208
S. MENGE et al.
pounds show up to 100 fold delayed isomerization rates compared to the corresponding oxopeptides, while the isomer ratio should not be affected by the substitution of oxygen by sulfur. The particularly extreme requirements of CZE of
peptide bond isomers resulted in the development of special methods. Moore and
Jorgenson (1995) describe the cis/trans isomer separation of proline containing
di- and oligopeptides (B-casomorphins) on extremely short capillaries with a
small inner diameter (6f!m) after derivatization with fluorescinisothiocyanate
and ultra short injection times (5-50 ms) and called this approach Fast-CZE. The
application of subzero temperatures was published by Ma et al.(1995). The separation of proline containing di- to hexapeptides was performed down to -17°C
with glycerol diminishing the viscosity of the borate buffer at such very low temperatures. Pep tides with two Xaa-Pro bonds could be resolved into a conformer
pattern of four peaks. The migration order cis before trans was confirmed by
accompanying RP-HPLC and molecular modeling studies.
The application of carboxymethylated B-Cyclodextrin (CMBCD) as additive to
the bulk electrolyte represents an approach to separate the cis/trans conformers
of prolyl peptides by inclusion complexation in CZE (Rathore and Horvath 1998).
In this way the complete resolution of angiotensin I conformers succeeded within
few minutes. A decrease of isomer mobilities with increasing CMBCD concentrations and a slight shift in the cis/trans ratio hint at the formation of peptidecyclodextrin complexes.
In conclusion, low temperature HPLC and CE supported by cavity interaction
has been found to be applicable in the detection of conformational changes in
proline containing peptides, for the determination of thermodynamic and kinetic
parameters of cis/trans interconversion and for the fractionation of cis and trans
conformers as valuable substrates for advanced studies. Shifts in equilibrium distribution of conformers by inclusion complexation or interactions to a hydrophobic, solid support have to be taken into consideration.
References
Beissinger M, Buchner J (1998) How chaperons fold proteins. BioI. Chern. 379: 245-259
Bohmer V (1995) Calixarene-Makrocyden mit (fast) unbegrenzten Moglichkeiten. Angew. Chern.
107: 785-818
Brandsch M, Thunecke F, Kiillertz G, Schutkowski M, Fischer G, Neubert K (1998) Evidence for the
absolute conformational specificity of the intestinal H+ Ipeptide symporter, PEPT!. J. BioI. Chern.
273: 3861-3864
Brandts )F, Halvorson HR, Brennan M (1975) Consideration of the possibility that the slow step in
protein denaturation is due to cis-trans isomerism of proline residues. Biochemistry 14: 4953-4963
Fischer G, Bang H, Mech C (1984) Nachweis einer Enzymkatalyse fUr die cis-trans Isomerisierung der
Peptidbindung in prolinhaltigen Peptiden. Biomed. Biochem. Acta 43: 11 04-1111
Fischer G (1994) Peptidyl-prolyl cis/trans isomerases and their effectors. Angew. Chern. Int. Ed. Engl.
33: 1415-1436
Friebe S, Krauss G), Nitsche H (1992) High-performance liquid chromatographic separation of cistrans isomers of proline containing peptides. I. Separation on cydodextrin silica. J. Chromatogr.
598: 139-145
Friebe S, Hartrodt B, Neubert K, Krauss G) (1994), High-performance liquid chromatographic separation of cis-trans isomers of proline containing peptides. II. Fractionation in different cydodextrin
systems. J. Chromatogr. A 661: 7-12
Friebe S, Gebauer S, Krauss G), Goermar G, Krueger) (1995) HPLC on calixarene bonded silica gels.
I. Characterization and applications of the p-tert-butyl-calix[4larene. ). Chromatogr. Sci. 33:
281-284
S. MENGE et al.
pounds show up to 100 fold delayed isomerization rates compared to the corresponding oxopeptides, while the isomer ratio should not be affected by the substitution of oxygen by sulfur. The particularly extreme requirements of CZE of
peptide bond isomers resulted in the development of special methods. Moore and
Jorgenson (1995) describe the cis/trans isomer separation of proline containing
di- and oligopeptides (B-casomorphins) on extremely short capillaries with a
small inner diameter (6f!m) after derivatization with fluorescinisothiocyanate
and ultra short injection times (5-50 ms) and called this approach Fast-CZE. The
application of subzero temperatures was published by Ma et al.(1995). The separation of proline containing di- to hexapeptides was performed down to -17°C
with glycerol diminishing the viscosity of the borate buffer at such very low temperatures. Pep tides with two Xaa-Pro bonds could be resolved into a conformer
pattern of four peaks. The migration order cis before trans was confirmed by
accompanying RP-HPLC and molecular modeling studies.
The application of carboxymethylated B-Cyclodextrin (CMBCD) as additive to
the bulk electrolyte represents an approach to separate the cis/trans conformers
of prolyl peptides by inclusion complexation in CZE (Rathore and Horvath 1998).
In this way the complete resolution of angiotensin I conformers succeeded within
few minutes. A decrease of isomer mobilities with increasing CMBCD concentrations and a slight shift in the cis/trans ratio hint at the formation of peptidecyclodextrin complexes.
In conclusion, low temperature HPLC and CE supported by cavity interaction
has been found to be applicable in the detection of conformational changes in
proline containing peptides, for the determination of thermodynamic and kinetic
parameters of cis/trans interconversion and for the fractionation of cis and trans
conformers as valuable substrates for advanced studies. Shifts in equilibrium distribution of conformers by inclusion complexation or interactions to a hydrophobic, solid support have to be taken into consideration.
References
Beissinger M, Buchner J (1998) How chaperons fold proteins. BioI. Chern. 379: 245-259
Bohmer V (1995) Calixarene-Makrocyden mit (fast) unbegrenzten Moglichkeiten. Angew. Chern.
107: 785-818
Brandsch M, Thunecke F, Kiillertz G, Schutkowski M, Fischer G, Neubert K (1998) Evidence for the
absolute conformational specificity of the intestinal H+ Ipeptide symporter, PEPT!. J. BioI. Chern.
273: 3861-3864
Brandts )F, Halvorson HR, Brennan M (1975) Consideration of the possibility that the slow step in
protein denaturation is due to cis-trans isomerism of proline residues. Biochemistry 14: 4953-4963
Fischer G, Bang H, Mech C (1984) Nachweis einer Enzymkatalyse fUr die cis-trans Isomerisierung der
Peptidbindung in prolinhaltigen Peptiden. Biomed. Biochem. Acta 43: 11 04-1111
Fischer G (1994) Peptidyl-prolyl cis/trans isomerases and their effectors. Angew. Chern. Int. Ed. Engl.
33: 1415-1436
Friebe S, Krauss G), Nitsche H (1992) High-performance liquid chromatographic separation of cistrans isomers of proline containing peptides. I. Separation on cydodextrin silica. J. Chromatogr.
598: 139-145
Friebe S, Hartrodt B, Neubert K, Krauss G) (1994), High-performance liquid chromatographic separation of cis-trans isomers of proline containing peptides. II. Fractionation in different cydodextrin
systems. J. Chromatogr. A 661: 7-12
Friebe S, Gebauer S, Krauss G), Goermar G, Krueger) (1995) HPLC on calixarene bonded silica gels.
I. Characterization and applications of the p-tert-butyl-calix[4larene. ). Chromatogr. Sci. 33:
281-284