A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD+/NADH in Streptococcus mutans
改进型显色试验测定变异链球菌中的游离胞内NAD+/NADH比率
发布: 2016年08月20日第6卷第16期 DOI: 10.21769/BioProtoc.1902 浏览次数: 14085
评审: Valentine V TrotterSwetha ReddyRolf Lood
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参见作者原研究论文
The authors used this protocol in:
Dec 2015
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Abstract
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 Streptococcus mutans. 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 et al. (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 S. mutans; B. Preparation of deproteinated cell extracts containing NADtotal or NADH; C. NAD+/NADH cycling assay. This method has proven robust in measuring the NAD+/NADH ratio in S. mutans under a variety of conditions, and should be applicable to other Gram-positive bacteria.
Figure 1. Flowchart illustrating protocol Procedure parts B-C
Bacteria (细菌)
Streptococci (链球菌)
Streptococcus mutans (变形链球菌)
Oxidative stress (氧化应激)
Materials and Reagents
- Glass test tubes, 18 x 150 mm (VWR International, catalog number: 60825-443 )
- 50 ml centrifuge tubes (Corning, catalog number: 430829 )
- Screw-top microcentrifuge tubes with O-ring, 2.0 ml (Laboratory Product Sales, catalog number: L233072 )
- Glass beads, 0.1 mm (BioSpec Products, catalog number: 11079101 )
- Centrifugal Filters, 0.5 ml, 10,000 MWCO (Merck Millipore Corporation, Amicon, catalog number: UFC501024 )
- Microcentrifuge tubes, 1.7 ml (Laboratory Product Sales, catalog number: L211511 )
- Streptococcus mutans bacterial strains of interest
- Brain Heart Infusion medium (BHI) (BD, DifcoTM, catalog number: 299070 )
- Bacto-agar (BD, DifcoTM, catalog number: 214010 )
- Tricine (Sigma-Aldrich, catalog number: T5816 )
- Sodium hydroxide (Sigma-Aldrich, catalog number: S5881 )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5134 )
- Sodium chloride (J.T. Baker, catalog number: 3624-01 )
- Thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: M2128 )
- Phenazine ethosulfate (PES) (Sigma-Aldrich, catalog number: P4544 )
- Alcohol dehydrogenase from Saccharomyces cerevisiae (Adh) (Sigma-Aldrich, catalog number: A3263 )
- Ethanol, 200 proof (VWR International, Koptec, catalog number: 64175 )
- Sodium bicarbonate (Sigma-Aldrich, catalog number: S8875 )
- Sodium carbonate (J.T. Baker, catalog number: 3602-01 )
- Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
- Nicotinamide (Sigma-Aldrich, catalog number: N3376 )
- Hydrochloric acid (J.T. Baker, catalog number: 9535 )
- β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH) (Sigma-Aldrich, catalog number: N8129 )
- 1 M tricine-NaOH (see Recipes)
- 40 mM EDTA solution (see Recipes)
- 6 M NaCl (see Recipes)
- 0.1 M NaCl (see Recipes)
- 4.2 mM MTT (see Recipes)
- 16.6 mM PES (see Recipes)
- 100 U/ml Adh (see Recipes)
- NAD+/NADH master mix (see Recipes)
- NAD+/NADH extraction buffer (see Recipes)
- 2 µM NADH (see Recipes)
Equipment
- 3 mm (1 µl) inoculation loops (VWR, catalog number: 50807-020 )
- CO2 incubator (LABEQUIP) (VWR Scientific, catalog number: 10810-744 )
- Erlenmeyer flasks, 250 ml (Pyrex)
- Mini Beadbeater 8 (BioSpec Products, model: Mini Beadbeater 8 )
- Microcentrifuge (Eppendorf, model: 5424 )
- Centrifuge (Fisher Scientific, model: Legend RT) with Thermo swinging bucket rotor (Thermo Fisher Scientific, model: TX-750 )
- 60 °C water bath
- 37 °C water bath
- Vortexer (Vortex Genie 2) (Scientific Industries, model: G560 )
- Dark room
Procedure
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文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Baker, J. L., Faustoferri, R. C. and Quivey, Jr, R. G. (2016). A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD+/NADH in Streptococcus mutans . Bio-protocol 6(16): e1902. DOI: 10.21769/BioProtoc.1902.
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分类
微生物学 > 微生物新陈代谢 > 其它化合物
微生物学 > 微生物生物化学 > 其它化合物
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