Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain

https://doi.org/10.1016/j.jchromb.2006.10.043Get rights and content

Abstract

Reversed-phase high-performance liquid radio-chromatography (radio-HPLC) was set up to detect the time course of labeled degradation product formation of the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (5P), which has oostatic effects in different insect species. The detection limit of the system was in the range of 80–150 Bq. To follow formation of the degradation products, three amino acid residues in 5P were independently tritiated: Tyr1, Pro3 and Pro5. Each of the three tritiated peptides was analyzed after incubation with fresh hemolymph or ovaries of Neobellieria bullata. In the incubation mixture, free terminal amino acids and shortened sequences of 5P were identified. A metabolite of tyrosine represented the only exception; it was finally identified as water using degradation of [3H]Tyr by tyrosinase. Metabolic degradation of [3H]Tyr-5P was found to be considerably quicker than that of H-[3H]Tyr-Asp-Pro-Ala-OH (4P). The degradation of 5P was considerably slower in ovaries in comparison to hemolymph.

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 1a1e [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 (2a2c), carboxy (2d, 2e) or both the termini (2f2h) 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 2a2h (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.

References (14)

  • R. Tykva et al.

    J. Chromatogr. A

    (2004)
  • J. Slaninová et al.

    Bioorg. Chem.

    (2004)
  • F. Fezza et al.

    Anal. Biochem.

    (2005)
  • J. Hlaváček et al.

    Bioorg. Chem.

    (1998)
  • E. Kaiser et al.

    Anal. Biochem.

    (1970)
  • J. Stenersen

    Chemical Pesticides: Mode of Action and Toxicology

    (2004)
  • D. Borovsky

    J. Exp. Biol.

    (2003)
There are more references available in the full text version of this article.

Cited by (0)

The nomenclature and symbols of amino acids follow Recommendations of IUPAC/IUB Joint Commission on Biochemical Nomenclature (1984).

View full text