Triton X-114を用いるミセル水性二相分配法による膜タンパク質の抽出に及ぼす荷電水溶性ポリマーの添加効果

charged polymer

The effect of charged water-soluble polymers on the extractability of membrane proteins in a Triton X-114-based aqueous micellar two-phase system was examined. Cytochrome b5 (Cyt.b5), NADH-cytochrome b5 reductase (fp1), and NADPH-cytochrome P450 reductase (fp2), used as model proteins, were to well-extracted into a surfactant-rich phase without polymers. In the presence of non-charged dextran, there were no changes in their extractability. However, by the addtion of cationic diethylaminoethyl-dextran at the concentration range of 0.01 ～　0.3%, the extractability of Cyt.B5 and fp2 was greatly reduced. In contrast, the use of anionic dextran sulfate at the same concentration range depressed the extraction of fp1 alone. A further increase in the polymer concentration or salt addition resulted in an increase in the extractability. From a calculation of net charge of the proteins, it was shown that Cyt.b5 and fp2 are negative and fp1 is positive at pH 7.4, where the extraction was conducted. Thus, the depressed extraction of the proteins in the presence of the charged polymers is attributable to the electrostatic interaction between the proteins and the charged polymers in the aqueous phase. These results indicate that charged polymers could be effective for the selective extraction of membrane proteins in a Triton X-114-based aqueous micellar two-phase system.