engleski

Structural characteristics of the local snake venom protein ammodytagin

Ammodytagin is a Zn2+-dependent metalloproteinase isolated from the venom of nose-horned viper (Vipera ammodytes ammodytes). Its molecular mass varies from 103 to 108 kDa, depending on the venom batch used for isolation. Ammodytagin exhibits strong hemorrhagic activity and and thus surely participates significantly in inducing local and systemic hemorrhages, the major pathological phenomena in humans after nose-horned viper bites. Partial amino acid sequencing by Edman degradation and MS/MS analysis identified sequences which classify this protein as metalloproteinase with disintegrin-like and cysteine-rich domains, which in addition to its heterodimeric nature allow classification of ammodytagin into the P-IIIc group of snake venom metalloproteinases (SVMPs).1 Te protein was found to have around 7.1 % of sugar content. Deglycosylation of ammodytagin with PNGase F resulted in a shift of both subunits in the SDS-PAGE gel indicating that both subunits are N-glycosylated. In order to gain more detailed information about sugar content of ammodytagin we used mass spectrometry (MS). MS measurements of ammodytagin after reduction, alkylation and/or de-N-glycosylation were hampered due to almost complete attachment of processed protein to the inner wall of the reaction tube which could not be overcome using a high content of organic solvent. Only a SDS containing buffer allowed the solubilization. Modification of the procedure and buffer content for reduction, alkylation and de-N-glycosylation resulted finally in satisfying MS signal revealing a large number of disulfide bridges in ammodytagin and heterogeneous N-glycosylation. Additional interesting feature of ammodytagin is its pI. Since it was isolated by cation exchange chromatography using basic conditions (pH 9.0) one would presume it to have a basic pI. Isoelectric focusing and non-equilibrium pH gradient electrophoresis revealed that ammodytagin has a basic pI (around 8) when native conditions are used but focuses around pH 6 when reduced with DTT or denaturated by urea. To investigate this uncommon result we tested binding of pure ammodytagin to both cation and anion exchanger and found that it binds to both. This indicates possible presence of surface patches of positive charge sufficient for binding to cation exchanger whereas overall charge is neutral to acidic. It has already been described that heterogeneous and asymmetric charge distribution over the exterior surface makes the net charge concepts in ion-exchange chromatography less relevant2. In addition, the influence of net charge and charge patches on binding of protein to the chromatographic stationary phase depends also on ligand density of the column material3. Detailed insight into ammodytagin structure will hopefully be elucidated by planned crystallization and X-ray structural studies.

ammodytagin; glycoprotein; mass spectrometry

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