• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the colorimetric peptide substrate Ac-Phe-Thiaphe-OH in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M.     et al. (1999) J. Biol. Chem.     274:30468. The specific activity is >4,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Leu17-Tyr417, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Leu17

• Structure / Form

  Pro form    
   

• Predicted Molecular Mass

  46 kDa

• SDS-PAGE

  43 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)

• Recombinant Human Carboxypeptidase A2/CPA2 (rhCPA2) (Catalog # 2896-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 (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhCPA2 to 100 µg/mL with 1.0 µg/mL Trypsin in Assay Buffer.

2. Incubate at room temperature for 60 minutes.

3. Dilute active rhCPA2 to 0.1 µg/mL in Assay Buffer.

4. 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.

5. Load 50 µL of the 0.1 µg/mL rhCPA2 into microplate. Include a Substrate Blank with 50 µL of Assay Buffer in place of rhCPA2.

6. Start the reaction by adding 50 µL of 200 µM Substrate into wells.

7. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.

8. 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:

• rhCPA2: 0.005 µg

• Substrate: 100 µM

• DTNB: 100 µM

Background: Carboxypeptidase A2/CPA2

Carboxypeptidase A2 encoded by the CPA2 gene cleaves the C-terminal amide or ester bond of peptides that have a free C-terminal carboxyl group (1). It prefers the C-terminal residues with aromatic side chains including Phe, Tyr, and Trp. The deduced amino acid sequence of human CPA2 consists of a signal peptide (residues 1 to 16), a pro region (residues 17 to 112), and a mature chain (residues 113 to 417). The purified rhCPA2 corresponds to the pro form, which can be activated and assayed under the conditions described in the Activity Assay Protocol.

• References:

1. Auld, D.S. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) p. 821, Academic Press, San Diego.

• Entrez Gene IDs:

  1358 (Human)

• Alternate Names:

  carboxypeptidase A2 (pancreatic); Carboxypeptidase A2; CPA2; EC 3.4.17; EC 3.4.17.15