Collagenases were extracted from the skeletal muscle of winter flounder (Pseudopleuronectes americanus) with Tris-HCl buffer, pH 7.4, containing 5 mM CaCl$ sb2.$ The crude extract in the active form was purified by ammonium sulfate fractionation, followed by a succession of column chromatographic steps which included ion-exchange, immobilized metal affinity and size-exclusion in the Fast Protein Liquid Chromatography (FPLC) system. The trypsin-like and chymotrypsin-like activities of the crude extract diminished with purification. A comparative study of the collagenase fraction from ion-exchange chromatography (IEX-1) and the commercial collagenase fraction from Clostridium histolyticum indicated that the two enzymes were similar with respect to their response to temperature but differed with respect to their response to pH. The enzymes differed slightly in terms of their thermal and pH stabilities. The winter flounder collagenase fraction from size-exclusion chromatography (SEC) had a higher optimum pH temperature than the IEX-1 fraction as well as the commercial collagenase. However, both SEC and the IEX-1 extracts had the same optimum pH. The collagenase fraction from SEC had a slightly lower thermal stability than the IEX-1 fraction and the commercial collagenase. / The higher catalytic efficiency (V$ sb{ rm max}$/K$ sb{ rm m}$') and the lower $ Delta$G values for C. histolyticum collagenases showed that bacterial collagenases are better catalysts than winter flounder skeletal muscle collagenases for the PZ-peptide hydrolase reaction at 37$ sp circ$C and pH 7.1. / Zymography revealed the presence of two collagenase isoenzymes from the fish muscle, designated as WFC-1 and WFC-2 with molecular weights of 79,600 and 75,500, respectively. WFC-1 was separated from WFC-2 by electrophoretic blotting onto the PVDF membrane. The amino acid composition of WFC-1 and WFC-2 were closely related. / The fish collagenases were inhibited by metal chelators, EDTA and 1,10-phenanthroline suggesting that these enzymes are metalloproteases. The enzyme activity in the presence of EDTA and 1,10-phenanthroline were recovered upon addition of low levels of calcium and zinc ions, respectively. Higher levels of these metal ions inhibited the isoenzymes. 2-Mercaptoethanol and dithiothreitol were also effective inhibitors.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.34467 |
| Date | January 1997 |
| Creators | Teruel, S. R. Luzette T. |
| Contributors | Simpson, B. K. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Food Science and Agricultural Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001566888, proquestno: NQ30402, Theses scanned by UMI/ProQuest. |
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