Tb3+ binding to bovine prothrombin and bovine prothrombin fragment 1.
The binding of Tb3+ to bovine prothrombin and the amino-terminal 156 residues of prothrombin (F-1) was studied. On the basis of various Tb3+ emission properties, three classes of Tb3+-binding sites were described. The first class contained three high affinity sites in the F-1 region. These sites were filled noncooperatively and were saturated with Tb3+ before the other classes of sites started to fill. Ho3+ quenching of Tb3+ emission showed that these sites were in close proximity to one another (estimated distances 6-12 A). The second class of sites contained three lower affinity sites, also in the F-1 region. These sites bound Tb3+ in a stoichiometric manner and saturated prior to metal binding to the final class of sites. The number of protein ligands binding Tb3+ in the high affinity sites decreased as this second set of sites was filled. Ho3+ quenching of Tb3+ emission suggested that these sites were closely spaced and/or close to the first set of sites. The third class of sites contained 4-6 low affinity sites unique to prothrombin (not in the F-1 region). These sites were not studied extensively, but Tb3+ did not appear to bind stoichiometrically and did not saturate these sites in a manner similar to the other two classes of sites. The emission properties of Tb3+ bound to F-1 were different in KCl versus NaCl containing buffer while the emission properties of Tb3+ bound to prothrombin were not. Optimum conditions for studying lanthanide binding to F-1 (i.e. when Tb3+ bound to F-1 showed emission properties similar to Tb3+ bound to prothrombin) were when F-1 experiments were done at low F-1 concentrations in buffer containing 0.1 M KCl.
D000818:Animals; D001665:Binding Sites; D002021:Buffers; D002417:Cattle; D006695:Holmium / Q000378:metabolism; D007700:Kinetics; D007811:Lanthanum / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011498:Protein Precursors; D011516:Prothrombin / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013725:Terbium / Q000378:metabolism*
D002021:Buffers; D010446:Peptide Fragments; D011498:Protein Precursors; D013725:Terbium; D012965:Sodium Chloride; D011189:Potassium Chloride; D007811:Lanthanum; C029733:prothrombin fragment 1; D011516:Prothrombin; D006695:Holmium