Hérve F, Gentin M, Rajkowski K M, Wong L T, Hsia C J, Cittanova N
UFR Biomédicale des Saints-Pères, Département de Biochimie, Paris, France.
J Steroid Biochem. 1990 Jul 4;36(4):319-24. doi: 10.1016/0022-4731(90)90224-g.
Rat fetal serum alpha 1-fetoprotein (AFP), a heterogeneous glycoprotein, binds estrogens with high affinity but at a fractional number of sites even after treatment with charcoal (n = 0.6), which may mean 60% of the protein has 1 site and the remainder none. To investigate the origin of this fractional number of sites the "native" protein (purified by negative affinity chromatography) was further purified (step 1) and fractionated (step 2) into its two main charge variants (electrophoretically "slow" and "fast") by a two-step fast-protein liquid chromatography method. The binding parameters for estrone and estradiol-17 beta of the "native" and "repurified" proteins and of each charge variant were determined by equilibrium microdialysis. The molar extinction coefficient at 278 nm of each sample was also determined. (1) The "repurified" AFP and each charge variant had a number of binding sites for estrogens close to unity. This increase in the number of sites could neither be explained by the loss of a non-binding isoform (corresponding to 40% of the protein) during chromatography, nor by the existence of complex negative modulatory interactions between isoforms. (2) The affinities for estrogens of the "repurified" protein and the two charge variants were slightly decreased compared to that of "native" AFP, except that the "fast" form had the "native" protein's high affinity for estrone--but not for estradiol-17 beta. (3) The molar extinction coefficients at 278 nm of the "repurified" AFP and the isoforms were much lower than that of the "native" protein. These results suggest that the presence of (an) inhibitor(s) of estrogen binding on the "native" protein which is/are removed by the ion-exchange fast protein liquid chromatography (FPLC) column. A ligand absorbing at 278 nm, which may or may not be the inhibitor, is also removed. The isoform heterogeneity with respect to estrone binding is discussed.
大鼠胎血清甲胎蛋白（AFP）是一种异质性糖蛋白，它能以高亲和力结合雌激素，但即使在用活性炭处理后，其结合位点数量也只是部分完整（n = 0.6），这可能意味着60%的蛋白质有1个位点，其余则没有。为了研究这种部分位点数量的起源，通过两步快速蛋白质液相色谱法对“天然”蛋白质（通过负亲和色谱法纯化）进行进一步纯化（步骤1）并分离（步骤2），得到其两个主要电荷变体（电泳“慢”型和“快”型）。通过平衡微透析测定“天然”和“再纯化”蛋白质以及每个电荷变体与雌酮和雌二醇-17β的结合参数。还测定了每个样品在278 nm处的摩尔消光系数。（1）“再纯化”的AFP和每个电荷变体对雌激素的结合位点数量接近完整。这种位点数量的增加既不能用色谱过程中无结合能力的异构体（占蛋白质的40%）的丢失来解释，也不能用异构体之间复杂的负调节相互作用的存在来解释。（2）与“天然”AFP相比，“再纯化”蛋白质和两个电荷变体对雌激素的亲和力略有降低，只是“快”型对雌酮具有“天然”蛋白质的高亲和力——但对雌二醇-17β则不然。（3）“再纯化”的AFP及其异构体在278 nm处的摩尔消光系数远低于“天然”蛋白质。这些结果表明，“天然”蛋白质上存在雌激素结合抑制剂，离子交换快速蛋白质液相色谱（FPLC）柱可将其去除。一种在278 nm处有吸收的配体（可能是也可能不是抑制剂）也被去除。讨论了雌酮结合方面的异构体异质性。
