Investigation of the NADH/NAD⁺ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox

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• 作者：Vijay Tejwani^a ; Franz-Josef Schmitt^b ; Svea Wilkening^b ; Ingo Zebger^b ; Marius Horch^b ; Oliver Lenz^b ; Thomas Friedrich^b ; ^{friedrich@chem.tu-berlin.de}
• 关键词：Ralstonia eutropha ; Fluorescence sensor protein ; NADH ; NAD+ ratio ; Fluorescence lifetime ; Decay-associated spectra ; Hydrogenase
• 刊名：Biochimica et Biophysica Acta (BBA) - Bioenergetics
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：1858
• 期：1
• 页码：86-94
• 全文大小：1006 K
• 卷排序：1858

文摘

The fluorescent sensor Peredox was utilized for the first time to measure the [NADH]:[NAD+] ratio in a bacterial organism, Ralstonia eutropha. The Peredox sensor operates close to its saturation level in aerobically grown R. eutropha cells indicating a high [NADH]:[NAD+] ratio. Comparing Peredox signals in vivo and in R. eutropha lysates, we estimate the free cytosolic [NADH] as 28 µM and [NAD+] as 1.9 mM. Spectrally resolved fluorescence lifetime analysis provides a novel, sensitive tool for determining the [NADH]:[NAD+] using Peredox.