The in vitro fibril assembly of calf skin collagen was examined as a function of ionic strength and temperature. In a 0.03 M NaPi, pH 7.0, buffer, fibril assembly required a minimum critical concentration of collagen. At nearly physiological ionic strengths and temperatures, the critical concentration was less than 1 microgram/mL and required a very sensitive method for measurement. Raising the ionic strength of the buffer resulted first in higher and then lower critical concentrations. Raising the temperature led to lower critical concentrations. A van't Hoff plot of the fibril growth constant calculated from the critical concentration gave positive enthalpy changes and positive heat capacity changes which indicate that the fibril growth is driven by both hydrophobic and ionic inter-collagen interactions. Sedimentation equilibrium studies showed the collagen to be monomeric at subcritical concentrations. Differential scanning microcalorimetric studies showed only one very sharp heat absorption peak for the fibril assembly which coincided with the appearance of solution turbidity. Within experimental error, the enthalpy changes of the fibril assembly measured with the microcalorimeter were of the same magnitude as the van't Hoff enthalpy changes. These results are discussed in light of a cooperative nucleation-growth mechanism of collagen fibril assembly proposed earlier.

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Type I collagen purified from calf skin was further separated into monomeric and oligomeric fractions and characterized with gel electrophoresis and measurement of solution viscosity. The thermal stabilities of the triple-helical structure of the collagen molecules of these preparations and the fibrils assembled therefrom were determined with differential UV spectroscopy and scanning microcalorimetry. The monomeric collagen was reduced with NaBH4-, and the kinetics and equilibrium of the reversible fibril assembly-disassembly were examined in detail. Fibril assembly and disassembly of the collagen induced by slow scans of temperature showed hysteresis. The assembly curve was very sharp whereas the disassembly curve was gradual. Equilibrium centrifugation showed the collagen disassembled from the fibrils to be predominantly monomers. However, unlike the unassembled collagen, the collagen disassembled from fibrils by cooling showed no lag phase in subsequent cycles of fibril assembly. The thermodynamic parameters of fibril growth were derived from a fibril disassembly curve. Fibril growth was weaker for the NaBH4-reduced monomeric collagen than the native crude collagen, perhaps due to the removal of oligomers and the changes in the molecular structure brought by the reduction. The results corroborated the strongly cooperative mechanism for the fibril assembly proposed in the preceding paper.