TY - JOUR
T1 - Elution of fibronectin proteolytic fragments from a hydroxyapatite chromatography column. A simple procedure for the purification of fibronectin domains
AU - Zardi, L.
AU - Carnemolla, B.
AU - Balza, E.
AU - Borsi, L.
AU - Castellani, P.
AU - Rocco, M.
AU - Siri, A.
PY - 1985
Y1 - 1985
N2 - Human plasma fibronectin is composed of at least five distinct domains which we refer to as Hep-1/Fib-1, Gel, Cell, Hep-2 and Fib-2 depending on their affinity for heparin (Hep), gelatin (Gel), the cell surface (Cell) or fibrin (Fib). These domains are aligned from the NH2 to the COOH terminus in the above order and can be separated from each other by mild proteolytic digestion. We have studied the elution of fibronectin thermolysin digest from a hydroxyapatite column using a linear gradient (0.5-190 mM) of sodium phosphate buffer. The five major fibronectin domains were eluted from the hydroxyapatite chromatography column in the following order: Gel, Fib-2, Cell, Hep-1/Fib-1 and Hep-2. They were identified on the basis of their molecular mass, affinity to different macromolecules and reaction with domain-specific monoclonal antibodies. All domains except the Cell and Hep-2 domains eluted as single homogeneous peaks. The Cell domains eluted as two different peaks and the Hep-2 domain eluted as four different peaks. This is the first time that heterogeneity of these two domains has been observed. Since chromatography of a fibronectin thermolysin digest on a hydroxyapatite column provides a good separation of the five major fibronectin domains, we have elaborated a procedure in which each fibronectin domain is purified by no more than two steps: hydroxyapatite and molecular exclusion chromatography. Fractionation of fibronectin proteolytic digest on a hydroxyapatite chromatography column should be of great value in the comparative analysis of fibronectin from different sources and in the study of fibronectin heterogeneity. Its use in combination with molecular exclusion chromatography offers a simple and high-yield method for the purification of large amounts of fibronectin domains.
AB - Human plasma fibronectin is composed of at least five distinct domains which we refer to as Hep-1/Fib-1, Gel, Cell, Hep-2 and Fib-2 depending on their affinity for heparin (Hep), gelatin (Gel), the cell surface (Cell) or fibrin (Fib). These domains are aligned from the NH2 to the COOH terminus in the above order and can be separated from each other by mild proteolytic digestion. We have studied the elution of fibronectin thermolysin digest from a hydroxyapatite column using a linear gradient (0.5-190 mM) of sodium phosphate buffer. The five major fibronectin domains were eluted from the hydroxyapatite chromatography column in the following order: Gel, Fib-2, Cell, Hep-1/Fib-1 and Hep-2. They were identified on the basis of their molecular mass, affinity to different macromolecules and reaction with domain-specific monoclonal antibodies. All domains except the Cell and Hep-2 domains eluted as single homogeneous peaks. The Cell domains eluted as two different peaks and the Hep-2 domain eluted as four different peaks. This is the first time that heterogeneity of these two domains has been observed. Since chromatography of a fibronectin thermolysin digest on a hydroxyapatite column provides a good separation of the five major fibronectin domains, we have elaborated a procedure in which each fibronectin domain is purified by no more than two steps: hydroxyapatite and molecular exclusion chromatography. Fractionation of fibronectin proteolytic digest on a hydroxyapatite chromatography column should be of great value in the comparative analysis of fibronectin from different sources and in the study of fibronectin heterogeneity. Its use in combination with molecular exclusion chromatography offers a simple and high-yield method for the purification of large amounts of fibronectin domains.
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U2 - 10.1111/j.1432-1033.1985.tb08690.x
DO - 10.1111/j.1432-1033.1985.tb08690.x
M3 - Article
C2 - 2982601
AN - SCOPUS:0022005352
VL - 146
SP - 571
EP - 579
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
SN - 0014-2956
IS - 3
ER -