Recombinant Human D-Amino Acid Oxidase His-tag Protein, CF

• Buffer: 50 mM Sodium Phosphate, pH 8.0
• Flavin Adenine Dinucleotide (FAD) (Sigma, Catalog # F6625), 10 mM stock in deionized water
• Recombinant Human D-Amino AcidOxidase His-tag (rhDAO) (Catalog # 10116-DO)
• Coupling Enzyme: Horseradish Peroxidase (HRP) (250-333 U/mg) (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
  
• Substrate Component 1: D-Alanine (Sigma, Catalog # A7377), 500 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. Prepare Assay Buffer (50 mM Sodium phosphate, 5 µM FAD, pH 8.0) by adding 10 mM stock of FAD to Buffer right before use.
2. Dilute rhDAO to 2 ng/µL in Assay Buffer.
3. Dilute D-Alanine to 40 mM in Assay Buffer.
4. Combine equal volumes of 2 ng/µL rhDAO and 40 mM D-Alanine.  Also, create a Substrate Blank by combining equal volumes of Assay Buffer and 40 mM D-Alanine.
5. Incubate the reactions for 10 minutes at 37 °C.
6. Prepare Substrate Mixture containing 2 units/mL HRP and 100 µM AUR in Assay Buffer.
7. Load 50 µL of the incubated reactions into the wells of a black well plate, and start the reaction by adding 50 µL of Substrate Mixture.
8. Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in endpoint mode. Note:  A cutoff must be set manually at a wavelength of 570 nm.
9. Calculate specific activity:

*Adjusted for Substrate Blank
**Derived using a fluorescent standard prepared by incubating 50 µM AUR, 1 unit/mL HRP, 10 mM D-Alanine, and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.

• rhDAO: 0.05 µg
• D-Alanine: 10 mM
• HRP: 1 unit/mL
• AUR: 50 µM