HEMOGLOBIN AND MYOGLOBIN KINETIC STUDIES

这项建议涉及对……的动力学和机理的研究 氧和一氧化碳与各种血红蛋白的结合、组装反应 以及组装体中中间体的功能性质 进程。在组装反应中，我们有兴趣确定 配基结合中的协同作用出现了哪些阶段和程度。 这些反应是在激光的光散射、荧光 各向异性停流、串流和流动闪蒸实验。从 光散射和荧光测量，相对种群 可以测定中间体。串列流动研究使我们能够捕获 瞬时中间体，并确定其功能性质。 结扎的详细停流研究旨在解开 人类T-状态的早期结扎和构象变化 血红蛋白。激光光解将探测双分子和双分子的细节 位于已知一氧化碳两个极端的血球蛋白中的一氧化碳复合 反应性。在一种血红蛋白中，可以对两种血红蛋白进行n秒的研究 T和R状态。这些研究将提供有关能源的信息 连接血红素腔和蛋白质通道的障碍，这可能 参与双分子反应。最新的合成进展使 美国将提议进行实验，旨在阐明 根效应中的阿尔法链和贝塔链，玻尔的最极端 效果。这是通过合成两种经过修饰的 血红蛋白和各种封闭的或价的杂交体，允许连接 在R和T两种状态下都要遵循的单反应链。这个 最简单的合作HBs，它是二聚体的，没有玻尔效应或 可检测到的α-β异质性，将被研究以尝试 提供最基本的合作单位的描述。在……里面 总而言之，这些研究都是为了改进和扩大我们的 了解蛋白质-蛋白质和蛋白质-配基相互作用； 特别是，研究亚基相互作用和氧的结合 和CO结合成不同的血红蛋白。

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.