Calcium ion-induced stabilization and refolding of agkisacutacin from Agkistrodon acutus venom studied by fluorescent spectroscopy.
Agkisacutacin isolated from the venom of Agkistrodon acutus is a coagulation factor IX / coagulation factor X-binding protein with marked anticoagulant- and platelet-modulating activities. Ca(2+) ion-induced stabilization and refolding of Agkisacutacin have been studied by following fluorescent measurements. Ca(2+) ions not only increase the structural stability of agkisacutacin against GdnHCl denaturation, but also induce its refolding. The GdnHCl-induced unfolding of the apo-agkisacutacin and the purified agkisacutacin is a single-step process with no detectable intermediate state. Ca(2+) ions play an important role in the stabilization of the structure of agkisacutacin. Ca(2+)-stabilized agkisacutacin exhibits higher resistance to GdnHCl denaturation than the apo-agkisacutacin. It is possible to induce refolding of the unfolded apo-agkisacutacin merely by adding 1 mM Ca(2+) ions without changing the concentration of the denaturant. The kinetic result of Ca(2+)-induced refolding provides evidences for that agkisacutacin consists of at least two refolding phases and the first phase of Ca(2+)-induced refolding should involve the formation of the compact Ca(2+)-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, PR China. xuxl@ustc.edu.cn
Chen
Jiexia
J
Zhang
Liyun
L
Wang
Shouye
S
Shen
Dengke
D
Liu
Qingliang
Q
D017836:Agkistrodon / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002413:Cations, Divalent / Q000737:chemistry; D003435:Crotalid Venoms / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D002413:Cations, Divalent; D003435:Crotalid Venoms; C401844:agkisacutacin; D002118:Calcium