Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-α-d-N-acetylneuraminate) substrate

Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors

“…The activity of rN2 and rN2-Tyr406Asp was monitored using a fluorometric assay based on methods from Potier et al 42 NA activity was monitored using 0-500 mM 4-methylumbelliferyl-Neu5Ac (J&K Scientific) with 33 mM MES and 4 mM CaCl 2 (pH 6.0). The final concentration of enzymes used for wild-type rN2 and rN2-Tyr406Asp were 1 and 167 nM, respectively.…”

“…A 4-methylumbelliferyl-Neu5Ac-based fluorometric NA assay was also used to test competitive NA inhibition 42 . Recombinant NA or live influenza virus was diluted until NA activity was in the appropriate range for fluorescence detection of liberated 4-methylumbelliferone on a SpectraMax M5 (Molecular Devices), using excitation and emission wavelengths of 355 and 460 nm, respectively.…”

Effect of substrate aglycon on enzyme mechanism in the reaction of sialidase from influenza virus

“…Sialidase activity, using Neu5Ace2MU as substrate, was assayed by a modification [7] of a previously reported fluorometric method [11]. The fluorescence measured was linear with respect to the concentration of 4-methylumbelliferone.…”

Intramuscularly administered neuraminidase inhibitor peramivir is effective against lethal H5N1 influenza virus in mice

“…We used a modified fluorescence-based NA enzyme inhibition assay (Potier et al, 1979). Briefly, NA inhibitors at concentrations ranging from 0.00005 μM to 10 μM were incubated with H5N1 influenza virus preparations representing a standard amount of NA activity before the addition of the substrate 2′-(4-methylumbelliferyl)-d-N-acetylneuraminic acid (MUNANA, Sigma, St. Louis, MO).…”