If fibril formation in a cold collagen solution (0.1 mg/ml) is initiated by warming to 26°C for 10 min, the temperature can be reduced to 4°C for up to 40 min during the turbidity lag period (and then returned to 26°C) without affecting the reaction half-time of 125 min. Any additional time at 4°C increases the half-time by the same amount. The product present after 50 min at 26°C, or 10 min at 26°C followed by 40 min at 4°C, is long thin filaments with minimal diameters of 2 to 4 nm. Therefore, fibril formation in vitro requires at least three steps. The first step, initiation, involves a temperature-dependent change which leads to an unidentified intermediate. The second step is linear growth of filaments by a process that is apparently temperature-independent. The third, step, which is associated with the turbidity change, is lateral association of filaments by a temperature-dependent process. The reaction times of both the second and third steps are inversely proportional to collagen concentration suggesting that both linear and lateral growth occur by accretion. If there is a nucleation step, it is not rate-limiting. However, it is likely that there is a temperature-dependent critical filament length for lateral assembly. Reduced collagen (unable to cross-link) behaves the same way except that the reaction half-time is 210 min and the turbidity increase can be reversed by cooling, yielding filaments indistinguishable from those formed in Step 2. The longer half-time and a very small apparent critical concentration suggest that Step 3 involves a series of stages leading to an increasingly stable fibril. Covalent cross-linking may increase the net rate of lateral assembly and decrease the critical filament length by decreasing the rate of disassembly in the early stages. The filaments obtained by cooling are stabel at 4°C but at 26°C they reassemble and form native fibrils again. Their structure is not known, but they may be the 5-fold helical microfibrils proposed from other evidence.
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