A study of the denaturation of human C-reactive protein in the presence ofcalcium ions and glycero-phosphorylcholine

Differential scanning calorimetry, fourier transform infrared spectroscopy and surface plasmon resonance spectrometry have been used to study the ther mal properties of C-reactive protein in its ligand-bound and ligand-free fo rms. Clear denaturation endotherms were detected during calorimeter heating scans. Upon binding either or both its ligands, Ca2+ and glycero-phosphory lcholine, the denaturation temperature of the protein and the enthalpy chan ge on denaturation were increased. Marked changes in the infrared spectrum of the protein were seen when it was heated. The main absorption peaks in t he spectrum, at 1651 and 1643 cm(-1) were replaced by a single peak at 1628 cm(-1). There was an isosbestic point at 1630 cm(-1). Comparison with the calorimetric data indicated that the changes in spectra were caused by prot ein denaturation. The spectra, like the calorimetric data, showed that the presence of glycero-phosphorylcholine raised the denaturation temperature o f the protein. However, the presence of calcium ions had little effect on t he spectra. This suggests that unlike glycero-phosphorylcholine, calcium bi nds to C-reactive protein in such a way that hydrogen bonding in the polype ptide backbone is not altered. Using surface plasmon resonance spectrometry the adsorption of C-reactive protein onto self-assembled phosphorylcholine monolayers was measured in the presence of the protein's ligands before an d after its denaturation. Monolayers were formed by reacting a phosphorylch oline alkyl thiol with the surface of silvered microscope slides. It was po ssible to demonstrate a specific interaction between C-reactive protein and the phosphorylcholine surface and show that this interaction did not take place after the protein had been denatured. (C) 1999 Elsevier Science B.V. All rights reserved.