Studies on anticoagulation factor II from Agkistrodon acutus venom by rareearth ion fluorescence probes

The anticoagulation factor II (ACF II) from Agkistrodon acutus venom has tw o Ca2+-binding sites and Ca2+ ions enhance the intrinsic fluorescence of AC F II. Rare earth ions (Nd3+, Sm3+, Eu3+, Gd3+, Tb3+) can replace the Ca2+ i ons in ACF II and have significant quenching effects on the fluorescence of ACF II. The characteristic fluorescence of Tb3+ is enhanced through the no nradiative energy transfer from Trp residues in ACF II to bound Tb3+. The f luorescence titration of ACF II with rare earth ions demonstrates that ACF II has two RE3+-binding sites, and the rare earth ions and Ca2+\ ions bind to ACF II competitively in the same two sites. Although the ionic radii of Nd3+, Sm3+, Eu3+, Gd3+ and Tb3+ are different, their apparent association c onstant K-1 or K-2 are similar, respectively. Linear free energy relationsh ips for Tb3+ and RE3+ (RE=Nd, Sm, Eu, Gd) indicate that there is not an obv ious size restriction for the binding of lanthanide ions at the two binding sites of ACF II, which reveals the conformational flexibility of the two b inding sites in ACF II. This conformational flexibility offers a possibilit y for Ca2+ ions, when they take place in the inducing conformational change s of ACF II and the binding of ACF II with the activated coagulation factor X.