多光谱技术结合分子模拟研究头孢唑林和头孢曲松与人血清白蛋白的相互作用

Study on the Interaction of Cefazolin and Ceftriaxone with Human Serum Albumin with Multi-Spectroscopic and Docking Methods

头孢唑林(CFZ)属第一代β-内酰胺类半合成头孢菌素,对革兰氏阳性菌和革兰氏阴性菌均有较强的抗菌作用。头孢曲松(CRO)属第三代β-内酰胺类广谱抗生素,对敏感致病菌导致的疾病及手术后期感染预防有一定作用。人血清白蛋白(HSA)作为生物体内循环系统中含量最丰富的蛋白质,可以与多种内源性和外源性化合物可逆性结合,起到储存和转运的作用。因此,研究CFZ和CRO与HSA的相互作用对了解CFZ和CRO的药代动力学行为具有重要意义。在模拟生理条件下,采用多种光谱法和分子对接技术研究CFZ和CRO与HSA的相互作用。结果表明,在298和310K条件下,CFZ和CRO与HSA分别形成复合物导致内源荧光猝灭,猝灭机制均为静态猝灭。在消除内滤光影响下,HSA-CFZ和HSA-CRO体系的猝灭常数(KSV)和结合常数(Ka)均随着温度的升高而降低,结合位点数约为1。根据Fster能量转移定律,CFZ和CRO与HSA结合距离分别为2.41和1.40nm。希尔系数(nH)值小于1,表明CFZ和CRO分别与HSA结合后存在药物间负协同作用。热力学参数(ΔH_(HSA-CFZ)=-22.67kJ·mol^(-1),ΔH_(HSA-CRO)=-39.56kJ·mol^(-1),ΔS_(HSA-CFZ)=-4.90J·mol^(-1)·K^(-1),ΔS_(HSA-CRO)=-37.28J·mol^(-1)·K^(-1))揭示,CFZ和CRO能自发地通过氢键和范德华力与HSA相结合。三维荧光光谱和圆二色谱法(CD)显示CFZ和CRO使HSA的微环境和构象发生改变。分子对接技术显示CFZ和CRO均结合在HSA的siteⅠ结合位点上,与取代实验结果一致。本研究有助于了解CFZ和CRO在机体内的作用机制及对HSA结构和功能的影响。

Cefazolin（CFZ）is the first generation ofβ-lactam semi-synthetic cephalosporin and which are mainly used to treat Gram positive and negative bacteria.Ceftriaxone（CRO）belongs to the third generation ofβ-lactam broad-spectrum antibiotic and are particularly useful in treating the disease caused by sensitive bacteria and post-surgery infection prevention.Human serum albumin（HSA）is the most abundant protein in plasma and can bind with a variety of endogenous and exogenous compounds reversibly,which is playing a role in storage and transport.Therefore,detailed investigating the interaction of CFZ and CRO with HSA are very important to understand the pharmacokinetic behavior of the CFZ and CRO.The interaction between CFZ and CRO with HSA were investigated with fluorescence spectroscopy,three-dimensional fluorescence spectroscopy,circular dichroism（CD）and molecular docking and so on under imitated physiological conditions.The results showed that CFZ and CRO could interact with HSA to form complex at 298 and 310K,which led to quench the intrinsic fluorescence of HSA via static quenching.After correcting the binding parameters,the Stern-Volmer quenching constants（KSV）and binding constants（Ka）of HSACFZ and HSA-CRO system inversely correlated with temperatures,and there was one binding site between HSA-CFZ or HSACRO system.Based on the Fster＇s theory of non-radiation energy transfer,the specific binding distance between HSA-CFZ or HSA-CRO system was 2.41 or 1.40 nm,respectively.Hill＇s coefficients（nH 1）proved that a negative cooperativity was found when CFZ or CRO bound to HSA.The thermodynamic parameters（ΔH（（HSA-CFZ））=-22.67kJ·mol^（-1）,ΔH（（HSA-CRO））=-39.56kJ·mol^（-1）,ΔS（（HSA-CFZ））=-4.90J·mol^（-1）·K^（-1）,ΔS（（HSA-CRO））=-37.28J·mol^（-1）·K^（-1））demonstrated that CFZ and CRO can spontaneously bind with HSA through hydrogen bonds and van der Waals forces.Three-dimensional fluorescence spectroscopy and circular dichroism（CD）showed that CFZ and CRO could change the micro-environment and conformation of HSA.The results of molecular docking revealed that CFZ and CRO were located in Sudlow＇s site Ⅰ of HSA proven bycompetitive binding experiments.This study will be helpful to understand the mechanism which affects the conformation and function of HSA with CFZ and CRO in biological processes.