Metal ion-induced stabilization and refolding of anticoagulation factor II from the venom of Agkistrodon acutus.
Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism (CD). Metal ions were found to increase the structural stability of ACF II against GdnHCl and irreversible thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF II and Tb(3+)-ACF II is a two-state process with no detectable intermediate state, while the GdnHCl-induced unfolding/refolding of holo-ACF II in the presence of 1 mM Ca(2+) follows a three-state transition with an intermediate state. Ca(2+) ions play an important role in the stabilization of both native and I states of holo-ACF II. The decalcification of holo-ACF II shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, while the reconstitution of apo-ACF II with Tb(3+) ions shifts the initial zone of the denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.1 M GdnHCl) at which refolding from the fully denatured state of apo-ACF II to the I state of holo-ACF II or to the native state of Tb(3+)-ACF II can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF II, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ion-induced refolding provide evidence for the fact that the first phase of Tb(3+)-induced refolding should involve the formation of the compact metal-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.
Xu
Xiaolong
X
Department of Chemistry, University of Science and Technology of China, Hefei 230026, Peoples Republic of China.
Liu
Qingliang
Q
Xie
Yongshu
Y
D017836:Agkistrodon; D000818:Animals; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification; D004546:Elapid Venoms / Q000737:chemistry*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013725:Terbium / Q000737:chemistry*; D013816:Thermodynamics
D003435:Crotalid Venoms; D004546:Elapid Venoms; C432745:anticoagulation factor II, Agkistrodon acutus; D013725:Terbium