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

增强水胶束两相分布的选择性，用于提取疏水性膜蛋白

• 批准号: 06453108
• 负责人: WATANABE Hiroto
• 金额: $ 4.42万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06453108/
• 关键词: 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

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.

水溶性聚合物如聚乙二醇（PEG）或二乙氨基乙基葡聚糖（DEAE-Dx）能诱导辛基-β-D-硫代葡萄糖苷（OTG）胶束水溶液在0 ℃下发生相分离，形成的两相之一是表面活性剂耗尽的水溶性聚合物水溶液（水相），另一相是浓缩的OTG溶液（表面活性剂相）。结果表明，在OTG和PEG的联合作用下，细胞色素P450（P450）和细胞色素b_5（b_5）被很好地萃取到表面活性剂相中，而没有明显的变性。P450的提取率略高于b_5。与PEG和Dx相比，DEAE-Dx对b_5的提取率明显降低，而对P450的提取率几乎没有变化。DEAE-Dx具有诱导相分离和减少b_5进入表面活性剂相的双重作用，从而实现了P450和b_5的高选择性分离。这些结果为在生理上可接受的温和条件下分离P450和b_5提供了一条更快、更有效的途径，从而避免了两种膜蛋白的变性。

项目成果

Hirofumi Tani: "Polymer-induced phase separation in aqueous micellar solutions of octyl-beta-D-thioglucoside for extraction of cytochromes P-450 and b5" Biotechnology and Bioengineering. (to be submitted). (1996)

Hirofumi Tani：“辛基-β-D-硫代葡萄糖苷胶束水溶液中聚合物诱导的相分离，用于提取细胞色素 P-450 和 b5”生物技术和生物工程。

Tohru Saitoh: "Equilibrium study on protein interactions with surfactant micelles by micellar electrokinetic chromatography" Analytical Sciences. (submitted). (1996)

Tohru Saitoh：“通过胶束电动色谱法对蛋白质与表面活性剂胶束相互作用的平衡研究”分析科学。

Tamio Kamidate: "Luminol Chemiluminescent Assay of Cytochrome b_5" Analytical Biochemistry. 223. 325-327 (1994)

Tamio Kamidate：“细胞色素 b_5 的鲁米诺化学发光测定”分析生物化学。

Tohru Saitoh: "Polymer-Induced Phase Separation in Aqueous Micellar Solutions of Alkylglucoside for Protein Extraction" Analytical Sciences. 10. 299-303 (1994)

Tohru Saitoh：“用于蛋白质提取的烷基糖苷胶束水溶液中聚合物诱导的相分离”分析科学。

Tohru Saitoh: "Polymer-induced phase separation in aqueous micellar solutions of alkylglucoside for proteinextraction" Analytical Sciences. Vol.10. 299-303 (1994)

Tohru Saitoh：“用于蛋白质提取的烷基糖苷胶束水溶液中聚合物诱导的相分离”分析科学。