Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Department of Cell & Molecular Sciences, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran.
Int J Biol Macromol. 2018 Jun;112:422-432. doi: 10.1016/j.ijbiomac.2018.01.067. Epub 2018 Jan 12.
β-Lactoglobulin (βLG) is a basic element of globular carrier protein, which is the major protein in the whey of ruminant milk and is of main interest in the dairy industry. In the present study, the simultaneous effects of both of the important anticancer drugs of 5-fluorouracil (5-FU) and oxali-palladium, on the structure of βLG were investigated using different spectroscopic methods of fluorescence and circular dichroism (CD) in combination with a molecular docking at two temperatures of 25 and 37°C. The resulted data from intrinsic fluorescence spectra of protein indicated that 5-FU and oxalli-palladium can quench the fluorescence intensity of βLG in dose-dependent manner via static mechanism of fluorescence quenching. Analysis of fluorescence quenching data in agreement with theoretical results have represented that there are I binding sites on βLG for binding of oxali-palladium and also II binding sites for 5-FU, at both temperatures of 25 and 37°C. Also, competitive binding results showed that the number of binding sites on protein for each of the drug when the protein incubated with one of the drug did not show any changes. The values of thermodynamic parameters of ΔH°, ΔS° and ΔG° illustrate that van der Waals and hydrogen-bond interactions have the main role in the binding of oxali-palladium and 5-FU to βLG, respectively. The analysis of circular dichroism spectra indicated reduction in stability of the protein and alteration in the secondary structure of protein with reduction of α-helical structure and increasing of β-sheet structure in the presence of increasing concentration of oxali-palladium and 5-FU. Also, the transition temperature (T) value of βLG indicated the significant decreasing in the presence of 5-FU and oxali-palladium. As a result, it can be concluded that both of the chemotherapeutic drugs of oxali-palladium and 5-FU can bind to independent binding sites on carrier protein of βLG, which can be used in design and simultaneous delivery of both drugs.
β-乳球蛋白（βLG）是球状载体蛋白的基本元素，是反刍动物乳清中的主要蛋白质，也是乳品行业的主要关注点。在本研究中，使用荧光和圆二色性（CD）的不同光谱方法，结合在 25 和 37°C 下的分子对接，同时研究了两种重要抗癌药物 5-氟尿嘧啶（5-FU）和奥沙利铂对βLG 结构的影响。来自蛋白质内源荧光光谱的数据表明，5-FU 和奥沙利铂可以通过静态荧光猝灭机制，以剂量依赖的方式猝灭βLG 的荧光强度。荧光猝灭数据的分析与理论结果一致，表明βLG 上有 I 个结合位点用于结合奥沙利铂，以及在 25 和 37°C 下有 II 个结合位点用于结合 5-FU。此外，竞争结合结果表明，当蛋白质与一种药物孵育时，蛋白质上每个药物的结合位点数量没有变化。ΔH°、ΔS°和ΔG°的热力学参数值表明，范德华力和氢键相互作用分别是奥沙利铂和 5-FU 与βLG 结合的主要作用力。圆二色光谱分析表明，在奥沙利铂和 5-FU 浓度增加的情况下，蛋白质的稳定性降低，蛋白质的二级结构发生变化，α-螺旋结构减少，β-折叠结构增加。此外，βLG 的转变温度（T）值表明，在存在 5-FU 和奥沙利铂的情况下，T 值显著降低。因此，可以得出结论，两种化疗药物奥沙利铂和 5-FU 可以结合到βLG 载体蛋白的独立结合位点上，这可用于同时设计和输送这两种药物。
