Effects of rare earth ions on the conformational stability of anticoagulation factor II from Agkistrodon acutus venom probed by fluorescent spectroscopy

摘要

Anticoagulation factor II (ACF II) isolated front the venom of Agkistrodon acutus is all activated coagulation factor X-binding protein in a Ca2+-dependent fashion with marked anticoagulant activity. The equilibrium unfolding of rare earth ions (RE3+)-reconstituted ACF It in guanidine hydrochloride (GdnNCl) solution was studied by fluorescence. The GdnHCl-induced unfolding of Re3+ (Nd3+, Sm3+, Eu3+, Gd3+)-reconstituted ACF II follows a three-state transition with a stable intermediate state: Substitutions of the RE3+ ions for Ca2+ in ACF II decrease the conformational stability of its native state but markedly increase the conformational stability of its intermediate state. The free energy change of RE3+-ACF II from the intermediate state to denatured state linearly increases with the increase of ionic potentials of bound metal ions (Ca2+, Nd3+, Sm3+, Eu3+, and Gd3+). The refolding of ACF II from the unfolded state to the intermediate state can be induced merely, by adding 10 mu M RE3+ ions without changing the concentration of the denaturant. The kinetic results of the RE3+-induced refolding provide evidence indicating that the intermediate state of RE3+-ACF II consists of at least two refolding phases and that the refolding rate constant values of the faster phase decrease with the increase of the difference between the radii of Ca2+ and RE3+, but the refolding rate constant values of the slower phase are similar to each other. The results of this study indicate that the size of metal ion is the major, factor responsible for the metal ion-induced conformational stabilization of the native ACF II, while the metal ionic potential plays a predominant role in stabilizing the conformation for the intermediate state. (c) 2006 Wiley Periodicals, Inc.