A Fluorescent Assay Method for GDP-L-Fuc: N-Acetyl-β-D-Glucosaminide α1-6Fucosyltransferase Activity, Involving High Performance Liquid Chromatography

Abstract

An assay method for GDP-L-Fuc: N-acetyl-β-D-glucosaminide α1-6fucosyltransferase (α1-6FucT; EC 2. 4. 1. 68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine γ-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. _??_ The substrate and guanosine diphospho-fucopyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HPLC. Quantitation of the product involved measurement of the fluorescence intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGnF-bi-Asn-PABA (product) were analyzed by ¹H NMR. The enzymatic product was also analyzed by ¹H NMR and was found to have α1-6fucose at the reducing end GlcNAc. This method is highly specific for α1-6FucT and is applicable for various experiments, including purification and cell culture ones.