Structural Energetics of Hemoglobin Intermediates

描述（由申请人提供）：本研究的目的是确定 分子机制和驱动力，调节通信之间 人类血红蛋白的氧结合位点。总的氧结合 血红蛋白的性质是有据可查的，然而， 积极的协同性，这是至关重要的生理功效，是不是 明白血红蛋白四聚体中的主要功能单元具有 最近才被鉴定为它的α-二聚体成分，如 中间态之间的结构能量分布。订单 氧结合，以及它的释放，遵循特定的组合 规则，优先结合四聚体内一个二聚体上的两个位点， 然后与剩余二聚体上的位点结合。这种功能上的不对称 血红蛋白四聚体中的蛋白质也在对杂交的初步研究中观察到。 四聚体，其中一个α-二聚体是正常的，但另一个二聚体含有 交叉二聚体界面中的单个氨基酸。修改的效果 对氧结合的合作自由能的影响仅在改性的 二聚体，而正常的二聚体没有显着影响。艾克斯医生计划 为了扩展这些使用重组血红蛋白的O2中间体的研究 设计了一系列改变的残基位点。亚基相互作用 重组系统将通过动力学、分析凝胶电泳、免疫荧光和免疫荧光等技术进行研究。 色谱法，低温等电聚焦，以及直接02 约束力由于Hb调节系统是一个重要的原型， 家庭的合作多位点调控组件，开发的方法 该方案具有广泛的适用性。更深入地了解人类 血红蛋白机制也是目前感兴趣的潜在设计的红细胞 替代氧载体和基于血红蛋白的药物递送系统。

DESCRIPTION (provided by applicant): The goal of this research is to determine the molecular mechanism and driving forces that regulate communication between the oxygen binding sites of human hemoglobin. The overall oxygen binding properties of hemoglobin are well-documented, however, the molecular basis of positive cooperativity, which is crucial to physiological efficacy, is not understood. The primary functional units within the hemoglobin tetramer have only recently been identified as its alphabeta dimer components, as shown by the distribution of structural energetics among intermediate states. The order of oxygen binding, as well as its release, follows specific combinatorial rules, binding preferentially to both sites on one dimer within the tetramer, then binding to the sites on the remaining dimer. This functional asymmetry within the hemoglobin tetramer is also observed in initial studies on hybrid tetramers, where one alphabeta dimer is normal but the other dimer contains a single amino acid in the cross-dimer interface. The effect of the modification on the cooperative free energy of oxygen binding is felt only on the modified dimer, while the normal dimer is not significantly affected. Dr. Ackers plans to extend these studies for the 02 intermediates using recombinant hemoglobins with designed sets of altered residue sites. Subunit interactions of the recombinant systems will be studied by techniques of kinetics, analytical gel chromatography, cryogenic isoelectric focusing, as well as by direct 02 binding. As the Hb regulatory system is an important prototype for a large family of cooperative multi-site regulatory assemblies, the methods developed by this program should have wide applicability. Deeper understanding of human Hb mechanisms is also of current interest to the potential design of red cell substitute oxygen carriers and hemoglobin-based drug delivery systems.