Recombinant Human Hydroxyacid Oxidase-1/HAO-1 Protein, CF

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

Recombinant Human Hydroxyacid Oxidase-1/HAO-1 Protein, CF Summary

• Details of Functionality: Measured by its ability to oxidize glyoxylate. The specific activity is >200 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Hydroxyacid Oxidase-1/HAO-1 protein
  Leu2-Ile370, with an N-terminal Met and 6-His tag
• Accession #: Q9UJM8
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HAO1
• Purity: >95%, 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: 42 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: 40 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 Sodium Acetate, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, pH 8.0
  • Recombinant Human Hydroxyacid Oxidase‑1/HAO‑1 (rhHAO-1) (Catalog # 6197-GO)
  • Coupling Enzyme: Horseradish Peroxidase (HRP) (250-330 U/mg) (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
  • Substrate Component 1: Sodium Glyoxylate (Sigma, Catalog # G4502), 400 mM stock in deionized water
  • Substrate Component 2: Amplex Ultra Red (AUR) (Molecular Probes, Catalog # A36006), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhHAO-1 to 0.5 ng/µL in Assay Buffer.
  2. Prepare the Substrate Mixture, 4 mM Sodium Glyoxylate, 2 units/mL HRP and 100 µM AUR, in Assay Buffer.
  3. In a plate, load 50 µL of 0.5 ng/µL rhHAO-1, and start the reaction by adding 50 µL of the Substrate Mixture (step 2). Include a Substrate Blank containing 50 µL of the Assay Buffer and 50 µL of the Substrate Mixture.
  4. Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in kinetic mode for 5 minutes. Note: A cutoff must be set manually at a wavelength of 570 nm.
  5. Calculate specific activity:

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

  *Adjusted for Substrate Blank
  **Derived using a fluorescent standard prepared by incubating 50 µM AUR, 1 unit/mL HRP, 2 mM Sodium Glyoxylate, and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.

  Per Well:
  • rhHAO-1: 0.025 µg
  • Sodium Glyoxylate: 2 mM
  • HRP: 1 unit/mL
  • AUR: 50 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 Hydroxyacid Oxidase-1/HAO-1 Protein, CF

• (S)-2-hydroxy-acid oxidase
• EC 1.1.3.15
• Glycolate oxidase
• GOX
• GOX1MGC142227
• GOXMGC142225
• HAO1
• HAO-1
• HAOX1
• hydroxyacid oxidase (glycolate oxidase) 1
• hydroxyacid oxidase 1
• Hydroxyacid Oxidase1
• Hydroxyacid Oxidase-1

Background

Glycolate oxidase is a member of the superfamily of the alpha ‑hydroxy acid oxidases (HAO), enzymes that are present in both plants and animals (1). It catalyzes the FMN-mediated oxidation of glycolate to glyoxylate and glyoxylate to oxalate with reduction of oxygen to hydrogen peroxide (2, 3). The co‑factor, FMN, is tightly bound but not covalently linked to the protein. In humans and other vertebrates, HAOs are found primarily in the peroxisomes of liver, kidney, and pancreas. Three HAOs have been identified in humans (4). HAO-1 is most highly expressed in liver and pancreas and is most active on two-carbon substrates such as glycolate. HAO-2 is expressed in liver and kidney and has greater activity against long-chain alpha ‑hydroxy acid substrates such as 2‑hydroxypalmitate. HAO-3 is expressed primarily in the pancreas. Recently, HAO has been identified as a major contributor to hyperoxaluria, a disorder in which large deposits of calcium oxalate form kidney stones (5).

1. Caroline, V. et al. (2007) Arch. Biochem. Biophys. 465:410.
2. Murray, M.S. et al. (2008) Biochemistry, 47:2439.
3. Pennati, A. and G. Gadda. (2009) J. Biol. Chem. 284:31214.
4. Jones, J.M. et al. (2000) J. Biol. Chem. 275:12590.
5. Monico, G.C. et al. (2002) Kidney Int. 62:392.
   

Bioinformatics

• Gene Symbol: HAO1
• Uniprot:
  • Q9UJM8(Human)