Recombinant Human Carboxypeptidase A4/CPA4 Protein, CF

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Assay Buffer: 50 mM Tris, pH 8.0
• Recombinant Human Carboxypeptidase A4/CPA4 (rhCPA4) (Catalog # 5906-ZN)
• Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)
• AEBSF (Catalog # EI001), 100 mM in deionized water
• Substrate: N-Acetyl-L-Phenylalanyl-L-3-Thiaphenylalanine (Peptides International, Catalog # STP-3621-PI),10 mM stock in DMSO
• 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
• 96 well clear plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhCPA4 to 100 µg/mL with 1.0 µg/mL rhTrypsin 3 in Activation Buffer.
2. Incubate at 37 °C for 2.5 hours.
3. Stop rhTrypsin 3 activity by adding AEBSF at a final concentration of 1 mM.
4. Incubate at room temperature for 15 minutes.
5. Dilute activated rhCPA4 to 0.2 µg/mL in Assay Buffer.
6. Dilute Substrate to 200 µM with 200 µM DTNB in Assay Buffer (prepare immediately before use).
7. Load 50 µL of the 0.2 µg/mL rhCPA4 into a plate, and start the reaction by adding 50 µL of Substrate/DTNB mixture. Include a Substrate Blank with 50 µL of Assay Buffer and 50 µL of Substrate/DTNB mixture.
8. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
9. Calculate specific activity using the following formula:

     *Adjusted for Substrate Blank 
     **Using the extinction coefficient 13260 M^-1cm^-1 
     ***Using the path correction 0.32 cm
     Note: the output of many spectrophotometers is in mOD Per Well:

• rhCPA4: 0.01 µg
• Substrate: 100 µM
• DTNB: 100 µM