CAPILLARY-ZONE-ELECTROPHORESIS OF OLIGONUCLEOTIDES AND PEPTIDES IN ISOELECTRIC BUFFERS - THEORY AND METHODOLOGY

The use of isoelectric buffers in capillary zone electrophoresis is re viewed. Such buffers allow application of extremely high voltage gradi ents (up to 1000 V/cm in relatively high bore capillary, e.g. 75 to 10 0 mu m internal diameter), permitting separations of the order of a fe w minutes and thus favoring high resolution due to minimal, diffusion- driven peak spreading. The fundamental properties of ampholytes are fi rst discussed, such as buffering power (beta) as a function of Delta p K, i.e. of the distance between the pI value and neighboring protolyti c groups. The highest possible relative beta value (= 2) is obtained f or amphoteres possessing a Delta pK = 0.6, a condition not met by exis ting amphoteric species. A novel parameter for ampholyte evaluation is then proposed, namely the beta/lambda ratio, i.e. the ratio between t he beta power and conductivity at the pI value. It is additionally sho wn that the pI is not a constant value, but depends on ampholyte conce ntration in solution. In addition, at constant concentration, the theo retical pI can change as a function of Delta pK. Isoelectric His and, to a lesser extent, Lys have been found to offer unique separations of oligonucleotides in sieving liquid polymers. In the absence of sievin g media, isoelectric Asp, in presence of 7 M urea (apparent pH 3.77), permits unique separations of oligonucleotides having the same length but different nucleotide composition. Isoelectric Asp (pI2.77 at 50 mM concentration) provides a medium of high resolving power for generati ng peptide maps. In difficult cases, of coincident titration curves, t he pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thu s eliciting separation of unresolved peptides at pH 2.77. This was ill ustrated by running peptide maps of tryptic digests of human beta-glob in chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) a llows generation of high resolution peptide maps.