Thermodynamics of porphyrin binding to human serum albumin using affinity capillary electrophoresis

Summary The interaction thermodynamics of heptacarboxylporphyrin (HCP) and protoporhyrin (PP) with human serum albumin (HSA) was studied by affinity capillary electrophoresis (ACE) over the temperature range of 25–50°C, where HCP and PP bound to HSAvia 1:1 molecular association. The binding equilibrium constants (pH 7.4, phosphate buffer) for the binding of HCP with HSA were found to decrease with an increase in temperature, whereas the binding constants of the PP/HSA system appeared to be independent of temperature changes over the range studied. The van’t Hoff relationship (25–50°C) was found to be linear for the interaction of either HCP or PP with HSA. However, the interaction thermodynamics for both of these porphyrins with HSA were found to be quite different. In particular, the interaction of HCP (a hydrophilic porphyrin) with HSA appeared to be based on an enthalpy-driven process, whereas the binding between PP (a hydrophobic porphyrin) and HSA driven by a favorable change in entropy. The ability of using ACE to evaluate the interaction thermodynamics of serum proteins (e.g., HSA) with ligands (e.g., porphyrins and related compounds) should aid in the development of new and more effective photosensitizers in the photodynamic therapy of cancer.     

Study of donepezil binding to serum albumin by capillary electrophoresis and circular dichroism

The interaction between human serum albumin (HSA) and the acetylcholinesterase inhibitor donepezil, has been studied by means of capillary electrophoresis frontal analysis (CE/FA) and circular dichroism. CE/FA enabled rapid and direct estimation of the quantity of free donepezil present at equilibrium with a physiological level of serum albumin (600 mol L^–1). Application of Scatchard analysis enabled estimation of the binding parameters of HSA towards donepezil, such as association constant and number of binding sites on one protein molecule. Furthermore, due to enantioseparation ability shown by HSA on donepezil in CE mode, displacement experiments were carried out using ketoprofen and warfarin as coadditives to the HSA based running buffer. The addition of these compounds reduced the enantioresolution of donepezil by HSA only when used at high concentration. These data were confirmed and corroborated by circular dichroism (CD) experiments. Using CD, bilirubin was also applied as a ligand specific to site III of HSA. The observed behaviour suggested that donepezil could be considered a ligand with independent binding to sites I and II; although site III is not the highest affinity site, indirect interaction (i.e. cooperative binding) can be assumed.     

毛细管电泳研究抗癌药物紫杉醇与人血清蛋白结合作用

采用毛细管区带电泳(CZE)技术, 研究了天然抗癌药物紫杉醇(Paclita-xel)与人血清白蛋白(HSA)的结合机制. 在以硼砂-碳酸钠(pH 10, 50 mmoL)为运行缓冲溶液, 运行电压21 kV, 进样时间5.0 s, 紫外检测器(214 nm)的条件下检测, 结合常数和结合位点数在298和310 K分别为K298 K=1.7×104 L/mol, n298 K=4.1, K310 K=3.4×104 L/mol, n310 K=3.0.     

Ultrasonic microdialysis coupled with capillary electrophoresis electrochemiluminescence study the interaction between trimetazidine dihydrochloride and human serum albumin

The paper describes a homemade ultrasonic microdialysis device coupled with capillary electrophoresis electrochemiluminescence (CE-ECL) for studying the interaction between human serum albumin (HSA) and trimetazidine dihydrochloride (TMZ). The time required for equilibrium by ultrasonic microdialysis was 45 min, which was far less than that by traditional dialysis (240 min). It took 80 min to achieve the required combination equilibrium by normal incubation and only 20 min by ultrasonic. Compared with traditional dialysis, the use of ultrasonic microdialysis simplified experimental procedures, shortened experimental time and saved consumption of sample. A simple, sensitive and selective determination of TMZ was developed using CE-ECL and the parameters that affected ECL intensity were optimized. Under the optimized conditions, the linear range of TMZ was from 0.075 to 80 μmol/L (r² = 0.9974). The detection limit was 26 nmol/L with RSD of 2.8%. The number of binding sites and binding constant were 1.54 and 15.17 L/mol, respectively.     

Study of interaction between drug enantiomers and human serum albumin by flow injection-capillary electrophoresis frontal analysis

Flow injection (FI)-CE coupled with frontal analysis (FA) was applied to the study of stereoselectivity binding of amlodipine (AL) to HSA. Under protein-drug binding equilibrium, the unbound concentrations of drug enantiomers were measured by plateau height. The stereoselectivity of AL binding to HSA was proved by the different free fractions of two enantiomers. In physiological phosphate solution (pH 7.4, ionic strength 0.17) when 200 microM (+/-)AL was equilibrated with 300 microM HSA, the concentration of unbound R-AL was about 1.5 times higher than that of its antipode. The binding constants of two enantiomers, KR-AL and KS-AL, were 9910-11200 and 90200-104000 M(-1), respectively. The results obtained by the method were compared with those determined by conventional equilibrium dialysis (ED)-CE and fluorescence spectra. Hydroxypropyl-beta-CD (HP-beta-CD) (10 mM) was used as a chiral selector in pH 3.7 phosphate buffer. L-tryptophan (L-try) and ketoprofen (Ket) were used as displacement reagents to investigate the binding sites of AL to HSA. A binding synergism effect between hydrochlorothiazide (QL) and AL was observed and the results suggested that QL can destroy binding equilibrium of R-AL and S-AL toward HSA and they can occupy the same binding site of HSA (site I). The reproducibility was confirmed by RSD (RSD<1.5%) of the plateau height determined by FI-CE frontal analysis (FI-CE-FA). The FI-CE-FA was a good method to study protein-drug interaction.     

Determination of the number of binding sites and binding constant between diltiazem hydrochloride and human serum albumin by ultrasonic microdialysis coupled with online capillary electrophoresis electrochemiluminescence

A simple, sensitive and selective determination of diltiazem hydrochloride (DLT) is described using capillary electrophoresis electrochemiluminescence (CE-ECL). The CE-ECL parameters that affect separation and detection were optimized. Under the optimized conditions, the linear range of DLT was from 0.02 to 100 μmol/L (r(2) = 0.9983), with the detection limit of 5.1 nmol/L (3σ). The relative standard deviations of ECL intensity and the migration time were <2% for 0.1 μmol/L and 22 μmol/L DLT (n = 11). A new technique for determining of the number of binding sites and binding constant between DLT and HSA was developed using ultrasonic microdialysis coupled with CE-ECL. The number of binding sites and binding constant were 5.9 and 6.3 × 10(4) L/mol, respectively. The time required for ultrasonic microdialysis was 10 times less than that for traditional dialysis. Compared with traditional dialysis, ultrasonic microdialysis is simple, rapid, and should be applicable to a wide range of interactions of drugs and biomacromolecules.     

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs

In this study, two capillary electrophoresis–based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l -tryptophan (l -TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l -TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.     

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.     

Study on the interactions of sulfonylurea antidiabetic drugs with normal and glycated human serum albumin by capillary electrophoresis‐frontal analysis

Diabetes is one of the most widespread diseases characterized by a deficiency in the production of insulin or its ineffectiveness. As a result, the increased concentrations of glucose in the blood lead not only to damage to many of the body's systems but also cause the nonenzymatic glycation of plasma proteins affecting their drug binding. Since the binding ability influences its pharmacokinetics and pharmacodynamics, this is a very important issue in the development of new drugs and personalized medicine. In this study, capillary electrophoresis‐frontal analysis was used to evaluate the affinities between human serum albumin or its glycated form and the first generation of sulfonylurea antidiabetics, since their inadequate concentration may induce hypoglycaemia or on the contrary hyperglycaemia. The binding constants decrease in the sequence acetohexamide > tolbutamide > chlorpropamide > carbutamide both for normal and glycated human serum albumins, with glycated giving lower values. These results provide a more quantitative picture of how these drugs bind with normal and modified human serum albumin and indicate capillary electrophoresis‐frontal analysis to be another tool for examining the changes arising from modifications of albumin, or any other protein, with all its benefits like short analysis time, small sample requirement, and automation.     

Acid-induced transient isotachophoretic stacking of basic drugs in co-electroosmotic flow capillary zone electrophoresis

Online sample concentration or stacking of basic drugs by transient isotachophoresis with the injection of an acid in co-electroosmotic flow capillary zone electrophoresis was studied experimentally and with computer simulation. The acid stacking strategy afforded an order of magnitude improvement in concentration sensitivity for model tricyclic antidepressant and β blocker drugs.     

Study of the interactions between fluoroquinolones and human serum albumin by affinity capillary electrophoresis and fluorescence method

The interactions between fluoroquinolones and human serum albumin (HSA) were investigated by affinity capillary electrophoresis (ACE) and fluorescence quenching technique. Based on the efficient separation of several fluoroquinolones using a simple phosphate buffer, the binding constants of fluoroquinolones with HSA were determined simultaneously during one set of electrophoresis by ACE method. The thermodynamic parameters were obtained from data at different temperatures, and the negative ΔH and ΔS values showed that both hydrogen bonds and van der Waals interaction played major roles in the binding of fluoroquinolones to HSA. The interactions were also studied by fluorescence quenching technique. The results of fluorescence titration revealed that fluoroquinolones had the strong ability to quenching the intrinsic fluorescence of HSA through the static quenching procedure. The binding site number n, apparent binding constant K_b and the Stern-Volmer quenching constant K_sv were determined. The thermodynamic parameters were also studied by fluorescence method, and the results were consonant with that of ACE.     

Capillary electrochromatography with a neutral monolithic column for classification of analytes and determination of basic drugs in human serum

Capillary electrochromatography (CEC) using a neutral hydrophobic polymer-based monolithic column has been developed for classification of analytes based on their acidity and charges of group. The monolithic columns were prepared by in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate without any charged monomers in the reaction mixture. The ionic analytes will only be driven by their electrophoretic mobilities and were separated on the basis of their differences in electrophoretic mobility and in hydrophobic interaction with the stationary phase. Only negatively or positively charged analyte (acid or basic) migrated toward detection window in a single run by applying negative or positive voltage. The CEC system was also applied for the analysis of basic drugs in human serum by internal standard method using acidic running buffer. The sample of human serum spiked with basic drugs was directly injected after a simple sample pretreatment. The calibration curves with regression coefficients (0.9995-0.9997) in the range of 0.318-80 microg/mL were obtained with the limits of detection being below 0.15 microg/mL. The intra- and inter-day precisions, determined as relative standard deviations, were less than 4.21%.     

A new CZE method for profiling human serum albumin and its related forms to assess the quality of biopharmaceuticals

We present a new CZE method, which uses a polyethylene oxide-coated capillary to separate native HSA from more than five of its structural variants. These variants include oxidized, truncated, and cysteinylated forms of HSA which can all be found in biopharmaceutical products. Both CE and MS confirmed the high degree of heterogeneity of HSA preparations. Recovery studies demonstrated that adsorption of HSA on the capillary was significantly reduced under the conditions we developed, which led to a satisfactory repeatability (RSD for migration times and relative peak areas were less than 0.2 and 7.0%, respectively). Assignment of the main peaks was attempted using in vitro degraded/stressed HSA. We used our method to test batch-to-batch comparability and detected slight quantitative differences in the proportion of native HSA in batches produced from different fractionation methods.     

Binding of brucine to human serum albumin

The feature of brucine binding to human serum albumin (HSA) was investigated via fluorescence and UV/vis absorption spectroscopy. The results revealed that brucine caused the fluorescence quenching of HSA by the formation of brucine–HSA complex. The hydrophobic interaction plays a major role in stabilizing the complex; the binding site number n and apparent binding constant K_A, corresponding thermodynamic parameters the free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) at different temperatures were calculated. The distance r between donor (HSA) and acceptor (brucine) was obtained according to fluorescence resonance energy transfer. The effect of brucine on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy and UV/vis absorption spectroscopy.     

Evaluation of the enantioselective binding of imazalil to human serum albumin by capillary electrophoresis

In this work, a methodology for the evaluation of enantioselective binding of imazalil (IMA) enantiomers to human serum albumin (HSA) that does not require the separation of free and bound to HSA fractions is developed. This methodology comprises the incubation of IMA–HSA designed mixtures for 30 min directly in the capillary electrophoresis system and the subsequent direct injection and chiral separation of IMA employing highly sulfated β‐cyclodextrin as chiral selector and the complete filling technique. Two mathematical approaches were used to estimate apparent affinity constants (K₁), protein binding and enantioselectivity (ES) for both enantiomers of IMA. Moderate enantioselective binding of IMA enantiomers to HSA (ES = 2.0) was shown by the 1:1 stoichiometry and log K₁ values of 3.4 ± 0.4 and 3.1 ± 0.3 for the first and second eluted enantiomers, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

毛细管区带电泳—前沿分析法测定苯甲酸钠与牛血清白蛋白的相互作用

将毛细管区带电泳-前沿分析技术(CZE-FA)用于测定苯甲酸钠与牛血清白蛋白(BSA)平衡体系中结合参数的研究.通过压力进样将苯甲酸钠和白蛋白混合液引入石英毛细管电泳柱,在20 mmol/L硼砂缓冲液(pH=9.2)及20kV运行电压条件下,未结合苯甲酸钠的浓度可通过电泳平台峰的高度定量,两者具有良好的线性关系(相关系...     

Using capillary electrophoresis mobility shift assay to study the interaction of CdTe quantum dots with bovine serum albumin

In this work, the capillary electrophoresis mobility shift assay （CEMSA） was first adopted to study the interaction of protein with quantum dots （QDs）. In this study, bovine serum albumin （BSA） and CdTe QDs were used as model samples. We observed that BSA was facilely adsorbed to CdTe QDs surface, and the QD-BSA complex was formed by a 1：1 stoichiometric ratio. A value of 2.17 4-0.27 × 10^6 mol^-1 L^-1 （at 25 ℃） for the association constant was obtained by CEMSA.     

Capillary electrophoresis with electrochemiluminescence detection for the analysis of quinolone drugs and pharmacokinetics study

A novel method for the determination of two quinolone drugs norfloxacin （NOR） and levofloxacin （LVX） was described by capillary electrophoresis with electrochemiluminescence detection. The good relationship （r ≥ 0.9991） between peak area and concentration of analytes was established over two orders of magnitude. The limits of detection （LOD, S/N = 3） in standard solution are 4.8 × 10^-7 mol/L for NOR and 6.4 × 10^-7 mol/L for LVX, respectively. The limits of quantitation （LOQ, S/N = 10） in real human urine samples are 1.2 × 10^-6 mol/L for NOR and 1.4 × 10^-6 mol/L for LVX, respectively. The present method was successfully applied to the determination of NOR and LVX in human urine and the studv of oharmacokinetics of NOR.     

Comparison of three methods for analyzing loureirin B and human serum albumin interaction using capillary electrophoresis

Loureirin B (LB), a bioactive drug, is widely used in the treatment of biological diseases. However, due to its poor solution in water, it is important to find the approach which helps LB to specific biological targets. As the most abundant protein in plasma, HSA plays the role of a carrier of numerous drug ligand. Thus, the interaction between LB and HSA was explored by ACE, CE frontal analysis, and pressure‐mediated ACE under simulated physiological conditions (pH 7.4). The binding constants were calculated as 13.14 × 10⁴ L/mol, 7.00 × 10⁴ L/mol, and 2.78 × 10⁴ L/mol for each method, respectively. At the same time, the binding site number (n = 1.429) could be only calculated by the CE frontal analysis method. Furthermore, good experimental repeatability was obtained by pressure‐mediated ACE with RSDs for retention times and peak areas within 2.149 and 1.228, respectively.     

The auxiliary determination of the binding site of berberine binding to human serum albumin by surface-enhanced Raman scattering

We report on the joint application of fluorescence, ultraviolet-visible (UV-Vis) and Raman spectroscopy to the study of berberine with human serum albumin (HSA). We propose the surface-enhanced Raman scattering (SERS) technique to improve the understanding of the quenching interaction caused by berberine which could be applied in recognition process of fluorescent drugs with large biomolecules. The fluorescence and UV-Vis spectroscopic results show that the fluorescence intensity of HSA is significantly decreased in the presence of berberine, and the quenching mechanism is static. The SERS technique demonstrates clear advantages over direct measurements in physiological conditions. By means of this method, we are able to deduce important information concerning the binding property of berberine when interacting with HSA. We show the nitrogen atom is free but the dioxolane is involved in the spontaneously electrostatic inducement and subsequently hydrophobic binding.