Binding of anticoagulation factor II from the venom of Agkistrodon acutus with activated coagulation factor X.
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.
Xu
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Department of Chemistry, University of Science and Technology of China, 230026, Hefei, China.
Liu
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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
D000925:Anticoagulants; D003435:Crotalid Venoms; C432745:anticoagulation factor II, Agkistrodon acutus; D005170:Factor X; D002118:Calcium