Payne D W, Katzenellenbogen J A
Endocrinology. 1979 Sep;105(3):745-53. doi: 10.1210/endo-105-3-743.
The binding specificity of rat alpha-fetoprotein (AFP) for a series of estrogen derivatives has been investigated and compared to that of the uterine cytosol estrogen receptor. In addition, several equilibrium and nonequilibrium binding techniques have been examined for their appropriateness in studying AFP-estrogen interactions under different experimental conditions. The ratio of association constants (RAC) of various estrogen derivatives (compared to 17 beta-estradiol; RAC = 100%) were determined using a rapid and convenient miniaturized equilibrium assay that utilizes the partitioning properties of Sephadex G-25. AFP has the highest affinity for estrone derivatives and is especially tolerant of small, nonpolar substituents in the D ring; it binds nonsteroidal estrogens and antiestrogens weakly or not at all. Thus, it has a binding specificity quite unlike that of the estrogen receptor. Several potential affinity labels were studied. Of these, 16-diazoestrone, a photoreactive compound, has the highest affinity for AFP (RAC = 121%). Another equilibrium technique, steady state polyacrylamide electrophoresis, effects a clear separation between AFP and albumin. It is superior to conventional polyacrylamide electrophoresis under conditions where dissociation is rapid. Charcoaldextran adsorption also rapidly removes estradiol that is bound to AFP (t1/2 = approximately 10 min at 0 C). The AFP-estradiol complex appears to be stabilized by adsorption to hydroxylapatite, however, so that hydroxylapatite can be employed in a batchwise manner to assay binding to AFP. This is the most appropriate technique when high concentrations of ligand are used. These studies highlight the marked differences in binding specificity between rat AFP and the uterine cytosol estrogen receptor and indicate the techniques most appropriate for binding measurements under different conditions. This work should facilitate the design of affinity labeling reagents for rat AFP as well.
已对大鼠甲胎蛋白（AFP）与一系列雌激素衍生物的结合特异性进行了研究，并与子宫胞液雌激素受体的结合特异性进行了比较。此外，还考察了几种平衡和非平衡结合技术在不同实验条件下研究AFP-雌激素相互作用的适用性。使用一种利用Sephadex G-25分配特性的快速便捷的微型平衡分析法，测定了各种雌激素衍生物的缔合常数比（RAC，与17β-雌二醇相比；RAC = 100%）。AFP对雌酮衍生物具有最高亲和力，对D环中的小的非极性取代基尤其耐受；它与非甾体雌激素和抗雌激素的结合较弱或根本不结合。因此，它具有与雌激素受体截然不同的结合特异性。研究了几种潜在的亲和标记物。其中，光反应性化合物16-重氮雌酮对AFP具有最高亲和力（RAC = 121%）。另一种平衡技术，稳态聚丙烯酰胺电泳，能使AFP和白蛋白得到清晰分离。在解离迅速的条件下，它优于传统聚丙烯酰胺电泳。活性炭葡聚糖吸附也能快速去除与AFP结合的雌二醇（0℃时t1/2约为10分钟）。然而，AFP-雌二醇复合物似乎通过吸附到羟基磷灰石上而得到稳定，因此羟基磷灰石可用于分批测定与AFP的结合。当使用高浓度配体时，这是最合适的技术。这些研究突出了大鼠AFP与子宫胞液雌激素受体在结合特异性上的显著差异，并指出了在不同条件下最适合进行结合测量的技术。这项工作也应有助于设计大鼠AFP的亲和标记试剂。
