Capillary Electrophoresis-frontal Analysis Research Articles

Investigating noncovalent interactions of rutin – serum albumin by capillary electrophoresis – frontal analysis

The application of capillary electrophoresis-frontal analysis (CE-FA) to study noncovalent interaction between rutin and serum albumin (bovine serum albumin, BSA and human serum albumin, HSA) in phosphate buffer solution (67 mM, pH 7.4) at 37 °C is presented. Using fixed HSA or BSA concentration and increasing rutin concentration, the number of primary binding sites per HSA or BSA molecules, and the affinity constants were obtained. Both affinity constants are in a comparable range suggesting the similarity of affinity properties of HSA and BSA towards rutin. The proposed CE-FA method is simple, rapid and cost-effective which may be useful in further high-throughput protein binding studies of multi-components in traditional herbal medicines for pharmacological effect evaluations.

Interaction of glycyrrhetinic acid, furosemide and hydrochlorothiazide with bovine serum albumin and their displacement interactions: capillary electrophoresis and fluorescence quenching study

Licorice is the most widely used crude drug in traditional Chinese medicine. Glycyrrhetinic acid (GA) is the metabolite of glycyrrhizic acid, which is the main bioactive ingredient of licorice. In this work, capillary electrophoresis-frontal analysis (CE-FA) was applied to study the binding of bovine serum albumin with GA and two diuretics: furosemide (FU) and hydrochlorothiazide (HZ). The binding parameters of GA were determined by Scatchard analysis, which showed that there are two kinds of binding sites in bovine serum albumin for GA. However, the results showed that the CE-FA method was not suitable for the interaction study of FU and HZ. Therefore, utracentrifugation-CE was used to probe the binding characteristic of these two drugs and the results showed only one kind of binding site for them under the studied conditions. Displacement interactions between these drugs were also investigated by utracentrifugation-CE method and the results showed that GA hardly displaces HZ while it can slightly displace FU and FU can slightly displace HZ. For comparison, the binding of these drugs was also studied by the fluorescence quenching method and the data were processed by the Stern-Volmer quenching equation. Results showed that the binding constants were basically consistent for two methods for all drugs studied. The number of binding sites on one protein molecule was well consistent for FU and HZ while it was quite different for GA.

Screening of phage displayed human liver cDNA library against dexamethasone

This paper described an attempt to establish a new method to screen the target biomolecule from phage displayed cDNA library against small molecule drug insoluble in water. Dexamethasone was selected as the model drug, and the screening was carried out in an Eppendorf tube packed with the drug. The whole procedure was monitored by PCR with the enriched specific phage clone as the template. After four rounds screening, the PCR products of selected phages with the lengths of 400 and 600 bp were sequenced, and revealed identical sequences with cytochrome c oxidase subunit III and albumin respectively by GenBank searching. Furthermore, frontal analysis-capillary electrophoresis (FA-CE) was performed to study the interaction between dexamethasone and albumin, and the binding constant was calculated to be 1.153 × 10 3, validating the weak specific interaction between the drug and the target protein. All these results demonstrated that with insoluble drug as the solid phase directly, the screening of target large molecule expressed in phage display cDNA library was feasible, which might pave an easy way to screen the candidate drug targets.

Frontal Analysis Capillary Electrophoresis Hyphenated to Electrospray Ionization Mass Spectrometry for the Characterization of the Antithrombin/Heparin Pentasaccharide Complex

The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. A new strategy based on the hyphenation of frontal analysis capillary electrophoresis (FACE) with electrospray ionization mass spectrometry (ESIMS) is reported for the characterization of protein/carbohydrate complexes. While most of the previously reported CE-MS experiments were performed using capillary electrophoresis in zone format, we report for the first time CE-MS experiments in which CE was performed in frontal analysis (FACE-MS). We showed that the frontal mode offered a better sensitivity than zone mode and was well suited for the CE-MS coupling. This FACE-MS coupling was applied to the analysis of the complex between antithrombin and the sulfated pentasaccharide reproducing the antithrombin-binding sequence in heparin. The mixture of coincubated antithrombin and heparin pentasaccharide was continuously injected into the capillary, and the electrophoretic separation of the free and bound forms of the protein was achieved. The intact noncovalent complex antithrombin/heparin pentasaccharide was detected on-line by ESIMS in positive ionization mode and in nondenaturing sheath liquid conditions. The complex stoichiometry was determined from the mass measurement of the complex. In addition, the characterization of the sulfated pentasaccharide ligand dissociated from the complex was performed in negative ionization mode using a denaturing sheath liquid, allowing the determination of its molecular mass and sulfation features. This FACE-ESIMS strategy opens the way to ligand fishing experiments performed on heterogeneous carbohydrate mixtures and subsequent characterization of specifically bound carbohydrates.

Characterization of antihistamine–human serum protein interactions by capillary electrophoresis

An important topic in the drug discovery and development process is the role of drug binding to plasma proteins. In this paper the characterization of the interaction between antihistamines (cationic drugs) towards human serum albumin (HSA) and α 1-acid glycoprotein (AGP) under physiological conditions by capillary electrophoresis-frontal analysis is presented. Furthermore, the binding of these drugs to all plasma proteins is evaluated by using ultrafiltration and capillary electrophoresis. Antihistamines present a wide-ranging behaviour with respect to their affinities towards plasma proteins. Orphenadrine, phenindamine, tripelenamine and tripolidine principally bind to HSA; carbinoxamine, dimetindene and etintidine principally bind to AGP; brompheniramine, chlorpheniramine and ranitidine present an important binding to lipoproteins and/or globulins and finally, chlorcyclizine, cinarizine, cyclizine, doxylamine, hydroxyzine, perphenazine and terfenadine do not bind to lipoproteins and/or globulins but bind to HSA and AGP in different extension. The interaction of antihistamines with HSA is determined by the hydrophobicity (direct relationship) and the polar surface area (indirect relationship) of the compounds. The steric parameters and hydrogen bonding character of compounds seems to be related with the binding of antihistamines to AGP. The antihistamine–HSA affinity constants were evaluated and the K 1 values ranged from 7 × 10 2 M −1 (for doxylamine) to 4 × 10 4 M −1 (for phenindamine).

Development of an ESI-MS screening method for evaluating binding affinity between integrin fragments and RGD-based peptides

Electrospray ionization mass spectrometry (ESI-MS) is used to evaluate binding affinities between integrin fragments and RGD-based peptide ligands. The integrin fragment peptides synthesized and studied were selected based on crystallographic and literature data which highlighted the specific binding pockets for the RGD motif in intact integrin proteins. Relative binding constants were determined by fitting the data obtained from ESI-MS titration experiments to a quadratic equation based on a 1:1 association model. Frontal analysis capillary electrophoresis (FACE) was used as a complementary solution phase technique to measure the absolute binding constants for the same host–guest systems and the results from both techniques were compared. Gas phase collision activated dissociation threshold measurements in an ion trap mass spectrometer also were carried out on the complexes to further validate binding constant measurements. Better correlation was observed between the MS/MS and CE results, than for ESI-MS titration. The ESI-MS titration results showed some correlation; however, it is apparent that there are important methodological shortcomings which need to be addressed. Several factors related to the titration method, ionization efficiencies, enthalpy/entropy contributions during phase transfer, and solvent effects are discussed. This work introduces a first approach to development of a high throughput ESI-MS-based method suitable for screening potential peptide-based drug compounds that target integrins on cancerous cells. It also serves as a cautionary tale to those interested in performing similar types of analyses.

Investigating noncovalent squarylium dye–protein interactions by capillary electrophoresis–frontal analysis

Noncovalent interactions between fluorescent probe molecules and protein analyte molecules, which typically occur with great speed and minimal sample handling, form the basis of many high sensitivity analytical techniques. Understanding the nature of these interactions and the composition of the resulting complexes represents an important area of study that can be facilitated by capillary electrophoresis (CE). Specifically, we will present how frontal analysis (FA) and Hummel-Dreyer (HD) methods can be implemented with CE to determine association constants and stoichiometries of noncovalent complexes of the red luminescent squarylium dye Red-1c with bovine serum albumin (BSA) and β-lactoglobulin A. By adjusting solution conditions, such as pH or ionic strength, it is possible to selectively modify the binding process. As such, conditions for optimal selectivity for labeling reactions can be established by capillary electrophoresis–frontal analysis (CE–FA) investigations.

Tudy on drug displacement interactions by capillary electrophoresis-frontal analysis

The interaction between 18-methyl norethindrone and ketoprofen including the displacement of ketoprofen from human serum albumin binding sites was investigated by capillary electrophoresis-frontal analysis method (CE-FA) at room temperature. A very large sample plug was introduced hydrostatically into the capillary (65 cm x 50 microm i.d., effective length of 35 cm) over 80 s at a height difference of 11 cm. The working conditions for CE-FA separation are as follows: operating voltage, 10 kV; running buffer, 67 mmol/L phosphate, pH 7.4. The unbound ketoprofen concentration was directly measured from the height of the frontal peak. When the concentration of 18-methyl norethindrone was increased from 0 to 200 micromol/L, the unbound ketoprofen concentration was found to increase from 22.4 to 26.4 micromol/L at 100 micromol/L total ketoprofen concentration and from 82.1 to 106.2 micromol/L at 200 micromol/L total ketoprofen concentration. From these data, it may be deduced that the administration of high concentration of 18-methyl norethindrone could displace ketoprofen from its secondary binding site.

Single run measurements of drug‐protein binding by high‐performance frontal analysis capillary electrophoresis and mass spectrometry

A novel drug-protein binding measurement method based on high-performance frontal analysis and capillary electrophoresis (HPFA/CE) is presented. A single run measurement approach is proposed to circumvent utilization of a calibration curve that is often performed with HPFA. A sensitive mass spectrometer is applied as a detector enabling the measurement of in vitro protein binding at lower drug concentrations. Unbound free fraction and binding constants can be determined by a single run measurement by consecutive injections of an internal drug standard, a buffer plug and a drug-protein mixture. Effects of injection volumes on peak height and plateau profile were investigated in two different separation systems, non-volatile buffer and volatile buffer, with UV and mass spectrometry detection, respectively. A simplified one-to-one binding model is employed to evaluate the proposed method by using both single and multiple drug concentrations to measure the unbound free fraction and calculate the binding constants of some selected compounds. The method is suitable for rapid and direct screening of the binding of a drug to a specific protein or drug-plasma protein binding.

Complexation between low-molecular-weight cationic ligands and negatively charged polymers as studied by capillary electrophoresis frontal analysis.

The potential of using capillary electrophoresis frontal analysis for the study of low-molecular-weight ligand-polyelectrolyte interactions was assessed. The interaction of the ligands 1-propylpyridinium bromide, 2-propylisochinolinium bromide, and paraquat with the polymer dextran sulfate was investigated as a function of polymer concentration and ionic strength of the buffer solution. Linear binding isotherms were obtained and association constants were determined. The complex formation was independent of the dextran sulfate concentration at low ionic strength. Ligand-polyelectrolyte interactions were strongly dependent on the ionic strength. The interaction of the divalent cation paraquat with the dextran sulfate was much stronger than the interactions of the monovalent cationic ligands with the polyelectrolyte. The binding data obtained were in accord with results obtained by equilibrium dialysis. Capillary electrophoresis frontal analysis has the potential to become a valuable tool for characterization of ligand-polyelectrolyte interactions in drug design as well as in other areas.

High-throughput capillary electrophoresis frontal analysis method for the study of drug interactions with human serum albumin at near-physiological conditions.

The application of the short-end capillary injection to capillary electrophoresis frontal analysis (CE-FA) to study the interaction between basic, neutral and acid drugs towards human serum albumin (HSA) at near-physiological conditions is presented. The compounds selected display a wide range of binding affinities and the results obtained were in good agreement with those reported in the literature. An equation for the estimation of the number of primary binding sites and their corresponding affinity constants is developed isolating the experimentally measured variables in just one axis. The proposed CE-FA method to screen drug interactions with HSA under physiological conditions is simple, rapid and cost-effective what may facilitate its implementation in the drug discovery process.

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 micromol 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.

Capillary electrophoresis frontal analysis: principles and applications for the study of drug-plasma protein binding.

Capillary electrophoresis is a well-established technique for the study of noncovalent interactions. Various approaches exist and capillary electrophoresis-frontal analysis provides an interesting alternative to the migration shift affinity capillary electrophoresis methods and conventional methods. The present work reviews the principles on which the frontal analysis method is founded. Advantages and limitations of capillary electrophoresis frontal analysis in comparison with both conventional and other capillary electrophoresis based methods for quantification of binding interactions are discussed. Investigations utilizing capillary electrophoresis-frontal analysis have focused on the interaction of drugs with plasma proteins. These studies, primarily addressing the binding of drugs to human serum albumin, alpha1-acid glycoprotein, and lipoproteins are reviewed together with some recent developments in capillary electrophoresis-frontal analysis methodology.

Complexation trends and binding constants between dextrin oligomers and small molecules as measured through affinity capillary electrophoresis.

Quantitative aspects of a previously reported pre-equilibration affinity/capillary electrophoretic methodology (Hong, M., et al., Anal. Chem. 1998, 70, 3590-3597) were studied using fluorescently labeled oligosaccharides and analgesic drugs. Frontal analysis capillary electrophoresis (FA-CE) was investigated for these interaction systems. In order to fulfill certain FA requirements, the analysis conditions of the sugar-drug combinations had to be adjusted individually. Only after the establishment of these conditions, acceptable binding data (association constants) could be obtained. The constants are in the range of weak hydrogen bonding and are comparable to the constants that were independently obtained through 1H nuclear magnetic resonance (NMR) spectroscopy.

Study on the protein binding of ketoprofen using capillary electrophoresis frontal analysis compared with liquid chromatography frontal analysis.

A method of capillary electrophoresis frontal analysis (CEFA) is developed for the first time to study the binding of ketoprofen to human serum albumin (HSA) and compared with high-performance liquid chromatography frontal analysis (LCFA). The separation is performed in an uncoated fused-silica capillary (60-cm x 75- micro m i.d., 50-cm effective length) with a phosphate buffer (pH 7.4, ionic strength of 0.17M) as the running buffer. The applied voltage is 13 kV and the detection is set at 254 nm. A trapezoidal peak of the unbound ketoprofen appears after HSA elution in the electropherogram. The plateau height of the peak is employed to determine the unbound concentration of ketoprofen in the HSA equilibrated sample solution. The CEFA method provides the advantages of small sample injection volume and rapidity and the disadvantage of low sensitivity compared with LCFA. CEFA is applicable to the binding parameter estimation of ketoprofen to the secondary binding site; an association constant (K(2)) of 0.24 x 10(6)M(-1) and the number for the binding site per molecule HSA of 2.54 is estimated. In contrast, LCFA measures parameters for both primary and secondary sites, which are 1.05 x 10(6)M(-1) and 0.94 for K(1) and n(1), respectively, and 0.12 x 10(6)M(-1) and 3.16 for K(2) and n(2), respectively. It is found that ketoprofen binds mainly at the primary site at a molecular ratio of ketoprofen versus HSA lower than 0.75, and the binding at the secondary site occurs at a higher ratio.

Capillary electrophoretic study on pH dependence of enantioselective disopyramide binding to genetic variants of human alpha1-acid glycoprotein.

A high-performance frontal analysis-capillary electrophoresis (HPFA-CE) method was applied to investigate the effect of pH on the drug binding properties of genetic variants of human alpha1-acid glycoprotein (AGP), A variant and a mixture of F1S variants. The unbound concentrations of a model basic drug, disopyramide (DP), in A variant solutions and in F1S variant solutions were measured by HPFA-CE to evaluate binding constants at pH 4.0, 5.0, 6.0 and 7.4. The binding between DP and A variant was gradually weakened by acidification of background buffer (from pH 7.4 to 4.0), while the binding between DP and FIS variants decreased at first (from pH 7.4 to 6.0), and then gained (from pH 6.0 to 4.0). Consequently, DP was more strongly bound to A variant than to FIS variants at pH 7.4, while at pH 4.0 DP was more strongly bound to F1S variants. At any pH (S)-DP was bound more strongly than (R)-DP, and the enantioselectivity of A variant was significantly higher than that of F1S variants. Electrophoretic mobilities of the AGP genetic variants decreased along with a decrease in pH. Fluorescent emission of these genetic variants indicated a distinct conformational change between pH 5.0 and 4.0. However, there was no significant difference in the electrophoretic mobility and the fluorescent emission spectrum between these variants at any pH. On the other hand, circular dichroism analyses revealed that beta-sheet content in FIS variants diminished as pH decreased, while that in A variant increased. These results suggest that the conformational change induced by acidification of background buffer differs between these genetic variants, and this causes the difference in DP bindability.

Effect of Dextran as a Run Buffer Additive in Drug−Protein Binding Studies Using Capillary Electrophoresis Frontal Analysis

The study of drug-protein interactions by capillary electrophoresis frontal analysis requires establishment of a sufficient mobility difference between the mobility of the ligand and protein. The potential utility of dextran as a run buffer additive to manipulate the electrophoretic mobilities of low molecular weight ligands and protein in capillary electrophoresis frontal analysis binding studies was assessed. It was demonstrated that dextran was effective in improving the separation between the ligands warfarin and flurbiprofen and human serum albumin. Separation of ligand and protein increased with the concentration of added dextran (0-7.5% (w/w)), while molecular weight of the additive (70,000-2,000,000) only had a minor effect. The effect of dextran addition on viscosity and electrophoretic and electroosmotic mobilites was systematically studied. Optimal frontal analysis settings were a compromise between achieving satisfactory separation and acceptable analysis times without loss of plateau peak conditions. No effect of dextran upon the drug-human serum albumin interactions could be detected for the model ligands. Introduction of dextran into the electrophoresis buffer expands the applicability of capillary electrophoresis frontal analysis in drug research to binding interactions between proteins and low molecular weight ligands possessing similar electrophoretic mobilities.

Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions.

Capillary electrophoresis frontal analysis was applied to 12 low molecular weight compounds including 8 drug substances displaying a range of different properties with respect to binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. It was found that capillary electrophoresis frontal analysis can be used as a general method to study and quantify drug-human serum albumin interactions. The binding parameters obtained were consistent with literature values. Dextran was in some cases added to the run buffer to improve separation of the drug and human serum albumin plateau peaks. Results indicate that mobility differences between free and complexed human serum albumin give rise to only minor errors. Capillary electrophoresis frontal analysis was also found applicable to the study of human serum albumin drug displacement reactions. Low sensitivity of the UV-detection system was found to be the major limitation of capillary electrophoresis frontal analysis. The method is simple, and minimal effort has to be put into method development, which makes it well suited for screening in early drug development.

Capillary electrophoresis for the determination of albumin binding capacity and free bilirubin in jaundiced neonates

This study develops a capillary electrophoresis/frontal analysis (CE/FA) method to separate free bilirubin from its albumin bound complex, with only small-sized samples. Under our optimized conditions, CE/FA is proven to be a simple and accurate method in assaying the concentration of free bilirubin in the jaundiced serum. Upon saturation of albumin-binding, the amount of bilirubin added to the serum bears a linear relationship with the absorbance at OD44OnM (R = .9987, P < .00001). Application of this method to study jaundiced neonates has yielded precise data on the residual binding capacity, which may be of significant therapeutic implications. The CE/FA method we have developed appears to be promising and applicable for clinical use as it is highly reproducible and it only requires a small sample size for tests. Our method should be an invaluable adjunct to the neonatal management of hyperbilirubinaemia.

Affinity capillary electrophoresis is a powerful tool to identify transthyretin binding drugs for potential therapeutic use in amyloidosis

In this work we used affinity capillary electrophoresis (ACE) to investigate the extent of interaction between a pool of drugs and wild-type transthyretin. After qualitative preliminary screening, attention was focused on the most promising molecules, flufenamic acid and flurbiprofen, which underwent a further stage of investigation, the determination of the binding constants, and, when possible, the assessment of the number of binding sites by ACE, frontal analysis (FA) capillary electrophoresis (CE) and parallel ultrafiltration (UF) experiments. Furthermore, our data demonstrate that FA CE is a suitable technique for identifying fibril ligands. This represents a novel CE application of pharmaceutical interest.