Aqueous Micellar Two-phase Systems Research Articles

Enhancement of the excluded-volume effect in protein extraction using triblock copolymer-based aqueous micellar two-phase systems

Triblock copolymer surfactants consisting of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), Pluronic L61 (PEO–PPO–PEO, L61) and Pluronic 25R2 (PPO–PEO–PPO, 25R2) were exploited in aqueous micellar two-phase systems for the protein extraction. The extraction was based on the phase separation into surfactant-depleted and -condensed phases (an aqueous and a surfactant-rich phases, respectively) upon warming aqueous micellar solutions of triblock copolymer. In both systems, hydrophilic proteins such as albumin were not extracted into the surfactant-rich phase. On the other hand, hydrophobic cytochrome b 5 was well extracted in the L61 system due to hydrophobic interaction. However, the extraction of cytochrome b 5 was not observed in the 25R2 system. This abnormal extractability of cytochrome b 5 in the 25R2 system was explained by the enhanced excluded-volume interaction between cytochrome b 5 and 25R2 micellar network in the surfactant-rich phase, which overcomes the hydrophobic interaction. Additionally, ionic surfactants were added into the systems for controlling extractability of proteins. In the 25R2 system, cationic tetradecyltrimethylammonium was effective for extracting anionic cytochrome b 5 against the excluded-volume effect, while not for anionic albumin because of its large molecular weight. In 25R2 system containing ionic surfactant, the partitioning of proteins were found to be governed by the hydrophobic, excluded-volume, and electrostatic interactions. Micellar network formed by 25R2 type of surfactant with a strong excluded-volume interaction could provide new selective extraction systems for the separation of proteins.

Novel bioseparations using two-phase aqueous micellar systems

We review our recent experimental and theoretical work aimed at investigating the potential use of two-phase aqueous micellar systems for the separation or concentration of hydrophilic biomaterials using the principle of liquid-liquid extraction. The systems studied include (1) a two-phase aqueous micellar system composed of the nonionic surfactant n-decyl tetra(ethylene oxide) (C(10)E(4)) and (2) a two-phase aqueous micellar system composed of the zwitterionic surfactant dioctanoyl phosphatidyl-choline (C(8)-lecithin). The experimental partitioning behavior of several hydrophilic proteins, including cytochrome (c), soybean trypsin inhibitor, ovalbumin, bovine serum albumin, and catalase, in two-phase aqueous C(10)E(4) and C(8)-lecithin micellar systems is reviewed. A theoretical formulation of the protein partitioning behavior, based on a description of excluded-volume interactions between the hydrophilic proteins and the micelles, is also reviewed. The theoretically predicted protein partitioning behavior is compared with that observed experimentally and is found to be in good agreement. The results of our investigation suggest that two-phase aqueous micellar systems of the type examined in this article are indeed potentially useful as extractant phases for the separation or concentration of proteins and other biomaterials.

Aqueous Micellar Two-Phase Systems for Protein Separation

The extractive technique for protein purification based on two-phase separation in aqueous micellar solutions (aqueous micellar two-phase system (AMTPS)) is reviewed. The micellar solution of a nonionic surfactant, such as polyoxyethyl-ene alkyl ether, which is most frequently used for protein extraction, separates into two phases upon heating above its cloud point. The two phases consist of a surfactant-depleted phase (aqueous phase) and a surfactant-rich phase. Hydrophilic proteins are partitioned to the aqueous phase and hydrophobic membrane proteins are extracted into the sur-factant-rich phase. Because of the methodological simplicity and rapidity, this technique has become an effective means, and thus has been widely used for the purification and characterization of proteins. In contrast to polyoxyethylene alkyl ether, micellar solutions of a zwitterionic surfactant, such as alkylammoniopropyl sulfate, separate below the critical temperature. Alkylglucosides can also separate into two phases upon adding water-soluble polymers. Recently, these two-phase systems have been exploited for protein separation. Additionally, hydrophobic affinity ligands, charged polymers, and ionic surfactants have been successfully used for controlling the extractability of proteins in AMTPS.

Separation of proteins and viruses using two-phase aqueous micellar systems

We discuss the utilization of a novel two-phase aqueous nonionic micellar system for the purification and concentration of biomolecules, such as proteins and viruses, by liquid–liquid extraction. The nonionic surfactant n-decyl tetra(ethylene oxide), C 10E 4, phase separates in water into two coexisting aqueous micellar phases by increasing temperature. The mild interactions of the C 10E 4 nonionic surfactant with biomolecules, combined with the high water content of the two coexisting micellar phases, suggest the potential utility of two-phase aqueous C 10E 4 micellar systems for the purification and concentration of biomolecules. In this paper, we review our recent experimental and theoretical studies involving the partitioning of several water-soluble proteins, including cytochrome c, soybean trypsin inhibitor, ovalbumin, bovine serum albumin, and catalase, in the two-phase aqueous C 10E 4 micellar system. In addition, we present results of our preliminary experimental investigation on the partitioning of bacteriophages, including φX174, P22, and T4.