Fabrication and Evaluation of Functional Lipid Bilayer Membranes which oxidize NADH.

氧化 NADH 的功能性脂质双层膜的制备和评估。

• 批准号: 03805071
• 负责人: MATSUE Tomokazu
• 金额: $ 1.15万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03805071/
• 关键词: NADH; Diaphorase; Monolayer; Bilayer Lipid Membrane; BLM; Ion Channel; Electron Transfer; Impedance; ボルタンメトリ-; 膜透過係数; シミュレ-ション; ジアフォラ-ゼ; 自己集積膜; 固定化酵素

1.Regeneration of NAD(H) at Diaphorase-Immobilized SurfacesDiaphorase was immobilized onto an Au electrode by step-by-step reaction with aminoethanethiol and glutaraldehyde. The surface concentration was found to be 6x10^<-12> mol/cm^2. Cyclic voltammetry with ferrocenylmethanol (FMA) as mediator showed a remarkable increase in the oxidation peak for FMA in the presence of NADH in solution. From the intercept and slope of the 1/i vs. exp[F(E-E)/RT] plot at the onset current region, the Michaelis constant and the molecular activity have been determined, respectively, to be 8x10^<-7> M and k_2=9s^<-1>.2.Ion-and Electron-Transport Across a Planer Bilayer Lipid Membrane (BLM)The electron-transport across the BLM containing tetracyanoquinodimethane(TCNQ) and the ion transport through the BLM with alamethicin ion channels were investigated by electrochemical methods. From the impedance spectroscopy of the BLM, heterogeneous electron-transfer rates between redox species and the BLM were determined. The results demonstrated that hydrophobic and electrostatic interactions have important roles in the electron-transfer. The selective determination of I^- transported through alamethicin channels was also investigated by measuring the oxidation current of I^- at a microdisk electrode located near the BLM. From the analysis of the oxidation current upon application of membrane potential, the flux of I^- through the BLM was determined.

1.心肌黄酶固定化表面再生NAD（H）的研究心肌黄酶以氨基乙醇和戊二醛分步固定在Au电极上。发现表面浓度为6x 10 ^<-12>mol/cm ^2。以二茂铁甲醇（FMA）为介体的循环伏安法表明，溶液中存在NADH时，FMA的氧化峰显著增加。由1/i vs. exp[F（E-E）/RT]曲线在起始电流区的截距和斜率确定了米氏常数为8 × 10 ~（-1）M，分子活性<-7>为k_2= 9 s ~（<-1>-1）。用电化学方法研究了丙甲霉素离子通道对BLM离子转运的影响。从阻抗谱的BLM，氧化还原物种和BLM之间的异质电子转移速率进行了测定。结果表明，疏水相互作用和静电相互作用在电子转移过程中起重要作用。通过测量I^-在位于BLM附近的微盘电极上的氧化电流，还研究了通过丙甲霉素通道转运的I^-的选择性测定。通过分析施加膜电位时的氧化电流，可以确定I^-通过BLM的通量。

项目成果

末永 智一: "マイクロ電極を用いた生体物質の極微量分析" BME. 5. 15-22 (1991)

Tomokazu Suenaga：“使用微电极对生物材料进行超痕量分析”BME 5. 15-22 (1991)。

H. Yamada: "Study of Permeation Through Planer Bilayer Lipid Membrane with a Microelectrode Located Near the Membrane." Denki Kagaku. 60. 664-665 (1992)

H. Yamada：“利用位于膜附近的微电极研究平面双层脂质膜的渗透。”

A. Kabasawa: "Study on Electrochemical Stability of Thiol Monolayer on Au and Ag Electrodes by AC Impedance and In situ Surface-Enhanced Raman Scattering" Denkikagaku. 60. 989-991 (1992)

A. Kabasawa：“通过交流阻抗和原位表面增强拉曼散射研究金和银电极上硫醇单层的电化学稳定性”Denkikagaku。

H.YAMADA: "A Microvoltammetric Study of Permeation of Ferrocene Derivatives Through a Planer Bilayer Lipid Membrane" Biochem.Biophys.Res.Commun. 180. 1330-1334 (1991)

H.YAMADA：“二茂铁衍生物通过平面双层脂质膜渗透的微伏安研究”Biochem.Biophys.Res.Commun。

T.SAWAGUCHI: "Catalytic Capability of DiAphorase Bound to a Self-Assembled Thiol Monolayer at a Gold Electrode." Bioelectrochem.Bioenerg.29. 127-133 (1992)

T.SAWAGUCHI：“DiAphorase 与金电极上的自组装硫醇单分子层结合的催化能力。”