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人类血红蛋白变体与配体反应的平衡和动力学来表征其功能特性。本项目的目的是为本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。 Core C 将使用定点诱变和在大肠杆菌中表达珠蛋白链来制备血红蛋白变体以及包含两种不同 Alphabeta 二聚体的血红蛋白（混合突变杂合血红蛋白）和包含不同金属卟啉的血红蛋白（混合金属杂合体），然后进行分离和组装。将测量溶液中氧与突变血红蛋白结合的平衡。将使用停流和闪光光解技术进行动力学研究，以检查突变对血红蛋白分子的 R 和 T 四元态特性的影响、这些状态的相对稳定性以及四聚体解离成 Alphabeta 二聚体的稳定性。这些材料的 T 态特性也将通过测量与聚乙二醇中生长的 T 态晶体以及嵌入硅胶中的 T 态分子结合的氧平衡来检查。 T四元态内血红蛋白变体的α和β链的特性以及部分配体血红蛋白变体的功能特性将通过检查对称和不对称混合金属和混合突变杂合血红蛋白的平衡和动力学特性来评估。