UTILITY OF HIGH-RESOLUTION CAPILLARY ELECTROPHORESIS FOR MONITORING PEPTIDE HOMO-DIMER AND HETERO-DIMER FORMATION

The monomer and disufide-linked homo-dimer of two different peptides, one with an amino-terminal cysteine, the other with a cysteine at the carboxy-terminal, are shown to be baseline resolved by capillary electrophoresis in less than 15 min. Time-course for homo-dimer formation with both peptides, either under mild (air) or stronger (hydrogen peroxide oxidizing conditions, was easily monitored. Confirmation that the second peak appearing under oxidizing conditions was indeed the homo-dimer was obtained with mass spectrometry. The possibility that stronger oxidizing conditions led to the production of the sulfonic acid derivative of the monomeric peptide, was ruled out through generation of the derivative by performic acid oxidation. As expected, the negative charge of the sulfonic acid moiety gives the peptide a slower electrophoretic mobility than both the monomer and the dimer. Moreover, as would be expected with a sulfonic acid derivative, oxidation to the dimeric form was not possible. This was consistent with the observation that the homo-dimer peak could be reduced to monomeric form in the presence of dithiotreitol. Co-oxidization of the amino- and carboxy-terminal peptides led to the expected production of both homo-dimers and the hetero-dimer, all of which were resolved.