Recombinant Mouse Carboxypeptidase A1/CPA1 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Carboxypeptidase A1/CPA1 Protein, CF Summary

• Details of Functionality: 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.
• Source: Mouse myeloma cell line, NS0-derived mouse Carboxypeptidase A1/CPA1 protein
  Asn17-Tyr419, with a C-terminal 10-His tag
• Accession #: Q7TPZ8
• N-terminal Sequence: Asn17
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Cpa1
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 47 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 42 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • 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:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Carboxypeptidase A1/CPA1 Protein, CF

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

Background

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.

1. Auld, D.S. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) p. 812  Academic Press, San Diego.

Bioinformatics

• Gene Symbol: Cpa1
• Uniprot:
  • Q7TPZ8()