叶酸受体靶向土大黄苷前药与HSA相互作用研究

Interaction between a novel folate receptor targeted rhaponticin conjugate and human serum albumin by molecular spectroscopy

目的从分子水平上研究叶酸受体靶向土大黄苷前药(FRHA)与人血清白蛋白(human serum albumin,HSA)的结合作用。方法模拟生理条件,采用荧光光谱和紫外-可见光谱研究FRHA与HSA结合反应的光谱学特征,探讨药物对蛋白质内源性荧光的淬灭机制,并结合三维、同步荧光光谱和圆二色谱分析FRHA对HSA微环境及构象的影响。结果 FRHAHSA复合物的形成将导致HSA发生荧光淬灭,298、302、306和310 K时FRHA与HSA相互作用的结合常数分别为1.4322×105、1.1793×105、0.9334×105和0.7896×105L/mol。由实验计算出热力学参数焓变ΔH为-38.772 k J/mol,熵变ΔS为-31.39 J/(mol·K)。结论 FRHA-HSA复合物的形成是自发进行的,范德华力和氢键作用力是使该复合物稳定的主要作用力,并且复合物的形成使HSA的微环境和构象发生改变,进而导致HSA发生荧光淬灭,淬灭机制为静态淬灭。

Objective To investigate the affinity and interaction of a folate receptor targeted rhapontion（RHA）conjugate（FRHA）with human serum albumins（HSA）. Method The interaction between FRHA and HSA under physiological conditions was investigated by fluorescence spectroscopy,UV-visual（vis）spectroscopy and circular dichroism（CD）spectroscopy. Great attempts were made to investigate their interaction mechanism regarding the quenching mechanism,the specific binding site,the type of interaction force,and the effect of FRHA on the micro-environmental and conformational changes in HSA molecules. Results The formation of the complex of FRHA-HSA would lead to fluorescence quenching. The corresponding values of Ka were 1.4322×105,1.1793×105,0.9334×105and 0.7896×105L/mol when the temperature were 298,302,306,and 310 K,respectively. The enthalpy change（ΔH）and entropy change（ΔS）were calculated to be-38.772 k J/mol and-31.39 J/（mol·K）,indicating that van der Waals force and hydrogen bonds played major roles in stabilizing the complex. Conclusion The interaction process of the formation of FRHA-HSA is spontaneous. The negative values of enthalpy change（ΔH）and entropy change（ΔS）indicate that van der Waals force and hydrogen bonds play major roles in stabilizing the complex. The conformational investigation reveals the α-helical structure is decreased and the microenvironment of HSA is changed upon the addition of FRHA. The fluorescence quenching of HSA caused by FRHA is static quenching. Furthermore,the results of site marker competitive experiment suggest that FRHA binds to the sub-domainⅡA of HSA.