A continuously monitored spectrophotometric assay of glycosidases with nitrophenyl glycosides

A general method for a continuously monitored spectrophotometric assay of glycosidases at all values of pH using p-nitrophenyl glycosides is presented. The method is demonstrated specifically by the development of a routine assay for α-galactosidase from fig and Mortierella vinacea using p-nitrophenyl galactopyranoside (NPG) at pH 3.9 and 5.8, respectively, and also for jack bean meal β-N-acetylhexosaminidase using p-nitrophenyl-β-2-acetamido-2-deoxy-d-glucopyranoside (NPADG) at pH 5.0. A number of different wavelengths may be used for the assay depending upon the criterion of the user; maximum sensitivity at a selected pH, determination of enzyme pH optima with a pH-independent difference extinction coefficient, or the reduction of background absorbance for kinetic studies at high substrate concentrations.