title: Binding of anticoagulation factor II from the venom of Agkistrodon acutus with activated coagulation factor X.
abstract: Anticoagulation factor II (ACF II) from the venom of Agkistrodon acutus has been identified as a binding protein to activated bovine coagulation factor X (FXa) by the method of polyacrylamide gel electrophoresis and high performance liquid chromatography. This protein formed a 1:1 complex with FXa in the presence of Ca2+ ions, and the maximal binding of ACF II to FXa occurred at the concentration of Ca2+ ions of about 1mM. The binding of Ca2+ ions to ACF II was analyzed by equilibrium dialysis and two Ca2+-binding sites with different affinities were identified. At pH 8.0, the apparent association constant K1 and K2 values for these sites were (1.1+/-0.3)x10(5) M(-1) and (1.7+/-0.4)x10(4) M(-1) (mean+/-SE, n=4), respectively. It was evident from the observation of Ca2+-induced changes in the intrinsic fluorescence of ACF II that ACF II underwent a conformational change upon binding of Ca2+ ions. The occupation of both Ca2+-binding sites in ACF II requires a concentration of Ca2+ ions of about 1mM, which is equal to the effective concentration of Ca2+ ions required for maximal binding of ACF II to FXa, and for the maximal Ca2+-induced enhancement of emission fluorescence of ACF II. It can be deduced from these results that the occupation of both Ca2+-binding sites in ACF II with Ca2+ ions and subsequent conformational rearrangement should be essential for its recognition of Ca2+-mediated conformational changes of FXa.
authors.affiliation: Department of Chemistry, University of Science and Technology of China, 230026, Hefei, China.
authors.forename: X
authors.initials: X
authors.lastname: Xu
authors.forename: Q
authors.initials: Q
authors.lastname: Liu
mesh_terms: D017836:Agkistrodon / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D005170:Factor X / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011517:Prothrombin Time; D013050:Spectrometry, Fluorescence
chemical_list: D000925:Anticoagulants; D003435:Crotalid Venoms; C432745:anticoagulation factor II, Agkistrodon acutus; D005170:Factor X; D002118:Calcium