Collagenase - Assay

Weigh 25 mg of Worthington bovine collagen into each of four test tubes. Include at least two tubes to serve as blanks which will contain no enzyme. Add 5.0 ml of 0.05 M TES buffer to the tubes and incubate at 37°C for 15 minutes. Start the reaction by adding 0.1 ml of enzyme dilution to appropriate tubes.

After 5 hours, stop the collagenase reaction by transferring 0.2 ml of solution (leaving behind the collagen) to test tubes containing 1.0 ml of ninhydrin-citric acid mixture. Include an enzyme blank (collagen incubated with 0.1 ml TES buffer in place of enzyme). Heat for 20 minutes in a boiling water bath. After cooling, dilute with 5 ml of 50% n-propanol. Let stand for 15 minutes and read absorbance at 600 nm. From an L-leucine standard curve determine micromoles amino acid equivalent to leucine liberated.

Non-specific protease activity (i.e. caseinase activity) is determined using the above assay and substituting 25 milligrams vitamin free casein for collagen. The reaction is stopped after 5 hours by the addition of 0.5 ml of 50% trichloroacetic acid. After centrifugation, 0.2 ml of the supernatant is transferred to 1.0 ml of ninhydrin and treated as above. Caseinase activity is calculated as collagenase activity.