[1H-NMR spectroscopy--a potent method for the determination of substrate specificity of sialidases (author's transl)].
We describe here the application of 1H-NMR spectroscopy to determine the substrate specificity of sialidases using a 1:1 mixture of NeuAc alpha 2-3Gal beta 1-4Glc and NeuAc alpha 2-6Gal beta 1-4Glc, one viral and five bacterial sialidases. This method utilizes the separate signals in NMR spectra, characteristic for the different alpha ketosidically linked NeuAc residues and also for bound and free NeuAc. The signals generally most suitable for these purposes are those of H3a, H3e and NCOCH3. By observation and integration of these signals we can follow--qualitatively and quantitatively--which and how many NeuAc residues of the substrates are hydrolized. In contrast to the generally used colorimetric tests it is now possible to investigate with this method substrates containing two or more NeuAc residues and to determine the corresponding rate constants for hydrolysis of the differently bound NeuAc molecules. The six sialidases used show large differences in their specificity as compared with our "model substrate": The sialidase from fowl plague virus hydrolizes NeuAc alpha 2-3Gal beta 1-4Glc nearly 18 times and the enzyme from Clostridium perfringens four times, from Vibrio cholerae two times faster than NeuAc alpha 2-6Gal beta 1-4Glc. On the contrary, the sialidase from Arthrobacter ureafaciens hydrolizes the alpha 2-6 linkage six times faster than the alpha 2-3 linkage. The sialidases from Bifidobacterium show no obvious differences in their specificities relative to the linkage.
D000191:Actinomycetaceae / Q000201:enzymology; D001173:Arthrobacter / Q000201:enzymology; D003016:Clostridium perfringens / Q000201:enzymology; D009980:Influenza A virus / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009439:Neuraminidase / Q000378:metabolism*; D013379:Substrate Specificity; D014734:Vibrio cholerae / Q000201:enzymology
D009439:Neuraminidase
1H-NMR-Spektroskopie--Eine aussagekräftige Methode zur Bestimmung der Substratspezifität von Sialidasen.