Novel assay method for dimethyl sulfoxide reductase using capillary electrophoresis

在DMSO存在的可见光下厌氧培养反硝化球形红杆菌绿色突变体，获得了二甲基亚砜还原酶。通过多种色谱柱技术纯化的酶在SDS PAGE上呈单条带。建立了毛细管电泳法测定DMSO还原酶的新方法。胶束电动力学色谱法在纳克级以下的定量分析中非常有效，无需对酶的反应混合物进行任何预处理。从毛细管的阳极端以静压模式（20sec， 5cm高度）引入约6nl的样品溶液。MEKC采用硼磷酸盐- sds缓冲液pH7。样品溶液在15kV下电泳，电流约为25 uA，在200nm的吸光度下进行柱上检测。将MEKC法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还原酶的定量分析。这种新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，发现DMSO依赖的氧化活性。活性化合物正在被提纯。

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.