| Project/Area Number |
06453108
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
工業分析化学
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
WATANABE Hiroto Hokkaido Univ., Fac. of Eng., Prof., 工学部, 教授 (80002856)
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| Co-Investigator(Kenkyū-buntansha) |
SEGAWA Tadashi Hokkaido Univ., Fac. of Eng., Research Associate, 工学部, 助手 (30196935)
SAITOH Tohru Hokkaido Univ., Fac. of Eng., Research Associate, 工学部, 助手 (40186945)
KAMIDATE Tamio Hokkaido Univ., Fac. of Eng., Assoc. Prof., 工学部, 助教授 (70185990)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1995: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1994: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Keywords | Cytochrome P-450 / Cytochrome b_5 / Extractive separation / Two-phases separation / Non-ionic surfactant / Micellar solution / Water-soluble polymer / Hydrophobic membrane proteins / チトクロームb_5 |
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| Research Abstract |
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.
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