A fluorometric procedure for measuring the neuraminidase activity: Its application to the determination of this activity in influenza and parainfluenza viruses

Abstract A fluorometric procedure for quantitating the amount of N -acetylneuraminic acid enzymatically released by the neuraminidase activity from N -acetylneuraminyl-lactose (sialyl-lactose) has been developed. The liberated lactose is hydrolyzed with β-galactosidase, and the released galactose is oxidized with galactose dehydrogenase and NAD + ; finally the NADH produced is measured by fluorometry (excitation at 340 nm and analysis of emitted light at 465 nm). The fluorometric assay is about 10-fold more sensitive than the spectrophotometric procedure that measures NADH at 340 nm. It readily measures amounts as little as 2 nmol of sialic acid, and does not require the use of radioactive isotopes. Interferences due to sucrose or other substances, which cause errors in some cases with the use of the periodate-thiobarbiturate method for neuraminidase activity determination, are avoided. The procedure reported here provides a sensitive, rapid, and relatively simple method (feasible with commercialized reagents) for measuring the neuraminidase activity not only in purified samples from different sources but also directly in biological materials such as viruses. The technique has been tested with some viruses recently isolated belonging to Orthomyxoviridae or Paramyxoviridae families, known to be rich in neuraminidase. Reciprocally, this method can also be employed for determining the sialic acid concentration in acylneuraminyl-lactose-containing compounds when using purified neuraminidase for hydrolysis.