Characterization of the individual collagenases from Clostridium histolyticum

Abstract

Six collagenases (.alpha., .beta., .gamma., .delta., .epsilon. and .zeta.), previously isolated from C. histolyticum, were characterized in detail. The MW determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis range from 68,000-125,000. Isoelectric focusing experiments demonstrate that the isoelectric points of the collagenases are in the 5.35-6.20 range. The subspecies of .alpha.- and .gamma.-collagenases (.alpha.1 vs. .alpha.2 and .gamma.1 vs. .gamma.2) have different isoelectric points but the same MW. Microheterogeneity was also observed for the .beta.- and .epsilon.-collagenases. The amino acid compositions of all 6 collagenases were determined, and analysis for neutral sugars and hexosamines shows that none of the enzymes have a significant carbohydrate content. Zn and Ca are the only metals that copurify with the collagenases. The purified enzymes contain .apprx. 1 mol of Zn/mol of protein and a Ca content that varies from .apprx. 2 mol/mol for .alpha.-collagenase to .apprx. 7 mol/mol for .beta.-collagenase. All of the collagenases are 5-10 times more active against gelatin than collagen. The .alpha.-, .beta.- and .gamma.-collagenases are significantly less active toward the synthetic peptide substrates examined than the .delta.-, .epsilon.- and .zeta.-collagenases. C. histolyticum produces 2 distinct classes of collagenase.