Nonlinear analysis of dynamic binding in affinity capillary electrophoresis demonstrated for inclusion complexes of beta-cyclodextrin

The stability of the molecular host-guest inclusion complexes of beta-cyclodextrin with benzoate and four different hydroxybenzoates is investigated. For the measurement of the binding constants an experimental method is devised that is based on affinity capillary electrophoresis (ACE) with indirect UV absorbance detection. We derive an explicit equation for effective mobilities in ACE experiments without violation of rigorous mass balance. This equation is employed in the nonlinear least-squares analyses of the experimental data yielding binding constants of 48+/-2 M(-1) for benzoate, 299+/-38 M(-1) for 2-hydroxybenzoate, 37+/-1 M(-1) for 3-hydroxybenzoate, 228+/-9 M(-1) for 4-hydroxybenzoate, and 895+/-110 M(-1) in the case of 2,4-dihydroxybenzoate.     

Nonlinear electrophoresis of colloidal particles

Advances over the past decade in nonlinear electrophoresis of charged, dielectric colloidal particles in aqueous electrolytes are reviewed. Here, the word nonlinear refers to the fact that the ratio of the electrophoretic speed of the particle to the magnitude of the applied electric field—the electrophoretic mobility—is not independent of field strength. This is in stark contrast to the vast majority of work on (linear) colloidal electrophoresis over the last century, where the mobility is assumed to be a material property dependent only on the particle–electrolyte combination. The present discussion is focused on: (i) experimental measurements of the field-dependent mobility; (ii) an asymptotic scheme to calculate the mobility in the common thin-Debye-layer limit; and (iii) computations of nonlinear electrophoresis from numerical solution of the electrokinetic equations. The article concludes with suggestions for future work in this evolving area of colloid science.     

Analysis of immune complexes by capillary electrophoresis

A simple method for immune complexes (IC) analysis by CE is described. This method combines the ease of precipitation of the IC by polyethylene glycol with the separation power of CE. The advantage of this method is a better quantitation of the IC, since it corrects and eliminates the interferences from other serum proteins. It also reveals the composition (monoclonality) of the precipitate. Three types of IC have been detected in this method: monoclonal, polyclonal and mixed (mono-polyclonal) IC. Furthermore, the method is rapid and simple.     

Nonlinear electrokinetics and "superfast" electrophoresis

Nonlinear and nonequilibrium electrophoresis of spherical particles of radius a is shown to be possible when the solid surface allows field or current penetration. At low particle Peclet numbers, transient capacitative charging occurs until the surface polarization completely screens the external field. For a DC applied field [see text], the resulting electrokinetic velocity reaches Dukhin's maximum value of [formula: see text], where [see text] and mu are the liquid permittivity and viscosity. At high Peclet numbers, electroosmotic convection of the electroneutral bulk stops the transient charging before complete field-line exclusion. For an ion-selective and conducting spherical granule, the polarization is then determined by the steady-state Ohmic current driven by the penetrated external field. The high-Peclet electrokinetic velocity is lower, diffusivity-dependent and scales as [see text].     

Nonlinear electrophoresis of dielectric particles in Newtonian fluids

In classical electrokinetics, the electrophoretic velocity of a dielectric particle is a linear function of the applied electric field. Theoretical studies have predicted the onset of nonlinear electrophoresis at high electric fields because of the nonuniform surface conduction over the curved particle. However, experimental studies have been left behind and are insufficient for a fundamental understanding of the parametric effects on nonlinear electrophoresis. We present in this work a systematic experimental study of the effects of buffer concentration, particle size, and particle zeta potential on the electrophoretic velocity of polystyrene particles in a straight rectangular microchannel for electric fields of up to 3 kV/cm. The measured nonlinear electrophoretic particle velocity is found to exhibit a 2(±0.5)-order dependence on the applied electric field, which appears to be within the theoretically predicted 3- and 3/2-order dependences for low and high electric fields, respectively. Moreover, the obtained nonlinear electrophoretic particle mobility increases with decreasing buffer concentration (for the same particle) and particle size (for particles with similar zeta potentials) or increasing particle zeta potential (for particles with similar sizes). These observations are all consistent with the theoretical predictions for high electric fields.     

Capillary electrophoresis of viruses, subviral particles and virus complexes

CZE and CIEF were so far applied to the analysis of tobacco mosaic virus, Semliki forest virus, human rhinovirus, adenovirus, norovirus and the bacteriophages T5 and MS2. The concentration of viral or subviral particles, of capsid proteins and viral genomes were determined, their electrophoretic mobilities and pI values were measured and bioaffinity reactions between viruses and antibodies, antibody fragments and receptor fragments were assessed. The role of detergents added to the BGE to obtain reproducible electrophoretic conditions was elucidated. The analytes were detected via their UV-absorbance or via fluorescence after derivatization of the viral capsid, the nucleic acid, or both. A new dimension to the detection is added by the possibility of making use of the viral infectivity. At least in theory, this allows for the unequivocal identification of a single infectious virus particle after collection at the capillary outlet. This review summarizes the 25 papers so far published on this topic.     

Analysis of immune complexes of cerebrospinal fluid by two-dimensional gel electrophoresis

From the cerebrospinal fluid of 32 patients with different neurological diseases immune complexes were isolated using protein A-Sepharose. The isolated heavy and light chains and their constituents were analyzed by two-dimensional gel electrophoresis. In addition to immunoglobulins, some proteins such as albumin, apolipoprotein A-I and a number of unknown proteins were detected in all preparations. A complex consisting of three proteins with molecular masses between 52-55 kDa reacted slightly with polyclonal antibodies to glial fibrillary acidic protein. Whether the linkage between these antigens and the Ig is due to the Fab region or the Fc region remains unknown in our study. In some immune complexes of neurological diseases such as amyotrophic lateral sclerosis, astrocytoma and multiple sclerosis, differences are easily recognizable in the gel pattern.     

Chiral separations of transition metal complexes using capillary zone electrophoresis.

Several buffer additives that may facilitate chiral separation for optically active transition metal (TM) systems are investigated using capillary zone electrophoresis. The TM complexes evaluated exhibit considerable heterogeneity with respect to total complex charge (0 to 4+), ligand type, and identity of the central metal including Ru2+, Ni2+, Cr3+, and Co3+, threo-D[+]-Isocitrate, potassium antimonyl-d-tartrate and dibenzoyl-L-tartrate are identified as the most efficient chiral selectors. Interestingly, TM complexes exhibiting a (3+) total complex charge exhibit a reversal of enantiomer elution order versus all other complexes when separated using the tartrate additives. Operating parameters including pH, temperature, and capillary length are discussed, and chiral separations of complex mixtures are demonstrated.     

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.     

Comparison of cyclodextrin-barbiturate noncovalent complexes using electrospray ionization mass spectrometry and capillary electrophoresis

Various noncovalent complexes between native and derivatized cyclodextrins (CDs) and barbiturates were studied using capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). This paper involves the study of four aspects of CD-barbiturate noncovalent inclusion complexes. The first study focused on determining the formation of CD-barbiturate inclusion complexes in ESI-MS. This determination was accomplished by the comparison of migration data from CE with ESI-MS inclusion complex peak abundances, which were found to be complementary. The second study found the possibility of predicting native beta-CD mediated CE elution orders for barbiturates using data from ESI-MS. A third study focused on the formation of barbiturate inclusion complexes with derivatized beta-CD and gamma-CD. As part of this study, the effect of the extent of side chain substitution on native CD complexation behavior was investigated. The results indicated that the number of side chains on the CD does not affect the formation of barbiturate complexes with the hydrophobic CD cavity. Finally, a comparison of the hydroxypropyl-beta-CD-barbiturate and hydroxypropyl-gamma-CD-barbiturate complexes in CE and ESI-MS was made to study the relationship between strength of drug-CD binding and enantioresolution. The results from the above studies indicated that the gas phase and the solution state complexes showed comparable behavior indicating that similar interactions played a role in stabilizing these complexes. While it was possible to use the ESI-MS data to determine drug binding to the CDs, it was not possible to predict whether a separation of the enantiomers of a chiral barbiturate would occur. However, the ESI-MS data could be used to eliminate certain CDs from consideration as chiral selectors.     

Comparison of local anesthetic-cyclodextrin non-covalent complexes using capillary electrophoresis and electrospray ionization mass spectrometry

Non-covalent complexes between three derivitized cyclodextrins (CD's) and six local anesthetics were studied using capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). The CE study was performed using the complete filling technique (CFT). A comparison between the migration data from CE and ESI-MS inclusion complex peak abundances was made representing the association between local anesthetics and CD's in the solution and the gas phase, respectively. The results from this study showed comparable behavior of the complexes in the CE and mass spectrometer, indicating similarity in the parameters controlling the stability of these complexes. Therefore, the formation of specific non-covalent complexes, as shown in this study, could be used to predict the behavior of a complexing agent with a substrate in the solution phase by observing data obtained from ESI-MS.     

Separation and kinetic study of chromium(III) chloro complexes by capillary electrophoresis

Summary Three Cr(III) species (dichlorotetraaquachromium (III), [CrCl₂(H₂O)₄]⁺; monochloropentaaquachromium(III), [CrCl(H₂O)₅]²⁺; and hexaaquachromium(III), [Cr(H₂O)₆]³⁺) have been separated and determined by capillary electrophoresis. The first two complexes could be detected in direct mode in phosphate buffer, but because the absorption of complex [Cr(H₂O)₆]³⁺ is poor in the UV range, indirect UV detection had to be used. For indirect detection 5 mM imidazole was added to the buffer solution. The formation and decomposition of the different Cr(III) complexes were monitored in time after the preparation of solutions of CrCl₃.6H₂O. The slowest process was the decomposition of [CrCl(H₂O)₅]²⁺; 300 h after preparation of a solution of CrCl₃.6H₂O of pH 1 the solution contained only [Cr(H₂O)₆]³⁺. The effects of pH and the content of some matrix ions on the rates of conversion of the complexes were studied. The kinetic characteristics of this complex system could be investigated adequately by means of capillary electrophoresis. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

A study of chromium(iii) sulphate complexes by electrophoresis on filter paper

Paper electrophoresis in combination with radioactive tracer techniques permits the investigation of the reactions of Cr⁺³ with SO₄^-2. According to our results green solutions contain invariably complexed SO₄^-2 groups. The kinetics of the uptake of SO₄^-2 was also studied at two different temperatures.     

Nonlinear optical properties of tetrapyrazinoporphyrazinato indium chloride complexes due to excited-state absorption processes

The multiphoton absorption properties of the axially substituted tetrapyrazinotetraazaporphyrinato complex Pyz(4)TAPInCl (1) are reported and interpreted. In particular, the nonlinear optical transmission of the complex and the excited states involved in the nonlinear absorption have been determined at the frequency of the second harmonic generation of a Nd:YAG laser in the nanosecond time regime. Pyz(4)TAPInCl has an excited-state absorption cross section larger than its ground state in the 460-540 nm spectral region, and it shows an optical limiting (OL) behavior at 532 nm, which derives from a sequential two-photon absorption with a larger absorption cross section of the excited triplet state with respect to the ground state. It results that the absorption cross section of 1 in the excited triplet state is 7.8 x 10(-18) cm(2) vs 0.9 x 10(-18) cm(2) of the ground state at the wavelength of OL analysis.     

Capillary electrophoresis as a probe of enantiospecific interactions between photoactive transition metal complexes and DNA

Recently, we have demonstrated the capacity to separate chiral transition metal (TM) complexes of the type [M(diimine)(3)](n+) using CE buffers containing chiral tartrate salts. In separate work, several chromium(III)-tris-diimine complexes in particular have been shown to bind enantioselectively with calf-thymus (CT) DNA, and a qualitative assessment of the relative strength and enantiospecificity of this interaction is of significant interest in the characterization of these complexes as potential DNA photocleavage agents. Here, we describe two convenient approaches to investigate such binding behavior using chiral CE. For complexes with lower DNA affinities exhibiting primarily surface binding, DNA itself is used as the chiral resolving agent in the electrophoretic buffer. In this approach, resolution of the TM complexes into their Lambda and Delta isomers is achieved with the isomer eluting later exhibiting superior binding affinity toward DNA. For more strongly bound TM complexes containing ligands known to intercalate with DNA, the [Cr(diimine)(3)](3+) complexes are preincubated with oligonucleotide and subsequently enantiomerically resolved in a dibenzoyl-L-tartrate buffer system that facilitates analysis of the unbound TM species only. Differences in isomer binding affinity are distinguished by the relative peak areas of the Lambda- and Delta-isomers, and relative binding strengths of different complexes can be inferred from comparison of the total amount of unbound complex at equivalent DNA/TM ratios.     

LC-nanospray-MS/MS analysis of hydrophobic proteins from membrane protein complexes isolated by blue-native electrophoresis

The application of two-dimensional electrophoresis for the identification of hydrophobic membrane proteins is principally hampered by precipitation of many of these proteins during first-dimension, isoelectric focusing. Therefore new strategies towards the identification and characterization of membrane proteins are being developed. In this work we present a direct and rapid approach from blue-native gels to mass spectrometry, which allows the analyses of complete complexes and prevents protein aggregation of hydrophobic regions during electrophoresis. We combine blue-native gel electrophoresis and liquid chromatography--nanospray-iontrap tandem mass spectrometry to analyze the composition of oxidative phosphorylation complexes I, III, IV and V from bovine-heart mitochondria as a model system containing a number of highly hydrophobic proteins. Bands from blue-native gels were subjected either to in-gel or to in-solution tryptic digestion. The obtained peptide mixtures were further analyzed by liquid chromatography--tandem mass spectrometry and the corresponding proteins were identified by database search. From a total of 86 proteins, 67 protein subunits could be identified including all highly hydrophobic components, except the ND4L and ND6 subunits of complex I. We demonstrate that liquid chromatography--tandem mass spectrometry combined to blue-native electrophoresis is a straightforward tool for proteomic analysis of multiprotein complexes, and especially for the identification of very hydrophobic membrane protein constituents that are not accessible by common isoelectric focusing/sodium dodecyl sulphate gel electrophoresis.