Ca(II)- and Tb(III)-induced stabilization and refolding of anticoagulation factor I from the venom of Agkistrodon acutus.
Anticoagulation factor I (ACF I) 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 I, holo-ACF I, and Tb(3+)-reconstituted ACF I in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism. Metal ions were found to increase the structural stability of ACF I against GdnHCl and thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF I and Tb(3+)-ACF I is a two-state process with no detectable intermediate state(s), whereas the GdnHCl-induced unfolding/refolding of holo-ACF I in the presence of 1 mM Ca(2+) follows a three-step transition, with intermediate state a (Ia) and intermediate state b (Ib). Ca(2+) ions play an important role in the stabilization of the Ia and Ib states. The decalcification of holo-ACF I shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, whereas the reconstitution of apo-ACF I with Tb(3+) ions shifts the initial zone of denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.0 M GdnHCl) at which refolding from the fully denatured state of apo-ACF I to the Ib state of holo-ACF I or to the native state of Tb(3+)-ACF I 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 I, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ions-induced refolding provide evidence that the compact Tb(3+)-binding region forms first, 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, China.
Liu
Qingliang
Q
Yu
Huaming
H
Xie
Yongshu
Y
D017836:Agkistrodon; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification; D004546:Elapid Venoms / Q000737:chemistry*; D019791:Guanidine; D006358:Hot Temperature; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000737:chemistry*; D013816:Thermodynamics
D003435:Crotalid Venoms; D004546:Elapid Venoms; D013725:Terbium; D019791:Guanidine; D002118:Calcium