Pauszek Raymond F, Stanley Robert J
Department of Chemistry, Temple University, 250B Beury Hall, 1901N. 13th Street, Philadelphia, PA, 19122, USA.
Methods Mol Biol. 2014;1146:443-66. doi: 10.1007/978-1-4939-0452-5_17.
Flavins and flavoproteins have been studied by a plethora of spectroscopic techniques. Beginning with the characterization of DNA photolyases and the discovery of the diversity of roles played by excited-state flavins in photobiology, the characterization of the electronic excited state of flavins has become increasingly important. In this protocol, we provide a guide to using Stark spectroscopy in obtaining the degree of electronic charge redistribution in simple flavins and in flavoproteins. Stark spectroscopy is technically simpler than more common approaches used to explore the structure of the excited state, considerably cheaper to implement, and yet very powerful in its scope. At the end of this guide, we present data taken on non-photobiological flavoproteins, glutathione reductase and lipoamide dehydrogenase, that suggest that Stark spectroscopy is a unique way to elucidate the electrostatic environment that the flavin cofactor experiences bound inside the protein.
黄素和黄素蛋白已通过大量光谱技术进行了研究。从DNA光解酶的表征以及激发态黄素在光生物学中所起作用的多样性的发现开始，黄素电子激发态的表征变得越来越重要。在本方案中，我们提供了一份指南，介绍如何使用斯塔克光谱法来获得简单黄素和黄素蛋白中电荷重新分布的程度。斯塔克光谱法在技术上比用于探索激发态结构的更常见方法更简单，实施成本要低得多，但其应用范围却非常强大。在本指南的结尾，我们展示了关于非光生物学黄素蛋白、谷胱甘肽还原酶和硫辛酰胺脱氢酶的数据，这些数据表明斯塔克光谱法是阐明黄素辅因子在蛋白质内部结合时所经历的静电环境的独特方法。
