| Assay Method Information | |
| | GRX Assay |
| Description: | Human GRX-1 (0.25 unit/mL) was incubated at 25 °C with increasing concentrations of 2-AAPA (25, 50, 100, and 200 μM) and BSA (1 mg/mL). Aliquots were withdrawn for determination of GRX activity at 3, 10, and 20 min. Control incubations without 2-AAPA were conducted in parallel. The aliquots were added to the GRX assay solution, and enzyme activity was determined from a coupled reaction with GR based on a literature reported procedure with minor modification. In this assay, a mixed disulfide between GSH and the mercaptoethanol moiety derived from 2-hydroxyethyldisulfide (HED) served as the substrate for GRX; briefly GSH (10 mM) and HED (7 mM) were premixed in water for 5 min before transferring onto ice. The final GRXassay solution contained GSH (1 mM), HED (0.7mM), GR (0.02 units/mL), NADPH (0.2 mM), and bovine serum albumin (BSA, 1 mg/mL) in Trisbuffer (pH 8, 0.1 M). Theactivity was determined by monitoring the disappearance of NADPH spectrophotometrically at λ = 340 nm [Holmgren et al., Meth. Enzymol., 252:283-292]. |
| Affinity data for this assay | |
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