1. Robbins P. W., D. F. Wirth and C. J. Hering. Expression of the Streptomyces enzyme endoglycosidase H in Escherichia coli. Biol Chem 256:10640-10644 (1981).
2. Robbins P. W., R. B. Trimble, D. F. Wirth, C. Hering, F. Maley , G. F. Maley, R. Das, B. W. Gibson, N. Royal and K. Biemann. Primary structure of the Streptomyces enzyme endo-beta-N-acetylglucosaminidase H. J Biol Chem 259:7577-7583 (1984).
3. Trimble R. B., A. L. Tarentino , G. E Aumick and F. Maley. Endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus. Methods Enzymol 83:603-610 (1982).
4. Trimble R. B. and F. Maley. Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H. Anal Biochem 141:515-522 (1984).
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Endo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.
Kit includes enzyme plus reaction buffers. Sufficient for up to 60 reactions.
Source: recombinant from Streptomyces plicatus in E.Coli
Alternative Names: Endo H, endo-β-N-acetylglucosaminidase H, Endoglycosidase H
Specific Activity: One unit of Endo H activity is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 µmole of denatured Ribonuclease B. Cleavage is monitored by SDS-PAGE (cleaved Ribonuclease B migrates faster).
60 µL aliquot of enzyme (300 mU) in 20 mM Tris-HCl, 25 mM NaCl, 1 mM EDTA (pH 7.5).
200 µL 5x Reaction Buffer 5.5 (250 mM sodium phosphate, pH 5.5)
Denaturation Solution - 2% SDS, 1 M Beta-mercaptoethanol
Specific Activity: >40 U/mg
Activity: >5 U/mL
Molecular weight: 29,000 daltons
pH range: 5-6, optimum 5.5
1. Add up to 200 µg of glycoprotein to Eppendorf tube. Adjust to 37.5 µL final volume with deionized water.
2. Add 10 µL 5X Endo H Buffer and 2.5 µL of Denaturation Solution (SDS/-ME). Heat at 100°C for 5 minutes.
3. Cool, and then add 2.0 µL of Endo H to the reaction. Incubate 3 hours at 37°C.
Storage: Store enzyme at 4°C.
Stability: Stable at least 12 months when stored properly. Several days exposure to ambient tempertures will not reduce activity.
Purity: Endoglycosidase H is tested for contaminating protease as follows; 10 µg of denatured BSA is incubated for 24 hours at 37°C with 2 µL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.
The production host strain has been extensively tested and does not produce any detectable glycosidases.