A nonradioactive 96-well plate assay for screening of trans-sialidase activity
| Autor: | Evelin Tiralongo, Roland Schauer, Silke Schrader, Gastón Paris, Teruo Yoshino |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Glycan
Glycoconjugate medicine.drug_class Trypanosoma congolense Trypanosoma cruzi Biophysics Neuraminidase Biology Monoclonal antibody Biochemistry Sensitivity and Specificity law.invention chemistry.chemical_compound law medicine Animals Fluorometry Molecular Biology Glycoproteins chemistry.chemical_classification Hydrolysis Temperature Galactosides Cell Biology Hydrogen-Ion Concentration biology.organism_classification N-Acetylneuraminic Acid Recombinant Proteins Sialic acid Enzyme chemistry Recombinant DNA Trypanosoma biology.protein Hymecromone |
| Zdroj: | Analytical biochemistry. 322(2) |
| ISSN: | 0003-2697 |
| Popis: | Trans-sialidase (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. 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-beta-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. |
| Databáze: | OpenAIRE |
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