Azocasein Assay

Total protease activity from each wound fluid was determined by the azocasein method as described by Tomarelli (19). Hundred μg of total protein from each wound fluid sample were added to 50 μl azocasein substrate (2% azocasein enzyme substrate in 10 mM Tris HCl, 8 mM CaCl₂, pH 7.4). The reaction mixture was incubated in 37 °C for 24 h. Thereafter, 240 ml 10% trichloroacetic acid was added and the samples mixed and allowed to stand for 15 min to ensure complete precipitation of undigested material. Tubes were centrifuged at 10,000 rpm (microfuge) for 5 min and 125 μl of the supernatant was transferred to wells of a 96-wells plate containing 125 μl 1.0 M NaOH. The absorbance at 440 nm was determined. Results given represent mean values from triplicate measurements.