Recombinant Human Glutathione Reductase Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Glutathione Reductase Protein, CF Summary

• Details of Functionality: Measured by its ability to reduce oxidized glutathione to glutathione in an NADPH-dependent manner. The specific activity is >15,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Glutathione Reductase protein
  Met44-Arg522, with an N-terminal Met and 6-His tag
• Accession #: P00390
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Proteins
• Gene: GSR
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 53 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 53 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol.
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Assay Buffer: 0.1 M Tris, pH 7.5
  • Recombinant Human Glutathione Reductase (rhGSR) (Catalog # 8866-GR)
  • NADPH (Sigma, Catalog # N7505), 10 mM stock in deionized water
  • Oxidized Glutathione (Amresco, Catalog # 0524), 50 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Create Substrate Mixture containing 0.2 mM NADPH and 1 mM Oxidized Glutathione in Assay Buffer.
  2. Dilute rhGSR to 0.4 µg/mL in Assay Buffer.
  3. Load 50 µL of the 0.4 µg/mL rhGSR to the plate, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate Mixture.
  4. Read plate in kinetic mode for 5 minutes at an absorbance of 340 nm.
  5. The specific activity can be calculated using the following equation:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x -1 x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate blank
  **Using the extinction coefficient 6220 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD.

  Per Well:
  • rhGSR: 0.02 µg
  • NADPH: 0.1 mM
  • Oxidized Glutathione: 0.5 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Glutathione Reductase Protein, CF

• EC 1.8.1
• EC 1.8.1.7
• GLUR
• Glutathione Reductase
• glutathione reductase, mitochondrial
• GR
• GRase
• GRD1
• GSR
• HEL-752
• MGC78522

Background

Glutathione Reductase (GR) is a homodimeric flavoprotein disulfide oxidoreductase that plays a critical role in the prevention of oxidative damage within the cell (1, 2). Glutathione Reductase helps to maintain appropriate levels of intracellular reduced glutathione (GSH) which is essential for maintaining the normal structure of red blood cells and for keeping hemoglobin in the ferrous state (3). Glutathione Reductase together with its co-factor, NADPH, catalyzes the reduction of oxidized glutathione (glutathione disulfide, GSSG) to glutathione. GSH is also a reactant for Glutathione Peroxidase, which converts hydrogen peroxide hydrogen peroxide (H2O2) into water (4). Glutathione Reductase is expressed as both mitochondrial and cytosolic isoforms, as determined by the presence or absence of an N-terminal transit peptide. Human Glutathione Reductase shares 86% and 76% amino acid sequence identity with mouse and rat Glutathione Reductase, respectively (5, 6). Alternative splicing of human Glutathione Reductase generates additional isoforms with deletions in the central pyridine nucleotide-disulfide oxidoreductase domain (7).

1. Deponte, M. (2013) Biochim. Biophys. Acta 1830:3217.
2. Fujii, J. et al. (2011) J. Clin. Biochem. Nutr. 49:70.
3. Tsantes, A.E. et al. (2006) Antioxid. Redox Signal. 8:1205.
4. Lubos, E. et al. (2011) Antioxid. Redox Signal. 15:1957.
5. Tutic, M. et al. (1990) Eur. J. Biochem. 188:523.
6. Krauth-Siegel, R.L. et al. (1982) Eur. J. Biochem. 121:259.
7. Satoh, N. et al. (2010) Biochem. Genet. 48:816.

Bioinformatics

• Gene Symbol: GSR
• Uniprot:
  • P00390()