Rodger Alison, Steel Michael J, Goodchild Sophia C, Chmel Nikola P, Reason Andrew
Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.
Department of Physics and Astronomy, Macquarie University, Sydney, NSW 2109, Australia.
QRB Discov. 2020 Oct 16;1:e8. doi: 10.1017/qrd.2020.11. eCollection 2020.
Infrared (IR) spectroscopy is increasingly being used to probe the secondary structure of proteins, especially for high-concentration samples and biopharmaceuticals in complex formulation vehicles. However, the small path lengths required for aqueous protein transmission experiments, due to high water absorbance in the amide I region of the spectrum, means that the path length is not accurately known, so only the shape of the band is ever considered. This throws away a dimension of information. Attenuated total reflectance (ATR) IR spectroscopy is much easier to implement than transmission IR spectroscopy and, for a given instrument and sample, gives reproducible spectra. However, the ATR-absorbance spectrum varies with sample concentration and instrument configuration, and its wavenumber dependence differs significantly from that observed in transmission spectroscopy. In this paper, we determine, for the first time, how to transform water and aqueous protein ATR spectra into the corresponding transmission spectra with appropriate spectral shapes and intensities. The approach is illustrated by application to water, concanavalin A, haemoglobin and lysozyme. The transformation is only as good as the available water refractive index data. A hybrid of literature data provides the best results. The transformation also allows the angle of incidence of an ATR crystal to be determined. This opens the way to using both spectral shape and spectra intensity for protein structure fitting.
红外（IR）光谱越来越多地用于探测蛋白质的二级结构，特别是对于高浓度样品以及复杂制剂载体中的生物制药。然而，由于光谱酰胺I区域中水的高吸光度，蛋白质水溶液透射实验所需的光程较短，这意味着光程无法准确得知，因此人们只考虑谱带的形状。这就舍弃了一个信息维度。衰减全反射（ATR）红外光谱比透射红外光谱更易于实施，并且对于给定的仪器和样品，能给出可重复的光谱。然而，ATR吸光度光谱会随样品浓度和仪器配置而变化，其波数依赖性与透射光谱中观察到的有显著差异。在本文中，我们首次确定了如何将水和蛋白质水溶液的ATR光谱转换为具有适当光谱形状和强度的相应透射光谱。通过应用于水、伴刀豆球蛋白A、血红蛋白和溶菌酶对该方法进行了说明。这种转换的效果仅取决于可用的水折射率数据。文献数据的混合能提供最佳结果。这种转换还能确定ATR晶体的入射角。这为利用光谱形状和光谱强度进行蛋白质结构拟合开辟了道路。
