title: Effects of terbium and pH on structure of anticoagulation factor II from Agkistrodon acutus venom probed by fluorescent spectroscopy and equilibrium dialysis.
abstract: Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein with marked anticoagulant activity. Present studies show that the pH has a marked effect on the fluorescence intensity of holo-ACF II; however, no appreciable shift of the emission maximum of holo-ACF II was observed in the pH range of 3-10. It was deduced from a relatively weak fluorescence emission of holo-ACF II at a neutral pH (6-7) that native holo-ACF II assumes a compactly folded structure in which the most interior Trp residues and quenchers are adjacent. Terbium ions can completely replace both Ca2+ ions in holo-ACF II as determined by equilibrium dialysis. Two Tb3+-binding sites with different apparent Tb3+ association constant values, (2.1 +/- 0.2) and (1.0 +/- 0.1) x 10(7) M(-1), were identified through Tb3+ fluorescence titration. In addition, it was confirmed from the titration of holo-ACF II and Tb3+-ACF II with N-bromosuccinimide (NBS) that only interior Trp residues are involved in the energy transfer to Tb3+ ions and all accessible Trp residues located in the surface of holo-ACF II have a similar affinity to NBS while those located in the surface of Tb3+-ACF II have two different kinds of affinity to NBS, which suggests a conformational change of holo-ACF II on the substitution of Tb3+ for Ca2+.
authors.affiliation: Department of Chemistry, University of Science and Technology of China, Hefei 230026, China.
authors.forename: Xiao Long
authors.initials: XL
authors.lastname: Xu
authors.forename: Qing Liang
authors.initials: QL
authors.lastname: Liu
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.forename: Yong Shu
authors.initials: YS
authors.lastname: Xie
mesh_terms: D003435:Crotalid Venoms; D003956:Dialysis / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D013725:Terbium / Q000494:pharmacology*
chemical_list: D003435:Crotalid Venoms; C432745:anticoagulation factor II, Agkistrodon acutus; D013725:Terbium