Description
1 set of this bundle includes 2,000,000 units of O-Glycosidase and 2,000 units of Neuraminidase.
O-Glycosidase, also known as Endo-α-N-Acetylgalactosaminidase, catalyzes the removal of
Core 1 and Core 3 O-linked disaccharides from glycoproteins.
Neuraminidase is the common name for Acetyl-neuraminyl hydrolase ( Sialidase ). This Neura-minidase catalyzes the hydrolysis of α2-3, α2-6, α2-8 linked N-acetyl-neuraminic acid residues
from glycoproteins and oligosaccharides.
Product Source
O-Glycosidase is cloned from Enterococcus faecalis and expressed in E.coli (1). Neuraminidase
is cloned from Clostridium perfringens (2) and overexpressed in E. coli (3).
Reagents Supplied
The following reagents are supplied with this product:
Properties and Usage
Unit Definition
One unit of O-Glycosidase is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of Neuraminidase digested, non-denatured fetuin in 1 hour at 37°C in a total reaction volume of 100 µl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively).
Non-denaturing Unit Definition of O-Glycosidase:
Two fold serial dilutions of O-Glycosidase are added to a reaction mixture of 5 mg of Neura-minidase digested fetuin with 1 X GlycoBuffer 2. The reaction is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by Morgan and Elson Assay (4). Note: Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent.
Unit Definition of Neuraminidase:
One unit of Neuraminidase is defined as the amount of enzyme required to cleave > 95% of the terminal α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl- coumarin (AMC), in 5 minutes at 37°C in a total reaction volume of 10 µl.
1X Glycoprotein Denaturing Buffer
0.5% SDS
40 mM DTT
1X NP-40
1% NP-40 in MilliQ-H2O
Reaction Conditions
1X GlycoBuffer 2
Incubate at 37°C
1X GlycoBuffer 2:
50 mM sodium phosphate
pH 7.5 @ 25°C
Heat Inactivation
Related Products
Companion Products
α2-3,6,8 Neuraminidase
O-Glycosidase
PNGase F
PNGase F (Glycerol-free)
Endo H
Endo Hf
Notes
1.Since O-Glycosidase is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present time. Double digest with Endo H must have NP-40 present (NP-40 does not inhibit Endo H).
2.To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
References
1.Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.
2.Roggentin, P. et al. (1988). FEBS Lett. 238, 31-34.
3.Guan, C. unpublished observations. New England Biolabs.
4.Morgan, W.T.J. and Elson, L.A. (1934). Biochem. J. 28, 988-995.
