POLYMER-INDUCED PHASE-SEPARATION IN AQUEOUS MICELLAR SOLUTIONS OF OCTYL-BETA-D-THIOGLUCOSIDE FOR EXTRACTION OF MEMBRANE-PROTEINS

A water-soluble polymer such as polyethylene glycol (PEG), Dextran T-5 00 (Dx), or diethylaminoethyl-Dextran (DEAE-Dx) induced aqueous micell ar solutions of octyl-beta-D-thioglucoside (OTG) to phase separation a t 0 degrees C. One of the two phases thus formed is a surfactant-deple ted aqueous solution (aqueous phase) of a water-soluble polymer and th e other a concentrated OTG solution (surfactant-rich phase). In a comb ination of OTG with PEG or Dx, cytochrome P450 (P450) and cytochrome b (5) (b(5)) were well extracted into the surfactant-rich phase. The ext raction yield of P450 was slightly greater than that of b(5). In contr ast to PEG and Dx, DEAE-Dx markedly reduced the extraction of b(5), wh ile that of P450 remained almost unchanged. DEAE-Dx served the dual fu nctions of inducing the phase separation and preventing the extraction of b(5) into the surfactant-rich phase. This depressed extraction of b(5) was reversed by the addition of potassium phosphate. DEAE-Dx and potassium phosphate proved effective in controlling the extractability of b(5). The polymer-induced phase separation provides a new basis fo r highly efficient extraction of membrane proteins under mild conditio ns that should be acceptable for thermolabile membrane proteins under physiological conditions. (C) 1997 John Wiley & Sons, Inc.