Brown-Mason A, Brown S A, Butcher N D, Woodworth R C
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405.
Biochem J. 1987 Jul 1;245(1):103-9. doi: 10.1042/bj2450103.
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.
在本论文中，已根据阿克尔斯和汤普森提出的吉尔伯特理论[（1965年）《美国国家科学院院刊》53, 342 - 349]对二价铁卵转铁蛋白（Fe2OTf）、卵转铁蛋白（OTf）的单个半分子以及半分子的等摩尔混合物进行了凝胶过滤研究。数据表明，半分子在溶液中可逆缔合，并能确定解离常数Kd' = 8.0（±2.7）微摩尔。为了评估在相同条件下每个半分子与鸡胚红细胞的结合情况，使用氯化铵来阻止从等摩尔差异碘标记的半分子（125I和131I）中去除铁，进行了平衡结合研究。已使用上述凝胶过滤实验得出的常数计算了一系列浓度下缔合半分子的量。对数据进行计算机化非线性最小二乘回归分析，得出了半分子的Kd*（OTf或半分子与鸡胚红细胞转铁蛋白（Tf）受体相互作用的表观解离常数）和Bmax（在无限游离配体浓度下的结合），其值与Fe2OTf的类似。最近的报道证实，OTf和人乳铁蛋白的两个铁结合结构域在溶液中以非共价方式缔合。我们的工作表明，OTf分离的半分子能够在溶液中重新缔合，并且这种重新缔合具有功能意义，因为它能使复合物被Tf受体识别。
