Interaction of haptoglobin with hemoglobin octamers based on the mutation αAsn78Cys or βGly83Cys.
Interaction of haptoglobin with hemoglobin octamers based on the mutation αAsn78Cys or βGly83Cys.
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DOI：
10.4236/ajmb.2012.21001
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发表时间：
2012-04-01
期刊：
影响因子：
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通讯作者：
Baudin-Creuza V
中科院分区：
文献类型：
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作者：
Brillet T;Marden MC;Yeh JI;Shen TJ;Ho NT;Kettering R;Du S;Vasseur C;Domingues-Hamdi E;Ho C;Baudin-Creuza V
八聚体血红蛋白是通过在 α 链或 β 链中引入表面半胱氨酸而开发的。最初设计为血液替代品，我们在此报告其结构和配体结合功能；此外还研究了与触珠蛋白的相互作用。具有α Asn78Cys 或β Gly83Cys 突变的重组Hbs (rHbs) 在半胱氨酸被氧化的条件下自发形成八聚体。氧结合曲线和 CO 动力学研究表明八聚体内四聚体的变构转变正确。两种 rHb 的晶体学研究显示每个八聚体有两个二硫键。还原剂可能引起四聚体解离，但八聚体在与新鲜人血浆混合时是稳定的，表明血浆的还原比溶解氧的氧化慢，这与增强的稳定性一致。八聚体 rHb 还与结合珠蛋白 (Hp) 溶液混合，结合珠蛋白 (Hp) 结合 Hb 二聚体：15 分钟的孵育时间几乎没有相互作用；然而，在更长的时间尺度上，形成了复合体。使用动态光散射来追踪 24 小时内 Hp 与 α Asn78Cys 八聚体的相互作用；观察到从 15 nm 的简单复合物到 60 nm 的最终尺寸的转变。结果表明 αβ 二聚体的特定方向可能对于结合珠蛋白很重要。
Octameric hemoglobins have been developed by the introduction of surface cysteines in either the alpha or beta chain. Originally designed as a blood substitute, we report here the structure and ligand binding function; in addition the interaction with haptoglobin was studied. The recombinant Hbs (rHbs) with mutations alpha Asn78Cys or beta Gly83Cys spontaneously form octamers under conditions where the cysteines are oxidized. Oxygen binding curves and CO kinetic studies indicate a correct allosteric transition of the tetramers within the octamer. Crystallographic studies of the two rHbs show two disulfide bonds per octamer. Reducing agents may provoke dissociation to tetramers, but the octamers are stable when mixed with fresh human plasma, indicating that the reduction by plasma is slower than the oxidation by the dissolved oxygen, consistent with an enhanced stability. The octameric rHbs were also mixed with a solution of haptoglobin (Hp), which binds the dimers of Hb: there was little interaction for incubation times of 15 min; however, on longer timescales a complex was formed. Dynamic light scattering was used to follow the interaction of Hp with the alpha Asn78Cys octamer during 24 hours; a transition from a simple complex of 15 nm to a final size of 60 nm was observed. The results indicate a specific orientation of the αβ dimers may be of importance for the binding to haptoglobin.