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

虽然我们已经证明了非共振拉曼光学活性（ROA），利用可见激发，从而避免了被研究生物样品中存在的发色团对入射激光辐射的吸收，已经发展成为一种强大的生物分子新光谱学。该技术的更广泛使用受到几个基本问题的阻碍，如样品荧光痕迹的光谱干扰和需要相对较高的样品浓度，因此需要相当长的测量时间。然而，通过对典型蛋白质的紫外光谱特征的详细实验研究，以及对这些特征对预期ROA信噪比（SNR）的影响的仔细检查，我们确信，通过采用预共振测量制度，紫外激发与不同吸收带包络末端之间的电子跃迁相吻合，可以消除这些限制。在低得多的样品浓度下，但在其他可比较的实验条件下，使用紫外而不是可见光激发大大减少了数据采集时间，可以实现相同的ROA信噪比。因此，我们建议开发一种新的计算机控制仪器，用于进行生物分子的预共振紫外ROA研究。提出的紫外ROA仪器有可能成为一种重要的通用生化和生物医学工具，用于研究几乎所有蛋白质和病毒的水溶液结构和行为。它的整体性能将通过执行不同的基准实验进行测试和严格评估；从芳香氨基酸侧链中测量到的高质量的预共振紫外ROA蛋白信号将被分析，以提取有关其绝对立体化学的有价值信息。

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.