Novel assay method for dimethyl sulfoxide reductase using capillary electrophoresis

毛细管电泳测定二甲亚砜还原酶的新方法

• 批准号: 05660115
• 负责人: OKUBO Akira
• 金额: $ 1.34万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660115/
• 关键词: capillary electrophoresis; DMSO reductase; CZE; molybdenum containing enzyme; micellar electrokinetic chromatography; MEKC

Dimethyl sulfoxide reductase was obtained by anaerobic cultivation of a green mutant of Rhodobacter sphaeroides f.s.denitrificans under visible light in the presence of DMSO.The purified enzyme through several column techniques gave a single band in SDS PAGE.A novel assay method for DMSO reductase was developed using capillary electrophoresis. A micellar electrokinetic chromatography mode was quite effective for a quantitative determination below the nanogram level without any pretreatment of the reaction mixture of the enzyme. About 6nl of sample solution was introduced in the hydrostatic mode (20sec, 5cm height) from the anodic end of the capillary. A boratephosphate-SDS buffer, pH7, was used for MEKC.The sample solution was electrophoresed at 15kV with an electric current of around 25 uA,on-column detection being done at an absorbance of 200nm. The enzymatic activity calculated by MEKC was compared with that by conventional optical absorption method. Both methods was in close agreement, indicating that MEKC is useful for a quantitative assay of DMSO reductase. This novel assay method established enabled us to search the electron donor for DMSO reductase. In the water soluble fraction of the cell homogenate, DMSO dependent oxidation activity was found. The active compound was now being purified.

二甲亚砜还原酶是通过在DMSO存在下，在可见光下厌氧培养球形红细菌f.s.反硝化绿色突变体而获得的。通过多种柱技术纯化的酶在SDS PAGE上显示出单一条带。利用毛细管电泳建立了一种新的DMSO还原酶测定方法。胶束电动色谱模式对于纳克水平以下的定量测定非常有效，无需对酶的反应混合物进行任何预处理。以静水模式（20秒，5cm高度）从毛细管的阳极端引入约6nl的样品溶液。 MEKC使用硼酸磷酸盐-SDS缓冲液，pH7。样品溶液在15kV下电泳，电流约25uA，在吸光度200nm处进行柱上检测。将MEKC计算的酶活性与常规光吸收法计算的酶活性进行比较。两种方法非常一致，表明 MEKC 可用于 DMSO 还原酶的定量测定。这种新颖的测定方法使我们能够寻找 DMSO 还原酶的电子供体。在细胞匀浆的水溶性部分中，发现了 DMSO 依赖性氧化活性。现在正在纯化活性化合物。

项目成果

M.Tachibana, H.onoe, A.Okubo, S.Yamazaki: "Application of micellar electrokinetic chromatography as a novel assay method for dimethyl sulfoxide reductase." Biosci.Biotech.Biochem.59. 282-284 (1995)

M.Tachibana、H.onoe、A.Okubo、S.Yamazaki：“胶束电动色谱法作为二甲亚砜还原酶的新型测定方法的应用。”

M.Tachibana,et al.: "Application of MEKC as a Novel Assay Method for DMSO-reductase" Biosci.Biotech.Biochem.59(印刷中). (1995)

M. Tachibana 等人：“MEKC 作为 DMSO 还原酶新型测定方法的应用”Biosci.Biotech.Biochem.59（出版中）。

M.Abo,et al.: "Enantioselective Deoxygenation of Alkyl Aryl Sulfoxide by DMSO-R from Rhodobacter" Bioorg.Med.Chem.(印刷中). (1995)

M.Abo 等人：“来自红细菌的 DMSO-R 对烷基芳基亚砜的对映选择性脱氧”Bioorg.Med.Chem.（出版中）。

M.Abo, M.Tachibana, A.Okubo, S.Yamazaki: "Enantioselective reduction of the sulfoxide in methyl phenyl sulfoxide by DMSO reductase from R.sphaeroides." Biosci.Biotech.Biochem.58. 596-597 (1994)

M.Abo、M.Tachibana、A.Okubo、S.Yamazaki：“通过来自 R.sphaeroides 的 DMSO 还原酶对甲基苯基亚砜中的亚砜进行对映选择性还原。”

M.Abo, M.Tachibana, A.Okubo, S.Yamazaki: "Enantioselective deoxigenation of alkyl aryl by DMSO reductase from R.sphaeroides." Bioorg.Med.Chem.(in press). (1995)

M.Abo、M.Tachibana、A.Okubo、S.Yamazaki：“通过来自 R.sphaeroides 的 DMSO 还原酶对烷基芳基进行对映选择性脱氧。”