The structural events associated with the binding of divalent cations to β-bungarotoxin

In order to address the mechanism why the Ca2+ was crucial for the manifestation of the phospholipase A2 (PLA2) activity of β-bungarotoxin (β-BuTx), four divalent cations were used to assess their influences on the catalytic activity and the fine structures of β-BuTx. Substitution Mg2+ or Sr2+ for Ca2+ in the substrate solution was found to cause a decrease in the PLA2 activity to approximately 15 or 6% of that in the presence of Ca2+. However, only marginally detectable PLA2 activity was observed with the addition of Ba2+. The nonpolarity of 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of β-BuTx markedly increased with the binding of cations to β-BuTx. The negative ellipticity noted with the CD spectra of β-BuTx increased upon the binding of cations too. With the exception of Ba2+, the order of the ability of cations to enhance the intensity of ANS fluorescence or increase the increment of negative ellipticity was Sr2+>Ca2+>Mg2+, which was the same order as the increase in their atomic radii. However, the energy transfer from Trp fluorescence emission to ANS was most effective upon the addition of Ca2+. Moreover, the extent of glutaraldehyde crosslinking between A chain and B chain decreased in the presence of cations. Nevertheless, the binding affinities of β-BuTx for the four cations were similar. These results, together with the findings that the ANS molecule binds at the active site of the A chain in β-BuTx, suggest that the binding of Ca2+ to β-BuTx induces subtly conformational changes occurred at the active site for exerting the activity of β-BuTx. Moreover, the change in the gross conformation induced by the binding of Ca2+ may affect the interaction between A chain and B chain, and consequently the activity of β-BuTx as well.