RAPID PURIFICATION AND MOLECULAR MODELING OF AALT PEPTIDES FROM VENOMOF ANDROCTONUS-AUSTRALIS

As recombinant viruses expressing scorpion toxins are moving closer to ward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological act ivity 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 s peed of the purification by employing a preparative reversed-phase col umn. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtai ned from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect s elective 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 wi th a dose of less than 1.3 mu g against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification pro cess, we implemented mass spectrometry in addition to bioassays to mon itor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectr ometric screening can unambiguously follow the purification process an d can greatly facilitate and expedite the downstream purification of A aIT and AaIT5 eliminating the number of bioassays required. Further, e lectrospray ionization was compared with matrix-assisted desorption/io nization and evaluated as a method of choice for mass spectrometric ch aracterization of fractions from the venom purification for it provide d higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant t oxin LqhIT2. Three-dimensional structure of AaIT5 was compared with st ructures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins. (C) 1998 Wiley-Liss, Inc.