An assay method for GDP-L-Fuc: N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase (alpha 1-GFucT; EC 2.4.1.68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine gamma-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained.
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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 H-1 NMR. The enzymatic product was also analyzed by H-1 NMR and was found to have alpha 1-glucose at the reducing end GlcNAc. This method is highly specific for alpha 1-GFucT and is applicable for various experiments, including purification and cell culture ones.