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.