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 cY-galactosidase from fig and Morlierella vinacea using p-nitrophenyl galactopyranoside (NPG) at pH 3.9 and 5.8, respectively, and also for jack bean meal /?-N-acetylhexosaminidase using p-nitrophenyl-,8-Z-acetamido-2deoxy-n-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.
A number of different methods are currently available for assaying glycosidases. Static, fixed time spectrophotometric assays (l-5) are most commonly used. Nitrophenyl glycosides, or similar chromogenic substrates, are incubated with enzyme at desired pH and substrate concentrations for a specified period of time; the reaction is then quenched, and the p-nitrophenolate ion is spectrophotometrically measured at alkaline pH, usually 10-11. Continuously monitored spectrophotometric assay techniques have been developed (6,7) for determining the activity of glycosidases under alkaline conditions. Other methods employed include static fluorometric assays (8,9), extensively or selectively labelled radioactive substrates (10,ll) , titrimetric monitoring of hydrogen fluoride liberated by glycosyl fluorides (12), static methods where the product sugar is measured by conventional techniques (13), and coupled enzyme assays (12,14).
Continuously monitored spectrophotometric assays are preferable to static, fixed time assays for a number of reasons (15,16). The change in reaction rate caused by decreasing substrate concentration and increasing product, if it inhibits the reaction, is not easily determined in 120