Luminescence Energy Transfer Studies of C-Reactive Protein. Binding of Terbium (III) Ions in C-Reactive Protein

Abstract

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.