The Studies Of Noncovalent Interactions Of Protein Based On SPR

As the most important material constituting of living organisms, protein has extensive and diverse biological functions in the life activities. Researchs on the interactions between biological macromolecules, small molecular compounds, metal ion and protein, have very important theoretical and practical significance in revealling the law of life activities and designing new drugs. Now there are a few experimental methods to study the molecular interactions, among which surface plasmon resonance (SPR) and fluorescence spectrometry are used widely. In this thesis, the interactions between proteins and other molecules had been comprehensively investigated by using multiple experimental methods as SPR, fluorescence spectrometry and so on. In the meanwhile, SPR and fluorescence spectrometry had been discussed in term of the experimental methodology.The main experimental work was described as follow.1) The interaction between a cationic porphyrin and bovine serum albumin (BSA) was studied by using SPR spectroscopy. which was combined with fluorescence quenching method and cyclic voltammetric method to confirm the binding kinetic results. The equilibrium association constants obtained from the above three methods were6.49x104M-1.10×105M-1and1.21×105M-1respectively. By comparing the three binding constants, the factors influencing the SPR and fluorescence quenching experiments had been discussed.2) The interaction between a cationic porphyrin and recombinant human prion protein (rPrPc) was comprehensively studied by using SPR. fluorescence, resonance light scattering (RLS) and circular dichroism (CD) spectroscopy. The experimental results showed that the interaction between the cationic porphyrin and rPrPc was pH dependent. The equilibrium association constants obtained from SPR spectroscopy were4.12×103M-1at pH4.0.1.74×105M-1at pH6.0, and5.98×105M-1at pH7.0respectively. The binding constants at298K obtained from the fluorescence quenching method were7.29×104M-1at pH4.0. and1.46×105M-1at pH6.0. respectively. The themodynamic parameters△H,△S and△G were calculated and the results indicated that hydrogen bonds and van der Waals interactions played a major role in the binding reaction. The results of the RLS experiment indicated that the cationic porphyrin could inhibit the aggregation of rPrPc. The results of the CD experiment indicated that porphyrin had no impact on the secondary structure of rPrPc at pH4.0and pH6.0. The results of the thermal denaturation experiment indicated that porphyrin could improve the thermal stability of rPrPc at pH6.0.3) The interaction between rPrPc and the metal ions, such as Cu2+, Zn2+, Ni2+, Mn2+, Ca2+and Mg2+, was comprehensively studied by using SPR and fluorescence spectroscopy. In the SPR experiment, it was showed that rPrPc had selective metal-binding ability, as the binding affinity for Cu2+was much higher than that of the other metal ions. rPrPc also showed binding affinity for the metal ions of Zn2+, Ni2+and Mn2+in the order of Zn2+> Ni2+> Mn2+, but little binding affinity for Ca2+and Mg2+. And the interactions between rPrPc and the metal ions were pH dependent, as the binding affinities at pH7.2and pH6.0were much higer than those at pH5.0and pH4.0. In the fluorescence experiment, the experimental results also showed that the binding affinity for Cu2+was much higher than that of the other metal ions. And the interaction between rPrPc and Cu2+was pH dependent, as the binding affinity at pH6.0was much higer than those at pH5.0and pH4.0. It was also found that the fluorescence of rPrPc could be enhanced by Zn24) Through the widely used EDC/NHS cross-linking reaction, Escherichia coli was immobilized onto the preformed MPA self-assembled monolayer (SAM) as the recognition element, to construct a SPR biosensor for phage T4detection. The bacteria immobilization was verified efficiently through the electrochemical measurements and fluorescence microscopy observations. The results of SPR experiment indicated that the phage adsorption process should be a simple first-order reaction. The specific adsorption was much stronger than the non-specific adsorption of phage T4binding to the biosensor surface, and the latter could be neglected. Finally, the experimental results indicated that tthe phage adsorption process should be an irreversible process, and values of the SPR signal after elution represented the amount of phage T4binding to the biosensor surface.