Monitoring conformational changes of immobilized RNase A and Lysozyme in reductive unfolding by surface plasmon resonance

A labeling-free surface plasmon resonance (SPR) sensor technique was used to monitor the conformational changes of immobilized globular proteins (RNase A and Lysozyme) in chemical unfolding and refolding. The conformational changes of proteins at solid/liquid interface are characterized as two-state transformation (S-shaped) curves through matrix-effect correction and theoretic estimation. By extrapolation with a Santoro–Bolen equation, the SPR results for both reductive immobilized proteins are estimated to 1.9 kcal mole−1 global free energy (ΔGU) in urea-induced unfolding. But the ΔGU for RNase A and Lysozyme in GdmCl-induced unfolding are 1.5 and 2.15 kcal mole−1, respectively. The disagreement in free energy is partially accounted for by the differences of intra-molecular interactions and immobilization.