Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins
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.
KeywordsAmmodytoxin (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.
- 2.Halassy B, Habjanec L, Brgles M, Lang Balija M, Leonardi A, Kovačić L, Prijatelj P, Tomašić J, Križaj I (2008) The role of antibodies specific for toxix sPLA2s and haemorrhagins in neutralizing potential of antisera raised against Vipera ammodytes ammodytes venom. Comp Biochem Physiol, Part C: Toxicol Pharmacol 148:178–183CrossRefGoogle Scholar
- 3.Ritonja A, Ferlan I, Gubenšek F (1978) Initial studies of the primary structure of lethal phospholipase A from Vipera ammodytes venom. Period Biol 80(suppl 1):37–43Google Scholar
- 9.de Hoffmann E, Stroobant V (2007) Mass spectrometry, principles and applications, 3rd edn. Wiley, ChichesterGoogle Scholar
- 15.Saul FA, Prijatelj-Znidarsic P, Vulliez-le Normand B, Villette B, Raynal B, Pungercar J, Krizaj I, Faure G (2009) Comparative structural studies of two natural isoforms of ammodytoxin, phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity. J Struct Biol 169:360–369CrossRefGoogle Scholar
- 23.Maestro S (2009) LLC. New York, NYGoogle Scholar
- 25.Case DA, Darden TA, Cheatham TE III, Simmerling CL, Wang J, Duke RE, Luo R, Crowley M, Walker RC, Zhang W, Merz KM, Wang B, Hayik S, Roitberg A, Seabra G, Kolossváry I, Wong KF, Paesani F, Vanicek J, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Mathews DH, Seetin MG, Sagui C, Babin V, Kollman PA (2008) AMBER 10. University of California, San FranciscoGoogle Scholar