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Use of a microplate reader in an assay of glutathione reductase using 5,5'-dithiobis(2-nitrobenzoic acid)
A E Cribb1, J S Leeder, S P Spielberg
1Division of Clinical Pharmacology and Toxicology, Research Institute, Hospital for Sick Children, University of Toronto, Ontario, Canada.
Abstract:
The use of 96-well microtiter plates and a programmable microplate reader to measure glutathione reductase in an assay based on reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by GSH generated from an excess of GSSG is described. Samples are prepared in 96-well plates and absorbance at 415 nm with a reference wavelength of 595 is determined every 30 s for 3 min. The rate of increase in absorbance is directly proportional to the amount of glutathione reductase in the sample. Activity in an unknown sample is determined from a standard curve. The assay is rapid and allows many small samples to be analyzed in replicates of two or more at the same time.