title: Determination of neuraminidase kinetic constants using whole influenza virus preparations and correction for spectroscopic interference by a fluorogenic substrate.
abstract: The influenza neuraminidase (NA) enzyme cleaves terminal sialic acid residues from cellular receptors, a process required for the release of newly synthesized virions. A balance of NA activity with sialic acid binding affinity of hemagglutinin (HA) is important for optimal virus replication. NA sequence evolution through genetic shift and drift contributes to the continuous modulation of influenza virus fitness and pathogenicity. A simple and reliable method for the determination of kinetic parameters of NA activity could add significant value to global influenza surveillance and provide parameters for the projection of fitness and pathogenicity of emerging virus variants. The use of fluorogenic substrate 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and cell- or egg-grown whole influenza virus preparations have been attractive components of NA enzyme activity investigations. We describe important criteria to be addressed when determining K(m) and V(max) kinetic parameters using this method: (1) determination of the dynamic range of MUNANA and 4-methylumbelliferone product (4-MU) fluorescence for the instrument used; (2) adjustment of reaction conditions to approximate initial rate conditions, i.e. ≤15% of substrate converted during the reaction, with signal-to-noise ratio ≥10; (3) correction for optical interference and inner filter effect caused by increasing concentrations of MUNANA substrate. The results indicate a significant interference of MUNANA with 4-MU fluorescence determination. The criteria proposed enable an improved rapid estimation of NA kinetic parameters and facilitate comparison of data between laboratories.
authors: Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
authors: Bindumadhav M
authors: BM
authors: Marathe
authors: Vincent
authors: V
authors: Lévêque
authors: Klaus
authors: K
authors: Klumpp
authors: Robert G
authors: RG
authors: Webster
authors: Elena A
authors: EA
authors: Govorkova
mesh_terms: D000818:Animals; D002642:Chick Embryo; D004285:Dogs; D057075:Enzyme Assays / Q000592:standards; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000737:chemistry*; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology*; D007700:Kinetics; D061985:Madin Darby Canine Kidney Cells; D009439:Neuraminidase / Q000737:chemistry*; D059629:Signal-To-Noise Ratio; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D014764:Viral Proteins; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D009439:Neuraminidase
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