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 Ca 2+ ions, and the maximal binding of ACF II to FXa occurred at the concentration of Ca 2+ ions of about 1 mM. The binding of Ca 2+ ions to ACF II was analyzed by equilibrium dialysis and two Ca 2+-binding sites with different affinities were identified. At pH 8.0, the apparent association constant K 1 and K 2 values for these sites were (1.1±0.3)×10 5 M −1 and (1.7±0.4)×10 4 M −1 (mean±SE, n=4), respectively. It was evident from the observation of Ca 2+-induced changes in the intrinsic fluorescence of ACF II that ACF II underwent a conformational change upon binding of Ca 2+ ions. The occupation of both Ca 2+-binding sites in ACF II requires a concentration of Ca 2+ ions of about 1 mM, which is equal to the effective concentration of Ca 2+ ions required for maximal binding of ACF II to FXa, and for the maximal Ca 2+-induced enhancement of emission fluorescence of ACF II. It can be deduced from these results that the occupation of both Ca 2+-binding sites in ACF II with Ca 2+ ions and subsequent conformational rearrangement should be essential for its recognition of Ca 2+-mediated conformational changes of FXa.