• 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 >1,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived His16-Tyr417, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His16

• Structure / Form

  Pro form    
   

• Predicted Molecular Mass

  47 kDa

• SDS-PAGE

  45 kDa, reducing conditions

Assay Procedure

Materials

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

• Assay Buffer: 50 mM Tris, 100 mM NaCl, pH 7.5

• Recombinant Human Carboxypeptidase B1/CPB1 (rhCPB1) (Catalog # 2897-ZN)

• Trypsin (Sigma, Catalog # T-1426)

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

• 96 well clear UV-transparent microplate (Corning, Catalog # 3635)

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

1. Dilute rhCPB1 to 100 μg/mL with 1.0 μg/mL Trypsin in Activation Buffer.

2. Incubate at room temperature for 30 minutes.

3. Dilute active rhCPB1 to 2 μg/mL in Assay Buffer.

4. Dilute Substrate to 2 mM in Assay Buffer.

5. Load 50 μL of the 2 μg/mL rhCPB1 into a plate. Include a Substrate blank with 50 μL of Assay Buffer.

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

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

8. Calculate specific activity using the following formula:

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

Per Well:

• rhCPB1: 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). Expressed mainly in pancreas, CPB1 is a useful serum marker for acute pancreatitis (2). The deduced amino acid sequence of human CPB1 consists of a signal peptide (residues 1 to 15), a pro region (residue 16 to 110), and a mature chain (residues 111 to 417). The purified rhCPB1 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, A.J. et al.) p. 831, Academic Press, San Diego.

2. Yamamoto, K.K. et al. (1992) J. Biol. Chem. 267:2575.

• 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