BINDING OF CALCIUM TO SYNTHETIC PEPTIDES CONTAINING GAMMA-CARBOXYGLUTAMIC ACID

The Ca2+ binding properties of various gamma-carboxyglutamic acid (gla)-containing synthetic peptides with counterpart sequences in human protein C were investigated employing potentiometry with a Ca2 + -selective electrode and titration calorimetric techniques. The shortest peptides, FL(gla)(gla)LR, DF(gla)(gla)AK, and the oxidized form of the cyclic hexapeptide CI(gla)(gla)IC, each of which contains one pair of gla residues, have a weak affinity for Ca2+, with some peptides probably involved in intermolecular bridging of the Ca2+. The best example of this is the oxidized form of the peptide, CI(gla)(gla)IC, where one g-atom of Ca2+ interacts with 2 mol of peptide (n = 0.5) with a K(d) value of 1.6 mm. A second g-atom of Ca2 + interacts with 2 mol of this same peptide (n = 0.5) and is characterized by a K(d) of 8.8 mm. A longer peptide containing this same sequence, viz. L(gla)R(gla)CI(gla)(gla)IC, possesses two binding sites (n = 2.0) for Ca2 + of K(d) = 16.1 mm, as well as a tighter site (n = 1), of K(d) = 0.4 mm. An increase in stoichiometry of tight binding sites as the peptide is elongated is observed from binding data obtained on a 38-residue peptide that possesses all nine of the gla-residues of protein C in their proper sequence positions. The strongest Ca2 + binding sites (n = 3-4) possess an average K(d) Of 0.4 mm, followed by another class of sites (n = 5-10, average K(d) = 1. 5-3.0 mm). The affinity and stoichiometry of these stronger sites mimic those observed for binding of Ca2+ to the gla region of prothrombin fragment 1. By selective [C-13]-labeling of the essential gla 16 residue of the 38-mer peptide, we demonstrate that this particular gla residue participates as a donor for a high-affinity Ca2+ site. These similarities in binding properties between the synthetic peptide containing the entire gla domain and the gla domain as it exists in proteins and protein fragments indicate suitably designed peptides of this type may constitute appropriate models for investigation of the binding of Ca2+ to intact gla-containing proteins.