What is the difference between the enzyme assays that are commonly included in a certificate of analysis?

Collagenase activity can be assessed by a number of different enzyme activity assays. The enzyme activity is defined by the substrate so 

  • peptidase activity is detected when Pz-peptide (Wunsch assay) or FALGPA is used as substrates;
  • gelatinase activity is detected when gelatin or azocoll are as substrates;
  • collagen degradation activity (CDA) is detected when using collagen fibers (Mandl assay) or  FITC collagen fibrils (kinetic fluorescent microplate CDA assay) as substrate.

C. histolyticum class II collagenase has about a 50 fold higher peptidase activity than a comparable amount of class I collagenase. Similarly, class I collagenase has about a 2-5 fold higher gelatinase activity than class II collagenase. This difference in enzyme activity profiles led to definition of class I and class II collagenase.

Recently it has been shown that the CDA activities are different depending on the assay and substrate used to detect this activity.  Worthington and Sigma use the Mandl CDA assay to detect this activity. Intact or truncated class I collagenase has about a 2-3 fold higher specific activity than intact class II collagenase. In contrast, in the kinetic fluorescent microplate CDA, the activity correlates with the number of collagen binding domains on collagenase. Intact class I collagenase with two collagen binding domains has 7 to 10 fold higher specific CDA (U/mg protein) than intact class II or truncated class I collagenase, each having one collagen binding domain.