Investigation of the Lag Phase of Collagen Fibrillogenesis Using Fluorescence Anisotropy

Abstract

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.