Synthetic peptide substrates for a conductimetric assay of Pseudomonas aeruginosa elastase.
Pseudomonas aeruginosa is a zinc metalloprotease which may be involved in many infection processes, especially in the lung. In order to evaluate the production of the enzyme in culture supernatants, we developed an assay using peptide derivatives; the conductimetric method was used for monitoring the enzymatic activities. Tetrapeptide derivatives were enzymatically synthesized by coupling Z-Ala2 and X-AlaR using either thermolysin or P. aeruginosa elastase itself. In these substrates, X could be phenylalanine, tyrosine, or leucine and C-protection was performed by either an amide (NH2) or a methyl (OMe) group. Z-Ala2-Phe-AlaNH2 was found to be the best substrate, giving a catalytic ratio kcat/KM of 8600 mM-1.s-1. The evaluation of the alkaline protease activity with this substrate showed that the catalytic ratio is 1000-fold lower. The sensitivity of the conductimetric method was also demonstrated with as little as 1 nM elastase (0.13 microgram), being easily and accurately detected (SD, 3.8% for 10 measurements). Furthermore, the enzymatic activity was measured in a culture supernatant from a clinical strain.
Besson
C
C
Laboratoire de Biochimie Analytique et de Synthèse Bioorganique, Institut de Chimie et de Biologie Moléculaire et Cellulaire, Bâtiment 303, Villeurbanne Cedex, 69622, France.
Saulnier
J
J
Wallach
J M
JM
D000595:Amino Acid Sequence; D003217:Conductometry / Q000379:methods*; D005069:Evaluation Studies as Topic; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D010196:Pancreatic Elastase / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000472:pathogenicity; D013379:Substrate Specificity
D009842:Oligopeptides; D010196:Pancreatic Elastase