Abstract
Publisher Summary This chapter describes the purification and assays of vertebrate collagenases. Isolation of collagenases can be achieved through extraction from tissues or by accummulation of enzyme in culture medium from explants or monolayer cultures of cells. Purifications of collagenase have involved ion-exchange chromatography, gel-filtration chromatography, and various types of specialized affinity chromatography. Optimal activation conditions must be determined experimentally for each source of collagenase to ensure detection of maximal activity. In general, purer preparations of collagenase require milder activation procedures (e.g., less activating proteinase) for less time or at lower temperature. Although several neutral proteases have been used successfully to activate latent collagenases in vitro, trypsin has been chosen most commonly. Assays for vertebrate collagenase active against types I, II, and III collagens must be based on the specificity of the enzyme cleavage of collagen. Assays using soluble substrate can measure the specific cleavage rate, and analysis of products can be adapted to identify the cleavage site.
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