秋水仙碱与人血清白蛋白相互作用的光谱学和电化学研究

用荧光光谱、紫外吸收光谱和电化学方法研究了在近似生理条件下秋水仙碱与人血清白蛋白的相互作用.研究表明,秋水仙碱对人血清白蛋白的荧光猝灭主要是静态猝灭过程,秋水仙碱使人血清白蛋白的构象发生变化.与此同时,在pH=7.4、含0.05%吐温-20的磷酸盐缓冲液中,秋水仙碱在玻碳电极上出现一不可逆的氧化峰,加入人血清白蛋白后秋水仙碱的氧化峰电位正移,峰电流下降.此外,利用光谱学方法和电化学方法测定的秋水仙碱与人血清白蛋白相互作用的结合常数和结合位点数吻合.     

A New Electrochemical Method for the Determination of Proteins with Cupferron‐Cadmium(II) Complex

A new electrochemical method was proposed for the determination of trace amounts of proteins based on the cupferron (Cup) and cadmium(II) complex [Cup‐Cd(II)] as the voltammetric probe. In the selected pH 6.5 Britton–Robinson (B–R) buffer solution, Cup can interact with Cd(II) to form a stable complex of [Cup‐Cd(II)], which had a sensitive linear sweep voltammetric reductive peak at ?0.654 V (vs. SCE). The addition of human serum albumin (HSA) into [Cup‐Cd(II)] complex solution could greatly decrease the reductive peak current without the change of the reductive peak potential, which indicated that HSA could interact with [Cup‐Cd(II)] complex to form a supramolecular biocomplex. The interaction mechanism was discussed and the decrease of reductive peak current was proportional to the concentration of HSA, which could be further used for the proteins determination. The optimal conditions of the binding reaction and the electrochemical detection were carefully investigated. Under the optimal conditions a new quantitative determination method for different kinds of proteins such as HSA, bovine serum albumin (BSA) and bovine hemoglobin (BHb) etc. was developed. The proposed method was simple, practical and relatively free from the interferences of coexisting substances, and it was further applied to the samples determination with satisfactory results. The binding constant (β_s) and the binding number (m) of HSA with [Cup‐Cd(II)] complex were calculated by the voltammetric data with the results as β_s=1.12×10⁶ and m=1.     

Voltammetric Studies on Chromotrope 2B‐Protein Interaction and Its Analytical Application

In this paper the interaction of chromotrope 2B (Ch2B) with proteins was studied by the electrochemical method. Ch2B is an azo dye and shows irreversible electrochemical responses on the mercury electrode in a pH 3.0 Britton‐Robinson (B‐R) buffer solution. After the addition of human serum albumin (HSA) into the Ch2B solution, an interaction took place, and a supramolecular complex was formed in the mixed solution. The electrochemical parameters of the Ch2B‐HSA interaction system were calculated and compared. The results showed that in the absence and presence of HSA in Ch2B solution, the electrochemical parameters such as the formal potential E⁰, the electrode reaction standard rate constant k_s, etc. showed no significant changes, which indicated that an electro‐inactive supramolecular biocomplex was formed. The free concentration of Ch2B in reaction solution was decreased, and this resulted in the decrease of the peak current. The binding constant and the binding ratio were calculated as 7.85 × 10⁹ and 1:2, respectively, and the interaction mechanism was discussed. Based on the decrease of the peak current, this new electrochemical method was proposed for the determination of HSA in the concentration range of 2.0?25.0 mg/L with the linear regression equation as ΔIp′ (nA) = 50.56C (mg/L) — 6.72 (γ = 0.995). This method was further used to determine other different kinds of proteins, such as bovine serum albumin (BSA), oval albumin, etc‥ The new method was successfully applied to detect the content of albumin in healthy human serum samples with the results in good agreement with the traditional Coomassie Brilliant Blue G‐250 spectrophotometric method.     

Spectroscopic and Molecular Docking Approaches for Investigation of Interaction of Phellopterin with Human Serum Albumin

The mechanism of interaction between human serum albumin (HSA) and natural product phellopterin (PL) from Angelica dahurica was investigated by spectroscopic techniques with molecular docking under simulated physiological conditions. The experimental results showed that the fluorescence of HSA was regularly quenched by PL, and the quenching constants (K_SV) decreased with increasing temperature, which indicated that the quenching mechanism was a static quenching procedure. The binding constants (K_A) were larger than 10^?5 M^?1 and the number of binding sites (n) was approximate to 1 at different temperatures, which indicated that the binding affinity was hige and there was just one main binding site in HSA for PL. According to thermodynamic parameters from Van't Hoff equation, the binding process of PL with HSA was spontaneous and exothermic process due to ΔG < 0, and the electrostatic force played major role in the binding between PL and HSA according to ΔH < 0 and ΔS > 0. The binding distance (r) was calculated to be about 3.35 nm, which implied that the energy transfer from HSA to PL occurred with high possibility according to the theory of Förster's non-radiation energy transfer. The microenvironment and conformation of HSA changed with the addition of PL based on the results of synchronous and three-dimensional fluorescence methods. The molecular docking analysis revealed the binding locus of PL to HSA in subdomain IIIA (Sudlow's site II).     

Investigation of the behavior of HSA upon binding to amlodipine and propranolol: Spectroscopic and molecular modeling approaches

The interaction between human serum albumin (HSA) and two drugs - amlodipine and propranolol - was investigated using fluorescence, UV absorption and circular dichroism (CD) spectroscopy. In addition, the binding site was established by applying molecular modeling technique. Fluorescence data suggest that amlodipine will quench the intrinsic fluorescence of HSA; whereas propranolol enhances the fluorescence of HSA. The binding constants for the interaction of amlodipine and propranolol with HSA were found to be 3.63×10(5)M(-1) and 2.29×10(4)M(-1), respectively. The percentage of secondary structure feature of each one of the HSA-bound drugs, i.e. the α-helix content, was estimated empirically by circular dichroism. The results indicated that amlodipine causes an increase, and that propranolol leads to a decrease in α-helix content of HSA. The spectroscopic analysis indicates that the binding mechanisms of the two drugs are different from each other. The data obtained by the molecular modeling study indicated that these drugs bind, with different affinity, to different sites located in subdomain IIA and IIIA.     

光谱-分子模拟法分析杨梅素与人血清白蛋白的分子作用机制

在模拟生理条件下,用多种光谱法结合分子对接法测定了杨梅素(MY)与人血清白蛋白(HSA)的相互作用.研究结果表明,MY能够明显猝灭HSA的荧光,MY与HSA的相互作用为复合式静态结合过程,结合强度较强.热力学和分子对接结果表明,MY与HSA是自发结合的,维持MY与HSA的相互作用力主要是氢键和范德华力.能量转移结果表明...     

Linear sweep voltammetric determination of protein based on its interaction with Alizarin Red S

In this paper, the electrochemical behavior of the interaction of Alizarin Red S (ARS) with bovine serum albumin (BSA) was investigated on the hanging mercury drop electrode (HMDE). In the acidic solution (pH 4.2), ARS can be easily reduced on the HMDE and it has a well-defined polarographic wave at −0.29 V (SCE). On addition of BSA or human serum albumin (HAS) into the ARS solution, the reduction peak current of ARS decreases without the movement of the peak potential and the appearance of new peaks. The study shows that a new electrochemically non-active complex is formed via intercalation of ARS with BSA or HSA, which can not be reduced on the Hg electrode. The decrease of reductive peak current of ARS is proportional to BSA and HSA concentration in the range of 2.0–60 and 2.0–40 mg l⁻¹, respectively. The detection limit of BSA and HSA is 1.0-mg l⁻¹. The analytical results of human serum and urine samples by this method were in good agreement with the Coomassie brilliant blue G-250 assay. The binding number and the binding interaction mechanism are also discussed.     

Application of arsenazo III for the polarographic detection of proteins

In this paper, a diazo dye of arsenazo III (AAIII) was selected as a new electrochemical probe for the determination of proteins. In Britton-Robinson (B-R) buffer solution of pH 2.4, AAIII had a sensitive second order derivative linear sweep voltammetric reductive peak at ?0.39 V (vs. SCE). After the addition of human serum albumin (HSA) into AAIII solution, an interaction was taken place in the mixed solution and a biosupramolecular complex was formed, which resulted in the decreased reductive peak currents of AAIII. Based on the observed decrease in peak current, a sensitive electrochemical method was proposed for the determination of different proteins such as HSA, bovine serum albumin (BSA) and bovine hemoglobin (BHb). The optimal conditions for the interaction and the interfering effects of coexisting substances on the detection were investigated. The proposed method was successfully applied to the determination of HSA in synthetic samples with the recoveries in the range of 99.13–100.50%. The stoichiometry of HSA-AAIII biocomplex was calculated by voltammetric data with a binding number of 2 and a binding constant of 7.53 × 10⁹.     

Binding of trazodone hydrochloride with human serum albumin: A spectroscopic study

The binding of trazodone hydrochloride (TZH) to human serum albumin (HSA) was investigated by spectroscopic techniques. Various binding parameters have been evaluated. Negative enthalpy and positive entropy values indicated that both hydrogen bond and hydrophobic forces played a major role in the binding of TZH to HSA. The distance, r between donor (HSA) and acceptor (TZH) was found to be 2.16 nm based on the Förster's theory of non-radiation energy transfer. The circular dichroism data indicated that the α-helicity of HSA decreased upon interaction with TZH. The binding constant of HSA–TZH was found to decrease in presence of common ions and hence, shortened the stored time of drug in blood plasma.     

Studies on the interaction of protein with acid chrome blue K by electrochemical method and its analytical application

An electrochemical investigation on the interaction of acid chrome blue K (ACBK) with protein on the mercury electrode with different electrochemical methods such as cyclic voltammetry and linear sweep voltammetry was reported in this paper. In pH 3.0 Britton-Robinson (B-R) buffer solution, ACBK has an irreversible voltammetric reductive peak at -0.23 V (vs. SCE). The addition of human serum albumin (HSA) into the ACBK solution resulted in the decrease of reductive peak currents without the change of the peak potential and no new peaks appeared on the cyclic voltammogram. In the absence and presence of HSA, the electrochemical parameters such as the formal potential E0, the electrode reaction standard rate constant k(s) and the charge transfer coefficient alpha of the interaction system were calculated and the results showed that there were no significant changes between each other. Thus, the interaction of ACBK with protein forms an electro-inactive supramolecular bio-complex, which induces the decrease of the free concentration of ACBK in the reaction solution, and the decrease of the reductive peak current of ACBK. The binding constant and the binding ratio are calculated as 1.29 x 10(8) and 1:2, respectively, and the interaction mechanism is discussed. Based on the binding reaction, this new electrochemical method is further applied to the determination of HSA with the linear range from 3.0-20.0 mg/L and the linear regression equation as deltaIp"(nA) = 10.08 + 19.90 C (mg/L). This method was further applied to determinate the content of protein in the healthy human serum samples with the results in good agreement with the traditional Coomassie brilliant blue G-250 spectrophotometric method.     

Voltammetric,spectroscopic and thermal investigations of the interaction of levofloxacin with cysteine at physiological pH

In this study, levofloxacin (LEVOF) hemihydrate interaction with L-cysteine (RSH) was investigated by using square-wave voltammetry (SWV) in Britton–Robinson (B–R) buffer pH 7.4. Addition of the LEVOF to RSH solution resulted in dropping of the main reduction peak current of RSH (the current of mercurous cysteine thiolate Hg₂(RS)₂). The vary in the peak current of Hg₂(RS)₂, after the adding of the LEVOF is indicated an interaction between RSH and LEVOF molecules. Moreover, the interaction between two molecules also confirmed with UV-Vis, FT-IR spectroscopic measurements and thermal analysis data. Binding constant of LEVOF with RSH was calculated by both voltammetric and UV-Vis spectroscopic data.     

以溴甲酚紫为电化学探针测定血清白蛋白

常见的蛋白质定量分析方法、电化学行为及其分析应用已有报道．本文基于酸性条件下蛋白质可与溴甲酚紫(BP)结合生成一种超分子复合物,使溴甲酚紫还原峰电流降低,建立了一种测定蛋白质的新方法,并用于测定人血清样品中的白蛋白含量．     

偶氮氯膦Ⅲ与人血清白蛋白相互作用的电化学研究

偶氮氯膦Ⅲ是一种具有电化学活性的染料,在pH3.5的Britton-Robinson缓冲溶液中,它可以与人血清白蛋白发生相互作用形成一种生物超分子复合物,使溶液中游离的染料浓度降低.以线性扫描二阶导数极谱法对偶氮氯膦Ⅲ-人血清白蛋白的相互作用体系进行了详细的研究,复合物的形成使偶氮氯膦Ⅲ在-0.124V(vs.SCE)的还原峰电流降低,考察了结合反应的最佳条件和测定条件,求解了结合常数和结合比.     

白杨素与不同构型人血清白蛋白的作用机制

采用多种光谱学手段研究了白杨素(CHR)和不同构型人血清白蛋白(HSA)相互作用的分子机制.研究表明,白杨素能使蛋白质荧光发射峰发生静态淬灭,同时,白杨素的紫外吸收谱带也发生了明显的位移,说明与蛋白质的结合可使白杨素分子中的酚羟基发生解离.蛋白质还可以引起白杨素荧光发射峰强度的明显增强.利用荧光淬灭和荧光增强两种模式计算得到的白杨素和人血清白蛋白在生理条件下(pH 7.4)的结合常数(KA)分别为(9.97±0.24)×104和(9.75±0.11)×104L mol-1,其结合比例为1:1.随着pH值的降低,蛋白质与白杨素的结合常数逐渐减小,这与蛋白质的构型变化有关.根据不同异构体血清蛋白质的结构特征,判定白杨素在蛋白质分子上的结合位置位于IIA亚域的Site I活性位点.结合分子模拟,讨论了白杨素与蛋白质分子的结合机制.     

In vitro study on the interaction between thiophanate methyl and human serum albumin

Thiophanate methyl (MT) is one of the widely used fungicides to control important fungal diseases of crops, which has led to potential toxicological risk to public health. Several different transport proteins exist in blood plasma, but albumin only is bound by a wide diversity of xenobiotics reversibly with high affinity. We studied the interaction of MT with human serum albumin by using spectroscopic methods including fluorescence quenching technology, UV and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The result of fluorescence titration revealed that MT could quench the intrinsic fluorescence of HSA. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses. In addition, the studies of FT-IR spectroscopy showed that the binding of MT to HSA changed molecular conformation of HSA. The results obtained from molecular modeling showed that the interaction between MT and HSA was dominated by hydrophobic force, and there was also hydrogen bond interaction between the pesticide and the residues of HSA, which was in good agreement with the result of binding mode.     

Polarographic Detection of Arenediazonium Ions. Optimization of the Method and Application for Investigating Dediazoniation Mechanisms

The use of differential pulse polarography (DPP) at the dropping mercury electrode (DME) to detect and to investigate the mechanisms for decomposition of arenediazonium ions, ArN₂⁺, under different experimental conditions is discussed. The effect of a number of experimental and instrumental parameters on the polarographic peaks of a model arenediazonium ion was explored and representative applications to investigate their reaction mechanisms are given. The composite data shows that DPP provides an alternative, relatively cheap, technique to monitor ArN₂⁺ decomposition in, for example, opaque systems where, for obvious reasons, spectroscopic detection does not work; meanwhile in homogeneous systems, DPP complements the results obtained by employing spectroscopic or chromatographic techniques. In addition, a variety of valuable mechanistic information such as detection of transient intermediates or estimation of the binding constants of aryl radicals with macromolecular systems, that otherwise cannot be easily obtained, is readily available by using DPP.     

Spectroscopic Investigation on the Binding of the Antitumoral Pd(II) Complex to Human Serum Albumin

The interaction of human serum albumin (HSA) with 1,10‐phenanthroline‐ethyldithiocarbamatopalladium(II) nitrate complex, [Pd(phen)(Et‐dtc)]NO₃, has been studied by using absorption, fluorescence and circular dichroism spectroscopic measurements. UV‐Vis studies imply that The peptide strands of protein molecules extended more (denatured) upon the addition of Pd(II) complex. This process is spontaneous and exothermic. A fluorescence quenching reaction of Pd(II) complex and HSA was observed and quenching mechanism was suggested as static quenching according to Stern‐Volmer equation. The number of binding sites (n) and apparent association constant (K_A) were calculated using fluorescence quenching data. The circular dichroism results revealed the conformational changes in secondary structure of protein upon its interaction with Pd(II) complex. In these interaction studies, several thermodynamic and binding parameters are also determined which may provide deeper insights into structural changes induced by an antitumor Pd(II) complex on the protein as the metal complex side effects.     

Study on the interaction of three structurally related cationic Pt(II) complexes with human serum albumin: importance of binding affinity and denaturing properties

Human serum albumin (HSA) primarily functions as a transport carrier for a vast variety of natural ligands and pharmaceutical drugs. In the present study, three structurally related cationic Pt(II) complexes ([Pt(ppy)(dppe)]CF₃CO₂: 1, Pt(bhq)(dppe)]CF₃CO₂: 2, and [Pt(bhq)(dppf)]CF₃CO₂: 3) were used to evaluate their interaction with HSA under different experimental setups, using UV–Vis absorption spectroscopy, fluorescence and circular dichroism techniques. The spectroscopic results suggest that upon binding to HSA, the Pt(II) complexes could effectively induce structural alteration of the protein. The complexes can bind to HSA with the binding affinities of the following order: 3 > 2 > 1. Also, thermodynamic parameters of binding between these complexes and HSA indicated the existence of entropy-driven spontaneous interaction which primarily dominated with the hydrophobic forces. Also, docking simulation study revealed the binding details of these complexes on HSA. Complex 3 with highest binding affinity for HSA indicates lowest denaturing effect on this protein. The low denaturation properties of 3 appear important in the terms of lower susceptibility of this platinum complex for possible development of deleterious side effects.     

Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

In order to investigate the involvement of lysine residues of human serum albumin (HSA) in nalidixic acid (NA) binding, various modified preparations of HSA such as 44% carbamylated (C₄₄), 83% carbamylated (C₈₃) and 85% acetylated (A₈₅) were made by treating the HSA solution with a different molar excess of potassium cyanate and acetic anhydride. The extent of modification, charge homogeneity and conformational changes of these derivatives were checked by TNBSA reaction method, polyacrylamide gel electrophoresis (PAGE) and gel filtration using Sephacryl S-200 HR column, respectively. Binding of NA to HSA and its derivatives was examined using fluorescence quenching titration method to determine the binding constant. The emergence of a single band in PAGE and single symmetrical peak in gel filtration results confirmed the charge and size homogeneity of these derivatives. Hydrodynamic properties such as Stokes radius and frictional ratio, as obtained from the analytical gel filtration results suggested molecular expansion in C₈₃ and A₈₅ HSAs while C₄₄ HSA retained the native conformation. Addition of NA to both native and modified HSA derivatives quenched the fluorescence intensity of the protein at 344 ​nm to a different extent. Whereas the values of the Stern-Volmer constant (K_SV) and bimolecular quenching rate constant (k_q) suggested, NA-HSA complex formation, binding constant (K_a) value suggested an intermediate binding affinity between NA and HSA. Furthermore, the decrease in the K_a value with the extent of modification was indicative of the involvement of lysine residues in NA-HSA interaction.     

光谱法结合分子模拟技术研究磺胺甲恶唑与左氧氟沙星的协同作用

在模拟人体生理酸度(pH=7.4)条件下,运用荧光光谱、紫外光谱和分子模拟技术,研究了磺胺甲恶唑(SMZ)和左氧氟沙星(LVFX)的协同作用.实验结果表明,SMZ、LVFX主要通过氢键和疏水作用与人血清白蛋白(HSA)发生相互作用形成复合物,导致HSA的内源荧光发生静态猝灭.SMZ、LVFX在HSA上有相同的结合位点,即Site I位.在HSA-SMZ(或HSA-LVFX)体系中分别加入LVFX(或SMZ),其结合常数均明显减小,表明LVFX(或SMZ)的存在削弱了HSA-SMZ(或HSA-LVFX)体系的结合能力,使得LVFX(或SMZ)更多被释放,血液中游离的LVFX(或SMZ)浓度增大,SMZ与LVFX共存能够协同增强药效.