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：这是一个修订后的申请继续资助， 血红蛋白分子的研究， 氧气载体（“血液替代品”）。 这个项目的长期目标是 是重新设计重组血红蛋白的血红素口袋以优化 O2亲和力，区别于CO结合，实现高速率 O2结合常数，并增加对自氧化的抵抗力， 氯化血红素损失和珠蛋白变性。 有四个具体目标： 1. 在最佳O2条件下构建肌红蛋白和人血红蛋白原型 亲和性和增加的抗自氧化性和与NO的反应 和H2 O2。 将研究三种类型的修改：（a）合理的 多个远端囊袋置换的设计;（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;和（c）随机突变体将被 用于尝试创建新的远端囊袋组合， 抗解折叠，更紧密的血红素结合，中等的O2亲和力，和 自发的化学诱导的自氧化的低速率。 2. 设计和评估肌红蛋白和血红蛋白突变体， 对氯化血红素的亲和力，但正常的配体结合特性。 一个 脱辅基肌红蛋白，先前由该小组设计和生产（H64 Y/V68 F） 将用于筛选高铁血红素与Mb和Hb的解离速率 在CD 3、E10、F7、FG角等处具有取代的突变体 对于稳定结合的假体来说似乎很重要的区域 组 3. 筛选具有更大抗性的脱辅基肌红蛋白和脱辅基血红蛋白突变体 to unfolding展开. 将通过固有色氨酸测量解折叠 荧光和圆二色性在变性剂滴定期间发生变化。 4. 血红蛋白和肌红蛋白检测试剂的研制 细胞外血红蛋白的生理作用和测量 氯化血红素损失、NO产生和生物系统中的CO水平。 这 实验室已经开发了一个图书馆， 多个肌红蛋白突变体，可用于设计更稳定的 用于测量氯高铁血红素解离和用于测定氯高铁血红素解离速率的蛋白质 内皮细胞悬浮液和酶系统中NO和CO的产生。