Enzyme kinetic measurements for a combinatorial library of inhibitors of Pseudomonas elastase

George Cathcart, Brendan Gilmore, Brian Walker School of Pharmacy, QUB, Belfast 10/2009

Pseudomonas elastase (pseudolysin, LasB) is a metalloprotease virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. As one of the main virulence factors of this bacterium, it contributes to chronic and intractable infection in various disease states from the cystic fibrosis lung, to chronic ulcers of the skin.


The LasB activity assay employs an internally quenched protease substrate Abz-peptide-Nba (2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide) with low signal. After cleavage of the peptide by LasB the fluorescent donor group and quencher are separated resulting in higher fluorescence emission.


The assay was validated on a BMG LABTECH microplate reader that ensured robust measurements and parallel inhibitor characterization. Furthermore, the analysis software offered convenient calculation of Km values.

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