The Dissolution of Insoluble Bovine Collagens by Cathepsin BI, Collagenolytic Cathepsin and Pepsin: The influence of collagen type, age and chemical purity on susceptibility

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Abstract
Insoluble collagens were obtained from different bovine tissues using an exhaustive extraction procedure. Each collagen preparation was tested for its susceptibility to degradation by bovine spleen cathepsin Bl, collagcnolytic cathepsin and pepsin at 25°C and 37°C. The extent of collagenolysis varied widely. At 25°C skin and tendon collagens were nearly completely degraded in 48 h while bone collagen was almost totally resistant. Most, but not all collagens were degraded more effectively at 37°C. Some differences were noted in the relative effectiveness of cathepsin B1 and collagenolytic cathepsin against specific collagens. Pepsin was relatively more effective than the cathepsins except against nasal cartilage collagen. The progress curves revealed that collagenolysis occurred in up to three stages. There was an initial fast release of highly susceptible collagen and a much slower, linear release of collagen in the second stage. The third stage was reached when the rate diminished abruptly and the substrate residues remained almost or totally resistant to further degradation. Stabilization of the intermolecular cross-links in collagen by reduction with potassium borohydride before extraction increased the resistance to enzymatic dissolution. Pre-treatment of nasal cartilage collagen with chon-droitinase ABC or trypsin-H2O2 increased its susceptibility, especially to pepsin. The effects of temperature, pH, ionic strength and enzyme concentration were examined for pepsin. In addition the effect of prior extraction with urea, guanidine HC1 or NaOH was tested with some resistant collagens. It was concluded that the number and location of the intermolecular cross-links and the type and quantity of associated proteoglycans were important factors in determining the degree of resistance of the collagen to enzymatic dissolution. The evidence obtained from these data indicated that the genetic type of collagen was of little importance in determining the extent of dissolution by these enzymes.