A continuous fluorescent assay for measuring protease activity using natural protein substrate.
A continuous caseinolytic activity assay has been developed and characterized with trypsin, a serine protease, and transin, a metalloproteinase. Beta-casein labeled with both N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and fluorescein isothiocyanate (FITC) is used as the substrate in this assay. The effect of proteolysis of the substrate is a reduction of the intermolecular energy transfer from DACM to FITC. The caseinolytic activity is then monitored by the fluorescence increase. The activities of both proteases obey Michaelis-Menten kinetics with Km = 1.6 +/- 0.2 microM for trypsin and Km = 13.2 +/- 1.9 microM for transin. Protease concentrations as low as 10 ng/mL can be utilized. The pH dependence of the caseinolytic activity has been determined for both enzymes.
Farmer
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Syntex Research, Mississauga, Ontario, Canada.
Yuan
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ZY
D002364:Caseins; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D007202:Indicators and Reagents; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000032:analysis; D010447:Peptide Hydrolases / Q000032:analysis*; D014357:Trypsin / Q000032:analysis
D002364:Caseins; D007202:Indicators and Reagents; D010447:Peptide Hydrolases; D014357:Trypsin; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D016650:Fluorescein-5-isothiocyanate