The lag phase of collagen fibrillogenesis (1.0 mg/mL collagen solution) with
an l-glutamine-l-arginine mixture (Glu-Arg) was monitored by the fluorescence
anisotropy of tyrosine residues in real time. A suitable concentration of Glu-Arg
(40 mmol/L) could control the aggregate ingredients in a collagen solution
effectively before fibrillogenesis, and the mechanism was found to be similar to
that with the monovalent ions. Fluorescence anisotropy analysis in the lag phase for
a 1.0 mg/mL collagen solution confirmed the formation of collagen nuclei in multiple
steps during the lag phase when the initial state of the collagen molecules was
monomeric. A comparison of the fibrillogenesis lag phase for collagen solutions of
0.25, 0.50, and 1.0 mg/mL with 40 mmol/L Glu-Arg suggested that the length of the
lag phase is inversely proportional to the increase in collagen concentration.
Atomic force microscopy was used to investigate the effect of collagen aggregates on
the fiber size. Based on the fluorescence anisotropy and atomic force microscopy
results, it was proposed that an equilibrium exists between collagen aggregates and
monomers accompanied by a nucleation of collagen monomers. A kinetic analysis for
0.25 and 1.0 mg/mL collagen with 40 mmol/L Glu-Arg at 18-38 °C indicated that
collagen nucleation in the lag phase was favored by increasing temperature, and a
corresponding activation energy of 76 and 97 kJ/mol was obtained for a collagen
fibrillogenesis lag phase of 0.25 and 1.0 mg/mL, respectively.
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