Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.
Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.
复制标题
由重组突变β-珠蛋白合成的人血红蛋白的功能特性。
DOI：
10.1021/bi00151a033
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Ackers,GK
中科院分区：
文献类型：
--
作者：
Doyle,ML;Lew,G;DeYoung,A;Kwiatkowski,L;Wierzba,A;Noble,RW;Ackers,GK
Ala、81瓦尔-* Met和添加突变81+ Met），它们与血红素和天然α链组装成n282四聚体，以及它们的X射线晶体学结构。在这里，我们已经测量了这些血红蛋白的平衡和动力学变构特性。我们的目标是评估其效用作为正常血红蛋白的替代品，从进一步的突变体可以进行结构-功能研究。通过对血红蛋白浓度范围内测量的多个氧合等温线进行全局回归分析，确定了合作氧合和二聚体-四聚体组装之间的热力学联系。发现氧与四聚体的结合是高度合作的（最大希尔斜率从3.1到3.2），并且O2-连接的亚基组装自由能的类似模式表明在α ′ 82界面处合作切换的共同模式。发现二聚体表现出与正常血红蛋白相同的非合作O2平衡结合特性。突变重组血红蛋白和正常血红蛋白之间氧平衡的最明显差异是略微降低的O2亲和力。通过停流和闪光光解技术测量CO结合和O2解离的动力学。在存在和不存在有机磷酸盐的情况下，用突变体和正常血红蛋白进行平行研究，以评估其对磷酸盐的变构反应。在不存在有机磷酸盐的情况下，发现突变体二聚体和四聚体的CO结合和O2解离动力学性质与正常血红蛋白的那些性质几乎相同。然而，有机磷酸盐对突变体的CO结合动力学性质的影响并不一致：发现81+ Met突变体偏离正常状态，而81瓦尔-*· Met突变体再现了天然变构反应。81瓦尔-·Met突变体的变构性质的进一步表征通过用张力测定法测量其总体氧亲和力的pH依赖性来进行。发现质子对氧亲和力的调节在pH 5.8至9.3范围内与正常血红蛋白几乎相同（在pH 7.4时，每结合一个氧释放0.52±0.07个质子）。本研究表明，重组81瓦尔-*·
Ala, 81 Val-* Met, and the addition mutation 81+ Met), their assembly with heme and natural a chains into «282 tetramers, and their X-ray crystallographicstructures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. Thethermodynamic linkagesbetween cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of 02-linkedsubunit assembly free energies indicated a common mode of cooperative switching at the a’82 interface. The dimers were found to exhibit the same noncooperative 02 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered 02 affinity. The kinetics of CO binding and 02 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and 02 dissociation kinetic properties of the muant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the 81+ Met mutant was found to deviate somewhat from normalcy, while the 81 Val-*· Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the 81 Val-» Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52±0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant 81 Val-*·