Thin-Layer Chromatography and Polyacrylamide Gel Electrophoresis-Based Assays for Sialyltransferases Using Tetramethylrhodamine-Labeled Acceptors

• Published in: Analytical biochemistry 2000-10, Vol.285 (1), p.92-99
• Main Authors: Hubl, Ulrike, Slim, George C., Zubkova, Olga V.
• Format: Article
• Language: English
• Subjects: Animals; Carbohydrate Sequence; Cattle; Chromatography, Thin Layer - methods; Electrophoresis, Polyacrylamide Gel - methods; polyacrylamide gel electrophoresis; Rats; Rhodamines - chemistry; Sensitivity and Specificity; sialyltransferase; Sialyltransferases - analysis; tetramethylrhodamine-labeled acceptor; thin-layer chromatography
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2000.4727

Two novel assay systems for the determination of sialyltransferase activity using a tetramethylrhodamine-labeled disaccharide Galβ1-4GlcNAc (2) as the acceptor are described. The TMR-labeled disaccharide 2 was synthesized by directly coupling Galβ1-4GlcNAc-O-(CH2)6NH2 (1) with 5-tetramethylrhodamine N-hydroxysuccinimide ester. The Km value for compound 2 obtained with α-2,6-sialyltransferase from rat liver (EC 2.4.99.1) was 160 ± 20 μM. After incubation of compound 2 with sialyltransferase the product and the unreacted acceptor substrate were separated either by thin-layer chromatography (TLC) on C-18 silica gel plates or by polyacrylamide gel electrophoresis (PAGE). The density of the spots on the TLC plates and the fluorescence of the bands on the gel were quantified. The assay conditions were optimized using crude bovine colostrum extract and also α-2,6-sialyltransferase from rat liver. The detection limits for the TLC and PAGE assays were 1 and 0.4 μU of the rat liver enzyme, respectively. Either assay allows the parallel investigation of several samples at a time and is useful for the testing of fractions during enzyme purification.