Doyle M L, Speros P C, LiCata V J, Gingrich D, Hoffman B M, Ackers G K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1991 Jul 23;30(29):7263-71. doi: 10.1021/bi00243a031.
The thermodynamic linkage between cooperative oxygenation and dimer-tetramer subunit assembly has been determined for cobaltous human hemoglobin in which iron(II) protoporphyrin IX is replaced by cobalt(II) protoporphyrin IX. The equilibrium parameters of the linkage system were determined by global nonlinear least-squares regression of oxygenation isotherms measured over a range of hemoglobin concentrations together with the deoxygenated dimer-tetramer assembly free energy determined independently from forward and reverse reaction rates. The total cooperative free energy of tetrameric cobalt hemoglobin (over all four binding steps) is found to be 1.84 (+/- 0.13) kcal, compared with the native ferrous hemoglobin value of 6.30 (+/- 0.14) kcal. Detailed investigation of stepwise cooperativity effects shows the following: (1) The largest change occurs at the first ligation step and is determined on model-independent grounds by knowledge of the intermediate subunit assembly free energies. (2) Cooperativity in the shape of the tetrameric isotherm occurs mainly during the middle two steps and is concomitant with the release of quaternary constraints. (3) Although evaluation of the pure tetrameric isotherm portrays identical binding affinity between the last two steps, this apparent noncooperativity is the result of a "hidden" oxygen affinity enhancement at the last step of 0.48 (+/- 0.12) kcal. This quaternary enhancement energy is revealed by the difference in subunit assembly free energies of the triply and fully ligated species and is manifested visually by the oxygenation isotherms at high versus low hemoglobin concentration. (4) Cobaltous hemoglobin dimers exhibit apparent anticooperativity of 0.49 (+/- 0.16) kcal (presumed to arise from heterogeneity of subunit affinities).(ABSTRACT TRUNCATED AT 250 WORDS)
已确定钴代人血红蛋白中协同氧合作用与二聚体 - 四聚体亚基组装之间的热力学联系，其中铁（II）原卟啉IX被钴（II）原卟啉IX取代。通过对一系列血红蛋白浓度下测得的氧合等温线进行全局非线性最小二乘回归，以及独立于正向和反向反应速率确定的脱氧二聚体 - 四聚体组装自由能，来确定该联系系统的平衡参数。发现四聚体钴血红蛋白的总协同自由能（在所有四个结合步骤中）为1.84（±0.13）千卡，而天然亚铁血红蛋白的值为6.30（±0.14）千卡。对逐步协同效应的详细研究表明：（1）最大变化发生在第一步结合时，并且基于与模型无关的理由，通过了解中间亚基组装自由能来确定。（2）四聚体等温线形状的协同作用主要发生在中间两步，并且与四级约束条件的释放同时发生。（3）尽管对纯四聚体等温线的评估显示最后两步之间具有相同的结合亲和力，但这种明显的非协同性是最后一步“隐藏”的氧亲和力增强0.48（±0.12）千卡的结果。这种四级增强能量通过三重和完全结合物种的亚基组装自由能差异揭示，并在高与低血红蛋白浓度下的氧合等温线上直观体现。（4）钴血红蛋白二聚体表现出0.49（±0.16）千卡的明显反协同性（推测源于亚基亲和力的异质性）。（摘要截断于250字）
