• 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 esterase activity. The specific activity is >2 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Ala19-Lys283, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala19

• Predicted Molecular Mass

  32 kDa

• SDS-PAGE

  33 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 12.5 mM Tris, 75 mM NaCl, pH 7.5

• Absorbance Buffer: 12.5 mM Tris, pH 8.0

• Recombinant Human Carbonic Carbonic Anhydrase IV/CA4 (rhCA4) (Catalog # 2186-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 rhCA4 to 40 ng/μL in Assay Buffer.

2. Dilute Substrate to 2.0 mM in Assay Buffer.

3. Load 50 μL of 40 ng/μL rhCA4 into plate and start the reaction by adding 50 μL of 2.0 mM Substrate. As a Substrate Blank combine 50 μL Assay Buffer with 50 μL Substrate.

4. Cover and incubate at room temperature for 60 minutes in the dark.

5. After incubation add 100 μL Absorbance Buffer and read plate immediately.

6. Read (top read) absorbance in endpoint mode at 410 nm.

7. Calculate specific activity:

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

Per Well:

• rhCA4: 2 μg

• Substrate: 0.5 mM

Background: Carbonic Anhydrase IV/CA4

Carbonic Anhydrase (CA) 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. CA4 is a GPI?anchored membrane enzyme expressed on the luminal surfaces of pulmonary (and certain other) capillaries and of proximal renal tubules. It functions as the principal CO₂ taste sensor (2). In addition, a genetic mutation (Arg 14 to Trp in the signal peptide) of CA4 was found to cosegregate with the RP17 form of retinitis pigmentosa in two large families and was not found in 36 unaffected family members or 100 controls (3).

• References:

1. Hewett-Emmett, D. and R.E. Tashian (1996) Mol. Phylogenet. Evol. 5:50.

2. Chandrashekar, J. et al. (2009) Science 326:443.

3. Rebello, G. et al. (2004) Proc. Natl. Acad. Sci. USA 101:6617.

• Entrez Gene IDs:

  762 (Human); 12351 (Mouse)

• Alternate Names:

  CA4; CAIV; CA-IV; Car4; Carbonate dehydratase IV; carbonic anhydrase 4; Carbonic Anhydrase IV; carbonic anhydrase IVRP17; carbonic dehydratase IV; EC 4.2.1.1; retinitis pigmentosa 17 (autosomal dominant); RP17