Recombinant Human Carboxypeptidase A1/CPA1 Protein, CF

• Assay Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35, pH 7.5 (TCNB)
• Recombinant Human Carboxypeptidase A1/CPA1 (rhCPA1) (Catalog # 2856-ZN)
• Trypsin (Sigma, Catalog # T-1426)
• Substrate: Ac-Phe-Thiaphe-OH (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 (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhCPA1 to 100 µg/mL with 1.0 µg/mL Trypsin in Assay Buffer.
2. Incubate at room temperature for 60 minutes.
   Dilute active rhCPA1 to 0.2 µg/mL in Assay Buffer.
3. Combine equal volumes of 10 mM Substrate and 10 mM DTNB. Then, dilute this mixture to 200 µM Substrate, 200 µM DTNB with Assay Buffer.
4. Load 50 µL of the 0.2 µg/mL rhCPA1 into a clear microplate. Include a substrate blank with 50 µL of Assay Buffer in place of rhCPA1.
5. Start the reaction by adding 50 µL of 200 µM Substrate into wells.
6. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
7. 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:

• rhCPA1: 0.010 µg
• Substrate: 100 µM
• DTNB: 100 µM