engleski

Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins

Ammodytoxins (Atxs) are neurotoxic components of the Vipera ammodytes ammodytes (Vaa) snake venom. There are three Atx isoforms ; A, B and C, which differ in only 5 amino acid positions at C-terminus but differ substantially in their toxicity. The aim of this study was to establish an analytical method for unambiguous identification of all three isoforms and to use the method for analysis of purification procedure 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) from affinity column followed by cation exchange on CIM (Convective Interaction Media) disks. The monitoring of the purification procedure was performed by means of reversed phase chromatography (RPC) and mass spectrometry (MS). Although previous cation exchange of pure isoforms showed separate elution of AtxA from B and C, separation of AtxA from Atxs mixture was not accomplished. RPC was not able to separate Atx isoforms, whereas MS based approach proved to be more powerful. Peptides resulting from tryptic digest of Atxs that enable differentiation between 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. Affinity of Atxs to make homodimers and heterodimers of similar stability was confirmed by molecular modeling.

Ammodytoxin (Atx); MALDI mass spectrometry; molecular modeling; chromatography; convective interaction media (CIM)

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