engleski

Characterization of purified ammodytoxins isolated from the venom of Vipera ammodytes ammodytes

Snake venoms are deadly cocktails, each comprising unique mixtures of more than 100 peptides and proteins as well as other compounds. Snake venomics in combination with biochemical research is one of the best approaches to unravel and outline the full map of native toxins that constitute the venom. Detailed knowledge of the identity and relative amounts of the different toxins in the venom is required for generating immunization protocols to elicit toxin-specific antibodies with greater specificity and effectiveness than currently used in conventional systems. Long-nosed viper (Vipera ammodytes) is the most poisonous European snake and Vipera ammodytes ammodytes (V.a.a.) – a subspecies – inhabits also Croatia. Its bite is a serious event that requires immediate hospital care. Only recently ammodytoxins were identified as the key molecules for the design of in vitro tests for the prediction of venom toxicity as well as for the prediction of the antivenom neutralization capacity. Venoms contain a plenty of phospholipase A2 (PLA2) homologues whereof ammodytoxin A, B and C (AtxA, AtxB and AtxC) from V.a.a. venom have been isolated for further characterization on the molecular level. Although PLA2 is a class of relatively small proteins (approx. 13 kDa) characterization is very challenging as AtxA, AtxB and AtxC differ only in 3 (AtxA – AtxB) or 2 (AtxA – AtxC) amino acid residues. To confirm the identity of the isolated protein(s) (or to find new forms) MALDI-TOF-, MALDI-TOF/RTOF- and ESI-QTOF-MS was used. The molecular masses of the intact proteins were measured with high precision confirming theoretical values (deduced from genomic data) for AtxC but not for AtxA and AtxB, respectively. To determine the primary structures of the proteins experimental conditions for formic acid cleavage and tryptic digestion had to be optimized to obtain a high sequence coverage which is of utmost importance. In combination with off-line nano-LC peptide mass fingerprinting and confirming by MS/MS experiments resulted in a sequence coverage (and therefore sequence confirmation) for AtxA/AtxB and AtxC of 96% and 100%, respectively.

ammodytoxins; mass spectrometry; Vipera ammodytes ammodytes

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