Effect of charged water-soluble polymers on the extraction of membrane proteins in a Triton X-114-based aqueous micellar two-phase system

The effect of charged water-soluble polymers on the extractability of membr ane proteins in a Triton X-114-based aqueous micellar two-phase system was examined. Cytochrome b(5) (Cyt.b(5)), NADH-cytochrome b(5) reductase (fp1), and NADPH-cytochrome P450 reductase (fp2), used as model proteins, were to be well-extracted into a surfactant-rich phase without polymers. In the pr esence of non-charged dextran, there were no changes in their extractabilit y. However, by the addition of cationic diethylaminoethyldextran at the con centration range of 0.01 similar to 0.3%, the extractability of Cyt.b(5) an d fp2 was greatly reduced. In contrast, the use of anionic dextran sulfate at the same concentration range depressed the extraction of fp1 alone. A fu rther increase in the polymer concentration or salt addition resulted in an increase in the extractability. From a calculation of net charge of the pr oteins, it was shown that Cyt.b(5) and fp2 are negative and fp1 is positive at pH 7.4, where the extraction was conducted. Thus, the depressed extract ion of the proteins in the presence of the charged polymers is attributable to the electrostatic interaction between the proteins and the charged poly mers in the aqueous phase. These results indicate that charged polymers cou ld be effective for the selective extraction of membrane proteins in a Trit on X-114-based aqueous micellar two-phase system.