A New Assay For Sialyltransferases Using Fluorescein-Labelled Acceptors



A novel HPLC assay system for sialyltransferase activity based on the use of fluorescein-labelled acceptor oligosaccharides is described. The fluorescein-labelled disaccharides βGal-(1→4)-βGlc-OR (4), βGal-(1→4)-βGlcNAc-OR (17), and βGal-(1→3)-βGlcNAc-OR (22) where OR consists of a six carbon spacer with fluorescein attached, were synthesised. Synthetic standard products were produced chemo-enzymatically on a preparative scale to yield fluorescein-labelled trisaccharides. The use of reverse phase HPLC with an ionpairing agent allowed the separation of starting materials from products and separation of the two isomeric trisaccharides αNeu5Ac-(2→3/6)-βGal-(1→4)-βGlcNAc-OR (24 and 25), so that the assay could be used to measure the different silalyltransferase activities in a mixture. The assay was successfully applied to the detection of sialyltransferase activity of commercially available enzymes and a crude preparation of bovine colostrum. The predominant sialyltransferase activity in bovine colostrum adds sialic acid α(2–6) to 17.