Structure/function relationships of bovine prothrombin fragment 1

The objective of this investigation was to evaluate the unique contribution of the N-terminal 33 residues of prothrombin, the Gla domain, to the Ca(II) and phospholipid binding properties of prothrombin. Prothrombin fragment 1 contains the Gla domain and is often used to study the Ca(II) and phospholipid binding properties of prothrombin. Two Gla domain peptides, 1-42 and 1-45, produced by chymotryptic cleavage of F-1 and isolated by anion-exchange chromatography were utilized to characterize the Gla domain of prothrombin. All experiments with Gla domain peptides was conducted at Ca(II) concentrations less than 2 mM since these peptides will precipitate from the solution at Ca(II) concentrations greater than 2 mM. Examinations of the properties of the Gla domain peptides was undertaken by several experimental approaches. In contrast to F-1, the intrinsic fluorescence of both 1-42 and 1-45 was not quenched upon the addition of 1 mM Ca(II) or any concentration of Mg(II). Equilibrium dialysis studies indicated that 1-42 binds three Ca(II) ions non-cooperatively. Similar studies using F-1 have shown positive cooperativity and seven Ca(II) ion binding sites. Gel permeation chromatography revealed that radioiodinated 1-45 dimerizes at the peptide concentration utilized in the equilibrium dialysis studies