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: 6537007
• 负责人: JOHN S. OLSON
• 金额: $ 25.78万
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537007
• 关键词: Escherichia coli; biomaterial development /preparation; blood /plasma substitute; capillary; chemical stability; chimeric proteins; circular dichroism; conformation; cytotoxicity; free radical scavengers; gene expression; heme; hemoglobin; hemoprotein; human tissue; hydrogen peroxide; intermolecular interaction; ionic bond; microspectrophotometry; mutant; myoglobin; nitric oxide; oxygen transport; protein engineering; recombinant proteins

DESCRIPTION: (Investigator's abstract) The long range goals are to develop O2 delivery pharmaceuticals based on extracellular recombinant hemoglobins (rHb). These next generation rHbs will be designed for optimal O2 transport, minimal interference with vasoregulation, enhanced resistance to denaturation, and increased expression levels in E. coli. Seven required properties are: (a) moderate O2 affinity (P50 5-30 mm Hg); (b) discrimination against CO binding; (c) large rate constants for oxygen binding and release; (d) significantly reduced rates of NO scavenging; (e) resistance to autooxidation and reactions with H2O2 (f) low rates of hemin dissociation; and (g) highly stable apoglobin structures. The mechanisms underlying these properties are being determined using sperm whale myoglobin as a simple prototype for the alpha and beta subunits of human hemoglobin, and the results are being used to develop strategies for solving specific problems in the clinical use and commercial development of extracellular hemoglobins (i.e., the hypertensive side effect and production costs). These mutagenesis studies will provide a database for evaluating more general principles for heme protein engineering. We will also address fundamental physiological questions about O2 transport and NO signaling and examine basic mechanisms of NO scavenging and O2 binding which apply to other key heme proteins, including flavohemoglobin NO dioxygenases. The specific aims for the next five years are to: (1) design new rHb products with low rates of NO scavenging and more efficient O2 transport properties to eliminate the hypertensive side effect of extracellular hemoglobin; (2) determine the relative importance of P50, oxygen dissociation rate constants, and cooperativity on O2 transport in capillaries in order to define the minimum requirements for efficacy; (3) examine systematically all physiologically relevant reactions of NO with the iron atom in hemoglobin to avoid other potential side effects; (4) improve the in vivo stability and in vitro shelf-life of recombinant hemoglobins by enhancing their resistance to autooxidation, reaction with H2O2, and heme loss; and (5) increase expression yields and lower cost of production in E. coli by enhancing the stability of apohemoglobin.

描述：（研究者摘要）长期目标是开发O2 基于细胞外重组血红蛋白（rHb）的药物递送。 这些下一代rHb将被设计为最佳的O2运输，最小 干扰血管调节，增强对变性的抵抗力，以及 在E.杆菌七个必需的属性是：（a） 中等O2亲和力（P50 5-30 mm Hg）;（B）对CO结合的辨别; (c)氧结合和释放的速率常数大;（d）显著 NO清除率降低;（e）抗自氧化和反应 （f）氯化血红素解离速率低;和（g）脱辅基珠蛋白高度稳定 结构.这些特性背后的机制正在被确定 使用抹香鲸肌红蛋白作为α和β的简单原型， 人类血红蛋白的亚基，结果被用于开发 解决临床使用和商业应用中特定问题的策略 细胞外血红蛋白的形成（即，高血压副作用 生产成本）。这些诱变研究将为 评价血红素蛋白质工程的更一般原则。我们还将 解决有关O2运输和NO信号的基本生理问题 并检查适用于NO清除和O2结合的基本机制， 其他关键血红素蛋白，包括黄素血红蛋白NO双加氧酶。 未来五年的具体目标是：（1）设计新的rHb产品 具有低的NO清除率和更有效的O2传输特性， 消除细胞外血红蛋白的高血压副作用;（2） 确定P50的相对重要性，氧解离速率常数， 和协同性对毛细血管中的O2运输，以确定最小 （3）系统地检查所有生理学要求; NO与血红蛋白中的铁原子的相关反应，以避免其他 潜在的副作用;（4）提高体内稳定性和体外 通过增强重组血红蛋白对 自氧化、与H2 O2反应和血红素损失;以及（5）增加表达 产量和生产成本较低。大肠杆菌的稳定性， 脱血红素