Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation.

摘要

OBJECTIVES: A collagen degradation activity (CDA) assay was developed to improve the biochemical characterization of purified collagenase used for islet isolation. MATERIALS AND METHODS: Purified class I collagenase (CI) or class II collagenase (CII) from Clostridium histolyticum cultures were used in all experiments. The CDA assay was performed by incubating 50 microg/mL of FITC fibrils with CI or CII for 60 minutes at 35 degrees C. The correlation of the molecular species of the enzyme to CDA was determined by fractionating CI:CII mixtures over an anion exchange column. Individual fractions were analyzed for A280, CDA activity, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to correlate chromatographic analysis of these enzyme mixtures to the molecular species of collagenase effective in collagen degradation. RESULTS: CI has approximately 6 to 17 x higher specific activity than CII in this assay. Assays of different individual fractions recovered after anion exchange chromatography showed that the CDA of collagenase was dependent on the molecular species of the enzyme. Only intact CII and CI with molecular weights >or=100 kDa could degrade collagen fibrils. CONCLUSIONS: This assay provides a more reliable assessment of the functional activity of collagenase enzymes than peptide substrates currently used today. Fractionation of purified collagenase mixtures by anion exchange chromatography followed by analysis of individual fractions by SDS-PAGE and CDA assays will provide a powerful tool to analyze the molecular species of CI and CII required for islet isolation.