Subunit dissociations in natural and recombinant hemoglobins.
Subunit dissociations in natural and recombinant hemoglobins.
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天然和重组血红蛋白中的亚基解离。
DOI：
10.1002/pro.5560050423
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Manning,JM
中科院分区：
文献类型：
--
作者：
Manning,LR;Jenkins,WT;Hess,JR;Vandegriff,K;Winslow,RM;Manning,JM
本文描述了一种利用Superose-12凝胶过滤来精确、快速地测定氧构象中一些天然和重组血红蛋白的四聚体-二聚体解离常数的方法。选择天然的镰刀状血红蛋白，通过与使用不基于凝胶过滤的独立方法报告的值进行比较，来验证结果的有效性。重组镰状血红蛋白以及在Val-6(β)受体位点发生替换的镰状双突变体具有与天然镰状血红蛋白大致相同的解离常数。在α-1-β-2亚单位界面氨基酸替换的两个重组血红蛋白中，一个被广泛解离，另一个被完全解离。此外，变构调节剂DPG和IHP对解离常数没有影响。因此，四聚体解离常数现在可以很容易地确定，并与其他标准一起用于表征血红蛋白及其与小调节分子的相互作用。
A precise and rapid procedure employing gel filtration on Superose‐12 to measure the tetramer‐dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val‐6(β) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in theα1β2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.