200
S. MENGE et al.
The partial double bond character and the energy barriers result in a delay of
the cis/trans isomerization with relaxation times of 10-1000 sec- 1 so that analytical methods with corresponding time scales of measurement are able to detect
both isomers in their aqueous solution. Quantitative data, describing this type of
conformational changes, can be determined by spectroscopic and kinetic methods (Fischer et al. 1984, Hubner et al. 1990).
Several papers report on the use of proline containing oligopeptides as model
substrates for the determination of the catalytic activity of PPI'ases from different natural sources. Peptide isomers can be used as models to study different biological processes including protein folding/refolding (Brandts et al. 1975),
immune response (Fischer et al. 1989, Schreiber 1991) or the assembly ofhormon
receptor complexes (Schmidt et al. 1995, Owens-Grillo et al. 1995).
New studies are published on the introduction of a protease-free assay for the
determination of the time course of cis/trans isomerization on the basis of different UV/vIS spectra of the conformational states (Kullertz et al. 1998, Janowski et
al. 1997). Special methods of NMR spectroscopy were used to study the cis/trans
ratio of different peptide structures and rates of isomerization depending on different conditions such as pH, as well as the ionization grade of substrates, the
role of phosphorylation, the content of organic solvent in the aqueous solution or
the interaction to micelles (Hubner et al. 1990, Kramer and Fischer 1997, Schutkowski et al. 1998).
In order to study whether rate and ratios of cis/trans isomerization may effect
biological processes high resolution chromatographic and electrophoretic methods have been applied, too. HPLC and CZE are suitable separation techniques
allowing the isolation and identification of peptide conformers.
2
HPLC of cis/trans Isomers of Proline Containing Peptides
2.1
Commonly Applied Separation Principles
In the early eighties, Melander and Horvath (1982) first reported on the appearance of a secondary equilibrium during the RP-HPLC of proline containing peptides. They observed that peak splitting and deformity of unprotected peptides
including proline in a C-terminal position are attributed to the dynamic interconversion of peptide bond isomers. During the chromatographic run the cis/
trans isomerization of prolyl peptides interferes with the second dynamic equilibrium: the distribution of both conformers between the mobile and the stationary phases (Kalman et al. 1996). This interference of the two dynamic processes
leads not only to an insufficient resolution of the isomer peaks, but also to an
influence on the rate of interconversion and the cis to trans ratio depending on
the operation conditions.
In several papers the fundamentals for the RP-HPLC of interconverting species
have been studied. They succeeded in the isomer separation of oligopeptides only
on the basis of slight differences in the overall hydrophobicity of both conformational species. Optimum separation of cis/trans isomers for given prolyl pep tides
S. MENGE et al.
The partial double bond character and the energy barriers result in a delay of
the cis/trans isomerization with relaxation times of 10-1000 sec- 1 so that analytical methods with corresponding time scales of measurement are able to detect
both isomers in their aqueous solution. Quantitative data, describing this type of
conformational changes, can be determined by spectroscopic and kinetic methods (Fischer et al. 1984, Hubner et al. 1990).
Several papers report on the use of proline containing oligopeptides as model
substrates for the determination of the catalytic activity of PPI'ases from different natural sources. Peptide isomers can be used as models to study different biological processes including protein folding/refolding (Brandts et al. 1975),
immune response (Fischer et al. 1989, Schreiber 1991) or the assembly ofhormon
receptor complexes (Schmidt et al. 1995, Owens-Grillo et al. 1995).
New studies are published on the introduction of a protease-free assay for the
determination of the time course of cis/trans isomerization on the basis of different UV/vIS spectra of the conformational states (Kullertz et al. 1998, Janowski et
al. 1997). Special methods of NMR spectroscopy were used to study the cis/trans
ratio of different peptide structures and rates of isomerization depending on different conditions such as pH, as well as the ionization grade of substrates, the
role of phosphorylation, the content of organic solvent in the aqueous solution or
the interaction to micelles (Hubner et al. 1990, Kramer and Fischer 1997, Schutkowski et al. 1998).
In order to study whether rate and ratios of cis/trans isomerization may effect
biological processes high resolution chromatographic and electrophoretic methods have been applied, too. HPLC and CZE are suitable separation techniques
allowing the isolation and identification of peptide conformers.
2
HPLC of cis/trans Isomers of Proline Containing Peptides
2.1
Commonly Applied Separation Principles
In the early eighties, Melander and Horvath (1982) first reported on the appearance of a secondary equilibrium during the RP-HPLC of proline containing peptides. They observed that peak splitting and deformity of unprotected peptides
including proline in a C-terminal position are attributed to the dynamic interconversion of peptide bond isomers. During the chromatographic run the cis/
trans isomerization of prolyl peptides interferes with the second dynamic equilibrium: the distribution of both conformers between the mobile and the stationary phases (Kalman et al. 1996). This interference of the two dynamic processes
leads not only to an insufficient resolution of the isomer peaks, but also to an
influence on the rate of interconversion and the cis to trans ratio depending on
the operation conditions.
In several papers the fundamentals for the RP-HPLC of interconverting species
have been studied. They succeeded in the isomer separation of oligopeptides only
on the basis of slight differences in the overall hydrophobicity of both conformational species. Optimum separation of cis/trans isomers for given prolyl pep tides