Separate and simultaneous binding effects of aspirin and amlodipine to human serum albumin based on fluorescence spectroscopic and molecular modeling characterizations: A mechanistic insight for determining usage drugs doses

The binding of aspirin (ASA) and amlodipine (AML) to human serum albumin (HSA) in aqueous solution was investigated by multiple techniques such as fluorescence quenching, resonance light scattering (RLS), three-dimensional fluorescence spectroscopy, FT-IR and zeta-potential measurements in an aqueous solution at pH=7.4. For the protein-ligand association reaction, fluorescence measurements can give important clues as to the binding of ligands to proteins, e.g., the binding mechanism, binding mode, binding constants, binding sites, etc. Fluorescence spectroscopy showed that ASA and AML could quench the HSA fluorescence spectra, and this quenching effect became more significant when both ASA and AML coexisted. The results pointed at the interaction between HSA and both drugs as ternary systems decreasing the binding constant and binding stability of the HSA-drug complex as a binary system. Therefore, by reducing the amount of drugs transported to their targets, the free drug concentration of the target would be reduced, lowering the efficacy of the drugs. It was demonstrated that there exists antagonistic behavior between the two drugs when it comes to binding of HSA. Furthermore, the fluorescence results also showed that the quenching mechanism of HSA-drug complexes as binary and ternary systems is a static procedure. The number of binding sites of HSA-ASA, (HSA-AML)ASA, HSA-AML and (HSA-ASA) AML were 1.31, 0.92, 1 and 0.93, respectively. Due to the existence of the antagonistic action between ASA and AML, the binding distance r was reduced. The results of synchronous fluorescence and three-dimensional fluorescence spectra showed that the antagonistic action between ASA and AML would alter the micro-environment around Trp and Tyr residues. Moreover, the simultaneous presence of ASA and AML during binding to HSA should be taken into account in multidrug therapy, as it induces the necessity of a monitoring therapy owing to the possible increase of uncontrolled toxic effects. Molecular dynamic studies showed that the affinity of each of the drugs to HSA was reduced in the presence of significant amounts of the other. In the interaction of HSA with both drugs, the zeta potential of the ternary system is more negative than its binary counterpart. The zeta-potential results suggested induced conformational changes on HSA that confirmed the experimental and theoretical results.     

Characterization of the Binding of Metoprolol Tartrate and Guaifenesin Drugs to Human Serum Albumin and Human Hemoglobin Proteins by Fluorescence and Circular Dichroism Spectroscopy

The interactions of metoprolol tartrate (MPT) and guaifenesin (GF) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins at pH?7.4 were studied by fluorescence and circular dichroism (CD) spectroscopy. Drugs quenched the fluorescence spectra of HSA and HMG proteins through a static quenching mechanism. For each protein-drug system, the values of Stern-Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were determined at 288.15, 298.15, 310.15 and 318.15 K. It was found that the binding constants of HSA-MPT and HSA-GF systems were smaller than those of HMG-MPT and HMG-GF systems. For both drugs, the affinity of HMG was much higher than that of HSA. An increase in temperature caused a negative effect on the binding reactions. The number of binding site on blood proteins for MPT and GF drugs was approximately one. Thermodynamic parameters showed that MPT interacted with HSA through electrostatic attraction forces. However, hydrogen bonds and van der Waals forces were the main interaction forces in the formation of HSA-GF, HMG-MPT and HMG-GF complexes. The binding processes between protein and drug molecules were exothermic and spontaneous owing to negative ?H and ?G values, respectively. The values of binding distance between protein and drug molecules were calculated from Förster resonance energy transfer theory. It was found from CD analysis that the bindings of MPT and GF drugs to HSA and HMG proteins altered the secondary structure of HSA and HMG proteins.     

光谱法研究抗氧化剂、DPPH与人血清蛋白三元体系的作用

选取几种天然抗氧化剂杨梅素、桑色素、辣椒碱、甜菜碱作为研究对象,运用荧光光谱法、同步荧光光谱法以及三维荧光光谱法研究了几种抗氧化剂以及DPPH自由基与人血清蛋白相互作用,结果表明辣椒碱、甜菜碱、V_C不与人血清蛋白发生猝灭反应,杨梅素、桑色素、DPPH均能够与人血清蛋白发生猝灭反,反应均为形成了稳定复合物而导致的静态猝灭,通过疏水作用力与HSA结合,结合位点数均为1,主要结合位点在色氨酸基团附近,DPPH与人血清蛋白猝灭过程改变了人血清蛋白结构的疏水性,引起蛋白质构象发生变化,而杨梅素、桑色素与人血清蛋白相互作用未造成其构象发生了变化。运用荧光光谱法研究了几种抗氧化剂抑制DPPH直接损伤人血清蛋白的能力,杨梅素、桑色素、辣椒碱、甜菜碱、V_C对DPPH损伤HSA的抑制率分别为25%,18.30%,85.38%,4.02%和84.58%。根据分子结构分析辣椒碱主要通过清除DPPH自由基作用从而抑制其损伤人血清蛋白,根据二元体系反应结果可知杨梅素与桑色素三元体系反应过程中两种抗氧化剂与DPPH竞争结合位点,因此杨梅素、桑色素主要通过占据结合位点的方式抑制DPPH损伤人血清蛋白,而甜菜碱既不能占据结合位点也不能清除自由基,因而抑制能力最弱。分析表明几种天然抗氧化剂的抑制能力与其分子结构中主要官能团结构密切相关。     

秋水仙碱与人血清白蛋白相互作用的谱学研究

采用紫外、荧光和圆二色光谱研究了秋水仙碱与人血清白蛋白之间的相互作用。结果发现秋水仙碱使人血清白蛋白的紫外吸收增强,特征荧光峰猝灭,并且随温度升高猝灭常数K_SV降低。求算了不同温度下秋水仙碱与人血清白蛋白相互作用的平衡常数与结合位点数。根据Van’t Hoff方程计算出ΔH=-11.66 kJ·mol-1,ΔS=51.507 J·(mol·K)-１,得出二者之间的作用力主要是静电作用力。圆二色光谱测得加入秋水仙碱后人血清白蛋白的α-螺旋降低,二级结构改变,表明秋水仙碱对人血清白蛋白的荧光猝灭机制属于形成配合物所引起的静态猝灭。     

三种异黄酮类药物与不同异构体人血清白蛋白的作用机制研究

利用荧光光谱研究了三种异黄酮类化合物染料木素、鸡豆黄素A和3’,4’,7-三羟基异黄酮与不同异构体人血清白蛋白的相互作用机制。计算了异黄酮与蛋白质形成复合物的各种结合参数(猝灭速率常数、结合常数及结合位点数),结果表明三种异黄酮类化合物在人血清白蛋白上只有一个结合位点,位于结合位点siteⅠ,结合常数在0.17×105～1.20×105 L.mol-1之间。荧光增强光谱结果显示,与蛋白质作用后药物的荧光强度明显增加,说明了药物与人血清白蛋白发生了结合,在此基础上讨论了三种异黄酮与人血清白蛋白的结合机理。     

Studies on the antagonistic action between chloramphenicol and quinolones with presence of bovine serum albumin by fluorescence spectroscopy

Chloramphenicol (CHL) and quinolone drugs like ofloxacin (OFLX), lomefloxacin (LMX) and ciprofloxacin (CPFX) can all quench the fluorescence of bovine serum albumin (BSA) in the aqueous solution of pH=7.40. This quenching effect becomes more significant when CHL and quinolone drugs coexist. Based on this, further studies on the interactions between CHL and quinolone drugs using fluorescence spectrum are established. The results showed that the interaction between the drugs would increase the binding constant and binding stability of the drug and protein, thus reducing the amount of drugs transported to their targets. Therefore, free drug concentration at targets would decrease, reducing the efficacy of the drugs. It indicated that there exists antagonistic action between drugs. The results also showed that the quenching mechanism of BSA by the drugs is a static procedure. The number of binding sites is 1 in various systems. Due to the existence of the antagonistic action between drugs, the binding distance r is reduced. Studies utilizing synchronous spectra showed that the antagonistic action between the drugs would affect the conformation of BSA, making protein molecules extend and hydrophobic decrease. The order of antagonistic action between CHL and quinolone drugs is: CPFX>OFLX>LMX with presence of BSA.     

头孢噻肟钠和氯霉素与牛血清白蛋白相互作用的荧光光谱分析

在pH=7.40 Tris-HCl缓冲溶液中,应用荧光光谱法分别研究了298,303,308 K时,头孢噻肟钠(CTX)、氯霉素(CHL)对牛血清白蛋白(BSA)荧光的猝灭反应.结果表明:药物与BSA的结合稳定常数Ka随温度增加而降低,两种药物对BSA的荧光猝灭皆为静态猝灭过程.标记竞争实验表明CTX、CHL在BSA中...     

芦丁与血清白蛋白结合作用的热力学研究(I)

在生理pH值条件下,研究了芦丁与牛血清白蛋白和人血清白蛋白之间的结合作用。通过荧光法确定了芦丁与血清白蛋白的荧光猝灭机制,根据热力学方程讨论了两者间的主要作用力类型。芦丁对血清白蛋白的荧光猝灭机制为静态猝灭。确定了不同温度下该结合反应的结合常数和结合位点数,并根据热力学方程求得了结合反应的热力学参数。两者结合的主要作用力类型是氢键和Vander Waals力。芦丁在体内能够被血清白蛋白存储和转运,且结合时对蛋白质构象无影响。     

Characterization of the interaction between human lactoferrin and lomefloxacin at physiological condition: Multi-spectroscopic and modeling description

The interaction between lomefloxacin (LMF) and human lactoferrin (Hlf) was studied by using fluorescence, circular dichroism (CD) spectroscopic and molecular modeling measurements. By the fluorescence quenching results, it was found that the binding constant K_A=8.69×10⁵ L mol⁻¹, and number of binding sites n=1.75 at physiological condition. Experimental results observed showed that the binding of LMF to Hlf induced conformational changes of Hlf. The participation of tyrosyl and tryptophanyl residues of protein was also estimated in the drug-Hlf complex by synchronous fluorescence. The quantitative analysis data of far-UV CD spectra from that of the α-helix 37.4% in free Hlf to 30.2% in the LMF-Hlf complex further confirmed that secondary structure of the protein was changed by LMF. Near-UV CD showed perturbations around tryptophan and tyrosine residues which involves perturbations of tertiary structure. The thermodynamic parameters like, ΔH° and ΔS°, have been calculated to be 63.411 kJ mol⁻¹ and 231.104 J mol⁻¹ K⁻¹, respectively. Thermodynamic analysis showed that hydrophobic interactions were the main force in the binding site but the hydrogen bonding and electrostatic interaction could not be excluded which in agreement with the result of molecular docking study. The distance r between donor and acceptor was obtained according to fluorescence resonance energy transfer (FRET) and found to be 1.78 nm. The interaction between LMF and Hlf has been verified as consistent with the static quenching procedure and the quenching mechanism is related to the energy transfer. Furthermore, the study of molecular modeling that LMF could bind to the α-helixes between Pro145-Asn152 and Phe167-Gln172 regions and hydrophobic interaction was the major acting force for the binding site, which was in agreement with the thermodynamic analysis.     

Transport of a Cancer Chemopreventive Polyphenol,Resveratrol: Interaction with Serum Albumin and Hemoglobin

Resveratrol is a natural phytoalexin with pharmacologic effects on several human diseases: carcinogenesis, coronary heart disease and neurodegenerative disease. Due to its poor water solubility, resveratrol must be bound to proteins to keep it at a high concentration in serum. In our work, the bindings of resveratrol to plasma proteins, human serum albumin (HSA) and hemoglobin (Hb), have been investigated systematically by fluorescence quenching technique, synchronous fluorescence, UV–vis absorption spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling method. The fluorescence data show that the binding of resveratrol to HSA or Hb is a static quenching procedure and each protein has only one binding site for the drug. The binding constant of resveratrol to HSA is larger than that of resveratrol to Hb at corresponding temperature, which indicates that the affinity of HSA toward the drug is higher than that of Hb. The CD spectroscopy indicates that the secondary structures of the proteins are changed in the presence of resveratrol with the reduction of α-helices, which decreased about 18.75% for HSA and 9.43% for Hb at the drug to proteins molar ratio of 2. Thermodynamic analysis and molecular modeling suggest that hydrophobic interaction plays a major role in the binding of resveratrol to HSA, and hydrogen bonding is the mainly binding force in the binding of resveratrol to Hb. The study of molecular modeling shows that resveratrol is located in the hydrophobic cavity between subdomain IB and IIA of HSA (the entrance of site I), or located in the central cavity of Hb (partial to the subunit A).     

Optical, Structural and Thermodynamic Studies of the Association of an Anti-leucamic Drug Imatinib Mesylate with Transport Protein

The interaction of an anti-leukemic drug, imatinib mesylate (IMT) with human serum albumin (HSA) was investigated by fluorescence, synchronous fluorescence, three-dimensional fluorescence, circular dichroism and UV–vis absorption techniques under physiological condition. The process of binding of IMT on HSA was observed to be through a spontaneous molecular interaction procedure. IMT effectively quenched the intrinsic fluorescence of HSA via static quenching. The values of binding constant, number of molecules that interact simultaneously with the binding site and thermodynamic parameters were evaluated by carrying out the interactions at three different temperatures. Based on thermodynamic parameters and displacement studies with site probes, it was proposed that the drug bound at Sudlow’s site I of subdomain IIA. The change in the conformation of HSA was evident from synchronous, three-dimensional fluorescence and circular dichroism studies. The distance between the donor (protein) and acceptor (drug) was calculated based on the Foster’s theory of resonance energy stransfer and it was found to be 1.30 nm. The effect of different metal ions on the binding of the drug to protein was also investigated.     

叶酸与人血清白蛋白结合作用的光谱研究

在不同温度下的pH 7.4的Tris-HCl缓冲溶液体系中, 采用荧光光谱、紫外吸收光谱和同步荧光光谱研究了人血清蛋白与叶酸的相互作用。研究表明,这种相互作用使人血清白蛋白发生内源荧光猝灭,属于静态猝灭机制。通过计算得到人血清蛋白与叶酸在17和37 ℃下静态猝灭的猝灭速率常数分别为7.396 6×104和7.2652×104 L·mol-1、结合常数分别为7.50×104和1.98×105 L·mol-1、结合位点数均为1。根据Förster非辐射能量转移机理,求算出给体(HSA)与受体(叶酸)间的作用距离和能量转移效率分别为1.77和0.052 65 nm,并结合热力学参数说明了叶酸分子与人血清白蛋白的作用以疏水作用为主,同时也存在静电引力。利用同步荧光光谱研究了人血清蛋白与叶酸的相互作用中HSA的构象变化,发现色氨酸残基所处环境的疏水性降低,说明叶酸分子进入了人血清白蛋白的疏水腔中。     

环丙沙星、氧氟沙星同时与牛血清白蛋白分子作用的荧光光谱

在pH=7.40的水溶液中,环丙沙星(CPFX)、氧氟沙星(OFLX)能够猝灭牛血清白蛋白(BSA)的荧光。当两种药物共存时BSA荧光被进一步猝灭。据此建立了利用荧光发光光谱法进行喹诺酮类药物CPFX与OFLX间相互作用的研究。结果表明:药物间存在相互作用,使药物与蛋白间的结合常数减小、结合稳定性下降,游离型药物含量增加,造成药效增强;药物对蛋白荧光的猝灭属于静态猝灭,药物与蛋白结合位点数约为1。根据Frster非辐射能量转移理论,确定了药物与蛋白之间的结合距离r7nm,属于非辐射能量转移。药物间的相互作用使r值增加,结合距离增大。同步荧光光谱研究表明药物间的相互作用对蛋白构象产生影响,使蛋白质分子伸展,疏水性降低。     

荧光法研究PTB与白蛋白的结合

利用药物对蛋白的荧光猝灭作用,用荧光法研究了N-苯酰甲噻唑溴(PTB)与牛血清白蛋白(BSA)及人血清白蛋白(HSA)的相互作用。测定发现BSA溶液的最大激发波长为280 nm,HSA溶液的最大激发波长为290 nm。分别向溶液中加入PTB后,原有的最大发射波长处的强度明显减弱。说明PTB对BSA和HAS有荧光猝灭作用。PTB与BSA,HSA有中等强度的结合。测得15 ℃时PTB与BSA,HSA的结合常数分别为3.66×103和3.83×103,结合位点数n分别为1.02和1.16;37 ℃时PTB与BSA,HSA的结合常数分别为3.58×103和3.35×103,结合位点数分别为0.95和0.87。根据热力学常数确定了PTB与BSA,HSA之间的主要作用力类型均为静电作用力。通过Fster偶级-偶级非辐射能量转移原理,得到BSA,HSA与PTB结合的位置距色氨酸残基的距离分别为7.5和7.9 nm。根据白蛋白的结构,可以推测BSA,HSA与PTB结合的位点在ⅡA亚结构域,靠近Try214的区域。     

光谱法研究碳量子点与人血清白蛋白的相互作用

近年来,纳米材料对环境和人类健康的潜在风险已经引起了人们的广泛关注。纳米颗粒尺寸小、反应活性高,当其进入机体后很可能与体内的生物大分子相互作用,使其原来的理化性质发生改变,更容易被细胞识别、吞噬,进而对组织、器官等产生危害;作用过程中生物大分子也会受到纳米颗粒的影响,可能导致其生理结构受到损伤,并干扰其特定功能的正常执行;因此研究纳米材料与蛋白质的相互作用对探究其生物安全性具有重要意义。实验通过多种光谱手段研究了碳量子点（CQDs）对人血清白蛋白（HSA）结构与功能性质的影响以及两者的相互作用机制。荧光光谱与紫外-可见吸收光谱结果显示,CQDs通过与HSA结合形成复合物导致蛋白质荧光猝灭;结合常数K_A与结合位点数n的计算结果表明CQDs在HSA上只有一个结合位点,且同步荧光和位点竞争实验发现,这个结合位点接近HSA中ⅡA亚结构域上的色氨酸残基;根据Förster共振能量转移(FRET)理论计算出两者的作用距离r=2.89 nm<8 nm,表明HSA与CQDs之间通过非辐射能量转移导致蛋白质内源性荧光猝灭;根据Van’t Hoff方程计算得到HSA-CQDs体系的热力学参数,由热力学参数计算结果推断相互作用过程中主要有氢键和范德华力参与;根据共振光散射（RLS）、三维荧光光谱和圆二色光谱（CD）结果显示,相互作用会导致HSA的二级、三级结构发生改变,蛋白质中α-螺旋结构含量增加,HSA进一步卷曲折叠,使色氨酸残基所处微环境的疏水性增大;而结构变化还进一步影响着蛋白质的聚集状态和一些生理功能,使得相互作用后HSA的聚集程度和类酯酶活性降低,不利于蛋白质的团聚,自由基清除能力略有提高。该研究可为纳米材料的生物与环境安全评价提供参考依据,也为探究纳米颗粒-蛋白质相互作用提供了一套较为系统的光谱分析方法。     

光谱法研究抗癌药物槲皮素和人血清白蛋白的相互作用

研究槲皮素与人血清白蛋白(HSA)的相互作用,以阐明槲皮素在体内的运输过程。利用荧光光谱和紫外吸收光谱进行实验,采用Stern-Volmer方程,Lineweaver-Burk双倒数方程和双对数回归曲线模型处理实验数据,求出了不同温度下槲皮素与HSA的结合常数、结合位点数和相关热力学参数。随槲皮素浓度的增大,HSA在210nm和280nm处的紫外吸收峰增强,HSA的内源荧光强度被猝灭。槲皮素和HSA作用后形成了新的复合物,静态猝灭是槲皮素对HSA荧光猝灭的主要原因。不同模型得到的结合常数和热力学参数有所不同,都表明槲皮素和HSA二者之间具有较强的作用力,其相互作用是自发进行的。抗癌药物槲皮素是可以被HSA所储存和运输的。     

抗艾滋病药物司他夫定与血液蛋白相互作用的研究

研究药物和血液中载体蛋白的相互作用对阐明药物在体内的转运、分布、代谢和药效等具有重要意义。运用稳态荧光、紫外-可见吸收光谱、动力学瞬态发射光谱和循环伏安法研究了抗艾滋病(HIV)药物司他夫定(stavudine,D4T)对人血清白蛋白(HSA)、牛血清白蛋白(BSA)和血红蛋白(Hb)三种血液蛋白的荧光猝灭机制,均为静态猝灭;得出不同温度(300 K,310 K,320 K)下D4T和载体的结合常数K_a(K_a的大小顺序为Hb＞HSA＞BSA)和结合位点数n(n均为1);分析二者结合过程的热力参数ΔH,ΔS和ΔG,三种血液蛋白均为ΔG＞0,ΔH＞0,说明D4T和载体的结合是一种自发的放热过程,同时由ΔH＜0,ΔS＜0,推测出D4T与HSA,BSA和Hb之间的结合力都为氢键和范德华力;根据Frster非辐射能量转移理论(FRET)分析了供体(蛋白)和受体(D4T)之间发生能量转移的可能性并计算了结合距离R₀和r,其中r＜7 nm且0.5R₀＜r＜1.5R₀,表明从HSA,BSA和Hb到D4T之间发生能量转移的可能性很大。同时利用同步荧光,三维荧光和圆二色谱法得出,D4T与载体结合时对载体(HSA,BSA和Hb)的二级结构无影响,且三级构象变化不大。通过本文实验可知,HSA,BSA和Hb三种血液蛋白均可作为运输D4T到靶位置的良好载体蛋白,这些结果为更深入研究D4T药物分子设计和抗HIV作用的应用提供有利的实验依据。     

3,3',4',7-四羟基黄酮醇与人血清白蛋白结合的光谱学表征

应用荧光光谱和紫外光谱对3,3’,4’,7-四羟基黄酮醇(FIS)与人血清白蛋白(HSA)的结合机理进行了表征。在生理pH7.4下,FIS对HSA的内源荧光有显著的猝灭现象,在实验浓度范围内(药物与蛋白质浓度比0.1至10之间)其荧光猝灭机理主要是静态猝灭。研究结果表明,FIS和HSA之间形成了1∶1的复合物,结合常数为(1.05±0.18)×105L·mol-1。利用紫外光谱研究了FIS在不同pH值条件下的解离行为,发现FIS在生理条件下以离子和中性分子的混合形式存在。与蛋白质的结合使FIS的紫外吸收光谱Ⅰ带发生了明显的红移(与中性分子相比红移幅度超过40nm),证明了药物分子离子状态以静电力与蛋白质发生结合。其紫外光谱的二阶导数谱显示,药物分子与蛋白质的结合可分为特征和非特征形式。由于激发态质子转移,与蛋白质的相互作用引起了FIS内源荧光发射峰强度的明显增加,进一步证实了它们与蛋白质的结合。     

Binding studies of costunolide and dehydrocostuslactone with HSA by spectroscopy and atomic force microscopy

Human serum albumin (HSA), a major plasma protein and plasma-derived therapeutic, interacts with a wide variety of drugs and native plasma metabolites. In this study the interactions of costunolide (CE) and dehydrocostuslactone (DE) with HSA were investigated by molecule modeling, atomic force microscopy (AFM), and different optical techniques. In the mechanism discussion, it was proved that fluorescence quenching of HSA by both of the drugs is a result of the formation of drug-HSA complexes. Binding parameters for the reactions were determined according to the Stern-Volmer equation and static quenching. The results of thermodynamic parameters ΔG⁰, ΔH⁰, and ΔS⁰ at different temperatures indicated that hydrogen bonding interactions play a major role in the drug-HSA associations process. The binding properties were further studied by quantitative analysis of CD, FTIR, and Raman spectra. Furthermore, AFM results showed that the dimension of HSA molecules became more swollen after binding with the drugs.     

光谱法和分子对接模拟技术研究左氧氟沙星与人血清白蛋白的相互作用

左氧氟沙星(LVFX)是临床上普遍使用的一种抗生素,对革兰氏阳性菌及革兰氏阴性菌引起的各种感染都有一定的作用。人血清白蛋白(HSA)是血液循环系统中最丰富的运输蛋白,能与多种内源及外源性物质结合,起着储存和转运的作用。因此详细研究LVFX与HSA间的相互作用对了解LVFX的药代动力学行为具有重要意义。运用光谱法和分子对接模拟技术研究左氧氟沙星和人血清白蛋白的相互作用。结果表明,LVFX对HSA的荧光淬灭作用为形成复合物导致的静态猝灭,结合常数为9.44×104 L·mol-1 (294 K)和2.72×104 L·mol-1 (310 K),结合位点数均为1,两者间的主要作用力为氢键和范德华力。取代实验表明,LVFX在HSA的Site Ⅰ,ⅡA 子域上有一个结合位点。根据Frster理论得到的LVFX和色氨酸(Trp)残基间的结合距离为3.66 nm,这一结果与分子对接模拟技术得到的结果相一致。紫外差谱,三维荧光光谱和红外光谱都进一步表明LVFX能够改变HSA的结构。采用傅里叶变换红外光谱法对LVFX与HSA作用前后HSA二级结构的变化进行了定量分析,结果表明,当加入LVFX后HSA的α-螺旋结构有所降低,β-折叠结构、β-转角结构和无规则卷曲有所上升,说明LVFX能使HSA的二级结构变得松散。