Jones C M, Ansari A, Henry E R, Christoph G W, Hofrichter J, Eaton W A
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1992 Jul 28;31(29):6692-702. doi: 10.1021/bi00144a008.
To determine the speed of communication between protein subunits, time-resolved absorption spectra were measured following partial photodissociation of the carbon monoxide complex of hemoglobin. The experiments were carried out using linearly polarized, 10-ns laser pulses, with the polarization of the excitation pulse both parallel and perpendicular to the polarization of the probe pulse. The substantial contribution to the observed spectra from photoselection effects was eliminated by isotropically averaging the polarized spectra, allowing a detailed comparison of the kinetics as a function of the degree of photolysis. These results show that prior to 1 microsecond both geminate ligand rebinding and conformational relaxation are independent of the number of ligands dissociated from the hemoglobin tetramer, as expected for a two-state allosteric model. After this time the kinetics depend on the ligation state of the tetramer. The conformational relaxation at 10 microseconds can be interpreted in terms of the two-state allosteric model as arising from the R to T quaternary conformational change of both unliganded and singly liganded molecules. These results suggest that communication between subunits requires about 1 microsecond and that the mechanism of the communication which occurs after this time is via the R to T conformational change. The optical anisotropy provides a novel means of accurately determining the extinction coefficients of the transient photoproduct. The decay in the optical anisotropy, moreover, provides an accurate determination of the rotational correlation time of 36 +/- 3 ns.
为了确定蛋白质亚基之间的通信速度，在血红蛋白一氧化碳复合物的部分光解离后测量了时间分辨吸收光谱。实验使用线性偏振的10纳秒激光脉冲进行，激发脉冲的偏振方向与探测脉冲的偏振方向平行和垂直。通过对偏振光谱进行各向同性平均，消除了光选择效应对观测光谱的重大贡献，从而能够详细比较作为光解程度函数的动力学。这些结果表明，在1微秒之前，双分子配体重新结合和构象弛豫均与从血红蛋白四聚体解离的配体数量无关，这与两态别构模型的预期一致。在此之后，动力学取决于四聚体的连接状态。10微秒时的构象弛豫可以根据两态别构模型解释为来自未结合和单结合分子的R到T四级构象变化。这些结果表明，亚基之间的通信需要大约1微秒，并且在此之后发生的通信机制是通过R到T构象变化。光学各向异性提供了一种准确测定瞬态光产物消光系数的新方法。此外，光学各向异性的衰减提供了36±3纳秒旋转相关时间的准确测定。
