The intestinal homogenates, along with purified proteases, were tested for general proteolytic activities (metallo-, serine, acid and sulfhydryl proteases) using casein derivative substrate (Enzchek Protease Assay Kit, E6639, Molecular Probes-Invitrogen, Carlsbad, CA).
The substrate in the Enzchek Protease Assay Kit, casein, is derivatized with pH-insensitive fluorophores. When undigested, the fluorophores are quenched and the molecule does not fluoresce. Upon cleavage by proteases, the peptides with these fluorophores will fluoresce. The level of measured fluorescence increases with the amount of peptide present. The fluorescence of the intestinal homogenates incubated with this substrate was measured in duplicates on a 96-well plate by a spectrophotometer (Spectromax Gemini XS) using the Softmax Pro software (Molecular Devices Corp., Sunnvale, CA) and expressed in relative fluorescent units (RFUs). The level of fluorescence increases with the number of sites within the casein molecule cleaved by the enzymes in the sample.
In each well of a multiwell plate, 5 μl of intestinal sample, 95 μl of digestion buffer and 100 μl of casein substrate were mixed. The plate was covered with aluminum foil to prevent evaporation, incubated at 37°C in a spectrophotometer (protected from light), and kinetic measurements were made over a period of 60 minutes.
The substrate in the Enzchek Protease Assay Kit, casein, is derivatized with pH-insensitive fluorophores. When undigested, the fluorophores are quenched and the molecule does not fluoresce. Upon cleavage by proteases, the peptides with these fluorophores will fluoresce. The level of measured fluorescence increases with the amount of peptide present. The fluorescence of the intestinal homogenates incubated with this substrate was measured in duplicates on a 96-well plate by a spectrophotometer (Spectromax Gemini XS) using the Softmax Pro software (Molecular Devices Corp., Sunnvale, CA) and expressed in relative fluorescent units (RFUs). The level of fluorescence increases with the number of sites within the casein molecule cleaved by the enzymes in the sample.
In each well of a multiwell plate, 5 μl of intestinal sample, 95 μl of digestion buffer and 100 μl of casein substrate were mixed. The plate was covered with aluminum foil to prevent evaporation, incubated at 37°C in a spectrophotometer (protected from light), and kinetic measurements were made over a period of 60 minutes.