A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction

Efrat Kessler*, Mary Safrin, Shmaryahu Blumberg, Dennis E. Ohman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Pseudomonas aeruginosa LasA protease is a secreted metalloendopeptidase that can lyse Staphylococcus aureus cells by cleaving the pentaglycine bridges of their peptidoglycan. It can also degrade elastin and stimulate shedding of cell-surface proteoglycans, activities implicated in pathogenesis of P. aeruginosa infections. The activity of LasA protease can be assayed spectrophotometrically by following the reduction in turbidity of S. aureus cell suspensions. This assay, however, does not permit kinetic studies and its reproducibility is poor. Here we describe a two-stage enzymatic reaction for the continuous measurement of LasA protease activity using a defined substrate, succinyl-Gly-Gly-Phe-4-nitroanilide, supplemented with Streptomyces griseus aminopeptidase. Cleavage of the Gly-Phe bond by LasA protease is followed by hydrolysis of the product Phe-4-nitroanilide by the aminopeptidase and the rate of release of the chromophore (4-nitroaniline) is measured spectrophotometrically using a 96-well microplate reader. Activity of nanogram amounts of LasA protease could be determined within a few minutes. Furthermore, this assay permitted the determination of Km and kcat values for LasA protease, which were 0.46mM and 11.8s-1, respectively. Pseudomonas elastase was also active in the assay. However, it was less effective than LasA protease and its activity was inhibited by phosphoramidon. The assay is highly sensitive and reproducible, providing a convenient tool for further studies of LasA protease function(s) and mechanism of action.

Original languageEnglish
Pages (from-to)225-232
Number of pages8
JournalAnalytical Biochemistry
Volume328
Issue number2
DOIs
StatePublished - 15 May 2004

Keywords

  • LasA protease
  • Lysostaphin
  • Pseudomonas aeruginosa elastase
  • Staphylolysin
  • Staphylolytic protease

Fingerprint

Dive into the research topics of 'A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction'. Together they form a unique fingerprint.

Cite this