Ansari A, Jones C M, Henry E R, 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.
Biophys J. 1993 Mar;64(3):852-68. doi: 10.1016/S0006-3495(93)81446-2.
Polarized photolysis experiments have been performed on the carbon monoxide complex of myoglobin to assess the effects of photoselection on the kinetics of ligand rebinding and to investigate the reorientational dynamics of the heme plane. The results are analyzed in terms of the optical theory developed in the preceding paper by Ansari and Szabo. Changes in optical density arising from rotational diffusion of the photoselected population produce large deviations from the true geminate ligand rebinding curves if measurements are made with only a single polarization. The apparent ligand rebinding curves are significantly distorted even at photolysis levels greater than 90%. These deviations are eliminated by obtaining isotropically-averaged optical densities from measurements using both parallel and perpendicular polarizations of the probe pulse. These experiments also yield the optical anisotropy, which gives a novel method for accurately determining the degree of photolysis, as well as important information on the reorientational dynamics of the heme plane. The correlation time for the overall rotational diffusion of the molecule is obtained from the decay of the anisotropy. The anisotropy prior to rotational diffusion is lower than that predicted for a rigidly attached, perfectly circular absorber, corresponding to an apparent order parameter of S = 0.95 +/- 0.02. Polarized absorption data on single crystals suggest that the decreased anisotropy results more from internal motions of the heme plane which take place on time scales shorter than the duration of the laser pulse (10 ns) than from out-of-plane polarized transitions.
已对肌红蛋白的一氧化碳复合物进行了偏振光解实验，以评估光选择对配体重新结合动力学的影响，并研究血红素平面的重取向动力学。根据安萨里和萨博在前一篇论文中提出的光学理论对结果进行了分析。如果仅用单一偏振进行测量，光选择群体的旋转扩散引起的光密度变化会导致与真正的双分子配体重新结合曲线产生很大偏差。即使在光解水平大于90%时，表观配体重新结合曲线也会明显扭曲。通过使用探测脉冲的平行和垂直偏振进行测量来获得各向同性平均光密度，从而消除了这些偏差。这些实验还得出了光学各向异性，它提供了一种准确测定光解程度的新方法，以及关于血红素平面重取向动力学的重要信息。分子整体旋转扩散的相关时间是从各向异性的衰减中获得的。旋转扩散之前的各向异性低于刚性附着的完美圆形吸收体所预测的值，对应于表观序参数S = 0.95±0.02。单晶的偏振吸收数据表明，各向异性的降低更多是由于血红素平面的内部运动，这些运动发生的时间尺度短于激光脉冲的持续时间（10纳秒），而不是由于平面外偏振跃迁。
