Alpha(2-3) Neuraminidase - α -D-N- α -D-mannopyranoside -D-galactopyranoside -D-galactopyranoside -D-N-acetylglucosaminide β β α α sodium -D-galactopyranosyl)- mM β -nitrophenyl- -nitrophenyl- -nitrophenyl- -nitrophenyl- -3-O-( D-galactopyranoside (2-3) Neuraminidase is defined as the (2-3) Neuraminidase is tested for (2-3) Neuraminidase is α α sodium phosphate (pH 6.0) provides the sodium phosphate (pH 6.0) provides -N-acetylgalactosaminidase p-nitrophenyl-2-acetamido-2-deoxy -N-acetylglucosaminidase High mannose oligosaccharides β α oligosaccharide to a tube. 5X Reaction buffer 6.0 - 250 5X Reaction buffer phosphate, pH 6.0 mM p -Mannosidase -Galactosidase p -Galactosidase p Contaminant Substrate Contaminant Substrate Endo- β Endo- α N-acetylglucosaminidase α ¾ 1. or 1 nmol of Add up to 100 µg of glycoprotein 2. Add water to a total of 14 µL. Procedure for De-sialylation Procedure One unit of Purity Each lot of amount of enzyme required to produce 1 µmole of to produce amount of enzyme required in 1 minute at 37°C, pH 5.0 methylumbelliferone MU-NANA [2’-(4-methylumbelliferyl)- from contaminating substances by incubating the enzyme contaminating substances indicated in the for 24 hours with the substrates is evident for any No detectable activity table below. The detection limit of these potential contaminants. of these assays is 5 µU/mL (IUB). BSA is 10 µg of denatured assay, For the protease of incubated for 24 hr with 2 µL of enzyme. Analysis should show no the BSA band after SDS-PAGE evidence of degradation. pH Range: 50 optimal buffer for enzyme activity with 3’-siayllactose, optimal buffer is substrate. If glycosidase treatment a standard performed at suboptimal pH because of glycoprotein expect some solubility or activity requirements, diminution in enzyme activity. Suggestions for Use Assay Reagents acetylneuraminic acid]. p (2-3) (2-3) sialic . It is α α (2-3,6,8,9) α (2-3) unbranched sialic α Streptococcus pneumonia 5 U/mL ≥ (2-8) linkages or on branched α sodium phosphate pH 7.5. Structural analysis of oligosaccharides Determining sialic acid linkages desialylation Glycoprotein glycoproteins from Removing heterogeneity mM 150 U/mg, (2-6) or (2-3) Neuraminidase (N-acetylneuraminate (2-3) Neuraminidase is isolated from culture (2-3) Neuraminidase is isolated from (2-3) Neuraminidase is useful for: ¾ ¾ ¾ ¾ Product Description Product Specifications acids (see Figure 1). acids (see Figure acid residues from complex carbohydrates and complex carbohydrates from acid residues activity on is no detectable There glycoproteins. α Molecular Weight ~75,000 Daltons Specificity terminal unbranched Only non-reducing Activity ≥ α EC 3.2.1.18) cleaves exclusively glycohydrolase terminal the non-reducing Storage at 4°C. Do not freeze. Store Formulation solution in as a sterile-filtered The enzyme is provided 50 Stability properly. Stable at least 12 months when stored will to ambient temperatures Several days exposure activity. not reduce Neuraminidase. α supernatants of linkages (Figure 1). To cleave all non-reducing 1). To linkages (Figure including branched sialic terminal sialic acid residues complex acids (linked to an internal from residue) use carbohydrates and glycoproteins, purified free of contaminating exo- and endo- purified free by chromatographic glycosidases and proteases methods standards. α Alpha(2-3) Neuraminidase Alpha(2-3) Alpha(2-3) Neuraminidase (1-4) Glc β 60 µL +4 3 2 (1-4) GlcNAc (2-3) Neuraminidase α β Package Size Temp.°C αα (1-4) Gal β (1-6) Glc NeuAc β structural studies of glycoconjugates. Anal Biochem structural studies of glycoconjugates. 100:1-14 (1979). Streptococcus from purification of five glycosidases 252:8615-8623 (1977). pneumonia. J. Biol Chem J Bacteriol 91:601-3 activity in Diplococcus pneumonia. (1965). sequencing based on exo- C. J. Edge. Oligosaccharide and endoglycosidase digestion and liquid J products. analysis of the chromatographic A 720:263-274 (1996). Chromatogr (1-4) GlcNAc β no cleavage 3. for Kobata, A. Use of endo- and exoglycosidases 4. L R., J. C. Paulson and R. L. Hill. Systematic Glasgow, 5. Neuraminidase and S. Farmer. D. Greiff R. T., Kelly, 6. and R. B. Parekh Venton, A. M. Prime, S. J. Dearnley, (1-3) Gal (1-3) GalNAc β Man β no cleavage Gal 6 (1-6) 2 (1-3) α α α (1-3) GlcNAc β NeuAc (1-4) β Gal (1-2) Man (1-2) Man β β (2-3) α (1-4) GlcNAc β NeuAc no cleavage (1-4) GlcNAc (1-4) GlcNAc (1-4) Glc β β β Gal (2-3) Neuraminidase αα (Streptococcus pneumonia) Gal 3 2 α (2-3) Neuraminidase. (2-3) α α (2-6) Gal (2-3) (1-4) Gal α α β NeuAc NeuAc (2-8) NeuAc NeuAc GalNAc α The specificity of viral and bacterial sialidases for The specificity of viral and sialic acids in alpha(2-3) and alpha(2-6)-linked 744:121-126 Biochim Biophys Acta glycoproteins. (1963). Analysis of glycoprotein-associated and R. B. Parekh. oligosaccharides. Ann Rev Biochem 62:65-100 (1993). 120055-1 Order Information Order Catalog No. Description Product 1. H. Higa, J. C. Paulson and R. Schauer. Cornfield, P., A. 2. M. R. Wormald Dwek, R. A., C. J. Edge, D. J. Harvey, 3. 6.0. 4 µL 5X Reaction Buffer Add 4. Add 2 µL of 5. Incubate at 37°C for 1 hour. Gal = Galactose; Glc = Glucose; Man = Mannose; GalNAc = N-acetylgalactosamine; GlcNAc = N-acetylglucosamine; NeuAc = N-acetylneuraminic Acid (Sialic Acid) De-sialylation may be monitored by SDS-PAGE if the by SDS-PAGE De-sialylation may be monitored and desialylated between native size differential for detection. is sufficient protein References Figure 1Figure (in bold) for - Linkage specificities showing cleavable residues NeuAc PN001606 Rev002