Reversible association of half-molecules of ovotransferrin in solution. Basis of co-operative binding to reticulocytes.
In the present paper, gel-filtration studies of diferric-ovotransferrin (Fe2OTf), the individual half-molecules of ovotransferrin (OTf) and equimolar mixtures of half-molecules have been interpreted according to the Gilbert theory as developed by Ackers & Thompson [(1965) Proc. Natl. Acad. Sci. U.S.A. 53, 342-349]. The data indicate that the half-molecules associate reversibly in solution and allow determination of a dissociation constant, Kd' = 8.0 (+/- 2.7) microM. Equilibrium binding studies have been performed using NH4Cl to block removal of iron from equimolar differentially iodine-labelled half-molecules (125I and 131I), in order to evaluate the binding of each to chick-embryo red blood cells under identical conditions. The amount of associated half-molecules over a range of concentrations has been calculated using the constant derived from the gel-filtration experiments described above. A computerized non-linear least-squares regression analysis of the data leads to determination of Kd* (the apparent dissociation constant for the interaction between OTf or half-molecules and the transferrin (Tf) receptors of chick-embryo red blood cells) and Bmax (binding at infinite free-ligand concentration) for the half-molecules similar to those found for Fe2OTf. Recent reports confirm that the two iron-binding domains of both OTf and human lactotransferrin associate non-covalently in solution. Our work shows that the isolated half-molecules of OTf are able to reassociate in solution and that this reassociation has functional significance by allowing the complex to be recognized by the Tf receptor.
Brown-Mason
A
A
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405.
Brown
S A
SA
Butcher
N D
ND
Woodworth
R C
RC
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002642:Chick Embryo; D002850:Chromatography, Gel; D003207:Conalbumin / Q000378:metabolism*; D004527:Egg Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D011485:Protein Binding / Q000187:drug effects; D012156:Reticulocytes / Q000378:metabolism*; D012996:Solutions
D004527:Egg Proteins; D008024:Ligands; D046911:Macromolecular Substances; D012996:Solutions; D000643:Ammonium Chloride; D003207:Conalbumin; D007501:Iron