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.
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 microl), 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 approximately 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.
Ridley
Helen
H
Institute for Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
Watts
Carys A
CA
Richardson
David J
DJ
Butler
Clive S
CS
D000838:Anions; D002462:Cell Membrane / Q000201:enzymology; D016972:Enterobacter cloacae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010088:Oxidoreductases / Q000378:metabolism*; D010269:Paraquat / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D000838:Anions; D010088:Oxidoreductases; C078108:selenate reductase; D010269:Paraquat