DEVELOPMENT OF BIOPHYSICAL METHODS FOR STUDYING BIOCHEMICAL REACTIONS

血红蛋白（Hb）的氧亲和力受到以下因素的显著影响： 几种变构效应物包括2，3-二磷酸甘油酸（2，3-DPG）， 质子氯化物和二氧化碳 几项研究 表征了这些变构效应物如何调节 血红蛋白。 氨甲酰血红蛋白（HbNHCOO-）一直是一个困难的 分子分离;因此，CO2的结构/功能关系 与血红蛋白的结合尚不清楚。 本研究的重点是血红蛋白与CO2结合的热力学 作为氯化物的函数。 CO2与-NH 2-末端缬氨酸基团结合 在血红蛋白的四条链中，β链具有3倍的 比相应的α链亲和力更高。 NMR研究和 使用修饰的血红蛋白已经表明无机阴离子， 特别是氯，也结合在相同的位点（瓦尔1 -Arg 141）和 其他几个位点（Lys 82 [DPG位点]，His 146-Asp 94）。 与 微量热法，我们已经确定了二氧化碳结合的依赖性， 血红蛋白作为氯化物浓度的函数。 一个落在 CO2-脱氧Hb相互作用的焓从0.01M氯开始， 在高浓度氯化物（1.00 M）下继续下降。 这 这一结果与CO2和氯离子竞争的事实是一致的 结合至相同位点（2）或附近位点的变构效应物，以及 随着血红蛋白在盐浓度下从四聚体解离成二聚体， 浓度超过0.2M。

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.