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

Abstract

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 m M concentration, exhibited an isoelectric point (p I) of 1.80; given a Δp K=0.6, the p K of the carboxyl was assessed as 2.1 and the p K of the sulphate group as 1.50. At 100 m M 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 p I 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 p K changes as an effect of urea addition. It was found that the minimum mass increment, for proper peptide separation, was Δ M r=ca. 1%. In case of simultaneous M r and p K changes, the minimum Δ M r is reduced to only 0.6%, provided that a concomitant minimum Δp K=0.08 took place. When separating large peptides (human globin chains) in 100 m M Cys-A, 30% HFP and 7 M urea, the β-chain was found to co-elute with the α-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.