KINETIC AND EQUILIBRIUM STUDIES OF LIGAND BINDING TO HEMOGLOBIN VARIANTS

与血红蛋白变体结合的配体的动力学和平衡研究

• 批准号: 6564615
• 负责人: ROBERT W NOBLE
• 金额: $ 17.7万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564615
• 关键词: Bohr effect; acidity /alkalinity; chemical kinetics; chemical models; crystallization; flash photolysis; hemoglobin; hemoprotein structure; human tissue; metalloporphyrins; oxygen tension; protein binding; protein isoforms; protein structure function; site directed mutagenesis; stereochemistry; stop flow technique

The aim of this project is the characterization of the functional properties of variants of human hemoglobin by examination of the equilibria and kinetics of their reactions with ligands. The purpose of this undertake is to contribute to the further development and testing of predictive stereochemical model which has been developed by this Program Project. Hemoglobin variants, as well as hemoglobins containing two different alphabeta dimers (mixed-mutant hybrid hemoglobins) and hemoglobins containing different metalloporphyrins (mixed-metal hybrids), will be prepared by Core C, using site directed mutagenesis and expression of globin chains in E. coli., followed by isolation and assembly. Equilibria of oxygen binding in solution to the mutant hemoglobins will be measured. Kinetic studies will be carried out using stopped-flow and flash photolysis techniques to examine the effects of mutations on the properties of the R and T quaternary states of the hemoglobin molecule, the relative stabilities of these states and the stability of the tetramer with respect to dissociation into alphabeta dimers. Properties of the T states of these materials will also be examined by measuring the equilibria of oxygen binding to T state crystals grown in polyethylene glycol as well as to T state molecules embedded in silica gels. Properties of the alpha and beta chains of hemoglobin variants within the T quaternary state, and the functional properties of partially liganded hemoglobin variants will be assessed by examining the equilibrium and kinetic properties of symmetric and asymmetric mixed-metal and mixed-mutant hybrid hemoglobins.

该项目的目的是通过检查其与配体的反应的平衡和动力学来表征人血红蛋白变体的功能特性。这项工作的目的是为该计划项目开发的预测立体化学模型的进一步开发和测试做出贡献。血红蛋白变异以及含有两个不同字母二聚体（混合杂种杂交血红蛋白）和含有不同金属核蛋白（混合 - 金属杂交）的血红蛋白，使用核心C制备，使用核心C在E. coli in E. coli in E. coli interiate and Coliation and Colleation and Collecty，然后使用核心C来制备。将测量与突变血红蛋白溶液中氧结合的平衡。动力学研究将使用停止流量和闪光光解技术进行，以检查突变对血红蛋白分子的R和T Quaternary态的影响，这些状态的相对稳定性以及四聚体稳定性与分离为Alphabeta Dimers的稳定性。这些材料的T态的性质也将通过测量与在聚乙烯乙二醇中生长的T态晶体的平衡以及嵌入在硅胶中的T态分子的平衡。 T季度内血红蛋白变体的α和β链的性能，以及部分配体血红蛋白变异的功能性能，通过检查对称和非对称的混合和混合含量和混合含量的氢包的平衡和动力学特性来评估。