Nonradioactive trans-sialidase screening assay.
Trans-sialidase (TS; E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3-linkage to galactose or N-acetylgalactosamine. In the absence of an appropriate acceptor, TS acts as a sialidase, hydrolytically releasing glycosidically linked sialic acid. Interest in TS has increased rapidly in recent years owing to its great relevance to the pathogenicity of trypanosomes and its possible application in the regiospecific synthesis of sialylated carbohydrates and glycoconjugates. Recently, the authors described a newly developed nonradioactive screening test for monitoring TS activity (1). In this highly sensitive and specific assay, 4-methylumbelliferyl-beta-D-galactoside is used as acceptor substrate and sialyllactose as donor to fluorimetrically detect enzyme activity in the low mU range (approximately 0.1-1 mU/mL possible). The test can be applied to screen a large number of samples quickly and reliably during enzyme purification, for testing inhibitors, and for monitoring TS activity during the production of monoclonal antibodies (2). This chapter focuses on the main steps of this assay and gives detailed instructions for performing a nonradioactive TS 96-well-plate fluorescence test. In addition, it describes the controls necessary when starting to monitor an unknown TS and facts to be considered when testing new substrates and inhibitors.
Schrader
Silke
S
Biochemisches Institut, University of Köln, Köln, Germany.
Schauer
Roland
R
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002138:Calibration; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D009439:Neuraminidase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D012680:Sensitivity and Specificity; D014348:Trypanosoma congolense / Q000201:enzymology; D014349:Trypanosoma cruzi / Q000201:enzymology
D004791:Enzyme Inhibitors; D005697:Galactosides; D006023:Glycoproteins; D006923:Hymecromone; C026845:4-methylumbelliferyl-galactopyranoside; C069715:trans-sialidase; D009439:Neuraminidase