Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

The free solution electrophoretic behavior of DNA‐protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA‐protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA‐methyl‐CpG‐binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA‐protein complexes. This method is based on the effective separation of free DNA and DNA‐protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide‐coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA‐MBD2b complexes, we found that the values of (μ₀/μ)‐1 were directly proportional to the charge‐to‐mass ratios of formed complexes, where the μ₀ and μ are the mobility of free DNA probe and DNA‐protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge‐to‐mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA‐binding properties of the purified MBD2b protein and help to analyze other DNA‐protein complexes. Additionally, this study enhances the understanding of the influence of the charge‐to‐mass ratios of formed DNA‐protein complexes on their separation and electrophoretic behaviors.     

Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility

Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm(3). Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water.     

SOLUBILIZATION OF PHOTOSYNTHETIC PIGMENT-PROTEIN COMPLEXES

Studies utilizing fractionation of photosynthetic pigment-protein complexes from the chloroplast thylakoid membrane often employ dodecylsulfate at a concentration of 10 mg mL^?1 to disrupt membrane structure prior to electrophoretic fractionation of the complexes. We investigated the effect of varying dodecylsulfate concentration on the solution/air interfacial surface tension in the absence and presence of the same concentrations of thylakoid membranes used by four different fractionation systems that have been commonly employed to fractionate photosynthetic pigment-protein complexes. Concentrations of dodecylsulfate in the range5–10 mg mL^?1, normally utilized to treat thylakoids prior to fractionation, were effective in reducing the interfacial surface tension to levels equivalent to control solutions without added thylakoid membranes. However, thylakoid membranes treated with these concentrations of dodecylsulfate are not resolved into discrete pigment-protein complexes when subjected to electrophoresis on an agarose gel, and do not produce significant amounts of pigment-containing complexes with a molecular size < 100 000 as measured by filtration with size-exclusion membranes. We conclude that many surfactant systems empirically developed to fractionate photosynthetic pigment-protein complexes may not fully solubilize the complexes prior to the electrophoretic step.     

Characterization of complexes between phenethylamine enantiomers and β‐cyclodextrin derivatives by capillary electrophoresis—Determination of binding constants and complex mobilities

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte‐cyclodextrin‐complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β‐cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x ‐reciprocal, y ‐reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β‐cyclodextrin, (2‐hydroxypropyl)‐β‐cyclodextrin, methyl‐β‐cyclodextrin and 6‐O‐α‐maltosyl‐β‐cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer‐cyclodextrin‐complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β‐cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes.     

Determination of the stability constants of lanthanide complexes with oxyacids using capillary electrophoresis

Different algorithms for calculating the stability constants of lanthanide complexes with hydroxycarbonic acids on the basis of experimental data obtained by capillary electrophoresis are compared. It is shown that the optimal approach is based on the consideration of the electrophoretic mobilities of all complex forms present in the solution, taking into account their fractions. The most satisfactory approximation of the experimental data by the calculated ones is achieved on the assumption that the electrophoretic mobilities of the complex species depend on their charge only, regardless of the particle size. The obtained stability constants of lanthanide complexes with lactic and 2-hydroxy-2-methylpropanoic acids agree with the literature data. The stability constants of lanthanide complexes with tartaric and citric acids were significantly corrected, while the constants for complexes with mandelic, 2-hydroxy-2-methylbutanoic, 2-hydroxy-2-propy-lpentanoic, and malic acid were determined for the first time.     

Electrokinetic behaviour of polymer colloids with adsorbed Triton X-100

An experimental study on the electrophoretic mobility (µ_e) of polystyrene particles after adsorption of Triton X-100 (TX100) is described. Three polystyrene particles with different functionality (sulphate, carboxyl and amidine) were used as solid substrate for the adsorption of the surfactant. The electrophoretic mobility of the polystyrene-TX100 complexes at different electrolyte concentrations has been studied versus the amount of adsorbed surfactant. The presence of TX100 onto the colloidal particles seems to produce a slight shifting of the slipping plane. This is observed for electrolyte concentrations above ~10^-3 M. On the other hand, the electrophoretic mobilities of the latex-surfactant complexes with maximum surface coverage were measured versus pH and salt concentration. Specific ion interactions between H⁺/carboxyl groups and OH^-/amidine groups appeared at extreme pH which explain the anomalous electrophoretic behaviour encountered in the region where surface charge change.     

Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses

This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.     

Chromatographic and electrophoretic studies of immune complexes in non-A, non-B hepatitis

Immune complexes isolated from two patients with chronic non-A, non-B hepatitis, one patient with acute non-A, non-B hepatitis and one patient with juvenile rheumatoid arthritis were examined by means of a combined chromatographic and electrophoretic method. Both analyses showed the presence of complexes consisting of IgG, IgM, complement c1q factor and albumin; no antigen constituents were detected. The IgG-to-IgM ratio varied from 1:1 to 4:1, suggesting that one could be dealing with complexes of both IgG-IgM and IgG-IgG types. Moreover, the detectable presence of c1q factor might indicate that such complexes were capable of activating complement.     

Capillary electrophoretic methods applied to the investigation of peptide complexes

This article gives an overview of the applications of capillary electrophoretic methods to investigate the non‐covalent interactions of peptides (peptide complexes) with variable middle‐ and high‐molecular‐mass receptors (ligands) as well as with small ions and molecules in the period 2007–2014. Different modes of capillary electrophoretic methods, such as mobility shift (vacancy) affinity capillary electrophoresis, multiple injection affinity capillary electrophoresis, partial filling affinity capillary electrophoresis, Hummel–Dryer method, vacancy peak method and (continuous) frontal analysis capillary electrophoresis, are briefly described and their applicability to determination of binding constants of peptide complexes is discussed. In addition, the detailed experimental conditions of individual applications and the values of binding constants of the particular peptide complexes are presented.     

Electrokinetic characterization of polystyrene–non-ionic surfactant complexes

An experimental study on the electrophoretic mobility (μ_e) of polystyrene particles after the adsorption of non-ionic surfactants with different chain lengths is described. Two sulphate latexes with relatively low surface charge densities (3.2 and 4.8 μC cm⁻²) were used as solid substrate for the adsorption of four non-ionic surfactants, Triton X-100, Triton X-165, Triton X-305 and Triton X-405, each one with 9–10, 16, 30 and 40 molecules of ethylene oxide (EO), respectively. The electrophoretic mobility of the polystyrene–non-ionic surfactant complexes was studied versus the amount of adsorbed surfactant (Γ). The presence of non-ionic surfactant onto particles surface seems to produce a slight shifting of the slipping plane because the mobilities of the different complexes display a very small decreasing. The increase in the number of EO chains in the surfactant molecule seems to operate as a steric impediment which decreases the number of adsorbed large surfactant molecules. The electrophoretic mobilities of the latex–surfactant complexes with maximum adsorption were measured versus the pH and ionic strength of the dispersion. While the different complexes showed a similar qualitative behaviour compared with that of the bare latex against the pH, the adsorption of the surfactant reduces the typical maximum in the μ_e−log[electrolyte].     

Electrostatic interactions between a protein and oppositely charged micelles

Micellar solutions made of a fully fluorinated surfactant, LiPFN, form water-soluble complexes with lysozyme in a wide concentration range. Such complexes are stabilized by electrostatic and, very presumably, double-layer interactions. The mixtures were investigated by combining electrophoretic mobility, DLS, and dielectric relaxation methods. The former gives information on the surface charge density of protein-micelle complexes and indicates that the resulting adducts retain a negative charge (i.e., charge neutralization is incomplete). The double-layer thickness of proteins, micelles, and protein-micelle complexes is also connected to the dielectric relaxation frequency. Changes in particle size (inferred by DLS), charge density, and double-layer thickness are closely interrelated to each other. A model was developed to quantify such properties.     

Time-resolved electrophoretic analysis of mobility shifts for dissociating DNA ligands

Intercalative binding of ligands to DNA can be demonstrated by helix unwinding, monitored by gel electrophoresis of supercoiled DNA, as electrophoretic mobility is sensitive to the topological DNA state. However, we show that an apparent lack of unwinding in an electrophoretic assay could be due to dissociation of the (intercalated) ligand during the analysis, rather than evidence for a nonintercalative mode of binding to DNA. Repetitive scanning during the electrophoresis ensures that release of the ligand during electrophoresis does not affect the measured degree of unwinding, based on the electrophoretic velocity being determined as a function of time. We use this assay to establish intercalation as a mode of binding to DNA for the cyanine dyes YO, YO-PRO as well as two enantiomeric forms of the ruthenium complexes [(phen)2 Ru(tatpp)Ru(phen)2]4+, and to support groove-binding for the new unsymmetrical cyanine dyes BOXTO and BOXTO-PRO. Groove-binding could be concluded from a lack of unwinding, because we could rule out that it is caused by release of the dye during the electrophoresis. The gel electrophoresis has the advantage over hydrodynamic techniques that much smaller sample amounts are required, and our time-resolved approach can be employed in all mobility-shift assays when applied to dissociating complexes.     

Mechanism of enantioseparation of DL-pantothenic acid in ligand exchange capillary electrophoresis using a diol-borate system

Borate complexes formed in the ternary system at pH 9.2 containing borate, (S)-3-amino-1,2-propanediol (SAP), and DL-pantothenic acid (DL-PTA) were identified by 13C and 11B NMR, and it is confirmed that the binary complexes, [B(OH)2(SAP)], [B(SAP)2]+ [B(OH)2(D- or L-PTA)]2-, and [B(D- or L-PTA)2]3- (including [B(D-PTA)(L-PTA)]3-), and the ternary complexes, [B(SAP)(D- or L-PTA)]-, coexist at equilibrium in the ternary system. Thermodynamic experiments by variable-temperature 11B NMR revealed that the ternary complex, [B(SAP)(D-PTA)]-, is entropically more stable than [B(SAP)(L-PTA)]-. Because two geometrical isomers are possible for the respective ternary complexes, semi-empirical molecular orbital calculations were performed by PM5, PM3, and AM1 methods in order to obtain the optimized structures. It is indicated from the calculated heats of formation and experimentally obtained thermodynamic parameters that the (S)-isomer is more probable for the respective ternary complexes with D- and L-PTA. In the optimized structure of (S)-[B(SAP)(D-PTA)]- in water, the SAP and D-PTA ligands were oppositely oriented to form a rather linear structure, while the diastereomer, (S)-[B(SAP)(L-PTA)]-, had a folded structure. Because such a difference in the solvated structure of the ternary complexes can give a different electrophoretic velocity in CE, the enantioseparation of DL-PTA in CE is reasonably attributed to a difference in the observed electrophoretic mobility for the equilibrated ternary systems containing the respective ternary complexes.     

Characteristics of complexes composed of sodium hyaluronate and bovine serum albumin

Complexes composed of sodium hyaluronate (NaHA) and bovine serum albumin (BSA) were studied to elucidate the exact composition of the complex, the phase separation, the electrophoretic mobility and the size using dynamic light scattering (DLS) and electrophoretic light scattering (ELS), etc. The phase diagram of the mixed solutions was determined. The complexes were soluble in neutral or weakly acidic pH regions and showed phase separation in the more acidic pH region. From the concentration of Na+ released from NaHA when it binds to BSA, the ratios of BSA to NaHA of the complexes were determined. In the region of soluble complexes, one BSA molecule was determined to bind with 15 carboxylic groups of NaHA and in the region of insoluble complexes to bind with 6 carboxylic groups. At the phase separation point, 117 BSA molecules bound with one NaHA molecule and 17% of the carboxylic groups of NaHA did not contribute to the binding of BSA. The sizes of the complexes decreased from several microm to several hundred nm as the binding ratio of BSA increases. Decreases in the viscosities of the mixed solutions were consistent with the decreases of the sizes. From these results, a model of complex formation is proposed.     

Study of Chlorambucil and Chlorambucil–Trimethyl-β-cyclodextrin Inclusion Complex by CE

The results of CE studies of solid inclusion complexes of chlorambucil (CHL) and trimethyl-β-cyclodextrin (TM-β-CD) are presented. Chlorambucil has low stability in aqueous solution, so the formation of complexes with β-CD is a good way to improve its stability and dissolution. The experimental results confirm the existence of inclusion complexes. CE can differentiate free CHL from CHL–TM-β-CD complexes due to their different electrophoretic mobilities.     

Adsorption Kinetics of Complexes Formed by Oppositely Charged Polyelectrolytes

The objective of this study is to gain an understanding of the kinetics of adsorption of complexes formed in aqueous solution by oppositely charged polyelectrolytes. The properties of the complexes were characterized by means of dynamic light scattering and electrophoretic mobility and the reaction stoichiometry was studied by titration. The stoichiometry in the complexes depends on the polymer weight ratio w(p). Their point of zero charge (pzc) is shifted toward lower w(p) when simple cations of higher valence are added to the solution. The adsorption kinetics of the complexes on silica was studied by stagnation point reflectometry. The sign of charge of the complexes as well as the valence and concentration of simple cations governs the occurrence of adsorption in the electrolytes studied. Five different types of adsorption kinetics were found. Copyright 2001 Academic Press.     

Capillary zone electrophoresis with UV-scanning detection for the analysis of metal species and their organic binding partners in biological systems

A capillary zone electrophoretic method with UV-scanning detection for the separation and identification of both free ligands and metal species is presented. The electrophoretic behavior of naturally occurring binding partners such as organic acids and amino acids was studied and compared with their metal-complexes. Copper(II) complexes of citrate and amino acids showed decreased electrophoretic mobilities and altered UV spectra. The optimized method was validated on ultrafiltered cow's milk and human milk samples. In cow's milk six low molecular weight substances, including citrate, orotate, and hippurate, could be separated. Metal supplementation with Cu(II), Ni(II), or Zn(II) decreased not only the citrate peak but also the orotate signal and in the case of copper(II) supplementation a new signal for the copper(II)-citrate complex appeared. In human milk samples various amino acids such as glutamate, phenylalanine, tyrosine, and tryptophan could be identified besides citrate. The electrophoretic mobilites and peak areas of the amino acids were also influenced by the metal supplementation.     

Interaction of bacterial endotoxine (lipopolysaccharide) with latex particles: application to latex agglutination immunoassays

The latex agglutination immunoassay technique uses polymer colloids as carriers for antibodies or antigens to enhance the immunological reaction. In this work, the interaction of a lipopolysaccharide (LPS) of Brucella Melitensis with two conventional latexes has been studied. Some experiments on the physical adsorption of the LPS onto these polystyrene beads have been performed and several complexes with different coverage degrees were obtained by modifying the incubation conditions. Regarding the application in the development of diagnostic test systems, it is advisable to study the latex-LPS complexes from an electrokinetic and colloidal stability point of view. The complexes were electrokinetically characterized by measuring the electrophoretic mobility under different redispersion conditions. The colloidal stability was determined by simple turbidity measurements. Experimental and theoretical data have been employed to study the molecular disposition of the LPS in the latex particle surface to compare with the outer membrane of bacterial cells. Latex complexes covered by different LPS amounts showed high colloidal stability and adequate immunoreactivity that remains for a long time period.     

Electrophoretic mobility of ester betulin derivatives and their complexation with γ-cyclodextrin studied by capillary electrophoresis in aqueous solutions at different pH values

In this article, capillary electrophoresis was used to measure the effective electrophoretic mobility of ester betulin derivatives as a pH function and to study their complexation with γ-cyclodextrin (γ-CD). The electrophoretic mobility of betulin 3,28-diphthalate (DPhB) and 3,28-disuccinate (DScB) changed unusually with decreasing pH: instead of decreasing, it first increased and then decreased. This fact as well as the turbidity of sample solutions at pH from 2.5 to 6, broadening of electrophoretic peaks and a decrease in the surface tension of the solutions indicates that these betulin derivatives, being amphiphilic compounds and weak acids, exist as micelles in aqueous solutions at pH 6 and below. The inclusion complexation of betulin derivatives with γ-CD at pH 9.18 and 4.5 was studied by mobility shift affinity capillary electrophoresis. At pH 9.18, the apparent binding (stability) constant logarithms for 1:1 γ-CD complexes of DPhB, betulin 3,28-disulfate (DSB) and DScB with 95% confidence interval limits were equal to 7.44 ± 0.02, 7.09 (7.01–7.19), and 6.97 (6.87–7.08) at 25°C, respectively. At pH 4.5, the binding constant for the DSB complex was slightly lower, while the micelle formation did not allow determining the exact values of the constants for the DPhB and DScB complexes.