Activity Determination of Glycosyltransferases

  • Kalyan R. Anumula
Part of the Methods in Molecular Biology book series (MIMB, volume 1934)


The unique chemistry of 2-aminobenzoic acid (2-AA, anthranilic acid, AA) for labeling glycans in aqueous buffer solutions was crucial in developing the assays for measuring the activity of transferases (Anumula, Anal Biochem 457:31–37, 2014). N-acetylglucosamine and N-acetyllactosamine were used as acceptors, and UDP-galactose and CMP-N-acetylneuraminic acid as donors for measuring the activity of β1-4 galactosyltransferases (GalT-1) and α2-6 sialyltransferase (ST-6), respectively. Products formed were labeled in situ with 2-AA and separated from the substrates on a normal-phase TSKgel Amide 80 column. Activity units were determined by comparison of the peak areas to the concomitantly derivatized standards (Galβ1-4GlcNAc and NANAα2-6Galβ1-4GlcNAc). Performance of the assays was determined by linearity (time and enzyme concentration), precision (intra- and inter-assay), and reproducibility. The fluorescence-based HPLC assay described here was highly sensitive and performed equal to or better than traditional radioactive sugar-based measurements. This assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling.

Key words

Glycosyltransferase Fluorescence Assay 2-AA Galactosyltransferase Sialyltransferase Oligosaccharides Glycans HPLC Fluorescence Anthranilic acid Profiling 


  1. 1.
    Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3:97–130CrossRefGoogle Scholar
  2. 2.
    Dwek RA (1996) Toward understanding the function of sugars. Chem Rev 96:683–720CrossRefGoogle Scholar
  3. 3.
    Jefferis R (2005) Glycosylation of recombinant antibody therapeutics. Biotechnol Prog 21:11–16CrossRefGoogle Scholar
  4. 4.
    Anthony RM, Nimmerjahn F, Ashline DJ et al (2008) Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320:373CrossRefGoogle Scholar
  5. 5.
    Nimmerjahn F, Ravetch JV (2008) Fc gamma receptors as regulators of immune responses. Nat Rev Immunol 8:34CrossRefGoogle Scholar
  6. 6.
    Dalziel M, Crispin M, Scanlan CN et al (2014) Emerging principles for the therapeutic exploitation of glycosylation. Science 3:343Google Scholar
  7. 7.
    Raju TS, Briggs JB, Chamow SM et al (2001) Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues. Biochemistry 40:8868–8876CrossRefGoogle Scholar
  8. 8.
    Anumula KR (2014) Single tag for total carbohydrate analysis. Anal Biochem 457:31–37CrossRefGoogle Scholar
  9. 9.
    Anumula KR (2012) New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection. Glycobiology 22:912–917CrossRefGoogle Scholar
  10. 10.
    Anumula KR (2012) Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc. J Immunol Methods 382:167–176CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kalyan R. Anumula
    • 1
  1. 1.Glycan TechnologiesNorth WalesUSA

Personalised recommendations