Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain☆
Introduction
In comparison to pesticides [1], oligopeptides may have advantages in insect pest control. Apart from easier synthesis and solubility in water, they cause considerably less or no pollution of the subsurface environment [2].
Our investigation proved the deteriorating effect of the decapeptide H-Tyr-Asp-Pro-Ala-Pro6-OH (10P) [3] isolated from the mosquito Aedes aegypti by Borovsky [4] on ovarian development (i.e. oostatic effect) of species Diptera, Orthoptera and Hemiptera [5]. The highest effect was found for C-terminally truncated analogues H-Tyr-Asp-Pro-Ala-Pro-OH (5P) and H-Tyr-Asp-Pro-Ala-OH (4P). It was shown previously [3] that after their application the hatchability was lower compared to controls and the development of egg chambers of the second gonadotrophic cycle was pathologically modified. The nuclei of follicular cells formed a multinuclear layer, which proliferated towards the inner part of the egg chamber. Such eggs were not able to complete their development and were later resorbed. To enrich our knowledge about the fate of these oostatic peptides after application to Neobellieria bullata (Diptera), metabolic degradation of 5P was analyzed in this paper.
A highly sensitive radio-HPLC [2], [6], [7] was selected to quantify small amounts of 5P metabolites in the presence of a relatively high background of other organic compounds. Three radiolabeled derivatives of 5P were prepared for this study. Selective tritiation of tyrosine and proline residues in the peptide sequence made it possible to resolve the time course of production of the individual degradation products. Radio-HPLC also enabled an analysis of a tyrosine metabolite found in the mixture of degradation products.
Previously, synthesis and characterization of precursors of the tritiated peptides, i.e. 3,4-dehydroproline analogues 1a–1e [8] and of the standards 4P and 5P [9] used also for tritiation 1f and 1g, have been described (Table 1).
In addition to the tritiated oostatic peptides, their non-labeled fragments truncated in the amino (2a–2c), carboxy (2d, 2e) or both the termini (2f–2h) were synthesized as standards for HPLC study (Table 2). As standards corresponding to the carboxy- and amino-terminus, amino acids proline and tyrosine were used, respectively.
Section snippets
Materials and methods
Tyrosine: C9H11NO3 (181.19), >99% (non-aqueous titration) [α]D20 − 11.5° (0.04 g/ml of 1N HCl) and proline: C5H9NO2 (115.13), >99% (non-aqueous titration) [α]D20 − 84.5 (0.05 g/ml of water) were obtained from Fluka Chemie AG (Buchs, Switzerland). Fmoc-Asp(OtBu)-OH: C23H25NO6 (411.5), 99.8%, mp 147–148 °C, [α]D20 − 23.8 (0.01 g/ml of DMF), 0.1% d-enantiomer and Fmoc-Tyr(tBu)-OH: C28H29NO5 (459.5), 99.9%, mp 151–152 °C, [α]D20 − 29.4 (0.01 g/ml of DMF), 0.1% d-enantiomer were purchased from Senn Chemicals AG
Peptide standards
The non-labeled peptides 2a–2h (Table 2) synthesized for the purpose of this study were characterized by different analytical methods (Table 4). Their qualities entitled them to be used as standards for identification of the radiochromatographic fractions (Fig. 1).
Radio-HPLC analysis
Radio-HPLC of tritiated pentapeptides was performed after 1, 30 and 60 min of incubation alone or with the hemolymph or ovaries. The samples were thawed and centrifuged for 5 min in an Eppendorf centrifuge, and an aliquot of the
Conclusion
The experiments showed the convenience of radio-HPLC for monitoring of the degradation of peptides in biological fluids. Radiolabeling in different positions of the peptide chain allows to determine the decisive step of the degradation; in our case, it is the splitting off the C-terminal proline from the pentapeptide 5P. In comparison to the LC/MS analysis of metabolites used in our previous paper [3], the radio-HPLC gives more precise and quite unambiguous results. The results also support our
Acknowledgement
This research was carried out under the project Z4 055 0506 and was supported by grants of the Czech Science Foundation Nos. 203/02/0247 and 203/06/1272.
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The nomenclature and symbols of amino acids follow Recommendations of IUPAC/IUB Joint Commission on Biochemical Nomenclature (1984).