Purification of nonspecific protease-free collagenase from Clostridium histolyticum

The collagenase (EC 3.4.24.3) produced by the bacterium Clostridium histolyticum has been purified free from nonspecific protease contaminants by a two-step procedure. The crude culture medium is chromatographed over heparin-Sepharose and Sephacryl S-200, and the resulting preparation has no activity versus noncollagenous proteins or N alpha-benzoyl-L-arginine ethyl ester, yet cleaves native thermally reconstituted collagen fibrils quite efficiently (specific activity, 3000 units/mg). The purification described may be useful for those investigators requiring substantially purified collagenase for applications such as cell culture or collagen quantitation in protein mixtures.