DESIGN OF HEME-PROTEIN BASED BLOOD SUBSTITUTES

基于血红素蛋白的血液替代品的设计

基本信息

批准号：
3366211
负责人：
JOHN S. OLSON
金额：
$ 12.82万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-08-01 至 1995-07-31
项目状态：
已结题

项目摘要

Our goal is to obtain the experimental data and mechanistic interpretations required to design the heme pocket in protein-based blood substitutes for optimal gas transport properties and maximum stability to autoxidation and heme loss. Five important properties of potential blood substitutes are: (1) cooperativity and moderately low )2 affinity; (2) discrimination against CO in favor of )2 binding (i.e., KCO/KO2 <200); (3) large association and dissociation rate constants for efficient gas transport in the microcirculation; (4) low rate of autooxidation; and (5) high affinity for heme and stability to denaturation. In another study, we have been examining the 02 and CO binding properties of a series of genetically engineered, heme pocket mutants of pig and sperm whale myoglobin and human hemoglobin. In this project, we propose to measure the autoxidation and heme stabilities of an expanded set of these mutant proteins. Because of complexities due to alpha and Beta subunit differences, tetramer dissociation into dimers, and quaternary conformational changes in human hemoglobin, the myoglobins will be used as model systems to investigate the relationships between oxygen binding parameters and those for iron oxidation and heme loss. The effects of amino acid replacements at positions 64(E7), 68(E11), 67(E10), 45(CD3), 43(CD1), 29(B10), 32(B13), and 107(G8) on the rates of autooxidation will be surveyed. Temperature, pH, and O2 concentration dependences will be measured for native and selected mutant proteins in order to determine the mechanism of this process. Experiments will also be carried out to measure rates of heme binding and dissociation for the same set of proteins. As a test of the utility of this approach, we will then attempt to construct a synthetic myoglobin with optimal O2 affinity, CO discrimination, ligand binding rate constants, and stability to oxidation and heme loss. Autooxidation and heme binding studies will also be carried out with mutants of human hemoglobin containing His(E7) to Gly and Gln and Val(E11) to Ala, Leu, and Ile substitutions in both the alpha and B subunits. the results of these studies and those for myoglobin will be used to design heme pockets in the alpha and B subunits of human hemoglobin with optimal O2 binding parameters and minimal rates of autoxidation and hemin dissociation. The data for the recombinant myoglobins and hemoglobins will also provide more complete structural interpretations of naturally occurring hemoglobinopathies associated with congenital Heinz body hemolytic anemia and elevated levels of methemoglobin.

我们的目标是获得实验数据和机制解释需要设计基于蛋白质的血液替代品中的血红素袋最佳的气体传输特性和最大的自氧化稳定性血红素损失。潜在血液替代品的五个重要特性是： (1) 协作性和适度低的 )2 亲和力； (2) 歧视反对 CO 有利于 )2 结合（即 KCO/KO2 <200）； (3)大有效气体传输的缔合和解离速率常数微循环； (4)自氧化率低； (5)高亲和力用于血红素和变性稳定性。在另一项研究中，我们检查一系列基因的 02 和 CO 结合特性猪和抹香鲸肌红蛋白和人类的工程血红素口袋突变体血红蛋白。在这个项目中，我们建议测量自氧化和这些突变蛋白的扩展集的血红素稳定性。由于由于 α 和 β 亚基差异、四聚体而导致的复杂性解离成二聚体，以及人类的四级构象变化血红蛋白、肌红蛋白将用作模型系统来研究氧结合参数与铁结合参数之间的关系氧化和血红素损失。氨基酸替代的影响位置 64(E7)、68(E11)、67(E10)、45(CD3)、43(CD1)、29(B10)、32(B13) 和 107（G8）关于自动氧化速率的调查将被调查。温度、pH、将测量原生和选定的 O2 浓度依赖性突变蛋白以确定这一过程的机制。还将进行实验来测量血红素结合率和同一组蛋白质的解离。作为实用性的测试通过这种方法，我们将尝试构建一种合成肌红蛋白最佳 O2 亲和力、CO 辨别力、配体结合速率常数，以及对氧化和血红素损失的稳定性。自氧化和血红素结合还将对人类血红蛋白突变体进行研究包含 His(E7) 至 Gly 和 Gln 以及 Val(E11) 至 Ala、Leu 和 Ile α 和 B 亚基均发生取代。这些结果研究和肌红蛋白研究将用于设计血红素口袋具有最佳 O2 结合参数的人血红蛋白 α 和 B 亚基以及最小的自氧化和血红素解离率。数据为重组肌红蛋白和血红蛋白也将提供更完整的自然发生的血红蛋白病的结构解释与先天性亨氏小体溶血性贫血及其水平升高有关高铁血红蛋白。