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A microtitre plate assay for measuring glycosidase activity

2008; Taylor & Francis; Volume: 23; Issue: 1 Linguagem: Inglês

10.1080/14756360701384252

1475-6374

Andrea L. Ball, Kirsty A. Chambers, Meera Hewinson, Sambavi Navaratnarajah, Lamia Samrin, Nesta Thomas, Abigail E. H. Tyler, Amanda J. Wall, Matthew D. Lloyd,

Lysosomal Storage Disorders Research

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 Ki 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.