Huang Y, Koestner M L, Ackers G K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Biophys Chem. 1997 Feb 28;64(1-3):157-73. doi: 10.1016/s0301-4622(96)02236-3.
Heterotropic effects of NaCl were studied using CN-met hemoglobin, which has been found to follow the same rules of homotropic cooperativity as CO-Hb and O2-Hb [Huang and Ackers, Biochemistry, 35 (1996) 704; Huang et al., Biophys. J., 71 (1996) 2094]. Modulation of heme site cooperativity by NaCl was determined in this study for all partially ligated CN-met intermediates by measuring their dimer-to-tetramer assembly free energies as a function of NaCl concentration (0.08-1.4 M; pH 7.4, T = 21.5 degrees C). Thermodynamic linkage analysis yielded the contributions to heme site binding cooperativity for all 16 reactions of the binding cascade, and also their apparent changes in bound salt. The principal findings were as follows: (i) At each [NaCl] the ten tetrameric species exhibited three discrete cooperative free energy levels; (ii) positional isomers of the doubly ligated tetramers were distributed among two of these levels according to their specific configurations of ligated sites, in conformity with the symmetry rule mechanism of hemoglobin cooperativity [Ackers et al., Science 255 (1992) 54]; (iii) the apparent moles of NaCl release followed the same configuration-specific distribution as that of heme site cooperativity, i.e., this parameter was synchronized according to the same response clusters. The system thus manifests both a "tertiary chloride effect" and a "quaternary chloride effect", which parallel the tertiary and quaternary Bohr effects [Daugherty et al., Biochemistry, 33 (1994) 10345; Perrella et al., Biochemistry, 33 (1994) 10358] and the tertiary and quaternary enthalpy effects [Huang and Ackers, Biochemistry, 34 (1995) 6316]. Comparison with findings on the stoichiometric O2-binding linkages over an identical range of conditions [Doyle et al., Biophys. Chem., 64 (1997)] revealed that the overall NaCl release upon ligating all four hemes is identical for O2 and CN-met, whereas the detailed distributions of apparent chloride release showed variations between the two ligands, i.e., CN-met Hb showed only a negligible quaternary enhancement at all [NaCl] conditions and a larger tertiary chloride effect compared with O2-Hb. Possible origins of these variations are considered.
利用氰化高铁血红蛋白研究了氯化钠的异促效应，已发现其遵循与一氧化碳血红蛋白和氧合血红蛋白相同的同促协同作用规则[Huang和Ackers，《生物化学》，35 (1996) 704；Huang等人，《生物物理杂志》，71 (1996) 2094]。在本研究中，通过测量所有部分连接的氰化高铁中间产物的二聚体到四聚体组装自由能随氯化钠浓度（0.08 - 1.4 M；pH 7.4，T = 21.5摄氏度）的变化，确定了氯化钠对血红素位点协同作用的调节。热力学耦合分析得出了结合级联中所有16个反应对血红素位点结合协同作用的贡献，以及它们结合盐的表观变化。主要发现如下：(i) 在每个[氯化钠]浓度下，十种四聚体物种表现出三个离散的协同自由能水平；(ii) 双连接四聚体的位置异构体根据其连接位点的特定构型分布在其中两个水平上，这与血红蛋白协同作用的对称规则机制一致[Ackers等人，《科学》255 (1992) 54]；(iii) 氯化钠释放的表观摩尔数遵循与血红素位点协同作用相同的构型特异性分布，即该参数根据相同的响应簇同步。因此，该系统表现出“三级氯化物效应”和“四级氯化物效应”，它们与三级和四级玻尔效应[Daugherty等人，《生物化学》，33 (1994) 10345；Perrella等人，《生物化学》，33 (1994) 10358]以及三级和四级焓效应[Huang和Ackers，《生物化学》，34 (1995) 6316]相似。与在相同条件范围内关于化学计量氧结合耦合的研究结果[Doyle等人，《生物物理化学》，64 (1997)]比较表明，对于氧和氰化高铁，连接所有四个血红素时氯化钠的总释放量是相同的，而表观氯化物释放的详细分布在两种配体之间存在差异
