Ultraviolet Resonance Raman Spectroscopy
Ultraviolet resonance Raman spectroscopy (UVRR) is a powerful technique that can be used to study protein biopharmaceutical materials. The technique is based on the same physical principles that normal Raman spectroscopy does but with the unique advantage that by tuning the excitation wavelength between 200 and 240 nm, moderate selectivity can be obtained in the resonance Raman spectrum, allowing Trp, Tyr, Phe or amide groups to be selectively enhanced. Additionally, the strong fluorescence that biological materials usually exhibit is avoided when exciting in the deep ultraviolet.
Structural and conformational information of these important aromatic residues is provided by UVRR when the excitation wavelength is set at 229 or 244 nm. The degree of hydrogen bonding of both, tyrosine and tryptophan, the relative orientation of the tryptophan side-chain and additionally, the degree of solvent exposure (relative hydrophobicity) of the tryptophan and tyrosine residues can be obtained.
Thus the local environment of tryptophan and tyrosine residues can be monitored in detail, making the technique highly complementary to fluorescence measurements and a complementary tool in formulation, stability and comparability studies.
229 nm UVRR spectra of native horse heart cytochrome C, molten state (A-state) and unfolded state (black, red and green spectrum respectively) showing the Raman spectral changes caused by the acid denaturation of cytochrome C. The A-state is a partially folded state induced at low pH and high salt concentration.
Facilities
UVRR spectra are obtained with a commercial Raman microscope, a Renishaw RM 1000, adapted for operation with deep UV laser wavelengths (229 and 244 nm laser excitation provided by an intra-cavity frequency-doubled argon ion laser).