关键词: 稀土离子, 抗凝血因子, 荧光探针, 金属离子结合位点

Abstract: The anticoagulation factor Ⅱ (ACF-Ⅱ) from Agkistrodon acutus venom has two Ca ²⁺ binding sites and Ca ²⁺ ions enhance the intrinsic fluorescence of ACF-Ⅱ. Rare earth ions (Nd ³⁺, Sm³⁺, Eu ³⁺, Gd ³⁺, Tb ³⁺ ) can replace the Ca ²⁺ ions in ACF-Ⅱ and have significant quenching effects on the fluorescence of ACF-Ⅱ. The characteristic fluorescence of Tb ³⁺ is enhanced through the nonradiative energy transfer from Trp residues in ACF-Ⅱ to bound Tb ³⁺. The fluorescence titration of ACF-Ⅱ with rare earth ions demonstrates that ACF-Ⅱ has two RE ³⁺ binding sites, and the rare earth ions and Ca ²⁺ ions bind to ACF-Ⅱ competitively in the same two sites. Although the ionic radii of Nd ³⁺, Sm ³⁺, Eu ³⁺, Gd ³⁺ and Tb ³⁺ are different, their apparent association constant K₁ or K₂ are similar, respectively. Linear free energy relationships for Tb ³⁺ and RE ³⁺ (RE=Nd, Sm, Eu, Gd) indicate that there is not an obvious size restriction for the binding of lanthanide ions at the two binding sites of ACF-Ⅱ, which reveals the conformational flexibility of the two binding sites in ACF-Ⅱ. This conformational flexibility offers a possibility for Ca ²⁺ ions, when they take place in the inducing conformational changes of ACF-Ⅱ and the binding of ACF-Ⅱ with the activated coagulation factor Ⅹ.

Key words: Rare earth ions, Anticoagulation factor Ⅱ, Fluorescence probe, Metal ion binding site