Baker Jonathon L, Faustoferri Roberta C, Quivey Robert G
Department of Microbiology & Immunology, University of Rochester School of Medicine and Dentistry, Rochester, USA.
Center for Oral Biology, University of Rochester School of Medicine and Dentistry Rochester, USA.
Bio Protoc. 2016 Aug 20;6(16). doi: 10.21769/BioProtoc.1902.
Nicotinamide adenine dinucleotide is a coenzyme present in all kingdoms of life and exists in two forms: oxidized (NAD) and reduced (NADH). NAD(H) is involved in a multitude of essential metabolic redox reactions, providing oxidizing or reducing equivalents. The ratio of free intracellular NAD/NADH is fundamentally important in the maintenance of cellular redox homeostasis (Ying, 2008). Various chromogenic cycling assays have been used to determine the ratio of NAD/NADH in both bacterial and mammalian cells for more than forty years (Bernofsky and Swan, 1973; Nisselbaum and Green, 1969). Here, we describe in detail an assay to determine the ratio of free intracellular NAD to NADH in This cycling assay is a modified version of the protocol first described by Bernofsky and Swan (Bernofsky and Swan, 1973), using the extraction buffer described by Frezza (2011), followed by the reduced MTT precipitation described by Gibbon and Larher (Gibon and Larher, 1997). As depicted in Figure 1, alcohol dehydrogenase is used to drive a series of redox reactions utilizing exogenously added ethanol and NAD from sample extracts as initial substrates, phenazine ethosulfate (PES) as an electron carrier, and thiazolyl blue tetrazolium bromide (MTT) as a terminal electron acceptor. 6 M NaCl is used to stop the reaction. The reduced MTT (formazan dye) is purple in color and can be quantified by measuring absorbance at 570 nm. This protocol is divided into three steps: A. Preparation of cell pellets of B. Preparation of deproteinated cell extracts containing NAD or NADH; C. NAD/NADH cycling assay. This method has proven robust in measuring the NAD/NADH ratio in under a variety of conditions, and should be applicable to other Gram-positive bacteria.
烟酰胺腺嘌呤二核苷酸是一种存在于所有生命王国中的辅酶，以两种形式存在：氧化型（NAD）和还原型（NADH）。NAD（H）参与众多重要的代谢氧化还原反应，提供氧化或还原当量。细胞内游离NAD/NADH的比例对于维持细胞氧化还原稳态至关重要（Ying，2008年）。四十多年来，各种显色循环测定法已被用于测定细菌和哺乳动物细胞中NAD/NADH的比例（Bernofsky和Swan，1973年；Nisselbaum和Green，1969年）。在此，我们详细描述一种测定[具体细胞或生物]中细胞内游离NAD与NADH比例的方法。这种循环测定法是对Bernofsky和Swan首次描述的方案（Bernofsky和Swan，1973年）的改进版本，使用Frezza（2011年）描述的提取缓冲液，随后是Gibbon和Larher（Gibon和Larher，1997年）描述的还原型MTT沉淀。如图1所示，乙醇脱氢酶用于驱动一系列氧化还原反应，利用样品提取物中外源添加的乙醇和NAD作为初始底物，吩嗪硫酸乙酯（PES）作为电子载体，噻唑蓝四唑溴化物（MTT）作为终端电子受体。6 M NaCl用于终止反应。还原型MTT（甲臜染料）呈紫色，可通过测量570 nm处的吸光度进行定量。该方案分为三个步骤：A. [具体细胞或生物]细胞沉淀的制备；B. 制备含有NAD或NADH的脱蛋白细胞提取物；C. NAD/NADH循环测定。该方法已被证明在各种条件下测量[具体细胞或生物]中的NAD/NADH比例时具有稳健性，并且应该适用于其他革兰氏阳性细菌。
