Prothrombin and factor X possess two high affinity and several low affinity lanthanide ion binding sites. In both proteins, the association constant of the high affinity sites is at least 50-fold greater than that of the low affinity sites. Moreover, metal bound to these high affinity sites is extremely difficult to displace. It has been proposed that one of the two high affinity sites in factor X involves Gla residues while the other involves β-hydroxyaspartic acid and no Gla residues. It is also known that ^H can be specifically incorporated into Gla residues at an acidic pH. We have determined that under nondenaturing conditions when Gla (synthetic or in proteins) is complexed to metal at pH 5.5, this specific 3H incorporation is blocked. Furthermore, we have found that β-hydroxyaspartic acid does not incorporate in the presence or absence of metal. When we incubated prothrombin or factor X (41 μM) with 3H2O in the presence of Tb3+ or Gd3+ (82 μM), we blocked 5.6 Gla residues per prothrombin and 5.5 Gla residues per factor X from 3H incorporation. Under these conditions, we calculated that >95% of the high affinity sites are occupied by metal. Thus, in prothrombin, an average of 2.8 Gla residues are involved in forming each high affinity site. If the Gla residues in factor X participate in forming only one of the two high affinity sites, then all 5.5 Gla residues blocked from incorporation must be involved in forming that site. However, this seems highly unlikely. We conclude that, as in prothrombin, both high affinity sites in factor X involve Gla residues (average 2.75/site). However, our data does not exclude the possibility of existence of a heterologous site containing both β-hydroxyaspartic acid and Gla residues.
Synthesis and Photoregulated Metal Coordination of Azobenzene Polymer Having Ion Binding Sites
