The Application Of Raman Ratio Spectrum In Aqueous Solution

Raman spectroscopy is often used to study the structure of molecules in solution. Because of the intermolecular hydrogen bands, proteins have their special structures. Since the function of protein is decided by the structure, it is important to study the secondary structure. Protein only shows biological activity in aqueous solution. As a key to analyze the secondary structure of protein, amide A band overlaps with the O-H stretching of water seriously. To solve the problem, this paper consists of two parts of study. In the 1st study, we presented a new analytical method of Raman ratio spectrum, which can extract the spectrum we usually could not get. Such as the amide A bands of proteins in water and the process of thermal denaturation of lysozyme. In the 2nd study, the non-uniform mixing of aqueous acetone was analyzed by Raman ratio spectrum. Specific studies are as follows:(1) A mathematical simulation of Raman ratio spectrum was employed. The simulated spectrum of water added a weak gaussian peak to simulate the spectrum of aqueous protein. The spectrum of amide A band is hard to extract. The Raman ratio spectrum was calculated, a single band was observed, both the Raman shift and the FWHF of this band agreed well with these of the simulated band. The general applicability of Raman ratio spectrum was checked in the whole O-H stretching regions.(2) The amide A band of lysozyme and a-Chymotrypsin in water were obtained by Raman ratio spectrum. Two kinds of protein, lysozyme and a-Chymotrypsin with different secondary structures were employed. In the region of amide I and amide A band, a-helix locates in a lower and higher wavenumber than β-sheet, respectively. The amide I band of lysozyme located at a lower wavenumber than that of a-chymotrypsin, which demonstrated a-helix structure of lysozyme in water were much more than that of a-chymotrypsin in water. The relationship agreed with the Raman spectra of solid samples. In the Raman ratio spectra, it was found that the frequency of the amide A band of the aqueous lysozyme with more a-helix was larger than that of the a-chymotrypsin with more β-sheet. The relationship also agreed with the Raman spectra of solid samples. The amide A bands of them in water were extracted from Raman ratio spectra.(3)The process of thermal denaturation of lysozyme was detected from Raman ratio spectrum. The Raman ratio spectra were recorded in the amide A region for aqueous lysozyme from 60℃ to 84℃. These temperature dependent frequencies were fitted with a sigmoid function. The denaturation temperature was determined to be-75 ℃. The temperature agreed with that from other technologies. These results demonstrated the Raman ratio spectra could be employed to study the amide A modes of proteins in water.(4)The study of the micro structure of aqueous acetone by Raman ratio spectrum. Acetone has the simplest structure of ketones, it is often seen in the lab and used as solvent. From our eyes, it seems that the uniform mixing of acetone and water can be observed. However, at the molecular level, whether acetone and water mixed over is the issue that people have been discussing. The solutions with different concentrations of acetone and water were studied by Raman Spectroscopy and Raman ratio spectrum. In order to ease the study of acetone-water system in the region of O-H stretching, deuterated Acetone which has similar physical properties as acetone was used instead of acetone, it was mixed with water to gain the solution. The Raman spectrum of hydration water around acetone was gained with the use of Raman raiio spectrum. After we gained the Raman spectrum of hydration water, the Gauss calculation was used to analyze the micro structure of water-acetone system with different mole fraction of them. The non-uniform mixing of aqueous acetone was observed and the special hydrogen bond of C-H......O is still studying.