Heparin binding is necessary, but not sufficient, for fibronectin aggregation. A fluorescence polarization study.
Analysis of parameters governing heparin binding to fibronectin indicates that heparin binding is a necessary, but insufficient, condition for fibronectin cryoprecipitation. Heparin binding to fibronectin is a rapid, readily reversible event which can occur under several conditions which prohibit fibronectin cryoprecipitation. While cryoprecipitation of fibronectin is abolished at temperatures in excess of 10 degrees C, appreciable heparin binding to fibronectin does occur even at 40 degrees C. While increasing ionic strength and pH inhibit both heparin binding and cryoprecipitation of fibronectin, heparin binding can still occur at high ionic strengths and pH values which completely abolish cryoprecipitation. Scatchard analysis of fluorescent polarization data reveals a biphasic heparin binding curve with high and low affinity Kd values of 3.5 X 10(-8) and 10(-6) M, respectively. In contrast to heparin binding, fibronectin aggregation is a cooperative phenomenon. Fibronectin cryoprecipitation is greatly reduced at temperatures above 10 degrees C, at pH values above pH 10, and at ionic strengths above 0.3 M. Thus, heparin binding and protein aggregation are separate events which occur during fibronectin cryoprecipitation. Results obtained here via fluorescence polarization in conjunction with other physical measurements suggest that a decrease in flexibility of the fibronectin molecule is associated with the protein aggregation step of cryoprecipitation. The role of heparin in the mechanism of fibronectin cryoprecipitation is discussed.
D000818:Animals; D005353:Fibronectins / Q000378:metabolism*; D005452:Fluoresceins; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine; D013816:Thermodynamics
D005353:Fibronectins; D005452:Fluoresceins; D046911:Macromolecular Substances; D006493:Heparin