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 Å). 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.