CAPILLARY ZONE ELECTROPHORESIS AT SUBZERO TEMPERATURES - SEPARATION OF THE CIS AND TRANS CONFORMERS OF SMALL PEPTIDES

The cis-trans conformers of two dipeptides, Phe-Pro and Leu-Pro, and t wo opioid heptapeptides containing one or two proline residues were se parated by capillary zone electrophoresis (CZE) in berate buffer at lo w temperatures down to -17 degrees C. Ar temperatures near ambient, th e relaxation time of the cis-trans isomerization is on the time-scale of minutes for the dipeptides and thus commensurate with the migration times in CZE under usual operating conditions. The conformers of both dipeptides could be separated with baseline resolution below 10 degre es C in neat aqueous 100 mM sodium berate (pH 8.4). The conformer peak s on the electropherograms were identified by using authentic samples of the cis and trans forms of Phe-Pro and Leu-Pro that were obtained b y reversed-phase HPLC at 0 degrees C, validated by NMR spectroscopy an d stored in liquid nitrogen. The interplay of the electrophoretic migr ation and on-column isomerization reaction in CZE of Phe-Pro under var ious conditions was analyzed in the light of the Damkohler number (Da) . The results showed that besides employing low temperature increasing the voltage and/or decreasing the capillary length also reduce the ma gnitude of Da to bring about the separation of interconverting species . In this work the use of low temperature in this work was preferred d ue to the experimental simplicity. The separation of cis-trans conform ers of two opioid heptapeptides was carried out by CZE at subzero temp eratures with aqueous sodium berate containing 23% (v/v) glycerol at p H 11.3 as measured with a glass electrode. The two conformers of Tyr- Pro-Phe-Asp-Val-Val-Gly-NH, were baseline separated at -12 degrees C a nd the four conformers of Tyr-Pro-Phe-Gly-Tyr-Pro-Ser-NH, due to the p resence of two peptidyl-proline bonds in the molecule, were also resol ved at -12 degrees C. From the electrophoretic mobilities, the hydrody namic radii of the cis-trans conformers of the dipeptides Phe-Pro and Leu-Pro were estimated. In both cases, the trans isomers had 1.3 times greater Stokes radii than the cir; conformers. This agrees with the o bserved migration order and molecular modeling results. The hydrodynam ic radii of the Phe-Pro conformers were smaller than those of the Leu- Pro isomers despite the lower molecular mass of the latter. The result s demonstrate that CZE is suitable for measuring certain molecular pro perties and suggest that the methods introduced here are applicable to the study of other systems of interconverting conformers.