Effects of metal ions and an inhibitor on the fluorescence and activity of acutolysin A from Agkistrodon acutus venom

Abstract

Acutolysin A, a protein isolated from the venom of Chinese Five-pace snake (Agkistrodon acutus) has shown marked hemorrhagic and proteolytic activities. In the present study, the effects of metal ions and an inhibitor EDTA on the fluorescence and function of autolysin A have been studied, by following fluorescence and activity measurements. Acutolysin A contains a Ca²⁺-binding site, which provides it with important structural stability, and a Zn²⁺-binding site, which is essential for its enzymatic activities. The removal of metal ions in acutolysin A by incubation with EDTA results in irreversible inhibition and complete denaturation, and a marked decrease in its fluorescence intensity. The fluorescence intensity of acutolysin A is also decreased in the presence of Cu²⁺, Co²⁺, Mn²⁺ or Mg²⁺, but does not change in the presence of Ca²⁺, Cd²⁺, or Tb³⁺. Caseinolytic activity of acutolysin A is enhanced by Co²⁺, Ca²⁺ and Mg²⁺, but is partly inhibited by Cu²⁺, Mn²⁺ and Tb³⁺, and completely inhibited by Cd²⁺. Both Zn²⁺ and Co²⁺ recover the loss of activity of the protein caused by Cd²⁺.