Investigation Of The Interaction Of ADR And ADR-Tf With DNA By Spectroscopy And Electroanalytic Chemistry Methods

Adriamycin (ADR) is an widely used clinical anthracycline antibiotic. It can intercalate in DNA base pairs and influence the replication of DNA then has an obvious curative effect on kinds of tumor while concomitant by its side effect. ADR was combined with Tf as oriented drug and became more efficient and with few side effects by the excessive expression of transferrin receptor (TfR) on tumor cell surface which can be interact with transferrrin (Tf) exclusively. In order to reveal the characteristic of the interaction between ADR-Tf and DNA, this thesis use spectroscopy and electrochemistry methods to discuss and compare the interaction of ADR, ADR-Tf and DNA.The binding reaction between ADR and Tf was investigated by fluorescence and ultraviolet visible absorption(UV-vis) spectroscopy methods. It was confirmed that the mechanism of the fluorescence quenching of Tf induced by ADR was static quenching. The equilibrium constant K=0.45~10.3×107 and the number of binding sites n=1.09~1.37 were determined at different concentration of ADR and Tf by fluorescence quenching method. The binding distance r=1.94nm was also obtained according to Forster theory of non-radiation energy transfer with 6.9 X 10~-5mol .L~-1ADR and 5.6×10~-6mol.L~-1Tf.The interaction of ADR, ADR-Tf and DNA was discussed by spectroscopy methods and the result showed that ternary compound of ADR-Tf-DNA was formed. The interaction constant K_ADR-DNA=1.10×10~6, K_ADR-Tf-DNA=1.95×10~6 and binding sites n_ADR-DNA=4.31,n_ADR-Tf-DNA=2.65 of ADR, ADR-Tf and DNA was obtained respectively using EB as fluorescence probe by Scatchard equation. Comparing to the interaction between EB and DNA, the conclusion was made that ADR combines DNA in inlaid way while ADR-Tf complex combines DNA in not only inlaid way but also electrostatic way. Furthermore, by using ADR as fluorescence probe to study the interaction of ADR,ADR-Tf and DNA, the equilibrium constant K_ADR-DNA=1.43× 10~6,K_ADR-Tf-DNA=1.44×10~6 and the number of binding sites n_ADR-DNA=4.53, n_ADR-Tf-DNA=3.40 of ADR, ADR-Tf and DNA were determined. The mechanism ofthe interaction of ADR, ADR-Tf and DNA was the same as that obtained by using EB as fluorescence probe. The influence of pH, ion strength on the interaction of ADR^ ADR-Tf complex and DNA was investigated and showed that higher temperature, strong acidic and alkaline condition, the existence of NaCl go against the interaction of ADR, ADR-Tf complex and DNA.The electrochemical behavior of ADR and ADR-Tf on bare gold electrode was studied by cyclic voltammetry and differential pulse voltammetry methods. Two reductive peaks of cyclic voltammetry curve of ADR at +0.17V and -0.64V were obtained in phosphatic buffer solution(pH=7.4) during the scan range from 0.6V to -1.0V while two corresponding oxidative peaks at +0.57V and -0.61V was obtained scanning reverse; the redox peak of ADR-Tf has the same potenal with ADR while its current was a little lower. And two reductive peaks of differential voltammetry curve of ADR at +0.25V and -0.60V were also obtained during the scan range from 0.6V to -0.1V and from -0.4V~-1.0V respectively; the peak potential of ADR-Tf was the same as that of ADR, the peak current was in some sort reduced. After comparison, we select the differential pulse voltammetry peaks at -0.60V of ADR as research object to study the influence of DNA on them and the results showed that the interaction of ADR-Tf and DNA is weaker than that of ADR and DNA. The influence of pH and ion strength on the interaction of ADR^ ADR-Tf and DNA were discussed, proving that strong acidic, alkaline condition and the existence of NaCl go against the interaction of ADR, ADR-Tf and DNA. Cyclic voltrammetry was used to study further the interaction of ADR, ADR-Tf and DNA, showing that DNA denaturalization can also be induced by ADR-Tf but the extent of DNA denaturalization is smaller than that induced by ADR and proving that the mode of the interaction of ADR-Tf and DNA was accordant to that provided by spectroscopy experiments.In conclusion, the mechanism of ADR-Tf and DNA is similar to that of ADR and DNA that ADR-Tf can also intercalate in DNA base pairs and change the conformation of DNA. But there is also electrostatic interaction between ADR-Tf andDNA. And because Tf is a biologic macromolecule then has the binding sites of ADR-Tf and DNA is lesser and the interaction is weaker than those of ADR and DNA.