Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography

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Abstract
Six collagenases present in the culture filtrate of C. histolyticum were purified to homogeneity. Chromatography over hydroxylapatite, Sephacryl S-200 and L-arginine-Affi-Gel 202 removes the brown pigment and the great majority of the contaminating proteinases active against casein, benzoyl-L-arginine ethyl ester and elastin. Reactive Red 120 dye ligand chromatography subdivides the collagenases, which have very similar physicochemical properties, among 4 fractions. The final purification is achieved by chromatography over DEAE-cellulose and SP-Sephadex. All 6 collagenases, designated .alpha., .beta., .gamma., .delta., .epsilon. and .zeta. by the order of their purification, are highly active against collagen and devoid of other proteolytic activities. Each exhibits a single band on sodium dodecyl sulfate-polyacrylamide gels. Two distinct subspecies of the .alpha. and .gamma. enzymes were isolated, which have the same MW and activity but different isoelectric points. There is some less pronounced microheterogeneity for the other collagenases. On the basis of their activities toward native collagen and the synthetic peptide 2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine (FALGPA), the 6 collagenases are divided into 2 classes. Class I collagenases (.alpha., .beta. and .gamma.) have high collagenase activity and moderate FALGPA activity, class II collagenases (.delta., .epsilon. and .zeta.) have moderate collagenase and high FALGPA activities. The relationship between these 6 collagenases and others reported to have been isolated in the literature was also examined.