Chromatography,mass spectrometry,and molecular modeling studies on ammodytoxins |
| |
Authors: | Email author" target="_blank">Marija?BrglesEmail author Branimir?Berto?a Wolfgang?Winkler Tihana?Kurtovi? Günter?Allmaier Martina?Marchetti-Deschmann Beata?Halassy |
| |
Institution: | (1) Institute of Immunology, 10000 Zagreb, Croatia;(2) Rudjer Bošković Institute, 10000 Zagreb, Croatia;(3) Institute of Chemical Technologies and Analytics, Vienna University of Technology, 1040 Vienna, Austria |
| |
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. |
| |
Keywords: | |
本文献已被 PubMed SpringerLink 等数据库收录! |
|