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 investigated by following H+ release as a function of metal ion concentration 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. M2+ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H+ release by M2+ is bell shaped and consistent with controlling pKa values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca2+ and Mg2+ titrations following H+ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M2+. 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 remains dimeric in the presence of Mg2+. These results and those for metal ion-dependent H+ release indicate that H+ release occurs concomitantly with association processes involving prothrombin.
Koehler
K A
KA
Surgery Department, Case Western Reserve University School of Medicine, MetroHealth Medical Center, Cleveland, Ohio 44109-1988, USA.
Jain
M K
MK
Gabriel
D A
DA
Chang
H Y
HY
Malhotra
O P
OP
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007834:Lasers; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D009994:Osmolar Concentration; D011199:Potentiometry; D011516:Prothrombin / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D012542:Scattering, Radiation; D013696:Temperature
D046911:Macromolecular Substances; D011522:Protons; D011516:Prothrombin; D008274:Magnesium; D002118:Calcium