Effects of metal ions on the conformation and activity of acutolysin D from Agkistrodon Acutus venom.

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A (1%) (280)) of acutolyisn D have been determined to be 47,850 +/- 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca(2+)-binding sites and two Zn(2+)-binding sites determined by atomic absorption spectrophotometer. Zn(2+) is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn(2+) significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)(2) precipitate formation. Ca(2+) is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca(2+) markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu(2+), Co(2+), Mn(2+) or Tb(3+) and inhibited completely by Cd(2+), is enhanced by Mg(2+). The fluorescence intensity of the protein decreases in the presence of Cu(2+), Co(2+), Cd(2+) or Mn(2+), but neither for Ca(2+), Mg(2+) nor for Tb(3+). Zn(2+), Ca(2+), Mg(2+), Cu(2+), Mn(2+), Co(2+ )and Tb(3+) have slight effects on its secondary structure contents. In addition, Cd(2+) causes a marked increase of antiparallel beta-sheet content from 45.5% to 60.2%.