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Infrared Absorption Intensity Analysis as a New Tool for Investigation of Salt Effect on Proteins
Heng Li, Yan-yan Xu, Yu-xiang Weng*
Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China
Abstract:
The native protein structures in buffer solution are maintained by the electrostatic force as well as the hydrophobic force, salt ions play an important role in maintaining the protein native structures, and their effect on the protein stability has attracted tremendous interests. Infrared spectroscopy has been generally used in molecular structure analysis due to its fingerprint resolution for different species including macromolecules as proteins. However spectral intensities have received much less attention than the vibrational frequencies. Here we report that the spectral intensities of protein amide I band, the finger prints for the protein secondary structures, are very sensitive to the local electric field known as Onsager reaction field caused by salt ions. IR absorbance thermal titrations have been conducted for a series of samples including simple water soluble amino acids, water soluble monomeric protein cytochrome c and dimeric protein DsbC and its single-site mutant G49R. We found that at lower temperature range (10-20 oC), there exists a thermal activated salting-in process, where the IR intensity increases with a rise in the temperature, corresponding to the ions binding of the hydrophobic surface of protein. This process is absent for the amino acids. When further raising the temperature, the IR intensity decreases, this is interpreted as the thermal activated breaking of the ion-protein surface binding. Applying Van't Hoff plot to the thermal titration curves, the thermodynamic parameters such as ?H and ?S for salting-in and ion unbinding processes can be derived for various protein secondary structural components, revealing quantitatively the extent of hydrophobic interaction as well as the strength of the ion-protein binding.
Key words:  Infrared intensity, Salt effect, Fourier transform infrared spectroscopy, Secondary structure, Thermodynamic constant
FundProject:
Infrared Absorption Intensity Analysis as a New Tool for Investigation of Salt Effect on Proteins
李恒, 徐妍妍, 翁羽翔*
中国科学院物理研究所软物质物理实验室,北京凝聚态物理国家实验室,北京100080
摘要:
通过观测红外光谱的强度变化研究了在不同温度下盐离子对谷氨酸和三种水溶性蛋白质(细胞色素C、DsbC和DsbC-G49R)的影响. 证实了Onsager反应场理论. 区分并且定义了随温度变化的蛋白质盐效应盐溶过程和解离过程. 同时利用Van't Hoff关系式对实验结果进行线性拟合,获得了蛋白质及其二级结构在上述两个过程中的热力学常数.
关键词:  红外吸收强度,盐效应,傅立叶红外光谱,二级结构,热力学常数
DOI:10.1088/1674-0068/22/06/556-562
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