Schneider A S, Harmatz D
Biochemistry. 1976 Sep 21;15(19):4158-62. doi: 10.1021/bi00664a004.
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.
报道了一种用于解释红细胞中血红蛋白原位异常吸收和圆二色性（CD）光谱的实验方法。通过在极短光路比色皿中使用高细胞浓度克服了吸收扁平化效应。使用能够进行可变检测几何结构的CD仪器解决了圆二色性的差分散射贡献。还使用高折射率介质与红细胞折射率匹配来解决散射效应。结果与红细胞CD光谱的平行计算分析一致，该分析预测了扁平化和差分散射CD贡献的相对大小。已获得消除了散射和扁平化的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。所述方法应广泛适用于天然环境中大分子的构象研究。
