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Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases.

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posted on 2013-10-24, 13:05 authored by J.K. Hayre, G. Xu, L. Borgianni, G.L. Taylor, Peter W. Andrew, J.D. Docquier, M.R. Oggioni
Background: Streptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate. Results: In this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p-NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses. Conclusion: We developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.

History

Citation

BMC Biochemistry, 2012, 13:19

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/School of Medicine/Department of Infection, Immunity and Inflammation

Version

  • VoR (Version of Record)

Published in

BMC Biochemistry

Publisher

BioMed Central Ltd

eissn

1471-2091

Copyright date

2012

Available date

2013-10-24

Publisher version

http://www.biomedcentral.com/1471-2091/13/19

Notes

PMCID: PMC3483245

Language

en

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