Chemical modification of bovine prothrombin fragment 1 in the presence of Tb³⁺ ions

The formaldehyde-morpholine method for the conversion of γ-carboxyglutamyl (Gla) residues to γ-methyleneglutamyl (γ-MGlu) residues has been applied to the modification of bovine prothrombin fragment 1. In the absence of Tb³⁺ ions or at Tb³⁺ ion concentrations of 2 K(m)(app) and 25 K(m)(app) the action of 10,000-fold molar excess of formaldehyde and morpholine, pH 5.0, converts the 10 Gla residues of the protein into 10 γ-MGlu residues. Modification of the protein using the same conditions but increasing the Tb³⁺ concentration to 100 K(m)(app) provided a homogeneous protein containing 3 γ-MGlu and 7 Gla residues, bovine 3 γ-MGlu-fragment 1. The modified protein binds the same number of Ca²⁺ ions (6-7) as bovine fragment 1. However, the positive cooperativity associated with Ca²⁺ binding is abolished and the overall affinity for Ca²⁺ ions is reduced. Fluorescence titrations of 3 γ-MGlu-fragment 1 using either Ca²⁺ or Mg²⁺ ions indicate that the modified protein retains a fluorescence quenching behavior similar to that of the native protein. The modified protein does not bind to phosphatidylserine/phosphatidylcholine vesicles in the presence of Ca²⁺ ions. Thus the metal ion-induced fluorescence transition exhibited by the bovine protein appears to be a necessary but not sufficient condition for phospholipid binding.