Detection of neutral and charged mutations in alpha- and beta-human globinchains by capillary zone electrophoresis in isoelectric, acidic buffers

A simple and reliable method, for screening for point mutations in alpha- a nd beta-human globin chains, is reported here, utilizing capillary zone ele ctrophoresis in isoelectric, acidic buffers. A solution of 50 mM iminodiace tic acid (pI 2.23) containing 7 M urea and 0.5% hydroxyethylcellulose (appa rent pH 3.2) is used as background electrolyte for fast separation of heme- free, denatured globin (alpha and beta) chains. Due to the low conductivity of such buffers, high voltage gradients (600 V/cm) can be applied, thus re ducing the separation time to only a few minutes. In presence of neutral to neutral amino acid substitutions, it is additionally shown that the inclus ion of 3% surfactant (Tween 20) in the sample and background electrolyte in duces the separation of the wild-type and mutant chains, probably by a mech anism of hydrophobic interaction of the more hydrophobic mutant with the de tergent micelle, via a mechanism similar to "micellar electrokinetic chroma tography". At this low operative pH, however, charged mutants, involving su bstitutions of acidic amino acids (Glu and Asp) are not detected, since the se residues are extensively protonated. Curiously, however, they are still separated in presence of detergent, due to the large variation in hydrophob icity involved in such mutations. Of the 19 mutants analyzed, all but one w ere resolved: Hb St Nazaire (beta 103 Phe-->Ile). This is due to the fact t hat the Delta G (in kcal/mol) in the substitution Phe-->Ile is zero, thus n o separation can possibly take place between two chains exhibiting the same hydrophobicity parameter. (C) 1999 Elsevier Science B.V. All rights reserv ed.