Investigation of the interaction of GdnHCl with poly-L-lysine, horseradish peroxidase and cytochrome c monitored via FTIR, CD and fluorescence spectroscopies

Spectroscopic probes were utilized to study the interaction of GdnHCl with three model systems: poly-L-lysine (PLL), horseradish peroxidase (HRP) and cytochrome c. Probing the chemically denatured states of proteins by FTIR is a novel technique, which has only recently been reported. To aid in the interpretation of the FTIR results, fluorescence and CD were employed. The use of deuterated solvents, i.e. Gdn-d₅-DCl and D₂O, simplified data processing and is recommended for FTIR studies of protein chemical denaturation. PLL, a random coil, was used to model the interaction between Gdn-d₅-DCl and random structures. Spectroscopic evidence strongly suggested that Gdnd₅-DCl associates with PLL, particularly with its lysyl side chains. The analysis of HRP indicated that it was not a suitable model due to complex time-dependent spectral changes. The study of cytochrome c in Gdn-d₅-DCl revealed that the denaturant preferentially interacted with random structures. The denatured states of horse and tuna c were characterized and subtle differences between the two species were reported. Curvefitting of the FTIR amide I′ mode (1600–1700 cm−1) revealed that tuna c contained more residual structure in Gdn-d₅-DCl than horse c. It was successfully demonstrated that FTIR is a powerful tool for probing the chemically denatured states of model protein systems, with potential applications for other biomolecules.