Recombinant Human Glyoxalase I Catalog Number: 4959-GL

DESCRIPTION Source E. coli­derived Ala2­Met184, with an N­terminal Met and 6­His tag Accession # NP_006699

N­terminal Sequence Met Analysis Predicted Molecular 22 kDa Mass

SPECIFICATIONS SDS­PAGE 25 kDa, reducing conditions

Activity Measured by its ability to catalyze the formation of S­D­lactoylglutathione 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.

Endotoxin Level <1.0 EU per 1 μg of the protein by the LAL method.

Purity >85%, by SDS­PAGE under reducing conditions and visualized by silver stain. Formulation Lyophilized from a 0.2 μm filtered solution in Tris­HCl and DTT. See Certificate of Analysis for details.

Activity Assay Protocol Materials l Assay Buffer: 0.1 M Sodium Phosphate, pH 7.0 l Recombinant Human Glyoxalase I (rhGlyoxalase I) (Catalog # 4959­GL) l Glutathione, Reduced (GSH) (Amresco, Catalog # 0399) l Methylglyoxal solution, 40% (Sigma, Catalog # M0252) l 96­well Clear UV Plate (Costar, Catalog # 3635) l Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Assay 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: Adjusted V * (OD/min) x Conversion Factor** (nmol/OD) Specific Activity (nmol/min/µg) = max amount of enzyme (µg) *Adjusted for Substrate Blank **Derived using calibration standard S­Lactoylglutathione (Sigma, Catalog # L7140).

Final Assay Per Well: Conditions l rhGlyoxalase I: 0.020 µg l Glutathione: 2 mM l Methylglyoxal: 2 mM

PREPARATION AND STORAGE 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 freeze­thaw cycles. l 6 months from date of receipt, ­20 to ­70 °C as supplied. l 3 months, ­20 to ­70 °C under sterile conditions after reconstitution.

Rev. 2/6/2018 Page 1 of 2

Recombinant Human Glyoxalase I Catalog Number: 4959-GL

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 S­D­lactoylglutathione from the hemimercaptal adduct that forms spontaneously between methylglyoxal and reduced GSH (1­4). The monomeric subunit for human Glyoxalase I is 184 amino acids (aa) in length. In the mature protein, the methionine at the N­terminus is removed. Human Glyoxalase I exists in three separable isoforms as homo­and hetero­dimers 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, 3­4). 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).

References: 1. Ridderstrom, M. & B. Mannervik (1996) Biochem. J. 314:463. 2. Marmstal, E. & B. Mannervik (1981) FEBS Lett. 131:301. 3. Kim, N­S. 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.

Rev. 2/6/2018 Page 2 of 2