Kiger L, Poyart C, Marden M C
Institut National de la Santé et de la Recherche Médicale U299, Hôpital de Bicêtre, France.
Biophys J. 1993 Sep;65(3):1050-8. doi: 10.1016/S0006-3495(93)81164-0.
The bimolecular and geminate CO recombination kinetics have been measured for hemoglobin (Hb) with over 90% of the ligand binding sites occupied by NO. Since Hb(NO)4 with inositol hexaphosphate (IHP) at pH below 7 is thought to take on the low affinity (deoxy) conformation, the goal of the experiments was to determine whether the species IHPHb-(NO)3(CO) also exists in this quaternary structure, which would allow ligand binding studies to tetramers in the deoxy conformation. For samples at pH 6.6 in the presence of IHP, the bimolecular kinetics show only a slow phase with rate 7 x 10(4) M-1 s-1, characteristic of CO binding to deoxy Hb, indicating that the triply NO tetramers are in the deoxy conformation. Unlike Hb(CO)4, the fraction recombination occurring during the geminate phase is low (< 1%) in aqueous solutions, suggesting that the IHPHb(NO)3(CO) hybrid is also essentially in the deoxy conformation. By mixing stock solutions of HbCO and HbNO, the initial exchange of dimers produces asymmetric (alpha NO beta NO/alpha CO beta CO) hybrids. At low pH in the presence of IHP, this hybrid also displays a high bimolecular quantum yield and a large fraction of slow (deoxy-like) CO recombination; the slow bimolecular kinetics show components of equal amplitude with rates 7 and 20 x 10(4) M-1 s-1, probably reflecting the differences in the alpha and beta chains. Samples of symmetric hybrids (a2NOI32Co or a2Co922NO) showed a lower (R-like) bimolecular yield and less slow phase for the CO bimolecular recombination, relative to the asymmetric hybrid or the triply NO species. The slower (T state) bimolecular rate of 7 x 104 M-1 s-1 was observed for CO rebinding to a chain.While oxygen equilibrium studies with 'HPHb(NO)3 were hampered by a high oxidation rate, it was possible to perform experiments with samples equilibrated with a mixed CO/oxygen atmosphere. Photodissociation of CO allows a temporary exposure of the binding sites to oxygen. The results confirm that IHPHb(NO)3 has a low oxygen affinity.
已经测量了超过90%的配体结合位点被NO占据的血红蛋白（Hb）的双分子和双生CO复合动力学。由于在pH低于7时，含有肌醇六磷酸（IHP）的Hb(NO)4被认为呈现低亲和力（脱氧）构象，实验的目的是确定IHP Hb-(NO)3(CO)物种是否也存在于这种四级结构中，这将允许对脱氧构象的四聚体进行配体结合研究。对于在pH 6.6且存在IHP的样品，双分子动力学仅显示出速率为7×10⁴ M⁻¹ s⁻¹的慢相，这是CO与脱氧Hb结合的特征，表明三重NO四聚体处于脱氧构象。与Hb(CO)4不同，在水溶液中双生相期间发生的复合分数很低（<1%），这表明IHP Hb(NO)3(CO)杂合体也基本上处于脱氧构象。通过混合HbCO和HbNO储备溶液，二聚体的初始交换产生不对称（αNOβNO/αCOβCO）杂合体。在低pH且存在IHP的情况下，这种杂合体也表现出高双分子量子产率和大部分缓慢（类似脱氧）的CO复合；缓慢的双分子动力学显示出速率为7和20×10⁴ M⁻¹ s⁻¹的等幅成分，这可能反映了α链和β链的差异。相对于不对称杂合体或三重NO物种，对称杂合体（α₂NOβ₂CO或α₂COβ₂NO）的样品显示出较低的（R样）双分子产率和较少的CO双分子复合慢相。观察到CO重新结合到α链的较慢（T态）双分子速率为7×10⁴ M⁻¹ s⁻¹。虽然用IHP Hb(NO)3进行的氧平衡研究因高氧化速率而受阻，但可以对用混合CO/氧气氛平衡的样品进行实验。CO的光解离允许结合位点暂时暴露于氧气。结果证实IHP Hb(NO)3具有低氧亲和力。
