Affinity capillary electrophoresis and fluorescence spectroscopy for studying enantioselective interactions between omeprazole enantiomer and human serum albumin

Baseline separation of omeprazole (OME) enantiomers was achieved by affinity capillary electrophoresis (ACE), using human serum albumin (HSA) as the chiral selector. The influence of several experimental variables such as HSA concentration, the type and content of organic modifiers, applied voltage and running buffer concentration on the separation was evaluated. The binding of esomeprazole (S‐omeprazole, S‐OME) and its R‐enantiomer (R‐omeprazole, R‐OME) to HSA under simulated physiological conditions was studied by ACE and fluorescence spectroscopy which was considered as a reference method. ACE studies demonstrated that the binding constants of the two enantiomers and HSA were 3.18 × 10³ M⁻¹ and 5.36 × 10³ M⁻¹, respectively. The binding properties including the fluorescence quenching mechanisms, binding constants, binding sites and the number of binding sites were obtained by fluorescence spectroscopy. Though the ACE method could not get enough data when compared with the fluorescence spectrum method, the separation and binding studies of chiral drugs could be achieved simultaneously via this method. This study is of great significance for the investigation and clinical application of chiral drugs.     

Effect of pH and small inorganic ions on binding of sulfadimethoxine and sulfaphenazole to human serum albumin measured by circular dichroism

The binding of sulfadimethoxine and sulfaphenazole to human serum albumin (HSA) has been shown by circular dichroism measurements to be dependent on the N-B transition. The secondary drug binding sites were found to be optically active in the B conformation form in HSA but optically inactive in the N form. Moreover, the drug-HSA interaction in Tris-HCl buffer seems to be more sensitive to the conformational change in HSA, compared with that in the phosphate buffer.     

聚乙烯亚胺对人血清白蛋白的构象和结合能力的影响

为了解基因载体材料聚乙烯亚胺（PEI）细胞毒性的分子作用机理,本文应用吸收光谱、荧光光谱、圆二色谱、动态光散射和zeta-电位测定分析平均相对分子量为1.8和25 kDa的PEI（记为PEI1.8k和PEI25k）对人血清白蛋白（HSA）构象的影响,同时以8-苯氨基萘-1-磺酸镁（ANS）和槲皮素为模型化合物,了解PEI对HSA结合能力的影响及机理。结果发现,PEI与HSA结合形成静态复合物,导致HSA流体动力学直径变小和分子内环境疏水性增强。PEI1.8k和低浓度的PEI25k引起HSA的a-螺旋结构增加,但是高浓度的PEI25k对HSA二级结构具有稳定作用。PEI对HSA结合能力的影响主要归因于PEI的竞争结合和PEI与HSA结合引起的蛋白质构象变化。PEI的竞争结合降低了HSA对ANS和槲皮素的结合效率,但是蛋白质的构象变化增强了HSA与ANS和槲皮素的结合能力。PEI与HSA的相互作用具有明显的分子尺寸效应,增加PEI的相对分子量可以增强对HSA构象和结合能力的影响。     

Binding of Dioxopromethazine Hydrochloride with Human Serum Albumin and Its Effect on the Conformation of the Protein

The interaction between dioxopromethazine hydrochloride (DPZ) and human serum albumin (HSA), in vitro, was investigated by means of fluorescence, absorption and circular dichroism (CD) spectroscopy. The results obtained from analysis of the fluorescence spectra indicate that DPZ has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. The HSA?CDPZ binding distance was determined to be less than 7?nm, suggesting that energy transfer from HSA to DPZ may occur. The thermodynamic parameters of the interaction between DPZ and HSA were measured according to the van??t Hoff equation. The negative enthalpy change (??H ⁰) and positive entropy change (??S ⁰) values indicate that hydrophobic and electrostatic interactions play major roles in the binding between DPZ and HSA. The binding process was a favorable process in which the Gibbs energy change (??G ⁰) is negative. The results of displacement experiments suggested that DPZ is located in site I of HSA within subdomain IIA. In addition, the results of absorption, synchronous fluorescence and CD spectra show that binding of DPZ with HSA can induce conformational changes in HSA.     

Evaluation of enantioselective binding of antihistamines to human serum albumin by ACE

The drug binding to plasma and tissue proteins is a fundamental factor in determining the overall pharmacological activity of a drug. HSA, together with alpha(1)-acid glycoprotein, are the most important plasma proteins, which act as drug carriers, with implications on the pharmacokinetic of drugs. Among plasma proteins, HSA possesses the highest enantioselectivity. In this paper, a new methodology for the study of enantiodifferentiation of chiral drugs with HSA is developed and applied to evaluate the possible enantioselective binding of four antihistamines: brompheniramine, chlorpheniramine, hydroxyzine and orphenadrine to HSA. This study includes the determination of affinity constants of drug enantiomers to HSA and the evaluation of the binding sites of antihistamines on the HSA molecule. The developed methodology includes the ultrafiltration of samples containing HSA and racemic antihistaminic drugs and the analysis of the free or bound drug fraction using the affinity EKC-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of antihistamines to HSA, the major plasmatic protein.     

Effect of glycation on the heterogeneity of human serum albumin analysed by reversed-phase high-performance liquid chromatography in a solvent containing formic acid.

Non-enzymic glycation of human serum albumin (HSA) induces a change in its charge heterogeneity that may account for its particular renal clearance in patients with early diabetic nephropathy. A new high-performance liquid chromatographic analysis for the study of HSA heterogeneity is described based on a high content of formic acid in the mobile phase combined with a concave gradient of isopropanol. Under these conditions, native HSA was separated into three individual components (I, II and III). When glycated HSA was analysed, it was found that although the present method is not suitable for the separation of glycated from non-glycated HSA, it shows the effect of glycation in producing changes in HSA heterogeneity that are different from those reported on surface change. This finding suggests an additional factor (probably conformational changes) that is contributing to the heterogeneity of glycated HSA.     

Chiral complexation and aggregation of bilirubin with serum albumin at a liquid/liquid interface

The chiral complexation of bilirubin (BR) with bovine and human serum albumin (BSA and HSA), and the aggregation of the complexes at the heptane+chloroform(5:1)/water interface were studied via UV/Vis absorption and circular dichroism (CD) measurements in combination with the centrifugal liquid membrane (CLM) method. The interfacial adsorptivities of BR, BSA and their complexes were also studied by performing interfacial tension measurements at the interface. The changes in the absorbances and the induced CD amplitudes of the interfacial BR-BSA complex provided insights into the mechanism of the conformational enantioselective complexation at the interface, and indicated that the chiral conversion induced by the complexation with BSA was from the P(+) form to the M(-) form of BR. The broadening of the 450 nm band and the appearance of a new shoulder at 474 nm further supported the formation of aggregates of the complexes at the interface. The dependence of the CD amplitude on the molar ratio of BSA to BR revealed that the composition of the complex was 1:1 BSA:BR. The probable interfacial reaction scheme was proposed, and the affinity constant of BR-BSA at the interface was found to be 4.67 x 10(8) M(-2). The interfacial complexation and aggregation of BR and HSA were weaker than those of the BR-BSA complex due to the different BR binding positions adopted for BSA and HSA and the binding effect of chloroform.     

A Thermodynamic Study on the Binding of Human Serum Albumin with New Synthesized Anti Cancer Pd (II) Complex

The thermodynamics of the interaction between new synthesized anti-cancer drug (2,2′-bipyridin n-butyl dithiocarbamato Pd (II), ButPd), and HSA was investigated at pH=7 by isothermal titration calorimetry. A new solvation model was used to reproduce the enthalpies of HSA interaction by ButPd within a broad range of complex concentrations. The solvation parameters attained from the new model were attributed to the structural change and biological activity of HSA. The binding parameters for the interaction of ButPd and HSA indicated that the considerable conformational changes in HSA were not observed after being bound with ButPd. It was found that HSA has three identical and cooperative binding sites for ButPd.     

利福布汀与人血清白蛋白相互作用的光谱研究

用荧光光谱法和圆二色谱法研究了利福布汀(RB)与人血清白蛋白(HSA)的相互作用. 结果表明, RB与HSA之间的相互作用主要是疏水作用, 作用机制是静态猝灭与动态猝灭的结合. 其结合常数(Ka)在106数量级, 说明RB和HSA有很强的结合. 此外, 探讨了金属离子(Cu2+, Zn2+, Mg2+ 和Ca2+)对RB与HSA结合常数的影响. 同步荧光光谱和圆二色谱数据表明, RB可导致HSA的构象改变.     

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.     

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase.

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin-azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.     

Enantioseparation of nuarimol by affinity electrokinetic chromatography-partial filling technique using human serum albumin as chiral selector

The present paper deals with the enantiomeric separation of nuarimol enantiomers by affinity EKC-partial filling technique using HSA as chiral selector. Firstly, a study of nuarimol interactions with HSA by CE-frontal analysis was performed. The binding parameters obtained for the first site of interaction were n(1) = 0.84; K(1) = 9.7 +/- 0.3x10(3 )M(-1) and the protein binding percentage of nuarimol at physiological concentration of HSA was 75.2 +/- 0.2%. Due to the moderate affinity of nuarimol towards HSA the possibility of using this protein as chiral selector for the separation of nuarimol using the partial filling technique was evaluated. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length was carried out. Separation of nuarimol enantiomers was obtained under the following selected conditions: electrophoretic buffer composed of 50 mM Tris at pH 7.3; 160 muM HSA solution applied at 50 mbar for 156 s as chiral selector; nuarimol solutions in the range of 2-8x10(-4) M injected hydrodynamically at 30 mbar for 2 s and the electrophoretic runs performed at 30 degrees C applying 15 kV voltage. Resolution, accuracy, reproducibility speed and cost of the proposed method make it suitable for quality control of the enantiomeric composition of nuarimol in formulations and for further toxicological studies. The results showed a different affinity between nuarimol enantiomers towards HSA.     

Hyphenation of ionic liquid albumin glassy carbon biosensor or protein label-free sensor with differential pulse stripping voltammetry for interaction studies of human serum albumin with fenoprofen enantiomers

A new biosensor or protein label-free sensor composed of 1-butyl-3-methylimidazolium hexafluorophosphates (BMIMPF₆)-human serum albumin (HSA) film on glassy carbon electrode (GCE) was produced. Unfortunately, the native proteins themselves are often unstable in physiological conditions. Here, we introduced conjugation with ionic liquid (IL) such as BMIMPF₆ which improved the stability and binding affinity of protein onto GCE. A rapid, simple and reliable method for the chiral discrimination and real time protein binding studies of fenoprofen enantiomers with HSA was developed by hyphenating ionic liquid albumin glassy carbon (ILAGC) biosensor with differential pulse cathodic stripping voltammetry under physiological conditions. The electrochemical behavior of chiral fenoprofen was monitored by cyclic voltammetry, from which large response was obtained from l-fenoprofen. The surface coverage of fenoprofen enantiomers was calculated by double potential-step chronocoulometry. The binding constants of chiral fenoprofen with HSA were estimated to be 3.2 × 10⁵ ± 0.3 L mol⁻¹ and 0.8 × 10⁴ ± 0.4 L mol⁻¹ for l- and d-fenoprofen, respectively giving acceptable precision (SD ≤ 0.4) and good agreement with the literature values. The competitive interactions of ibuprofen with fenoprofen enantiomers–HSA were studied giving a significant decreasing in the binding degrees of analytes to HSA. The reciprocal competitive experiments indicated that l-fenoprofen replaced d-fenoprofen from HSA. The proposed electrochemical biosensor holds great potential for chiral discrimination and real time binding studies of drugs with protein.     

Studies of Interaction between Ergosta‐4,6,8(14),22‐tetraen‐3‐one (Ergone) and Human Serum Albumin by Molecular Spectroscopy and Modeling

Our previous experimental results have shown that ergosta‐4,6,8(14),22‐tetraen‐3‐one (ergone) is one of the main bioactive components of Polyporus umbellatus. The efficacy of ergone binding to human serum albumin (HSA) is critical for pharmacokinetic behavior of ergone. The interactions between ergone and HSA under simulative physiological conditions were investigated by the methods of fluorescence spectroscopy, absorption and circular dichroism spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by ergone was the result of the formation of the ergone‐HSA complex. According to the modified Stern‐Volmer equation, the binding constants (K_a) between ergone and HSA were determined. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be 0.989 kJ mol^‐1 and 11.214 J mol^‐1 K^‐1, indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding of ergone to HSA. The conformational investigation showed that the presence of ergone decreased the α‐helical content of HSA and induced the slight unfolding of the polypeptides of protein. Furthermore, displacement experiments using warfarin and ibuprofen indicated that ergone could bind to site I of HSA, which was also in agreement with the results of the molecular modeling.     

Resonance energy transfer and ligand binding studies on pH-induced folded states of human serum albumin

Human serum albumin (HSA) is a very important transporter protein in the circulatory system. It is a multi-domain binding protein, which binds a wide variety of ligands in its multiple binding sites and aids in transport, distribution and metabolism of many endogenous and exogenous ligands. With change in pH, HSA is known to undergo conformational transformation, which is very essential for picking up and releasing them at sites of differing pH inside physiological system. Hence, the characterization of ligand binding to these pH-induced conformers is extremely important. We have explored binding interaction of a ligand protoporphyrin IX (PPIX), which is demonstrated (X-ray crystallography) to reside in domain-IB at the various pH-induced folded states of HSA. The ligand PPIX is found to remain attached to all the HSA conformers which offers an opportunity to use Förster’s resonance energy transfer (FRET) between an intrinsic donor fluorophore (Trp214) located in domain-IIA to the acceptor ligand PPIX to characterize the inter-domain separation between IB and IIA. Additionally FRET between an extrinsic fluorophore 2-p-toluidinylnaphthalene-6-sulfonate (TNS) located in domain-IIIA and PPIX is also undertaken to quantify the inter-domain separation between IB and IIIA. Circular dichroism (CD) and dynamic light scattering (DLS) studies have been done in conjunction with picosecond time resolved fluorescence and polarization-gated spectroscopy to determine, respectively, the secondary and tertiary structures of various pH-induced folded states of the protein. Severe structural perturbation including swelling of the protein is observed in the low pH-induced conformer of HSA as evidenced from all the techniques used.     

Binding of bezafibrate to human serum albumin: insight into the non-covalent interaction of an emerging contaminant with biomacromolecules

In recent years, bezafibrate (BZF) has been frequently detected in environmental media. In order to reveal the toxicity of such an emerging pollutant, its interaction with human serum albumin (HSA) was studied by fluorescence spectrometry, circular dichroism, and equilibrium dialysis. Fluorescence data showed that the fluorescence quenching of HSA by BZF resulted from the formation of HSA-BZF complex. The binding constants were determined to be 3.33 × 103, 2.84 × 103 M?1 at 298 and 309.5 K, respectively. The thermodynamic determination indicated that the hydrophobic and electrostatic interaction were the dominant binding force. The conformational investigation showed that the presence of BZF increased the α-helix content of HSA and induced the slight unfolding of the polypeptides of protein. Finally, the equilibrium dialysis showed that 0.56 mM BZF decreased the binding of vitamin B? to HSA by 29%.     

Application of liquid pre-column capillary electrophoresis technique to the study of interaction between drug enantiomers and human serum albumin

Based on the chiral separation of several basie drugs, dimetindene, tetryzoline, theodrenaline and verapamil, the liquid pre-colunm capillary electrophoresis (LPC-CE) technique was established. It was used to determine free concentrations of drug enantiomers in mixed solutions with human serum albumin (HSA). To prevent HSA entering the CE chiral separation zone, the mobility differences between HSA and drugs under a specific pH condition were employed in the LPC. Thus, the detection confusion caused by protein was totally avoided. Further study of binding constants determination and protein binding competitions was carried out. The study proves that the LPC technique could be used for complex media, particularly the matrix of protein coexisting with a variety of drugs.     

Spectroscopic studies on the effect of temperature on pH-induced folded states of human serum albumin

Human serum albumin (HSA) is a very important multi-domain transporter protein in the circulatory system responsible for carriage of various kinds of ligands within the physiological system. HSA is also known to undergo conformational transformation at different pH(s) and temperatures. In this report we have studied the binding interactions of a photosensitizing drug, protoporphyrin IX (PPIX) with various conformers of HSA at different temperatures using picosecond time-resolved fluorescence spectroscopy. Also, using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy we have followed the structural transition of various conformers of HSA at different temperatures. Ensuring the intact binding of PPIX to various conformers of HSA at different temperatures as revealed through time-resolved fluorescence anisotropy decay and significant spectral overlap of emission of Trp214 residue (donor) in domain-IIA and absorption of PPIX (acceptor) bound to domain-IB of HSA, we have applied F?rster's resonance energy transfer (FRET) technique to determine the interdomain separation under various environmental conditions. The alkali-induced conformer of HSA shows almost no change in donor-acceptor distance in contrast to the native and acid-induced conformers of HSA, which show a decrease in distance with increase in temperature. Through this study the non-covalently bound PPIX is shown to be an efficient FRET probe in reporting the different temperature-induced folded states of HSA in buffer solutions of widely differing pH values.     

Potential of human serum albumin as chiral selector of basic drugs in affinity electrokinetic chromatography-partial filling technique

The enantiomeric resolution of compounds using HSA by means of affinity EKC (AEKC)-partial filling technique is the result of a delicate balance between different experimental variables such as protein concentration, running pH (background electrophoretic buffer (BGE), protein, and compound solutions), and plug length. In this paper, the possibility of using HSA as chiral selector for enantioseparation of 28 basic drugs using this methodology is studied. The effect of the physicochemical parameters, the structural properties of compounds, and compound-HSA protein binding percentages over their chiral resolution with HSA is outlined. Based on the results obtained, a decision tree is proposed for the "a priori" prediction of the capability of HSA for enantioseparation of basic drugs in AEKC. The results obtained indicated that enantioresolution of basic compounds with HSA depends on the hydrophobicity, polarity, and molar volume of compounds.     

Development of a disulfiram-modified human serum albumin-based HPLC column

Summary A human serum albumin (HSA)-based HPLC column has been modified in situ by disulfiram, an alcohol-deterrent drug reported to bind cys₃₄, the only free cysteine in HSA, under physiological conditions. The reversible and covalent binding of disulfiram was found to change the binding properties of the protein, giving rise to a new selector which performed differently from the native albumin-based stationary phase. When low concentrations of disulfiram were used as mobile phase modifier, reversible binding resulted in a cooperative allosteric effect with improved selector performance. Covalent modification resulted in markedly reduced affinity for binding of the drugs to sites I or II, while still maintaining enantioselectivity. This study has enabled the monitoring of interactions of disulfiram with potentially coadministered drugs, and the preparation of a chiral selector with different drug affinity and enantioselectivity.