The role of cross-linkages in the solubilization of insoluble collagen

To determine the relative contributions of interchain hydrogen bonds and covalent cross-linkages to the stability of collagen, the solubilization process was compared with the thermal shrinkage in a variety of solvents, some of which compete with internal protein hydrogen bonds (e.g., 2.0 M KCNS, 6.0 M urea). The solubilization process in all solvents clearly occurred in two stages showing the existence of at least two species of molecules in the intact structure. This was confirmed by coacervation-fractionation of the soluble gelatins. The thermodynamics of the activation step in solubilization were determined by rate studies. In water, at T_Extraction < T_Shrinkage, ΔF₅₀ ≠ = 27.7 kcal., ΔH₅₀ ≠ = 42.5 kcal., ΔS₅₀ ≠ = 45.6 e.u.; at T_E > T_S, ΔF₇₀ ≠ = 27.5, ΔH₇₀ ≠ = 5.9, ΔS₇₀ ≠ = -63. In 2.0 M KCNS ΔF₅₀ ≠ = 25.9, ΔH₅₀ ≠ = 13.3, ΔS₅₀ ≠ = -38.9 while ΔF₇₀ ≠ = 27.0, ΔH₇₀ ≠ = 6.7, ΔS₇₀ ≠ = -59.2. Since ΔS₆₀ ≠ = +361 e.u. and ΔH₆₀ ≠ 141 for shrinkage, it is evident that imbibition of solvent rather than thermal shrinkage is essential for extraction. The discontinuity in ΔS ≠ occurred when the molecular species being extracted was changed rather than at T_S as would have been expected if H-bonds were the primary cause of the insolubility of collagen. The origin and distribution of the cross-linkages is discussed.