Manning L R, Jenkins W T, Hess J R, Vandegriff K, Winslow R M, Manning J M
Northeastern University, Department of Biology, Boston, MA 02115, USA.
Protein Sci. 1996 Apr;5(4):775-81. doi: 10.1002/pro.5560050423.
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(beta) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the alpha 1 beta 2 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.
描述了一种精确且快速的方法，该方法采用Superose - 12凝胶过滤来测量一些处于氧合构象的天然和重组血红蛋白的四聚体 - 二聚体解离常数。选择天然镰刀型血红蛋白，通过将结果值与使用不基于凝胶过滤的独立方法所报告的值进行比较，来验证结果的有效性。重组镰刀型血红蛋白以及在Val - 6(β)受体位点有一个替代的镰刀型双突变体，其解离常数与天然镰刀型血红蛋白大致相同。在α1β2亚基界面有氨基酸替换的两种重组血红蛋白中，一种被发现有广泛解离，另一种则完全解离。此外，还证明了变构调节剂DPG和IHP对解离常数没有影响。因此，现在可以很容易地确定四聚体解离常数，并将其与其他标准一起用于血红蛋白的表征及其与小调节分子的相互作用。
