【作者】 孙艳涛；

【导师】 张寒琦；

【作者基本信息】 吉林大学 ， 分析化学， 2009， 博士

【摘要】 本论文主要应用荧光光谱法和紫外-可见光谱法研究了药物测定及药物小分子与生物大分子的相互作用。1、应用不同的计算公式研究了小分子探针和生物大分子相互作用的结合参数和结合位点数。2、通过蛋白内源荧光的猝灭研究了陈皮中两种活性成分同BSA和HSA的相互作用。利用推导的计算结合常数的方程和计算机计算程序得到了橙皮苷和柚皮苷同蛋白作用的结合常数和结合位点数,并与两种常见计算结合常数公式得到的结果进行了比较。结果发现,利用本文所建立的方法计算快速、结果合理。同时,用荧光和吸收光谱法研究了常见四种金属离子对橙皮苷和柚皮苷同两种蛋白相互作用的影响。3、利用荧光法测定了牛蒡子苷。根据DNA与牛蒡子苷共存时的荧光光谱和紫外光谱。建立了一种新的计算结合常数的方法,得到了牛蒡子苷与DNA的结合常数。为了进一步阐明牛蒡子苷与DNA的结合机理,进行了竞争结合实验和熔解实验。研究结果表明,牛蒡子苷与DNA的结合应当是沟区结合。荧光法的结果还表明单链DNA的猝灭效应大于双链DNA,这意味着牛蒡子苷与DNA的作用不属于嵌入结合。4、利用荧光光谱法测定了鬼臼毒素、甘草苷和红景天苷,建立了测定这些化合物的方法。研究并选择了它们的最佳激发和荧光发射参数,详尽地考察了乙醇浓度、温度、放置时间、pH值和共存离子对荧光强度的影响。在最佳实验条件下,鬼臼毒素、甘草苷和红景天苷的荧光强度与其浓度分别在0.04～8.29μg·mL^-1,0.04～8.37μg·mL^-1和0.04～18.03μg·mL^-1范围内呈良好的线性关系,检测下限分别为0.024,0.017和0.019μg·mL^-1。更多还原

【Abstract】 Protein and deoxyribonucleic acid（DNA） are the basic composition of all organisms,and play important roles in all kinds of life activities.DNA is the carrier of genetic materials which acts the main role to the biological evolution,and protein is the expresser of these genetic materials.The investigation of interaction between small drug molecules with biomacromolecules,such as bovine serum albumin（BSA）, human serum albumin（HSA） and DNA,is helpful for us to understand the mechanism of function for drugs and exploit new drugs.1.The structure and function of BSA,HSA and DNA were introduced,and the study on the interaction between drug molecules and biomacromolecules by fluorescence and UV-visible spectrophotometry,especially by the fluorescence quenching,was summarized.2.The interaction of the phenylbutazone and ibuprofen with bovine serum albumin was studied by fluorescence spectrometry.The thermodynamic parameters for the binding were obtained by the fluorescence quenching.The reasonable results were obtained by programmed calculation based on the calculating equation established.Compared with the results obtained by the other calculating method,it is proved that the satisfactory results can be obtained by the proposed method.Based on F（o|¨）rster theory of non-radiation energy transfer,the binding distance r of the bovine serum albumin with phenylbutazone and ibuprofen was 1.57 and 1.54 nm, respectively.In addition,the effect of common ions on the binding of the drugs and bovine serum albumin was also examined.The emission intensity of ethidium bromide（EB） in the presence of DNA can be greatly enhanced.The interaction of the DNA with EB was studied and binding parameters,including binding constant and binding sites,were also obtained. 3.Protein has intrinsic fluorescence and can be used as a donor to react with an acceptor.Because of forming of the coordination compound,the protein intrinsic fluorescence can be quenched.Because the bound and unbound drug concentration is difficult to be know during the experiment,the unbound drug concentration is often replaced by the total drug concentration in calculating the binding parameters.The interaction of the two main active components in fruit of Citrus aurantium L.with BSA and HSA was studied by the quenching of intrinsic fluorescence protein.The binding constants and sites of the hesperidin and naringin to proteins were obtained by an improved calculating method and the programmed calculation process.For comparison,other two calculation methods also were applied.The reasonable results were rapidly obtained by programmed calculation process.The△H,△S and△G obtained indicate that the hydrophobic force plays a major role in the interaction of drugs and BSA.The main acting force between naringin and HSA is electrostatic force.However,van der Waals or hydrogen bond plays a major role in the interaction of hesperidin with HSA.Based on the F（o|¨）rster’s theory,the binding average distance,r between the protein and drug was evaluated and found to be less than 3 nm.At the same time,the interaction of hesperidin and naringin with BSA and HSA in the presence of four common metal ions was studied by fluorescence and absorption spectrometry.4.Arctiin was determined by fluorescence method in the various different experiment conditions.At the optimal conditions,the good linear response was observed in the arctiin concentration range from 0.05～10.69μg·mL^-1.The interaction between arctiin and DNA was also studied by fluorescence and ultraviolet absorption spectrometry.The biomolecule is possessed of a fluorophore and many binding sites, and the drug molecules are quencher in most studies of biomolecule binding with the small molecule.However,in the work DNA is quencher in the interaction of the DNA with EB.A new method for calculating the binding constants and binding sites was developed and the comparison of the propsed method with the conventional method was made.The reasonable results were obtained by the proposed method.The competitive binding and melting temperature（T_m） were carried out to investigate binding mechanism.In addition,the binding characteristics of arctiin and DNA were further studied by fluorescence method and experimental results showed that the fluorescence of arctiin was quenched easier by single stranded DNA（ssDNA） than that by double stranded DNA（dsDNA）,which indicated that the interaction between fluoroquinolone and dsDNA does not belong to intercalation.5.The podophyllotoxin,liquiritin and salidroside were determined by fluorescence spectrometry.The effects of ethanol concentration,temperature,placing time,pH values and foreign substances on the determination of the drugs were examined.At optimal conditions,the good linear responses were observed in the concentration range from 0.04～8.29μg·mL^-1,0.04～8.37μg·mL^-1 and 0.04～18.03μg·mL^-1,for liquiritin and 0.9996 for salidroside and detection limits were 0.024μg·mL^-1,0.017μg·mL^-1 and 0.020μg·mL^-1,respectively.更多还原