Structural comparisons of meizothrombin and its precursor prothrombin in the presence or absence of procoagulant membranes

A stable form of meizothrombin derived from an active-site (Ser528---Ala) mutant of recombinant bovine prothrombin [Pei et al. (1991) J. Biol. Chem. 266, 9598-9604] has been used to determine the physical properties and conformation of meizothrombin both in solution and when bound to a procoagulant membrane. As determined with quasi-elastic light scattering, meizothrombin and prothrombin had similar molecular dimensions normal to a membrane (9.4 +/- 1.0 nm) and similar binding affinities to procoagulant membranes (1.8 +/- 0.2 microM at 0.4 M NaCl). However, meizothrombin had a greater tendency to form oligomers or aggregates in solution. The enhanced oligomerization of meizothrombin was also evidenced by a high apparent z-weighted molecular weight in equilibrium sedimentation experiments at low spin speeds. However, velocity sedimentation experiments performed at high spin speeds demonstrated the same sedimentation coefficient for meizothrombin (s20,w(0) = 4.7 +/- 0.2 S) as for prothrombin (s20,w(0) = 4.7 +/- 0.15 S). Circular dichroism measurements revealed minor differences in protein secondary structure between meizothrombin and prothrombin either in the presence or in the absence of phospholipid membranes, as reflected in an increased theta 222/theta 208 ratio in meizothrombin relative to prothrombin. The main endotherm of the meizothrombin thermal denaturation profile in a Ca(2+)-containing buffer, as determined by differential scanning calorimetry, was indistinguishable from that of prothrombin. However, in the presence of phosphatidylserine-containing membranes, the peak temperatures of denaturation profiles of meizothrombin were distinct from those of prothrombin.