HPAEC-PAD Analysis of Monosaccharides Released by Exoglycosidase Digestion Using the CarboPac MA1 Column

  • Michael Weitzhandler
  • Jeffrey Rohrer
  • James R. Thayer
  • Nebojsa Avdalovic
Part of the Springer Protocols Handbooks book series (SPH)


Exoglycosidases are useful reagents for the structural determination of glycoconjugates. Their anomeric, residue, and linkage specificity for terminal monosaccharides have been used to assess monosaccharide sequence and structure in a variety of glycoconjugates (1). Their usefulness depends on the absence of contaminating exoglycosidases and an understanding of their specificity. Digestions of oligosaccharides with exoglycosidases give two classes of products: monosaccharides and the shortened oligosaccharides. Most assays of such reactions have monitored the reaction by following oligosaccharides that are labeled at their reducing ends. In these assays, after exoglycosidase digestion the shortened oligosaccharide retains the label at its reducing end. The other digestion product, the released monosaccharide, does not carry a label and thus cannot be quantified. Additionally, identification of any other monosaccharide that could be the result of a contaminating exoglycosidase activity would not be possible. Quantitative measurement of all products (all released monosaccharide [s] as well as the shortened oligosaccharide product) would be useful because it would enable the determination of any contaminating exoglycosidase activities by determining the extent of release of other monosaccharides. High pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) detects the appearance of monosaccharide product(s), the shortened oligosaccharide product(s) as well as the disappearance of the oligosaccharide substrate(s) in a single chromatographic analysis without labeling. Thus, HPAEC-PAD has been used extensively to monitor the activities of several different exoglycosidases on glycoconjugates, usually using the CarboPac PA1 column to separate the digestion products (see refs. 1-8).


Digestion Product Jack Bean Terminal GlcNAc Therapeutic Glycoprotein Exoglycosidase Digestion 
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  1. 1.
    Jacob, J. S. and Scudder, P. (1994) Glycosidases in structural analysis. Meth. Enzymol. 230, 280–299.PubMedCrossRefGoogle Scholar
  2. 2.
    Scudder, P., Neville, D. C. A., Butters, T. D., Fleet, G. W. J., Dwek, R. A., Rademacher, T. W., and Jacob, G. S. (1990) The isolation by ligand affinity chromatography of a novel form of α-L-fucosidase from almond. J. Biol. Chem. 265, 16,472–16,477.PubMedGoogle Scholar
  3. 3.
    Butters, T. D., Scudder, P., Willenbrock, F. W., Rotsaert, J. M. V., Rademacher, T., Dwek, R. A., and Jacob, G. S. (1989) A serial affinity chromatographic method for the purification of charonia lampas α-L-fucosidase, in Proceedings of the 10th International Symposium on Glycoconjugates, Sept. 10-15 (Sharon, N., Lis, H., Duksin, D., and Kahane, I., eds.), Magnes, Jerusalem, Israel, pp. 312,313.Google Scholar
  4. 4.
    Davidson, D. J. and Castellino, F. J. (1991) Structures of the asparagine-289-linked oligosaccharides assembled on recombinant human plasminogen expressed in a Mamestra brassicae cell line (IZD-MBO503). Biochemistry 30, 6689–6696.PubMedCrossRefGoogle Scholar
  5. 5.
    Grollman, E. F., Saji, M., Shimura, Y., Lau, J. T., and Ashwell, G. (1993) Thyrotropin regulation of sialic acid expression in rat thyroid cells. J. Biol. Chem. 268, 3604–3609.PubMedGoogle Scholar
  6. 6.
    Willenbrock, F. W., Neville, D. C. A., Jacob, G. S., and Scudder, P. (1991) The use of HPLC-pulsed amperometry for the characterization and assay of glycosidases and glycosyltransferases. Glycobiology 1, 223–227.PubMedCrossRefGoogle Scholar
  7. 7.
    Weitzhandler, M., Hardy, M., Co, M. S., and Avdalovic, N. (1994) Analysis of carbohydrates on IgG preparations,. J. Pharm. Sci. 83, 1670–1675.PubMedCrossRefGoogle Scholar
  8. 8.
    Lin, A. I., Philipsberg, G. A., and Haltiwanger, R. S. (1994) Core fucosylation of highmannose-type oligosaccharides in GlcNAc transferase I-deficient (Lec 1) CHO cells. Glycobiology 4, 895–901.PubMedCrossRefGoogle Scholar
  9. 9.
    Hardy, M. R., Townsend, R. R., and Lee, Y. C. (1988) Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. Analyt. Biochem. 170, 54–62.PubMedCrossRefGoogle Scholar
  10. 10.
    Chou, T. Y., Dang, C. V., and Hart, G. W. (1995) Glycosylation of the c-Myc transactivation domain,. Proc. Natl. Acad. Sci. USA 92, 4417–4421.PubMedCrossRefGoogle Scholar
  11. 11.
    Lin, A. I., Polk, C., Xiang, W. K., Philipsberg, G. A., and Haltiwanger, R. S. (1993) Novel fucosylation pathways in parental and GlcNAc transferase I deficient (Lec 1) CHO cells. Glycobiology 3, 524 (Abstract).Google Scholar
  12. 12.
    Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130.PubMedCrossRefGoogle Scholar
  13. 13.
    Yamashita, K., Ohkura, T., Yoshima, H. and Kobata, A. (1981) Substrate specificity of Diplococcal β-N-acetylhexosaminidase, a useful enzyme for the structural studies of complex type asparagine-linked sugar chains. Biochem. Biophys. Res. Commun. 100, 226–232.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Michael Weitzhandler
    • 1
  • Jeffrey Rohrer
    • 1
  • James R. Thayer
    • 1
  • Nebojsa Avdalovic
    • 2
  1. 1.Dionex CorporationLife Science Research GroupSunnyvale
  2. 2.Dionex Corporation, Life Science Research GroupSunnyvale

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