An assay for the enzyme N-acetyl-beta-D-glucosaminidase (NAGase) based on electrochemical detection using
R M Pemberton1, J P Hart, T T Mottram
1Centre for Research in Analytical, Materials and Sensors Science (CRAMSS), Faculty of Applied Sciences, University of the West of England, Bristol, UK.
Abstract:
An electrochemical assay for the enzyme N-acetyl-beta-D-glucosaminidase (NAGase) is described, using bare screen-printed carbon electrodes (SPCEs). The enzyme substrate, 1-naphthyl-N-acetyl-beta-D-glucosaminide, was added to the NAGase-containing sample under hydrodynamic conditions and was hydrolysed to 1-naphthol, which was monitored amperometrically at an Eapp of +650 mV versus SCE. A pH study revealed the apparent Vmax for the assay to occur at pH 4.5. corresponding to an apparent substrate Km of 0.28 mM. In order to be compatible with the analysis of biological fluids, a final operating pH of 5.4 was selected, and, using a data recording time of 100 s post-substrate addition, the assay gave a linear response (r2 = 0.988) over the range 3.1 to 108 mU ml(-1) NAGase (RSD = 15.4%). This assay has the potential to monitor NAGase levels in a number of application areas.
