Chemiluminescence determination of proteases by flow injection using immobilized isoluminol

Abstract

A flow-injection system for protease determination is described. A tripeptide–isoluminol derivative was immobilized on an affinity support, and incorporated into the flow system. Injected enzyme sample solutions (0.025 cm3) catalysed the hydrolysis of some of the derivatives at pH 8.47, and the released isoluminol was detected by the chemiluminescence produced by its cobalt-catalysed oxidation with hydrogen peroxide. Linear calibration results were obtained for α-chymotrypsin (from 5 × 10–4 to 0.41 mg dm–3), trypsin (from 4 × 10–2 to 4 mg dm–3) and a commercial protease (from 2 × 10–3 to 4 mg dm–3). The limits of detection were 2.7 × 10–4, 4 × 10–2 and 2 × 10–3 mg dm–3, respectively.