An experimental method correcting for absorption flattening and scattering in suspensions of absorbing particles: circular dichroism and absorption spectra of hemoglobin in situ in red blood cells.
An experimental method correcting for absorption flattening and scattering in suspensions of absorbing particles: circular dichroism and absorption spectra of hemoglobin in situ in red blood cells.
复制标题
校正吸收颗粒悬浮液中的吸收平坦和散射的实验方法：红细胞中原位血红蛋白的圆二色性和吸收光谱。
作者：
A. S. Schneider;D. Harmatz
本文报道了一种解释红细胞中血红蛋白原位反常吸收和圆二色光谱的实验方法。吸收平坦化效应已通过在非常短的光路比色皿中使用高细胞浓度来克服。差分散射贡献圆二色性已经解决了使用CD仪器能够可变检测几何。散射效应也已经使用高折射率的介质来解决，以匹配红细胞的折射率。结果是一致的并行计算分析的红细胞CD光谱，预测的相对大小的平坦化和微分散射CD的贡献。在消除散射和平坦化的情况下，获得了红细胞中血红蛋白的实验吸收光谱。这定量地模拟了血红蛋白溶液的光谱。所描述的方法应广泛适用于大分子在其天然环境中的构象研究。
An experimental approach to interpretation of the anomalous absorption and circular dichroism (CD) spectra of hemoglobin in situ in red blood cells is reported. Absorption flattening effects have been overcome by use of high cell concentratons in very short light path cuvettes. Differential scattering contributions to circular dichroism have been resolved using a CD instrument capable of variable detection geometry. Scattering effects have also been resolved using media of high refractive index to match that of the red blood cell. The results are in agreement with a parellel calculational analysis of red blood cell CD spectra, which predicted the relative magnitudes of the flattening and differential scattering CD contributions. An experimental absorption spectrum has been obtained for hemoglobin in the red blood cell with scattering and flattening eliminated. This quantitatively simulates the spectrum of a hemoglobin solution. The methods described should be widely applicable to conformational studies of macromolecules in their native environment.