RECOMBINANT HUMAN HEMOGLOBINS

重组人血红蛋白

• 批准号: 6110153
• 负责人: CLARA FRONTICELLI
• 金额: $ 23.77万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110153
• 关键词: allosteric site; autooxidation; blood /plasma substitute; chemical substitution; chimeric proteins; circular dichroism; conformation; crosslink; gene mutation; genetic manipulation; globin; hemoglobin; nuclear magnetic resonance spectroscopy; plasmids; polymerase chain reaction; polymerization; polymers; protein engineering; protein structure function; recombinant proteins; respiratory oxygen

The objective of this project is to investigate approaches for altering the oxygen affinity of human hemoglobin in ways which should be relevant to the rationale design of clinically useful blood substitute. To this end we will use site directed mutagenesis to attempt to construct derivatives of human hemoglobin with an oxygen affinity and retention time in circulation similar to blood. The tetrameric Hb molecule will be stabilized by constructing a plasmid in which the two beta-globins are joined by a crosslinker peptide (beta1- (aa)n-beta2). The crosslinker peptide will be designed to introduce in the Hb molecule a decreased oxygen affinity. Intermolecular crosslinker will be designed on the model of Hb Porto Alegre beta(9)Ser-Cys, in which S-S bonds are formed between Hb molecules - (Sbeta1 beta2S)-(Sbeta1 beta2S)-. We will attempt to decrease the oxygen affinity of this derivative by amino acid substitutions which should introduce the mechanism of oxygen affinity modulation by the C1 of the solvent, present in the beta-chains of bovine Hb. Alternatively site specific mutation known to decrease the oxygen affinity of natural Hb can be introduced. Combination of the intra and intermolecular crosslinks will be used to engineer octameric (beta2-(aa)n-beta1S)-(Sbeta1-(aa)n-beta2) or superpolymeric derivatives - (Sbeta1-(aa)n-beta2S)-(Sbeta1-(aa)n[beta2S)- (Sbeta1-(aa)n-beta2S)- The larger size of these hemoglobin derivatives and the increased stabilization produced by the intra and intermolecular crosslinks should greatly increase their retention time in circulation. While an isooncotic solution of Hb has a concentration of 7%, polymeric hemoglobins can be used at higher concentration, with an increase in the oxygen carrying capacity of the solutions. The proposed system will produce hemoglobin derivatives which are retained in circulation and have an oxygen delivery capacity similar to blood.

该项目的目的是调查更改的方法 人类血红蛋白的氧亲和力应相关的方式 临床上有用的血液替代品的理由设计。 对此 结束我们将使用定向诱变的站点尝试构建 具有氧亲和力的人血红蛋白的衍生物 循环时间类似于血液。 四聚体HB分子将通过构建质粒稳定 其中两个β-珠蛋白通过交联肽连接（beta1- （aa）n-beta2）。 交联肽将被设计为引入 HB分子A氧气亲和力降低。 分子间交联 将以HB Porto Alegre Beta（9）ser-cys的模型进行设计，其中 S-S键在HB分子之间形成 - （SBETA1 beta2s） - （SBETA11 beta2s） - 。 我们将尝试降低此的氧气亲和力 氨基酸取代的衍生物应引入 溶剂的C1的氧亲和力调节机制，存在 在牛HB的β链中。 替代地点特定突变 已知可以降低天然HB的氧亲和力。 内部和分子间交联的组合将用于 工程师八聚体（beta2-（aa）n-beta1s） - （sbeta1-（aa）n-beta2）或 超聚合物衍生物 - （sbeta1-（aa）n-beta2s） - （sbeta1-（aa）n [beta2s） - （sbeta1-（aa）n-beta2s） - 这些血红蛋白衍生物的较大尺寸 并增加了分子内和分子产生的稳定性 交叉链接应大大增加其循环中的保留时间。 虽然Hb的等综合溶液的浓度为7％，但聚合物 血红蛋白可以以较高的浓度使用，并增加 溶液的氧气承载能力。 拟议的系统将 产生保留在循环中并具有的血红蛋白衍生物 氧气输送能力类似于血液。