O-Glycosidase

Part number E-G001
Source Recombinant Streptococcus pneumoniae in E.Coli
EC 3.2.1.97

Contents
60 µl aliquot of enzyme (75 mU) in 50 mM sodium phosphate, pH 7.5.
5x Reaction Buffer 5.0 (250 mM sodium phosphate, pH 5.0)

Specific Activity >12 U/mg
Activity 1.25 U/ml

Molecular weight 180,000 daltons
pH range 5-7, optimum 5.0

Suggested usage
1. Add up to 100 µg of glycoprotein to a tube.
2. Add de-ionized water to a total of 13 µl.
3. Add 4 µl of 5x Reaction Buffer 5.0.
4. Add 1 µl of Sialadase(a 2-,3,6,8,9), part number E-S001.
5. Add 2 µl of O-Glycosidase.
6. Incubate for 1 hour at 37°C.

Specifictity Cleaves only unsubstituted Gal-ß(1-3)GalNAc-alpha disaccharides attached to the serine or threonine residues of glycoproteins or glycopeptides. Substitutions such as sialic acid, galactose, fucose or N-acetylglucosamine must first be removed with the appropriate exoglycosidase prior to treatment with Endo-O-Glycosidase.
At minimum, a sialadase such as Sialidase(alpha-2-3,6,8,9), part number E-S001, is almost always required to remove sialic acids

Specific Activity Defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pNP) in 1 minute at 37°C, pH 5.0 from p-nitrophenyl- 2-acetamido-2-deoxy-3-O-(ß-D-galactopyranosyl)-alpha- D-galactopyranoside.

Storage Store enzyme at 4°C.

References
Bhavanandan, V.P., J. Umemoto and E.A. Davidson. Characterization of an endo-alpha-N-acetylgalactosaminidase from Diplococcus pneumoniae. Biochem Biophys 70: 738-74 5 (1976 ).

Fan, J.Q., K. Yamamoto, H. Kumagai and T. Tochikura. Induction and efficient purification of endo-alpha-N-acetyl- D-galactosaminidase from Alcaligenes sp. Agric Biol Chem 54: 233-23 4 (1990 ).

Glasgow, L R., J.C. Paulson and R.L. Hill. Systematic purification of five glycosidases from Streptococcus pneumoniae. J Biol Chem 252: 8615-8 623 (1977).

Iwase, H. and K . Hotta. Release of O-linked glycoprotein glycans by endo-alpha-N-acetyl-D-galactosaminidase. Meth Mol Biol 14: 151-159 (1993).

Unemoto, J., V.P. Bhavanandan and E.A. Davidson. Purification and properties of an endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae. J Biol Chem 252: 8609-8 614 (1977).