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

S. F. Wright, P. Berkowitz, D. W. Deerfield, P. A. Byrd, D. L. Olson, R. S. Larson, G. C. Hinn, K. A. Koehler, L. G. Pedersen, R. G. Hiskey

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)10598-10605
Number of pages8
JournalJournal of Biological Chemistry
Volume261
Issue number23
StatePublished - 1986
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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