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

虽然我们已经证明，非共振拉曼光学活性（罗阿），使用可见光激发，从而避免了在研究中的生物样品中存在的发色团对入射激光辐射的吸收，已经发展成为生物分子的强大的新光谱学，该技术的更广泛的应用受到几个基本问题的阻碍，例如样品荧光痕量的光谱干扰和需要相对更高的样品浓度和因此更长的测量时间。然而，从典型蛋白质的紫外（UV）光谱特性的详细实验研究，并在仔细检查这些特性对预期的罗阿信噪比（SNR）的影响后，我们相信，通过采用紫外激发与不同吸收带包络尾端之间的电子跃迁相一致的预共振测量制度，这些限制可以取消。在低得多的样品浓度下，但在其他方面相当的实验条件下，相同的罗阿SNR可以实现与显著减少的数据采集时间，采用UV而不是可见光激发。因此，我们建议开发一种新的计算机控制的仪器进行预共振紫外罗阿研究的生物分子。拟议的UV罗阿仪器有可能成为一个重要的通用生物化学和生物医学工具的调查几乎所有的蛋白质和病毒的水溶液的结构和行为。将通过进行不同的基准实验来测试和严格评估其整体性能;将分析来自芳香族氨基酸侧链的足够高质量的测量的预共振UV罗阿蛋白信号，以提取关于其绝对立体化学的有价值的信息。

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.