Five sepharose‐bound ligands for the chromatographic purification of Clostridium collagenase and clostripain

Two of the extracellular proteolytic enzymes produced by the anaerobic microorganism Clostridium histolyticum have been studied in particular because of their restricted specificity: collagenase (clostridiopeptidase A, EC 3.4.24.3) which cleaves native collagen preferentially at the amino group of glycine residues and clostripain (clostridiopeptidase B, EC 3.4.22.8) which cleaves proteins exclusively at the carboxyl of arginine residues. Several methods have been proposed for their separation and purification: the highest specific esterolytic activity values as yet obtained for clostripain were 1 .l. pkat.mg-’ [l] and 2.72 pkat.mg-’ (after activation) [2] ; the highest value of specific activity of purified collagenase on a synthetic substrate [3] was 0.27 pkat.mg-’ [4]. In both cases these values can still not be considered as upper limits. In this paper we describe several chromatographic separations using Sepharose-bound ligands: compounds which act as competitive inhibitors of clostripain, such as arginine [5], polyarginine [l] , benzamidine [6] and soybean trypsin inhibitor [7], the SH-binding mercurial p-aminophenylmercuric acetate [8] and acridme dye Rivanol which is known to inhibit collagenase [9] .