Spectroscopic investigation of calcium binding sites in the neurotoxin Vipoxin and its components-relation with the X-ray structure

Vipoxin is a neurotoxin from the venom of Vipera ammodytes meridionalis, th e most toxic snake in Europe. It is a unique complex of a toxic phospholipa se A(2) (PLA(2)) and a non-toxic PLA(2)-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 thi s metal ion with the neurotoxic complex and its separated components was in vestigated using the fluorescent probe ANS. Vipoxin binds two calcium ions, one per each subunit. The X-ray model of the Ca2+-free neurotoxin shows th at the potential metal-binding sites require minor structural changes to bi nd calcium. The dissociation constants K-Ca(2+) of the calcium complexes of Vipoxin and its components, PLA(2) and Inh, were determined to be 16, 10 a nd 9 mM, respectively. The affinity for calcium of Vipoxin is reduced in co mparison to those of PLA(2) and Inh. The X-ray model shows that the potenti al 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-Ca(2+) are 20 and 30 mM. The saturation of Ca2+-binding sites increased the melting point T-m of Vipoxin by 11 degreesC and the activatio n energy for the thermal deactivation of the excited tryptophans E-a by 11 kJ mol(-1). Ca2+ is important not only for the enzymatic activity of Vipoxi n but also for its thermostability. (C) 2000 Elsevier Science B.V. All righ ts reserved.