HEMOGLOBIN AND MYOGLOBIN KINETIC STUDIES

该提案的长期目标是研究平衡和 血红蛋白和其他蛋白质中配体结合和蛋白质组装的动力学 系统. (1)特殊停流条件下的酶促耗氧 比色皿将允许连续测定氧平衡， 多种血红蛋白 双波长和多波长分光光度法将 用于获得脱氧过程中中间产物的光谱 用于Adair常量的全局拟合。 一个实际的目标是 开发一种快速简便的测定氧结合的方法 突变和工程血红蛋白中的常数。 待研究的血红蛋白 包括人血红蛋白、天然T-状态的血红蛋白、二聚血红蛋白， 蚯蚓高分子量手镯血红蛋白。 停流和激光 将对一些血红蛋白进行光解研究，以确定 构象变化的动力学，对于二聚体系统， 根据MWC模型获得完整的动力学描述。 (2)EXAFS 将继续对模型R和T的氧、CO和脱氧形式进行研究 状态Hbs，以及锁定到R和T状态的二溴血红素Hbs， 定量这些部位的结构变化，以与伴随的 在Fe的变化。 (3)生物素标记的探针、蛋白质和 结合到抗生物素蛋白的核苷酸将通过荧光各向异性来测量 停止流动。 将开发化学物质以提供新的程序 用于制备各种血红蛋白的链。 动力学、化学计量和Tm 与荧光标记的寡核苷酸与模型的结合有关 将通过荧光各向异性的变化来研究DNA。 这些 程序最终可能为人类提供方便的第一步， 基因组计划，提供了一种替代印迹技术，并与 扩增，允许快速检测可能相关的病毒DNA 用于艾滋病研究。 (4)涉及溴同位素的热原子化学将是 用于开发光亲和性测定的替代方法， 生物结构。 (5)NMR和光散射停止流将是 用于研究蛋白质的组装和解离， 与配体结合的聚集状态。 (6)将探讨新的程序 用于微分方程的积分系统，以辅助建模 动力学过程

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.