Thin-layer chromatography and polyacrylamide gel electrophoresis-based assays for sialyltransferases using tetramethylrhodamine-labeled acceptors

Two novel assay systems for the determination of sialyltransferase activity using a tetramethylrhodamine-labeled disaccharide Gal beta 1-4GlcNAc (2) a s the acceptor are described, The TMR-labeled disaccharide 2 was synthesize d by directly coupling Gal beta 1-4GlcNAc-O-(CH2)(6)NH2 (1) with 5-tetramet hylrhodamine N-hydroxysuccinimide ester. The K-m value for compound 2 obtai ned with alpha-2,6-sialyltransferase from rat liver (Ee 2.4.99.1) was 160 /- 20 mu M. After incubation of compound 2 with sialyltransferase the produ ct and the unreacted acceptor substrate were separated either by thin-layer chromatography (TLC) on C-18 silica gel plates or by polyacrylamide gel el ectrophoresis (PAGE). The density of the spots on the TLC plates and the fl uorescence of the bands on the gel were quantified. The assay conditions we re optimized using crude bovine colostrum extract and also alpha-2,6-sialyl transferase from rat liver. The detection limits for the TLC and PAGE assay s were 1 and 0.4 mu U of the rat liver enzyme, respectively. Either assay a llows the parallel investigation of several samples at a time and is useful for the testing of fractions during enzyme purification. (C) 2000 Academic Press.