This paper presents holo/apo conversion two-dimensional urea polyacrylamide gel electrophoresis (HAC-2D urea PAGE) as a novel method for speciating Fe3+-bound transferrin (Tf) species in biological samples, with a combination of metal ion contaminant sweeping (MICS) technique and Fe3+ detection PAGE. In the HAC-2D urea MICS-PAGE approach, HAC was performed to dissociate all the Fe3+ ions bound to Tf from the Fe-Tf species, during a two-step urea PAGE. Using this method, Fe2-Tf, FeN-Tf, and FeC-Tf (holo-Tf, Fe3+-bound Tf attached to N-lobe, and Fe3+-bound Tf attached C-lobe, respectively) were completely isolated based on the difference in the higher-order structure of Tf, visible as horizontally aligned spots off the diagonal. The Fe3+ ions bound to Tf in each gel fraction were determined using PAGE with a fluorescent probe. Without the MICS technique, which electrophoretically removes all contaminant Fe3+ ions from the gel medium to ensure accurate determination of the Fe3+ concentration, it becomes challenging to precisely measure the distribution of metalloprotein species owing to the contaminants. Finally, the distribution of each Fe-bound Tf in a standard human serum sample was successfully determined by complete separation from large amounts of coexisting proteins, and the free Fe3+ concentration in the serum was estimated.

中文翻译：

---

Holo/apo 转换二维尿素 PAGE 用于血清中 Fe3+ 结合转铁蛋白的形态分析。

本文介绍了全息/apo 转换二维尿素聚丙烯酰胺凝胶电泳 (HAC-2D 尿素 PAGE) 作为一种结合金属离子污染物清除 (MICS) 来确定生物样品中 Fe3+ 结合转铁蛋白 (Tf) 物种的新方法技术和 Fe3+ 检测 PAGE。在 HAC-2D 尿素 MICS-PAGE 方法中，HAC 用于在两步尿素 PAGE 过程中从 Fe-Tf 物质中解离与 Tf 结合的所有 Fe3+ 离子。使用此方法，根据差异完全分离出 Fe2-Tf、FeN-Tf 和 FeC-Tf（分别为 Holo-Tf、附着于 N 叶的 Fe3+ 结合 Tf 和附着于 C 叶的 Fe3+ 结合 Tf）在 Tf 的高阶结构中，可见为偏离对角线的水平排列的点。使用 PAGE 和荧光探针测定每个凝胶组分中与 Tf 结合的 Fe3+ 离子。如果没有 MICS 技术（通过电泳从凝胶介质中去除所有污染物 Fe3+ 离子以确保准确测定 Fe3+ 浓度），精确测量污染物造成的金属蛋白种类的分布就变得具有挑战性。最后，通过与大量共存蛋白质的完全分离，成功测定了标准人血清样品中每种 Fe 结合 Tf 的分布，并估算了血清中游离 Fe3+ 的浓度。