Abstract

Binding of iprodione (IPR), a dicarboxamide fungicide, to the carrier protein in bovine circulation, bovine serum albumin (BSA) was characterized with the help of fluorescence, absorption, circular dichroism and Fourier transform infrared (FTIR) spectral measurements in combination with computational analysis. The increase in the K_SV (Stern–Volmer constant) value with temperature and absorption spectral results characterized the IPR-induced quenching of BSA fluorescence as dynamic quenching. On the other hand, higher value (> 10¹¹ M⁻¹ s⁻¹) of the bimolecular quenching rate constant (k_q) suggested complex formation between IPR and BSA. In view of it, increase in K_SV value with temperature can be considered as an indication of the involvement of endothermic apolar (hydrophobic) interactions in stabilizing the IPR–BSA complex, as these forces are maximized at higher temperature. A weak binding affinity was anticipated from the values of the binding constant (K_a = 0.83–2.69 × 10³ M⁻¹) for IPR–BSA association. Thermodynamic analysis of the binding data further supported contribution of hydrophobic interactions in the IPR–BSA association process. This result was validated by the computational docking analysis. Spectral results from three-dimensional fluorescence and circular dichroism demonstrated microenvironmental changes around BSA fluorophores and protein’s structural (secondary and tertiary) alterations, respectively, upon IPR binding to the protein. Slight variation in the secondary structures of BSA in the presence of IPR was also verified from FTIR spectral results. IPR was found to bind to both site I (subdomain IIA) and site II (subdomain IIIA) of BSA, showing more preference toward site II, as identified by the competitive drug displacement results and supported by the computational analysis.

Graphic abstract

中文翻译：

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探索二甲酰胺类杀菌剂异丙洛酮与牛血清白蛋白的相互作用机理

摘要

异丙基丙二酮（IPR）（一种二甲酰胺类杀菌剂）与牛循环中的载体蛋白结合，通过荧光，吸收，圆二色性和傅立叶变换红外（FTIR）光谱测量结合计算来表征牛血清白蛋白（BSA）分析。在增加ķ _SV随温度和吸收光谱的结果（斯特恩-沃尔默常数）值，其特征在于BSA荧光作为动态猝灭的IPR诱导猝灭。另一方面，双分子猝灭速率常数（k _q）的值较高（＞ 10 ¹¹  M ^-1 s ^-1）表明IPR和BSA之间形成复合物。鉴于此，增加K_SV随温度的变化可以认为是吸热非极性（疏水）相互作用与稳定IPR-BSA复合物有关的指示，因为这些力在较高温度下会最大化。从结合常数的值（K _a  = 0.83–2.69×10 ³  M ^-1），用于IPR–BSA协会。结合数据的热力学分析进一步支持了疏水相互作用在IPR-BSA缔合过程中的贡献。通过计算对接分析验证了该结果。三维荧光和圆二色性的光谱结果表明，IPR与蛋白质结合后，BSA荧光团周围的微环境变化和蛋白质的结构（二级和三级）改变。FTIR光谱结果也证实了在存在IPR的情况下BSA二级结构的轻微变化。发现IPR与BSA的位点I（子域IIA）和位点II（子域IIIA）都结合，显示出对位点II的更多偏好，这由竞争性药物置换结果确定并得到了计算分析的支持。

图形摘要