• 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 >400 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Gly17-Tyr420, with a C-terminal 10-His tag Accession # Q6P8K8

• Accession #

• N-terminal Sequence    
  Analysis

  Gly17

• Predicted Molecular Mass

  47 kDa

• SDS-PAGE

  48 kDa, reducing conditions

Assay Procedure

Materials

• 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 Mouse Carboxypeptidase A4/CPA4 (rmCPA4) (Catalog # 2899-ZN)

• Trypsin (Sigma, Catalog # T-1426)

• AEBSF (Catalog # ), 100 mM in deionized water

• 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 rmCPA4 to 100 µg/mL with 1.0 µg/mL Trypsin in Activation Buffer.

2. Incubate at room temperature for 1 hour.

3. Stop Trypsin activity by adding AEBSF at a final concentration of 1 mM.

4. Incubate at room temperature for 15 minutes.

5. Dilute active rmCPA4 to 6 µg/mL in Assay Buffer.

6. Dilute Substrate to 200 µM with 200 µM DTNB in Assay Buffer.

7. Load 50 µL of the 6 µg/mL rmCPA4 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:

• rmCPA4: 0.3 µg

• Substrate: 100 µM

• DTNB: 100 µM

Background: Carboxypeptidase A4/CPA4

Carboxypeptidase A4 encoded by the CPA4 gene is a member of the metallocarobxypeptidase family that cleaves the C-terminal amide or ester bond of peptides that have a free C-terminal carboxyl group. The deduced amino acid sequence of mouse CPA4 consists of a signal peptide (residues 1 to 16), a pro region (residue 17 to 113), and a mature chain (residues 114 to 420). The purified recombinant mouse CPA4 corresponds mainly to the pro form, which can be activated and assayed under the conditions described in the Activity Assay Protocol.

• Entrez Gene IDs:

  51200 (Human); 71791 (Mouse)

• Alternate Names:

  Carboxypeptidase A3; Carboxypeptidase A4; CPA3EC 3.4.17; CPA4; EC 3.4.17.-; EC 3.4.17.1