Solid-supported single- and multi-enzyme amplified assays for clostridiopeptidase A

A rapid and sensitive spectrophotometric assay for Clostridium histolyticum clostridiopeptidase A (collagenase) was accomplished by measuring the activity of an alkaline phosphatase indicator enzyme released into solution from insoluble, covalently linked alkaline phosphatase indicator enzyme released into solution from activity of the alkaline phosphatase was monitored spectrophotometrically using either p-nitrophenyl phosphate as substrate or more sensitively by a signal amplification system consisting of NAD⁺, alcohol dehydrogenase, diaphorase and INT-Violet. Under the reaction conditions the amount of indicator enzyme produced is directly proportional to the concentration of collagenase. With p-nitrophenyl phosphate as substrate the magnitude of the signal was 0.003 abs. min^?1 per 100 ng ml^?1 collagenase whereas with the multienzyme amplification system it was 0.035 abs. min^?1, i.e. approximately as 12-fold increase. The method consists in first incubating the substrate with the bacterial collagenase for 20 min, then up to 96 samples of the released alkaline phosphatase can be analysed in 2 min using a microtitre plate reader run in the kinetic mode.