Binding of calcium to synthetic peptides containing γ‐carboxyglutamic acid

The Ca 2+ binding properties of various γ‐carboxyglutamic acid (gla)‐containing synthetic peptides with counterpart sequences in human protein C were investigated employing potentiometry with a Ca 2+ ‐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 Ca 2+ , with some peptides probably involved in intermolecular bridging of the Ca 2+ . The best example of this is the oxidized form of the peptide, CI(gla)(gla)IC, where one g‐atom of Ca 2+ interacts with 2 mol of peptide ( n = 0.5) with a K d value of 1.6 mM. A second g‐atom of Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ to the gla region of prothrombin fragment 1. By selective [ 13 C]‐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 Ca 2+ 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 Ca 2+ to intact gla‐containing proteins.