Differentiation of metal ion-induced transitions of prothrombin fragment 1

The intrinsic fluorescence of human and bovine prothrombin fragment 1 is quenched (approximately -35%) when calcium ions are bound. The intrinsic fluorescence of prothrombin is also quenched by the binding of Ca2+ but to a lesser extent (total change is approximately -6%). The prothrombin fragment 1 fluorescence transition is also effected by the binding of Mg2+, Mn2+, Gd3+, and Tb3+. With all ions the fluorescence quenching is readily reversed by titration with EDTA. Titration with any of the above ions results in a sigmoidal titration curve. The fluorescence transition midpoints (expressed as Tm) occur at the following concentrations of ions: Ca2+, 0.22 (0.35) mM; Mg2+, 0.22 (0.45) mM; Mn2+, 12.6 (12.6) muM; and Gd3+, 3.6 (5.3) muM (the values in parentheses indicate the concentration of the respective ions bound to bovine prothrombin fragment 1). The presence of phospholipid steepens the Ca2+ titration curve, significantly more so for the bovine system than for the human. Tm values for Ca2+ binding in the presence of phospholipid are 0.21 mM and 0.22 mM for binding to human and bovine prothrombin fragment 1, respectively. Sedimentation equilibrium and velocity studies indicate that prothrombin fragment 1 which is monomeric in the presence of EDTA undergoes concentration-dependent association in the presence of Ca2+. A plot of fraction monomer versus Ca2+ concentration is sigmoidal, with a Tm of approximately 1 mM Ca2+. Mg2+ is only marginally effective in promoting the dimerization, and Mn2+ and Gd3+ are even less effective. However, sedimentation studies performed in the presence of 1 mM Mg2+ at varying Ca2+ concentrations shifted the apparent dimerization transition markedly to the left, the midpoint of the transition occurring at 0.25 mM. Kinetic studies reveal that while Mg2+ by itself does not promote prothrombin activation to thrombin, Mg2+ will partially substitute for Ca2+ in prothrombin activation. In the presence of 1 mM Mg2+ maximal activation rates are attained with 0.3 mM Ca2+; higher Mg2+ concentrations (at this Ca2+ concentration) are inhibitory. Only Ca2+ and Gd3+ permit significant prothrombin-phospholipid binding. Scatchard plots derived from equilibrium dialysis experiments performed with bovine prothrombin fragment 1 suggest marked cooperativity in calcium binding and the existence of approximately six Ca2+ binding sites. Similar studies, but with 1 mM Mg2+ also present in the dialysate, suggested that Mg2+ promotes a greater degree of cooperativity in Ca2+ binding to prothrombin fragment 1 with an apparent decrease in the number of Ca2+ sites occupied. It therefore appears that prothrombin fragment 1 has two classes of metal ion binding sites. One class, probably comprising two of six sites, is apparently fairly nonselective with respect to which metal ion is bound; binding of metal ion to these sites is responsible for the fluorescence change and apparently triggers a conformational transition...