• 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 >2,500 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Asn17-Tyr419, with a C-terminal 10-His tag Accession # Q7TPZ8

• Accession #

• N-terminal Sequence    
  Analysis

  Asn17

• Structure / Form

  Pro form

• Predicted Molecular Mass

  47 kDa

• SDS-PAGE

  42 kDa, reducing conditions

Assay Procedure

Materials

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

• Recombinant Mouse Carboxypeptidase A1/CPA1 (rmCPA1) (Catalog # 2765-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 rmCPA1 to 100 µg/mL with 1.0 µg/mL Trypsin in Assay Buffer.

2. Incubate at room temperature for 60 minutes.

3. Dilute active rmCPA1 to 0.2 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 0.2 µg/mL rmCPA1 into a plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.

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 0.320 cm  
     Note: the output of many spectrophotometers is in mOD. Per Well:

• rmCPA1: 0.01 μg

• Substrate: 100 µM

• DTNB: 100 µM

Background: Carboxypeptidase A1/CPA1

Carboxypeptidase A1 encoded by the CPA1 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 or branched aliphatic side chains including Phe, Tyr, Trp, Leu or Ile. It is important in the degradation of food proteins to produce essential amino acids such as Phe and Trp. The deduced amino acid sequence of mouse CPA1 consists of a signal peptide (residues 1 to 16), a pro region (residue 17 to 110), and a mature chain (residues 111 to 419). The purified recombinant CPA1 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. 812  Academic Press, San Diego.

• Entrez Gene IDs:

  1357 (Human); 109697 (Mouse)

• Alternate Names:

  carboxypeptidase A1 (pancreatic); Carboxypeptidase A1; CPA; CPA1; EC 3.4.17; EC 3.4.17.1