Premium
Ca(II)‐ and Tb(III)‐induced stabilization and refolding of anticoagulation factor I from the venom of Agkistrodon acutus
Author(s) -
Xu Xiaolong,
Liu Qingliang,
Yu Huaming,
Xie Yongshu
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.4130102
Subject(s) - chemistry , guanidine , circular dichroism , native state , equilibrium unfolding , denaturation (fissile materials) , crystallography , hydrochloride , venom , biophysics , nuclear chemistry , biochemistry , biology
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 μM 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.