A microtitre plate assay for measuring glycosidase activity

Abstract

Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC₅₀ and K_i values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors.

Keywords::

• Enzyme kinetics
• galactosidases
• glucosidases
• glucuronidases
• inhibitors
• microtitre plate colourmetric assay
• 4-Nitrophenol

Acknowledgements

We thank Dr P. J. Wood and Dr K. D. E. Boardman (University of Bath, U.K.) for assistence and Dr A. Watts (University of Bath, U.K.) for critically reading this manuscript. This work was funded by a University of Bath undergraduate project grant to MDL.