Detection of neutral and charged mutations in α- and β-human globin chains by capillary zone electrophoresis in isoelectric, acidic buffers

Arianna Saccomani, Cecilia Gelfi, Henri Wajcman, Pier Giorgio Righetti

Research output: Contribution to journalArticlepeer-review

Abstract

A simple and reliable method, for screening for point mutations in α- and β-human globin chains, is reported here, utilizing capillary zone electrophoresis in isoelectric, acidic buffers. 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 (α and β) chains. Due to the low conductivity of such buffers, high voltage gradients (600 V/cm) can be applied, thus reducing the separation time to only a few minutes. In presence of neutral to neutral amino acid substitutions, it is additionally shown that the inclusion of 3% surfactant (Tween 20) in the sample and background electrolyte induces the separation of the wild-type and mutant chains, probably by a mechanism of hydrophobic interaction of the more hydrophobic mutant with the detergent micelle, via a mechanism similar to 'micellar electrokinetic chromatography'. At this low operative pH, however, charged mutants, involving substitutions of acidic amino acids (Glu and Asp) are not detected, since these residues are extensively protonated. Curiously, however, they are still separated in presence of detergent, due to the large variation in hydrophobicity involved in such mutations. Of the 19 mutants analyzed, all but one were resolved: Hb St Nazaire (β103 Phe→Ile). This is due to the fact that the ΔG (in kcal/mol) in the substitution Phe→Ile is zero, thus no separation can possibly take place between two chains exhibiting the same hydrophobicity parameter.

Original languageEnglish
Pages (from-to)225-238
Number of pages14
JournalJournal of Chromatography A
Volume832
Issue number1-2
DOIs
Publication statusPublished - Jan 5 1999

Keywords

  • Globins
  • Haemoglobin
  • Proteins

ASJC Scopus subject areas

  • Analytical Chemistry

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