Collagenase/Elastase

Clostridium histolyticum, Bacterial collagenases, collagenase, Porcine pancreatic elastase, Protease, ColH, ColG, Chymotrypsin-like elastase family member 1, Elastase-1

Collagenase/Elastase is partially purified and 0.22μm filtered and contains 22,500 units of collagenase type 1 and 30 units of elastase per vial. Collagenase type 1 has the original balance of collagenase, caseinase, clostripain and tryptic activities. 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. Porcine pancreatic elastase has a molecular weight of 25.9 kDa, and a pH optimum of 8.5. Suitable for the isolation of Type II lung cells.

Store vial at 2 - 8 ° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. Dilute only prior to immediate use.

Elastase is assayed using a method adapted from that of Feinstein et al., Biochem. Biophys. Res. Comm., 50, 1020 (1973) and using the more soluble substrate of Bieth et al., Biochem. Med., 11, 350 (1974) . 1 SucAla3NA unit is approximately equivalent to 6 elastin digestion units. Aqueous liquid suspensions should be aseptically handled to avoid bacterial contamination. Due to the viscous nature of the aqueous suspension the vial should be rinsed to recover contents. One Unit cleaves one micromole of N-succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide per minute at 25°C, pH 8.0. The collagenase assay is a modification of Mandl 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. Collagenase is typically used at concentrations from 0.05 % to 0.5 % (w/v) in balanced salt solutions such as Hank’s, Earle’s and others. Specific conditions for reactivity should be optimized by the end user.