Abstract
A coupled-enzyme assay for determining viral neuraminidase activity is described. All reactants—viral neuraminidase, the initial substrate (fetuin), N-acetylneuraminic acid aldolase, lactic acid dehydrogenase, and reduced nicotinamide adenine dinucleotide—are combined in a single cuvette. Thus, in a single coupled system neuraminidase releases N-acetylneuraminic acid, which is cleaved to N-acetyl-D-mannosamine and pyruvic acid; finally, pyruvate is reduced to lactate as reduced nicotinamide adenine dinucleotide is oxidized. The rate of change of absorbance at 340 nm, as reduced nicotinamide adenine dinucleotide is oxidized, is a measure of the rate of reaction of the coupled system. This procedure, which measures the rate of release of N-acetylneuraminic acid by neuraminidase, is an alternate method for those procedures which require multistep, colorimetric determinations.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- AMINOFF D. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem J. 1961 Nov;81:384–392. doi: 10.1042/bj0810384. [DOI] [PMC free article] [PubMed] [Google Scholar]
- COMB D. G., ROSEMAN S. The sialic acids. I. The structure and enzymatic synthesis of N-acetylneuraminic acid. J Biol Chem. 1960 Sep;235:2529–2537. [PubMed] [Google Scholar]
- Kendal A. P., Madeley C. R. A comparative study of influenza virus neuraminidases, using automated techniques. Biochim Biophys Acta. 1969 Jul 8;185(1):163–177. doi: 10.1016/0005-2744(69)90292-7. [DOI] [PubMed] [Google Scholar]
- RAFELSON M. E., Jr, SCHNEIR M., WILSON V. W., Jr STUDIES ON THE NEURAMINIDASE OF INFLUENZA VIRUS. II. ADDITIONAL PROPERTIES OF THE ENZYMES FROM THE ASIAN AND PR 8 STRAINS. Arch Biochem Biophys. 1963 Dec;103:424–430. doi: 10.1016/0003-9861(63)90432-6. [DOI] [PubMed] [Google Scholar]
- WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
- Webster R. G., Laver W. G. Preparation and properties of antibody directed specifically against the neuraminidase of influenza virus. J Immunol. 1967 Jul;99(1):49–55. [PubMed] [Google Scholar]