Observations on the different substrate behavior of tropocollagen molecules in solution and intermolecularly cross-linked tropocollagen within insoluble polymeric collagen fibrils.
Bacterial collagenase was used to compare the extent of digestion of tropocollagen monomers in solution and in reconstituted fibrils with that of tropocollagen molecules intermolecularly cross-linked within insoluble polymeric collagen fibrils obtained from mature tendons at given time-intervals. The extent of digestion of tropocollagen monomers in solution was directly proportional to the enzyme concentration (a range of enzyme substrate molar ratios 1:200 to 1:10 was used). The extent of digestion of polymeric collagen was followed by measuring the solubilization of fluorescent peptides from fluorescent-labelled insoluble polymeric collagen fibrils. The extent of digestion of tropocollagen within polymeric collagen was linear over a very small range of enzyme concentrations, when the enzyme/substrate ratio in the reaction mixture was less than 1:400 on a molecular basis. The behavior of tropocollagen in the form of reconstituted collagen fibrils, which had been matured at 37 degrees C for 8 weeks, was intermediate between the behaviour of solutions of tropocollagen and insoluble polymeric collagen fibrils. The significance of the results is discussed in terms of the structure of polymeric collagen fibrils and the protection against enzymic attack provided by tropocollagen molecules on the circumference of the fibril. The results suggest that assays of collagenase activities based on tropocollagen as substrate cannot be directly related to the ability of these enzymes to degrade mature insoluble collagen fibrils.
D000818:Animals; D002417:Cattle; D003094:Collagen / Q000378:metabolism*; D005452:Fluoresceins; D003012:Microbial Collagenase / Q000378:metabolism; D012235:Rhodamines; D012995:Solubility; D014332:Tropocollagen / Q000378:metabolism*; D014783:Viscosity
D005452:Fluoresceins; D012235:Rhodamines; D014332:Tropocollagen; D003094:Collagen; D003012:Microbial Collagenase