INTRAMOLECULAR DOMAIN-DOMAIN INTERACTIONS AND INTERMOLECULAR SELF-ASSOCIATION IN BOVINE PROTHROMBIN - A POTENTIOMETRIC AND LASER LIGHT-SCATTERING STUDY

The interaction of bovine prothrombin with Ca2+ and Mg2+ ions was inve stigated by following H+ release as a function of metal ion concentrat ion at pH 6 and pH 7.4 at high and low ionic strength, Prothrombin Ca2 + and Mg2+ binding is characterized by high- and low-affinity sites. M (2+) binding at these sites is associated with intramolecular conforma tional changes and also with intermolecular self-association. The pH d ependence of H+ release by M(2+) is bell shaped and consistent with co ntrolling pK(a) values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca2+ and Mg2+ titrations following H+ release clearly show indep endent low- and high-affinity binding sites. Laser light scattering re veals that at pH 7.4 and low ionic strength, and at pH 6.0 and high io nic strength, the prothrombin molecular weight is between 73 and 98 kD . At pH 7.4 and high ionic strength, prothrombin is monomeric in the a bsence of metal ions, but appears to dimerize in the presence of M(2+) At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca2+ and r emains dimeric in the presence of Mg2+. These results and those for me tal ion-dependent H+ release indicate that H+ release occurs concomita ntly with association processes involving prothrombin.