Oxidation Products of Tryptophan and Proline in Adipokinetic Hormones-Artifacts or Post-Translational Modifications?

Adipokinetic hormones (AKHs) regulate important physiological processes in insects. AKHs are short peptides with blocked termini and Trp in position 8. Often, proline occupies position 6. Few post-translational modifications have been found, including hydroxyproline ([Hyp⁶]) and kynurenine. Our recent data suggest that the Hyp- and Kyn-containing AKHs occur more often than originally thought and we here investigate if they are natural or artifactual.

From crude extracts of the corpora cardiaca (CC) of various insect species, AKHs were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS). Synthetic [Hyp⁶]-AKHs were tested in an in vivo metabolic assay. Freshly dissected Periplaneta americana and Blaberus atropos CCs (with precautions taken against oxidation) were analyzed. B. atropos CC were placed into a depolarizing saline and the released AKHs were measured.

Hyp was detected in several decapeptides from cockroaches. The modified form accompanied the AKH at concentrations below 7%. The [Hyp⁶]-AKHs of B. atropos were present in fresh CC preparations and were shown to be releasable from the CC ex vivo. Synthetic [Hyp⁶]-containing peptides tested positively in a hypertrehalosemic bioassay. Hydroxyprolination was also detected for Manto-CC from the termite Kalotermes flavicollis and for Tetsu-AKH of the grasshopper, Tetrix subulata. Oxidized Trp-containing forms of Nicve-AKH were found in species of the burying beetle genus Nicrophorus.

Trp oxidation is known to occur easily during sample handling and is likely the reason for the present findings. For hydroxyprolination, however, the experimental evidence suggests endogenous processes.