Exploring the evaluation of net charge, hydrodynamic size and shape of peptides through experimental electrophoretic mobilities obtained from CZE

This work explores the validity of simple CZE models to analyze the electrophoretic mobilities of 102 peptides reported in literature. These models are based mainly on fundamental physicochemical theories providing analytical expressions amenable to relatively simple numerical analysis. Thus, the Linderstr?m-Lang capillary electrophoresis model (LLCEM) and its perturbed version (PLLCEM), proposed and applied previously to the CZE of globular proteins, are adapted and used here for peptides. Also the effects of pK-shifts on net charge, hydration and hydrodynamic size and shape of peptides are analyzed and discussed. Emphasis is placed on the fact that these parameters are physically coupled, and thus a variation in the net charge may produce an appreciable change in the hydrodynamic size of peptides. Within the framework of CZE, peptides may be assumed as having a hydrodynamic volume associated with either spherical or spheroidal particles. The effects on peptide net charge and hydrodynamic size, of electrostatic interaction between a pair of charged groups in the chain and electrical permitivitty around the peptide domain are studied. The predictions of the PLLCEM and LLCEM are in good agreement with results reported previously in the literature. Several limitations concerning these models and some needs for further research are also described.     

Hydration, charge, size, and shape characteristics of peptides from their CZE analyses

A CZE model is presented for peptide characterization on the basis of well-established physicochemical equations. The effective mobility is used as basic data in the model to estimate relevant peptide properties such as, for instance, hydration, net and total electrical charge numbers, hydrodynamic size and shape, particle average orientation, and pH-microenvironment from the charge regulation phenomenon. Therefore 102 experimental effective mobilities of different peptides are studied and discussed in relation to previous work. An equation for the estimation of peptide hydration as a function of ionizing, polar, and non-polar amino acid residues is included in the model. It is also shown that the shape-orientation factor of peptides may be either lower or higher than one, and its value depends on a complex interplay among total charge number, molar mass, hydration, and amino acid sequence.     

Determination of cationic mobilities and pKa values of 22 amino acids by capillary zone electrophoresis

The effective mobilities of the cationic forms of common amino acids--mostly proteinogenic--were determined by capillary zone electrophoresis in acidic background electrolytes at pH between 2.0 and 3.2. The underivatized amino acids were detected by the double contactless conductivity detector. Experimentally measured effective mobilities were fitted with the suitable regression functions in dependence on pH of the background electrolyte. The parameters of the given regression function corresponded to the values of the actual mobilities and the mixed dissociation constants (combining activities and concentrations) of the compound related to the actual ionic strength. McInnes approximation and Onsager theory were used to obtain thermodynamic dissociation constants (pK(a)) and limiting (absolute) ionic mobilities.     

Objective testing for the dependence of electrophoretic mobilities upon size in capillary zone electrophoresis

Summary Eighteen peptides have been modeled. From the volumetric data derived, and published mobilities, the relationship between electrophoretic mobility (μ_ep) and the hydrodynamic radius(r) has been examined. Objective testing with respect to size has been achieved by the log-log version of generalized relationship. (1) From the gradient of the plot versus log r(2.02) there is good support for the inverse square law (μ_ep α 1/r²). Equivalent calculations using molecular weight (M_r) and the number of amino acid residues (n) similarly lead to μ_ep α 1/M _r ^2/3 and μ_ep α 1/n^2/3, respectively. However, the strength of the correlation is diminished as the precision of the representation of size is degraded. (2) An examination of the effect of size at fixed charge and a statistical analysis of the charge distribution on the peptides leads to the conclusion that deviations from the averaged behaviour arise from a charge-induced volumetric effect. Taken together, (1) and (2) indicate that whilst net charge and total size can describe average electrophoretic behaviour well, these parameters are inadequate to describe the specific mobilities of individual analytes. Objective analysis of alkylpyridine data indicates μ_ep α 1/r^x where x=2.6–2.8 (depending upon the nature of the r values utilized), but is certainly ≠1 as may have been presumed. A very small range of values may be responsible for this surprising result.     

Individual electrophoretic mobilities of liposomes and acidic organelles displaying pH gradients across their membranes

This report focuses on measuring the individual electrophoretic mobilities of liposomes with different pH gradients across their membrane using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The results from the individual analysis of liposomes show that, using surface electrostatic theories and the electrokinetic theory as the first approximation, zeta potential contributes more significantly to the electrophoretic mobility of liposomes than liposomal size. For liposomes with an outer pH 7.4 (pH(o) 7.4) and a net negative outer surface charge, the most negative electrophoretic mobilities occur when the inner pH (pH(i)) is 6.8; at higher or lower pH(i), the electrophoretic mobilities are less negative. The theories mentioned above cannot explain these pH-induced electrophoretic mobility shifts. The capacity theory, predicting an induced electrical charge on the surface of liposomes, can only explain the results at pH(i) > 6.8. In this report, we hypothesize that there is a flip-flop process of phospholipids, which refers to the exchange of phospholipids between the outer and inner layers of the membrane. This flip-flop is caused by the pH gradient and membrane instability and results in the observed electrophoretic mobility changes when pH(i) is <6.8. Furthermore, it is found that the mobilities of acidic organelles are consistent with the predictions of liposome models we used here.     

On the electrophoretic mobilities of partially charged oligosaccharides as a function of charge patterning and degree of polymerization

Fully or partially charged oligosaccharide molecules play a key role in many areas of biology, where their fine structures are crucial in determining their functionality. However, the separation of specific charged oligosaccharides from similar moieties that typically coexist in extracted samples, even for those that are unbranched, and in cases where each saccharide moiety can only carry a single charge or not, is far from trivial. Typically such molecules are characterized by a degree of polymerization n and a number m (and distribution) of charged residues, and must be separated from a plethora of similar species possessing different combinations of n and m. Furthermore, the separation of the possible isomers of each species of fixed n and m is a formidable challenge to analytical chemists. Herein, we report the results of molecular dynamics simulations that have been performed in order to calculate the free solution electrophoretic mobilities of galacturonides and charged oligosaccharides derived from digests of the important plant cell‐wall polysaccharide pectin. The simulations are compared with an experiment and are found to correctly predict the loss of resolution of fully charged species above a critical degree of polymerization n and the ionic strength dependence of the electrophoretic mobilities of different partially charged oligosaccharides. It is expected that having a predictive tool for the calculation of the electrophoretic mobilities of differently charged oligosaccharide species in hand will allow experimental conditions that optimize the resolution of particular species to be ascertained and understood.     

Capillary electrophoretic separation of amino acids: Fraction collection

A simple method is described which enables solutes to be collected at an electrically isolated exit after they have been separated by a free solution capillary electrophoretic system. The method is illustrated by the separation of dansyl amino acids using multiple separation capillaries.     

Sensitivity enhancement by on-line preconcentration and in-capillary derivatization for the electrophoretic determination of amino acids

This study describes an application of on-line preconcentration by large-volume stacking in combination with in-capillary derivatization for enhancing spectrophotometric detection sensitivity in capillary electrophoresis. The method is illustrated by an example dealing with the determination of amino acids with 1,2-naphthoquinone-4-sulfonate as a labelling agent. Samples are dissolved in water in order to create a stacking process based on differences in the conductivity between this medium and a concentrated running buffer. The in-capillary derivatization is accomplished following a sandwich procedure in which the sample is inserted between two segments of reagent. Amino acid derivatives are obtained and separated in a fused-silica capillary with a sodium borate electrolyte buffer using 2-propanol as an organic modifier. The method is applied to the analysis of amino acids in pharmaceutical and feed samples. A good concordance between the predicted values and those obtained with the standard method is observed, with overall quantification error below 5%. The proposed procedure allows the detection limits sensitivity to be enhanced in 1000-fold with respect to conventional precapillary derivatization.     

Capillary zone electrophoretic (CZE) study of uranium(VI) complexation with humic acids

The interaction of UO₂ ²⁺ with various humic acids (HA's) has been studied by capillary zone electrophoresis (CZE). The experiments were done in 10 mM acetate buffer with pH 3.3 and 4.0, to avoid hydrolysis of uranium. It was found that in slightly acidic media and low HA concentration (<3 mM), two complexes with uranium(VI) are formed by fast kinetics and uranyl migrates as cationic species. Electrophoretic mobilities are decreasing with the increasing HA/uranium ratio and a low soluble neutral compound is also formed. In addition, it was found that at HA concentrations higher than 3 mM negatively charged species are formed. Similar results were obtained for HA's of different origin (soil, peat, coal derived, IHSS standards). Conditional stability constants of the complexes UO₂ ²⁺-HA for Fluka I HA, were estimated to be log ₁ = 4.18±0.06 and log ₂ = 7.28±0.18.     

Determination of pKa values of tenoxicam from H NMR chemical shifts and of oxicams from electrophoretic mobilities (CZE) with the aid of programs SQUAD and HYPNMR

In this work it is explained, by the first time, the application of programs SQUAD and HYPNMR to refine equilibrium constant values through the fit of electrophoretic mobilities determined by capillary zone electrophoresis experiments, due to the mathematical isomorphism of UV-vis absorptivity coefficients, NMR chemical shifts and electrophoretic mobilities as a function of pH. Then, the pK_a values of tenoxicam in H₂O/DMSO 1:4 (v/v) have been obtained from ¹H NMR chemical shifts, as well as of oxicams in aqueous solution from electrophoretic mobilities determined by CZE, at 25 °C. These values are in very good agreement with those reported by spectrophotometric and potentiometric measurements.     

Determination of erythrocyte amino acids by gas chromatography

Erythrocyte amino acid levels were determined, by gas chromatography, in a group of 34 normal human adults. No significant sex or age correlations were noted. A method for the quantitative gas chromatographic analysis of free amino acids in erythrocytes is described. Following hemolysis and deproteinization the amino acids were isolated on a cation-exchange resin. Glutathione was removed from the amino acid mixture by adsorption on an anion-exchange resin. Following conversion to their N-acetyl-n-propyl esters, 19 amino acids were separated and quantitated by gas chromatography on a single column in 18 min. Typical reproducibility data indicate that a coefficient of variation of 2-5% is attainable.     

Selectivity control in the non-aqueous capillary electrophoretic separation of amino acids

The separation of dansylated amino acids and underivatized amino acids in non-aqueous electrolytes was evaluated with direct and indirect UV detection. Different migration orders were achieved for dansylated amino acids in methanol compared to aqueous electrolyte systems. A reversed migration order was observed for some dansylated amino acids. Separation selectivity was different under acidic and basic conditions and was also a function of the solvation properties of the solvent. Underivatized amino acids were separated in basic and acidic electrolytes in methanol; different separation selectivities and, for some amino acids, a reversed migration order were also observed in these electrolyte systems. Analytical merits of the separation of both derivatized and underivatized amino acids were briefly evaluated; detection limits for dansylated amino acids were in the range of 2·10⁻⁷–4·10⁻⁷ mol/l and, for underivatized amino acids, were 2·10⁻⁶–4·10⁻⁵ mol/l.     

反相流动注射-化学发光法测定氨基酸

基于碱性条件，氨基酸对次氯酸钠-一鲁米诺化学发光体系的显著增敏作用，建立了反相流动注射-化学发光法测定氨基酸的新方法。该法不需对氨基酸进行衍生或转化，检出限为0．016μg/mL，采样频率为150次／h，对1μg／L的组氨酸进行连续12次平行测定，其RSD为0．9％。对氨基酸注射液样品中的氨基酸测定的回收率在98．6％～102．1％之间，并和经典的茚三酮比色法进行了对照。     

Identification of the electrophoretic peaks of the phenylthiohydantoin derivatives of amino acids

Two techniques for identifying the peaks of phenylthiohydantoin (PTH) amino acids separated under the conditions of micellar electrokinetic chromatography were compared. The first technique is linear regression analysis, in which the retention time of an amino acid is a function of the retention times of two retention-time standards. The second technique takes into account hydrophobicity constants logD′, which were calculated using the ACD/LC Simulator 8.0 program package from ACDLabs (Canada). These constants provide an opportunity to calculate the relative migration times of PTH amino acids taking into account the velocity of the electroosmotic flow. The first technique allows us to identify the electrophoretic peaks of all 16 amino acids separated; the second procedure allows us to predict the elution order of the electrophoretic peaks; the use of a correlation equation gives better results.     

Determination of plasma amino acids by gas chromatography

A complete methodology, incorporating a novel clean-up technique, for quantitative determination of amino acids in plasma by gas chromatography is described. Glucose, a component causing major analytical interference, is removed by an enzymic reaction included in the pre-chromatographic clean-up. The procedure for derivatisation of amino acid standards is shown to be reproducible down to a level of 2.5 micrograms for each amino acid, relative standard deviations for all amino acids except arginine and histidine being 3% or lower. For the entire procedure applied to plasma, relative standard deviations for most amino acids are below 5% with recoveries ranging from 90 to 120%. Normal values, obtained using eighteen plasma samples, are in reasonable agreement with published data. Plasma amino acid values were determined simultaneously by gas chromatographic and ion-exchange chromatographic techniques. Statistical evaluation shows there to be no significant difference between corresponding values for eleven amino acids. Values for tyrosine, histidine and particularly phenylalanine show significant differences (p less than 0.001).     

Ultrafast charge separation driven by differential particle and hole mobilities

The process of a local excitation evolving into an intramolecular charge-separated state is followed and compared for several systems by directly simulating the time propagation of the electronic wavefunction. The wavefunction and Hamiltonian are handled using the extended second-order algebraic diagrammatic construction (ADC(2)-x), which explicitly accounts for electron correlation in the dynamic many-particle state. The details of the charge separation can be manipulated according to the chemical composition of the system; atoms which dope the conjugated system with either particles or holes are shown to effect whether the particle or hole is more mobile. Initially, the charges oscillate between the ends of linear molecules (with different rates), separating periodically, but, at long times, both charges tend to spread over the whole molecule. Charge separation is also shown to occur for asymmetric systems, where it may eventually be experimentally feasible to excite a localized resonance (nonstationary state) on one end of the molecule preferentially and follow the ensuing dynamics.