X-ray diffraction analysis of tendon collagen at ambient and cryogenic temperatures: role of hydration.

Equatorial (d) and meridional (D) spacings of native rat tail tendon (RTT) and unmineralized native turkey leg tendon (UTLT) were measured at ambient and liquid-nitrogen temperatures, using X-ray diffraction. Cooling of air-dried RTT or UTLT caused little change in d, which was approximately equal (1.1 nm) in the two tissues before and after cooling. For fully hydrated RTT or UTLT, cooling caused the familiar broad equatorial diffraction pattern to increase in sharpness to more resemble the pattern seen in dehydrated tissue. The d-spacings of hydrated RTT and UTLT fell by 0.12 nm (8.5%) and 0.19 nm (13%), respectively to values seen (at ambient temperature) when the tissue water content is the maximum possible in the absence of unbound water (0.5 g water/g dry collagen). These results can be explained by the movement of water within the fibril. In tissue with a water content of greater than 0.5 g/g dry collagen the spacings reflecting the lateral packing of the axially-linked tropocollagen molecules comprising a collagen fibril are determined partly by the unbound component of intermolecular hydration. As the bulk water between the fibrils freezes, this mobile component remains initially unfrozen as the tissue is cooled below zero degree C. It diffuses from the intermolecular spaces into the interfibrillar spaces where it also rapidly freezes. This allows the d-spacing to decrease to the value appropriate for the presence of bound intermolecular water only. The mechanism is likely to be an energetically favourable relaxation of the lateral positions of the tropocollagen molecules from a quasi-hexagonal arrangement to hard-disk liquid-like packing. The results and methods of this study may be applicable in the elaboration of more complex collagenous systems.