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.

我们的目标是获取实验数据和机理解释在基于蛋白质的血液替代品中设计血红素口袋所需最佳气体传输特性和对自氧化的最大稳定性和血红素损失。潜在血液替代品的五个重要特性是：（1）合作性和中度低）2亲和力；（2）歧视反对CO支持）2结合（即KCO/KO2 <200）；（3）大有效气体传输的关联和解离速率常数微循环；（4）自氧化率低；（5）高亲和力对于血红素和变性的稳定性。在另一项研究中，我们一直检查一系列遗传学的02和CO结合特性经过工程，猪和抹香鲸肌红蛋白和人类的血红素袋装突变体血红蛋白。在这个项目中，我们建议衡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Alpha和Beta亚基差异而引起的复杂性，四聚体分解为二聚体和人类的第四纪构象变化血红蛋白，肌球蛋白将用作模型系统来研究氧结合参数与铁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）将对自氧化率进行调查。温度，pH，将测量和选择的O2浓度依赖突变蛋白是为了确定该过程的机制。还将进行实验以衡量血红素结合的速率和相同蛋白质的解离。作为对实用程序的测试这种方法，然后我们将尝试与最佳O2亲和力，CO歧视，配体结合速率常数和氧化和血红素损失的稳定性。自氧化和血红素结合研究还将用人血红蛋白的突变体进行将他的（E7）包含到Gly，Gln和Val（E11）到Ala，Leu和Ile 在alpha和B亚基中取代。这些结果研究和肌红蛋白的研究将用于设计血红素口袋最佳O2结合参数的人血红蛋白的α和B亚基以及自氧化和HEMIN解离的最小速率。数据的数据重组肌球蛋白和血红蛋白也将提供更完整的天然存在的血红蛋白病的结构解释与先天性亨氏体溶血性贫血和升高水平相关高铁血红蛋白。