Submitted to: Methods in Enzymology
Publication Type: Book / Chapter
Publication Acceptance Date: April 19, 2001
Publication Date: January 1, 2002
Citation: Smith, A.D., Levander, O.A. 2002. High throughput 96-well microplate assay for determining the specific activities of glutathione peroxidase and thioredoxin reductase. Methods in Enzymology. Interpretive Summary: Glutathione peroxidase (GPX) and thioredoxin reductase (TR) are selenocysteine-containing enzymes that are constituents of the cellular antioxidant defense system. Conventional cuvette-based assays for GPX and TR are laborious and time consuming. High throughput can be achieved with assays designed to work in a 96-well microplate reader providing an alternative methodology for high throughput with reduced instrumentation cost. However, due to differences in the light pathlength when using a 96-well format, the values obtained cannot be compared directly with those obtained using a 1-cm cuvette. Described here are three assays modified to work in a 96-well format. The first is an enhanced microplate version of the BCA protein assay that has increased sensitivity and working range. Also described are assays for GPX and TR, modified to work in a 96-well format that incorporates light pathlength determinations into the assays. The values obtained using a high throughput 96-well enzyme assay format in conjunction with pathlength determinations are in agreement with those obtained using a standard 1-cm cuvette. While spectrophotometrically derived pathlengths are the most accurate, calculated pathlengths based on assay volume and well size can be used with only a small amount of error introduced.
Technical Abstract: Glutathione peroxidase (GPX) and thioredoxin reductase (TR) are proteins that help protect our bodies against oxidative stress. Conventional assays used to study these proteins are laborious and time consuming. Improved efficiency in the laboratory can be achieved using assays designed to work in a special format where 96 samples can be assayed at one time using an instrument called a microplate reader. Described here are three assays modified to work with a microplate reader. The first is an enhanced protein assay that has increased sensitivity and working range. Also described are assays for GPX and TR, modified to work with the microplate reader. The values obtained using the microplate reader match those obtained using conventional assay. The advantage is that many more samples can be tested with less work using the new assay method.