Pseudomonas aeruginosa protease IV enzyme assays and comparison to other pseudomonas proteases

Pseudomonas aeruginosa secretes multiple proteases that have been implicate d as virulence factors and the detection of each specific enzyme can be dif ficult to determine. Unlike the three Pseudomonas enzymes that have been we ll characterized (elastase A, elastase B, and alkaline protease), the activ ity of protease TV in multiple assays has yet to be described. This study d efines new assays for Pseudomonas proteases and compares protease IV activi ty to the activities of elastase A, elastase B, and alkaline protease. Six in vitro assays were studied: zymography, elastin congo red assay, staphylo lytic assay, colorimetric peptide assay, solid-phase colorimetric peptide a ssay, and poly-L-lysine degradation. Casein zymography distinguished protea se IV from elastase B and alkaline protease, and gelatin zymography differe ntiated all four proteases. The elastin congo red assay detected mainly ela stase B while the staphylolytic assay was specific for elastase A. Protease IV activity was assayed specifically by the colorimetric assay and two new assays, the solid-phase colorimetric assay and degradation of poly-L-lysin e in the presence of EDTA. Alkaline protease could be specifically assayed by poly-L-lysine degradation in the presence of N-alpha -p-to-syl-L-lysine chloromethyl ketone. The results identified three specific assays for prote ase TV, a new assay specific for alkaline protease, and showed that proteas e TV has a distinct enzymatic specificity relative to the three other Pseud omonas proteases. (C) 2001 Academic Press.