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 separation of oligo- and polypeptides by capillary zone electrophoresis: cysteic acid (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, this 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 conductivity. In Cys-A-HFP buffer, small peptides exhibited a mobility closely following the Offord equation, i.e., proportional to the ratio M-r(2/3)/Z). With addition of 4-5 M urea, there was an inversion in the mobility of some peptides, suggesting strong pK changes as an effect of urea addition. It was found that the minimum mass increment, for proper peptide separation, was Delta M-r=ca. 1%. In case of simultaneous M-r and pK changes, the minimum Delta 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 with the alpha-chain, suggesting a subtle interplay between the helix forming (HFP) and helix breaking (urea) agents. When HFP was omitted, the original globin separation could be restored. (C) 1999 Elsevier Science B.V. All rights reserved.