Purification and characterization of tadpole back-skin collagenase with low gelatinase activity.

A collagenase secreted by tadpole (Rana catesbiana) back-skin explants in culture has been purified to electrophoretic homogeneity by successive chromatography on sulfopropyl Sephadex, Sephacryl S-200, collagen Sepharose, and heparin Sepharose. The purified enzyme has a molecular weight of approximately 49,000 and an isoelectric pH of 5.0. The enzyme is more active versus soluble collagen than reconstituted fibrils and exhibits very low activity against gelatin (specific activities: Type I collagen, 7660 units/mg; Type I gelatin, 66 units/mg). The collagenase obeys simple Michaelis-Menten kinetics using soluble type I collagen (Km), 0.35 microM; Vm, 1380 units/mg, at 25 degrees C and pH 7.4) and is inhibitable by chelating agents specific for transition metals. Methylene blue catalyzes the photoinactivation of this collagenase, suggesting the presence of essential histidine, tryptophan, tyrosine, or methionine residues.