C. histolyticum collagenase and many other bacterial neutral proteases are metallo-proteases. They require excess calcium to maintain their enzyme activity. One mM calcium ion is sufficient to protect this activity.
Cation chelating agents such as EDTA or EGTA should not be used in the presence of these enzymes. As many of you know, these chelating agents are routinely used in the isolation of mammalian hepatocytes using Seglen’s two step procedure. However, the solutions containing chelating agents are used in a separate step of this procedure and are washed out of the tissue before perfusion of the collagenase solution in a calcium containing buffered media.
Phosphate buffered saline enzyme solution can be used for cell isolation but prolonged or frozen storage of the enzyme in this solution will lead to loss of collagenase or protease activity. Phosphate chelates calcium, leading to loss of enzyme activity.