title: Spectroscopic investigation of calcium binding sites in the neurotoxin vipoxin and its components-relation with the X-ray structure.
abstract: Vipoxin is a neurotoxin from the venom of Vipera ammodytes meridionalis, the most toxic snake in Europe. It is a unique complex of a toxic phospholipase A2 (PLA2) and a non-toxic PLA2-like protein inhibitor (Inh) which probably evolved from the enzyme and reduces its activity and toxicity. The enzymatic activity of Vipoxin is Ca2+-dependent and the interaction of this metal ion with the neurotoxic complex and its separated components was investigated using the fluorescent probe ANS. Vipoxin binds two calcium ions, one per each subunit. The X-ray model of the Ca2+-free neurotoxin shows that the potential metal-binding sites require minor structural changes to bind calcium. The dissociation constants K(2+)Ca of the calcium complexes of Vipoxin and its components, PLA2 and Inh, were determined to be 16, 10 and 9 mM, respectively. The affinity for calcium of Vipoxin is reduced in comparison to those of PLA2 and Inh. The X-ray model shows that the potential Ca2+-binding sites in the two components are partially 'shielded' in the complex. The affinity of the neurotoxin to Sr2+ and Ba2+ is lower and the respective K(2+)Ca are 20 and 30 mM. The saturation of Ca2+-binding sites increased the melting point Tm of Vipoxin by 11 degrees C and the activation energy for the thermal deactivation of the excited tryptophans Ea by 11 kJ mol(-1) x Ca2+ is important not only for the enzymatic activity of Vipoxin but also for its thermostability.
authors.affiliation: Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia.
authors.forename: D N
authors.initials: DN
authors.lastname: Georgieva
authors.forename: C
authors.initials: C
authors.lastname: Betzel
authors.forename: B
authors.initials: B
authors.lastname: Aleksiev
authors.forename: N
authors.initials: N
authors.lastname: Genov
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014757:Viper Venoms / Q000737:chemistry*; D017819:Viperidae
chemical_list: D014757:Viper Venoms; C033134:vipoxin; D002118:Calcium