Spectroscopic Studied On The Interaction Between LYSO With Flavonoid Compounds

Protein is an essential life active substances in the living organisms and plays an important part in life process. Lysozyme is an alkaline protein and is widely distributed in organisms. It is commonly used as a model system to study the protein conformation. Small flavoind drug molecules are capable of a wide range of pharmacological activities, such as antioxidant, anti-virus, anti-cancer, anti-tumor, anti-inflammatory and anti-bacterial, etc. Studies on the interaction of drugs and biomacromolecules have very important reference value in the field of the target molecules for drugs, new drugs design, anti-cancer drugs in the pharmacological and toxicological research.In this thesis, fluorescence, time-resolved fluorescence, UV absorption, synchronous fluorescence spectroscopy and circular dichroism (CD) spectrometries are employed to study the interaction mechanism between lysozyme (LYSO) and flavonoid compounds, such as baicalein, chrysin, myricetin. Finally, the fluorescence quenching mechanism of interaction between drugs and protein, it's binding constant, binding sites, thermodynamic binding parameters, and combination distance, the impact on the protein conformational changed and activity of enzyme by flavonoid compounds, the effect of solution pH value, the metal ions and urea are demonstrated. This thesis is written in four sections.In the first section, we mainly summarize the significance of study of interaction between protein and small molecules and the research methods, and give the research content of the thesis.In the second section, we study the mechanism between lysozyme with baicalein using fluorescence under simulative physiologicl conditions. The quenching mechanism of baicalein and lysozyme is static quenching in lower concentration of baicalein, and complex quenching in higher concentration. Lifetime measured by time-resolved fluorescence spectroscopy proves that the fluorescence quenching is mainly static quenching, the binding constants is 1.636×106 L/mol in the lower concentration of baicalein. The enthalpy changes (ΔH0) and entropy changes (ΔS0) indicated are 67.84 kJ/mol and 338 J/mol·K, respectively. The results indicate that the dominant intermolecular forces is hydrophobic force. According to F?rster non-radiation energy transfer theory, the average binding distance r is 4.19nm between donor and acceptor. In addition , the alterations of lysozyme conformation in the presence of baicalein are confirmed by the evidences from synchronous fluorescence, UV absorption and circular dichroism spectroscopy. The results show that the content ofα-helicity structures of lysozyme increase in the presence of baicalein.In the third section, the fluorescence quenching mechanism between lysozyme and chrysin is studied by steady-state and time-resolved fluorescence spectroscopy. The quenching mechanism of lysozyme by chrysin is mainly static quenching. Under simulative physiological condition, there is a single primary chrysin binding site on lysozyme with a binding constants of 1.298×105 L/mol. The enthalpy changes (ΔH0) and entropy changes (ΔS0) are -68.64 kJ/mol and -123.96 J/mol·K, respectively. The result showes that the vander Waals force and hydrogen bond are the predominant forces in the binding process. According to F?rster theory, the distance r is 2.47nm between donor and acceptor. The results of synchronous fluorescence, UV absorption and circular dichroism spectroscopy show that the conformation of lysozyme is changed.In the fourth section, the interaction of myricetin with lysozyme is studied by using steady-state and time-resolved fluorescence spectroscopy. The results reveal that myricetin cause the fluorescence quenching of lysozyme through a static quenching procedure. The quenching constant was obtained at three different temperatrue. The binding power between myricetin and lysozyme is mainly the vander Waals force and hydrogen bond according to the thermodynamic. The distance r is 4.77 nm between donor and acceptor based on F?rster's non-radiation energy transfer mechanism. It is proved that myricetin has effection on the second conformation of lysozyme by synchronous fluorescence, UV absorption and circular dichroism spectroscopy, and the content ofα-helicity structures of lysozyme decrease.