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

• 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:

*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.

• rhHAO-1: 0.025 µg
• Sodium Glyoxylate: 2 mM
• HRP: 1 unit/mL
• AUR: 50 µM