Biomaterial Database

Fluid-phase interaction between human plasma fibronectin and gelatin determined by fluorescence polarization assay. 1983

H Forastieri, and K C Ingham

Previous studies of the binding properties of fibronectin (Fn) have utilized methods whereby one or the other macromolecule was immobilized on a solid phase. In order to examine the interaction between human plasma Fn and gelatin in solution, the latter was labeled with fluorescein isothiocyanate (FITC) whose fluorescence polarization (P) served as a sensitive indicator of the formation of soluble complexes. Changes in P were detectable at Fn concentrations below 10(-9) M and continued up to concentrations above 10(-6) M at pH 7.3 and 25 degrees C. Fractionation of FITC-gelatin by exclusion chromatography and titration of selected fractions revealed a trend towards higher affinity with increasing size. A high-molecular-weight fraction comprised of beta and gamma components and a low-molecular-weight fraction comprised primarily of alpha chains exhibited sigmoidal increases in P (apparent positive cooperativity) with 50% saturation near 10(-9) and 10(-8) M Fn, respectively. By contrast, a 42-kDa chymotrypsin-generated Fn fragment which retains the ability to adhere to gelatin-Sepharose exhibited normal (noncooperative) binding to both gelatin fractions with Kd = 7 X 10(-7) M. In all cases, the increase in P could be reversed by addition of excess unlabeled gelatin or urea. The interaction of FN with FITC-gelatin provides the basis for a fast and sensitive determination of Fn levels in plasma and other fluids. Interference caused by other proteins such as albumin, which has an affinity for the fluorescein moiety, could be minimized by addition of 1.0 M NaCl which had no effect on the interaction between Fn and gelatin.

UI	MeSH Term	Description	Entries
D007201	Indicator Dilution Techniques	Methods for assessing flow through a system by injection of a known quantity of an indicator, such as a dye, radionuclide, or chilled liquid, into the system and monitoring its concentration over time at a specific point in the system. (From Dorland, 28th ed)	Dilution Techniques,Dilution Technics,Indicator Dilution Technics,Dilution Technic,Dilution Technic, Indicator,Dilution Technics, Indicator,Dilution Technique,Dilution Technique, Indicator,Dilution Techniques, Indicator,Indicator Dilution Technic,Indicator Dilution Technique,Technic, Dilution,Technic, Indicator Dilution,Technics, Dilution,Technics, Indicator Dilution,Technique, Dilution,Technique, Indicator Dilution,Techniques, Dilution,Techniques, Indicator Dilution
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D002846	Chromatography, Affinity	A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D005353	Fibronectins	Glycoproteins found on the surfaces of cells, particularly in fibrillar structures. The proteins are lost or reduced when these cells undergo viral or chemical transformation. They are highly susceptible to proteolysis and are substrates for activated blood coagulation factor VIII. The forms present in plasma are called cold-insoluble globulins.	Cold-Insoluble Globulins,LETS Proteins,Fibronectin,Opsonic Glycoprotein,Opsonic alpha(2)SB Glycoprotein,alpha 2-Surface Binding Glycoprotein,Cold Insoluble Globulins,Globulins, Cold-Insoluble,Glycoprotein, Opsonic,Proteins, LETS,alpha 2 Surface Binding Glycoprotein
D005454	Fluorescence Polarization	Measurement of the polarization of fluorescent light from solutions or microscopic specimens. It is used to provide information concerning molecular size, shape, and conformation, molecular anisotropy, electronic energy transfer, molecular interaction, including dye and coenzyme binding, and the antigen-antibody reaction.	Anisotropy, Fluorescence,Fluorescence Anisotropy,Polarization, Fluorescence,Anisotropies, Fluorescence,Fluorescence Anisotropies,Fluorescence Polarizations,Polarizations, Fluorescence
D005780	Gelatin	A product formed from skin, white connective tissue, or bone COLLAGEN. It is used as a protein food adjuvant, plasma substitute, hemostatic, suspending agent in pharmaceutical preparations, and in the manufacturing of capsules and suppositories.	Gelafusal
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012996	Solutions	The homogeneous mixtures formed by the mixing of a solid, liquid, or gaseous substance (solute) with a liquid (the solvent), from which the dissolved substances can be recovered by physical processes. (From Grant & Hackh's Chemical Dictionary, 5th ed)	Solution