Luminescence energy transfer studies of C-reactive protein. Binding of terbium (III) ions in C-reactive protein.

The acute phase of inflammation is characterized by numerous changes in blood composition, perhaps the most dramatic of these being the elevation of C-reactive protein levels. C-reactive protein (CRP) is known to bind to molecules containing phosphocholine-substituents following reaction with Ca2+ ions. Luminescence energy transfer (LET) has been used effectively to study the Ca2+ and Mg2+ binding properties of many proteins by employing appropriate lanthanides (III). We have used Tb3+ as an isomorphous analogue to study Ca2+ binding to CRP. Energy transfer occurs effectively and demonstrates the importance of aromatic residues (viz., tyrosine and tryptophan) in the binding of Tb3+. The binding of Tb3+ is remarkably dependent on the pH and indicates the requirement of a deprotonated residue in the pH range 6.4 +/- 0.2 for effective Tb3+ binding. A 50-fold molar excess of Ca2+ is sufficient to displace the Tb3+ suggesting that Tb3+ is bound with greater affinity to CRP than the natural analogue Ca2+. We propose that Tb3+ (by inference Ca2+) binding takes place near the CRP subunit disulfide bond, where two histidine residues are present. The pH dependency of Tb3+ binding is best explained by the deprotonation of a histidine residue(s) in CRP.