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-(14)-βGlc-OR (4), βGal-(14)-βGlcNAc-OR (17), and βGal-(13)-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-(23/6)-βGal-(14)-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.