title: Capillary zone electrophoresis at subzero temperatures. I. Separation of the cis and trans conformers of small peptides.
abstract: The cis-trans conformers of two dipeptides, Phe-Pro and Leu-Pro, and two opioid heptapeptides containing one or two proline residues were separated by capillary zone electrophoresis (CZE) in borate buffer at low temperatures down to -17 degrees C. At temperatures near ambient, the 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 degrees C in neat aqueous 100 mM sodium borate (pH 8.4). The conformer peaks on the electropherograms were identified by using authentic samples of the cis and trans forms of Phe-Pro and Leu-Pro that were obtained by reversed-phase HPLC at 0 degree C, validated by NMR spectroscopy and stored in liquid nitrogen. The interplay of the electrophoretic migration and on-column isomerization reaction in CZE of Phe-Pro under various conditions was analyzed in the light of the Damköhler number (Da). The results showed that besides employing low temperature increasing the voltage and/or decreasing the capillary length also reduce the magnitude of Da to bring about the separation of interconverting species. In this work the use of low temperature in this work was preferred due to the experimental simplicity. The separation of cis-trans conformers of two opioid heptapeptides was carried out by CZE at subzero temperatures with aqueous sodium borate containing 23% (v/v) glycerol at pH* 11.3 as measured with a glass electrode. The two conformers of Tyr-Pro-Phe-Asp-Val-Val-Gly-NH2 were baseline separated at -12 degrees C and the four conformers of Tyr-Pro-Phe-Gly-Tyr-Pro-Ser-NH2 due to the presence of two peptidyl-proline bonds in the molecule, were also resolved at -12 degrees C. From the electrophoretic mobilities, the hydrodynamic 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 cis conformers. This agrees with the observed migration order and molecular modeling results. The hydrodynamic radii of the Phe-Pro conformers were smaller than those of the Leu-Pro isomers despite the lower molecular mass of the latter. The results demonstrate that CZE is suitable for measuring certain molecular properties and suggest that the methods introduced here are applicable to the study of other systems of interconverting conformers.
authors.affiliation: Department of Chemical Engineering, Yale University, New Haven, CT 06520, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ma
authors.forename: F
authors.initials: F
authors.lastname: Kálmán
authors.forename: A
authors.initials: A
authors.lastname: Kálmán
authors.forename: F
authors.initials: F
authors.lastname: Thunecke
authors.forename: C
authors.initials: C
authors.lastname: Horváth
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003080:Cold Temperature; D004151:Dipeptides / Q000302:isolation & purification; D019075:Electrophoresis, Capillary; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000302:isolation & purification*; D011487:Protein Conformation; D013237:Stereoisomerism; D014783:Viscosity
chemical_list: D004151:Dipeptides; D009842:Oligopeptides