Spectrophotometric assay for amidolytic activity of alkaline protease from Pseudomonas aeruginosa

A spectrophotometric method was applied to assay Pseudomonas aeruginosa alkaline protease (EC 3.4.24.40) activity using N-carbobenzyloxyarginylarginyl-para-nitroanilide (Z-Arg-Arg-pNA). With the same substrate the activity of P. aeruginosa elastase (PsE) was 250-fold lower, this selectivity being even more increased in the presence of 100 μM phosphoramidon. This dipeptide derivative, which is an inhibitor of metalloproteases such as PsE was demonstrated to have no effect on alkaline protease (AP). The method was successfully applied for measurement of the enzyme activity in a culture supernatant from a clinical strain.