In this context, translational studies in clinical specimens (BAL and sputum) from patients with CF indicate that lipidated FRET reporters designed to measure protease activity at the surface of inflammatory cells, such as NEmo-2 and LaRee-1 [16] 16], may be sensitive novel tools to identify CF patients with the greatest risk to develop severe lung damage.
To measure protease activity in the spent medium, the medium was mixed in 1∶ 4 ratio with freshly prepared casein (0.625% w/v) solution and incubated at 37°C for 30 min. The reaction was stopped by addition of 5 ml of 5% (w/v) trichloroacetic acid and the protease activity was measured by determining the rate of enzymatic hydrolysis of casein by the Folin Ciocalteu method [24], [25].
Protein concentrations ranged from 0.08 to 1.7 mg/mL and measured protease activities were normalised against these values and expressed as units of trypsin/mg protein.
For measuring protease and lipase activity the raw enzyme used for the assay was isolated from the culture broth following separation of cells.
This assay measures a distinct protease activity associated with cytotoxicity and uses a fluorogenic peptide substrate (bis-alanyl-alanyl-phenylanlanyl-rhodamine 110 (bis-AAF-R110)) to assess protease activity that has been released from cells that have lost membrane integrity (Promega, Mannheim, Germany).
Unfortunately at the moment, further therapeutic options to reduce or inhibit measured increased protease activity are still missing.
To measure alkaline protease activity, one unit of protease activity was defined as the amount of enzyme required to liberate 1 μg of tyrosine per minute under the experimental conditions used.
To measure OmpT protease activity, the change in fluorescence emission at 430 nm of the cleavable fluorogenic peptide (Abz-Ala-Arg-Arg-Ala-Tyr- NO2 -NH2) (CAbz-Ala-Arg-Arg-Ala-Tyr- NO2 -NH2, U.K). wAbz-Ala-Arg-Arg-Ala-Tyr- NO2 -NH25 nm.
Bacterial protease activity could release media components that are attractive, so we measured protease production of Bcc strains that vary in nematode preference (Table 2) but found no correlation between these traits.
The MultiTox-Fluor Assay simultaneously measures two protease activities; one is a marker of cell viability, and the other is a marker of cytotoxicity.
