Recombinant Human Glyoxalase II Protein, CF

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

Recombinant Human Glyoxalase II Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze S-lactoylglutathione. The specific activity is >25,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Glyoxalase II protein
  Met1-Asp260, with a C-terminal 6-His tag
• Accession #: NP_001035517
• N-terminal Sequence: Met1
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HAGH
• 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: 30 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: 29-30 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl 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: 50 mM Tris, 250 mM NaCl, pH 7.5
  • Recombinant Human Glyoxalase II (rhGlyoxalase II) (Catalog # 5944-GO)
  • Substrate: S-Lactoylglutathione (Sigma, Catalog # L7140), 100 mM stock in deionized water
  • 5, 5’-dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D8130), 10 mM stock in DMSO
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhGlyoxalase II to 0.4 ng/µL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer with 400 µM DTNB to form Substrate Mixture.
  3. Load 50 µL of 0.4 ng/µL rhGlyoxalase II into a plate and start the reaction by loading 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate Mixture.
  4. Read at 405 nm (absorbance) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) 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 13260 M^-1cm^-1
  ***Using the path correction 0.320 cm
  Note: the output of many spectrophotometers is in mOD. Per Well:
  • rhGlyoxalase II: 0.020 µg
  • S-Lactoylglutathione: 1 mM
  • DTNB: 200 µM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human Glyoxalase II Protein, CF

• EC 3.1.2.6
• GLO2hydroxyacyl glutathione hydrolase
• Glx II
• GLX2
• GLXII
• Glyoxalase II
• HAGH
• HAGH1GLX2
• hydroxyacylglutathione hydrolase
• hydroxyacylglutathione hydrolase, mitochondrial
• hydroxyacylglutathione hydroxylase

Background

The glyoxalase system consists of two enzymes, Glyoxalase I and Glyoxalase II. The purpose of the glyoxalase system is to detoxify alpha -ketoaldehydes, particularly methylglyoxal, a by-product of glycolysis (1). Glyoxalase II catalyzes the hydrolysis of S-lactoylglutathione, the product of the glyoxalase I reaction, to glutathione and lactate (2). Glyoxalase II exists as two isoforms, one cytosolic and one mitochondrial (3). The enzyme is believed to be important for the response of cells to stress (4).

1. Thornalley, P.J. (1996) Gen. Pharmacol. 27:565.
2. Ridderström, M. et al. (1996) J. Biol. Chem. 271:319.
3. Cordell, P.A. et al. (2004) J. Biol. Chem. 279:28653.
4. Xu, Y. and X. Chen (2006) J. Biol. Chem. 281:26702.

Publications for Glyoxalase II/HAGH (5944-GO)(1)

Publication using 5944-GO

• M Everett, N Sprynski, A Coelho, J Castandet, M Bayet, J Bougnon, C Lozano, DT Davies, S Leiris, M Zalacain, I Morrissey, S Magnet, K Holden, P Warn, F De Luca, JD Docquier, M Lemonnier Discovery of a Novel Metallo-�-Lactamase Inhibitor, which can Potentiate Meropenem Activity against Carbapenem-Resistant Enterobacteriaceae Antimicrob. Agents Chemother., 2018-04-26;0(0):. 2018-04-26 [PMID: 29530861] (Bioassay, Human)

Bioinformatics

• Gene Symbol: HAGH
• Uniprot:
  • NP_001035517()