Frontal Analysis Continuous Capillary Electrophoresis Research Articles

Study of Interactions between Antigens and Polymeric Adjuvants in Vaccines by Frontal Analysis Continuous Capillary Electrophoresis.

Vaccine adjuvants are used to enhance the immune response induced by antigens that have insufficient immunostimulatory capabilities. The present work aims at developing a frontal analysis continuous capillary electrophoresis (FACCE) methodology for the study of antigen-adjuvant interactions in vaccine products. After method optimization using three cationic model proteins, namely lysozyme, cytochrome c, and ribonuclease A, FACCE was successfully implemented to quantify the free antigen and thus to determine the interaction parameters (stoichiometry and binding constant) between an anionic polymeric adjuvant (polyacrylic acid, SPA09) and a cationic vaccine antigen in development for the treatment for Staphylococcus aureus. The influence of the ionic strength of the medium on the interactions was investigated. A strong dependence of the binding parameters with the ionic strength was observed. The concentration of the polymeric adjuvant was also found to significantly modify the ionic strength of the formulation, the extent of which could be estimated and corrected.

Effect of Dendrigraft Generation on the Interaction between Anionic Polyelectrolytes and Dendrigraft Poly(l-Lysine).

In this present work, three generations of dendrigraft poly(l-Lysine) (DGL) were studied regarding their ability to interact with linear poly (acrylamide-co-2-acrylamido-2-methyl-1-propanesulfonate) (PAMAMPS) of different chemical charge densities (30% and 100%). Frontal analysis continuous capillary electrophoresis (FACCE) was successfully applied to determine binding constants and binding stoichiometries. The effect of DGL generation on the interaction was evaluated for the first three generations (G2, G3, and G4) at different ionic strengths, and the effect of ligand topology (linear PLL vs. dendrigraft DGL) on binding parameters was evaluated. An increase of the biding site constants accompanied with a decrease of the DGL-PAMAMPS (n:1) stoichiometry was observed for increasing DGL generation. The logarithm of the global binding constants decreased linearly with the logarithm of the ionic strength. This double logarithmic representation allowed determining the extent of counter-ions released from the association of DGL molecules onto one PAMAMPS chain that was compared to the total entropic reservoir constituted by the total number of condensed counter-ions before the association.

Interactions between Oppositely Charged Polyelectrolytes by Isothermal Titration Calorimetry: Effect of Ionic Strength and Charge Density

In this study, binding of linear poly(l-lysine) to a series of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonate copolymers was examined by isothermal titration calorimetry (ITC). Binding constant and stoichiometry were systematically determined at different ionic strengths and for different polyanion charge densities varying between 15% and 100%. The range of investigated ionic strengths was carefully adjusted according to the polyanion charge densities to get measurable binding constants (i.e., formation binding constant typically comprised between 104 and 106 M-1) by isothermal titration calorimetry (ITC). The number of released counterions during the polyelectrolyte complex formation was determined from the log-log dependence of the binding constant according to the ionic strength and was compared to the total number of condensed counterions estimated from the Manning theory. Experimental results obtained by ITC are in very good agreement with those previously obtained by frontal analysis continuous capillary electrophoresis (FACCE) and can be used to model and predict the binding parameters at any ionic strength or any polyanion charge density. Thermodynamic parameters of the complexation between the oppositely charged polyelectrolytes confirm that the complex formation was entropically driven together with a favorable (but minor) enthalpic contribution. For the first time, specificities, advantages/disadvantages of ITC, and FACCE techniques for studying polyelectrolyte complexations are compared and discussed, using the same experimental conditions.

Modelling and predicting the interactions between oppositely and variously charged polyelectrolytes by frontal analysis continuous capillary electrophoresis.

In this work, a systematic study of the interactions between poly(l-lysine) and variously charged statistical copolymers of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonate (PAMAMPS) has been carried out by frontal analysis continuous capillary electrophoresis (FACCE). FACCE was successfully implemented to obtain the interaction parameters (binding constant and stoichiometry) at different ionic strengths and for different PAMAMPS charge densities varying between 15% and 100%. The range of investigated ionic strengths was carefully adjusted according to the PAMAMPS charge density to obtain measurable binding constants by FACCE (i.e. formation binding constant typically comprised between 104 and 106 M-1). The number of released counter-ions during the polyelectrolyte complex formation was systematically quantified via the ionic strength dependence of the binding constant and was compared to the total condensed counter-ion reservoir according to Manning theory on counter-ion condensation. A descriptive and predictive model relating the physico-chemical properties of the two partners, the binding constant and the ionic strength is proposed in the framework of multiple independent interaction sites of equal energy.

Taking Advantage of Electric Field Induced Bacterial Aggregation for the Study of Interactions between Bacteria and Macromolecules by Capillary Electrophoresis.

The quantification of interaction stoichiometry and binding constant between bacteria (or other microorganism) and (macro)molecules remains a challenging issue for which only a few adapted methods are available. In this paper, a new methodology was developed for the determination of the interaction stoichiometry and binding constant between bacteria and (macro)molecules. The originality of this work is to take advantage of the bacterial aggregation phenomenon to directly quantify the free ligand concentration in equilibrated bacteria-ligand mixtures using frontal analysis continuous capillary electrophoresis. The described methodology does not require any sample preparation such as filtration step or centrifugation. It was applied to the study of interactions between Erwinia carotovora and different generations of dendrigraft poly-L-lysines leading to quantitative information (i.e., stoichiometry and binding site constant). High stoichiometries in the order of 10(6)-10(7) were determined between nanometric dendrimer-like ligands and the rod-shaped micrometric bacteria. The effect of the dendrimer generation on the binding constant and the stoichiometry is discussed. Stoichiometries were compared with those obtained by replacing the bacteria by polystyrene microbeads to demonstrate the internalization of the ligands inside the bacteria and the increase of the specific surface via the formation of vesicles.

Studying glycosaminoglycan-protein interactions using capillary electrophoresis.

Methods for studying interactions between glycosaminoglycans (GAGs) and proteins have assumed considerable significance as their biological importance increases. Capillary electrophoresis (CE) is a powerful method to study these interactions due to its speed, high efficiency, and low sample/reagent consumption. In addition, CE works effectively under a wide range of physiologically relevant conditions. This chapter presents state-of-the-art on CE methods for studying GAG-protein interactions including affinity capillary electrophoresis (ACE), capillary zone electrophoresis (CZE), frontal analysis (FA)/frontal analysis continuous capillary electrophoresis (FACCE), and capillary electrokinetic chromatography (CEC) with detailed experimental protocols for ACE and CZE methods.

Effect of Dendrimer Generation on the Interactions between Human Serum Albumin and Dendrigraft Polylysines

This work aims at studying the interaction between human serum albumin and different generations of dendrigraft poly-L-lysine (DGL) in physiological conditions. The binding constants and stoichiometry of the interaction were successfully determined using frontal analysis continuous capillary electrophoresis. The effect of generation on the interaction was evaluated for the five first generations of DGL. An increase of the binding constant accompanied with a decrease of the HSA:DGL (1:n) stoichiometry and a decrease of the cooperativity with dendrimer generation was observed. These findings were in good agreement with the increase of ligand (DGL) size, the increase of electrostatic ligand-ligand repulsion, and the localization of two negatively charged interaction sites on the HSA. The effect of the ligand topology (linear vs dendrigraft) on the HSA interaction revealed that linear poly(L-lysine) leads to much lower stoichiometry compared to DGL of similar molar mass due to much higher flexibility and contour length.

Simulation of the effects of complex‐ formation equilibria in electrophoresis: I. Mathematical model

Simul 5 Complex is a one-dimensional dynamic simulation software designed for electrophoresis, and it is based on a numerical solution of the governing equations, which include electromigration, diffusion and acid-base equilibria. A new mathematical model has been derived and implemented that extends the simulation capabilities of the program by complexation equilibria. The simulation can be set up with any number of constituents (analytes), which are complexed by one complex-forming agent (ligand). The complexation stoichiometry is 1:1, which is typical for systems containing cyclodextrins as the ligand. Both the analytes and the ligand can have multiple dissociation states. Simul 5 Complex with the complexation mode runs under Windows and can be freely downloaded from our web page http://natur.cuni.cz/gas. The article has two separate parts. Here, the mathematical model is derived and tested by simulating the published results obtained by several methods used for the determination of complexation equilibrium constants: affinity capillary electrophoresis, vacancy affinity capillary electrophoresis, Hummel-Dreyer method, vacancy peak method, frontal analysis, and frontal analysis continuous capillary electrophoresis. In the second part of the paper, the agreement of the simulated and the experimental data is shown and discussed.

Frontal Analysis Continuous Capillary Electrophoresis Study on the Interaction of an Amphiphilic Alternating Copolymer with Triton X-100

The interaction of amphiphilic alternating copolymers of sodium maleate and dodecyl vinyl ether (Mal/C12) with a nonionic surfactant, Triton X-100 (TX), was investigated by frontal analysis continuous capillary electrophoresis (FACCE). The binding isotherms obtained from FACCE data were indicative of weak cooperative interaction for all the polymers examined. The cooperative interaction was also analyzed by the Hill model, and the results were compared with the previous results on the interaction of statistical copolymers of sodium 2-acrylamido-2-methylpropanesulfonate and N-dodecylmethacrylamide with TX.

Investigation of the Interaction Between Lidocaine and the Components of Hyaluronic Acid Using Frontal Analysis Continuous Capillary Electrophoresis

The frontal analysis continuous capillary electrophoresis (FACCE) technique was used for the characterziation of the interaction between lidocaine-HCl (Lido) and components of Na-hyaluronic acid (HA). N-Acetylglucosamine (GlcNAc) and glucuronic acid (GluA) were the components of Na-hyaluronic acid. For the investigations fused silica capillaries were used. The FACCE method was compared to affinity capillary electrophoresis (ACE). The association constants between lidocaine-HCl and the components of Na-hyaluronic acid were determined using FACCE. It was observed that only an interaction between Lido and GluA exhibited. The association constant (Klido-GluA) between Lido and GluA was 26 ± 0.3 L mol−1.

Cooperative Binding of Nonionic Surfactant to Hydrophobically Modified Polyanions as Studied by Frontal Analysis Continuous Capillary Electrophoresis

The binding of a nonionic surfactant, Triton X-100 (TX), to amphiphilic copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and N-dodecylmethacrylamide (C12) (p(A/C12(x)), where x denotes the mol % content of C12) was investigated by frontal analysis continuous capillary electrophoresis (FACCE) combined with dynamic light scattering focusing on the effect of the hydrophobe content on the binding in a wide range of x (5-60 mol %). From binding isotherms obtained from FACCE data, the binding was found to be cooperative in the whole range of x. Furthermore, a significant change in the binding behavior, i.e., cooperativity, was found to occur in a relatively narrow range of x (38-50 mol %), which is attributable to a change in the self-association behavior of p(A/C12(x)) in this x range.

Determination of the binding parameters for antithrombin–heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis

The suitability of affinity capillary electrophoresis (ACE) and frontal analysis continuous capillary electrophoresis (FACCE) for binding constant determination was investigated for complexes between heparin fragments and antithrombin III, one of the main target proteins in the coagulation cascade. In a 100 mM ionic strength phosphate buffer (pH 7.4), ACE was suitable to determine weak to medium interactions developed by short oligomeric heparin fragments, but it failed for decasaccharide, which presents a more complex irreversible interaction. However FACCE allowed evaluating the binding constant for these longer oligomeric fragments. Both experimental approaches were complementary for a wide variety of heparinic fragments.

A need for caution in the use of frontal analysis continuous capillary electrophoresis for the determination of ligand binding data

Attention is drawn to a need for caution in the determination of binding data for protein–polyelectrolyte interactions by frontal analysis continuous capillary electrophoresis (FACCE). Because the method is valid only for systems involving comigration of complex(es) and slower-migrating reactant, establishing conformity with that condition is clearly a prerequisite for its application. However, that requirement has not been tested in any published studies thus far. On the basis of calculated FACCE patterns, presented to illustrate features by which such comigration of complex(es) and slower-migrating reactant can be identified, the form of the published pattern for a β-lactoglobulin–poly(styrenesulfonate) mixture does not seem to signify the migration behavior required to justify its consideration in such terms. Additional experimental studies are therefore needed to ascertain the validity of FACCE as a means of determining binding data for the characterization of protein–polyelectrolyte interactions.

Characterization of polyanion–protein complexes by frontal analysis continuous capillary electrophoresis and small angle neutron scattering: Effect of polyanion flexibility

The binding constant ( K obs) for the β-lactoglobulin–poly(vinylsulfate) (BLG–PVS) complex was measured by frontal analysis continuous capillary electrophoresis at pH values above the isoelectric point of BLG, and the persistence length ( L p) of PVS was measured by small angle neutron scattering, to examine the effect of polyelectrolyte chain stiffness on its binding efficiency to proteins. The values of K obs and L p were compared with those of BLG–PSS and BLG–PAMPS (poly(2-acrylamido-2-methylpropanesulfonate)) reported previously. The relationship between K obs and L p was reciprocal, indicating that protein binding is enhanced by the flexibility of the polyanion, at least in the case where the net protein charge is negative. In addition, at a fixed pH, the polymer systems displayed a similar ionic strength dependence of K obs. This similarity was consistent with the proposal that the binding properties of PVS and PAMPS polyanions are governed purely by electrostatic interactions and are independent of their molecular structure.

Determination of the aggregation threshold of non–UV-absorbing, neutral or charged surfactants by frontal- and vacancy-frontal analysis continuous capillary electrophoresis

Supplementing our recent work on UV-absorbing anionic surfactants, new protocols based on frontal analysis continuous capillary electrophoresis (FACCE) were developed for the investigation of the aggregation threshold of non-UV absorbing anionic, cationic and neutral surfactants, and exemplified with sodium dodecyl sulfate (SDS), tetradecyltrimethylammonium bromide (TTABr) and Brij 35. Contrary to UV-absorbing surfactants, the critical micelle concentration (CMC) determination of non-UV absorbing surfactants requires the use of a marker providing adequate detection capabilities. UV-absorbing markers were selected, according to the charge of the studied surfactant (neutral for SDS and TTABr, anionic for Brij 35). In all cases, the free marker concentration was quantified as a function of the total surfactant concentration. In addition, a modified implementation of FACCE, that we called vacancy FACCE (VFACCE), was employed for the case of the neutral surfactant. VFACCE entails first filling the capillary with the system components to be studied in the background electrolyte, next continuously introducing the plain BGE electrokinetically. The salient theoretical features of FACCE and VFACCE were compared. These new protocols were successfully applied to yield reliable CMC values within short operational time and with low sample consumption.

Determination of the aggregation threshold of non?UV-absorbing, neutral or charged surfactants by frontal- and vacancy-frontal analysis continuous capillary electrophoresis

Supplementing our recent work on UV-absorbing anionic surfactants, new protocols based on frontal analysis continuous capillary electrophoresis (FACCE) were developed for the investigation of the aggregation threshold of non-UV absorbing anionic, cationic and neutral surfactants, and exemplified with sodium dodecyl sulfate (SDS), tetradecyltrimethylammonium bromide (TTABr) and Brij 35. Contrary to UV-absorbing surfactants, the critical micelle concentration (CMC) determination of non-UV absorbing surfactants requires the use of a marker providing adequate detection capabilities. UV-absorbing markers were selected, according to the charge of the studied surfactant (neutral for SDS and TTABr, anionic for Brij 35). In all cases, the free marker concentration was quantified as a function of the total surfactant concentration. In addition, a modified implementation of FACCE, that we called vacancy FACCE (VFACCE), was employed for the case of the neutral surfactant. VFACCE entails first filling the capillary with the system components to be studied in the background electrolyte, next continuously introducing the plain BGE electrokinetically. The salient theoretical features of FACCE and VFACCE were compared. These new protocols were successfully applied to yield reliable CMC values within short operational time and with low sample consumption.

Determination of aggregation thresholds of UV absorbing anionic surfactants by frontal analysis continuous capillary electrophoresis

Aggregation of anionic surfactants was investigated by frontal analysis continuous capillary electrophoresis (FACCE), a method involving the continuous electrokinetic introduction of the surfactant sample into the separation capillary. This process results in a partial separation of the monomeric and aggregated forms without perturbing the monomer-aggregate equilibrium. The critical micelle concentration (CMC) can then be easily derived from the height of the firstly detected migration front, corresponding to the monomeric form. This approach is exemplified with octyl and dodecylbenzenesulfonates and compared with conductimetry and surface tension measurements. FACCE turns out to be an effective method for the determination of CMC and intermediate aggregation phenomena with very small sample and short time requirements.

Frontal analysis continuous capillary electrophoresis for protein-polyelectrolyte binding studies.

A novel technique, frontal analysis continuous capillary electrophoresis (FACCE), has been described as an effective way to study protein-polyelectrolyte binding. FACCE involves continuous sampling, integrating sample injection and separation into one process that provides advantages over conventional frontal chromatography. The method provides rapid and precise determination of binding isotherms, and allows for quantitative binding analysis in terms of binding constant and the binding-site size by considering the protein as the ligand and allowing the polyelectrolyte to bind to a number of proteins with variable levels of cooperativity. FACCE is particularly suitable for binding systems involving rapid binding kinetics because it allows for the determination of the concentrations of free or bound ligands under conditions that avoid perturbation of the binding equilibrium. This chapter focuses on studies of the binding of bovine serum albumin (BSA) to heparin using FACCE. These investigations are demonstrated within the context of this chapter as representative of a model protein-polyelectrolyte system from which extensions to other systems can be made.

Quantification of the interactions between beta-lactoglobulin and pectin through capillary electrophoresis analysis.

Biopolymer interactions have many potential applications in pharmaceutical, cosmetic, nutraceutical, and functional food industries. Attractive interactions between proteins and polysaccharides can lead to the formation of complexes. Binding parameters of beta-lactoglobulin (beta-lg)/pectin complexes were determined using frontal analysis continuous capillary electrophoresis and the overlapping binding site model. At pH 4, approximately 23 beta-lg molecules were cooperatively complexed on low-methoxyl pectin, where each beta-lg molecule covered an average of 12 galacturonic acid residues. The calculated binding constant was 1431 M(-1). The interactions between pectin and four selected peptides located on the outer surface of the beta-lg were investigated in order to identify which part of the protein was likely to interact with the pectin. The peptide beta-lg 132-148, which corresponds to the alpha-helix zone, and the peptides beta-lg 76-83, 41-60, and 1-14 would be involved in the interaction with the pectin.

Ionic strength dependence of protein-polyelectrolyte interactions.

The effect of univalent electrolyte concentration on protein-polyelectrolyte complex formation has been measured by frontal analysis continuous capillary electrophoresis (FACCE) and turbidimetry for the interaction of bovine serum albumin (BSA) with a synthetic hydrophobically modified polyacid, for BSA with (porcine mucosal) heparin (Hp), a highly charged polyanion, and for Hp and insulin. All three highly diverse systems display maxima or plateaus in complex formation in the range of ionic strength 5 < I < 30 mM, confirmed in the case of BSA-Hp by multiple techniques. Similar maxima are reported in the literature, but with little discussion, for BSA-poly(dimethyldiallylammonium chloride), lysozyme-hyaluronic acid, and lysozyme-chondroitin sulfate, always in the I range 5-30 mM. While inversion of salt effect has been discussed specifically for the interaction of gelatin and sodium polystyrenesulfonate with gelatin(28) and with beta-lactoglobulin,(10) the general nature of this phenomenon, regardless of polyelectrolyte origin, molecular weight, and charge sign has not been recognized. The position of the maxima and their occurrence when protein and polyelectrolyte have the same net charge imply that they arise when Debye lengths extend, at low I, beyond half the protein diameter so that addition of salt screens repulsions, as well as attractions. This appears to be a general effect caused by electrostatic repulsions that can coexist simultaneously with hydrophobic interactions. Modeling of protein electrostatics via Delphi is used to visualize this effect for BSA, lysozyme, insulin, and beta-lactoglobulin.