DESIGN OF HEME-PROTEIN BASED BLOOD SUBSTITUTES

我们的目标是获得实验数据和机械解释 在蛋白质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， 血红素损失 潜在血液替代品的五个重要特性是： (1)合作性和中等低）2亲和力;（2）歧视性 有利于）2结合（即，KCO/KO 2 <200）;（3）大 缔合和解离速率常数，用于有效的气体输运， 微循环;（4）低自氧化速率;（5）高亲和力 血红素和变性的稳定性。 在另一项研究中， 检查一系列遗传学上的O2和CO结合特性， 猪和抹香鲸肌红蛋白和人肌红蛋白的血红素口袋突变体 血红蛋白。 在这个项目中，我们建议测量自氧化， 这些突变蛋白质的扩展集的血红素稳定性。 因为 由于α和β亚基的差异，四聚体 解离成二聚体，以及人体中的四级构象变化 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（CD 3）、43（CD 1）、29（B10）、32（B13）和 107（G8）对自氧化率的影响将被调查。 温度、pH值、 和O2浓度依赖性将被测量为天然的和选择的 突变蛋白，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， 对同一组蛋白质进行解离。 为了检验 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识别、配体结合速率常数，以及 稳定氧化和血红素损失。 自氧化和血红素结合 还将对人类血红蛋白的突变体进行研究 含有His（E7）至Gly和Gln以及瓦尔（E11）至Ala、Leu和Ile 在α和B亚基两者中的取代。 的结果予以 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O2结合参数的人血红蛋白的α和B亚基 以及最小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 自然发生的血红蛋白病的结构解释 与先天性海因茨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.