Enhanced selectivity in aqueous micellar two-phases distribution for extraction of hydrophobic membrane proteins

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A water-soluble polymer such as polyethylene glycol (PEG) or diethylaminoethyl-Dextran (DEAE-Dx) could induce aqueous micellar solutions of octyl-beta-D-thioglucoside (OTG) to phase separate at 0 ﾟC.One of the phases thus formed wasa surfactant-depleted aqueous solution (aqueous phase) of a water-soluble polymer and the other a concentrated OTG solution (surfactant phase). In combination of OTG with PEG,cytochrome P450 (P450) and cytochrome b_5 (b_5) were well extracted into the surfactant phase without significant denaturation. The extraction yield of P450 was slightly higher than that of b_5. In contrast to PEG and Dx, DEAE-Dx yields a marked decrease in the extraction of b_5, while that of P450 remained almost unchanged. DEAE-Dx could serve the dual functions of inducing the phase separation and of reducing theentry of b_5 into the surfactant phase, thus resulting in the highly selective separation of P450 and b_5. These results would provide a faster and more effective route for isolation of P450 and b_5 under mild condition taht would be physioligically acceptableso as to prevent denaturation of the two membrane proteins.