Separation of peptides in isoelectric cysteic acid buffer and hydro-organic solvents (hexafluoro-2-propanol-urea)

A novel amphoteric, isoelectric, acidic buffer is here reported for separat ion of oligo- and polypeptides by capillary zone electrophoresis: cysteic a cid (Cys-A). Cys-A, at 200 mM concentration, exhibited an isoelectric point (pI) of 1.80; given a Delta pK=0.6, the pK of the carboxyl was assessed as 2.1 and the pK of the sulphate group as 1.50. At 100 mM concentration, thi s buffer provided an extraordinary buffering power: 140.10(-3) equiv./l per pH unit. In presence of 30% (v/v) hexafluoro-2-propanol (HFP), this buffer did not change its apparent pi value, but drastically reduced its conducti vity. In Cys-A-HFP buffer, small peptides exhibited a mobility closely foll owing the Offord equation, i.e., proportional to the ratio M-r(2/3)/Z). Wit h addition of 4-5 M urea, there was an inversion in the mobility of some pe ptides, suggesting strong pK changes as an effect of urea addition. It was found that the minimum mass increment, for proper peptide separation, was D elta M-r=ca. 1%. In case of simultaneous M-r and pK changes, the minimum De lta M-r is reduced to only 0.6%, provided that a concomitant minimum Delta pK=0.08 took place. When separating large peptides (human globin chains) in 100 mM Cys-A, 30% HFP and 7 M urea, the beta-chain was found to co-elute w ith the alpha-chain, suggesting a subtle interplay between the helix formin g (HFP) and helix breaking (urea) agents. When HFP was omitted, the origina l globin separation could be restored. (C) 1999 Elsevier Science B.V. All r ights reserved.