| This study is two groups of protein-protein interaction from egg white and its influence on functional properties. During the study, lysozyme and ovalbumin, Ovotransferrin and ovalbumin from egg white is selected. A series of experiments was undertaken to study Several aspects including Qualitative analysis, the secondary structure changing, the changing of protein nature function, antimicrobial synergy determination and kinetic constants of PPI. The main findings are as follows:(1) The experiment to Qualitative determination of the interaction between two group proteins was undertaken by Cyclic voltammetry polarization and dual polarization interferometer. During Cyclic voltammetry experiments, electrode surface was Modified, and the protein was fixed on the electrode surface sequently. Both of ovalbumin and ovotransferrin, ovalbumin and lysozyme did found interactions exist varying the electrical signal. Dualpolarization polarization interferometer method can reflect the process changing real-time of protein interaction of two group proteins. After the reaction,the result of Dualpolarization polarization interferometer between ovalbumin and lysozyme is that thickness of the chip surface increases3.4874nm, the quality increase1.625027ng/mm2, the density increase0.46597g/cm3. The result of Dualpolarization polarization interferometer between ovalbumin and Ovotransferrin is that thickness of the chip Surface increases4.783581nm, the quality increase1.48436ng/mm2, the density increase0.310303g/cm3.(2) Through the above research, further study to determine the structure changing after protein-protein interactions were undertaken. Using infrared spectroscopy and Circular Dichroism methods determinate protein secondary structure of two group proteins. After48h interactions, experimental results show that the secondary structure of proteins a helix and β fold symbolize orderly structure decreased, disordered random coil structure was increased.(3) lysozyme and Ovotransferrin are important antimicrobial proteins in egg white, in order to further explore the impact on the antibacterial properties of protein-protein interactions, on the basis of these studies, we use an inverted microscope fluorescence and flow cytometry for further study of its antibacterial properties. In order to determine the best time of antibacterial protein, first, the bacterial growth curve was determinated under protein solution, the result shows that antibacterial effect is most obvious when3h later. Use inverted fluorescence microscope observed the initial state of the bacteria, compared with microscope at3h cultured in antimicrobial proteins, we can found that interaction between proteins have synergy effects on bacteriostatic action. The result of Fluorescence flow cytometry consistent with the inverted microscope.(4) Based on these studies, quantitative determination of protein-protein interactions by surface plasmon resonance and isothermal titration calorimetry was undertaken to obtain the kinetic constants. The result show that KA value between Ovalbumin and lysozyme is498, KA value between Ovalbumin and Ovotransferrin is152which is slightly lower than the value of Ovalbumin and lysozyme. Both the two group proteins interaction way are fast binding and fast dissociation. This also means that the way of interaction of two group proteins are strong reversibility. At the same time, we can know bonding type of interaction is hydrophobic interactions by KA value. Isothermal titration calorimetry donot occurs intense heat changing, we conclude that the interaction may be caused by reversible. Meanwhile there have a serious baseline drift phenomenon, this may be related to the nature of the protein itself or the interaction product.(5) At last, Lywallzyme activity and transferrin activity were determinated, the changing of Lywallzyme activity of Lysozyme is not obvious. But transferrin activity varied with different temperatures, it increased under4℃and22℃, when it decreased under37℃. |
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