interaction is among the main driving forces for the transfer of hemoglobin into the "water pools" of microemulsion.Hydrophilic ionic liquid-polyvinyl chloride (PVC) hybrids were prepared by immobilizing N-methylimidazole (N-mim) to PVC chains in toluene. The NmimCl-PVC hybrids were characterized by FT-IR,1H NMR. The immobilization ratio, i.e., the percentage of chloride on PVC chain reacting with N-mim to form the hybrid, was improved from3.3% to15.1%by increasing the N-mim/PVC molar ratio. The most distinct feature of the hybrid is its excellent selectivity for adsorbing basic proteins by effective suppression of the non-specific protein adsorption by pure PVC, and a higher immobilization ratio facilitates better selectivity. In Tris-HCl buffer, basic proteins of100μg mL-1, i.e., lysozyme (Lys), cytochrome c (Cyt-c) and hemoglobin (Hb), were favorably adsorbed with efficiencies of97%,98%and94%by the hybrid with an immobilization ratio of15.1%, while the adsorption of acidic proteins, i.e., bovine albumin serum (BSA), transferrin (Trf) and immuneglobulin G (IgG) were negligible. The retained Lys, cyt-c and Hb could be readily recovered by elution with phosphate buffer, carbonate buffer and SDS solution with efficiencies of89%,87%and84%, respectively. Another feature of the hybrid is the significant improvement on the biocompatibility characterized by the maintenance of the activity of hemoglobin after adsorption and elution process. The practical usefulness of the hybrid was demonstrated by selective isolation of hemoglobin from human whole blood.The fluorescence behavior of the immobilized ionic liquids NmimCl-PVC was investigated. A red shift for both the excitation and fluorescence maximum was observed in comparison with pure ionic liquid attributing to the confinement of ionic liquid by PVC chain. The fluorescence maximum shifts with excitation wavelength due to the existence of the various associated structures of the ionic liquids. The fluorescence intensity of NmimCl-PVC increases with the grafting ratio of ionic liquid. In addition, adsorption of hemoglobin on NmimC1-PVC causes significant quenching of the fluorescence intensity of NmimCl-PVC. It concludes dynamic quenching interaction and energy transfer quenching between NmimC1-PVC and hemoglobin. A solid surface fluorimetry for the direct determination of hemoglobin adsorbed on the surface of NmimCl-PVC was thus established. The linear equation is F/F0=-0.0213C+0.989(R2=0.9948) and a linear dynamic range for the determination of hemoglobin within0.3-26.2μg mg-1is obtained. The detection limit is0.1ug mg-1with a relative standard deviation of2.6%at7.2μg mg-1of hemoglobin.The interactions of bovine serum albumin (BSA) with three imidazolium ionic liquids, e.g.,1,3-dibutylimidazolium chloride,1-butyl-3-methylimidazolium chloride as well as1-butyl-3-methylimidazolium nitrate, have been studied by monitoring the spectral behaviors of the systems. The UV-vis spectral differences arise from the disturbances of the micro-environment of the polypeptide of the protein. The observed fluorescence quenching of BSA by imidazolium ionic liquid at λex=230nm is either due to dynamic quenching (BbimCl, BmimCl) or a combination of dynamic and static quenching process (BmimNO3) with a quenching constant at the level of102L mol-1. The order of the quenching effect of IL is BbimCl≈BmimCl<BmimNO3. Thermodynamics reveal that the interaction is entropy driven with predominantly hydrophobic forces, as well as electrostatic interaction. The conformational changes of BSA due to the interaction were investigated qualitatively with synchronous fluorescence spectra along with CD spectra, and an unfolding of polypeptides was observed. Further, molecular docking studies revealed that imidazolium ionic liquids were bound at domain Ⅲ with hydrophobic interactions and also by electrostatic interactions and the ILs are far away from the Trp212residue of BSA located in the subdomain IIA.