Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions.
The formaldehyde-morpholine method for the conversion of gamma-carboxyglutamyl (Gla) residues to gamma-methyleneglutamyl (gamma-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 Km app and 25 Km 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 gamma-MGlu residues. Modification of the protein using the same conditions but increasing the Tb3+ concentration to 100 Km app provided a homogeneous protein containing 3 gamma-MGlu and 7 Gla residues, bovine 3 gamma-MGlu-fragment 1. The modified protein binds the same number of Ca2+ ions (6-7) as bovine fragment 1. However, the positive cooperatively associated with Ca2+ binding is abolished and the overall affinity for Ca2+ ions is reduced. Fluorescence titrations of 3 gamma-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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism*; D011498:Protein Precursors; D011516:Prothrombin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000494:pharmacology*
D000596:Amino Acids; D010446:Peptide Fragments; D011498:Protein Precursors; D013725:Terbium; C029733:prothrombin fragment 1; D011516:Prothrombin; D008274:Magnesium; D002118:Calcium