Electronic and molecular structures of size-enhanced Hbs and their metabolites

尺寸增强的 Hb 及其代谢物的电子和分子结构

• 批准号: 6654248
• 负责人: JACK PEISACH
• 金额: $ 38.74万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-05 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6654248
• 关键词: biomaterial development /preparation; biotechnology; blood /plasma substitute; crosslink; dextrans; electron spin resonance spectroscopy; hemoglobin; hemoprotein structure; nitric oxide; photolysis; polyethylene glycols; protein engineering; site directed mutagenesis; viscosity

DESCRIPTION (provided by applicant): This project proposes a series of continuous wave and advanced EPR studies of mutant and PEGylated hemoglobins (Hbs) whose results will aid in the optimization of new design of blood substitutes. The first of these investigations addresses the intrinsic stability of the Hb moiety of these molecules towards autoxidation and subsequent denaturation to form hemichromes, both reversible or reconstitutable, and irreversible. Included will be in vitro studies of identification and kinetics of formation as well as investigations of Hb stability in animal models. A second series of experiments addresses specific aspects of the reaction of NO and hemoglobin, with reference to distance measurement of heme to non-heme binding sites and the role of protein in governing the nature of these sites. Low-temperature photolysis of Co(II)O2, Fe(II)NO, and Co(II)NO forms of Hb-based blood substitutes, followed by EPR, will be used to measure distances between photolyzed ligand and metal centers. Mixed-metal Fe-Co complexes will be examined to delineate differences in alpha and beta chains in accommodating NO away from heme. T1 measurements using spin echo-detected saturation recovery techniques are planned with Fe(II)NO Hb and NO, while ESEEM studies will be carried out with Fe(II)NO Hb and deoxyCo(II) Hb containing 13CO. A third set of studies planned of a series of singly and doubly spin-label PEGylated Hbs are to measure distances between the Hb and appended PEG moieties using the double electron-electron resonance (DEER) method. These experiments will be used to test a hypothesis concerning the effect of PEGylation on the intrinsic viscosity as due to the entrapment of a water channel between the appended PEG and the Hb moiety.

描述（由申请人提供）： 本项目提出了一系列连续波和先进的EPR研究的突变体和 聚乙二醇化血红蛋白（Hbs），其结果将有助于优化新的血液设计 替代品这些研究中的第一个解决了血红蛋白部分的内在稳定性， 这些分子趋向于自动氧化和随后的变性以形成半色素， 既可以是可逆的，也可以是可重构的，还有不可逆的。包括体外研究， 鉴定和形成动力学以及动物模型中Hb稳定性的研究。 第二系列的实验解决了NO和N2的反应的具体方面。 血红蛋白，参照血红素到非血红素结合位点的距离测量， 蛋白质在控制这些位点的性质中的作用。低温光解 将使用基于Hb的血液替代品的Co（II）O2、Fe（II）NO和Co（II）NO形式，然后是EPR 用于测量光解配体和金属中心之间的距离。铁钴混合金属 复合物将被检查，以描绘α和β链的差异， 不离不弃，使用自旋回波检测饱和度的T1测量 计划采用Fe（II）NO Hb和NO回收技术，同时进行ESEEM研究 Fe（Ⅱ）NO Hb和含~（13）CO的脱氧Co（Ⅱ）Hb。第三组研究计划， 一系列单和双自旋标记的PEG化Hb用于测量Hb之间的距离， 和附加的PEG部分。 这些实验将用于测试关于PEG化对细胞的影响的假设。 由于附加的PEG之间的水通道的截留，特性粘度 和Hb部分。