Application Of Hexafluoroisopropanol-based New Deep Eutectic Solvents In Protein Purification And Analysis

The purification and analysis of protein have always attracted much attention.The development of efficient and green technique for protein purification and analysis is of great importance.Deep eutectic solvent?DES?,as a green solvent,owns many unique properties and is widely used in protein field.DES-based aqueous two-phase system?ATPS?is an environmentally friendly method for protein extraction.However,there is no effective back-extraction method to remove DES and achieve efficient protein purification.Capillary electrophoresis?CE?is a powerful method for protein analysis,and how to effectively inhibit protein wall adsorption is a key problem to be solved.Hexafluoroisopropanol?HFIP?is a perfluorinated alcohol with superior properties and can be used as hydrogen bond donor to form superior DES?HFIP-DES?with quaternary ammonium salts.In this paper,HFIP-DES based ATPS and CE dynamic coating technology were studied and applied to protein purification and analysis,respectively.The main work is as follows:?1?DESs were prepared with HFIP as hydrogen bond donor and different quaternary ammonium salts as hydrogen bond.ATPS consisting of HFIP-DES and salt was developed for the extraction of protein.Choline chloride?Ch Cl?-HFIP DES with strong phase separation capability and outstanding extraction ability was selected as extraction solvent while K₂HPO₄ was served as phase separation salt for the extraction process.A simple back-extraction method was proposed for the first time to free protein from DES-rich phase using water as solvent.Bovine serum albumin?BSA?as model protein,single factor experiments and response surface method were combined to optimize the extraction and back-extraction conditions.Under the optimal conditions,more than 99%of BSA was extracted into the DES-rich phase,the back-extraction efficiency was greater than 90%,and BSA product with a purity higher than 91%could be obtained through ultra-filtration and freeze-drying treatment.This method had also been successfully applied to purify BSA from a real sample of calf serum.UV,FTIR,circular dichroism?CD?spectra and detection of enzyme activity were carried out to characterize the structure and activity of prepared protein after extraction and back-extraction process.Seven proteins?BSA,lysozyme,bovine hemoglobin,ovalbumin,?-chymotrypsin A,pepsin,gluten?presented different results:the secondary structure and activity of hydrophilic proteins remained unchanged while those of hydrophobic proteins changed.Dynamic light scattering?DLS?and transmission electron microscopy?TEM?were combined to explore the mechanism,and the results indicated that proteins could aggregate with DES through interactions such as hydrogen bond and hydrophobic interaction,forming DES-protein aggregates in the extraction process while the destruction of DES-protein aggregates by water?back-extraction solvent?led to the release of protein,and the low solubility of released protein in HFIP-DES aqueous solution promoted the precipitation of protein.?2?DESs were prepared with HFIP as hydrogen bond donor and different cationic surfactant?CS,including decyl,dodecyl,tetradecyl,hexadecyltrimethylammonium bromide?as hydrogen bond.HFIP-CS DESs were used for the first time as buffer additives to prepare CE dynamic coatings,six acidic and basic proteins??-chymotrypsin A,trypsin,cytochrome C,lysozyme,BSA,bovine hemoglobin?were then simultaneously separated.Several factors,including the composition and concentration of HFIP-CS DES,the concentration and p H of buffer were investigated for their effects on the abilities of DES dynamic coating in electroosmotic flow?EOF?regulation and protein separation.Under the optimal separation conditions,the dynamic coating prepared by DESs composed of dodecyl,tetradecyl,cetyltrimethylammonium bromide and HFIP showed high separation efficiency?19 000-207 000 plates/m?and recovery?91.89%-101.36%?for proteins,and also good reproducibility of migration time?RSD?1.96%?.This suggested that HFIP-CS DES dynamic coating could effectively suppress protein wall adsorption and significantly improve CE separation efficiency of protein.The investigation of mechanism showed that HFIP-CS DES,as running buffer additive,could promote the formation of compact,homogeneous and stable surfactant bilayer coating.And the hydrogen bond,electrostatic interaction and hydrophobic interaction between cationic surfactant and HFIP played a crucial role in the formation of superior bilayer coating.