Study On The Interactions Between Lavonoids And Their Phosphorylated Productions And The Purification Of HSP70 From Human's Lung Cancer Tissues

Nucleic acids as proteins are all big biologic information molecular, it play an important role in the life process such as the growth, reproduce, heredity and aberrance of the organisms. Many small molecular can interact with nucleic acid, make it rupture and affect the gene's function of adjust, control and expression. So more scientists pay attention to the study of the interaction of small molecular and nucleic acids, and it is very important for researchers to study the functional mechanism of many anti-tumor medicines and develop new factionlized medicine to surmount many difficult diseases. Lavonoid compounds are polyphenol compound. The research discovers that flavones compounds have many potential pharmaceutical usages, such as their anti-oxidation, anti-virus and anti-tumor effects.The interactions between 7-hydroxyflavone, chrysin and their phosphorylated productions and calf thymus DNA(ctDNA) were investigated by fluorimetry spectra and UV spectra in the first part of paper. The result showed that 7-hydroxyflavone, chrysin and their phosphorylated productions could all bind to ctDNA at some range of the concentration, but the phosphorylated flavonoid showed higher binding affinity with ctDNA than 7-hydroxyflavone and chrysin did. The binding constants of 7-hydroxyflavones, chrysin, their phosphorylated productions with ctDNA were determined according the formula, the binding constants was 0.865×105 L/mol, 1.40×105 L /mol, 1.52×105 L/mol and 9.32×105 L/mol, respectively. The quenching constants of addition of 7-hydroxyflavones,chrysin ,their phosphorylated productions to the ctDNA-EB system at different temperature were also determined by Stern-Volmer equation, they were K25℃=601 L /mol, K37℃=547 L/mol; K25℃=2576 L/mol, K37℃=2238 L/mol; K25℃=1381 L/mol, K37℃=1253 L/mol; K25℃=30860 L/mol, K37℃=27760 L /mol, respectively. Experiments demonstrated that the higher the temperature was, the lower the slop of quenching curve of ctDNA-EB was in presence of different amounts of 7-hydroxy flavones, chrysin and their phosphorylated productions. It was confirmed that the bindings of ctDNA with 7-hydroxy flavones, chrysin and their phosphorylated productions were a single static quenching process, and 7-hydroxy flavone, chrysin and their phosphorylated productions could form non-covalent complexes with ctDNA. According to Scatchard equation, when the 7-hydroxy flavones, chrysin and their phosphorylated productions were present, the intrinsic association constants K to a site of ctDNA were 2.07×104 L /moK2.94×104 L/mol, 3.19×104 L/mol, 5.31×104 L/mol respectively and the number of binding sites per nucleic acid phosphate n were 0.54, 0.37, 0.45, 0.29 respectively. Besides, it could judge that 7-hydroxy flavone, chrysin and their phosphorylated productions could bind to ctDNA by groove mode according to the experiment data.It could be concluded from the experiments that:1. The binding constant of chrysin with ctDNA was bigger than the 7-hydroxy flavone did, which showed that the binding of flavones with ctDNA strengthen with the increase of hydroxy. The binding constant of diethyl flavone-5,7-yl phosphate with ctDNA was bigger than diethyl flavone-7-yl phosphate did, which showed that the binding affinity between phosphorylated flavonoids and ctDNA was related to the amount of phosphoryl.2. Diethyl flavone-7-yl phosphate and diethyl flavone-5,7-yl phosphate showed higher binding affinity with ctDNA than 7-hydroxy flavone and chrysin did.3. The interactions of diethyl flavone-7-yl phosphate and diethyl flavone-5'7-yl phosphate with ctDNA are very important in understanding their antitumor activities and the mechanism of their antitumor activities.In the second part of paper, a method of the purification of the HSP70 from the human's lung cancer tissues has been established. The HSP70 was separated successively by fast protein liquid chromatography (FPLC) system. The procedures included ConA-Sepharose columnand DEAE ino-exchange chromatography. The molecular weight and the identity of the puried HSP70 were confirmed by SDS-PAGE and Western-blot, and the puried HSP70 was quantified by Bradford standard curve method. The result showed that a protein with a molecular weight of about 70000 obtained was confirmed to be HSP70; The final protein recovery was 53.2μg per lg lung cancer tissue(wet weight). It could conclude that HSP70 with higher purity was obtained using FPLC system with ConA-Sepharose column, and the method is very useful to study further the reactions between flavones and HSP70.