Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran.
Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
Spectrochim Acta A Mol Biomol Spectrosc. 2024 Mar 15;309:123815. doi: 10.1016/j.saa.2023.123815. Epub 2023 Dec 24.
In this work, the interaction of human serum albumin (HSA) and human holo-transferrin (HTF) with the prepared Nano-Kaempferol (Nano-KMP) through oil-in-water procedure was investigated in the form of binary and ternary systems by the utilization of different spectroscopy techniques along with molecular simulation and cancer cell experiments. According to fluorescence spectroscopy outcomes, Nano-KMP is capable of quenching both proteins as binary systems by a static mechanism, while in the form of (HSA-HTF) Nano-KMP as the ternary system, an unlinear Stern-Volmer plot was elucidated with the occurrence of both dynamic and static fluorescence quenching mechanisms in the binding interaction. In addition, the two acquired K values in the ternary system signified the existence of two sets of binding sites with two different interaction behaviors. The binding constant values of HSA-Nano KMP, HTF-Nano-KMP, and (HSA-HTF) Nano-KMP complexes formation were (2.54 ± 0.03) × 10, (2.15 ± 0.02) × 10 and (1.43 ± 0.04) × 10Mat the first set of binding sites and (4.68 ± 0.05) × 10 M at the second set of binding sites, respectively. The data of thermodynamic parameters confirmed the major roles of hydrogen binding and van der Waals forces in the formation of HSA-Nano KMP and HTF-Nano KMP complexes. The thermodynamic parameter values of (HSA-HTF) Nano KMP revealed the dominance of hydrogen binding and van der Waals forces in the first set of binding sites and hydrophobic forces for the second set of binding sites. Resonance light scattering (RLS) analysis displayed the existence of a different interaction behavior for HSA-HTF complex in the presence of Nano-KMP as the ternary system. Moreover, circular dichroism (CD) technique affirmed the conformational changes of the secondary structure of proteins as binary and ternary systems. Molecular docking and molecular dynamics simulations (for 100 ns) were performed to investigate the mechanism of KMP binding to HSA, HTF, and HSA-HTF. Next to observing a concentration and time-dependent cytotoxicity, the down regulation of PI3K/AkT/mTOR pathway resulted in cell cycle arrest in SW480 cells.
在这项工作中，通过利用不同的光谱技术以及分子模拟和癌细胞实验，以二元和三元体系的形式研究了人类血清白蛋白 (HSA) 和人全转铁蛋白 (HTF) 与通过油包水程序制备的纳米山奈酚 (Nano-KMP) 的相互作用。根据荧光光谱结果，Nano-KMP 能够通过静态机制猝灭两种蛋白质作为二元体系，而在 (HSA-HTF) Nano-KMP 作为三元体系的形式下，揭示了非线性 Stern-Volmer 图，表明在结合相互作用中存在动态和静态荧光猝灭机制。此外，三元体系中获得的两个 K 值表明存在两组具有两种不同相互作用行为的结合位点。HSA-Nano-KMP、HTF-Nano-KMP 和 (HSA-HTF) Nano-KMP 配合物形成的结合常数值分别为 (2.54±0.03)×10、(2.15±0.02)×10 和 (1.43±0.04)×10 M，在第一组结合位点，在第二组结合位点分别为 (4.68±0.05)×10 M。热力学参数数据证实了氢键和范德华力在 HSA-Nano-KMP 和 HTF-Nano-KMP 配合物形成中的主要作用。(HSA-HTF) Nano-KMP 的热力学参数值表明，在第一组结合位点中氢键和范德华力占主导地位，而在第二组结合位点中疏水作用力占主导地位。共振光散射 (RLS) 分析显示，在三元体系中存在 Nano-KMP 时，HSA-HTF 复合物存在不同的相互作用行为。此外，圆二色性 (CD) 技术证实了蛋白质二级结构的构象变化作为二元和三元体系。进行了分子对接和分子动力学模拟（100 ns），以研究 KMP 与 HSA、HTF 和 HSA-HTF 结合的机制。除了观察到浓度和时间依赖性细胞毒性外，PI3K/AkT/mTOR 通路的下调导致 SW480 细胞的细胞周期停滞。
