比色法测定大肠杆菌呼吸链脱氢酶活性

Colorimetric assay for respiratory chain dehydrogenaseactivity in Escherichia coli

为了研究大肠杆菌在不同生理状态下的代谢活力,提出一种测定大肠杆菌内呼吸链脱氢酶的活力方法. 大肠杆菌细胞膜呼吸链中的脱氢酶将氯化碘硝基四氮唑(INT)还原为暗红色水不溶性物质,用有机溶剂提取暗红色物质并用比色法测定,得到脱氢酶的活力. 研究脱氢酶催化氯化碘硝基四氮唑的反应条件, 考察显色反应的各种影响因素.得到反应的最优条件为:检测波长为490nm, 测定的菌体质量浓度为0.1～0.4g/L细胞干重, INT质量分数为0.05%, pH7.2, 温度32 ℃. 结果表明,该测定方法能够正确地表达大肠杆菌细胞呼吸链脱氢酶活性,可用于研究多种因素对大肠杆菌代谢活力的影响.

In order to investigate the metabolic activity of Escherichia coli (E. coli), under varying physiological conditions, an analytical method based on measurement of the respiratory chain dehydrogenase activity in E. coli was proposed. Iodonitrotetrazolium chloride (INT) was reduced by the respiratory chain dehydrogenase in E. coli to a dark-red water-insoluble substrate, then the color substrate was extracted by organic solvent and detected with the spectrophotometer. The activity of dehydrogenase in the respiratory chain of E. coli was determined, and some factors affecting the INT reduction reaction with respiratory chain dehydrogenase and the coloration conditions were studied. The optimum conditions were obtained as the detecting wavelength at 490 nm, the cell concentration range was at 0.1-0.4 g/L dry weight, the mass fraction of INT was 0.05%, pH 7.2 and 32°C for the reaction. The results indicate that the method can characterize quantitatively the activity of dehydrogenase in the respiratory chain of E. coli and is suitable for determining the variations of metabolic activity in E. coli.