Study On The Electrostatic Interaction-dependent Aqueous Micellar Two-phase Systems

The recent progresses and key problems of aqueous two-phase partitioning (ATPP) were reviewed. The characteristics and utilization of two-phase aqueous micellar systems were introduced especially. It was well known that elctrostatic interaction played important role in aqueous two-phase partitioning, especially in two-phase aqueous micellar systems composed of polysoap. This work consisted of three parts: preparation of hyamine hydrophobically modified ethylene oxides (HM-EO) and their measuring method, micellar properties and liquid-liquid equilibria behavior of HM-EO and its hyamine modified polymers, and partitioning behavior and purification process of proteins in aqueous two-phase systems (ATPS) composed of these polymers.Hyamine modified HM-EO (N-R-HM-EO, R= Me, Et, n-Bu) was prepared using the reaction between HM-EO solution and superfluous alkyl halide (CH₃Br, CH₃CH₂Br and n-CH₃(CH₂)₃Br) under the conditions of low temperature, stiring and ultrasonic treatment. By using acetone as the cosolvent, and coomassic brilliant blue G₂₅₀ as color-developing agent, fast and reliable spectrophotometric method was established to determine the concentration of polymers in aqueous solution. The dosage of the color-developing agent and reacttion time was optimized. It was found that pH and inorganic salts such as NaCl, KBr and Na₂SO₄ and so on in the samples had no disturb, but surfactant such as SDS could had an remarkable effect on the mensurement. The accuracy and precision of the method was higher than that of gravimetric method.In order to understand the phase behavior and electrochemical partitioning of aqueous micellar two-phase systems composed of polysoaps, micellar properties of HM-EO and its hyamine modified polymers were determined with the fluorescence probing method. The CMC values of hyamine modified polymers were higher than those of tertiary amine polymers, but the micellar aggreation number of hyamine modified polymers were less than those of hyamine modified polymers owing to the ionization of ionic group of hyamine modified polymers. The influence of salts, pH and temperature on micellar properties of four polysoaps were studied. The micellar aggreation numbers increased and the CMC values decreased with increasing salts concentration, because salts made the elctrostatic interaction between ionic group of polymers stronger. The pH of polysoaps solution had obvious effect on micelles composed of tertiary amine polymers, but had little effect on the micelles composed of hyamine modified polymers. The CMC values increased and the micellar aggreation numbers decreased slightly with incresing the temperature. The micellar properties of HM-EO-SDS mixtures were also discussed.The properties of mixed micelle depended on the proportion of SDS and surfaceactive units of HM-EO.HM-EO and its hyamine modified polymers were used to form aqueous two-phase system with only one phase-forming composition. It was found that the phase separation could be achieved under room temperature. The phase behavior and charge properties of aqueous micellar two-phase systems composed of these polysoaps were studied systematically. The amphiphilic properties of polysoaps molecule resulted in the micellization and aggregation of micellars in water. The micellar aggregates form in the bottom phase could be charged, and the net-charge could be adjusted by the pH and conductivity of bulk solution and adding sodium dodecyl sulfate (SDS). The increase of pH would improve the phase separation of HM-EO aqueous two-phase system. The effect of salts on the phase behavior was observed. The influence of cations followed as K+> Na , and the anions as SO4 >F">Cl">Br'>T. In addition, the interaction between HM-EO polymer and anion surfactant SDS was studied. The results indicated that the addition of SDS caused the formation of mixed micellar aggregates of SDS and HM-EO which changed significantly the charge property of HM-EO aqueous two-phase systems.Aqueous micellar two-phase systems composed of HM-EO and its hyamine modified polymers were introduced for the partitioning of proteins. The change of SDS concentration, pH, conductivity and temperature of system could affect the partitioning behavior of BSA and lysozyme. Mechanism analysis on the interaction between HM-EO and SDS showed that the networks of charged mixed micelles aggregates in bottom phase resulted in larger (p between phases and the electrostatic interaction was a main driven force to affect the proteins partitioning between two phases. Lower LCST and high molecular weight of HM-EO caused that proteins partitioning could be carried through at relatively low temperature. It was possible to attract the proteins with positive charges to the polymer-enriched bottom phase and repulse the proteins with negative charges to the polymer-depleted top phase in polysoaps systems. The interactions between proteins and micelles in N-Me-HM-EO-buffer system were much stronger than those micelles composed of other polymers. It could be concluded that N-Me-HM-EO-buffer system is promising to be one of powerful ATPS for protein separation. A partitioning model was applied to correlate the partitioning coefficients of model proteins. The results demonstrated that the model could provide a good representation of partitioning behavior with varying salts concentrations.The partitioning of cytochrome P450bm-3 in N-Me-HM-EO-buffer aqueous two-phasesystems was investigated. The possibility of using aqueous two-phase extraction with only one phase-forming component to separate P450bm-3 from cell extract supernatant was explored. There were many factors that influence the partitioning coefficient of cytochrome P450 BM-3, such as biomass addition, pH, temperature and salts. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to direct the proteins with different charges to the polymer-rich phase or the polymer-depleted phase by changing pH and addition of salts. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction in N-Me-HM-EO-buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The enzyme recovery was 73.3% with a purification factor of about 2.54.