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

Abstract

Six collagenases present in the culture filtrate of Clostridium histolyticum have been 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 four fractions. The final purification is achieved by chromatography over DEAE-cellulose and SP-Sephadex. All six 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 have been isolated, which have the same molecular weight 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 six collagenases are divided into two classes. Class I collagenases (alpha, beta, and gamma) have high collagenase activity and moderate FALGPA activity while the class II collagenases (sigma, epsilon, and sigma) have moderate collagenase and high FALGPA activities. The relationship between these six collagenases and other reported to have been isolated in the literature has also been examined.

The Recognition Sites of the Integrins α1β1 and α2β1 within Collagen IV Are Protected against Gelatinase A Attack in the Native Protein

“…A unique Nterminal sequence could not be detected. For further experiments, the commercial bacterial collagenase was subjected to several chromatographic purification steps to remove contaminating proteases (25). This highly purified enzyme was then used to digest preparation A.…”

A Study of the Collagen-binding Domain of a 116-kDaClostridium histolyticum Collagenase

“…Binding of ColH was not inhibited by low temperature unlike activity, suggesting that binding is independent of hydrolysis. In a commercial preparation of C. histolyticum collagenase, Bond and Van Wart (22)(23)(24) reported the presence of multiple forms of collagenases which showed similar peptide maps and amino acid analyses. They suggested that this organism harbors multiple collagenase genes (4).…”

Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase

“…Apart from the importance of this enzyme as a virulence factor, much attention has been paid to its biochemical properties. It is a zinc-metalloprotease capable of degrading both native and denatured collagen fibrils, unlike eukaryotic collagenases, which cleave only specific sites in native collagens (6,23). Because of its broad substrate specificity and its abundance in culture filtrates, it has been widely used for the disintegration of connective tissue and separation of tissue culture cells (26) and also for cleaving a collagen-like peptide in fusion proteins (25).…”