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 >25,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .
Source
E. coli-derived Pro42-Gly403, with an N-terminal Met and 6-His tag Accession # BAA00852
Accession #
N-terminal Sequence
AnalysisMet
Predicted Molecular Mass
40 kDa
SDS-PAGE
42 kDa, reducing conditions
5084-NM |
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Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. | ||
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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Assay Procedure
Materials
Assay Buffer: 50 mM Sodium Acetate, 150 mM NaCl, pH 4.5
Recombinant M. viridifaciens Neuraminidase (rMvNA) (Catalog # 5084-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
Dilute rMvNA to 0.2 ng/µL in Assay Buffer.
Dilute Substrate to 400 µM with Assay Buffer.
Load into plate 50 µL of 0.2 ng/µL rMvNA and start the reaction by adding 50 µL of 400 µM Substrate. Include a Substrate Blank containing 50 µL of Substrate and 50 µL of Assay Buffer.
Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate specific activity:
Specific Activity (pmoles/min/µg) = | Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU) |
| amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard 4-Methylumbelliferone (Sigma, Catalog # M1381).
rMvNA: 0.010 µg
Substrate: 200 µM
Background: Bacterial Neuraminidase
Neuraminidase is the common name for N-acetyl-neuraminyl hydrolase (Sialidase). Micromonospora viridifaciens Neuraminidase catalyzes the hydrolysis of alpha 2-3 and alpha 2-6 and alpha 2-8 linked N-acetyl-neuraminic acid residues from glycoproteins and glycolipids (1). The architecture of the full-length protein includes a canonical neuraminidase enzymatic domain, a linker domain and a C-terminal galactose binding domain (2). The full-length protein contains 647 amino acids and has a molecular weight of 68 kDa. It is easily degraded to 52 kDa and 41 kDa fragments (3). The expressed enzyme only contains the enzymatic domain.
References:
Aisaka, K. & Uwajima, T. (1987) FEBS Microbiol. Lett. 44:289.
Gaskell, A. et al. (1995) Structure 3:1197.
Sakurada, K. et al. (1992) J. Bacteriol. 174:6896.
Alternate Names:
Bacterial Neuraminidase
