Goss D J, Parkhurst L J, Görisch H
Biochemistry. 1975 Dec 16;14(25):5461-4. doi: 10.1021/bi00696a012.
The kinetics of the pH-induced dissociation of the 3 X 10(6) mol wt hemoglobin from Lumbricus terrestris (the earthworm) have been studied in a light-scattering stopped-flow apparatus. The ligand dependent dissociation data were fit well by a simple sequential model. The data for CO and oxyhemoglobin are consistent with Hb12 leads to 2Hb6 leads to 12Hb. Methemoglobin at pH 7 appears to be hexameric and the dissociation is consistent with the model: Hb6 leads to 6Hb. In a sequential decay scheme for which light-scattering changes are monitored, the relative amounts of rapid and slow phase are determined by the rate constants as well as the molecular weights of intermediate species. Assignment of the hexameric intermediate is supported by an investigation of the sensitivity of the theoretical kinetic curves to the molecular weights of the intermediates. This assignment is further supported by the following: (1) the same model will fit the data for oxy- and CO-hemoglobin at all three temperatures (a 24-29-fold variation in rate constants), (2) evidence from electron microscopy shows hexameric forms, and (3) methemoglobin is apparently stable as a hexamer at pH 7. When CO replaces O2 as the ligand, the dissociation rate increases by a factor of four. The met is about 20 times faster than the initial oxyhemoglobin dissociation rate, but perhaps more relevant for comparing dissociation of the hexamer, the met rate was respectively 100 times and 500 times faster than that for the assumed hexameric forms of CO- and oxy-hemoglobin. The activation energies for the dodecamer to hexamer dissociation and for the dissociation of the hexamer to smaller forms were about 30 kcal/mol for oxy-, CO-, and methemoglobin.
利用光散射停流装置研究了pH诱导的来自蚯蚓的3×10⁶摩尔质量血红蛋白解离的动力学。配体依赖性解离数据通过简单的顺序模型拟合良好。一氧化碳和氧合血红蛋白的数据与Hb₁₂→2Hb₆→12Hb一致。pH 7时的高铁血红蛋白似乎是六聚体，其解离与模型：Hb₆→6Hb一致。在监测光散射变化的顺序衰减方案中，快速和慢速相的相对量由速率常数以及中间物种的分子量决定。对理论动力学曲线对中间物种分子量的敏感性进行研究，支持了六聚体中间体的归属。以下内容进一步支持了这一归属：(1) 相同的模型在所有三个温度下都能拟合氧合血红蛋白和一氧化碳血红蛋白的数据（速率常数有24 - 29倍的变化），(2) 电子显微镜证据显示有六聚体形式，(3) 高铁血红蛋白在pH 7时作为六聚体显然是稳定的。当一氧化碳取代氧气作为配体时，解离速率增加了四倍。高铁血红蛋白的解离速率比初始氧合血红蛋白解离速率快约20倍，但也许对于比较六聚体的解离更相关的是，高铁血红蛋白的解离速率分别比假定的一氧化碳血红蛋白和氧合血红蛋白六聚体形式的解离速率快100倍和500倍。氧合血红蛋白、一氧化碳血红蛋白和高铁血红蛋白从十二聚体到六聚体解离以及从六聚体到更小形式解离的活化能约为30千卡/摩尔。
