DESIGN OF HEME PROTEIN BASED BLOOD SUBSTITUTES

描述：这是一份修订后的继续资助申请 设计用于尽可能无细胞的血红蛋白分子的研究 氧气携带者(“血液替代品”)。这个项目的长远目标是 是重新设计血红素口袋中的重组血红蛋白，以优化 O2亲和力，歧视CO结合，实现高转化率 氧结合的常数，并增加对自氧化的抗性， 氯化血红素丢失和珠蛋白变性。具体目标有4个： 1.构建具有最佳氧气的肌红蛋白和人血红蛋白原型 亲和力和增强的对自氧化的抵抗力以及与NO的反应 和过氧化氢。将研究三种类型的修改：(A)合理 设计多个远端口袋替换；(B)自然发生 在模拟的动物血红蛋白中发现的远端口袋结构 重组肌红蛋白和血红蛋白；以及(C)随机突变将是 用于尝试创建新的远端口袋组合，具有更大的 耐展开，更紧密的血红素结合，中等的O2亲和力，以及 自发性化学诱导自氧化的比率很低。 2.设计和评估肌红蛋白和血红蛋白突变体 与氯化高铁血红素有亲和力，但具有正常的配体结合特性。一个 无肌红蛋白，以前由该小组设计和生产(H64Y/V68F) 将用于筛选从Mb和Hb解离氯化血红素的速率 在CD3、E10、F7、FG角和其他位置发生替换的突变体 对固定固定假体来说很重要的区域 一群人。 3.筛选抗药性更强的脱脂肌红蛋白和脱脂血红蛋白突变体 为展开干杯。展开将通过内源性色氨酸进行测量 变性剂滴定过程中荧光和圆二色谱发生变化。 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效应及其测定 生物系统中氯化血红素的损失、NO的产生和CO水平。这 实验室已经开发了一个包含200多首单曲和 多个肌红蛋白突变体，可以用来设计更稳定的 用于测量氯化血红素解离和测定氯化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。

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.