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A nonradioactive 96-well plate assay for screening of trans-sialidase activity

2003; Elsevier BV; Volume: 322; Issue: 2 Linguagem: Inglês

10.1016/j.ab.2003.07.016

1096-0309

Silke Schrader, Evelin Tiralongo, Gastón Paris, Teruo Yoshino, Roland Schauer,

Biochemical and Molecular Research

Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably α2,3-linked sialic acid to another glycan or glycoconjugate, forming a new α2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-β-d-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.