KINETIC AND EQUILIBRIUM STUDIES OF LIGAND BINDING TO HEMOGLOBIN VARIANTS

这个项目的目的是通过检查人血红蛋白变体与配体反应的平衡和动力学来表征它们的功能特性。这项承诺的目的是为进一步开发和测试预测立体化学模型做出贡献，该模型已经由该计划项目开发。核心C将利用定点突变和在大肠杆菌中表达珠蛋白链的方法制备血红蛋白变体，以及含有两种不同的α-β二聚体的血红蛋白(混合突变杂化血红蛋白)和含有不同金属卟啉的血红蛋白(混合金属杂化血红蛋白)，然后分离和组装。将测量突变血红蛋白溶液中氧结合的平衡。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R和T四态性质的影响，这些态的相对稳定性，以及四聚体相对于解离成Alphabeta二聚体的稳定性。还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查这些材料的T态的性质。将通过检测对称和不对称混合金属和混合突变混合血红蛋白的平衡和动力学性质来评估T四元状态下血红蛋白变体的α和β链的性质，以及部分连接的血红蛋白变体的功能性质。

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.