Interaction of lanthanide ions with bovine factor X and their use in the affinity chromatography of the venom coagulant protein of Vipera russelli.
The substitution of trivalent lanthanide ions for Ca(II) in the Ca(II)-DEPENDENT ACTIVATION OF BOVINE Factor X by the coagulant protein of Russell's viper venom was studied at pH 6.8. Factor X contains two high affinity metal binding sites which bind Gd(III), Sm(III), and Yb(III) with a Kd of about 4 X 10-7 M and four to six lower affinity metal binding sites which bind Gd(III), Sm(III) with a Kd of about 1.5 X 10-5M. In comparison, 1 mol of Factor X binds 2 mol of Ca(II) with a Kd of 3 X 10-4M and weakly binds many additional Ca(II) ions. No binding of Gd(III) to the venom protein was observed. Dy(III), Yb(III), Tb(III), Gd(III), Eu(III), La(III), AND Nd(III) cannot substitute for Ca(II) in the Ca(II)-dependent activation of Factor X by the venom protein at pH 6.8. Kinetic data consistent with the models of competitive inhibition of Ca(II) by Nd(III) yielded a Ki of 1 to 4 X 10-6M. The substitution of lanthanide ions for Ca(II) to promote protein complex formation of Factor X-metal-venom protein without the activation of Factor X facilitated the purification of the coagulant protein from crude venom by affinity chromatography. Using a column containing Factor X covalently bound to agarose which was equilibrated in 10 mM Nd(III), Tb(III), Gd(III), or La(III), the coagulant protein was purified 10-fold in 40% yield from crude venom and migrated as a single band on gel electrophoresis in sodium dodecyl sulfate. These data suggest that lanthanide ions complete with Ca(II) for the metal binding sites of Factor X and facilitate the formation of a nonproductive ternary complex of venom protein-Factor X-metal. Tb(III) fluorescence, with emission maxima at 490 and 545 nm, is enhanced 10,000-fold in the presence of Factor X. The study of the participation of an energy donor intrinsic to Factor X in energy transfer to Tb(III) may be useful in the characterization of the metal binding sites of Factor X.
D000818:Animals; D001665:Binding Sites; D002118:Calcium; D002417:Cattle; D002846:Chromatography, Affinity; D004419:Dysprosium; D004591:Electrophoresis, Polyacrylamide Gel; D005063:Europium; D005170:Factor X / Q000302:isolation & purification / Q000378:metabolism; D005682:Gadolinium; D007700:Kinetics; D007811:Lanthanum; D009354:Neodymium; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification; D012493:Samarium; D012910:Snake Venoms; D013725:Terbium; D014688:Venoms; D015018:Ytterbium
D011506:Proteins; D012910:Snake Venoms; D014688:Venoms; D013725:Terbium; D004419:Dysprosium; D009354:Neodymium; D012493:Samarium; D005063:Europium; D007811:Lanthanum; D005170:Factor X; D005682:Gadolinium; D015018:Ytterbium; D002118:Calcium