Human globin chain separation by capillary electrophoresis in acidic isoelectric buffers

Abstract

A simple and reliable method, utilizing capillary electrophoresis in uncoated capillaries in acidic isoelectric buffers, is reported for screening for thalassemia and other defects on the synthesis of human globin chains. A solution of 50 mM iminodiacetic acid (pI 2.23), containing 7 M urea and 0.5% hydroxyethylcellulose (apparent pH 3.2) is used as background electrolyte for fast separation of heme-free, denatured globin (alpha, beta and gamma) chains. Due to the low conductivity of such a buffer, high voltage gradients (600 V/cm) can be applied, thus reducing the separation time to only a few minutes. It is additionally shown that inclusion of 2% surfactant (Tween 20) in the background electrolyte induces the splitting of the gamma chains into two zones, called Agamma and Ggamma, which represent the products of two genes coding for Ala or Gly as residue 136 of the chain.

An N‐methylpolyvinylpyridinium cationic polymer for capillary coating in electrophoresis of proteins and peptides

“…First of all, it would appear that, for best performance, such cationic polymers, adsorbed onto the silica wall, should be coupled with a BGE made of isoelectric buffers, in particular Asp. The use of Asp had been in fact advocated long ago in the separation of wheat gliadins [36], of maize zeins [37] and of human globin chains [38]; while it appeared to quench interactions of proteinaceous analytes to the wall, it also allowed, due to isoelectric conditions, delivering high-voltage gradients to the silica tube, without generation of excessive joule heating. But there could be more to it: the fact of using amphoteric molecules, in particular a free amino acid, might help in quenching potential interactions of analytes with the silica wall.…”

CE‐based analysis of hemoglobin and its applications in clinical analysis

“…1. Righetti et al [48][49][50] then presented a simple and reliable method utilizing CZE in isoelectric and acidic buffers for identification of the point mutations in some a-and bglobin chains. The possibility of a reliable prediction of the mobility on the basis of the peptide charge-to-mass ratio makes CZE an attractive method for the tryptic digestion of a-or/and b-chains of Hbs [51][52][53][54][55], which will be described below (cf.…”

Synthesis and characterization of quaternary ammonium dicarboxylic acid isoelectric buffers and their use in pH-biased isoelectric trapping separations

“…Next, the ionic strength-corrected ionic mobilities for the monocationic and monoanionic forms of QDBA (m IEB1 and m IEB2 ) were obtained according to the Onsager-Fuoss approach (using Eq. (1) [19][20][21][22][23] of [57] at the pH and ionic strength values of the buffers, from m IEB1 and m IEB2 , the hydronium activity (from the pH), the activity coefficients and the initial estimates for the unknown pK a1 and pK a2 values. Next, the differences between the measured effective mobilities (m (DpK a ,3).…”

“…Subsequently, Righetti's group used both HIS [9][10][11][12][13][14][15] and iminodiacetic acid (IDA) as IEBs [16,17] for the CE separation of both oligonucleotides and double-stranded DNA. Righetti also obtained peptide and protein separations in acidic IEBs, namely, in isoionic solutions of IDA [18][19][20][21][22], aspartic acid [23][24][25][26][27][28], cysteic acid [29,30], and glutamic acid [31]. Bean and Lookhart [32,33] and Solinova et al [34] also used some of these IEBs, aspartic acid, IDA, and glycine for the rapid CE separation of proteins and peptides, respectively.…”