美满霉素(Minocycline, MC)是一种半合成四环素类广谱抗生素, 具有较强的抗菌作用, MC经口服后迅速被吸收, 与血清白蛋白结合率为76%～83%。 研究牛血清蛋白(bovine serum albumin, BSA)与 MC之间的结合机理, 有利于在分子层面探讨MC与BSA的相互作用机制, 进一步了解MC与BSA的结构和功能关系, 并为MC的药理毒性、 药效的深入研究提供必要数据支持。 在模拟生理条件和不同的温度条件下, 采用荧光光谱、 圆二色谱、 紫外光谱和分子对接模拟技术研究MC和BSA之间的相互作用及机理。 研究结果表明, MC对BSA的荧光有猝灭作用, 且猝灭常数KSV随温度升高而降低, 说明MC与BSA的猝灭机理为静态猝灭。 利用Stern-Volmer方程和静态猝灭双对数公式对荧光结果进行计算, 结果表明MC与BSA之间的结合位点数n都接近于1。 根据Van’t Hoff热力学方程式在298, 303和308 K下求得热力学参数结果为焓变ΔH=-34.14 kJ·mol-1, 熵变ΔS=32.55 J·(mol·K)-1, 吉布斯自由能ΔG=-43.84 kJ·mol-1(298 K), -43.88 kJ·mol-1(303 K), -44.17 kJ·mol-1 (308 K), 证明二者之间的主要作用力为范德华力和氢键, 其作用过程为自发、 放热过程。 通过MC与BSA的紫外可见吸收光谱分析, 发现BSA的吸收峰位置有明显的红移, 表明BSA的构象发生了变化。 根据Frster’s非辐射能量转移理论得到的MC和BSA的结合距离为r=1.873 nm, 表明在MC和BSA之间发生了非辐射能量转移。 同步荧光光谱的实验结果表明, 当MC和BSA相互作用时, BSA的构象发生了变化, 结合位点位于色氨酸(Trp)残基上。 此外, 三维荧光光谱和圆二色谱分析都进一步表明MC与BSA相互作用时会使BSA的构象变化且BSA中的色氨酸(Trp)残基周围微环境极性减小, 疏水性增强。 通过MC与BSA作用前后圆二色谱二级结构的定量分析可知: BSA中α-螺旋结构占比为31.75%, 逐步加入MC后, α-螺旋结构占比依次变为47.10%(ｃBSA∶ｃTRO=1∶1), 54.39%(ｃBSA∶ｃTRO=1∶5), 说明α-螺旋结构含量占比, BSA的结构以α-螺旋结构为主。 分子对接模拟表明MC结合在BSA的site I(亚域IIA)位置, MC分子与BSA中的氨基酸残基以范德华力结合作用的有: PHE508, LYS535, HIS534, PHE501, GLN579, VAL546, MET547, LEU528, PHE508, 以氢键结合作用的有: LYS524和LEU531, 产生超共轭效应的残基有: ALA527, VAL575, LEU531, PHE508, 这些氨基酸与MC分子紧密结合, MC使BSA的二级结构发生改变。 本实验获得的数据有助于了解MC和BSA的相互作用机制, 同时也有助于了解MC在人体的储运过程中对BSA构象的影响。

Minocycline (MC) is a semisynthetic tetracycline broad-spectrum antibiotic with stronger antibacterial activity, which is absorbed rapidly after oral administration, and the binding rate to serum protein ranges from 76% to 83%. The study of the binding mechanism between Bovine serum albumin (BSA) and MC is helpful to explore the interaction mechanism between MC and BSA at the molecular level, to further understand the structure and functional relationship of BSA and MC, and to provide necessary data support for the further study of pharmacological toxicity and efficacy of MC. The interaction between MC and BSA has been investigated by fluorescence spectroscopy, circular dichroism, ultraviolet spectroscopy and molecular docking simulation at different temperature and simulated physiological conditions. The results show that MC quenches the fluorescence of BSA, and the quenching constant decreases with the increase of temperature. This indicates that the quenching mechanism of MC and BSA is static quenching. The fluorescence results were calculated using the Stern-Volmer equation and the static quenching double logarithmic formula, and the results showed that the number of binding sites n between MC and BSA is close to 1. According to Van’t Hoff thermodynamic equation at 298, 303 and 308 K, the thermodynamic parameters were obtained as follows: enthalpy change ΔH=-34.14 kJ·mol-1, entropy change ΔS=32.55 J·(mol·K)-1, Gibbs free energy ΔG=-43.84 kJ·mol-1 (298 K), -43.88 kJ·mol-1 (303 K), -44.17 kJ·mol-1 (308 K), which proved that the main force between MC and BSA is the van der Waals and hydrogen bonding, and the process of its action is the spontaneous and exothermic process. Through the UV-visible absorption spectrum analysis of BSA and MC, the position of the absorption peak of BSA has a significant red shift, indicating that the conformation of BSA has changed. According to Frster’s theory of non-radiative energy transfer, the binding distance r between MC and BSA is 1.873 nm, which indicates that non-radiative energy transfer occurs between MC and BSA. In addition, the experimental results of synchronous fluorescence spectroscopy showed that the conformation of BSA changed when MC interacted with BSA, and the binding site was on tryptophan (Trp) residues. The results also showed that the conformation of BSA changed by three-dimensional fluorescence spectroscopy and circular dichroism, and (Trp) the polarity of the surrounding environment decreased and hydrophobicity increased. Quantitative analysis of secondary structure of circular dichroism before and after the interaction of MC and BSA showed that the content of alpha-helix structure in BSA was 31.75%. After adding MC gradually, the content of α-helix structure changed to 47.10% (cBSA∶cTRO=1∶1) and 54.39% (cBSA∶cTRO=1∶5), indicating that the content of α-helix structure increased, and the structure of BSA was mainly α-helix structure. Molecular docking simulation showed that MC interacts into the site I (subdomain IIA) of BSA, it forms van der Waals interaction between MC and the amino acid residues PHE508, LYS535, HIS534, PHE501, GLN579, VAL546, MET547, LEU528, PHE508 of BSA, hydrogen bonds formed between MC and the amino acid residues LYS524 and LEU531, and super conjugation also formed between MC and the amino acid residues ALA527, VAL575, LEU531, PHE508. These amino acids bind closely with MC molecules, and MC changes the secondary structure of BSA. The data obtained in this experiment are helpful to understand the interaction mechanism between MC and BSA, as well as the effect of MC on BSA conformation during storage and transportation.