Recombinant Human Glyoxalase I Protein, CF

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

Recombinant Human Glyoxalase I Protein, CF Summary

• Details of Functionality: Measured by its ability to catalyze the formation of S-D-lactoylglutathione from the hemimercaptal adduct that forms spontaneously between methylglyoxal and reduced glutathione. The specific activity is >100 nmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived human Glyoxalase I protein
  Ala2-Met184, with an N-terminal Met and 6-His tag
• Accession #: NP_006699
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GLO1
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 22 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: 25 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Tris-HCl and DTT.
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile, deionized water.
• Assay Procedure:
  • Assay Buffer: 0.1 M Sodium Phosphate, pH 7.0
  • Recombinant Human Glyoxalase I (rhGlyoxalase I) (Catalog # 4959-GL)
  • Glutathione, Reduced (GSH) (Amresco, Catalog # 0399)
  • Methylglyoxal solution, 40% (Sigma, Catalog # M0252)
  • 96-well Clear UV Plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 100 mM GSH in deionized water. Note: Prepare fresh.
  2. Dilute 40% (6.48 M) Methylglyoxal solution to 100 mM in Assay Buffer. Note: Prepare fresh.
  3. Combine 1420 µL Assay Buffer, 40 µL 100 mM GSH, and 40 µL 100 mM Methylglyoxal to make the Substrate Mixture.
  4. Incubate at room temperature for 15 minutes.
  5. Dilute rhGlyoxalase I to 0.4 ng/µL in Assay Buffer.
  6. Load 50 µL of 0.4 ng/µL rhGlyoxalase I in a plate, and start the reaction by loading 150 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 150 µL of Substrate Mixture.
  7. Read at 240 nm (absorbance) in kinetic mode for 5 minutes. Include a 5 second mix before the first read and a 3 second mix between reads.
  8. Calculate specific activity:

  Specific Activity (nmol/min/µg) = (Adjusted Vmax* (OD/min) x Conversion Factor** (nmol/OD)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard S-Lactoylglutathione (Sigma, Catalog # L7140).

  Per Well:
  • rhGlyoxalase I: 0.020 µg
  • Glutathione: 2 mM
  • Methylglyoxal: 2 mM

Notes

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

Alternate Names for Recombinant Human Glyoxalase I Protein, CF

• Aldoketomutase
• EC 4.4.1.5
• GLO1
• GLOD1
• Glx I
• GLYI
• glyoxalase domain containing 1
• Glyoxalase I
• glyoxalase Ialdoketomutase
• Ketone-aldehyde mutase
• lactoyl glutathione lyase
• lactoylglutathione lyase
• Methylglyoxalase
• S-D-lactoylglutathione methylglyoxal lyase

Background

Glyoxalase I (also lactoylglutathione lyase, methylglyoxalase, and glx I) is a 21 kDa member of the Glyoxalase I family. The enzyme is an isomerase that catalyzes the formation of S-D-lactoylglutathione from the hemimercaptal adduct that forms spontaneously between methylglyoxal and reduced GSH (1-4). The monomeric subunit for human Glyoxalase I is 184 amino acids (aa) in length. In the mature protein, the methionine at the N-terminus is removed. Human Glyoxalase I exists in three separable isoforms as homo-and hetero-dimers of two allelic subunit variants, which differ in charge (1). The isoforms are formed when residue 19 is changed from cysteine to tyrosine and residue 111 is changed from glutamine to alanine. Each subunit binds one Zn²⁺ atom (1, 3-4). The protein is made up of multiple beta strands and alpha helical regions. Human Glyoxalase I shares 91% and 90% aa sequence identity with rat and mouse Glyoxalase I, respectively. The enzyme is ubiquitously expressed and is also present in many tumor cell lines, in which its concentration is often upregulated (1). The biological role of the enzyme remains unclear, but the glyoxalase system detoxifies the precursors of advanced glycation end products, which take part in the pathogenesis of vascular, diabetic, and uremic complications (5).

1. Ridderstrom, M. & B. Mannervik (1996) Biochem. J. 314:463.
2. Marmstal, E. & B. Mannervik (1981) FEBS Lett. 131:301.
3. Kim, N-S. et al. (1993) J. Biol. Chem. 268:11217.
4. Ranganathan, S. et al. (1993) J. Biol. Chem. 268:5661.
5. Kalousova, M. et al. (2007) Ann. N. Y. Acad. Sci. 1126:268.

Publications for Glyoxalase I (4959-GL)(4)

Publications using 4959-GL

• QA Al-Balas, ML Al-Smadi, MA Hassan, GA Al Jabal, AM Almaaytah, KH Alzoubi Multi-Armed 1,2,3-Selenadiazole and 1,2,3-Thiadiazole Benzene Derivatives as Novel Glyoxalase-I Inhibitors Molecules, 2019-09-04;24(18):. 2019-09-04 [PMID: 31487813] (Bioassay, Human)
• MA Al-Sha'er, QA Al-Balas, MA Hassan, GA Al Jabal, AM Almaaytah Combination of pharmacophore modeling and 3D-QSAR analysis of potential glyoxalase-I inhibitors as anticancer agents Comput Biol Chem, 2019-03-25;80(0):102-110. 2019-03-25 [PMID: 30947068] (Bioassay, Human)
• J Li, T Yakushi, F Parlati, AL Mackinnon, C Perez, Y Ma, KP Carter, S Colayco, G Magnuson, B Brown, K Nguyen, S Vasile, E Suyama, LH Smith, E Sergienko, AB Pinkerton, TD Chung, AE Palmer, I Pass, S Hess, SM Cohen, RJ Deshaies Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11 Nat. Chem. Biol, 2017-02-28;0(0):. 2017-02-28 [PMID: 28244987] (Bioassay)
• Jo-Watanabe A, Ohse T, Nishimatsu H, Takahashi M, Ikeda Y, Wada T, Shirakawa J, Nagai R, Miyata T, Nagano T, Hirata Y, Inagi R, Nangaku M Glyoxalase I reduces glycative and oxidative stress and prevents age-related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation. Aging Cell, 2014-02-24;13(3):519-28. 2014-02-24 [PMID: 24612481] (Bioassay, Rat)

Bioinformatics

• Gene Symbol: GLO1
• Uniprot:
  • NP_006699()