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Interaction of haptoglobin with hemoglobin octamers based on the mutation <i>α</i>Asn78Cys or <i>β</i>Gly83Cys

American Journal of Molecular Biology 2012年第1期2卷 1-10页

作者： Thomas Brillet Michael C. Marden Joanne I. Yeh Tong-Jian Shen Nancy T. Ho Regina Kettering Shoucheng Du Corinne Vasseur elisa domingues-hamdi Chien Ho Véronique Baudin-Creuza Department of Biological Sciences Carnegie Mellon University Pittsburgh USA Department of Structural Biology School of Medicine University of Pittsburgh Pittsburgh USA Inserm U779 Université Paris XI et VII CHU Bicêtre Le Kremlin-Bicêtre France

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.

关键词： Octamers Hemoglobin Haptoglobin Allosteric Transition Crystallography

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