Collagenase Type 4

Collagenase type 4 is partially purified and 0.22µm filtered, contains higher relative levels of protease activity, particularly clostripain. The collagenase assay is a modification of the Mandl collagen digestion procedure wherein collagenase is incubated for five hours with native collagen and the extent of collagen breakdown is determined using the Moore and Stein, JBC, 176, 367, (1948) colorimetric ninhydrin method. Amino acids released are expressed as micromoles L-leucine per milligram collagenase in 5 hours at 37°C, pH 7.5. Caseinase activity, a measure of non-specific proteolytic activity, is determined using the above assay and substituting 25 milligrams vitamin free casein for the collagen substrate. Caseinase activity is calculated as for collagenase activity. Clostripain activity is measured after activation in 2.5 mM dithiothreitol (DTT). One unit hydrolyzes one micromole of BAEE per minute at 25°C, pH 7.6, after activation. Tryptic activity is assayed using the same BAEE method as clostripain, but without activation.