HEMOGLOBIN AND MYOGLOBIN KINETIC STUDIES

这项建议的长期目标是研究均衡和 血红蛋白等的配基结合和蛋白质组装动力学 系统。(1)在特殊的停流中酶促耗氧 试管将允许连续测定氧平衡 不同的血红蛋白。双波长和多波长分光光度将 用于获得脱氧过程中中间体的光谱 用于Adair常量的全局配件。一个实际的目标是 建立一种快速简便的氧结合测定方法 突变和基因工程血红蛋白中的常量。有待研究的血红蛋白 包括人类HbS、自然处于T态的HbS、二聚体HbS和 来自地龙的高分子量手镯Hb。停流与激光 将对一些HBS进行光解研究，以确定 构象变化的动力学，对于二聚体体系， 根据MWC模型获得完整的动力学描述。(2)EXAFS 将继续研究R和T型的氧合、一氧化碳和脱氧形式 状态HBS，以及锁定到R和T态的二溴血红素HBS 量化这些部位的结构变化，以与伴随的 在Fe的变化。(3)生物素化探针、蛋白质和 结合亲和素的核苷酸将通过荧光各向异性来测量 停止流动。这种化学将被开发以提供一种新的方法 准备一系列不同的哈佛商学院。动力学、化学计量学和TM的 与荧光标记寡核苷酸与模型的结合有关 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一个方便的第一步 基因组计划，提供了印迹技术的替代方案，并且，随着 扩增，允许快速检测可能相关的病毒DNA 用于艾滋病研究。(4)涉及溴的同位素的热原子化学将是 用于开发一种替代光亲和力测定的方法 生物结构。(5)核磁共振和光散射停止流 用于研究蛋白质的组装和解离以及相关 具有配体结合的聚集态。(6)探索新程序 用于对微分方程组进行积分以辅助建模 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.