Date of Award
Master of Science in Pharmacology and Toxicology
Pharmacology and Toxicology
George C. Fuller
The classical assay for detection of collagenolytic activity has been the release of soluble peptide fragments from reconstituted radioactive gels following incubation with the enzyme. Because this assay system is dependent on diffusion of the enzyme through the gel, examination of the kinetic parameters of the enzyme has been hampered. In the present study, a soluble substrate for collagenase was developed by coupling purified Type I collagen to the fluorophor, 2-methoxy-2,4-diphenyl-3(2H) furanone (MDPF). MDPF-labeled collagen was incubated with varying amounts of collagenase for two hours and electrophoresed on polyacrylamide gels. The gels were scanned on a Gilford spectrophotometer and the resulting peaks quantitated on a Neumonic Electronic planimeter. It was found that the relationship between substrate disappearance and product disappearance was not stoichiometrical. After treatment of the fluorophor-labeled substrate with pepsin, the formation of product and breakdown of substrate was stoichiometrical. To test the integrity of the triple-helix, MDPF-labeled Type 1 collagen was treated with trypsin. The results indicate that substrate treated with pepsin was more resistant to trypsin degradation than fluorescent collagen not pretreated with pepsin.
LaPosta-Frazier, Nancy A., "DEVELOPMENT OF A MAMMALIAN COLLAGENASE ASSAY USING A SOLUBLE SUBSTRATE" (1980). Open Access Master's Theses. Paper 201.