The glutathione reductase (GR) fluorescent activity kit determines GR activity by the amount of GSH generated from the reduction of GSSG with a non-fluorescent molecule, ThioStar, which covalently binds to the free thiol group on GSH to produce a highly fluorescent product. The most commonly used procedure to measure GR is to monitor the oxidation of NADPH as a decrease in absorbance at 340 nm. However, many biological molecules absorb light at 340 nm, and the detection system gives very low OD readings.
The Arbor Assays Glutathione Reductase Fluorescent Activity Kit overcomes these problems of measuring GR activity by directly detecting GSH formed from oxidized glutathione. Glutathione reductase (GR) plays an indirect but essential role in preventing oxidative damage within the cell by helping to maintain adequate levels of intracellular glutathione (GSH). GSH, along with the enzyme glutathione peroxidase (GP), is the active reductant responsible for minimizing harmful cellular levels of hydrogen peroxide. The regeneration of GSH is catalyzed by GR.
GR is a 100-120 kDa ubiquitous dimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione, using ß-nicotinamide dinucleotide phosphate (NADPH) as a hydrogen donor. Molecules like NADPH act as hydride donors in a variety of enzymatic processes. It has been suggested that NADPH also acts as an antioxidant that operates indirectly, given its role in re-reducing GSSG to GSH and thus maintaining the antioxidant power of glutathione.
Components (edit) 96-well half-area black plate
Glutathione Reductase Standard 40 μL Glutathione Reductase at 200 mU / mL in a special stabilizer solution.
ThioStar Detection Reagent 1 vial ThioStar Thiol Detection Substrate stored in a desiccator. Reconstitute with dry DMSO.
Dry DMSO 2 ml of dry dimethylsulfoxide solvent over molecular sieves. It can be stored at room temperature.
Assay Buffer Concentrate 60 mL A 2x concentrated phosphate buffer containing detergents and stabilizers.
NADPH 1 lyophilized vial of ß-nicotinamide dinucleotide 2′-phosphate and reduced adenine with stabilizers stored in a desiccator.
NADPH Diluent 5 mL A phosphate buffer containing detergents and stabilizers.
Oxidized glutathione 3 mL Oxidized glutathione (GSSG) in a special stabilizer solution.
Material not included
Repeater pipette with disposable tips capable of dispensing 15 μL and 25 μL. 96-well fluorescence plate reader capable of reading fluorescent emission at 510 nm, with excitation at 390 nm.
Software to convert raw relative fluorescent unit (FLU) readings from the plate reader and perform four-parameter logistic curve fitting (4PLC). Set the plate parameters for a Corning Costar 3686 96-Well Plate.