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 b5 was well extracted in the L61 system due to hydrophobic interaction. However, the extraction of cytochrome b5 was not observed in the 25R2 system. This abnormal extractability of cytochrome b5 in the 25R2 system was explained by the enhanced excluded-volume interaction between cytochrome b5 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 b5 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.