Recombinant Human Carbonic Anhydrase VA Protein, CF

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

Recombinant Human Carbonic Anhydrase VA Protein, CF Summary

• Details of Functionality: Measured by its esterase activity. The specific activity is >100 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived human Carbonic Anhydrase VA/CA5A protein
  Ala40-Ser305, with a C-terminal 10-His tag
• Accession #: NP_001730
• N-terminal Sequence: Ala40
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CA5A
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 31 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: 32 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 under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 12.5 mM Tris, 75 mM NaCl, 0.05% (v/v) Brij-35, pH 7.5
  • Recombinant Human Carbonic Anhydrase VA (rhCA5A) (Catalog # 3049-CA)
  • Substrate: 4-Nitrophenyl acetate (4-NPA) (Sigma, Catalog # N8130), 100 mM stock in acetone
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhCA5A to 20 ng/µL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. In a plate, load 50 µL of 20 ng/µL rhCA5A, and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 2 mM Substrate.
  4. Read at a wavelength of 400 nm (bottom read) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x Conversion Factor** (pmol/OD)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using calibration standard 4-Nitrophenol (Sigma, Catalog # 241326).

  Per Well:
  • rhCA5A: 1 µg
  • Substrate: 1 mM

Notes

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

Alternate Names for Recombinant Human Carbonic Anhydrase VA Protein, CF

• CA5A
• CA5carbonic anhydrase 5A, mitochondrial
• Carbonate dehydratase VA
• carbonic anhydrase V, mitochondrial
• Carbonic Anhydrase VA
• carbonic anhydrase VA, mitochondrial
• carbonic dehydratase
• CAV
• CAVA
• CA-VA
• EC 4.2.1.1

Background

Carbonic Anhydrase catalyzes the reversible reaction of CO₂ + H₂O = HCO₃^- + H⁺, which is fundamental to many processes such as respiration, renal tubular acidification and bone resorption (1). Topics in a CA meeting (6^th International Conference on the CAs, June 20 - 25, 2003, Slovakia) ranged from the use of CAs as markers for tumor and hypoxia in the clinic, as a nutritional supplement in milk, and as a tool for CO₂ removal and mosquito control in industry. Carbonic Anhydrase VA encoded by the CA5A gene is a mitochondrial protein (2, 3). In comparison with another mitochondrial CA (CA5B), CA5A has different tissue distribution and chromosomal location (4, 5). Expression and inhibitor studies of different CAs in the rat pancreatic beta cells indicate that CA5A may be involved in the regulation of insulin secretion (6). CA5A may also participate in the detoxification of ammonia produced in the gastrointestinal tract by providing bicarbonate to carbamyl phosphate synthetase I (7). The amino acid sequence of recombinant human CA5A (residues 40 to 305) is 79%, 77%, and 76% identical to that of canine, bovine, and rat/mouse.

1. Hewett-Emmett, D. and R.E. Tashian (1996) Mol. Phylogenet. Evol. 5:50.
2. Nagao, Y. et al. (1993) Proc. Natl. Acad. Sci. USA 90:7623.
3. Nagao, Y. et al. (1995) Genomics 28:477.
4. Shah, G.N. et al. (2000) Proc. Natl. Acad. Sci. USA 97:1677.
5. Fujikawa-Adachi, K. et al. (1999) J. Biol. Chem. 274:21228.
6. Parkkila, A.K. et al. (1998) J. Biol. Chem. 273:24620.
7. Saarnio, J. et al. (1999) J. Histochem. Cytochem. 47:517.

Bioinformatics

• Gene Symbol: CA5A
• Uniprot:
  • NP_001730()