Recombinant Human Glyoxalase I Protein, CF
Recombinant Human Glyoxalase I Protein, CF Summary
Product Specifications
Ala2-Met184, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
4959-GL
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris-HCl and DTT. |
| Reconstitution | Reconstitute at 0.5 mg/mL in sterile, deionized water. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
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
- Prepare 100 mM GSH in deionized water. Note: Prepare fresh.
- Dilute 40% (6.48 M) Methylglyoxal solution to 100 mM in Assay Buffer. Note: Prepare fresh.
- Combine 1420 µL Assay Buffer, 40 µL 100 mM GSH, and 40 µL 100 mM Methylglyoxal to make the Substrate Mixture.
- Incubate at room temperature for 15 minutes.
- Dilute rhGlyoxalase I to 0.4 ng/µL in Assay Buffer.
- 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.
- 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.
- 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
Reconstitution Calculator
Background: Glyoxalase I
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).
- Ridderstrom, M. & B. Mannervik (1996) Biochem. J. 314:463.
- Marmstal, E. & B. Mannervik (1981) FEBS Lett. 131:301.
- Kim, N-S. et al. (1993) J. Biol. Chem. 268:11217.
- Ranganathan, S. et al. (1993) J. Biol. Chem. 268:5661.
- Kalousova, M. et al. (2007) Ann. N. Y. Acad. Sci. 1126:268.
Citations for Recombinant Human Glyoxalase I Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 4
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Combination of pharmacophore modeling and 3D-QSAR analysis of potential glyoxalase-I inhibitors as anticancer agents
Authors: MA Al-Sha'er, QA Al-Balas, MA Hassan, GA Al Jabal, AM Almaaytah
Comput Biol Chem, 2019;80(0):102-110.
Species: Human
Sample Types: Protein
Applications: Bioassay -
Multi-Armed 1,2,3-Selenadiazole and 1,2,3-Thiadiazole Benzene Derivatives as Novel Glyoxalase-I Inhibitors
Authors: QA Al-Balas, ML Al-Smadi, MA Hassan, GA Al Jabal, AM Almaaytah, KH Alzoubi
Molecules, 2019;24(18):.
Species: Human
Sample Types: Synthetic Compounds
Applications: Bioassay -
Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11
Authors: 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
Nat. Chem. Biol, 2017;0(0):.
Species: N/A
Sample Types: Protein
Applications: Bioassay -
Glyoxalase I reduces glycative and oxidative stress and prevents age-related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation.
Authors: 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
Aging Cell, 2014;13(3):519-28.
Species: Rat
Sample Types: Cell Culture Supernates
Applications: Bioassay
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