DEVELOPMENT OF BIOPHYSICAL METHODS FOR STUDYING BIOCHEMICAL REACTIONS

研究生化反应的生物物理方法的发展

• 批准号: 3757629
• 负责人: R L BERGER
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3757629
• 关键词: biophysics; calorimetry; carbon dioxide; chemical association; chemical binding; chemical reaction; chemical structure function; chlorine; dimer; hemoglobin; hydrogen; method development; molecular site; nuclear magnetic resonance spectroscopy; oxygen; thermodynamics

The oxygen affinity of hemoglobin (Hb) is significantly influenced by several allosteric effectors including 2,3-diphosphoglycerate (2,3-DPG), protons, chloride, and carbon dioxide. Several studies have characterized how these allosteric effectors modulate the reactivity of hemoglobin. The carbamino hemoglobin (HbNHCOO-) has been a difficult molecule to isolate; thus, the structure/function relationship of the CO2 binding to hemoglobin is not well understood. This study focuses on the thermodynamics of the CO2 binding of hemoglobin as a function of chloride. CO2 binds to the -NH2- terminal valine groups of the four chains of hemoglobin, with the beta-chains having a 3 fold higher affinity than the corresponding alpha-chains. NMR studies and the use of modified hemoglobins have shown that inorganic anions, specifically chloride, also bind at the same site (Val 1 -Arg141) and at several other sites (Lys82 [DPG site], His146 -Asp94). With microcalorimetry, we have determined the dependence of CO2 binding to hemoglobin as a function of chloride concentration. A drop-off in the enthalpy of the CO2-deoxy Hb interaction begins at 0.01M chloride and continues downward at high concentrations of chloride (1.00M). This result is consistent with the fact that CO2 and chloride are competitive allosteric effectors binding to the same site(2) or nearby site(s) and with the dissociation of the hemoglobin from tetramers to dimers at salt concentrations over 0.2M.

血红蛋白 (Hb) 的氧亲和力受以下因素影响显着： 几种变构效应物，包括 2,3-二磷酸甘油酸酯 (2,3-DPG)、 质子、氯化物和二氧化碳。 多项研究已 表征了这些变构效应器如何调节反应性 血红蛋白。 卡氨基血红蛋白 (HbNHCOO-) 一直是一种困难的 待分离的分子；因此，CO2 的结构/功能关系 与血红蛋白的结合尚不清楚。 本研究重点关注血红蛋白与 CO2 结合的热力学 作为氯化物的函数。 CO2 与 -NH2- 末端缬氨酸基团结合 血红蛋白的 4 条链中，β 链的 3 倍 比相应的α链具有更高的亲和力。 核磁共振研究和 改良血红蛋白的使用表明无机阴离子， 特别是氯化物，也结合在同一位点 (Val 1 -Arg141) 和 其他几个位点（Lys82 [DPG 位点]、His146 -Asp94）。 和 微量热法，我们确定了 CO2 结合的依赖性 血红蛋白作为氯化物浓度的函数。 下车在 CO2-脱氧 Hb 相互作用的焓从 0.01M 氯化物开始，并且 在高浓度氯化物 (1.00M) 下继续向下。 这 结果与 CO2 和氯化物具有竞争性的事实一致 变构效应子结合到同一位点（2）或附近位点，并且 随着盐作用下血红蛋白从四聚体解离为二聚体 浓度超过0.2M。