Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain.
Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, Zaragoza, Spain.
Methods Mol Biol. 2021;2280:135-155. doi: 10.1007/978-1-0716-1286-6_9.
Anaerobic stopped-flow (SF) spectrophotometry is a powerful biophysical tool that allows a complete kinetic characterization of protein interactions with other molecules when they are in different redox states, as well as of the redox processes consequence of such interactions. Differences in the absorption spectroscopic properties of oxidized, semiquinone and hydroquinone states of flavoproteins, as well as the appearance of transient spectroscopic features produced by the flavin cofactor during substrate binding and electron transfer processes, have made SF a suitable technique for kinetically dissecting their mechanisms of reaction. In addition, SF coupled to photodiode array detection, enables kinetic data collection in a wavelength range. When such type of data are available for a flavoprotein reaction, they allow for obtaining detailed information of individual reaction steps, including intermolecular dissociation constants as well as electron transfer rate constants. Methodologies for the mechanistic characterization of flavoproteins involved in redox processes by SF spectrophotometry are described in this chapter.
厌氧停流（SF）分光光度法是一种强大的生物物理工具，当蛋白质与其他分子处于不同氧化还原状态时，它可以完全对蛋白质与其他分子的相互作用进行动力学特征分析，也可以对这种相互作用导致的氧化还原过程进行分析。黄素蛋白的氧化态、半醌态和氢醌态在吸收光谱特性上存在差异，以及黄素辅因子在底物结合和电子转移过程中产生的瞬态光谱特征的出现，使得 SF 成为一种适合于动力学剖析其反应机制的技术。此外，SF 与光电二极管阵列检测相结合，能够在波长范围内收集动力学数据。当黄素蛋白反应有这种类型的数据时，它们可以提供有关单个反应步骤的详细信息，包括分子间离解常数和电子转移速率常数。本章描述了通过 SF 分光光度法对参与氧化还原过程的黄素蛋白进行机制特征分析的方法。
