Terbium probe of calcium-binding sites on the prothrombin-membrane complex.
Terbium was used as a probe of Ca2+-binding sites on the prothrombin-phospholipid complex. Stoichiometric titrations of prothrombin binding to phospholipid vesicles with either Tb3+ or Ca2+ showed that a minimum of 8 metal ions were needed for binding prothrombin to vesicles (3 Mn2+ + 5 Ca2+ for prothrombin or 8 Tb3+ for F-1). When Ca2+ alone was used, a total of about 11 metal ions were needed for complete binding. These stoichiometries indicated 3 classes of metal ions: one class needed to induce the conformational change, a second required for protein-membrane contact, and a third class bound at other sites on the protein that are not involved in membrane binding. By adding Tb3+ to solutions containing both protein and phospholipid, undesirable Tb3+-induced events, such as irreversible aggregation of prothrombin or vesicle fusion, were avoided. Protein-vesicle binding apparently prevented protein aggregation or vesicle fusion. The protein-vesicle binding affinity was severalfold greater in the presence of Tb3+ compared to Ca2+. CoEDTA quenching of Tb3+ bound to the prothrombin-phospholipid complexes indicated that all metal ions were at least partially exposed to the quencher. Some populations of Tb3+ showed lower quenching constants when all of the prothrombin was bound. Tb3+ emission lifetimes revealed that some Tb3+ ions in the protein-membrane complex were in a different environment from those bound to the protein alone. The results indicated that the metal ions in the prothrombin-membrane complex are relatively open to the solvent yet do affect the characteristics of the protein-membrane binding equilibrium.
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004492:Edetic Acid; D005170:Factor X / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D008027:Light; D008433:Mathematics; D011486:Protein C; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism*; D012542:Scattering, Radiation; D013725:Terbium / Q000378:metabolism*
D006023:Glycoproteins; D011486:Protein C; D013725:Terbium; C028222:cobalt-ethylenediamine tetraacetic acid chelate; D011516:Prothrombin; D005170:Factor X; D004492:Edetic Acid; D002118:Calcium