Alexander V. Stoyanov

Capillary electrophoresis of peptides and proteins in isoelectric buffers: an update.

Capillary electrophoresis in acidic, isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and little interaction of macromolecules with the untreated wall occurs. In addition, due to the low conductivity of quasi‐stationary, isoelectric buffers, high‐voltage gradients can be applied (up to 800 V/cm) permitting fast peptide analysis with a high resolving power due to minimal diffusional peak spreading. Four such buffers are here described: cysteic acid (Cys‐A, pI 1.85), iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). A number of applications are reported, ranging from food analysis to the study of folding/unfolding transitions of proteins.

Fundamental properties of isoelectric buffers for capillary zone electrophoresis

Abstract Different isoelectric buffers are analysed theoretically, taking into account a fundamental parameter, i.e., the ratio between intrinsic buffering power and conductivity (R=β/λ). For a model ampholyte, the above parameter is analysed both as a function of the pI and the pKb–pKa values. For natural pH gradients, the variation of R, connected with approaching the isoelectric point, is evaluated. A case of oligo-protic ampholytes is also considered.

Generation of tryptic maps of α- and β-globin chains by capillary electrophoresis in isoelectric buffers

A novel method for generating peptide maps, following tryptic digests of proteins, is reported here: capillary zone electrophoresis in the presence of isoelectric buffers as the sole buffering species. A typical buffer composition comprises 50 mM aspartic acid (pH=pI=2.77), 0.5% hydroxyethyl cellulose (added as a dynamic coating agent for preventing peptide adsorption to weakly ionized silanols), 5% trifluoroethanol and 1% zwitterionic detergent (CHAPS). With this buffer composition, a high-voltage gradient can be applied (typically 600 V/cm in 75 μm I.D. and 900 V/cm in 50 μm I.D. capillaries), thus drastically reducing the analysis times. The method is applied to the generation of peptide maps of α- and β-globin chains from human adult hemoglobin. In the case of β-peptides, at an operative pH of 2.77, which represents a cross-over point in the titration curve of peptides T2 and T9, the two analytes merge into a single peak. However it is shown that it is possible to change the pH of the zwitterionic buffer by adjusting its concentration in solution. In 30 mM Asp (pH 3.0) or 20 mM Asp (pH 3.1) resolution of these two peptides is fully restored. Isoelectric, amphoteric buffers thus seem to represent a novel, powerful buffer system able to offer high resolution and high selectivity.

Dissociation of polyvalent electrolytes

Understanding of the dissociation mechanism for polyvalent electrolytes remains a matter of critical importance. Different theoretical approaches could result in different values for the microscopic parameters of the system under analysis, as well as for its integral characteristics (such as conductivity and buffering power). A unified classification of dissociation schemes is proposed here. Some examples of hybrid type schemes are considered and the expressions relating the macroscopic and microscopic constants are given. The possibility of applying the equation of Linderstrøm-Lang to multi-dissociating systems with non-constant total concentration is considered. The problem of proton binding curves (so-called titration curves) modeling is discussed.

Properties of buffer systems with charges immobilized on a gel matrix and their potential use in capillary electrophoresis

Some physicochemical properties, such as conductivity and buffering capacity, are evaluated and compared for two types of buffer systems: soluble, amphoteric, isoelectric species and immobilized weak buffering ions and counter-ions grafted onto a polyacrylamide gel (Immobiline technology). Theoretical calculations are performed for a model system comprising two Immobilines in a wide range of concentrations and compared with the properties of model ampholyte solutions. The results obtained show that the conductivity of any system composed of ampholytes dissolved in pure water is mostly connected with the contributions of the hydrogen and hydroxyl ions, and that the intrinsic ampholyte contribution is negligible at any value of concentration, although it increases when the ampholyte isoelectric point (pI) is removed from neutrality. The buffering capacity behaviour is also analysed in the range of small ampholyte (Immobilines) concentrations as a function of ΔpK values (i.e., how far the pK values are removed from the pI of the amphotere). Due to the high flexibility in the preparation of isoelectric, Immobiline buffers (which can cover essentially any pH value), their potential use as background electrolytes for separations in capillary electrophoresis is evaluated.

Steady-state electrolysis of a solution of nonamphotheric compounds

The problem of stationary electrolysis of a solution of nonamphotheric compounds (acids or bases) is considered. The analysis is performed by taking into account the mobility dependence on pH. The properties of such a system are also compared with the ones pertaining to the solution of an amphoteric substance. It is anticipated that steady‐state electrolysis of free acids and bases, in a convection‐free system, might be useful for creating narrow pH gradients in rather acidic and alkaline milieus, which might be adopted for focusing without resorting to conventional carrier ampholytes or immobilized pH gradients.

Buffer properties of biopolymer solutions, as related to their behaviour in electrokinetic methodologies

Abstract The buffer properties of biopolymers (proteins and nucleic acids), with special reference to electrokinetic methodologies, are considered. The action of biopolymers as potential titrants is also analyzed. Since the ‘buffering capacity’ value ( β ), that is conventionally used, is a local value, we propose to use the concept of ‘buffer reserve’. The latter is obtained by integrating the β value over the necessary pH interval, and thus coincides with the concentration of strong titrant needed to induce the appropriate pH shift. With this parameter any buffer system may be characterized and its value will be a function of the ΔpH shift. The theoretical calculations performed for two real amphoteric substances show that the resistivity limit of such two buffers with the same initial β level may be quite different even in case of rather small pH shifts (several tenths of pH unit).