MECHANISM OF IRON REMOVAL FROM TRANSFERRIN

从转铁蛋白中去除铁的机制

• 批准号: 2139591
• 负责人: WESLEY R HARRIS
• 金额: $ 13.09万
• 依托单位: UNIVERSITY OF MISSOURI-ST. LOUIS
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-08-06 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2139591
• 关键词: active sites; allosteric site; aluminum; chelating agents; chemical binding; chemical kinetics; circular dichroism; conformation; electrophoresis; gallium; indium; intracellular transport; ion transport; iron; mass spectrometry; recombinant proteins; spectrometry; thermodynamics; transferrin

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.

血清转铁蛋白是负责运输Fe 3+的蛋白质 在吸收、利用和储存的地点之间。 它还结合Ca 3+， 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于 了解细胞内铁释放的机制， 开发治疗铁超载的新药，并用于 了解该组化合物的药理学和毒理学 13种金属离子。 根据该项目以前的结果，建议铁 清除过程通过两个平行的途径进行，一个似乎是 受到蛋白质构象变化的限制， 在配体中为一级配位。 该研究将进一步 评估此机制与其他实验室的替代方案。 Fe 3+、Al 3+、Ga 3+和In 3+的金属离子去除速率将为 然后使用电子光谱和电泳来确定 金属离子对绝对交换率和 这两条道路的相对重要性。 详细比较 中性和阴离子配体将被用来评估阴离子的作用 配体作为转铁蛋白的变构修饰剂。 无机物的影响 将测量两种除铁途径上的盐， 阴离子诱导的转铁蛋白的构象变化将被表征 通过镧系元素-Tf复合物的圆偏振发光。 阴离子和金属与脱铁转铁蛋白结合的额外研究 将进行与细胞内铁释放相关的低pH， 通过热力学方法确定阴离子是否能促进铁的释放 竞争共同的结合位点。 这表明，阳离子氨基酸侧链附近的 金属结合位点可能是选择性阴离子作用于 除铁动力学 重组转铁蛋白将在 哪些残基被怀疑参与阴离子结合， 选择性地改变。 阴离子结合的热力学 将测量重组蛋白以确定涉及的残基 在阴离子结合，和铁释放的动力学从重组 将评估蛋白质与一系列中性和阴离子配体的结合。 此外，还研究了配体和非配位阴离子对反应的影响。 Fe-HCO 3-Tf中协同作用的碳酸氢根阴离子的交换速率将 随后是一种涉及[13 C]质谱检测的新技术 二氧化碳。