DESIGN OF HEME-PROTEIN BASED BLOOD SUBSTITUTES

我们的目标是获得实验数据和机械解释 在以蛋白质为基础的血液替代品中设计血红素口袋所需的 最佳的气体传输性能和最大的自氧化稳定性 亚铁血红素丢失。潜在的血液替代品的五个重要特性是： (1)协作性和适中的低亲和力；(2)歧视 反对CO，赞成)2结合(即，KCO/KO2&lt；200)；(3)大 天然气高效输运的缔合和解离速率常数 微循环；(4)低自氧化率；(5)高亲和力 用于血红素和稳定性，以变性。在另一项研究中，我们一直在 检测一系列遗传基因的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 一组扩展的这些突变蛋白的血红素稳定性。因为. 由于α和β亚基差异而产生的复杂性，四聚体 解离成二聚体和人类的四次构象变化 血红蛋白，肌红蛋白将作为模型系统来研究 氧结合参数与铁结合参数的关系 氧化和血红素流失。氨基酸替代对肉鸡的影响 (E7)、68(E11)、67(E10)、45(CD3)、43(CD1)、29(B10)、32(B13)和 107(G8)将对自氧化速率进行调查。温度、酸碱度、 将测量原生和选定的O2浓度依赖关系 以确定这一过程的机制。 还将进行实验，以测量血红素结合率和 同一组蛋白质的解离。作为对的效用的测试 这种方法，然后我们将尝试构建合成肌红蛋白与 最佳的O2亲和力、CO识别率、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的突变进行研究。 包含His(E7)到Gly和Gln以及Val(E11)到Ala、Leu和Ile α亚基和B亚基中的替换。这些研究的结果 研究和肌红蛋白的研究将被用来设计血红素口袋 具有最佳氧结合参数的人血红蛋白α和B亚基 以及最低的自氧化和氯化血红素解离速率。的数据。 重组肌红蛋白和血红蛋白也将提供更完整的 对自然发生的血红蛋白疾病的结构解释 与先天性亨氏小体溶血性贫血和水平升高有关 高铁血红蛋白。

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.