J. W. van Nispen

HPLC analysis of phenylthiocarbamyl (PTC) amino acids. I. Evaluation and optimization of the procedure

SummaryAn HPLC system has been developed in which 23 phenylthiocarbamyl (PTC) amino acids are distinctly separated. Applying this system, the preceding steps in HPLC amino acid analysis i.e. gas-phase hydrolysis of the protein/peptide sample and the pre-column derivatization, were evaluated and optimized. PTC amino acids in HPLC buffer solution are stable for at least 16 hours if stored at 5°–8°C. The sensitivity of the method is in the low pmol range.

Capillary electrophoresis of peptides : Analysis of adrenocorticotropic hormone-related fragments

Capillary electrophoresis can be used successfully to analyse small peptides to give additional information to that obtained using high-performance liquid chromatography (HPLC). The separation of a modified adrenocorticotropic hormone (4-9) fragment (Org 2766) and several of its fragments was investigated using capillary zone electrophoresis. Prediction of migration in aqueous systems using pKa-related data and the migration behaviour using sodium dodecyl sulphate in the buffer are discussed, as is the choice of buffer systems. The electrophoretic patterns are compared with the HPLC separation.

Structural modifications of the ACHT-(4–9) analog ORG 2766 yields peptides with high biological activity

The behavioral effects of two peptides (HOE 427) and ORG 31433) related to the ACTH-(4-9) analog ORG 2766 were investigated in Wistar rats in a number of tests in which Org 2766 is active. Subcutaneous administration of HOE 427 in a dose of 0.5 ng/kg or ORG 31433 in doses of 0.5-5.0 ng/kg facilitated passive avoidance behavior whereas these peptides attenuated the avoidance response in doses of 25 ng/kg and 250 ng/kg respectively. ORG 31433 (0.1 - 1.0 microgram/kg) decreased motor activity of group housed rats tested under low light conditions. Furthermore subcutaneous (1.0- 10.0 ng/kg) or oral (10 microgram/kg) administration of ORG 31433 accelerated functional recovery from 6-hydroxydopamine (6-OHDA)-induced lesions in the nucleus accumbens which cause motor hypoactivity. The experiments show that as compared to ORG 2766 the peptides HOE 427 and ORG 31433 induce qualitatively similar responses but are approximately 10 to 100 times more potent. These data may imply that substitution of the C-terminal COOH group of ORG 2766 yields neuropeptides with increased potency.

Complementary information from isotachophoresis and high-performance liquid chromatography in peptide analysis

Reversed-phase high-performance liquid chromatography is a valuable analytical technique to support the synthesis, isolation and purification of peptides, as is illustrated by some critical separations. In addition to this technique, capillary isotachophoresis can give useful information on the purity determination of peptides and on the presence of ionic compounds of a non-peptidic nature. With regard to the latter aspect, isotachophoresis proved to be a suitable technique as a check on the effective removal of salts after preparative high-performance liquid chromatography.

HPLC analysis of phenylthiocarbamyl (PTC) amino acids II. Application in the analysis of (poly)peptides

SummaryThe composition of 10 known (poly)peptides of widely different nature was determined by a previously optimized HPLC-PTC amino acid analysis; for the gasphase hydrolysis and the derivatization step use was made of a Pico-Tag Workstation. Results are in good agreement with those of the classical Moore and Stein technique.Strong points of the new method are its sensitivity which is in the low pmol range (nmol for the classical procedure), and its ease of use. The complete procedure can be carried out within one working day by applying an accelerated hydrolysis step.Finally, the (gas-phase) hydrolysis time of peptides containing IIe/Val sequences could be reduced to 24 hours when hydrolysis was carried out at a higher temperature.

Ion-pair high-performance liquid chromatography separation of β-endorphin-(6–17) from fourteen fragments

SummaryIn order to support metabolism studies of the proposed antipsychotic compound β-endorphin-(6–17), (Org 5878), the HPLC separation of this parent compound from fourteen peptide fragments was studied. The addition to the mobile phase of hydrophobic ion-pairing agents proved to be necessary to obtain adequate separation. The influence of the chromatographic parameters pH, type and concentration of the pairing agent, buffer concentration and temperature were investigated systematically. As a result the complete separation of Org 5878 and its fourteen fragments is reported.

Inhibition of [gamma]-endorphin generating endopeptidase activity of rat brain by peptides: Structure activity relationship

Abstract γ-Endorphin generating endopeptidase (γEGE) activity is an enzyme activity which converts β-endorphin into γ-endorphin and β-endorphin-(18–31). The inhibitory potency on γEGE activity of neuropeptides and analogues or fragments of neuropeptides was tested. Dynorphin-(1–13) (IC50: 0.14 μM), human β-endorphin-(1–31) (IC50: 15.5 μM), porcine ACTH-(1–39) (IC50: 6.3 μM), and substance P (IC50: 26 μM) had an inhibitory activity on γEGE activity. β-Endorphin-(18–31) (IC50: 0.35 μM) but not γ-endorphin potently inhibited γEGE activity. The IC50 of poly (Lys)40–60 was 0.8 μM. It is concluded that 1) γEGE activity is strongly inhibited by its product β-endorphin-(18–31), 2) the enzyme is strongly inhibited by peptides with an aromatic amino acid at the NH2-terminal and/or basic amino acids in the COOH-terminal of the peptide chain.

Abstracts of lectures symposium disposition and delivery of peptide drugs

ConclusionsA number of different processes clear peptides from the circulation. These will be described and illustrated using examples from a range of peptide hormones and analogues.