光谱学与光谱分析, 2019, 39 (3): 851, 网络出版: 2019-03-19  

MCR-ALS结合分子对接研究隐色孔雀石绿与BSA的作用过程

Studies on the Interaction between Leucomalachite Green with Bovine Serum Albumin by MCR-ALS and Molecular Docking
作者单位
1 南昌大学食品科学与技术国家重点实验室, 江西 南昌 330047
2 南昌大学化学系, 江西 南昌 330031
3 南昌大学药学院, 江西 南昌 330031
摘要
孔雀石绿(LMG)在治理鱼卵中霉菌和杀灭鱼体寄生虫等方面的效果显著, 广泛应用于水产运输和养殖。 孔雀石绿进入动物机体后, 通过生物转化代谢为脂溶性的隐色孔雀石绿(LMG), LMG的毒性超过MG; LMG能快速在组织中蓄积, 具有致癌、 致畸、 致突变等毒副作用。 白蛋白可与多种内源和外源化合物结合, 是血浆中含量最丰富的载体蛋白, 也是药物发挥作用的重要载体和靶标。 模拟pH 7.4的生理条件, 通过荧光光谱和圆二色谱法(CD)采集两种不同滴加方式的LMG与牛血清白蛋白(BSA)动态作用过程中的多维数据, 并应用化学计量学多元曲线分辨-交替最小二乘法(MCR-ALS)对多维波谱数据进行解析和描述, 从重叠严重的光谱数据中同时得到作用体系的定量和定性信息。 从解析得到的浓度趋势图中, 说明体系在LMG∶BSA=2∶1时达到动态平衡, 并可确认复合物LMG2-BSA的生成; 解析得到的与所测量的BSA荧光和CD图符合, 印证由MCR-ALS获得的浓度趋势图的可靠性和正确性; 通常由重叠光谱中无法辨别的LMG2-BSA复合物荧光光谱和CD谱图也可由数学解析获得, 进一步印证了复合物的存在。 原子力显微镜(AFM)测量结果表明BSA与LMG结合后, BSA的形貌发生改变, 表面粗糙度(RMS)由(1.24±0.28) nm增至(13.47±0.53) nm; 同时由CD实验结果可知LMG与BSA作用达到平衡时, α-螺旋结构的含量从46.5%降低到42.3%, 推测是BSA所处微环境和构象发生变化所致。 荧光探针实验发现经典site Ⅰ标记物华法林加入后, LMG-BSA的猝灭常数由2.65×106 L·mol-1降低为1.88×106 L·mol-1, 但加入site Ⅱ标记物布洛芬后, LMG-BSA的猝灭常数变化不明显, 由此推断LMG可能结合在蛋白质的亚域ⅡA, 即site Ⅰ位。 分子对接证实BSA的Ⅰ位有足够的空间容纳LMG, 且LMG与BSA之间的主要作用是疏水作用力。 该研究从分子水平了解LMG与生物大分子的作用机制, 并为LMG的毒副作用研究提供重要的信息。
Abstract
Leucomalachite Green (LMG) is a major metabolite of malachite green (MG). It has a long residence period in edible fish tissues. At present, the use of MG has been banned in some countries for its increased risk of carcinogenesis, mutagenesis and other adverse effects to human health. However, MG is still widely used in aquaculture, aquatic transport and storage for its low price. The interaction between LMG and bovine serum albumin (BSA) under simulative physiological conditions was investigated by spectroscopy. Two spectroscopic approaches (fluorescence and circular dichroism) and two different experiments were used for monitoring the biological dynamic process. Qualitative and quantitative information was obtained with the resolution of the data matrices by chemometrics method - multivariate curve resolution-alternating least squares (MCR-ALS). Atomic force microscope (AFM) was executed in order to verdict the particle morphology and dimensions of the LMG-BSA conjugates. The root mean square (RMS) roughness of the individual BSA molecule was found to be (1.24±0.28) nm. The BSA molecule particle was observed to be looser on the mica substrate upon interaction with LMG. The RMS was changed to be (13.47±0.53) nm for the LMG-BSA interaction. The calculated result of circular dichroism (CD) spectra revealed that the α-helical content for the LMG-BSA complex was 42.5%, which has a slight decrease compared with the free BSA (46.3%). The results of AFM and CD spectra showed that the binding of LMG to BSA induced micro-environmental and conformational changes of BSA molecules. In order to identify the LMG-binding site on BSA, site marker competitive experiments were carried out, using drugs which were specifically bound to site Ⅰ (warfarin) and site Ⅱ (ibuprofen) on BSA. The binding constant of the system with warfarin (1.88×106 L·mol-1) was almost 70% of that without warfarin (2.65×106 L·mol-1), while the constants of the systems with and without ibuprofen had only a small difference, indicating that LMG was bound to site Ⅰ of BSA. The molecular docking gave more intuitive understanding of the binding of LMG and BSA. It was recognized that LMG binds within the sub-domain ⅡA pocket in domain Ⅱ of BSA. These values showed that hydrophobic forces were the main interactions in the binding of LMG to BSA and the stabilization of the complex. It can be expected that the study will have great significance in helping to further clarify the metabolism and distribution of LMG in vivo and the mechanism of toxicological effects and pharmacokinetics from molecular level.

张秋兰, 谢立昕, 杨林慧, 庹浔, 倪永年. MCR-ALS结合分子对接研究隐色孔雀石绿与BSA的作用过程[J]. 光谱学与光谱分析, 2019, 39(3): 851. ZHANG Qiu-lan, XIE Li-xin, YANG Lin-hui, TUO Xun, NI Yong-nian. Studies on the Interaction between Leucomalachite Green with Bovine Serum Albumin by MCR-ALS and Molecular Docking[J]. Spectroscopy and Spectral Analysis, 2019, 39(3): 851.

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