Carrier Ampholytes For Isoelectric Focusing Research Articles

Mass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results

In an attempt to prepare quasi-isoelectric buffers as BGEs for CE, carrier ampholytes (CAs) (Ampholine, pH 7-9; Servalyt, pH 7-9; Bio-Lyte, pH 8-10 and Pharmalyte, pH 8-10.5) have been subdivided with the Rotofor into 20 fractions, of ca. 0.1 pH unit span, whose composition has been studied by CZE-MS. The results have allowed identifying the number of different molecular mass compounds present in every commercial brand, as well as the number of isoforms (having identical mass, but representing positional isomers) associated with a given M(r) value. Ampholine is composed of 29 species, for a total of 85 different isoforms; Bio-Lyte is made of 43 compounds, for a total of 136 isoforms; Pharmalyte comprises 58 different M(r) chemicals, for a total of 102 isoforms and Servalyt is constituted by 65 species, for a total of 306 compounds (all of these values to be considered as minimum numbers, as detected by the present methodology). Surprisingly, and contrary to theory, a very large proportion (up to 70%) of these species are 'poor carrier ampholytes', in that they are unable to focus and are evenly distributed along the generated pH gradient in the electric field. Paradoxically, the pH gradient is created and sustained by the minority of species (30% for three brands, up to 50% for Pharmalyte) that appear to focus at their pI position into reasonably sharp zones. Even in the narrowest pI fraction, up to 20 different compounds can be detected. It is concluded that very few amines with different useful pK values are utilized for the synthesis and that a new generation of CAs with a more diversified population of amines with proper pK values within the given pH intervals should be sought. Ampholine, the poorest of the commercial brands, appears to be still made with the original synthesis devised by Vesterberg, i.e. by reacting a concoction of oligoamines with alpha,beta-unsaturated acids.

Preparative high-resolution two-dimensional electrophoresis enables the identification of RNA polymerase B transcription factor 3 as an apoptosis-associated protein in the human BL60-2 Burkitt lymphoma cell line.

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified RNA polymerase B transcription factor 3 (BTF3), which is associated with anti-IgM antibody-mediated apoptosis, using a subclone of the human Burkitt lymphoma cell line BL60. To identify the transcription factor BTF3, which is expressed only in minor amounts, we used preparative high-resolution two-dimensional gel electrophoresis (2DE) employing carrier ampholytes for isoelectric focusing. Comparison of the 2DE protein patterns from apoptotic and nonapoptotic cells showed BTF3 as a predominantly altered protein spot. The characterization of the differentially expressed transcription factor and 13 marker proteins described in this study were performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The proteome analysis was significantly improved by performing the newly developed preparative high-resolution two-dimensional gels employing high protein concentrations.

24] Synthesis of carrier ampholytes for isoelectric focusing

24] Synthesis of carrier ampholytes for isoelectric focusing

High-molecular-weight carrier ampholytes for isoelectric focusing of peptides

A method is described for the synthesis of high-molecular-weight carrier ampholytes for preparative isoelectric focusing of peptides. A giant polyethylene imine ( M r 40 000–60 000 ) is mixed with a linear gradient of acrylic acid in a flow-through system and let to react at 80°C for 70 h. Giant carrier ampholytes ( M r range 50 000–90 000 ) are thus obtained. These compounds interact very strongly among themselves, probably not by hydrogen bonds or hydrophobic interactions but ionic bonds. In fact, the aggregates are split by high salt (NaCl) or by zwitterionic compounds (Gly, taurine) or at acidic or alkaline pHs. They appear to interact only weakly and reversibly with proteins and no interactions are apparent with model dipeptides (His-Ser, His-Met, His-Phe and His-Lys).

Molecular weight distribution of carrier ampholytes for isoelectric focusing

By osmolarity, gel filtration an dialysis experiments it has been demonstrated that Ampholine carrier ampholytes have relative molecular masses ( M r) ranging from 600 daltons in the alkaline range (pH 8–10) up to 900 daltons in the acidic range (pH 3.5–), with graded intervals for the intermediate ranges. The most acidic range (pH 2.5–4) has a considerably smaller M r (325 daltons), suggesting that it is synthesized with different polyamines and/or acids. There is no indication that Ampholines with M r > 1000 daltons exist. Evidence is presented that carrier ampholytes aggregate in solution, giving rise to complexes with higher apparent M r.

Further study on distribution of carrier ampholytes in isoelectric focusing. Theoretical proof of the law of pH monotony.

Further study on distribution of carrier ampholytes in isoelectric focusing. Theoretical proof of the law of pH monotony.

Molarity and ionic strength of focused carrier ampholytes in isoelectric focusing

Focused 1% carrier ampholytes in the pH range 3.5–10 have a molarity of 9–1 0 m M, as determined by osmolarity measurements of fractions focused in free water. From electrophoretic and isoelectric focusing data of red blood cells, it has been demonstrated that the corresponding ionic strength is 0.5 mg-ions/1. Also theoretical considerations and conductivity measurements point to a value in the range of 0.5–1.0 mg-ions/1. The following equations for ionic strength ( I) calculations have been derived: I = 1 20 C amph + C H in the pH range 2.5–7 and I = 1 20 C amph + C OH in the pH range 7–11.

Distribution of carrier ampholytes in isoelectric focusing - A mathematical approach.

Distribution of carrier ampholytes in isoelectric focusing - A mathematical approach.

Binding of polyanions to carrier ampholytes in isoelectric focusing

The binding of carrier ampholytes to polyanions is markedly pH-dependent: it is very strong at pH 3, rather weak at pH 5 and abolished at pH 7. Binding is affected by the type of negative charge, its density and spatial orientation on the polyanion. On the basis of the type of negative charge, the binding strength decreases in the following order: polyphosphate > polysulphate > polycarboxylate. Given the same type of negative charge, the binding is dependent on charge density and its space orientation: thus polyglutamic acid forms stronger complexes than polygalacturonic acid. The minimum length of the polyanion eliciting a measurable binding appears to be of the order of about six negative charges, as demonstrated with hexametaphosphate.

Distribution of carrier ampholytes in isoelectric focusing

Distribution of carrier ampholytes in isoelectric focusing

Itaconic acid carrier ampholytes for isoelectric focusing

Commercial carrier ampholytes, obtained by coupling polyethylene polyamines to acrylic acid, exhibit a conductivity minimum in the pH range 5.5–6.5 owing to the lack of appropriate p K values of the polyamine in this pH region. By replacing acrylic with itaconic acid, it has been possible to effect substantial improvements in the pH range 5.5–6.5 as itaconic acid has a p K 2 value of 5.45. Upon coupling, the p K of the γ-carboxyl group remains virtually unaltered. With itaconic acid carrier ampholytes it has been possible to improve the conductivity in the pH range 5.5–6.5 by as much as 400% compared with conventional carrier ampholytes. It is suggested that the commercial products should be supplemented with itaconic acid carrier ampholytes in order to obtain a more uniform conductivity and buffering capacity in the pH range 3–10.

Characterization of synthetic carrier ampholytes for isoelectric focusing

The synthesis of carrier ampholytes suitable for isoelectric focusing is described. The mixture of of hexamethylenetetramine (HMTA), triethylenetetramine (TETA0. tetraethylenepentamine (TEPA) and pentaethylenehexamine (PEHA0 ampholytes closely resembles commercial Ampholine, and covers the pH range 3–9.5. We have been able to detect focused ampholytes in a gel slab, taking advantage of their different refractive indices, and to assess their relative amounts along the pH gradient. PEHA ampholytes contain up to 20% of chromophoric structures, with two UV peaks at 368 and 315 nm, in a pH-dependent equilibrium, associated with a very weak nitrogen function having a pK of 1.1. This could be the p K 6 of the last amino group in PEHA. However, NMR spectra failed to reveal any nitrogen heterocyclic structure formed during the synthesis. This mixture of ampholytes exhibits good conductivity, produces smooth pH gradients and allows sharp protein separations in the pH range 3–9.5. Their synthesis is very easy and their cost is extremely low. Their availability should make feasible large-scale preparative isoelectric focusing, and attract more interest to continuous-flow techniques, where large amounts of ampholytes are required.

Fractionation of carrier ampholytes for isoelectric focusing

A simple method for fractionating synthetic carrier ampholytes is reported, based on the principle of continuous-flow isoelectric focusing in gel-stabilized layers. An 8% ampholyte solution, encompassing the pH range 3–9.5, is separated into 12 fractions in a chamber filled with Sephadex G-100 by a continuous-flow technique. We are thus able to obtain ampholytes of narrow pH range, encompassing approximately 2 ph units, whose resolving power is comparable with that obtained with commercial Ampholine covering similar pH ranges.

Some optical properties of carrier ampholytes for isoelectric focusing

Some optical properties of carrier ampholytes are herewith described. Newly synthesized Ampholines do not possess asymmetric carbon atoms. pH 3–5 and pH 8–10 ranges, synthesized before 1970, rotate the plane of polarized light since they were “reinforced” with glutamic and aspartic acid, in the acidic side, and lysine in the basic region. The various pH ranges possess characteristic chromophores, whose absorbance is strongly pH dependent. These chromophores, when excited at appropriate wavelengths, exhibit a fluorescence emission spectrum, typically reproducing the pH dependence of the corresponding uv spectra. The optical properties here described can be useful in studying Ampholinesmacromolecules interactions. Due to the widespread use of optical scanning in situ of focusing systems, care has to be taken not to mistake Ampholine peaks at 285, 310, 315, 340, and 365 nm for the substance under study.