Analytical and Bioanalytical Chemistry

, Volume 402, Issue 9, pp 2737–2748 | Cite as

Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins

  • Marija Brgles
  • Branimir Bertoša
  • Wolfgang Winkler
  • Tihana Kurtović
  • Günter Allmaier
  • Martina Marchetti-Deschmann
  • Beata Halassy
Original Paper


The ammodytoxins (Atxs) are neurotoxic phospholipases which occur in Vipera ammodytes ammodytes (Vaa) snake venom. There are three Atx isoforms, A, B, and C, which differ in only five amino acid positions at the C-terminus but differ substantially in their toxicity. The objective of this study was to establish an analytical method for unambiguous identification of all three isoforms and to use the method to assess a procedure for purification of the most toxic phospholipase, AtxA, from the venom. Isolation procedure for AtxA consisted of isolation of Atx-cross-reactive material (proteins recognized by anti-Atx antibodies), by use of an affinity column, then cation exchange on CIM (Convective Interaction Media) disks. The purification procedure was monitored by means of reversed-phase chromatography (RPC) and mass spectrometry (MS). Although previous cation exchange of the pure isoforms enabled separate elution of AtxA from B and C, separation of AtxA from Atxs mixture was not accomplished. RPC was not able to separate the Atx isoforms, whereas an MS based approach proved to be more powerful. Peptides resulting from tryptic digestion of Atxs which enable differentiation between the three isoforms were successfully detected and their sequences were confirmed by post-source decay (PSD) fragmentation. Separation of Atx isoforms by ion-exchange chromatography is most presumably prevented by Atxs heterodimer formation. The tendency of Atxs to form homodimers and heterodimers of similar stability was confirmed by molecular modeling.


Ammodytoxin (Atx) MALDI mass spectrometry Molecular modeling Chromatography Convective Interaction Media (CIM) 



The authors thank Professor I. Križaj for providing pure Atxs. This work was supported by the Bilateral Cooperation Grant Croatia–Austria (HR 13/2010) and the Croatian Ministry of Science, Education, and Sports (grants 021-0212432-2033, 098-1191344-2860). CIM disks were kindly provided by BIA Separations.

Supplementary material

216_2012_5754_MOESM1_ESM.pdf (315 kb)
ESM 1 (PDF 314 kb)


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Marija Brgles
    • 1
  • Branimir Bertoša
    • 2
  • Wolfgang Winkler
    • 3
  • Tihana Kurtović
    • 1
  • Günter Allmaier
    • 3
  • Martina Marchetti-Deschmann
    • 3
  • Beata Halassy
    • 1
  1. 1.Institute of ImmunologyZagrebCroatia
  2. 2.Rudjer Bošković InstituteZagrebCroatia
  3. 3.Institute of Chemical Technologies and AnalyticsVienna University of TechnologyViennaAustria

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