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  • Title: Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H.
    Author: Trimble RB, Maley F.
    Journal: Anal Biochem; 1984 Sep; 141(2):515-22. PubMed ID: 6437277.
    Abstract:
    The ability of endo-beta-acetylglucosaminidase H (Endo H) from Streptomyces plicatus to hydrolyze high-mannose oligosaccharides from glycoproteins is influenced by numerous factors, including the tertiary structure of the substrate glycoproteins, the amount of Endo H used, the time of incubation, and the presence or absence of reagents that affect protein configuration. Endo H levels below 10 to 20 milliunits/ml may incompletely hydrolyze oligosaccharides, regardless of the incubation time, because even though the enzyme remains active, it becomes trapped or sequestered and is unavailable. Endo H activity can be potentiated by first denaturing substrate glycoproteins in a 1.2-fold weight excess of sodium dodecyl sulfate prior to hydrolysis. However, low levels of Endo H are sensitive to inactivation by sodium dodecyl sulfate, with considerable activity being lost over 4 h when the unbound detergent concentration exceeds protein by 0.02% (0.2 mg/ml). Other denaturants such as the Tritons, the zwittergents, the Brij series, or octylglucoside do not enhance or inhibit Endo H removal of oligosaccharides, but the chaotropic salt sodium thiocyanate at 0.5 M enhances Endo H action on some glycoproteins, particularly bovine thyroglobulin. Under denaturing conditions, proteolytic contaminants are a potential problem. Addition of 1 mM phenylmethylsulfonyl fluoride to Endo H incubations completely inhibits the residual Endo H-associated protease(s). Furthermore, Endo H is unaffected by a wide range of proteolytic inhibitors that may be used to protect substrate glycoproteins.
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