A fluorescent assay for the determination of UDP-GlcNAc: Gal beta 1,3GalNAc-R (GlcNAc to GalNAc) beta 1,6-N-acetylglucosaminyltransferase activity.

A fluorescent assay for UDP-GlcNAc: Gal beta 1,3Gal-NAc-R beta 1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) activity has been developed involving dansylation of the enzyme reaction product. Core 2 GlcNAc-T detection was performed using unlabeled UDP-GlcNAc as the donor and Gal beta 1,3GalNAc alpha-pAp as the acceptor. The product, Gal beta 1,3(GlcNAc beta 1,6)-GalNAc alpha-pAp, was quantitatively derivatized with dansyl chloride at the NH2 moiety of the pAp group and the resultant fluorescent trisaccharide was separated on a Spherisorb ODS2 HPLC column. This method, rapid and economical, was found to be sensitive enough for the detection of 1 pmol of reaction product and therefore represents a reliable alternative to assays which use radiolabeled substrates. Additionally, the approach described here can be adapted for the assay of other glycosyltransferases, where the acceptor substrate has a pAp group as a hydrophobic aglycon linker.