Barbara Verzola

Protein adsorption to the bare silica wall in capillary electrophoresis quantitative study on the chemical composition of the background electrolyte for minimising the phenomenon.

A novel method is reported for quantifying protein adsorption to naked silica tubings and for assessing the efficacy of amino quenchers added to the background electrolyte. It consists of flushing a fluorescently-labelled protein (myoglobin) into a capillary equilibrated in Tris-acetate buffer, pH 5.0, until full saturation of the potential adsorbing sites. Desorption is then affected by driving electrophoretically sodium dodecyl sulphate (SDS) micelles into the capillary from the cathodic reservoir: the peak of eluted material is quantified fluorometrically by using a dual laser beam instrument able to read the fluorescein-isothiocyanate-labelled myoglobin at 520 nm and the internal standard (sulphorodamine) at 630 nm. As potential quenchers, a series of monoamines have been investigated (triethylamine, triethanolamine, ethylamine), followed by diamines (putrescine, cadaverine and hexamethonium bromide) and finally by oligoamines [spermidine, spermine and TEPA (tetraethylenepentamine), i.e., a tri- a tetra- and a pentamine, respectively]. Two values of molarities have been derived: a value at 50% (a kind of a dissociation constant) and a value at 90% inhibition of binding of macromolecules to the silica surface. According to these figures of merit, mono- and diamines are rather poor quenchers of interaction with the wall, since the 50% values are of the order of 50-100 mM and the 90% values reach as high as 560 mM. On the contrary, oligoamines, especially spermine and TEPA, are most effective, since the 50% molarities are in the sub-millimolar range and the 90% values are of the order of ca. 1 mM. Figures of merit have also been derived for different washing procedures. Those most commonly adopted in routine practice, i.e., of washing with either 1 M NaOH or with 1 M HCl, or with both, leave behind traces of proteins still bound to the wall, whereas the SDS micelle electrophoretic desorption seems to be 100% effective.

Folding/unfolding/refolding of proteins: present methodologies in comparison with capillary zone electrophoresis.

A series of techniques for monitoring protein folding/unfolding/misfolding equilibria are here assessed and compared with capillary zone electrophoresis (CZE). They include spectroscopic techniques, such as circular dichroism, intrinsic fluorescence, nuclear magnetic resonance, Fourier transform infrared and Raman spectroscopy, small‐angle X‐ray scattering, as well as techniques based on biological assays, such as limited proteolysis and immunochemical analysis of different conformational states. Some unusual probes, such as mass spectrometry for probing unfolding transitions, are also discussed. Size‐exclusion chromatography is also evaluated in view of the fact that this technique, like all electrophoretic techniques, and unlike spectroscopic probes, which can only see an average signal in mixed populations, can indeed physically separate folded vs. unfolded macromolecules, especially in the case of slow equilibria. Particular emphasis is devoted to electrophoretic techniques, such as gel‐slab electrophoresis in transverse urea or thermal gradients, and CZE. In the latter case, a number of applications are shown, demonstrating the excellent correlation of CZE with more traditional probes, such as intrinsic fluorescence monitoring. It is additionally shown that CZE can be used for measuring the δG° of unfolding over the pH scale, in good agreement with theoretical calculations on the electrostatic free energy of folding vs. pH, as calculated with a linearized Poisson‐Boltzmann equation. Finally, it is demonstrated that CZE can probe also aggregate formation in the presence of helix‐inducing agents, such as trifluorethanol.

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.

Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: Measurement ofG and its variation along the pH scale

Free‐solution capillary zone electrophoresis (CZE) can be used to monitor folding/unfolding transitions of proteins and to construct the classical sigmoidal transition curve describing this isomerization process. By performing a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea]1/2, which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an idea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneously unfold even in the absence of a denaturant. The equation describing the sigmoidal folding/unfolding transition can be used for deriving the term ΔGo, which refers to the intrinsic difference in the Gibbs free energy between the (total or partial) denatured state and the reference state, taken usually as the native configuration of a protein. The variation of ΔGo between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically calculated) to be of the order of 7–10 kcal/mol and is here interpreted by assuming that at pH 2.5 and below there is an additionally stretching of the polypeptide coil due to coulombic repulsion, as the unfolded chain looses its zwitterionic character and assumes a pure (or very nearly so) cationic surface. Given the minute amounts of sample required, the fully automated state of the analysis, the rapidity and ease of operation, it is hoped that the CZE technique will become more and more popular in the years to come for monitoring folding/unfolding transitions of proteins.

Chapter 9 Capillary zone electrophoresis

Publisher Summary This chapter reviews the development of capillary zone electrophoresis (CZE), from early home-made instruments to the most sophisticated commercial equipment in use at present, exploiting a battery of channels for parallel, massive operations, such as DNA sequencing. A general problem with most of the CZE instruments is the reading length; it is difficult to achieve the sequencing of > 500 bases with > 99% accuracy, although it would be highly desirable to extend this value to at least 800 bases. However, the electric field strength gradients coupled to column temperatures of 60 o C allow the extension of the reading length with almost linear transit times up to 800 bases. One of the main inventions has been the development of the fused-silica column at the Hewlett-Packard Company. The discovery of fused-silica capillaries eliminated the intermediate step of using flexible quartz. The chapter discusses the four modes of performing CZE: frees CZE, CZE in sieving liquid polymers, CZE in the isoelectric focusing mode, and CZE under isotachophoretic conditions.

Chapter 15 Electrophoresis of proteins and peptides

Publisher Summary This chapter focuses on those methods of the electrophoresis of proteins and peptides that are widely adopted in modern separation science. These techniques are divided into two categories: gel-based and free-solution methodologies. Gel-based techniques include (1) conventional isoelectric focusing (IEF) in soluble carrier ampholyte buffers, (2) IEF in immobilized pH gradients (IPG), (3) sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and (4) two-dimensional maps as engendered by an orthogonal combination of IEF and IPG. The free-solution method is the capillary zone electrophoresis (CZE), which is the only useful approach currently used for performing fast separations of proteins/peptides in a free solution, although size separations in capillaries filled with appropriate solutions of sieving polymers can be easily performed. The chapter focuses on models for predicting the migration of peptides and presents recent studies aimed at assessing the folding/unfolding/misfolding processes that proteins undergo during denaturation/renaturation cycles.