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: 2392678
• 负责人: JOHN S. OLSON
• 金额: $ 16.12万
• 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/2392678
• 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：这是一份修订后的持续资助申请 设计用于尽可能无细胞的血红蛋白分子的研究 O2 载体（“血液替代品”）。 该项目的长期目标 就是重新设计重组血红蛋白的血红素袋来优化 O2亲和力，区别CO结合，实现高速率 O2 结合常数，并增加对自氧化的抵抗力， 血红素损失和珠蛋白变性。 具体目标有4个： 1. 构建具有最佳 O2 的肌红蛋白和人血红蛋白原型 亲和力以及对自氧化和与 NO 反应的抵抗力增强 和H2O2。 将研究三种类型的修改：（a）合理的 多个远端口袋替换的设计； (b) 自然发生的 在动物血红蛋白中发现的远端口袋结构 重组肌红蛋白和血红蛋白； (c) 随机突变体将是 用于尝试创建一个新的远端口袋组合，具有更大的 抗解折叠、更紧密的血红素结合、中等 O2 亲和力，以及 自发化学诱导的自氧化率低。 2. 设计和评估肌红蛋白和血红蛋白突变体，增加 对氯高铁血红素有亲和力，但具有正常的配体结合特性。 一个 无辅基肌红蛋白，之前由该小组设计和生产（H64Y/V68F） 将用于筛选血红素与 Mb 和 Hb 的解离率 在 CD3、E10、F7、FG 角和其他位置进行替换的突变体 对于稳定绑定假体似乎很重要的区域 团体。 3. 筛选脱辅基肌红蛋白和脱辅基血红蛋白突变体以获得更大的抵抗力 到展开。 解折叠将通过内在色氨酸来测量 变性剂滴定过程中荧光和圆二色性发生变化。 4. 血红蛋白和肌红蛋白检查试剂的开发 细胞外血红蛋白的生理效应及其测量 生物系统中血红素损失、NO 产生和 CO 水平。 这 实验室已经开发了一个包含 200 多个单一和 多种肌红蛋白突变体可用于设计更稳定的 用于测量血红素解离和测定速率的蛋白质 内皮细胞悬浮液和酶系统中 NO 和 CO 的产生。