title: Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins.
abstract: 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.
authors.affiliation: Institute of Immunology, Zagreb, Croatia. mbrgles@gmail.com
authors.forename: Marija
authors.initials: M
authors.lastname: Brgles
authors.forename: Branimir
authors.initials: B
authors.lastname: Bertoša
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Winkler
authors.forename: Tihana
authors.initials: T
authors.lastname: Kurtović
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
authors.forename: Martina
authors.initials: M
authors.lastname: Marchetti-Deschmann
authors.forename: Beata
authors.initials: B
authors.lastname: Halassy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography / Q000379:methods*; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000633:toxicity; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010740:Phospholipases / Q000737:chemistry* / Q000302:isolation & purification* / Q000633:toxicity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014757:Viper Venoms / Q000737:chemistry* / Q000633:toxicity; D017819:Viperidae
chemical_list: D003039:Cobra Neurotoxin Proteins; D007527:Isoenzymes; D014757:Viper Venoms; D010740:Phospholipases