Recombinant Human Arginase 1/ARG1 Protein, CF

• Assay Diluent: Deionized Water
• Recombinant Human Arginase 1/ARG1 (rhARG1) (Catalog # 5868-AR)
• Substrate Buffer: 125 mM L-Arginine, 625 mM Glycine, pH 10.5
• Manganese Choride, 1 M stock in deionized water
• o-Phthaldialdehyde (oPA), (Sigma, Catalog # P0657), 50 mg/mL (373 mM) stock in DMSO
• N-(1-Naphthyl)ethylene-diamine dihydrochloride (NED) (Sigma, Catalog # N9125), 500 mM stock in DMSO
• 50 mM Boric Acid, 1 M Sulfuric Acid, 0.03% Brij-35 (w/v) [Caution: highly acidic, neutralize before disposal]
• Urea, 100 mM stock in deionized water
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhARG1 to 0.25 µg/mL in deionized water.
2. Prepare a standard curve from the 100 mM Urea stock. Dilute 100 µL of 100 mM Urea with 900 µL of deionized water to make a 10 mM Urea solution. Use this for the first point of the curve.
3. Perform six additional one-half serial dilutions of the 10 mM Urea stock in deionized water. The standard curve has a range of 7813 to 500,000 pmol per well.
4. Load 50 µL of each dilution of the standard curve into a plate. Include a blank containing only deionized water.
5. Load 40 µL of the 0.25 µg/mL rhARG1 into the plate. Load multiple wells as some will be used as enzyme blanks.
6. From the Substrate Buffer and Manganese Chloride stocks, prepare a solution of 100 mM Arginine, 500 mM Glycine, 1.25 mM MnCl₂, pH 10.5. Do not prepare this solution until immediately before use as the manganese will gradually precipitate out of solution.
7. Add 10 µL of 100 mM Arginine, 500 mM Glycine, 1.25 mM MnCl₂, pH 10.5 to the wells containing 0.25 µg/mL rhARG1 (exclude the blanks). Mix well.
8. Cover the plate and incubate at 37 °C for two hours.
9. Dilute oPA stock to 4 mM in 50 mM Boric Acid, 1 M Sulfuric Acid, 0.03% Brij-35 (w/v).
10. Dilute NED stock to 4 mM in 50 mM Boric Acid, 1 M Sulfuric Acid, 0.03% Brij-35 (w/v).
11. Combine equal volumes of 4 mM oPA and 4 mM NED to form a solution of 2 mM oPA with 2 mM NED.
12. Add 200 µL of the 2 mM oPA, 2 mM NED solution to all wells, including the standard curve.
13. Prepare a fresh solution of 100 mM Arginine, 500 mM Glycine, 1.25 mM MnCl₂, pH 10.5. Add 10 µL to each well used as an enzyme blank.
14. Cover the plate and incubate at room temperature for 20 minutes.
15. Read plate at 520 nm (absorbance) in endpoint mode.
16. Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted Urea Detected* (OD)
	Incubation time (min) x amount of enzyme (µg)

     *Derived from the urea standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank.

• rhARG1: 0.01 µg (10 ng)
• Arginine: 4 mM
• oPA & NED: 1.6 mM
• Urea Curve: 500000, 250000, 125000, 62500, 31250, 15625 and 7813 pmol