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 (A1%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 Ca2+-binding sites and two Zn2+-binding sites determined by atomic absorption spectrophotometer. Zn2+ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn2+ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)2 precipitate formation. Ca2+ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca2+ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu2+, Co2+, Mn2+ or Tb3+ and inhibited completely by Cd2+, is enhanced by Mg2+. The fluorescence intensity of the protein decreases in the presence of Cu2+, Co2+, Cd2+ or Mn2+, but neither for Ca2+, Mg2+ nor for Tb3+. Zn2+, Ca2+, Mg2+, Cu2+, Mn2+, Co2+ and Tb3+ have slight effects on its secondary structure contents. In addition, Cd2+ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.