双背景法扣除溶剂水峰及蛋白质热变性的研究

研究蛋白质水溶液的红外光谱(IR)谱时，由于溶剂水的强吸收与蛋白质的吸收峰会发生严重重叠，极大地干扰对蛋白质吸收峰的识别、定性、定量和结构分析。尝试利用杂化光谱法扣除溶剂水峰。采用双背景方法，用空白ATR晶体（背景样品1）与ATR水层（背景样品2）合成了单光束杂化背景谱，通过控制对背景样品1和背景样品2的扫描次数，合成的单光束杂化背景谱中水的信号强度可任意调节，成功实现了牛血清白蛋白（BSA）水溶液中溶剂峰的在线扣除。与光谱差减技术比较，杂化光谱扣除法具有显著的优势：水3 400 cm-1峰强度接近于零，1 700～1 800 cm-1区间得到近似平滑直线，水1 640 cm-1峰彻底扣除，观察到了高质量的酰胺I带吸收峰。将杂化谱法获得BSA红外光谱二次微分，得到蛋白质二级结构大量信息，与文献报道高度吻合。杂化光谱法应用到蛋白质热变性研究中，成功获得没有溶剂水峰干扰的蛋白质红外光谱，变性前后，酰胺Ⅰ带光谱发生明显改变，峰形变化显著，吸收峰往低波数方向移动，吸收强度显著减小。杂化光谱ATR法扣除溶剂水峰简单、易操作、效果令人满意。

The Removal of Water Peaks From IR Spectra of BSA Aqueous Solution by Two ATR Background Samples and Thermal Behavior of Proteins

In the infrared spectroscopic study of aqueous protein solution, the characteristic peaks of solvent water and protein overlap partially or completely, which seriously affects the quantitative and structural analysis of protein. In this study, the solvent single beam spectra with arbitrary intensity were successfully synthesized by using two background samples. Therefore the solvent peaks in protein solution can be removed completely. The results of the hybrid spectrum were compared with those of the subtraction spectrum, and it was found that the hybrid spectrum has obvious advantages. The secondary structure information obtained from the second derivative of the hybrid spectra of BSA is in good agreement with that reported in the literature. Hybrid spectroscopy has also been used to study the thermal behavior of aqueous protein solutions. Without the interference of water peaks, the resolution of the amide Ⅰ and amide Ⅱ bands become easier. As the temperature increased, the peak position of the protein shifted. After high-temperature treatment, the bioactivity of BSA is lost.