Combined fluorescence and electrochemical investigation on the binding interaction between organic acid and human serum albumin

Human serum albumin （HSA） is a plasma protein responsible for the binding and transport of fatty acids and a variety of exogenous chemicals such as drugs and environmental pollutants. Such binding plays a crucial role in determining the ADME （absorption, distribution, metabolism, and excretion） and bioavailability of the pollutants. The binding interaction between HSA and acetic acid （C2）, octanoic acid （C8） and dodecanoic acid （C12） has been investigated by the combination of site-specific fluorescent probe, tryptophan intrinsic fluorescence and tyrosine electrochemistry. For the study of the fatty acid interaction with the two drug-binding sites on HSA, two fluorescent probes, dansylamide and dansyl-L-proline were employed in the displacement measurements. Intrinsic fluorescence of tryptophan in HSA was monitored upon addition of the fatty acids into HSA. Electrocatalyzed response of the tyrosine residues in HSA by a redox mediator was used to investigate the binding interaction. Qualitatively, observations from these three approaches were very similar. HSA did not show any change in the fluorescence and electrochemical experiments after mixing with C2, suggesting there is no significant interaction with the short-chain fatty acid. For C8, the measured signal dropped in a single-exponential mode, indicating an independent and non-cooperative binding. The calculated association constant and binding ratio were 3.1 × 10^6 L/mol and 1 with drug binding Site Ⅰ, 1.1 × 107 L/mol and 1 with Site Ⅱ, and 7.0× 0^4 L/mol and 4 with the tryptophan site, respectively. The measurements with C12 displayed multiple phases of fluorescence change, suggesting cooperativity and allosteric effect of the C12 binding. These results correlate well with those obtained by the established methods, and validate the new approach as a viable tool to study the interactions of environmental pollutants with biological molecules.

Non-covalent binding analysis of sulfamethoxazole to human serum albumin:Fluorescence spectroscopy,UV-vis,FT-IR,voltammetric and molecular modeling

This study was designed to examine the interaction of sulfamethoxazole （SMZ） with human serum albumin（HSA）. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of human serum albumin by SMZ was static mechanism. The binding constant values for the SMZ-HSA system were obtained to be 22,500 L/mol at 288 K, 15,600 L/mol at 298 K, and 8500 L/mol at 308 K. The distance r between donor and acceptor was evaluated according to the theory of Foster energy transfer. The results of spectroscopic analysis and molecular modeling techniques showed that the conformation of human serum albumin had been changed in the presence of SMZ. The thermodynamic parameters, namely enthalpy change （ΔH^0） - 36.0 kJ/mol, entropy change （ΔS^0） - 41.3 Jim01 K and free energy change （ΔG^0） - 23.7 kJ/ mol, were calculated by using van＇t Hoff equation. The effect of common ions on the binding of SMZ to HSA was tested.

Investigation of binding behaviour of procainamide hydrochloride with human serum albumin using synchronous,3D fluorescence and circular dichroism

Interaction of procainamide hydrochloride（PAH） with human serum albumin（HSA） is of great significance in understanding the pharmacokinetic and pharmacodynamic mechanisms of the drug. Multi-spectroscopic techniques were used to investigate the binding mode of PAH to HSA and results revealed the presence of static type of quenching mechanism. The number of binding sites, binding constants and thermodynamic parameters were calculated. The results showed a spontaneous binding of PAH to HSA and hydrophobic interactions played a major role. In addition, the distance between PAH and the Trp–214 was estimated employing the F？rster＇s theory. Site marker competitive experiments indicated that the binding of PAH to HSA primarily took place in subdomain IIA（Sudlow＇s site I）. The influence of interference of some common metal ions on the binding of PAH to HSA was studied. Synchronous fluorescence spectra（SFS）, 3D fluorescence spectra and circular dichroism（CD） results indicated the conformational changes in the structure of HSA.

Fluorescence spectroscopy of osthole binding to human serum albumin

The interaction of human serum albumin （HSA） with osthole was investigated by fluorescence spectroscopy. Osthole can quench the fluorescence of HSA and the quenching mechanism is a static process. The binding site number n and apparent binding constant K were measured at different temperatures. The thermodynamic parameters △H^0, △G^0 and △S^0 were calculated at different temperatures. The results indicated that electrostatic forces played a major role in the interaction of osthole with HSA. Results of osthole synchronous fluorescence and UV absorption spectra showed that the microenvironment and conformation of HSA were changed.

Fluorescence Spectroscopy and Molecular Docking Approach to Probe the Interaction between Dehydroeburicoic Acid and Human Serum Albumin

The interaction between dehydroeburicoic acid (DeEA), a triterpene purified from medicinal fungi and the major transport protein, human serum albumin (HSA), were systematically studied by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy and molecular docking approach under simulated physiological conditions. The intrinsic fluorescence of HSA was quenched through the combination of static and dynamic quenching mechanism. DeEA cannot be stored and carried by HSA in the body at higher temperature. The hydrogen bonding, hydrophobic force and van der Waals force were major acting forces. The site II was the major binding site. The energy transfer could occur with high probability and the binding distance was 3.29 nm. The binding process slightly changed the conformation and microenvironment of HSA. The DeEA molecule entered the hydrophobic cleft of HSA and formed the hydrogen bonding with Glu-492 and Lys-545.

Characterization of Gallic Acid Interaction with Human Serum Albumin by Spectral and Molecular Modeling Methods

The binding of drugs with human serum albumin（HSA） is a crucial factor influencing the distribution and bioactivity of drugs in the body.To understand the action mechanisms between gallic acid（GA,3,4,5-trihydroxybenzoic acid） and HSA,the binding of GA with HSA was investigated by a combined experimental and computational approach.The fluorescence properties of HSA and the binding parameters of GA collectively indicate that the binding is characterized by static quenching mechanism at one high affinity binding site.According to the estimated molecular distance between the donor（HSA） and the acceptor（GA）,the binding is related to the fluorescence resonance energy transfer.As indicated by the thermodynamic parameters,hydrophobic interaction plays a major role in the GA-HSA complex.Further,the experimental results reveal that GA is bound in the large hydrophobic cavity of subdomain IIA in the site I of HSA,which is well approved by molecular docking.

Study on the Interaction between Human Serum Albumins and Methyl Pheophorbide-a-Gd

The binding reaction between methyl pheophorbide-a-Gd (MPA-Gd) and Human Serum Albumins (HSA) was studied by fluorescence and UV-Vis absorption spectra. The results indicated that the binding reaction of them was a single static quenching process, MPA-Gd strongly bound HSA, the binding equilibrium constant K0=2.298×105 L·mol-1 at 25 ℃. The shortest binding distance(r) and energy transfer efficiency(E) between donor (HSA) and acceptor (MPA-Gd) was obtained by Frster′s nonradiative energy transfer mechanism as follows: r=4.03 nm, E=0.12. The enthalpy change (ΔH) and entropy change (ΔS) were calculated at 25 and 37 ℃. The results indicated that the hydrogen bonds played major role in the reaction. Furthermore, the displacement experiments indicated that MPA-Gd could bind to the site Ⅱof HSA.

Interaction between a novel intramolecular charge transfer fluorescent probe PFEP and human serum albumin

The interaction mechanism between human serum albumin(HSA) and 1-phenyl-3(fluorenone-2-yl)-5-(9-ethylcarbazole-3-yl)-2- pyrazoline(PFEP) was investigated by fluorescence and absorption titration techniques in combination with molecular modeling method.Stern-Volmer plots at different temperatures proved that PFEP could quench the intrinsic fluorescence of HSA attributed to a static quenching procedure.The association constants were calculated in the range of 1×10~5-8×10~5mol^(-1) at different pH conditions,a...

Investigation of the interaction between indigotin and two serum albumins by spectroscopic approaches

The binding characteristics of indigotin with human serum albumin （HSA） and bovine serum albumin （BSA） have been investigated by various spectroscopic techniques. Spectroscopic analysis revealed that the quenching mechanism between indigotin and HSA/BSA belonged to the static quenching. The displacement experiments suggested that indigotin primarily bound to tryptophan residues on proteins within site I. The thermodynamic parameters indicated that the binding of indigoti^HSA/BSA mainly depended on the hydrophobic interaction. The binding distance of indigotin to HSA/BSA was evaluated. The results by synchronous fluorescence, three- dimensional fluorescence, Fourier Transform Infrared spectroscopy （FT-IR） and circular dichroism （CD） spectra showed that the conformation of proteins altered in the presence of indigotin.

Insight into the interaction of inhaled corticosteroids with human serum albumin: A spectroscopic-based study

It is well known that the safety and efficacy profile of an inhaled cortocosteroid(ICS) is influenced by the pharmacokinetic properties and associated pharmacodynamic effects of the drug. Freely circulating,protein unbound, and active ICS can cause systemic adverse effects. Therefore, a detailed investigation of drug-protein interaction could be of great interest to understand the pharmacokinetic behaviour of corticosteroids and for the design of new analogues with effective pharmacological properties. In the present work, the interaction between some corticosteroids and human serum albumin(HSA) has been studied by spectroscopic approaches. UV–Vis spectroscopy confirmed that all the investigated corticosteroids can bind to HSA forming a protein-drug complex. The intrinsic fluorescence of HSA was quenched by all the investigated drugs, which was rationalized in terms of a static quenching mechanism. The thermodynamic parameters determined by the Van't Hoff analysis of the binding constants(negative ΔH and ΔS values) clearly indicate thathydrogen bonds and van der Waals forces play a major role in the binding process between albumin and betamethasone, flunisolide and prednisolone, while hydrophobic forces may play a major role in stabilizing albumin-triamcinolone complexes.

Binding interactions of pefloxacin mesylate with bovine lactoferrin and human serum albumin

The binding of pefloxacin mesylate （PFLX） to bovine lactoferrin （BLf） and human serum albumin （HSA） in dilute aqueous solution was studied using fluorescence spectra and absorbance spectra. The binding constant K and the binding sites n were obtained by fluorescence quenching method. The binding distance r and energy-transfer efficiency E between pefloxacin mesylate and bovine lactoferrin as well as human serum albumin were also obtained according to the mechanism of Forster-type dipole-dipole nonradiative energy-transfer. The effects of pefloxacin mesylate on the conformations of bovine lactoferrin and human serum albumin were also analyzed using synchronous fluorescence spectroscopy.

(E)-2-Benzylidenecycloalkanones XII.*Kinetic Measurement of Bovine and Human Serum Albumine Interaction with Selected Chalcones and Their Cyclic Chalcone Analogues by UV Spectrophotometry

UV-VIS spectroscopic investigations of interaction of bovine and human serum albumin with selected chalcones (1) and their cyclic chalcone analogues: (E)-2-(4’-X-benzylidene-1-tetralones (3), benzosuberones (4) with dimethylamino and methoxy substituents and (E)-2-(2’,4’-dimethox- ybenzylidene)-1-indanone (2) were performed in polar respiration medium. Absorption maxima of the tested compounds were investigated in the presence of bovine and human serum albumin at the 0, 10, 30 and 60 minute timepoints of the interaction. The absorbance of all studied compounds in the presence of proteins decreased after one hour of the reaction. Molecule 4a showed the strongest and fastest kinet initial interaction with both albumins.

Analysis of Binding Interaction between Captopril and Human Serum Albumin

The interaction between captopril, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by captopril. The binding constants and the number of binding sites can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG0 reveals that the binding process is a spontaneous process. According to the van’t Hoff equation, the standard enthalpy change (ΔH0) and standard entropy change (ΔS0) for the reaction were calculated to be 35.98 KJ●mol-1 and 221.25 J●mol-1 K. It indicated that the hydrophobic interactions play a main role in the binding of captopril to human serum albumin. In addition, the distance between captopril (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 1.05 nm according to the F?rster’s resonance energy transfer theory. The results obtained herein will be of biological significance in pharmacology and clinical medicine.

Multi-spectroscopic characterization of bovine serum albumin upon interaction with atomoxetine

The quenching interaction of atomoxetine(ATX) with bovine serum albumin(BSA) was studied in vitro under optimal physiological condition(pH=7.4) by multi-spectroscopic techniques. The mechanism of ATX-BSA system was a dynamic quenching process and was confirmed by the fluorescence spectra and lifetime measurements. The number of binding sites, binding constants and other binding characteristics were computed. Thermodynamic parameters ΔH^0 and ΔS^0 indicated that intermolecular hydrophobic forces predominantly stabilized the drug-protein system. The average binding distance between BSA and ATX was studied by F?rsters theory. UV-absorption, Fourier transform infrared spectroscopy(FT-IR), circular dichroism(CD), synchronous spectra and three-dimensional(3D) fluorescence spectral results revealed the changes in micro-environment of secondary structure of protein upon the interaction with ATX. Displacement of site probes and the effects of some common metal ions on the binding of ATX with BSA interaction were also studied.

环氧虫啶与人血清白蛋白的相互作用

新烟碱类杀虫剂环氧虫啶对非靶标生物具有潜在危害,但对其毒理机理、其在人体内的运输机制等方面缺乏研究。本研究运用荧光光谱法、分子对接、分子动力学、结合自由能计算和丙氨酸扫描等方法,研究了环氧虫啶与人血清白蛋白(HSA)的结合模式。结果表明:1)环氧虫啶能有效猝灭HSA荧光,不同温度下环氧虫啶与HSA的结合常数在0.76×10^(5)~1.57×10^(5)L/mol之间,具有较强的结合能力;2)环氧虫啶结合在HSA的IIA疏水腔内,有1个结合位点;3)结合自由能分析和热力学参数计算显示,环氧虫啶和HSA结合的主要作用力是范德华力和氢键作用;4)分子动力学模拟结果显示,二者结合自由能为-25.90 kJ/mol,形成了相对稳定的复合物;5)丙氨酸突变扫描结果显示,氨基酸残基Gln459是环氧虫啶与HSA结合的关键氨基酸。该研究结果可为阐明环氧虫啶在人体内的毒性机理提供理论依据。

荧光增白剂与人血清白蛋白相互作用的多种光谱法及分子对接技术研究

通过多种光谱方法与分子对接技术,从分子水平上对两种荧光增白剂(C.I.135、C.I.185)与人血清白蛋白(HSA)的相互作用机制进行研究。稳态荧光光谱及紫外-可见光谱实验结果表明,C.I.135、C.I.185与HSA均形成了基态复合物(HSA-C.I.135、HSA-C.I.185),猝灭机理为静态猝灭机理,热力学参数表明氢键与范德华力是结合作用的主要作用力,并且结合过程都是自发进行。三维荧光光谱结果表明C.I.135、C.I.185可以改变HSA的氨基酸残基微环境和构象,C.I.185对HSA产生的影响是最大的。分子模拟实验进一步验证上述实验结果。

新型黄酮探针检测人血清白蛋白

人血清白蛋白是人血浆中的主要蛋白质,在人体循环系统中发挥着重要的生理作用,例如调节血液渗透压、运输营养物质和代谢物、抗氧化等。研究表明白蛋白含量异常与多种疾病有关,例如心血管疾病、糖尿病、肝癌等。因此,白蛋白含量的检测具有十分重要的意义。本工作通过有机合成获得新型黄酮类荧光探针,该探针本身在水溶液中不发射荧光,当白蛋白存在时,荧光增强超过1 000倍,探针在10 min内完成响应,检测限为8.36 nM。通过计算机辅助分子对接计算分析验证了探针的响应机理,结果表明,探针能够进入白蛋白的疏水空腔,形成分子间氢键,限制了分子的自由旋转,从而实现荧光增强。

3,4-二甲氧基查尔酮荧光探针用于人血清白蛋白的检测

人血清白蛋白(HSA)是人血清中含量最丰富的蛋白质,它参与许多重要的生理过程,也与某些疾病息息相关,是临床诊断的重要生物标志物.本文以结构简单、合成简便且荧光性质易调控的查尔酮化合物为荧光探针母体,通过在查尔酮的A环引入2个甲氧基获得一种用于HSA检测的D-π-A型荧光探针3,4-二甲氧基查尔酮(DDP);经1H NMR和13C NMR及MS表征确证了其结构.查尔酮A环甲氧基的引入可以调控探针的推拉电子效应及其对HSA的荧光响应性能.进一步利用紫外-可见吸收光谱和荧光光谱等方法研究了DDP的光谱性质及其与HSA的相互作用.DDP的荧光光谱分析结果表明,其荧光对溶剂的极性非常敏感,其荧光发射波长随溶剂极性增大而发生红移.DDP与HSA响应后荧光增强73倍,响应前后的荧光量子产率分别为0.2%和4.0%.DDP对HSA的响应有优异的选择性,响应速度快且响应后荧光稳定.经Job’s plot和荧光滴定实验确定了DDP与HSA结合的化学计量比为1∶1,检出限(LOD)为40.32 nmol/L,结合常数为3.95×10^(5) L/mol;并通过置换实验证明了DDP的结合位点是HSA的DS2区.该探针可实现对HSA的高灵敏度、高选择性检测.

Spectroscopic Investigation of the Interaction between Human Serum Albumins and 10-Hydroxycamptothecin

The binding reaction between 10-hydroxycamptothecin （10-HCPT） and human serum albumins （HSA） is studied by means of fluorescence spectroscopy, UV-Vis absorption spectrum, IH NMR spectrum, and molecular simulation. The results indicate that the binding reaction of 10-HCPT and HSA is a single static quenching process, and the binding equilibrium constant for 10-HCPT binding with HSA is estimated K0-4.93×10^4 L · mol-I at 25 ℃ with the molar ratio of I ： 1. The distance （r） and energy transfer efficiency（E） between donor （HSA） and acceptor （10-HCPT） are obtained as follows, r=3.51 nm; E-0.27. The enthalpy change （△Hφ） and entropy change （△Sφ） are calcu- lated at different temperatures, and the hydrophobic force and shidipole force are the functions in the reaction. The results show that 10-HCPT binds within the subdomain II A of HSA by the hydrophobic force, and the 10-OH and 20-OH of 10-HCPT bind with both residue Leu-238 of HSA and Ala 291 of HSA by hydrogen bonds.

荧光光谱法研究三种金属卟啉与人血清白蛋白的结合反应

近年来,卟啉及金属卟啉在光动力疗法、抗癌药物研究方面备受关注,有些已被批准临床使用。人血清白蛋白(HSA)能结合、运输许多药物活性分子,详细研究金属卟啉与HSA的结合机制,对阐明卟啉类药物的作用机制具有重要的意义。研究合成了3种新型6-氯烟酸修饰的自由卟啉(4,5,6)及锌卟啉(4-Zn,5-Zn,6-Zn),并通过测试表征和理论计算的方法进行了结构确定。理论计算结果表明:3种锌卟啉中的侧链6-氯烟酸基团均远离卟啉环平面;4-Zn构型比连有取代基的5-Zn、6-Zn构型更稳定。在模拟生理条件下,采用荧光光谱法研究了3种锌卟啉与HSA之间的键合方式,通过Stern-Volmer方程、双对数方程和Van’t Hoff方程对测定结果进行计算。结果表明:(1)3种锌卟啉均能有效猝灭HSA的荧光,Stern-Volmer方程计算得到锌卟啉与HSA在不同温度下的K_(q)值均大于2.0×10^(10) L·mol^(-1)·s^(-1),表明猝灭类型为静态猝灭;(2)采用双对数方程得到锌卟啉与HSA的结合常数,除5-Zn在318 K时外,其他温度条件下的结合常数K_(A)均大于10^(3) L·mol^(-1),结合位点数n均接近1,说明形成了1∶1的复合物;(3)根据Van’t Hoff方程计算,3种锌卟啉与HSA结合的热力学参数均小于0,如4-Zn与HSA结合的热力学参数为ΔH=-123.9 kJ·mol^(-1),ΔS=-322.9 J·mol^(-1)·K^(-1),ΔG=-27.7 kJ·mol^(-1)(298 K),由此得出反应过程为自发进行,反应的主要作用力为范德华力和氢键。实验获得的数据可以为研究金属卟啉与生物小分子的作用机制提供一些有用的信息。