Terbium(III) fluorescence probe studies on metal ion-binding sites in anticoagulation factor I from Agkistrodon acutus venom.
Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein with marked anticoagulant activity. Present studies show that holo-ACF I assumes a compactly folded structure in the range of pH 5-6, in which the most interior Trp residues and quenchers are adjacent. Tb3+ ions can completely replace both Ca2+ ions in holo-ACF I, as determined by equilibrium dialysis. Although the two Tb3+ ions in Tb3+-ACF I have slightly different luminescence efficiencies, both have similar quenching effects on the intrinsic fluorescence, suggesting that probably there are same numbers of Trp residues close to both Tb3+-binding sites. Two Tb3+-binding sites with similar apparent Tb3+ association constant values, (1.69 +/- 0.02) x 10(7) M(-1) and (1.42 +/- 0.01) x 10(7) M(-1), respectively, were further identified through Tb3+ fluorescence titration. In addition, it has been confirmed from the titration of holo-ACF I and Tb3+-ACF I with NBS that only interior Trp residues are involved in the energy transfer to Tb3+ ions and that all accessible Trp residues located in the surface of holo-ACF I have similar affinity to NBS, while those located in the surface of Tb3+-ACF I have two different kinds of affinity to NBS, which strongly suggests a conformational change of holo-ACF I upon substitution of Tb3+ for Ca2+. The results show that although the Tb3+-altered conformation of ACF I cannot support the binding of Tb3+-ACF I with FXa, determined by nondenaturing PAGE, Tb3+ ions are effective and useful fluorescence probes to analyze the structures and properties of Ca2+-binding sites in ACF I.
Xu
Xiaolong
X
Department of Chemistry, University of Science and Technology of China, Hefei Anhui.
Liu
Qingliang
Q
Liu
Yi
Y
Xie
Yongshu
Y
D017836:Agkistrodon; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013725:Terbium; D013793:Thallium; D014364:Tryptophan
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D008670:Metals; C465285:anticoagulation factor I, Agkistrodon acutus; D013725:Terbium; D014364:Tryptophan; D013793:Thallium