Development of a viologen-based microtiter plate assay for the analysis of oxyanion reductase activity: Application to the membrane-bound selenate reductase from Enterobacter cloacae SLD1a-1
- 作者：HelenRidley ; Carys A. Watts ; David J. Richardson and Clive S. Butler
- 关键词：Methyl viologen ; Oxyanion reductase activity ; Microtiter plate ; Selenate ; Enterobacter cloacae
- 刊名：Analytical Biochemistry
- 出版年：2006
- 期刊代码：93_00032697
- 类别：imm
- 出版时间：15 November 2006
- 卷：358
- 期：2
- 页码：289-294
- 文件大小：239 K
摘要
The membrane-bound selenate reductase of Enterobacter cloacae SLD1a-1 is purified in low yield and has relatively low activity in the pure form compared to that of other oxyanion reductases, such as the membrane-bound and periplasmic nitrate reductases. A microtiter plate assay based on the original quartz cuvette viologen assay of Jones and Garland (R.W. Jones, P.B. Garland, Biochem. J 164 (1977) 199–211) was developed specifically for analysis of such low-abundant, labile oxyanion reductases. The plate assay detects the enzyme-dependent reoxidation of reduced methyl viologen spectrophotometrically at 600 nm. The assay is quick, uses a minimal sample volume (<5 μl), can simultaneously test a range of alternative substrates, and permits activity measurements on multiple samples. We demonstrate the accuracy and versatility of the microtiter plate assay by application to the kinetic analysis, inhibition, and pH optimization of the membrane-bound selenate reductase from E. cloacae SLD1a-1. Results show that the membrane–bound selenate reductase has optimum activity at pH 8 and its active site is able to accommodate larger inhibitory complexes resulting in mixed-type inhibition, in the presence of selenate and potassium thiocyanate.