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: 6107917
• 负责人: ROBERT W NOBLE
• 金额: $ 20.96万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107917
• 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.

这个项目的目的是表征人类血红蛋白变异的功能特性，通过检查平衡和动力学的反应配体。本项目的目的是为了进一步发展和测试本项目开发的预测性立体化学模型。在大肠杆菌中，利用位点定向诱变和表达球蛋白链，Core C将制备血红蛋白变体，以及含有两种不同α二聚体的血红蛋白（混合突变型杂交血红蛋白）和含有不同金属卟啉的血红蛋白（混合金属杂交）。，然后进行隔离和组装。将测量溶液中氧与突变血红蛋白的结合平衡。动力学研究将使用停止流动和闪光光解技术进行，以检查突变对血红蛋白分子R和T四元态性质的影响，这些状态的相对稳定性以及四聚体相对于解离成α二聚体的稳定性。这些材料的T态性质也将通过测量氧与聚乙二醇中生长的T态晶体以及嵌入硅胶中的T态分子的结合平衡来检验。通过研究对称和非对称混合金属和混合突变的杂交血红蛋白的平衡和动力学特性，将评估T季态血红蛋白变体的α链和β链的特性，以及部分配体血红蛋白变体的功能特性。