RAPID AND SENSITIVE ASSAY FOR SOME PROTEASE ENZYMES USING A FLUOROSUBSTRATE-IMMOBILIZED BIOREACTOR

Protease enzymes are commonly used in industry; exposure of the workfo rce to these enzymes may lead to respiratory sensitization. Assays are therefore needed which can be coupled to air sampling devices to prod uce real-time monitoring systems which are sufficiently sensitive and rapid to provide an early warning system that indicates that the airbo rne concentrations of enzyme have exceeded occupational exposure limit s. Current methods based on spectrophotometric end-points are too slow and insensitive for this application. An assay is reported that is ba sed on the release of fluorescein covalently attached to porcine thyro globulin which is itself immobilized onto glass beads in a bioreactor. Exposure of the bioreactor to the enzymes, subtilisin, trypsin, alcal ase and esperase produces a fluorescent peak in a flow through cell wi thin 3-4 min. The resulting signal is linear (r = 0.999, n = 5) over t he range 2-50 ng for subtilisin and the reproductibility of the assay (s(r)) at 5 ng is 0.8% for this enzyme.