Frank Thunecke

Kinetic study on the cis-trans isomerization of peptidyl-proline dipeptides

The equilibrium and kinetic parameters of cis-trans interconversion of dipeptides containing peptidyl-proline moiety were investigated using the in-column incubation method with both CZE and HPLC and the ad hoc dissolution method. The use of the latter was possible because the conformational make-up of the solid peptides, and consequently of their ad hoc solution, was sufficiently different from that of the solution at equilibrium. This method with 1H-NMR and CZE analyses yielded very similar results for the cis-trans isomerization of Phe Pro in aqueous sodium borate, pH 8.4, at 10 degrees C with an average value of 0.34 and 6.6 x 10(-5) s-1 for the equilibrium and rate constant, respectively. The in-column incubation method is performed by CZE or HPLC so that the conformers are separated in the first half of the column and then incubated in column where they interconvert and reach equilibrium. Subsequent separation in the second half of the column yielded four peaks. Thus by measuring the conformer composition as a function of the reaction time, the rate constant can be evaluated. The forward rate constant of 1.42 x 10(-4) s-1 determined by the CZE in-column incubation method for Phe-Pro isomerization at 10 degrees C was twice of the value obtained by the ad hoc dissolution method. It is believed that the inner wall of fused-silica capillaries has a catalytic effect on the isomerization. Computer simulation was also employed to gain further insight on the catalytic activity of the capillary inner wall on such isomerization. Whereas the experimental and simulation profiles of Phe-Pro in aqueous borate buffer, pH 8.4, with a 37 cm long capillary were in excellent agreement, a four times faster interconversion rate had to be used to match the experimental profile obtained with a 57 cm long capillary under otherwise identical conditions. The catalytic effect of the octadecyl silica stationary phase on the isomerization was confirmed by the in-column incubation method with HPLC. The overall rate of the cis-trans isomerization of Phe-Pro, which entails the reaction both on the stationary phase and in the mobility phase, was about six times faster at 0 degree C than the rate measured by NMR in free solution using the mobile phase containing 65% (v/v) sodium phosphate, pH 6.5, and 35% (v/v) methanol. The results presented here serve as a caveat that the effect of the wall in CZE or the stationary phase in HPLC on the reaction cannot be ignored.

Interaction of cyclophilin and cyclosporins monitored by affinity capillary electrophoresis

The affinity capillary electrophoretic separation of the complex of the enzyme cyclophilin (Cyp) with the immunosuppressive drug cyclosporin A (CsA) from uncomplexed Cyp and CsA in phosphate buffer (pH 8) under non-denaturing conditions by equilibrium-mixture analysis is reported. Using a new approach combining mobility-shift analysis and electrophoretically mediated microanalysis the binding constant of rhCyp18 to CsA and derivatives was estimated.

Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods

A screening procedure for protein‐protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophoresis (ACE) was established. GFP was fused as a fluorescent indicator to the C‐terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster. Cyclophilins (Cyps) belong to the ubiquitously distributed enzyme family of peptidyl‐prolyl cis/trans isomerases (PPIases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPIase activity of the GFP fused rDmCyp20 as well as the high affinity to CsA remain intact. Using native gel electrophoresis and ACE mobility‐shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protein p24 of HIV‐1 was detectable in the case of rDmCyp20 fused to the fluorescent tag. For the p24 / rDmCyp20‐GFP binding an ACE method was established which allowed to determine a dissociation constant of Kd = 20 ± 1.5×10−6 M. This result was verified by size‐exclusion chromatography and is in good agreement with published data for the nonfused protein. Moreover the fusion protein was utilized to screen rDmCyp20‐protein interactions by capillary electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility‐shift assays using native gel electrophoresis with fluorescence imaging and ACE with laser‐induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for moderate protein‐protein interactions in biological samples.

Investigations of cyclophilin interactions with oligopeptides containing proline by affinity capillary electrophoresis.

Affinity capillary electrophoresis using mobility-shift analysis was utilized to characterize the binding of peptide ligands to cyclophilins, which are members of the enzyme family of peptidyl-prolyl cis/trans isomerases. Peptides derived from the human immunodeficiency virus capsid protein p24 exhibited different affinities to the isoenzymes cyclophilin18 and cyclophilin20. For the interaction of the peptide hormone bradykinin with cyclophilin18, a dissociation constant of 1.4 +/- 0.1 mM was determined. Finally, the affinity of cyclophilin20 to peptides from a cellulose-bound peptide library scanning the sequence of Drosophila melanogaster protein cappuccino was investigated. The affinities of selected peptides to cyclophilin20 and a green fluorescent fusion protein with cyclophilin20 were compared.

119Sn-NMR-spektroskopische Untersuchungen an Tri-n-butylzinnderivaten von N-Acetylaminosäuren

SummaryEleven compounds have been prepared by azeotropic destillation of water from toluene solutions of bis(tri-n-butyltin)oxide and N-acetyl amino acids. All derivatives are white solids.119Sn-NMR-spectra of the tri-n-butyltin compounds have been studied in coordinating and non coordinating solvents. The chemical shifts and the coupling constants1J(119Sn,13C) depend significantly on the coordination number of the tin atom and on the properties of the substituents. The data for the compounds are discussed in comparison with those for other tri-n-butyltin compounds.

Characterization of alcohols, thiols and carboxylic acids in mixtures using the 119Sn-chemical shift of their tri-n-butyltin derivatives

SummaryMixtures of tri-n-butyltin thiolates, -alcoholates and -carboxylates were investigated. A characterization of diastereomeric compounds using their 119Sn-NMR-spectra was of special interest. It is shown that 119Sn-NMR spectroscopy is a useful method for the analysis of these mixtures.