DESIGN OF HEME PROTEIN BASED BLOOD SUBSTITUTES

基于血红素蛋白的血液替代品的设计

• 批准号: 2685381
• 负责人: JOHN S. OLSON
• 金额: $ 16.77万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2685381
• 关键词: biomaterial development /preparation; blood /plasma substitute; chemical stability; chimeric proteins; circular dichroism; conformation; heme; hemoglobin; hemoprotein; human tissue; myoglobin; oxygen transport; protein engineering

DESCRIPTION: This is a revised application for continued funding for studies of hemoglobin molecules designed for use as possible cell-free O2 carriers ("blood substitutes"). The long-range goal of this project is to redesign the heme pocket of recombinant hemoglobins to optimize O2 affinity, discriminate against CO binding, achieve high rate constants for O2 binding, and increase resistance to autooxidation, hemin loss, and globin denaturation. There are 4 specific aims: 1. Construct myoglobin and human hemoglobin prototypes with optimal O2 affinity and increased resistance to autooxidation and reactions with NO and H2O2. Three types of modifications will be studied: (a) rational design of multiple distal pocket replacements; (b) naturally occurring distal pocket structures found in animal hemoglobins modeled in recombinant myoglobins and hemoglobins; and (c) random mutants will be used to attempt to create a new distal pocket combination with greater resistance to unfolding, tighter heme binding, moderate O2 affinity, and low rates of spontaneous chemically-induced autooxidation. 2. Design and evaluate myoglobin and hemoglobin mutants with increased affinities for hemin but normal ligand binding properties. An apomyoglobin, previously designed and produced by this group (H64Y/V68F) will be used to screen the rates of hemin dissociation from Mb and Hb mutants with substitutions at CD3, E10, F7, the FG corner, and other regions which appear important for stabilizing the bound prosthetic group. 3. Screen apomyoglobin and apohemoglobin mutants for greater resistance to unfolding. Unfolding will be measured by intrinsic tryptophan fluorescence and circular dichroism changes during denaturant titration. 4. Development of hemoglobin and myoglobin reagents for examination of the physiological effects of extracellular hemoglobin and for measuring hemin loss, NO production, and CO levels in biological systems. This laboratory already has developed a library of over 200 single and multiple myoglobin mutants which can be used to design more stable proteins for measuring hemin dissociation and for determining rates of NO and CO production in endothelial cell suspensions and enzyme systems.

描述：这是持续资助的修订申请 研究旨在使用无细胞的血红蛋白分子的研究 O2载体（“血液替代品”）。 这个项目的远程目标 是重新设计重组血红蛋白的血红素口袋以优化 O2亲和力，歧视CO绑定，达到高率 O2结合的常数，并增加对自氧化的抗性， hemin损失和球蛋白变性。 有4个具体目标： 1。用最佳O2构建肌红蛋白和人血红蛋白原型 亲和力和对自氧化和反应的抵抗力增加 和H2O2。 将研究三种类型的修改：（a）理性 设计多个远端口袋更换； （b）天然发生 在模拟的动物血红蛋白中发现的远端口袋结构 重组肌球蛋白和血红蛋白； （c）随机突变体将是 用于创建更大的新远端口袋组合 抵抗展开，更紧密的血红素结合，中等O2亲和力和 自发性化学诱导的自氧化速率低。 2。设计和评估肌红蛋白和血红蛋白突变体随增加 血统的亲和力，但配体结合特性正常。 一个 Apomyoglobin，以前由该组设计和生产（H64Y/V68F） 将用于筛选出与MB和HB的Hemin解离速率 在CD3，E10，F7，FG角和其他方面取代的突变体 对于稳定约束假肢似乎很重要的区域 团体。 3。屏幕叠肌球蛋白和apohemoglobin突变体，以获得更大的耐药性 展开。 展开将通过固有的色氨酸来衡量 变性滴定期间的荧光和圆形二色性变化。 4。用于检查的血红蛋白和肌红蛋白试剂的开发 细胞外血红蛋白和测量的生理影响 Hemin损失，无生产和CO水平。 这 实验室已经开发了200多个单曲的库， 多个可用于设计更稳定的肌红蛋白突变体 用于测量Hemin分离的蛋白质和确定速率 内皮细胞悬浮液和酶系统中的NO和CO生产。