All-in-one assay for β-d-galactoside sialyltransferases: Quantification of productive turnover, error hydrolysis, and site selectivity

Abstract

Sialyltransferases are important enzymes of glycobiology and the related biotechnologies. The development of sialyltransferases calls for access to quick, inexpensive, and robust analytical tools. We have established an assay for simultaneous characterization of sialyltransferase activity, error hydrolysis, and site selectivity. The described assay does not require expensive substrates, is very sensitive (limit of detection=0.3 μU), and is easy to perform. It is based on sialylation of nitrophenyl galactosides; the products thereof are separated and quantified by ion pair reversed phase high-performance liquid chromatography with ultraviolet detection.

Keywords: Assay; Donor hydrolysis; Ion pair chromatography; Sialyltransferase; Site selectivity.