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Extraction, Purification and Sequencing of Adipokinetic/Red Pigment-Concentrating Hormone-Family Peptides

  • Gerd Gäde
Part of the Chromatographic Society Symposium Series book series (CSSS)

Summary

The adipokinetic and hypertrehalosaemic hormones of insects produced and stored in, and released from, the major neurohaemal organ of an insect, the corpora cardiaca, have been shown to be structurally related and belonging to what is now termed the adipokinetic hormone/red pigment-concentrating hormone peptide family (AKH/RPCH-family). Recently, we have elucidated the structures of new members of this family. Here a step-by-step account on the methods and techniques involved in the extraction, separation and sequencing of such peptides is given. The peptides have all been purified from corpus cardiacum material of different insect species. Various methods of extraction are compared. During isolation, bioassays in migratory locusts and American cockroaches were performed to monitor hyperlipaemic and hypertrehalosaemic effects. Separation from other compounds in the corpora cardiaca was achieved by a single-step method using reversed-phase high performance liquid chromatography (RP-HPLC). Examples are given for the use of HPLC systems in the isocratic or gradient mode. Separations on different support media are compared, and important features of detection are discussed. After separation, the amino acid composition of the purified peptides can be determined with pre- or post-column derivatization methods (phenylisothiocyanate or ninhydrin). Elucidation of the primary sequence by fast atom bombardment mass spectrometry or, after deblocking the pyroglutamate at the N-terminus, by gas-phase sequencing employing Edman degradation is outlined.

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Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • Gerd Gäde
    • 1
  1. 1.Institut für Zoologie, Lehrstuhl für TierphysiologieHeinrich-Heine-UniversitätDüsseldorf 1Germany

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