engleski

Isolation of ammodytoxin A from Vipera ammodytes ammodytes venom using two step chromatography

Ammodytoxins (Atxs) are phospholipases A2 acting as presynaptic neurotoxins. Three isoforms (A, B and C) have been identified in Vipera ammodytes ammodytes venom. The amount of Atxs in the venom correlates with venom's toxicity and immunogenicity. AtxA (the most toxic isoform) differs from AtxB in three and from AtxC in two amino acid residues, resulting in 2 and 33 Da mass differences. Isoelectric points of AtxA, AtxB and AtxC are 10.2, 10.0 and 9.4, respectively. Chromatography of pure Atx isoforms on CM-CIM disk under alkaline conditions resulted in separate elution of AtxA from AtxB and AtxC. We hypothesized that this allows the isolation of pure AtxA from crude venom so cation exchange chromatography of crude venom combined with affinity chromatography was performed, but this procedure did not result in isolation of pure AtxA. Mass spectrometry characterization (molecular mass determination of intact proteins, peptide mass fingerprinting and tandem mass spectrometry for sequence determination) of purified Atx fractions enabled identification of all three isoforms.

Ammodytoxins; CIM disk; Mass spectrometry

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