A fluorometric assay for glycosyltransferase activities using sugars aminated and tagged with 7-hydroxycoumarin-3-carboxylic acid as substrates and high performance liquid chromatography

Abstract

We developed a novel fluorometric assay method for the measurement of glycosyltransferase activities using mono- and di-saccharides aminated and tagged with 7-hydroxycoumarin-3-carboxylic acid (coumarin) as substrates, N- acetylglucosamine (GlcNAc)-coumarin for β1,4-galactosyltransferase from bovine milk and Galβ1-4GlcNAc-coumarin for α2,3- and α2,6- sialyltransferases from rat liver. Using Galβ1-3GlcNAc and Galβ1-4GlcNAc- coumarin, α1, 3/4 /4- and 1/4 ,3-fucosyltransferase activities were also determined, respectively. These enzymatic products liberated by the reactions of glycosyltransferases in the presence of sugar nucleotides, were separated by a normal phase or an ion-pair reversed phase HPLC with an isocratic elution and fluorescence detection. We applied this assay method to determine the glycosyltransferase activities in 8 kinds of human tumor cell lines, including the cell lines derived from hepatocytes (HuH-7, HepG2), colonic cells (Colo205, HT-29), myelocytes (HL-60, U-937), B-lymphocytes (Daudi) and T-lymphocytes (Jurkat). This assay method is accurate and easy compared with other isotopic and non-isotopic assay methods, and is sensitive enough to measure glycosyltransferase activities in cell homogenates.