Jalali Fahimeh, Rasaee Gelareh
Department of Chemistry, Razi University, 67346 Kermanshah, Iran.
Department of Chemistry, Razi University, 67346 Kermanshah, Iran.
Int J Biol Macromol. 2015 Nov;81:427-34. doi: 10.1016/j.ijbiomac.2015.08.025. Epub 2015 Aug 14.
Possible interaction between immunosuppressive drug, azathioprine, and calf thymus DNA was explored by cyclic voltammetry, spectrophotometry, competitive spectrofluorimetry, circular dichroism spectroscopy (CD), and viscosity measurements. Cyclic voltammetry showed negative shift in the reduction peak of azathioprine in the presence of DNA, and large decrease in peak current, referring to the predominance of electrostatic forces. The binding constant was calculated to be 1.22×10(3)M(-1). Absorption hyperchromism without shift in wavelength was observed when DNA was added to azathioprine solution. Competitive fluorescence experiments were conducted by using Hoechst 33258 and methylene blue as probes for minor groove and intercalation binding modes, respectively. The studies showed that azathioprine could release Hoechst 33258, while negligible effect was detected in the case of methylene blue. Stern-Volmer quenching constant (KSV) and complex formation constant (Kf) were obtained from the fluorescence measurements to be 7.6×10(3)M(-1) and 7.76×10(4)M(-1), respectively, at 298K. Enthalpy and entropy changes during the interaction between azathioprine and DNA were calculated from Van't Hoff plot (ΔH=-20.2kJmol(-1); ΔS=26.11Jmol(-1)K(-1) at 298K) which showed an exothermic spontaneous reaction, and involvement of electrostatic forces in the complex formation with DNA. Moreover, circular dichroism studies revealed that azathioprine induced detectable changes in the negative band of DNA spectrum. Viscosity of DNA solution decreased in the presence of azathioprine, showed a non-intercalative mode of interaction. Finally, molecular docking calculations showed that in the lowest energy level of drug-DNA complex, azathioprine approaches the minor grooves of DNA.
通过循环伏安法、分光光度法、竞争荧光光谱法、圆二色光谱法（CD）和粘度测量，研究了免疫抑制药物硫唑嘌呤与小牛胸腺DNA之间可能的相互作用。循环伏安法表明，在DNA存在下，硫唑嘌呤的还原峰出现负移，且峰电流大幅降低，这表明静电力占主导地位。计算得出结合常数为1.22×10³M⁻¹。当向硫唑嘌呤溶液中加入DNA时，观察到吸收增色现象且波长无偏移。分别使用Hoechst 33258和亚甲基蓝作为小沟结合模式和嵌入结合模式的探针进行竞争荧光实验。研究表明，硫唑嘌呤可释放Hoechst 33258，而对亚甲基蓝的影响可忽略不计。在298K下，通过荧光测量得到的Stern-Volmer猝灭常数（KSV）和络合物形成常数（Kf）分别为7.6×10³M⁻¹和7.76×10⁴M⁻¹。根据范特霍夫图计算出硫唑嘌呤与DNA相互作用过程中的焓变和熵变（298K时，ΔH = -20.2kJmol⁻¹；ΔS = 26.11Jmol⁻¹K⁻¹），结果表明这是一个放热的自发反应，且静电力参与了与DNA形成络合物的过程。此外，圆二色性研究表明，硫唑嘌呤可引起DNA光谱负带的可检测变化。在硫唑嘌呤存在下，DNA溶液的粘度降低，表明其相互作用模式为非嵌入模式。最后，分子对接计算表明，在药物-DNA络合物的最低能量水平下，硫唑嘌呤靠近DNA的小沟。
