• 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 fluorogenic substrate, 2’-(4-Methylumbelliferyl)-alpha -D-N-acetylneuraminic acid. The specific activity is >80,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Cys2-Gln382, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  44 kDa

• SDS-PAGE

  40 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM MES, 100 mM NaCl, 0.05% (w/v) Brij-35, pH 6.5

• Recombinant C. perfringens Neuraminidase (rCpNA) (Catalog # 5080-NM)

• Substrate: 2’-(4-Methylumbelliferyl)-alpha -D-N-acetylneuraminic acid (Sigma, Catalog # M8639), 10 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rCpNA to 0.04 ng/μL in Assay Buffer.

2. Dilute Substrate to 400 μM in Assay Buffer.

3. Load 50 μL of 0.04 ng/μL rCpNA into a plate, and start the reaction by adding 50 μL of 400 μM Substrate.

4. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard 4-Methylumbelliferone (Sigma, Catalog # M1381).

Per Well:

• rCpNA: 0.002 μg

• Substrate: 200 μM

Background: Bacterial Neuraminidase

Neuraminidase and Sialidase are the common names for N?acetyl?neuraminyl hydrolase (1-3). The enzyme catalyzes the hydrolysis of alpha 2-3 and alpha 2-6 linked N?acetyl?neuraminic acid residues from glycoproteins and glycolipids. It has little activity against the alpha 2-8 linked N?acetyl?neuraminic acid residues.

• References:

1. Christensen, S. et al. (2005) Biotechnol. Appl. Biochem. 41:225.

2. Roggentin, P. et al. (1988) FEBS Lett. 238:31.

3. Corfield, A. et al. (1983) Biochim. Biophys. Acta 744:121.

• Alternate Names:

  Bacterial Neuraminidase