A NEW ASSAY FOR SIALYLTRANSFERASES USING FLUORESCEIN-LABELED ACCEPTORS

A novel HPLC assay system for sialyltransferase activity based on the use of fluorescein-labelled acceptor oligosaccharides is described. Th e fluorescein-labelled disaccharides beta Gal-(1-->4)-beta Glc-OR (4), beta Gal-(1-->4)-beta GlcNAc-OR (17), and beta Gal-(1-->3)-beta GlcNA c-OR (22) where OR consists of a six carbon spacer with fluorescein at tached, were synthesised. Synthetic standard products were produced ch emo-enzymatically on a preparative scale to yield fluorescein-labelled trisaccharides. The use of reverse phase HPLC with an ion-pairing age nt allowed the separation of starting materials from products and sepa ration of the two isomeric trisaccharides alpha Neu5Ac-(2-->3/6)-beta- beta Gal-(1-->4)-beta GlcNAc-OR (24 and 25), so that the assay could b e used to measure the different sialyltransferase activities in a mixt ure. The assay was successfully applied to the detection of sialyltran sferase activity of commercially available enzymes and a crude prepara tion of bovine colostrum. The predominant sialyltransferase activity i n bovine colostrum adds sialic acid alpha(2-6) to 17.