• 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 a colorimetric peptide substrate, Hippuryl-Arg. The specific activity is >5,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Ser16-Tyr415, with a C-terminal 10-His tag Accession # NP_083982

• Accession #

• N-terminal Sequence    
  Analysis

  Ser16

• Predicted Molecular Mass

  47 kDa

• SDS-PAGE

  43 kDa and 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: 25 mM Tris, 0.1 M NaCl, pH 7.5

• Recombinant Mouse Carboxypeptidase B1/CPB1 (rmCPB1) (Catalog # 2898-ZN)

• Trypsin (Sigma, Catalog # T-1426)

• AEBSF (Catalog # ), 100 mM stock in DMSO

• Substrate: Hippuryl-Arg (Sigma, Catalog # H-2508), 25 mM in diH2O

• 96-well Clear UV Plate (Corning, Catalog # 3635)

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

1. Activate rmCPB1 at 100 µg/mL with 1 µg/mL Trypsin in Activation Buffer.

2. Incubate at room temperature for 30 minutes.

3. Add AEBSF at a final concentration of 1 mM to stop reaction.

4. Dilute activated rmCPB1 to 2 µg/mL in Assay Buffer.

5. Dilute Substrate to 2 mM in Assay Buffer.

6. In a plate, load 50 µL of 2 µg/mL rmCPB1, and include a Substrate Blank containing 50 µL Assay Buffer.

7. Start the reaction by adding 50 µL of 2 mM Substrate into wells.

8. Read in kinetic mode for 5 minutes at an absorbance of 254 nm.

9. Calculate specific activity using the following formula:

     *Adjusted for Substrate Blank
     **Derived using calibration standard Hippuric acid (Sigma Catalog # 112003).

Per Well:

• rmCPB1: 0.1 µg

• Substrate: 1 mM

Background: Carboxypeptidase B1/CPB1

Carboxypeptidase B1, encoded by the CPB1 gene, specifically cleaves the C-terminal Arg and Lys residues with a preference for Arg (1). The deduced amino acid sequence of mouse CPB1 consists of a signal peptide (residues 1 to 15), a pro region (residue 16 to 108), and a mature chain (residues 109 to 415). The purified rmCPB1 corresponds to the pro form, which can be activated by trypsin, the only pancreatic protease capable of generating active enzyme from the zymogen in vitro (1).

• References:

1. Aviles, F.X. and J. Vendrell (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) pp. 831, Academic Press, San Diego.

• Entrez Gene IDs:

  1360 (Human); 76703 (Mouse)

• Alternate Names:

  carboxypeptidase B; carboxypeptidase B1 (tissue); Carboxypeptidase B1; CPB; CPB1; DKFZp779K1333; EC 3.4.17; EC 3.4.17.2; Pancreas-specific protein; PASP; PCPB; procarboxypeptidase B