A nonradioactive 96-well plate assay for screening of trans-sialidase activity.
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.
Biochemisches Institut, Christian-Albrechts-Universität zu Kiel, 24098, Kiel, Germany. schauer@biochem.uni-kiel.de
Silke
S
Schrader
Evelin
E
Tiralongo
Gastón
G
Paris
Teruo
T
Yoshino
Roland
R
Schauer
D000818:Animals; D001671:Biochemistry / Q000295:instrumentation* / Q000379:methods*; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D005697:Galactosides / Q000032:analysis / Q000378:metabolism; D006023:Glycoproteins; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D009439:Neuraminidase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013696:Temperature; D014348:Trypanosoma congolense / Q000201:enzymology; D014349:Trypanosoma cruzi / Q000201:enzymology
C488471:2'-(4-methylumbelliferyl)-N-acetylneuraminic acid-alpha2,3-galactose; D005697:Galactosides; D006023:Glycoproteins; D011994:Recombinant Proteins; D006923:Hymecromone; C069715:trans-sialidase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid