Effects of terbium and pH on structure of anticoagulation factor II from Agkistrodon acutus venom probed by fluorescent spectroscopy and equilibrium dialysis.
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+.
Xu
Xiao Long
XL
Department of Chemistry, University of Science and Technology of China, Hefei 230026, China.
Liu
Qing Liang
QL
Wu
Bin
B
Xie
Yong Shu
YS
D003435:Crotalid Venoms; D003956:Dialysis / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D013725:Terbium / Q000494:pharmacology*
D003435:Crotalid Venoms; C432745:anticoagulation factor II, Agkistrodon acutus; D013725:Terbium