Endoglycosidase H (Endo-b-N-acetylglucosaminidase H)

Suitable for studies of glycopeptide and glycoprotein with пїЅHigh MannoseпїЅ sugar chains. \n\nEnzyme Commission No: EC 3.2.1.96\n\nTrivial Name: Endo-b-N-acetylglucosaminidase H\n\nSystematic Name: Mannosyl-glycoprotein-1, 4-N-acetamidodeoxy-b-D-glycohydrolase \n\nStabilizer: < 1mg/0.1u BSA (bovine serum albumin \n\nActivity: (same/more than)0.1units\n\nProtein: (same/more than)6ug/vial\n \nSpecific Activity: \n~30 units/mg protein \n\nContaminants: \nNo detectable amount of various exoglycosidases or proteases\n\nQuality Control: Endoglycosidase H (0.01 unit) was incubated with appropriate p-nitrophenylglycosides (0.1mg) in 0.2ml of citrate-phosphate buffer (pH 5.0) at 37 degrees C for 12 hours. No activites of a- and b-galactosidase or a-L-fucosidase were observed. Absence of contaminating a-mannosidase was also confirmed by using oligosaccharides from ovalbumin and yeast mannan.\n\nEndoglycosidase H (0.01 unit) was incubated with 10mg casein in 1ml of 0.1M Tris-CHl buffer at 37 degrees C for 20 hours. 1ml of 10% TCA was added. Release of acid soluble material was below the limit of detection (OD275 less than 0.05).\n\nTo inhibit potential protease activity during extended Endo-H digestions, 1mM PMSF and/or 10mM pepstatin A may be added to incubations. PMSF is stable as a 2mM stock solution in absolute methanol. \n\nOptimum pH: 5.0 \n\nKm Value: 0.44mM toward [14C]-acetyl-Asn-GlcNAc2Man6 \n\nUnit Definition: \nOne unit is that amount of enzyme required to hydrolyze 1mmole (Man)5-\n(GlcNAc)2Asn- [14C]-acetyl, pH 5.0 and 37 degrees C per minute at the saturated substrate concentration using MuramatsuпїЅs method.\n\nStorage and Stability: \nLyophilized powder may be stored at -20 degrees C. Stable for 12 months at -20 degrees C. Reconstitute with sterile ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20 degrees C. Reconstituted product is stable at pH 5.0пїЅ9.0 at 37 degrees C for 20 hours. Stable for 6 months at -20 degrees C. Repeated freezing and thawing may decrease the enzyme acitivity.

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SPECIFICATIONS

Catalog Number

E2290-10

Size

100mU

Form

Supplied as a lyophilized powder from 10mM Tris-HCl, pH 7.2 Reconstitute with 100ul sterile ddH2O.

Purity

Purified from the culture fluid of Streptococcus griseus using DEAE-Sephadex and Sephadex G-75.

References

1. Arakawa, M. and Muramatsu, T., (1974) J. Biochem. 76:307. 2. Tarentino, A. L. and Maley, F., (1974) J. Biol. Chem. 249:811. 3. Tarentino, A. L., et al., (1974) J. Biol. Chem. 249:818. 4. Koide, N. and Muramatsu, T. (1974) J. Biol. Chem. 249:4897. 5. Muramatsu, T. et al., (1978) Methods in Enzymol. 50:555. 6. Spiro, R. G., (1973) Adv. Protein Chem. 27:349. 7. Ashwell, G. and Morell, A. G., (1974) Advan Enzymol. 41:99. 8. Muramatsu, T., (1971) J. Biol. Chem. 246:5535. 9. Tai, T., et al., (1975) J. Biol. Chem. 250:8569. 10. Tarentino, A. L., et al., (1972) J. Biol. Chem. 247:2629. 11. Tarentino, A. L., et al., (1973) J. Biol. Chem. 245:5547. 12. Muramatsu, T., et al., (1976) J. Biol. Chem. 251:4673.