Size exclusion and diffusion of fluoresceinated probes within collagen fibrils

The diffusion of fluoresceinated probes inside single collagen fibrils was investigated by imaging the migration of fluorescence along the fibrils in oil and by monitoring fluorescence recovery after photobleaching (FRAP). Probes were excluded from the fibrils according to their size. Probes that were not excluded diffused in the fibrils, but FRAP occurred 6x10(-4) times more slowly than in water due to binding interactions between collagen and the probes. The dissociation constant of the fluorescein-collagen complex was determined (K-D=1.8+/-0.1 muM).