Methodology article
Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases
- † Equal contributors
1 Dipartimento di Biotecnologie, Università degli Studi di Siena, I-53100, Siena, Italy
2 Department of Respiratory Medicine, The Second Affiliated Hospital, Nanchang University, Nanchang, Jiangxi, 330006, China
3 Department of Pulmonary Medicine, Beijing 301 Hospital, Beijing, 100853, China
4 Biomedical Sciences Research Complex, University of St Andrews, St Andrews, KY16 9ST, UK
5 Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, LE1 9HN, UK
BMC Biochemistry 2012, 13:19 doi:10.1186/1471-2091-13-19
Published: 2 October 2012Abstract
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.
