New continuous and specific fluorometric assays for Pseudomonas aeruginosa elastase and LasA protease.
A highly sensitive assay based on new internally quenched fluorogenic peptide substrates has been developed for monitoring protease activities. These novel substrates comprise an Edans (5-(2-aminoethylamino)-1-naphthalenesulfonic acid) group at the C terminus and a Dabsyl (4-(dimethylamino)azobenzene-4'-sulfonyl chloride) fluorophore at the N terminus of the peptide chains. The Edans fluorescence increases upon peptide hydrolysis by Pseudomonas aeruginosa proteases, and this increase is directly proportional to the amount of substrate cleaved, i.e., protease activity. The substrates Dabsyl-Ala-Ala-Phe-Ala-Edans and Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans were used for testing the peptidasic activities of P. aeruginosa elastase and LasA protease, respectively. Elastase and LasA kinetic parameters were calculated and a sensitive assay was designed for the detection of P. aeruginosa proteases in bacterial supernatants. The sensitivity and the small sample requirements make the assay suitable for high-throughput screening of biological samples. Furthermore, this P. aeruginosa protease assay improves upon existing assays because it is simple, it requires only one step, and even more significantly it is enzyme specific.
Elston
Caroline
C
Laboratoire de Biochimie Analytique & Synthèse Bioorganique, UFR Chimie-Biochimie, Université Claude Bernard Lyon 1, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
Wallach
Jean
J
Saulnier
Joëlle
J
D001426:Bacterial Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000737:chemistry; D010196:Pancreatic Elastase / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000737:chemistry
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010455:Peptides; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; C010572:4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride; D004124:p-Dimethylaminoazobenzene; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C066371:staphylolytic protease