Recombinant Human PAM Protein, CF

• Assay Buffer: 50 mM MES, pH 6.0
• Recombinant Human Peptidylglycine alpha ‑Amidating Monooxygenase/PAM (rhPAM) (Catalog # 4837-AM)
• Substrate: Sodium Hippurate [0.5 M Hippuric Acid (Sigma, Catalog # 112003) in 0.5 M NaOH]
• Standard: Sodium Glyoxylate
• Glyoxylate Development Solution
• Catalase (Sigma, Catalog # C30), 100,000 units/mL stock in 50 mM MES, pH 6.5
• L-Ascorbic Acid (Sigma, Catalog # 255564), 0.5 M stock in deionized water
• Copper (II) Chloride (Sigma, Catalog # 222011), 1 M stock in deionized water
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute the standard to 2000 μM in Assay Buffer. Prepare the standard curve by performing six ½ serial dilutions of the 2000 μM standard. The standard curve has a range of 0.625 to 40 nmol per well.
2. Dilute rhPAM to 5 ug/mL in cold Assay Buffer. Keep on ice.
3. Dilute Substrate to 20 mM in Assay Buffer.
4. Prepare a Reaction Mixture containing 4000 Units/mL of Catalase, 0.2 μM of Copper Chloride and 10 mM of Ascorbic Acid in cold Assay Buffer. Keep on ice. (Note: Dilute Copper Chloride to 10 μM in deionized water before adding to the mixture to prevent precipitation.)
5. In microtubes, combine 75 μL of rhPAM with 150 μL of Reaction Mixture. As a Substrate Blank, combine 75 μL of Assay Buffer with 150 μL of Reaction Mixture. For the standard curve, combine 75 μL of each dilution of the standard curve with 150 μL of Reaction Mixture.
6. Start the reaction by adding 75 μL of 20 mM Substrate to all vials.
7. Incubate at 37 °C for 1 hour.
8. Add 300 μL of Glyoxylate Development Solution to each vial, mix briefly and incubate at 95‑100 °C for exactly 2 minutes.
9. Cool the vials on ice for 3 minutes.
10. Into the plate, pipette 160 μL of Substrate Blank, each dilution of the standard curve, and reactions to empty wells in triplicate.
11. Read plate in endpoint mode at excitation and emission wavelengths of 330 nm and 415 nm (top read), respectively.
12. Calculate specific activity:

     *Derived from a sodium glyoxylate standard curve using linear fitting and adjusted for Substrate Blank.

•  rhPAM: 0.1 μg
• Standard Curve: 0.625, 1.25, 2.5, 5, 10, 20, 40 nmol