亲和毛细管电泳法研究锌离子与人血清白蛋白的结合反应机制

建立了研究金属离子与人血清白蛋白(Human serum albumin,HSA)相互作用的亲和毛细管电泳(Affinity capillary electrophoresis,ACE)方法。生理条件下,构建配体(Zn2+)-受体(HSA)相互作用模型,以N,N-二甲基甲酰胺(N,N-Dimethylformamide,DMF)为内标物,基于Scatchard方程,依据有效淌度的变化,通过非线性模拟方程计算Zn2+-HSA结合反应的表观结合常数KB,定量表征了Zn2+-HSA相互作用的强度,并解析电泳谱图获得了Zn2+-HSA结合反应为一快平衡体系的结论。结果表明,建立的ACE方法简捷、有效,Zn2+-HSA相互作用的强度与Zn2+浓度之间存在明显的量效关系。     

考拉维酸对人血清白蛋白结构的影响

利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法,表征了模拟生理条件下一种植物药活性组分考拉维酸(KA)影响人血清白蛋白(HSA)的结构信息.同步荧光及紫外光谱证实考拉维酸的存在影响了HSA的微环境;二维及三维荧光光谱表明考拉维酸可以猝灭HSA的内源荧光,使其构象发生变化.荧光偏振的测定提供了考拉维酸与HSA作用后生成的配合物弛豫时间与聚集特性的信息,揭示KA的存在使HSA的流动性和微粘度发生变化.定量求得不同温度下(298、308和318 K)考拉维酸与HSA作用的键合参数和热力学参数.分子模拟表明考拉维酸键合位点于HSA分子的疏水腔内,并与赖氨酸Lys195和天冬氨酸Asp451形成三个氢键,与HSA的键合模式主要是疏水作用;位点竞争实验证明考拉维酸在HSA亚结构域的位点II位发生作用.另外,获得的相关物理化学参数从分子水平上揭示了考拉维酸与HSA相互作用的机制.结果表明,HSA对考拉维酸有较强的结合能力,提示人血清白蛋白对考拉维酸可起到储存和转运的作用.     

考拉维酸对人血清白蛋白结构的影响

利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法, 表征了模拟生理条件下一种植物药活性组分考拉维酸(KA)影响人血清白蛋白(HSA)的结构信息. 同步荧光及紫外光谱证实考拉维酸的存在影响了HSA的微环境; 二维及三维荧光光谱表明考拉维酸可以猝灭HSA的内源荧光, 使其构象发生变化. 荧光偏振的测定提供了考拉维酸与HSA作用后生成的配合物弛豫时间与聚集特性的信息, 揭示KA的存在使HSA的流动性和微粘度发生变化. 定量求得不同温度下(298、308 和318 K)考拉维酸与HSA作用的键合参数和热力学参数. 分子模拟表明考拉维酸键合位点于HSA分子的疏水腔内, 并与赖氨酸Lys195 和天冬氨酸Asp451 形成三个氢键, 与HSA的键合模式主要是疏水作用; 位点竞争实验证明考拉维酸在HSA亚结构域的位点Ⅱ位发生作用. 另外, 获得的相关物理化学参数从分子水平上揭示了考拉维酸与HSA相互作用的机制. 结果表明, HSA对考拉维酸有较强的结合能力, 提示人血清白蛋白对考拉维酸可起到储存和转运的作用.     

Binding of human serum albumin to N-(p-ethoxy-phenyl)-N'-(1-naphthyl)thiourea and synchronous fluorescence determination of human serum albumin.

The binding of N-(p-ethoxy-phenyl)-N'-(1-naphthyl)thiourea (EPNT) to human serum albumin (HSA) was investigated under simulative physiological conditions by fluorescence spectra in combination with UV absorption spectroscopy and a molecular modeling method. A strong fluorescence quenching reaction of EPNT to HSA was observed, and the quenching mechanism was suggested to be static quenching according to the Stern-Volmer equation. The binding constants (K) at different temperatures as well as thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated according to relevant fluorescent data and the vant' Hoff equation. This indicated that a hydrophobic interaction was a predominant intermolecular force for stabilizing the complex, which is in agreement with the results of molecule modeling study. The effects of energy transfer and other ions on the binding constant were considered. In addition, synchronous fluorescence technology was successfully applied to the determination of HSA added into the EPNT solution.     

Characterization of basic drug-human serum protein interactions by capillary electrophoresis

Drug-protein interactions are determining factors in the therapeutic, pharmacodynamic and toxicological drug properties. The affinity of drugs towards plasmatic proteins is apparently well established in bibliography. Albumin (HSA) especially binds neutral and negatively charged compounds; alpha(1)-acid glycoprotein (AGP) binds many cationic drugs, lipoproteins bind to nonionic and lipophilic drugs and some anionic drugs while globulins interact inappreciably with the majority of drugs. In this paper, the characterization of the interaction between cationic drugs, beta-blockers and phenotiazines towards HSA, AGP, and both HSA + AGP mixtures of proteins under physiological conditions by CE-frontal analysis is presented. Furthermore, the binding of these drugs to all plasmatic proteins is evaluated by using ultrafiltration and CE. The results indicate that the hydrophobic character of compounds seems to be the key factor on the interaction between cationic drugs towards proteins. In fact, hydrophobic basic drugs bind in great extension to HSA, while hydrophilic basic drugs present low interactions with proteins and bind especially to AGP.     

A study of intramolecular H-complexes of novel bis(heptamethine cyanine) dyes

Near-infrared (NIR) bis(heptamethine cyanine) (BHmC) dyes containing a flexible polymethylene linker between the two cyanine subunits are a novel class of compounds with versatile spectroscopic properties. The first bis-cyanine of this type is BHmC-10 (with a decamethylene bridge) that has been reported by us recently [G. Patonay, J.S. Kim, R. Kodagahally, L. Strekowski, Appl. Spectrosc., in press]. As part of this work, additional bis-cyanines BHmC-4, BHmC-6, and BHmC-8 were synthesized and their spectral properties were evaluated for the dyes free in solution and in the presence of human serum albumin (HSA). These bis-cyanines undergo H-type aggregation, mainly H-type intramolecular complexation between the two cyanine subunits, when free in aqueous solution. This H-type interaction in phosphate buffer (pH 7.2) is characterized by hypsochromic (H) absorption at 700 nm, low extinction coefficient, and low fluorescence quantum yield. By contrast, an analogous monomeric cyanine exhibits strong fluorescence under similar conditions. Upon binding with HSA, the fluorescence of BHmC-6 changes negligibly, that for BHmC-8 shows a slight increase, and the fluorescence of BHmC-4 is greatly increased. It is suggested that BHmC-4 binds with HSA in the open form exclusively, while the H-type intramolecular interaction in BHmC-6 is mostly retained in the complex with HSA. Bis-cyanine BHmC-4 may be of significant bioanalytical utility due to its negligible fluorescence in aqueous solution and a strong increase in fluorescence upon binding with a protein.     

Insight into the Interaction Mechanism of HSA with Aztreonam: A Multispectroscopic and Computational Approach

Aztreonam is a Gram-negative bacteria-targeting synthetic monobactam antibiotic. Human serum albumin (HSA) plays an important role in the transference of pharmaceuticals, hormones, and fatty acids, along with other compounds, determining their biodistribution and physiological fate. Using several biophysical and in silico approaches, we studied the interaction of aztreonam with HSA under physiological environments in this study. Results confirm the formation of HSA-aztreonam complex where aztreonam showed moderate affinity towards HSA. A static mode of quenching was confirmed from the steady state fluorescence data. FRET findings also showed that there was a significant feasibility of energy transfer between HSA and aztreonam. Site marker displacement experimental conclusion suggested the binding site of aztreonam was the sub-domain IB of HSA. Circular dichroic spectroscopic analysis suggested that aztreonam interaction decreases the α-helical content of HSA. Changes in microenvironment were studied through synchronous fluorescence data. According to molecular docking results, the HSA-aztreonam complex is mostly maintained by non-covalent forces, with a binding energy of 7.7 kcal mol⁻¹. The presence of a hydrogen bond, van der Waal interaction, and pi-anion interaction in the binding process, as well as conformational changes in HSA after binding with aztreonam, are all confirmed by molecular dynamic simulation.     

Electrochemical Study on the Interaction of Protein with Bromothymol Blue and Its Analytical Application

The interaction of bromothymol blue（BB） with human serum albumin（HSA） was studied by electrochemical techniques and a sensitive method for proteins assay was developed. When BB interacted with HSA, the voltammetric peak current value of BB decreased linearly with the concentration of HSA in a range of 1.0--40.0 mg/L, and the peak potential shifted negatively. Based on the results, a sensitive assay method for proteins, such as HSA, bovine serum albumin（BSA）, and egg albumin etc. was established. This method was further applied to determining the HSA in healthy human blood samples, and the results are not significantly different from those obtained by the classic Coomassie Brilliant Blue G-250 spectrophotometic method. The detecting conditions of this method were optimized and the interaction mechanism was discussed. The results show that the electrochemical parameters（formal potential E^0, standard rate constant of the electrode reaction ks, parameter of kinetic nα） of BB have no obvious changes before and after the interaction, which indicate that BB can interact with HSA, forming an electrochemical non-active complex. The equilibrium constant（βs） and the binding ratio（m） for this complex were calculated. The m is 4 and βs is 1.41 × 10^19. This method is fast, simple, highly sensitive, and has good selectivity, which can be used in clinical measurements.     

Investigation on the interaction between a heterocyclic aminal derivative, SBDC, and human serum albumin

The interaction between a novel promising drug (spiro[(2R,3R,4S)-4-benzyloxy-2,3-isopropylidene-dioxy-1-oxa-cyclopentane-5,5′-(2-benzoylmethylene-1,3-diaza-cyclohexane)] (SBDC)) and human serum albumin (HSA) under physiological conditions has been investigated by using fluorescence, absorption, and circular dichroism (CD) spectroscopic techniques in combination with protein–ligand docking study. It was observed that SBDC has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The association constants of SBDC with HSA were determined at different temperatures based on fluorescence quenching results. The negative ΔH and positive ΔS values in case of SBDC–HSA complex showed that apart from an initial hydrophobic association, both van der Waals interactions and hydrogen bonding play a vital role in the binding of SBDC to HSA. The quantitative analysis data of CD spectra showed that the binding of SBDC to HSA induced conformational changes in HSA and the α-helix of 52.1% in free HSA increased to 55.7% in HSA–SBDC complex. The distance between donor (HSA) and acceptor (SBDC) was obtained according to the Förster's theory of non-radiation energy transfer. Data obtained by spectroscopic techniques and protein–ligand docking study suggested that SBDC binds to residues located in subdomain IIA of HSA.     

Binding Models of Copper(II) Thiosemicarbazone Complexes with Human Serum Albumin: A Speciation Study

Copper(II) complexes of thiosemicarbazones (TSCs) often exhibit anticancer properties, and their pharmacokinetic behavior can be affected by their interaction with blood transport proteins. Interaction of copper(II) complexes of an {N,N,S} donor α-N-pyridyl TSC (Triapine) and an {O,N,S} donor 2-hydroxybenzaldehyde TSC (STSC) with human serum albumin (HSA) was investigated by UV–visible and electron paramagnetic resonance spectroscopy at physiological pH. Asp-Ala-His-Lys and the monodentate N-methylimidazole were also applied as binding models. Conditional formation constants were determined for the ternary copper(II)-TSC complexes formed with HSA, DAHK, and N-methylimidazole based on the spectral changes of both charge transfer and d-d bands. The neutral N-methylimidazole displays a similar binding affinity to both TSC complexes. The partially negatively charged tetrapeptide binds stronger to the positively charged Triapine complex in comparison to the neutral STSC complex, while the opposite trend was observed for HSA, which demonstrates the limitations of the use of simple ligands to model the protein binding. The studied TSC complexes are able to bind to HSA in a fast process, and the conditional constants suggest that their binding strength is only weak-to-moderate.     

Crosslink effect and albumin adsorption onto chitosan/alginate multilayered systems: an in situ QCM-D study

The adsorption of HSA onto CHI/ALG multilayer assemblies was assessed in situ using QCM-D. It was found that the behavior of HSA on biomaterials surface can be tuned by adjusting parameters of the polyelectrolyte system such as pH, layer number, crosslinker and polymer terminal layer. Our results confirmed the key role of electrostatic interactions during HSA adsorption, since oppositely charged surfaces were more effective in promoting protein adhesion. QCM-D data revealed that crosslinking (CHI/ALG)(5) CHI films allows HSA to become adsorbed in physiological conditions. Our results suggested that the biological potential of biopolymers and the mild conditions of the LbL technique turn these natural nanoassemblies into a suitable choice to be used as pH-sensitive coatings.     

毛细管电泳法考察全氟辛酸与人血清白蛋白的相互作用

利用毛细管电泳（CE）技术建立了全氟辛酸（PFOA,C₈HF₁₅O₂）与人血清白蛋白（HSA）相互作用的分析方法。在生理条件下构建配体（PFOA）-受体（HSA）相互作用模型,通过淌度移动法、区段-区段动力学（plug-plug kinetic,PPK）法、简化的Hummel-Dreyer（HD）法研究其与HSA的相互作用。简化的HD法运用非线性方程、Scatchard方程和Klotz方程获得PFOA-HSA体系的相互作用参数,进而分析了模型适用度。结果表明,淌度移动法、PPK法、简化的HD法均适用于PFOA-HSA体系相互作用的分析,其中简化的HD法最优。模型适用度分析得出非线性回归方程为最适理论模型。相互作用参数测试表明PFOA-HSA相互作用体系之间发生的结合反应只有单一类型的结合位点且结合稳定。相关工作阐明了人血清白蛋白与PFOA的相互作用机制,可为PFOA毒理机制的深入研究提供有益参考。     

Experimental Approaches to Adsorption-Desorption Dynamism of Human Serum Albumin (HSA) onto Crosslinked N,N′-Diethylaminoethyl (DEAE) Dextran Microbeads

Crosslinked N,N′-Diethylaminoethyl (DEAE) groups containing dextran microbeads have been used in human serum albumin (HSA) adsorption-desorption studies. For the HSA adsorption onto positively charged hydrophilic DEAE dextran microbeads, the adsorption kinetic was slightly decreased by the changing concentration of the protein solution. Adsorption kinetics and equilibrium isotherms for the adsorption of HSA on crosslinked DEAE dextran have been determined experimentally. Modeling of the adsorption processes on DEAE dextran microbeads were realized by applying different adsorption isotherms. Among the several isotherm equations, Langmuir and Freundlich adsorption isotherms were investigated depending on the two temperatures. These were only slightly dependent on the initial concentration of HSA but were considerably affected by the pH of the medium. The HSA adsorption capacity factor and the adsorption equilibrium constant were obtained and mathematical modeling of adsorption, adsorption rate constants and maximum adsorption were determined. Besides the adsorption mechanism, optimum ionic strength and optimum pH also were investigated. Desorption studies and desorption ratio of the system were determined for optimum medium conditions. It was been proved both experimentally and theoretically that human HSA is adsorbed by electrostatic attraction, ion-exchange, hydrophobic interaction and/or hydrogen bonding.     

Human serum albumin self-assembly on weak polyelectrolyte multilayer films structurally modified by pH changes

Adsorption of proteins onto film surfaces built up layer by layer from oppositely charged polyelectrolytes is a complex phenomenon, governed by electrostatic forces, hydrogen bonds, and hydrophobic interactions. The amounts of the interacting charges, however, both in polyelectrolytes and in proteins adsorbed on such films are a function of the pH of the solution. In addition, the number and the accessibility of free charges in proteins depend on the secondary structure of the protein. The subtle interplay of all these factors determines the adsorption of the proteins onto the polyelectrolyte film surfaces. We investigated the effect of these parameters for polyelectrolyte films built up from weak "protein-like" polyelectrolytes (i.e., polypeptides), poly(L-lysine) (PLL), and poly(glutamic acid) (PGA) and for the adsorption of human serum albumin (HSA) onto these films in the pH range 3.0-10.5. It was found that the buildup of the polyelectrolyte films is not a simple function of the pure charges of the individual polyelectrolytes, as estimated from their respective pKa values. The adsorption of HSA onto (PLL/PGA)n films depended strongly on the polyelectrolyte terminating the film. For PLL-terminated polyelectrolyte films, at low pH, repulsion, as expected, is limiting the adsorption of HSA (having net positive charge below pH 4.6) since PLL is also positively charged here. At high pH values, an unexpected HSA uptake was found on the PGA-ending films, even when both PGA and HSA were negatively charged. It is suggested that the higher surface rugosity and the decrease of the alpha-helix content at basic pH values (making accessible certain charged groups of the protein for interactions with the polyelectrolyte film) could explain this behavior.     

When are Two Waters Worse Than One? Doubling the Hydration Number of a Gd–DTPA Derivative Decreases Relaxivity

The synthesis of a novel ligand, based on N-methyl-diethylenetriaminetetraacetate and containing a diphenylcyclohexyl serum albumin binding group (L1) is described and the coordination chemistry and biophysical properties of its Gd(III) complex Gd-L1 are reported. The Gd(III) complex of the diethylenetriaminepentaacetate analogue of the ligand described here (L2) is the MRI contrast agent MS-325. The effect of converting an acetate to a methyl group on metal-ligand stability, hydration number, water-exchange rate, relaxivity, and binding to the protein human serum albumin (HSA) is explored. The complex Gd-L1 has two coordinated water molecules in solution, that is, [Gd(L1)(H2O)2]2- as shown by D-band proton ENDOR spectroscopy and implied by 1H and 17O NMR relaxation rate measurements. The Gd-H(water) distance of the coordinated waters was found to be identical to that found for Gd-L2, 3.08 A. Loss of the acetate group destabilizes the Gd(III) complex by 1.7 log units (log K(ML) = 20.34) relative to the complex with L2. The affinity of Gd-L1 for HSA is essentially the same as that of Gd-L2. The water-exchange rate of the two coordinated waters on Gd-L1 (k(ex) = 4.4x10(5) s(-1)) is slowed by an order of magnitude relative to Gd-L2. As a result of this slow water-exchange rate, the observed proton relaxivity of Gd-L1 is much lower in a solution of HSA under physiological conditions (r1(obs) = 22.0 mM(-1) s(-1) for 0.1 mM Gd-L1 in 0.67 mM HSA, HEPES buffer, pH 7.4, 35 degrees C at 20 MHz) than that of Gd-L2 (r1(obs) = 41.5 mM(-1) s(-1)) measured under the same conditions. Despite having two exchangeable water molecules, slow water exchange limits the potential efficacy of Gd-L1 as an MRI contrast agent.     

吡虫啉与人血清白蛋白相互作用热力学行为

在模拟人体生理条件下,综合利用荧光光谱、紫外吸收光谱、圆二色谱和分子模拟等方法,研究了吡虫啉(IMI)和人血清白蛋白(HSA)相互作用的热力学行为。荧光光谱和紫外吸收光谱的分析表明：吡虫啉能有效猝灭HSA的内源荧光,猝灭机制为静态猝灭;通过所获取的相互作用热力学参数,可知两者之间的相互作用是一个吉布斯自由能降低的自发过程,且二者之间的主要作用力为氢键和范德华力。位点竞争实验和分子模拟的结果表明：吡虫啉在HSA的主要结合位置为位点?。圆二色谱、同步荧光光谱和三维荧光的分析发现：吡虫啉引起HSA的构象发生改变,其α-螺旋含量降低,无规卷曲含量升高,肽链结构在吡虫啉的作用下有所伸展。     

腺苷与人血清白蛋白间相互作用的荧光光谱及分子模型法研究

在模拟人体生理条件下,结合紫外光谱和分子对接模型运用荧光光谱研究了腺苷与人血清白蛋白(HSA)间的键合作用。腺苷有较强的能力猝灭人血清白蛋白的内源荧光,且根据Stern-Volmer方程判断出猝灭机制为静态猝灭。本文运用相应的荧光值和Vant’Hoff热力学方程求得了不同温度下的结合常数(K)以及一些热力学参数,如焓变(ΔH)和熵变(ΔS)。结果表明：键合过程中疏水作用力对新化合物的稳定性起主要作用,这与分子对接模型方法研究的结果基本一致。另外还研究了常见离子对结合常数的影响。     

Spectral study of coumarin-3-carboxylic acid interaction with human and bovine serum albumins

Fluorescence spectroscopy and circular dichroism (CD) spectroscopy were used to investigate the interaction of coumarin-3-carboxylic acid with human serum albumin (HSA) and bovine serum albumin (BSA) under physiological conditions in a buffer solution of pH 7.4.      

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.     

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.