HEMOGLOBIN AND MYOGLOBIN KINETIC STUDIES

血红蛋白和肌红蛋白动力学研究

• 批准号: 3234617
• 负责人: Lawrence JOHN Parkhurst
• 金额: $ 12.56万
• 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 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3234617
• 关键词: Agnatha; Annelida; Bohr effect; binding proteins; bioenergetics; carbon monoxide; chemical association; chemical reaction; chemical structure function; fluorescence; globin; heme; hemoglobin; hemoprotein biosynthesis; hemoprotein structure; human tissue; laser spectrometry; ligands; light scattering; molecular rearrangement; myoglobin; oxygen transport; stop flow technique; temperature jump

This proposal is concerned with a study of: the kinetics and mechanisms of the binding of oxygen and CO to various hemoglobins, the assembly reactions of Hb, and the functional properties of intermediates in the assembly process. In the assembly reactions, we are interested in determining at what stages and to what extent co-operativity in ligand binding appears. These reactions are followed in laser light-scattering, fluorescence anisotropy stopped-flow, tandem flow, and flow-flash experiments. From the light-scattering and fluorescence measurements, relative populations of intermediates can be determined. Tandem flow studies allow us to trap the transient intermediates and determine their functional properties. Detailed stopped-flow studies of ligation are directed toward unravelling the early phases of ligation and conformational changes in T-state human hemoglobin. Laser photolysis will probe details of geminal and bimolecular CO recombination in hemo-globins which are at the two extremes of known CO reactivity. In one hemoglobin, n-sec studies can be carried out on both the T and R states. These studies will provide information on energy barriers for ligation on the heme cavity and for protein channels which may be involved in the bimolecular reactions. Recent synthetic advances allow us to propose experiments directed toward elucidating the roles of the alpha and beta chains in the Root effect, the most extreme of Bohr effects. This is to be accomplished by synthesizing both the modified hemoglobin and various blocked or valency-hybrids, permitting ligation to the single reactive chain to be followed in both R and T states. The simplest of co-operative Hbs, one that is dimeric, without a Bohr effect or detectable alpha-beta heterogeneity, will be studied in an attempt to provide a description of the most elementary co-operative unit. In general, these studies are all directed toward improving and enlarging our understanding of protein-protein and protein-ligand interactions; in particular, the study is of subunit interactions and the binding of oxygen and Co to diverse hemoglobins.

该提议涉及一项研究： 氧和CO与各种血红蛋白的结合，组装反应 HB的及组装中间体的功能特性 过程。 在大会反应中，我们有兴趣确定 在配体结合中出现了什么阶段以及在多大程度上的合作性。 这些反应在激光散射，荧光中遵循 各向异性停止流动，串联流和流动闪存实验。 来自 散落和荧光测量，相对种群 可以确定中间体。 串联流量研究使我们能够捕获 瞬态中间体并确定其功能特性。 详细的停止连接研究针对解开 T-State人的结扎和构象变化的早期阶段 血红蛋白。 激光光解会探测Geminal和Bimolocular的细节 血红蛋白中的CO重组，在已知CO的两个极端 反应性。 在一个血红蛋白中，可以对两者进行N-SEC研究 T和R状态。 这些研究将提供有关能源的信息 在血红素腔和蛋白质通道上连接的障碍 参与双分子反应。 最近的合成进步允许 我们提出了针对阐明该角色的实验 根部效应中的alpha和beta链，是玻尔最极端的 效果。 这是通过合成修改的 血红蛋白以及各种封闭或价杂种，允许连接到 在R和T状态下遵循的单个反应链。 这 最简单的合作HBS，一个是二聚体，没有玻璃效应或 可以研究可检测的α-beta异质性，以尝试 提供最基本的合作单元的描述。 在 一般，这些研究都致力于改善和扩大我们的 了解蛋白质 - 蛋白质和蛋白质 - 配体相互作用；在 特别是，该研究是亚基相互作用和氧的结合 并与多样的血红蛋白共同。