Assay of sialyltransferase activity by reversed-phase ion-pair high-performance liquid chromatography

Sialyltransferases (CMP-N-acetylneuraminic acid:glycoprotein sialyltransferases, EC 2.4.99.1) are involved in the transfer of a sialic acid moiety from CMP-N-acetylneuraminic acid (CMP-NeuAc) to an oligosaccharide side-chain of an acceptor, asialoglycoprotein (AGP), according to the following reaction: CMP-NeuAc + AGP----NeuAc-O-AGP + CMP. This enzyme occurs in elevated levels in the sera of patients with a wide variety of neoplastic diseases and its assay might be useful in monitoring treatment. Radioactive CMP-NeuAc has been used in assays and the radioactive sialylated product separated and counted by liquid scintillation spectrometry. This study shows that a simple, rapid, non-radiochemically based high-performance liquid chromatographic method developed for the analysis of CMP-sialic acid synthetase can be used for the quantitation of sialyltransferase activity by monitoring simultaneously the utilization of CMP-NeuAc and the release of CMP. We describe the application of this method to assay of commercially available sialyltransferase activity and to activities from synovial, ascites and gastric fluids.