Ioffe Intstitute, St. Petersburg 194021, Russia.
Institute of Nanobiotechnologies, Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia.
Biomolecules. 2023 Jan 30;13(2):256. doi: 10.3390/biom13020256.
Thorough study of composition and fluorescence properties of a commercial reagent of active equine NAD-dependent alcohol dehydrogenase expressed and purified from has been carried out. Several experimental methods: spectral- and time-resolved two-photon excited fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fast protein liquid chromatography, and mass spectrometry were used for analysis. The reagent under study was found to contain also a number of natural fluorophores: free NAD(P)H, NADH-alcohol dehydrogenase, NADPH-isocitrate dehydrogenase, and pyridoxal 5-phosphate-serine hydroxymethyltransferase complexes. The results obtained demonstrated the potential and limitations of popular optical methods as FLIM for separation of fluorescence signals from free and protein-bound forms of NADH, NADPH, and FAD that are essential coenzymes in redox reactions in all living cells. In particular, NADH-alcohol dehydrogenase and NADPH-isocitrate dehydrogenase complexes could not be optically separated in our experimental conditions although fast protein liquid chromatography and mass spectrometry analysis undoubtedly indicated the presence of both enzymes in the molecular sample used. Also, the results of fluorescence, fast protein liquid chromatography, and mass spectrometry analysis revealed a significant contribution of the enzyme-bound coenzyme pyridoxal 5-phosphate to the fluorescence signal that could be separated from enzyme-bound NADH by using bandpass filters, but could effectively mask contribution from enzyme-bound FAD because the fluorescence spectra of the species practically overlapped. It was shown that enzyme-bound pyridoxal 5-phosphate fluorescence can be separated from enzyme-bound NAD(P)H and FAD through analysis of short fluorescence decay times of about tens of picoseconds. However, this analysis was found to be effective only at relatively high number of peak photon counts in recorded fluorescence signals. The results obtained in this study can be used for interpretation of fluorescence signals from a mixture of enzyme-bound fluorophores and should be taken into consideration when determining the intracellular NADH/FAD ratio using FLIM.
对从 表达和纯化的商业试剂的活性马 NAD 依赖性醇脱氢酶的组成和荧光性质进行了深入研究。使用了几种实验方法：光谱和时间分辨双光子激发荧光、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、快速蛋白质液相色谱和质谱分析。研究用试剂还含有一些天然荧光团：游离 NAD(P)H、NADH-醇脱氢酶、NADPH-异柠檬酸脱氢酶和吡哆醛 5-磷酸-丝氨酸羟甲基转移酶复合物。结果表明，流行的光学方法（如 FLIM）具有分离还原反应中所有活细胞中必需辅酶 NADH、NADPH 和 FAD 的游离和蛋白结合形式的荧光信号的潜力和局限性。特别是，尽管快速蛋白质液相色谱和质谱分析无疑表明在所用分子样品中存在这两种酶，但在我们的实验条件下，NADH-醇脱氢酶和 NADPH-异柠檬酸脱氢酶复合物无法通过光学方法分离。此外，荧光、快速蛋白质液相色谱和质谱分析的结果表明，酶结合辅酶吡哆醛 5-磷酸对荧光信号有显著贡献，可通过使用带通滤波器从酶结合的 NADH 中分离出来，但由于这些物质的荧光光谱实际上重叠，因此可以有效地掩盖酶结合的 FAD 的贡献。结果表明，通过分析约数十皮秒的短荧光衰减时间，可以将酶结合的吡哆醛 5-磷酸荧光与酶结合的 NAD(P)H 和 FAD 分离。然而，这种分析仅在记录荧光信号中的峰值光子计数数较高时才有效。本研究的结果可用于解释酶结合荧光团混合物的荧光信号，在使用 FLIM 确定细胞内 NADH/FAD 比时应考虑这些结果。
