低密度脂蛋白与阴离子配基的相互作用研究
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摘要
由于低密度脂蛋白（Low Density Lipoprotein, LDL)与动脉粥样硬化有着密切的联系，因而降低血液中LDL的含量已经成为目前预防及治疗动脉粥样硬化的主要措施，其中LDL的体外去除方法因其简便高效的特点受到了人们的广泛关注。LDL吸附材料是体外去除LDL的研究重点，而LDL吸附配基的选择决定着吸附材料的吸附效果。因此，吸附配基与LDL相互作用的研究对LDL吸附材料的研制具有十分重要的指导意义。本文选取了可以通过静电作用吸附LDL的三种阴离子配基（谷胱甘肽、葡聚糖硫酸酯与肝素），考察了LDL与这三种配基的相互作用。
首先，本文通过微量热的方法考察LDL与上述三种配基的相互作用，获得了LDL与三种配基相互作用的热力学参数。结果表明：LDL与三种配基的结合反应为焓驱动，结合反应能够自发进行。谷胱甘肽、葡聚糖硫酸酯、肝素与LDL的相互作用常数K值分别为3.42×106、2.08×106、1.31×106，从而得到三种配基与LDL的结合强度强弱：谷胱甘肽>葡聚糖硫酸酯>肝素。在此基础上，本文通过光谱分析，进一步考察配基对LDL结构的影响。荧光光谱结果表明：谷胱甘肽、葡聚糖硫酸酯和肝素的加入分别使LDL荧光最大发射峰位蓝移6nm、5nm与2nm，说明配基与LDL的结合使LDL中荧光氨基酸的微环境疏水性增加，改变了LDL的构象，其构象改变程度：谷胱甘肽>葡聚糖硫酸酯>肝素；圆二色谱结果表明：三种配基与LDL相互作用时，LDL的二级结构（α-螺旋含量）发生改变，其中谷胱甘肽对LDL二级结构影响最大，使LDL中α-螺旋的含量降低了3.4%，葡聚糖硫酸酯次之，肝素最小。光谱的分析结果与微量热的方法结果一致。说明谷胱甘肽与LDL相互作用最强，对LDL的结构影响最大。
The level of low density lipoprotein (LDL) in the human blood isclosely related to the atherosclerosis disease. Therefore, the removal ofLDL in blood is considered as the main route for the medical preventionand treatment of atherosclerosis. Among varies LDL removing methods,the removal of LDL in vitro has attracted wide attention for their efficiencyand conveniency. The development of new LDL adsorbents is still inurgent needs, and the selection of LDL adsorption ligands determines theLDL removal efficiency. So the studies on the interaction between ligandsand LDL have great significance in developing of LDL adsorbents. Herewe selected three kinds of anionic ligands (glutathione, dextran sulfate andheparin) that could adsorb LDL through electrostatic interaction, andinvestigated their interactions with LDL.
Firstly, we studied the interaction between the three kinds of anionicligands and LDL by isothermal titration calorimetry (ITC), andthermodynamic parameters were obtained. The results showed that thethree kinds of ligands and LDL binding reactions were enthalpy-driven,and could occur spontaneously. The interaction constant values ofglutathione, dextran sulfate, heparin with LDL were3.42×106,2.08×106and1.31×106respectively, and the corresponding strength decreased in the following order: glutathione, dextran sulfate, and heparin. And spectralanalysis was carried out to further study the structural change of LDL afterligand combination. Fluorescence results showed the same trendconcerning the structural change of LDL after the interaction. Themaximum LDL fluorescence emission peak blue shifted6nm,5nm and2nm for glutathione, dextran sulfate and heparin respectively, whichindicated the increased hydrophobicity in the microenvironment of thefluorescent amino acid. Circular dichroism results also revealed the LDLsecondary structure (α-helix content) change after LDL interaction with thethree ligands, among which glutathione was the most effective one with anα-helix content decrease of3.4%followed by dextran sulfate and heparin.The spectral results indicated the glutathione had the strongest interactionwith LDL and the greatest impact on LDL structure change, which wereconsistent with the results of ITC.
首先，本文通过微量热的方法考察LDL与上述三种配基的相互作用，获得了LDL与三种配基相互作用的热力学参数。结果表明：LDL与三种配基的结合反应为焓驱动，结合反应能够自发进行。谷胱甘肽、葡聚糖硫酸酯、肝素与LDL的相互作用常数K值分别为3.42×106、2.08×106、1.31×106，从而得到三种配基与LDL的结合强度强弱：谷胱甘肽>葡聚糖硫酸酯>肝素。在此基础上，本文通过光谱分析，进一步考察配基对LDL结构的影响。荧光光谱结果表明：谷胱甘肽、葡聚糖硫酸酯和肝素的加入分别使LDL荧光最大发射峰位蓝移6nm、5nm与2nm，说明配基与LDL的结合使LDL中荧光氨基酸的微环境疏水性增加，改变了LDL的构象，其构象改变程度：谷胱甘肽>葡聚糖硫酸酯>肝素；圆二色谱结果表明：三种配基与LDL相互作用时，LDL的二级结构（α-螺旋含量）发生改变，其中谷胱甘肽对LDL二级结构影响最大，使LDL中α-螺旋的含量降低了3.4%，葡聚糖硫酸酯次之，肝素最小。光谱的分析结果与微量热的方法结果一致。说明谷胱甘肽与LDL相互作用最强，对LDL的结构影响最大。
The level of low density lipoprotein (LDL) in the human blood isclosely related to the atherosclerosis disease. Therefore, the removal ofLDL in blood is considered as the main route for the medical preventionand treatment of atherosclerosis. Among varies LDL removing methods,the removal of LDL in vitro has attracted wide attention for their efficiencyand conveniency. The development of new LDL adsorbents is still inurgent needs, and the selection of LDL adsorption ligands determines theLDL removal efficiency. So the studies on the interaction between ligandsand LDL have great significance in developing of LDL adsorbents. Herewe selected three kinds of anionic ligands (glutathione, dextran sulfate andheparin) that could adsorb LDL through electrostatic interaction, andinvestigated their interactions with LDL.
Firstly, we studied the interaction between the three kinds of anionicligands and LDL by isothermal titration calorimetry (ITC), andthermodynamic parameters were obtained. The results showed that thethree kinds of ligands and LDL binding reactions were enthalpy-driven,and could occur spontaneously. The interaction constant values ofglutathione, dextran sulfate, heparin with LDL were3.42×106,2.08×106and1.31×106respectively, and the corresponding strength decreased in the following order: glutathione, dextran sulfate, and heparin. And spectralanalysis was carried out to further study the structural change of LDL afterligand combination. Fluorescence results showed the same trendconcerning the structural change of LDL after the interaction. Themaximum LDL fluorescence emission peak blue shifted6nm,5nm and2nm for glutathione, dextran sulfate and heparin respectively, whichindicated the increased hydrophobicity in the microenvironment of thefluorescent amino acid. Circular dichroism results also revealed the LDLsecondary structure (α-helix content) change after LDL interaction with thethree ligands, among which glutathione was the most effective one with anα-helix content decrease of3.4%followed by dextran sulfate and heparin.The spectral results indicated the glutathione had the strongest interactionwith LDL and the greatest impact on LDL structure change, which wereconsistent with the results of ITC.
引文
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