Sialidase assay by luminescence in the low picomole-range of sialic acid. Its application to the measurement of this activity in influenza virus.
A new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.
D006868:Hydrolysis; D009980:Influenza A virus / Q000201:enzymology*; D008163:Luminescent Measurements; D008832:Microchemistry; D019158:N-Acetylneuraminic Acid; D009243:NAD / Q000032:analysis; D009439:Neuraminidase / Q000032:analysis*; D012794:Sialic Acids / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014764:Viral Proteins / Q000032:analysis
D012794:Sialic Acids; D014764:Viral Proteins; D009243:NAD; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid