Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis

Yoshiaki Nakagawa, Martin Sadilek, Elisabeth Lehmberg, Rafael Herrmann, Revital Herrmann, Haim Moskowitz, Young Moo Lee, Beth Ann Thomas, Ryo Shimizu, Masataka Kuroda, A. Daniel Jones, Bruce D. Hammock

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 μg against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins.

Original languageEnglish (US)
Pages (from-to)53-65
Number of pages13
JournalArchives of Insect Biochemistry and Physiology
Volume38
Issue number2
StatePublished - 1998

Keywords

  • AaIT
  • AaIT5
  • Heliothis virescens
  • HPLC
  • Mass spectrometry
  • Molecular modeling
  • Scorpion toxin

ASJC Scopus subject areas

  • Insect Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physiology
  • Physiology (medical)

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  • Cite this

    Nakagawa, Y., Sadilek, M., Lehmberg, E., Herrmann, R., Herrmann, R., Moskowitz, H., Lee, Y. M., Thomas, B. A., Shimizu, R., Kuroda, M., Jones, A. D., & Hammock, B. D. (1998). Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis. Archives of Insect Biochemistry and Physiology, 38(2), 53-65.