A modified procedure for the purification of clostridial collagenase

A method is described for the purification of clostridial collagenase from a crude enzyme preparation employing cation exchange chromatography on SP Sephadex, anion exchange chromatography on DEAE cellulose and gel filtration on Sephacryl S-200. Emphasis was placed on purity using continuous shallow gradients for the ion exchange separations to increase resolution and monitoring eluates both with respect to ultraviolet light absorption at 230 nm and analytical disc gel acrylamide electrophoresis. In addition, protein fractions were assayed for collagenolytic and non-specific proteolytic activity. The purity of the final preparation was assessed by acrylamide electrophoresis, gel filtration and amino acid analysis. The isolated enzyme hydrolyzed between 30 and 40% of rat tail tendon collagen in 1 h at 37 degrees C and lacked measurable trypsin or elastase-like activity.