Activity Determination of Glycosyltransferases
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 wordsGlycosyltransferase Fluorescence Assay 2-AA Galactosyltransferase Sialyltransferase Oligosaccharides Glycans HPLC Fluorescence Anthranilic acid Profiling
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