The preparation of a bacterial collagenase containing negligible non-specific protease activity.
A three stage procedure for the purification of crude bacterial collagenase is described. The three stages were ion exchange chromatography on SP-Sephadex and DEAE-cellulose, followed by molecular sieve chromatography on Sephadex G-200. The end product was eluted from Sephadex G-200 as a single peak of absorbance at 280 nm, and a single zone of activity against gelatin. The active eluate was divided into two halves, designated fraction 1 and 2 collagenase. Their activities were greater than those of commercially available collagenases when assayed viscometrically against pepsin solubilized collagen from guinea-pig skins. The non-specific protease activities in both fractions were much less than in the commercially available purified collagenases, and fraction 1 collagenase liberated only 2.6% of a [3H]-tryptophan label from a substrate of 2 mg of labelled chick embryo proteins, after an 18 hour incubation. When polymeric collagen was incubated with fraction 1 collagenase, at a final enzyme : substrate ratio of 1:160, the collagen was digested, resulting in the loss of 99.8% hydroxyproline as dialysable material.
D000818:Animals; D002642:Chick Embryo; D003013:Clostridium / Q000201:enzymology*; D005069:Evaluation Studies as Topic; D005780:Gelatin; D006168:Guinea Pigs; D006909:Hydroxyproline / Q000032:analysis; D003012:Microbial Collagenase / Q000302:isolation & purification*; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D012867:Skin
D005780:Gelatin; D010447:Peptide Hydrolases; D003012:Microbial Collagenase; D006909:Hydroxyproline