Division of Pharmaceutics, University of Erlangen-Nuremberg, Germany.
J Pharm Biomed Anal. 2010 Jan 5;51(1):39-45. doi: 10.1016/j.jpba.2009.07.031. Epub 2009 Aug 4.
The aim of this study was to develop a quick and objective method for the determination of changes in protein secondary structure by Fourier transform infrared spectroscopy (FTIR). Structural shifts from native regions (alpha-helix, intramolecular beta-sheet) to aggregated strands (intermolecular beta-sheet) were used to evaluate protein damage. FTIR spectra of 16 different proteins were recorded and quantified by peak fitting of the non-deconvolved and baseline corrected amide I bands. The resulting percentile secondary structures were correlated with the shape and intensity of the area normalized amide I bands using an interval partial least squares algorithm (iPLS). Structural elements were focused on the following regions: alpha-helix 1660-1650 cm(-1), intramolecular beta-sheet 1695-1683 cm(-1) and 1644-1620 cm(-1), intermolecular beta-sheet 1620-1595 cm(-1). Three calibration curves were created from the data sets. Calculated alpha-helix content ranged from 0% to 79.59%, intramolecular beta-sheet from 10.64% to 63.89% and intermolecular beta-sheet from 0.23% to 9.70%. The linear relationship between actual values (as determined by peak fitting) and calculated values was evaluated by correlation coefficient and root mean square error of calibration while cross-validation was performed to detect possible outliers. Results were verified by including two proteins as validation standards and comparing the calculated values to peak fitting and X-ray data. Structural changes of human serum albumin (HSA) due to elevated temperatures and the fibrillation of glucagon were quantified by calibration curve analysis. Performance and reliability of the iPLS algorithm were evaluated by comparing calculated secondary structure elements with results from peak fitting and circular dichroism. Different methods for the determination of secondary structure gave slightly different results but overall tendencies concurred. Additionally, formation of HSA aggregates could be linked to increasing beta-sheet content by comparing SEC-HPLC and turbidity analysis with results from the FTIR calibration curves. In summary, quantification of the alpha-helix to beta-sheet transition by iPLS analysis proves to be a feasible and objective way for the determination of damage to protein secondary structure.
本研究旨在开发一种快速、客观的方法，通过傅里叶变换红外光谱（FTIR）测定蛋白质二级结构的变化。利用从天然区域（α-螺旋、分子内β-折叠）到聚集链（分子间β-折叠）的结构位移来评估蛋白质损伤。记录了 16 种不同蛋白质的 FTIR 光谱，并通过对非去卷积和基线校正的酰胺 I 带进行峰拟合进行定量。使用区间偏最小二乘算法（iPLS）将所得百分比二级结构与归一化酰胺 I 带的形状和强度相关联。结构要素集中在以下区域：α-螺旋 1660-1650 cm（-1），分子内β-折叠 1695-1683 cm（-1）和 1644-1620 cm（-1），分子间β-折叠 1620-1595 cm（-1）。从数据集创建了三个校准曲线。计算的α-螺旋含量范围为 0%至 79.59%，分子内β-折叠含量为 10.64%至 63.89%，分子间β-折叠含量为 0.23%至 9.70%。通过相关系数和校准的均方根误差评估实际值（通过峰拟合确定）与计算值之间的线性关系，同时进行交叉验证以检测可能的异常值。通过将两种蛋白质作为验证标准并将计算值与峰拟合和 X 射线数据进行比较来验证结果。通过校准曲线分析定量测定人血清白蛋白（HSA）因温度升高和胰高血糖素纤维化而导致的结构变化。通过比较计算的二级结构元素与峰拟合和圆二色性的结果，评估 iPLS 算法的性能和可靠性。不同的二级结构测定方法给出了略有不同的结果，但总体趋势一致。此外，通过比较 SEC-HPLC 和浊度分析与 FTIR 校准曲线的结果，可以将 HSA 聚集物的形成与β-折叠含量的增加联系起来。总之，通过 iPLS 分析定量测定α-螺旋向β-折叠的转变，证明是一种可行且客观的方法，可用于测定蛋白质二级结构的损伤。
