Preparation and Characterization of Functional Domains of Porcine Plasma Fibronectin

The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine and human plasma fibronectins were adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0M, indicating that both fibronectins have very high surface hydrophobicities. Limited proteolysis of porcine plasma fibronectin with thermolysin yielded five fragments, of 140-150kDa, 43kDa, 25kDa, 17kDa and 14kDa. Analysis of the digests by high performance hydrophobic interaction chromatography and affinity chromatography on heparinor gelatin-Sepharose, together with previous results, indicated that the 140-150kDa fragment has cell-attachment and heparin-binding domains, the 43kDa fragment a collagen-binding domain, the 25kDa fragment a heparin-binding domain, the 17kDa fragment a fibrin-binding domain and the 14kDa fragment a heparin-binding domain. The three heparin-binding fragments were found to have a wide range of surface hydrophobicitie s, of which the 140-150kDa fragment had the lowest, the 25kDa fragment higher, and the 14kDa fragment the highest among all the fragments. The 43kDa and 17kDa fragments had surface hydrophobicities as high as that of fibronectin. Carbohydrate analysis and lectin binding studies using horseradish peroxidase conjugated lectins showed that porcine plasma fibronectin has 10 biantennary N-linked carbohydrate chains on the 43kDa and 140-150kDa fragments. It is noteworthy that the 43kDa collagenbinding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates (60% of the total carbohydrates)