The Studies On The Interaction Between Genetic Recombination Angiogenesis Inhibitor Kringle5and Small Ligands

At present, using antiangiogenesis drugs to restrain vessel rebirth has been an important method for curing tumor and other angiogenesis-depend diseases. Among many antiangiogenesis factors, Kringle5, the fifth kringle of plasminogen, is the one which has the strongest activity in restraining the growth of vascular endothelial cell, less toxic and side effect, higher specificity and stable quality so far. Therefore, Kringle5has a great potential and application value in curing tumor and other angiogenesis-depended diseases. By combining the experimental methods of fluorescence spectra and affinity chromatograph, in addition of theoretical forecasting methods of molecular docking, thermodynamics and kinetics simulation, this work studied the denaturants-induced structure change procedure of antiangiogenesis Kringle5, and the interaction as well as recognition between Kringle5and some ligands, which provided some new vision on illustrating the effect mechanism of Kringle5and regulating its pharmacology and efficacy.1. Comprehensively using fluorescence spectra, fluorescence phase diagram, fluorescent probe and fluorescence quenching, the denaturants-induced structure change of antiangiogenesis Kringle5during unfolding/refolding procedures were characterized. The result indicates that there is no folding intermediate in both unfolding and refolding of Kringle5, which means that both the GuHCl-and urea-induced unfolding/refolding processes follow the "two-state model". During the GuHCl-and urea-induced unfolding of Kringle5, the hydrophobic region originally existing in the internal of protein molecules would expose to the surface of the molecules, the proportion of fluorophores exposing to the surface of Kringle5and quenched by KI in all the fluorophores of Kringle5increased from59.2%to85.5%and from59.2%to83.3%, respectively; During the GuHCl-and urea-induced refolding of Kringle5, the hydrophobic region reformed in the internal of protein molecule, and the proportion of fluorophores exposing to the surface of Kringle5and quenched by KI in all the fluorophores of Kringle5decreased from88.3%to60.2%and from84.1%to61.1% respectively. All the results obtained from this part of the work indicate that the unfolding and refolding procedures of Kringle5induced by no matter GuHCl or urea are reversible processes.2. By using spectroscopy, molecular docking technology in addition of thermodynamics and kinetics simulation, this work studied the recognition and the interaction between Kringle5and five ligands (L-lysine, EACA,7-AHA, AMCHA and Benzylamine) at different temperatures in terms of their quenching types, association constants, binding sites information, types of interact force, converages as well as the distribution and docking of amino acid residues within Kringle5molecule and function groups within ligands. The result showed that, firstly, all the Kq values obtained from the binding procedures of Kringle5and five lignads were all larger than2×1010L·mol-1s-1, at the same time all the Ksv values decreased with a rise of experiment temperature, which indicated that all the binding procedures are static quenching process caused by the formation of non-fluorescigenic complex between Kringle5and five ligands; secondly, both binding constants and binding sites number of Kringle5and five ligands at different temperatures were determined, and it can be found that the interaction strength between Kringle5and five ligands from strong to weak are:AMCHA>EACA>Benzylamine>7-AHA>L-lysine; thirdly, it can be obtained that△G,△H and AS during the binding process were all negative, which indicated all the binding procedure between Kringle5and five ligands are spontaneous, exothermic reaction taking hydrogen bond and van der waals force as main force; fourthly, by using Forster non-radiative energy transfer theory, it can be found that all the five ligands had non-radiative energy transfer effect with Kringle5and the distances between Kringle5to ligands were close to each other; fifthly, through molecular docking, it can be discovered that there are following combination differences between five ligands and Kringle5:AMCHA and Kringle5were combined with two hydrogen bonds and a cation-7t interaction force, EACA and Kringle5were also combined with two hydrogen bonds and a cation-π interaction force, Benzylamine and Kringle5were combined with a hydrogen bond and a π conjugation interaction and two cation-π interaction forces,7-AHA and Kringle5were combined with one hydrogen bond and a cation-π interaction, L-lysine and Kringle5were combined with a hydrogen bond and a cation-π interaction. Lastly, by comparing the differences between five ligands’ structure, we try to discuss and find out the ligand type which is more suitable to interact with Kringle5.3. By using affinity chromatography, Kringle5was bonded to the silica gel base material through diazo reaction, and a method of studying the interaction of Kringle5and ligands with affinity chromatography was preliminarily established. The result shows that, the separation condition was good after Kringle5was bonded to the silica gel base material; the order of magnitude and the pecking order of association constants between Kringle5and five ligands obtained by affinity chromatography mentioned above were as the same as the results obtained from spectroscopy in chapter two. The results obtained in this part of work indicated that the affinity chromatography method mentioned can be used for studying the interaction between Kringle5and ligands, and laid a good foundation and direction for further study.