Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen.
Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.
D001665:Binding Sites; D003094:Collagen / Q000378:metabolism*; D005353:Fibronectins / Q000097:blood*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005780:Gelatin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D013861:Thiocyanates
D005353:Fibronectins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D013861:Thiocyanates; D005780:Gelatin; D003094:Collagen; D016650:Fluorescein-5-isothiocyanate