A New Assay For Sialyltransferases Using Fluorescein‐Labelled Acceptors

Abstract

Abstract 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 .