The effect of divalent metal ions on the electrophoretic mobility of bovine prothrombin and bovine prothrombin fragment 1

Examination of metal ion-dependent effects on the electrophoretic mobility of bovine prothrombin and fragment 1 provides a useful and sensitive method for investigation of conformational processes in these proteins. Utilization of this method reveals a conformational change in bovine prothrombin and fragment 1 which occurs at low metal ion concentrations. Equilibrium dialysis studies indicate that the metal ion-induced shape change occurs concomitant with binding of a single calcium ion/molecule of prothrombin or fragment 1. Mixed metal electrophoretic mobility studies with Mg²⁺ and Ca²⁺ have demonstrated the 'synergistic' effect for fragment 1 observed by others. Mixed metal equilibrium dialysis has provided experimental support for this observation and allows us to conclude that two tight Ca²⁺ sites are not affected by low Mg²⁺ concentrations and that the third Ca²⁺ site is also a tight site for Mg²⁺. Thus, at low Mg²⁺ concentrations and upon the addition of Ca²⁺, there are effectively three tight sites; consequently more Ca²⁺ will bind to the protein at lower total Ca²⁺ ion concentrations.