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.     

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.     

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 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.     

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...     

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.     

Fast enantiomeric separation of propranolol by affinity capillary electrophoresis using human serum albumin as chiral selector: application to quality control of pharmaceuticals

In the last years, capillary electrophoresis (CE) has gained considerable interest in pharmaceutical laboratories for controlling the chiral purity of drugs. This paper describes a simple and fast method for resolution of propranolol enantiomers by affinity capillary electrophoresis (ACE) using human serum albumin (HSA) as chiral selector. The effect of several experimental variables such as HSA concentration, temperature, chiral selector plug length and addition of organic modifiers, on the separation is evaluated. Complete enantioresolution of R- and S-propranolol was achieved in less than 5 min when the capillary was completely filled with 100 μM HSA solution and the electrophoresis was carried out with 67 mM phosphate buffer (pH 7.4) at 20 kV and 35 °C. Peaks were assigned to each propranolol enantiomer according to their relative affinities to HSA. The proposed method was applied to the analysis of pharmaceutical preparations containing propranolol. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed method make it suitable for quality control of the enantiomeric composition of propranolol in pharmaceuticals.     

pH对氟喹诺酮药物与BSA之间相互作用影响的研究

采用毛细管区带电泳法,通过测定在不同pH值、不同牛血清白蛋白(BSA)浓度缓冲溶液的条件下药物迁移时间的变化,并分别计算出了pH为6.8、7.4和8.0时培氟沙星、左氧氟沙星、氧氟沙星、环丙沙星等四种氟喹诺酮类药物与BSA相互作用的结合常数.结果表明:pH对结合常数有较大影响,四种药物分子结合常数的最大值均出现在pH=6.8时,并随着pH的增大,结合常数值明显下降.根据实验结果,还对四种氟喹诺酮类药物与BSA之间相互作用的类型、作用位置进行了分析探讨.研究结果对于进一步阐明药用机理并迅速开发出更高效的广谱抗菌药物具有较强的理论意义.     

Fluorescence study on the interaction of human serum albumin with bromsulphalein

The binding of bromsulphalein (BSP) with human serum albumin was investigated at different temperatures, 298 and 308 K, by the fluorescence spectroscopy at pH 7.24. The binding constant was determined by Stern-Volmer equation based on the quenching of the fluorescence HSA in the presence of bromsulphalein. The effect of various metal ions on the binding constants of BSP with HSA was investigated. The thermodynamic parameters were calculated according to the dependence of enthalpy change on the temperature as follows: DeltaH and DeltaS possess small negative (9.3 kJ mol(-1)) and positive values (22.3 J K(-l)mol(-l)), respectively. The experimental results revealed that BSP has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The binding constants between BSP to HSA were remarkable and independent on temperature. The binding constants between HSA and BSP decreased in the presence of various ions, commonly decreased by 30-55%. The hydrophobic force played a major role in the interaction of BSP with HSA. All these experimental results and theoretical data clarified that BSP could bind to HSA and be effectively transported and eliminated in body, which could be a useful guideline for further drug design.     

Determination of L-tryptophan-serum albumin binding by high-performance liquid chromatography

Summary Two high-performance liquid chromatography (HPLC) techniques were developed for the determination of binding constants in the interaction of serum albumin with L-tryptophan: internal calibration and external calibration. The results obtained were compared with those obtained by the classical method of equilibrium dialysis and by gel filtration. While all the methods are equally reliable, the internal and external calibration techniques seem to be superior in their simplicity, speed and convenience.     

Spectroscopic studies on binding of Puerarin to human serum albumin

The interaction between Puerarin with human serum albumin has been studied for the first time by spectroscopic methods including fluorescence quenching technology, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that Puerarin can strongly quench the intrinsic fluorescence of HSA by static quenching and there is a single class of binding site on HSA. In addition, the studies of CD spectroscopy and FT-IR spectroscopy showed that the binding of Puerarin to HSA changed slightly molecular conformation of HSA. Furthermore, the thermodynamic functions ΔH⁰ and ΔS⁰ for the reaction were calculated to be −9.067 kJ mol⁻¹ and 54.315 J mol⁻¹ K⁻¹ according to van’t Hoff equation. These data suggested that both hydrogen bond and hydrophobic interaction play a major role in the binding of Puerarin to HSA, which is in good agreement with the result of molecular modeling study.     

Evaluation of the enantioselectivity of glycogen-based dual chiral selector systems towards basic drugs in capillary electrophoresis

Several chiral reagents including cyclodextrins (CDs) and derivatives, crown ethers, proteins, chiral surfactants and polymers have been involved in dual selector systems for enantioseparation of a series of chiral compounds by capillary electrophoresis (CE). In comparison to the chiral reagents above-mentioned, there is no report concerning the use of polysaccharides in dual chiral CE system. In this paper we first investigate the enantioselectivity of polysaccharide-based dual selector systems towards some chiral drugs. During our recent work, glycogen belonging to the class of branched polysaccharides has been used as a novel chiral selector in CE. In this study, three glycogen-based dual chiral CE systems have been established for enantiomeric separations of several racemic basic drugs consisting of duloxetine, cetirizine, citalopram, sulconazole, laudanosine, amlodipine, propranolol, atenolol and nefopam. These three dual systems combined glycogen (neutral polysaccharide) with chondroitin sulfate A (CSA, ionic polysaccharide), β-CD and HP-β-CD, respectively. It was found that the dual system of glycogen/CSA exhibited good enantioselective properties toward the tested drugs. More importantly, compared to the single selector systems, synergistic effect was observed when glycogen was used with CSA for most of the analytes. This indicated the enhancement of enantioseparation observed for these analytes in glycogen/CSA system might be due to some favorable interaction effects between glycogen and CSA. Moreover, in order to evaluate the stereoselectivity of glycogen/CSA, the influences of buffer pH and selector concentration on enantioseparation of the studied drugs were also investigated.     

Direct determination of sialic acids in serum by capillary electrophoresis with UV detection

Summary A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) has been developed using high-performance capillary electrophoresis with UV detection at 195 nm, without pre or post-column derivatisation. The acids were separated in a 50-cm, fused-silica capillary (50μ i.d, 45.5-cm effective length) with Na₂B₄O₇−Na₂HPO₄ buffer. The detection limit for NANA is a concentration of 9.6×10⁻⁶ M or, in terms of mass:3.879×10⁻¹⁴ mol (39 fmol). This method is applicable to determination of NANA in normal human serum. The results were also compared with those of the colorimetrie method.     

Qualitative study of the competition of drugs in binding to serum albumin

Summary The competitive binding of five drugs and a detergent to bovine serum albumin at pH 7.4 and room temperature was studied by Hummel-Dreyer method in high performance liquid chromatography. The five drugs are: warfarin, sulfinpyrazone, aspirin, quinidine gluconate and lidocaine and the detergent is the sodium dodecyl sulfate (SDS). While the quantitative techniques of Hummel-Dreyer method have been well developed during last twenty years, this paper reports, perhaps for the first time, the qualitative techniques of Hummel-Dreyer method as an analytic tool to ascertain whether a drug would bind to protein and how one drug would affect another drug in binding, if the binding does occur. The results on the basis of qualitative observation indicate the strength of binding in the following order: warfarin > aspriin > lidocaine > sulfinpyrazone > quinidine gluconate. The SDS has capacity to disturb the binding site on the surface of protein.