MECHANISM OF IRON REMOVAL FROM TRANSFERRIN

血清转铁蛋白是负责Fe3+转运的蛋白质 在吸收、利用和储存的地点之间。它还能结合Ca3+， In~(3+)和Al~(3+)，当这些金属离子进入血液时。表征 这些金属离子与转铁蛋白的相互作用对 了解细胞内铁释放的机制，对于 治疗铁超载的新药的开发，以及 了解该组化合物的药理和毒理学 13种金属离子。 根据该项目以前的结果，提出了铁 清除通过两条平行的路径进行，其中一条似乎是 受蛋白质构象变化的限制，以及第二条途径 这在配位体中是一级的。拟议的研究将进一步 将此机制与其他实验中的替代方案进行比较。 Fe3+、Al3+、Ga3+和In3+的金属离子去除速率为 然后用电子光谱和电泳法测定 金属离子对绝对交换率的影响 这两条道路的相对重要性。详细比较了 将制作中性和阴离子配体来评估阴离子的作用 作为转铁蛋白变构修饰剂的配体。无机物的影响 将测量两条除铁路径上的盐分，并 阴离子诱导的转铁蛋白的构象变化将被表征 通过稀土-Tf络合物的圆偏振发光。 关于阴离子和金属与载脂蛋白结合的补充研究 与细胞内铁释放相关的低pH值将被引导到 用热力学方法确定阴离子是否能促进铁的释放 竞争一个共同的结合部位。 提出了阳离子氨基酸侧链位于基团附近 金属结合部位可能是阴离子选择性作用的部位。 除铁的动力学。重组转铁蛋白将在#年制备 被怀疑参与阴离子结合的残基将是 有选择地改变。阴离子与金属离子结合的热力学 将测量重组蛋白以确定所涉及的残留物 在阴离子结合中，以及从重组菌中释放铁的动力学 将对一系列中性和阴离子配体的蛋白质进行评估。 此外，配体和非配位阴离子对酶活性的影响 协同碳酸氢根阴离子在Fe-HCO3-Tf体系中的交换速率 紧随其后的是一种新的技术，涉及[13C]的质谱学检测 二氧化碳。

Serum Transferrin is the protein responsible for the transport of Fe3+ between sites of uptake, utilization, and storage. It also binds Ca3+, In3+, and Al3+ when these metal ions enter the blood. Characterization of the interactions of these metal ions with transferrin is important for understanding the mechanism of intracellular iron release, for the development of new drugs for treating iron overload, and for understanding the pharmacology and toxicology of compounds of the group 13 metal ions. Based on previous results from this project, it is proposed that iron removal proceeds through two parallel pathways, one which appears to be limited by a conformational change in the protein, and a second pathway which is first-order in ligand. The proposed research will further evaluate this mechanism versus alternative proposals from other labs. Rates of metal ion removal for Fe3+, Al3+, Ga3+, and In3+ will be followed using electronic spectroscopy and electrophoresis to determine the effect of the metal ion on absolute rates of exchange and on the relative importance of the two pathways. Detailed comparisons between neutral and anionic ligands will be made to assess the role of anionic ligands as allosteric modifiers of transferrin. The effects of inorganic salts on the two pathways for iron removal will be measured, and anion-induced conformational changes in transferrin will be characterized by circularly polarized luminescence of lanthanide-Tf complexes. Additional studies of anion and metal binding to apotransferrin at the low pH associated with intracellular iron release will be conducted to determine whether anions can promote iron release by thermodynamic competition for a common binding site. It is proposed that cationic amino acid side chains near the metal-binding site may be the locus for selective anion effects on the kinetics of iron removal. Recombinant transferrins will be prepared in which residues suspected to be involved in anion binding will be selectively altered. The thermodynamics of anion binding to the recombinant proteins will be measured to determine the residues involved in anion binding, and the kinetics of iron release from the recombinant proteins to a series of neutral and anionic ligands will be evaluated. In addition, the effect of ligands and non-coordinating anions on the rate of exchange of the synergistic bicarbonate anion in Fe-HCO3-Tf will be followed by a new technique involving mass spectral detection of [13C) CO2.