Article (Scientific journals)
Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases
Hayre, J. K.; Xu, G.; Borgianni, L. et al.
2012In BMC Biochemistry, 13 (1)
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Keywords :
Chromogenic sialic acids; Kinetic assay; Neuraminidase; Sialidase; Streptococcus pneumoniae; NanA protein; NanB protein; NanC protein; Biocatalysis; Buffers; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; N-Acetylneuraminic Acid; Recombinant Proteins; Spectrophotometry, Ultraviolet; Substrate Specificity
Abstract :
[en] Backgrounds. 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. © 2012 Hayre et al.; licensee BioMed Central Ltd.
Disciplines :
Biochemistry, biophysics & molecular biology
Microbiology
Author, co-author :
Hayre, J. K.;  Dipartimento di Biotecnologie, Universit Degli Studi di Siena, I-53100, Siena, Italy
Xu, G.;  Department of Respiratory Medicine, Second Affiliated Hospital, Nanchang University, Nanchang, Jiangxi, 330006, China, Department of Pulmonary Medicine, Beijing 301 Hospital, Beijing, 100853, China
Borgianni, L.;  Dipartimento di Biotecnologie, Universit Degli Studi di Siena, I-53100, Siena, Italy
Taylor, G. L.;  Biomedical Sciences Research Complex, University of St Andrews, St Andrews, KY16 9ST, United Kingdom
Andrew, P. W.;  Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, LE1 9HN, United Kingdom
Docquier, Jean-Denis ;  Université de Liège - ULiège > Département des sciences de la vie > Centre d'ingénierie des protéines
Oggioni, M. R.;  Dipartimento di Biotecnologie, Universit Degli Studi di Siena, I-53100, Siena, Italy
Language :
English
Title :
Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases
Publication date :
2012
Journal title :
BMC Biochemistry
eISSN :
1471-2091
Publisher :
BioMed Central, United Kingdom
Volume :
13
Issue :
1
Peer reviewed :
Peer Reviewed verified by ORBi
European Projects :
FP7 - 222983 - PNEUMOPATH - A comprehensive dissection of pneumococcal-host interactions
Funders :
EU - European Union [BE]
Available on ORBi :
since 19 November 2020

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