METHOD OF DETERMINATION OF NEUTRAL COLLAGENASE ACTIVITY Russian patent published in 1995 - IPC

FIELD: enzymology. SUBSTANCE: collagen specimen is treated with 0.5% dye solution - Orange active at pH 8.0 and temperature 40 C for 2 h (ratio of dye and substance to be stained is from 20 to 200 wt.-%), washed off with water from dye excess and dried at the room temperature. Reaction mixture is prepared at the following reagent concentrations, mg%: collagenase tested specimen 5 ± 0.2; stained collagen 500 + 20; tris 240 + 20; calcium chloride 110 + 10; sodium chloride 170 + 20, and distilled water - the rest. Mixture is incubated at optimal values of temperature and pH medium. After reaction completion specimens were diluted with tris-buffer by 5-fold, filtered, and optical density of stained solutions is determined at 510 nm. Collagenase activity is calculated by the formula: A = 377.8 x [CE/at]^0,56 where A - collagenase activity, kU/mg; E - optical density of filtrate at 5-fold dilution; a - optical density of filtrate after complete dissolving of 1 mg stained collagen; C - protein concentration in tested specimen, wt. -%; t - time reaction, min. EFFECT: improved method of determination. 3 tbl