Studies On The Interaction Between Vanillin, Metal Ions And Lysozyme

Protein is one of the most important biomacromolecules. It directly expresses biologic characters and performs a wide variety of physiological functions in the life. Exploring the interaction mechanisms between small molecules and proteins is of current interest in many research areas such as biology, chemistry, medicine and so on. Therefore, further investigating the binding mechanisms between small molecules (especially for those drug molecules) and proteins is worthy to be done. This work not only helps us to clarify the secret of life, understand the distribution, transportion, metabolism, efficacy of small molecules in the body and structural features of protein, but also provides valuable information in drug designing and screening, or disease diagnosis.In this thesis, the small molecules such as vanillin (VAN) and metal ions (Men+) interaction with lysozyme (LYS) were studied using molecule spectroscopy. The paper was consisted of the following parts:In the first part, the properties of VAN, the structures and functions of LYS were introduced briefly. The methods of the research on the interaction between small molecules and proteins were summarized.In the second part, the interaction of VAN with LYS in near physiological buffer condition was investigated by UV-Vis absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The fluorescence analysis showed that VAN could regularly quench the endogenous fluorescence of LYS and the quenching was initiated from compound formation, it was a static quenching mechanism. The quenching constants and apparent binding constants were obtained at different temperature, and every VAN combines to one LYS molecule. It showed that the binding power between VAN and LYS was mainly the hydrogen bond and van der waals force. The binding distance was an area 3.01nm away from LYS based on F?rster's non-radiation energy transfer theory. The conformation of LYS was analyzed by CD spectroscopy and synchronous fluorescence spectroscopy, the results indicated that conformation of LYS changed with the addition of VAN, and theα-helix was more tightly linked. The effect on the activity of LYS after treated by VAN was studied. We found that VAN can increase its enzymic activity.In the third part, the interaction of different Men+ with LYS was investigated. It was found out that these Men+ all can increase the UV absorption and quench the fluorescence intensity of LYS. The quenching constants, binding constants and the number of binding sites were calculated according to the fluorescence effect and quenching equation, and the quenching mechanism belongs to static quenching between Fe³⁺ or Cu²⁺ and LYS. The main sorts of binding force can be known according to the thermodynamic parameters.In the forth part, the effect of the coexistence of VAN and Men+ on the interaction characteristics among VAN, Men+ and LYS were investigated such as the change of quenching mechanism of fluorescence, the number of binding sites, the interaction forces, the conformation of enzyme. The experimental results showed that VAN and Men+ can further quench the fluorescence of LYS and enhance the interaction. The number of binding sites also changed compared with the binary system.