Huang Y, Koestner M L, Ackers G K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biophys J. 1996 Oct;71(4):2106-16. doi: 10.1016/S0006-3495(96)79409-2.
Dimer-tetramer assembly reactions of the 10 CN-met ligation microstates of hemoglobin (Hb) were analyzed as a function of NaCl concentration while maintaining constant water activity by the addition of compensating sucrose. The assembly free energy for fully ligated cyanomet Hb and for fully oxygenated Hb becomes less favorable by 1.8 kcal when [NaCl] is increased from 0.08 to 0.7 M, whereas that of unligated Hb is practically insensitive to changes in [NaCl]. Values of 1.6 and 0.3 mol chloride release were found for the assembly of fully ligated and deoxy Hb, respectively; i.e., a net release of 1.3 mol chloride is coupled to the ligation of tetramers for both oxygen and cyanomet ligation. The ligation-linked salt component at constant water activity was evaluated to be 1.0 mol for the full oxygenation of the Hb tetramer in agreement with the overall value previously reported. When the detailed salt linkages accompanying all 16 stepwise cyanomet ligation reactions were experimentally resolved, only two "chloride" effects were found. The first chloride effect correlates with the ligation steps, which create tertiary constraint, and the second effect is coupled to the six switchpoints of quaternary T-->R transition. The distribution of these chloride effects agrees closely with predictions of the "symmetry rule mechanism." The total chloride release for CN-met ligation is in good agreement with that for oxygenation. Free energy contributions to assembly and cooperativity arising from the osmotic effects of chloride were found to be small for all ligation species.
在通过添加补偿性蔗糖保持水活性恒定的情况下，分析了血红蛋白（Hb）的10种氰化高铁连接微状态的二聚体 - 四聚体组装反应与NaCl浓度的关系。当[NaCl]从0.08 M增加到0.7 M时，完全连接的氰化高铁Hb和完全氧合Hb的组装自由能变得更不利，减少了1.千卡，而未连接的Hb对[NaCl]的变化几乎不敏感。完全连接的和脱氧Hb的组装分别发现有1.6摩尔和0.3摩尔氯离子释放；即，对于氧合和氰化高铁连接，1.3摩尔氯离子的净释放与四聚体的连接相关。在恒定水活性下，Hb四聚体完全氧合时连接相关的盐成分经评估为1.0摩尔，与先前报道的总值一致。当通过实验解析所有16步氰化高铁连接反应伴随的详细盐键时，仅发现两种“氯离子”效应。第一种氯离子效应与产生三级约束的连接步骤相关，第二种效应与四级T→R转变的六个转换点相关。这些氯离子效应的分布与“对称规则机制”的预测非常吻合。氰化高铁连接的总氯离子释放与氧合的总氯离子释放非常吻合。对于所有连接物种，发现氯离子渗透效应对组装和协同性的自由能贡献很小。
