Interaction of manganese with bovine prothrombin and its thrombin-mediated cleavage products

The binding of the paramagnetic metal, Mn(II), to bovine prothrombin and the thrombin-mediated cleavage products of prothrombin, i.e. fragment 1 and the prethrombin 1 has been investigated. Analysis of the Scatchard plots of the binding data reveals that prothrombin has two high affinity Mn(II) binding sites with a Kd of 1.2 +/- 1.0 X 10(-5) M and approximately two to three lower affinity Mn(II) sites with a Kd of 1.3 +/- 1.0 X 10(-4) M. Positive cooperativity in Mn(II) binding to prothrombin was observed for the strong sites. Fragment 1, the phospholipid-binding region of prothrombin, possesses two high affinity Mn(II) sites with a Kd of 2.2 +/- 1.0 X 10(-5) M and at least two lower affinity sites with a Kd of approximately 2.5 +/- 1.0 X 10(-4) M. Positive cooperativity was not observed for the binding of Mn(II) to fragment 1. Prethrombin 1 binds one Mn(II) with a Kd of 3.2 +/- 1.0 X 10(-4) M. Using the values of free Mn(II) concentration, as determined by EPR measurements and the observed enhancements of the water proton relaxation rates at various concentrations of Mn(II) and protein, the binary enhancement values (epsilon b) of the metal-protein complexes were obtained. The extrapolated values are 11 +/- 0.4 for the initial prothrombin-binding sites, and 10 +/- 0.3 for the tight binding sites of fragment 1. The unique epsilon b value obtained for prethrombin 1 was 5.3 +/- 0.7. When Mn(II) was used in a Factor Xa-metal ion-phospholipid system for activation of prothrombin, the rate of generation of thrombin was less than or equal to 5% of that obtained when Ca(II) was employed in this activation system. Addition of Mn(II) to the same activation system containing Ca(II) resulted in a marked decrease in the rate of thrombin generation, suggesting that Mn(II) probably competes for the same sites on prothrombin as Ca(II). In agreement with this is the observation that the Mn(II) sites on prothrombin could be displaced by Ca(II) at high concentrations of Ca(II).