Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad 9175687119, Iran.
Molecules. 2013 Jan 11;18(1):789-813. doi: 10.3390/molecules18010789.
The interactions between cyclophosphamide (CYC) and lysozyme (LYZ) in the presence of different cyclodextrins (CDs) were investigated by UV absorption, fluorescence spectroscopy, circular dichroism (CD), and molecular modeling techniques under imitated physiological conditions. The UV absorption results showed the formation of complexes between CYC and LYZ in the presence of different CDs. Fluorescence data show that CYC has a stronger quenching effect on LYZ, and the red shifts suggested that the microenvironment of Trp residues was changed and became more hydrophilic. The interaction of CYC with LYZ and quenching properties of the complexes caused strong static fluorescence quenching in binary and ternary systems. The binding affinities as well as the number of binding sites were obtained from interaction between CYC and LYZ in the presence of different CDs as binary and ternary systems by modified Stern-Volmer plots. The Resonance Light Scattering (RLS) technique was utilized to investigate the effect of drug and CDs on conformational changes of LYZ as separate and simultaneous. The results suggested that the enhancement of RLS intensity was attributed to the formation of a complex between drug and protein in absence and presence of CDs. The effect of CYC and cyclodextrins on the conformation of LYZ was analyzed using synchronous fluorescence spectroscopy. Our results revealed that the fluorescence quenching of LYZ originated from the Trp and Tyr residues, and demonstrated conformational changes of LYZ with the addition of CYC and CDs. The molecular distances between the donor (LYZ) and acceptor (CYC and CDs) in binary and ternary systems were estimated according to Forster's theory and showed static quenching for protein with CYC in the presence of CDs. The CD spectra indicated that the binding of the CYC induced secondary structural changes in LYZ in binary and ternary systems. Molecular modeling suggested the binding sites of CYC in the ternary systems differ from those in the binary systems. estimated the distance between CYC and Trp residues in binary and ternary systems in the presence of CDs and confirmed the experimental results.
在模拟生理条件下，用紫外吸收光谱、荧光光谱、圆二色性（CD）和分子建模技术研究了环磷酰胺（CYC）和溶菌酶（LYZ）在不同环糊精（CDs）存在下的相互作用。紫外吸收结果表明，在不同 CD 存在下，CYC 与 LYZ 形成了配合物。荧光数据表明，CYC 对 LYZ 的猝灭效应更强，红移表明色氨酸残基的微环境发生了变化，变得更加亲水。在二元和三元体系中，CYC 与 LYZ 的相互作用和配合物的猝灭特性导致了强静态荧光猝灭。通过改进的 Stern-Volmer 图，从二元和三元体系中 CYC 与 LYZ 的相互作用获得了结合亲和力和结合位点数。共振光散射（RLS）技术用于研究药物和 CD 对 LYZ 构象变化的影响，分别和同时进行。结果表明，RLS 强度的增强归因于药物与蛋白质在 absence 和 presence 下形成复合物。用同步荧光光谱法分析了 CYC 和环糊精对 LYZ 构象的影响。结果表明，LYZ 的荧光猝灭源于色氨酸和酪氨酸残基，并证明了在添加 CYC 和 CDs 后 LYZ 的构象发生了变化。根据福斯特理论，估算了二元和三元体系中供体（LYZ）和受体（CYC 和 CDs）之间的分子距离，结果表明，在 presence 下，CDs 中的 CYC 对蛋白质具有静态猝灭作用。CD 谱表明，CYC 的结合导致 LYZ 在二元和三元体系中发生二级结构变化。分子建模表明，在三元体系中，CYC 的结合位点与二元体系中的不同。在 presence 下，估算了二元和三元体系中 CYC 与色氨酸残基之间的距离，并证实了实验结果。
