HEMOGLOBIN AND MYOGLOBIN KINETIC STUDIES

血红蛋白和肌红蛋白动力学研究

• 批准号: 2139767
• 负责人: Lawrence JOHN Parkhurst
• 金额: $ 18.95万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-12-15 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2139767
• 关键词: Chelonia; Cyprinidae; DNA; Invertebrata; alternatives to animals in research; avidin; chemical association; chemical binding; chemical kinetics; chemical structure function; circular dichroism; conformation; fluorescence; fluorescent dye /probe; heme; hemoglobin; hemoprotein structure; human tissue; ligands; molecular rearrangement; nuclear magnetic resonance spectroscopy; nucleic acid hybridization; nucleic acid probes; oxygen; photolysis; stop flow technique; thermodynamics

The long-term objectives of the proposal are to study the equilibrium and kinetics of ligand binding and protein assembly in hemoglobins and other systems. (1) The enzymatic depletion of oxygen in a special stopped-flow cuvette will allow continuous determinations of oxygen equilibria in diverse hemoglobins. Dual and multi-wavelength spectrophotometry will be used to obtain spectra of intermediates during the deoxygenation process for use in global fittings for Adair constants. A practical aim is to develop a rapid and convenient procedure for determining oxygen binding constants in mutant and engineered hemoglobins. Hemoglobins to be studied include human Hbs, Hbs that are naturally T-state, dimeric Hbs, and the high molecular weight bracelet Hb from Lumbricus. Stopped-flow and laser photolysis studies will be carried out on a number of the Hbs to determine the kinetics of conformational changes, and for the dimeric systems, to obtain a complete kinetic description in terms of the MWC model. (2) EXAFS studies will be continued on oxy, CO, and deoxy forms of model R and T state Hbs, as well as on di-Br heme Hbs locked into R and T states to quantitate structural changes at these sites to compare with concomitant changes at the Fe. (3) The kinetics of biotinylated probes, protein and nucleotides binding to avidin will be measured by fluorescence anisotropy stopped-flow. The chemistry will be developed to provide a new procedure for preparing chains of diverse Hbs. The kinetics, stoichiometry, and Tm's related to the binding of fluorescently-labeled oligonucleotides to model DNA's will be studied by changes in fluorescence anisotropy. These procedures may eventually provide a convenient first step for the human genome project, provide an alternative to blotting techniques, and, with amplification, allow rapid detection of viral DNA, of possible relevance for AIDS research. (4) Hot-atom chemistry involving isotopes of Br will be used to develop an alternative to photoaffinity determinations of biological structures. (5) NMR and light-scattering stopped-flows will be used to study protein assembly and dissociation and to correlate aggregation state with ligand binding. (6) New procedures will be explored for integrating systems of differential equations for aid in modeling kinetic processes.

该提案的长期目标是研究平衡和 配体结合和蛋白质组装的动力学在血红蛋白和其他 系统。 （1）特殊停止流中氧气的酶促消耗 混音将允许连续确定氧气中的氧气平衡 多样的血红蛋白。 双重和多波长分光光度法将是 用于在脱氧过程中获得中间体的光谱 用于Adair常数全球配件。 一个实际的目的是 制定快速方便的程序来确定氧结合 突变和工程血红蛋白的常数。 血红蛋白要研究 包括人类HB，自然是T型状态的HB，二聚体HB和 来自Lumbricus的高分子手镯Hb。 停止流量和激光 光解研究将在许多HBS上进行以确定 构象变化的动力学以及二聚体系统的动力学 根据MWC模型获得完整的动力学描述。 （2）EXAFS 研究将继续以R和T的Oxy，Co和Deoxy形式进行。 状态HBS以及锁定在R和T状态的Di-Br Heme HBS上 量化这些位点的结构变化以与伴随 FE的变化。 （3）生物素化探针，蛋白质和 将通过荧光各向异性测量与抗原蛋白结合的核苷酸 停止流。 化学将开发以提供新的程序 用于准备各种HB的链条。 动力学，化学计量学和TM 与荧光标记的寡核苷酸与建模的结合有关 DNA将通过荧光各向异性的变化来研究。 这些 程序最终可能为人类提供方便的第一步 基因组项目，提供了印迹技术的替代方案，并提供 放大，允许快速检测病毒DNA，可能相关性 用于艾滋病研究。 （4）涉及BR同位素的热原子化学将是 用于开发替代光性测定的替代方案 生物结构。 （5）NMR和散落的停止流将是 用于研究蛋白质组装和解离并相关 配体结合的聚集状态。 （6）将探讨新程序 用于集成微分方程的系统以辅助建模 动力学过程。