Development of an ultraviolet Raman optical activity instrument for pre-resonant protein and virus studies

Although we have demonstrated that non-resonant Raman optical activity (ROA), using visible excitation and consequently avoiding absorption of the incident laser radiation by chromophores present in biological samples under investigation, has evolved into a powerful new spectroscopy for biomolecules, more widespread use of the technique is hampered by several basic problems such as spectral interference of traces of sample fluorescence and the need for relatively high sample concentrations and consequently rather long measurement times. However, from detailed experimental studies of the ultraviolet (UV) spectroscopic characteristics of typical proteins and after careful examination of the influence of these characteristics on the expected ROA signal-to-noise ratio (SNR), we are convinced that, by adopting a pre-resonant measurement regime with UV excitation coinciding with electronic transitions in between the tail ends of distinct absorption band envelopes, these restrictions could be eliminated. With much lower sample concentrations but under otherwise comparable experimental conditions, the same ROA SNR could be achieved with significantly reduced data acquisition times employing UV rather than visible excitation. We therefore propose to develop a novel computer controlled instrument for conducting pre-resonant UV ROA studies of biomolecules. The proposed UV ROA instrument has the potential to become an important universal biochemical and perhaps biomedical tool for investigations into the the aqueous solution structure and behaviour of virtually all proteins and viruses. Its overall performance will be tested and critically assessed by performing different benchmark experiments; measured pre-resonant UV ROA protein signals of sufficiently high quality from aromatic amino acid sidechains will be analysed in order to extract valuable information about their absolute stereochemistry.