Liao D, Jiang J, Zhao M, Ferrone F A
Department of Physics and Atmospheric Science, Drexel University, Philadelphia, Pennsylvania 19104.
Biophys J. 1993 Nov;65(5):2059-67. doi: 10.1016/S0006-3495(93)81268-2.
We have extended the method of modulated excitation, a small perturbation kinetic method, to study ligand binding and conformational change of hemoglobin tetramers with a single ligand bound. To restrict the excitation to the first ligand, only 1% of the hemes have bound CO, and the remainder are kept unliganded. A detailed theory is presented which agrees well with the experimental observations. This method of observing ligand recombination also provides a novel and simple method for determination of hemoglobin concentration. Additional relaxation processes are also observed. By fitting independently determined spectra to the spectra associated with the relaxations, these processes are assigned as thermal excitation and thermally driven protonation/deprotonation reactions. These added relaxations arise from the deoxy-Hb portion of the samples, and demonstrate that modulated excitation can be used effectively for temperature perturbation in the absence of photodissociation. The spectra observed are not well described by the spectra of allosteric change, however, and we conclude that there is no significant mixing of quaternary states at the first ligation step. In an appendix we present a derivation of the particular features seen in thermally modulated protonation reactions.
我们扩展了调制激发方法，一种小扰动动力学方法，用于研究结合单个配体的血红蛋白四聚体的配体结合和构象变化。为了将激发限制在第一个配体上，仅1%的血红素结合了CO，其余保持未结合配体状态。本文提出了一个详细的理论，该理论与实验观察结果吻合良好。这种观察配体重组的方法还为血红蛋白浓度的测定提供了一种新颖且简单的方法。还观察到了其他弛豫过程。通过将独立测定的光谱与弛豫相关的光谱进行拟合，这些过程被确定为热激发和热驱动的质子化/去质子化反应。这些额外的弛豫源于样品中的脱氧血红蛋白部分，并表明在不存在光解离的情况下，调制激发可有效地用于温度扰动。然而，观察到的光谱不能很好地用别构变化的光谱来描述，我们得出结论，在第一次结合步骤中不存在四级状态的显著混合。在附录中，我们推导了热调制质子化反应中出现的特定特征。
