Coupled-Enzyme System for Measuring Viral Neuraminidase Activity

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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.

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