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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   

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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

Keywords: NADH oxidase(NADH氧化酶), Bacteria(细菌), Streptococci(链球菌), Streptococcus mutans(变形链球菌), Oxidative stress(氧化应激)

Materials and Reagents

  1. Glass test tubes, 18 x 150 mm (VWR International, catalog number: 60825-443 )
  2. 50 ml centrifuge tubes (Corning, catalog number: 430829 )
  3. Screw-top microcentrifuge tubes with O-ring, 2.0 ml (Laboratory Product Sales, catalog number: L233072 )
  4. Glass beads, 0.1 mm (BioSpec Products, catalog number: 11079101 )
  5. Centrifugal Filters, 0.5 ml, 10,000 MWCO (Merck Millipore Corporation, Amicon, catalog number: UFC501024 )
  6. Microcentrifuge tubes, 1.7 ml (Laboratory Product Sales, catalog number: L211511 )
  7. Streptococcus mutans bacterial strains of interest
  8. Brain Heart Infusion medium (BHI) (BD, DifcoTM, catalog number: 299070 )
  9. Bacto-agar (BD, DifcoTM, catalog number: 214010 )
  10. Tricine (Sigma-Aldrich, catalog number: T5816 )
  11. Sodium hydroxide (Sigma-Aldrich, catalog number: S5881 )
  12. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  13. Sodium chloride (J.T. Baker, catalog number: 3624-01 )
  14. Thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: M2128 )
  15. Phenazine ethosulfate (PES) (Sigma-Aldrich, catalog number: P4544 )
  16. Alcohol dehydrogenase from Saccharomyces cerevisiae (Adh) (Sigma-Aldrich, catalog number: A3263 )
  17. Ethanol, 200 proof (VWR International, Koptec, catalog number: 64175 )
  18. Sodium bicarbonate (Sigma-Aldrich, catalog number: S8875 )
  19. Sodium carbonate (J.T. Baker, catalog number: 3602-01 )
  20. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  21. Nicotinamide (Sigma-Aldrich, catalog number: N3376 )
  22. Hydrochloric acid (J.T. Baker, catalog number: 9535 )
  23. β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH) (Sigma-Aldrich, catalog number: N8129 )
  24. 1 M tricine-NaOH (see Recipes)
  25. 40 mM EDTA solution (see Recipes)
  26. 6 M NaCl (see Recipes)
  27. 0.1 M NaCl (see Recipes)
  28. 4.2 mM MTT (see Recipes)
  29. 16.6 mM PES (see Recipes)
  30. 100 U/ml Adh (see Recipes)
  31. NAD+/NADH master mix (see Recipes)
  32. NAD+/NADH extraction buffer (see Recipes)
  33. 2 µM NADH (see Recipes)

Equipment

  1. 3 mm (1 µl) inoculation loops (VWR, catalog number: 50807-020 )
  2. CO2 incubator (LABEQUIP) (VWR Scientific, catalog number: 10810-744 )
  3. Erlenmeyer flasks, 250 ml (Pyrex)
  4. Mini Beadbeater 8 (BioSpec Products, model: Mini Beadbeater 8 )
  5. Microcentrifuge (Eppendorf, model: 5424 )
  6. Centrifuge (Fisher Scientific, model: Legend RT) with Thermo swinging bucket rotor (Thermo Fisher Scientific, model: TX-750 )
  7. 60 °C water bath
  8. 37 °C water bath
  9. Vortexer (Vortex Genie 2) (Scientific Industries, model: G560 )
  10. Dark room

Procedure

  1. Preparation of S. mutans cell pellets (Note 1)
    1. The S. mutans bacterial strains of interest were streaked for isolation from frozen culture stocks, on BHI plates (37 g/L BHI; 15 g/L Bacto agar) using 1 µl loops and incubated overnight at 37 °C in a 5% (v/v) CO2/95% air incubator.
    2. Using sterile technique, an isolated colony was picked with a 1 µl loop and used to inoculate 5 ml BHI in a sterile glass test tube. Cultures were grown overnight 37 °C in a 5% (v/v) CO2/95% air incubator (Note 2).
    3. Using sterile technique, the 5 ml overnight culture was used to inoculate 45 ml BHI in a sterile 125 ml culture flask. Cultures were grown at 37 °C in a 5% (v/v) CO2/95% air incubator until the absorbance of the culture at 600 nm (OD600) reached ~0.6 (mid-log phase growth, roughly 4 h for type strain UA159).
    4. Cultures were transferred to 50 ml conical centrifuge tubes and cells were harvested by centrifugation at 4,000 rpm (2,272 x g) for 15 min at 4 °C. Cell pellets were placed at -80 °C until the assays were performed.

  2. Preparation of cell extracts from S. mutans
    1. Frozen cell pellets were thawed on ice and resuspended in 1 ml ice-cold NAD+/NADH extraction buffer (Frezza et al., 2011).
    2. Samples were transferred into 2.0 ml screw-capped microcentrifuge tubes containing 0.5 g glass beads.
    3. Samples were homogenized twice in a Mini Beadbeater on the “homogenize” speed setting at 4 °C for a 30 sec cycle, followed by a 2 min incubation on ice, then another 30 sec cycle.
    4. Homogenized samples were centrifuged at 4 °C in a microcentrifuge at 14,000 x g for 5 min.
    5. Supernatants were removed and transferred to a 10,000 MWCO centrifugal filter (Note 3).
    6. Supernatant was passed through the filter by centrifugation at 14,000 x g at 4 °C. With the sample size used, the filtration typically took about 30 min.
    7. The filtrate was split into two equal volumes in fresh microcentrifuge tubes (~400 µl each), one for determination of total NAD/NADH (NADtotal), and one for determination of NADH. From this point until step C7, the protocol must be carried out in a dark room (Note 4).
    8. The sample aliquot for NADH only was incubated in a 60 °C water bath for 30 min to remove NAD+ then transferred to ice (NAD+ is heat labile, NADH is not). The aliquot for determination of NADtotal was maintained on ice throughout this step (Note 5).

  3. NAD+/NADH cycling assay
    1. Aliquot 500 µl of master mix (Gibon and Larher, 1997) into an appropriate number of microcentrifuge tubes (Note 5).
    2. Add 100 µl of sample, NADH standard, or NAD+/NADH extraction buffer (for blank) to each microcentrifuge tube containing master mix.
    3. Bring each reaction volume to 900 µl with 0.1 M NaCl and vortex briefly.
    4. Incubate reactions in a 37 °C water bath for 5 min (Note 6).
    5. Add 100 µl of Adh (containing 10 U) to each reaction and vortex briefly.
    6. Incubate reactions in a 37 °C water bath for 40 min.
    7. Add 500 µl of 6 M NaCl to stop each reaction and precipitate the reduced MTT. Vortex briefly. The reactions are no longer light sensitive and the remainder of the protocol may be carried out under standard lighting conditions.
    8. Centrifuge stopped reactions at 4 °C and 10,000 x g for 5 min.
    9. Decant tubes (Note 7) and resuspend pellets in 1 ml 200 proof ethanol.
    10. Analyze samples and standards for absorbance at 570 nm, using the sample containing 100 µl NAD+/NADH extraction buffer as a blank. Generate a standard curve using known quantities of NADH (added in step C2 above) and use to calculate the concentration of unknown samples. Calculate NAD+ via NADtotal-NADH.

Notes

  1. Part A of this protocol describes preparing S. mutans cells grown in batch culture, however the authors expect the downstream portion of the protocol (Part B and further) to function for any Gram-positive bacterial species and growth condition. In addition to S. mutans batch cultures, the authors have also successfully performed the assay on samples of S. mutans grown in continuous cultures.
  2. As a facultative anaerobe, Streptococcus mutans is typically grown without agitation.
  3. This step removes cell debris and protein from the cell extracts, preventing sequestration, degradation, oxidation, or reduction of the NAD(H).
  4. From this point forward until step C7, the protocol must be performed in a dark room, with only an indirect light source to prevent oxidation of NAD(H), MTT, and PES.
  5. The NAD+/NADH master mix and NADH standards were prepared fresh for every assay as described in Recipes during the 30 min incubation described in step B8. An assay should be performed on several dilutions of each sample to ensure that the resulting absorbance values fall in within the linear range of the generated standard curve. A representative standard curve is displayed in Figure 2. The authors used solutions of 0 pmol, 10 pmol, 25 pmol, 50 pmol, 100 pmol, and 200 pmol to generate the standard curve. As there is generally a greater proportion of free intracellular NAD+ than NADH, it is likely the NADtotal samples will need to be diluted to a greater extent than the NADH samples. In addition to 100 µl of non-diluted sample, the authors recommend performing assays on 100 µl of 1:5, 1:10, and 1:50 dilutions of NADtotal samples and 100 µl of 1:2, 1:5 and 1:10 dilutions for NADH samples. For each sample and standard, three replicate assays are performed. A negative control reaction must also be performed (to use as a blank in the spectrophotometer) using 100 µl NAD+/NADH extraction buffer.


    Figure 2. Example of standard curve generated by several known concentrations of NADH

  6. Incubation at 37 °C results in oxidization of all the NADH into NAD+.
  7. Samples are no longer highly light-sensitive following this step and the remainder of the protocol can be performed in standard lighting conditions.

Recipes

  1. 1 M tricine-NaOH (pH 8)
    Add 17.92 g tricine to 80 ml H2O
    Adjust pH to 8.0 with NaOH
    Bring volume of solution to 100 ml with H2O. Store at RT.
  2. 40 mM EDTA
    Add 1.49 g EDTA to 80 ml H2O
    Bring volume of solution to 100 ml with H2O. Store at RT.
  3. 6 M NaCl
    Add 35.04 g NaCl to 80 ml H2O
    Bring volume of solution to 100 ml with H2O. Store at RT.
  4. 0.1 M NaCl
    Add 0.584 g NaCl to 80 ml H2O
    Bring volume of solution to 100 ml with H2O. Store at RT.
  5. 4.2 mM MTT
    Add 0.0087 g MTT to 5 ml H2O
    Prepare fresh each time the protocol is performed and keep in the dark.
  6. 16.6 mM PES
    Add 0.028 g PES to 5 ml H2O
    Prepare fresh each time the protocol is performed and keep in the dark.
  7. 100 U/ml Adh
    Add appropriate amount of Adh powder to 0.1 M tricine-NaOH (pH 8) to get 100 U/ml (units of activity per mg solid can be calculated from provided total units and mg solid provided on the label of each tube of enzyme).
    Prepare fresh each time the protocol is performed.
  8. NAD+/NADH master mix
    Prepare fresh each time the protocol is performed.
    Prepare a volume based upon the number of reactions to be performed (500 µl for each reaction).
    Must be mixed and stored in the dark.
    Per reaction add:
    100 µl 1 M tricine-NaOH (pH 8)
    100 µl 40 mM EDTA
    100 µl 0.1 M NaCl
    100 µl 4.2 mM MTT
    100 µl 16.6 mM PES
    100 µl 200 proof ethanol
  9. NAD+/NADH extraction buffer pH 10.3 (100 ml)
    To 80 ml H2O add:
    0.168 g sodium bicarbonate (final concentration of 20 mM)
    1.06 g sodium carbonate (final concentration of 100 mM)
    50 µl Triton-X 100 (final concentration of 0.05%)
    0.122 g nicotinamide (final concentration of 10 mM)
    After reagents are dissolved, adjust pH to 10.3 with 1 M HCl.
    Bring to 100 ml final volume.
    Store at RT.
  10. 2 µM NADH (for standard curve preparation)
    Add 0.007 mg to 5 ml H2O
    This quantity results in a 1,000x solution, so 3 serial dilutions (100 µl into 900 µl H2O) are necessary in order to get a 1x working stock.
    Standard curve
    0 pmol NADH = 1 ml H2O
    10 pmol NADH = 5 µl of 2 µM NADH into 995 µl H2O
    25 pmol NADH = 12.5 µl of 2 µM NADH into 987.5 µl H2O
    50 pmol NADH = 25 µl of 2 µM NADH into 975 µl H2O
    100 pmol NADH = 50 µl of 2 µM NADH into 950 µl H2O
    200 pmol NADH = 100 µl of 2 µM NADH into 900 µl H2O

Acknowledgments

This work was supported by the Training Program in Oral Sciences, NIH/NIDCR T90-DE021985 (J. L. B.), and by NIH/NIDCR DE-13683 (R. G. Q), NIH/NIDCR DE-17425 (R. G. Q.).
This cycling assay is a modified version of the protocol first described by 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 (1977).

References

  1. Bernofsky, C. and Swan, M. (1973). An improved cycling assay for nicotinamide adenine dinucleotide. Anal Biochem 53(2): 452-458.
  2. Frezza, C., Zheng, L., Tennant, D. A., Papkovsky, D. B., Hedley, B. A., Kalna, G., Watson, D. G. and Gottlieb, E. (2011). Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival. PLoS One 6(9): e24411.
  3. Gibon, Y. and Larher, F. (1997). Cycling assay for nicotinamide adenine dinucleotides: NaCl precipitation and ethanol solubilization of the reduced tetrazolium. Anal Biochem 251(2): 153-157.
  4. Nisselbaum, J. S. and Green, S. (1969). A simple ultramicro method for determination of pyridine nucleotides in tissues. Anal Biochem 27(2): 212-217.
  5. Ying, W. (2008). NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal 10(2): 179-206.

简介

烟酰胺腺嘌呤二核苷酸是存在于生命的所有王国中的辅酶,并且以两种形式存在:氧化(NAD +)和还原(NADH)。 NAD(H)参与多种必需的代谢氧化还原反应,提供氧化或还原等价物。游离细胞内NAD + /NADH的比率在维持细胞氧化还原稳态中是根本重要的(Ying,2008)。已经使用各种显色循环测定来测定细菌和哺乳动物细胞中NAD +/NAD +/NADH的比率超过四十年(Bernofsky和Swan,1973; Nisselbaum和Green,1969)。 />  在这里,我们详细描述了测定变异链球菌中游离细胞内NAD + 与NADH的比率的测定法。该循环测定是Bernofsky和Swan(Bernofsky和Swan,1973)首先描述的方案的修改版本,使用Frezza等人描述的提取缓冲液。 (2011),随后是由Gibbon和Larher描述的减少的MTT降水(Gibon和Larher,1997)。如图1所示,使用乙醇脱氢酶来驱动一系列氧化还原反应,利用来自样品提取物的外源添加的乙醇和NAD +作为初始底物,吩嗪乙硫酸盐(PES)作为电子载体,以及噻唑基蓝色四唑溴化物(MTT)作为末端电子受体。使用6M NaCl来停止反应。还原的MTT(甲an染料)是紫色的,并且可以通过测量570nm处的吸光度来定量。该方案分为三个步骤:A.制备S的细胞沉淀。 mutans ; B.含有NAD总NAD或NADH的去蛋白的细胞提取物的制备; NAD +循环测定。这种方法已经证明在测量NAD中的NAD + /NADH比例是稳健的。变异体,并应适用于其他革兰氏阳性菌。





图1.流程图说明协议过程部分BC >

关键字:NADH氧化酶, 细菌, 链球菌, 变形链球菌, 氧化应激

材料和试剂

  1. 玻璃试管,18×150mm(VWR International,目录号:60825-443)
  2. 50ml离心管(Corning,目录号:430829)
  3. 带有O形环的螺旋顶微量离心管,2.0ml(Laboratory Product Sales,目录号:L233072)
  4. 玻璃珠,0.1mm(BioSpec Products,目录号:11079101)
  5. 离心过滤器,0.5ml,10,000MWCO(Merck Millipore Corporation,Amicon,目录号:UFC501024)
  6. 微量离心管,1.7ml(Laboratory Product Sales,目录号:L211511)
  7. 感兴趣的变异链球菌菌株
  8. 脑心浸液培养基(BHI)(BD,Difco TM ,目录号:299070)
  9. 细菌琼脂(BD,Difco TM ,目录号:214010)
  10. Tricine(Sigma-Aldrich,目录号:T5816)
  11. 氢氧化钠(Sigma-Aldrich,目录号:S5881)
  12. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E5134)
  13. 氯化钠(J.T.Baker,目录号:3624-01)
  14. 噻唑基蓝四唑溴化物(MTT)(Sigma-Aldrich,目录号:M2128)
  15. 吩嗪硫酸乙酯(PES)(Sigma-Aldrich,目录号:P4544)
  16. 来自酿酒酵母的醇脱氢酶(Adh)(Sigma-Aldrich,目录号:A3263)
  17. 乙醇,200proof(VWR International,Koptec,目录号:64175)
  18. 碳酸氢钠(Sigma-Aldrich,目录号:S8875)
  19. 碳酸钠(J.T.Baker,目录号:3602-01)
  20. Triton X-100(Sigma-Aldrich,目录号:T8787)
  21. 烟酰胺(Sigma-Aldrich,目录号:N3376)
  22. 盐酸(J.T.Baker,目录号:9535)
  23. β-烟酰胺腺嘌呤二核苷酸,还原二钠盐水合物(NADH)(Sigma-Aldrich,目录号:N8129)
  24. 1 M tricine-NaOH(见配方)
  25. 40 mM EDTA溶液(参见配方)
  26. 6 M NaCl(见配方)
  27. 0.1 M NaCl(见配方)
  28. 4.2 mM MTT(参见配方)
  29. 16.6 mM PES(见配方)
  30. 100 U/ml Adh(参见配方)
  31. NAD + /NADH主混合物(参见配方)
  32. NAD + /NADH提取缓冲液(参见配方)
  33. 2μMNADH(参见配方)

设备

  1. (1μl)接种环(VWR,目录号:50807-020)
  2. CO 2培养箱(LABEQUIP)(VWR Scientific,目录号:10810-744)
  3. 锥形烧瓶,250ml(Pyrex)
  4. Mini Beadbeater 8(BioSpec Products,型号:Mini Beadbeater 8)
  5. 微量离心机(Eppendorf,型号:5424)
  6. 用Thermo摆动转子(Thermo Fisher Scientific,型号:TX-750)离心(Fisher Scientific,型号:Legend RT)
  7. 60°C水浴
  8. 37°C水浴
  9. Vortexer(Vortex Genie 2)(Scientific Indudtries,型号:G560)
  10. 暗室

程序

  1. 制备S。 mutans 细胞沉淀(注1)
    1. S。 使用1μl环在BHI平板(37g/L BHI; 15g/L Bacto琼脂)上将感兴趣的变异菌株感兴趣的菌株划线,以从冷冻培养物储备液中分离,并在37℃下在5% (v/v)CO 2/95%空气培养箱
    2. 使用无菌技术,用1μl环挑取分离的菌落,并用于在无菌玻璃试管中接种5ml BHI。 培养物在37℃下在5%(v/v)CO 2/95% 空气培养箱(注2)。
    3. 使用无菌技术,将5ml过夜培养物用于在无菌125ml培养瓶中接种45ml BHI。 培养物在37℃下在5%(v/v)CO 2/95%空气培养箱中生长,直到培养物在600nm处的吸光度(OD大于600) 达到〜0.6(中期对数期生长,对于菌株UA159约4小时)
    4. 将培养物转移到50ml锥形离心管中,通过在4℃下以4,000rpm(2,272×g)离心15分钟收获细胞。 将细胞沉淀置于-80℃直至进行测定
  2. 来自S的细胞提取物的制备。 变异
    1. 将冷冻的细胞沉淀在冰上解冻,并重悬浮于1ml冰冷的NAD + +/NADH提取缓冲液中(Frezza等人,2011)。
    2. 将样品转移到含有0.5g玻璃珠的2.0ml带螺旋盖的微量离心管中
    3. 将样品在Mini Beadbeater中在4℃下"匀化"速度设置均化30秒循环,随后在冰上孵育2分钟,然后再在另一个30秒循环中匀化两次。
    4. 将匀化的样品在4℃下在微量离心机中以14,000×g离心5分钟。
    5. 除去上清液并转移到10,000MWCO离心过滤器(注3)
    6. 将上清液在4℃以14,000×g离心通过过滤器。使用样品尺寸,过滤通常需要约30分钟。
    7. 将滤液在新鲜的微量离心管(每个〜400μl)中分成两个等体积,一个用于测定总NAD/NADH(NAD总共),一个用于测定NADH。从这一点直到步骤C7,协议必须在暗室中进行(注4)。
    8. 仅将NADH的样品等分试样在60℃水浴中温育30分钟以除去NAD +,然后转移至冰(NAD +是热不稳定的,NADH不是 )。 在整个步骤(注释5)中,将用于测定NAD总量的等分试样保持在冰上。

  3. NAD + /NADH循环试验
    1. 将500μl主混合物(Gibon和Larher,1997)分装到适当数量的微量离心管(注5)中。
    2. 向含有主混合物的每个离心管中加入100μl样品,NADH标准品或NAD + /NADH提取缓冲液(空白)。
    3. 用0.1 M NaCl使每个反应体积为900μl,短暂涡旋
    4. 在37℃水浴中孵育反应5分钟(注6)
    5. 向每个反应中加入100μlAdh(含10U),短暂涡旋
    6. 在37℃水浴中孵育反应40分钟
    7. 加入500微升6 M NaCl停止每个反应,沉淀还原的MTT。 短暂涡旋。 反应不再对光敏感,并且方案的其余部分可以在标准光照条件下进行
    8. 离心机在4℃和10,000xg下停止反应5分钟
    9. 滗析管(注7),并将颗粒重悬于1ml 200级乙醇中
    10. 使用含有100μlNAD + sup/NAD//NADH提取缓冲液的样品作为空白,分析样品和标准物在570nm处的吸光度。 使用已知量的NADH(在上述步骤C2中添加)产生标准曲线,并用于计算未知样品的浓度。 通过NAD - -NADH计算NAD +

笔记

  1. 本协议的A部分描述了制备S。 但是作者期望方案的下游部分(部分B和进一步)用于任何革兰氏阳性细菌物种和生长条件。 除了 S。 突变体分批培养,作者还成功地对 S样品进行了测定。 变异体在连续培养中生长
  2. 作为兼性厌氧菌,变异链球菌通常在无搅拌下生长。
  3. 这一步骤从细胞提取物中除去细胞碎片和蛋白质,防止多价螯合,降解,氧化或NAD(H)的减少。
  4. 从这一点直到步骤C7,协议必须在暗室中进行,只有间接光源,以防止NAD(H),MTT和PES的氧化。
  5. 如步骤B8中所述的30分钟孵育期间的配方中所述,对于每个测定新鲜制备NAD +主要混合物和NADH标准品。应对每个样品的几个稀释液进行测定,以确保所得吸光度值落在所生成的标准曲线的线性范围内。代表性的标准曲线显示在图2中。作者使用0pmol,10pmol,25pmol,50pmol,100pmol和200pmol的溶液以产生标准曲线。由于通常比NADH具有更大比例的游离细胞内NAD +,因此可能NAD +总样品将需要比NADH样品更大程度的稀释。除了100μl的非稀释样品外,作者建议在100μl的1:5,1:10和1:50稀释的NAD 总样品和100μl的1: 2,1:5和1:10稀释的NADH样品。对于每个样品和标准品,进行三次重复测定。还必须使用100μlNAD + /NADH提取缓冲液进行阴性对照反应(以在分光光度计中用作空白)。

    图2.由几种已知浓度的NADH
    产生的标准曲线的实例
  6. 在37℃孵育导致所有NADH氧化为NAD + sup/+。
  7. 在此步骤之后,样品不再具有高的光敏性,并且可以在标准照明条件下进行方案的其余部分

食谱

  1. 1M Tris-NaOH(pH8)
    向80ml H 2 O中加入17.92g三辛胺 用NaOH调节pH至8.0 用H 2 O使溶液体积为100ml。 在RT存储。
  2. 40mM EDTA
    将1.49g EDTA加入到80ml H 2 O中 用H 2 O使溶液体积为100ml。 存储在RT。
  3. 6 M NaCl
    将35.04g NaCl加入到80ml H 2 O中 用H 2 O使溶液体积为100ml。 存储在RT。
  4. 0.1 M NaCl
    将0.584g NaCl加入到80ml H 2 O中 用H 2 O使溶液体积为100ml。 存储在RT。
  5. 4.2 mM MTT
    将0.0087g MTT加入到5ml H 2 O中 每次执行协议时保持新鲜,并保持在黑暗中。
  6. 16.6mM PES
    将0.028g PES加入到5ml H 2 O中 每次执行协议时保持新鲜,并保持在黑暗中。
  7. 100 U/ml Adh
    将适当量的Adh粉末加入到0.1M tricine-NaOH(pH8)中以获得100U/ml(每mg固体的活性单位可以从提供的总单位和在每个酶管的标签上提供的mg固体计算)。
    每次执行协议时准备新鲜。
  8. NAD + /NADH主混合物
    每次执行协议时准备新鲜。
    基于待进行的反应的数量(每个反应500μl)制备体积。
    必须混合并储存在黑暗中。
    每个反应加:
    100μl1M tricine-NaOH(pH8)
    100μl40mM EDTA
    100μl0.1M NaCl
    100μl4.2mM MTT
    100μl16.6mM PES
    100μl200标准乙醇
  9. NAD +提取缓冲液pH10.3(100ml) 向80ml H 2 O中加入:
    0.168g碳酸氢钠(终浓度20mM) 1.06g碳酸钠(终浓度100mM) 50μlTriton-X 100(终浓度0.05%) 0.122g烟酰胺(终浓度为10mM) 试剂溶解后,用1M HCl调节pH至10.3 加至最终体积为100ml。
    存储在RT。
  10. 2μMNADH(用于标准曲线制备)
    加入0.007mg至5ml H 2 O x/v 该量产生1000倍的溶液,因此需要3个连续稀释(100μl加入900μlH 2 O)以获得1倍工作原料。
    标准曲线
    0pmol NADH = 1ml H 2 O 2 / 10pmol NADH =5μl2μMNADH加入995μlH 2 O中
    25pmol NADH =12.5μl的2μMNADH加入到987.5μlH 2 O中
    50pmol NADH =25μl的2μMNADH加入到975μlH 2 O中
    100pmol NADH =50μl的2μMNADH加入到950μlH 2 O中
    200pmol NADH =100μl2μMNADH加入900μlH 2 O中

致谢

这项工作得到了NIH/NIDCR T90-DE021985(J.L.B。)培训计划和NIH/NIDCR DE-13683(R.G.Q),NIH/NIDCR DE-17425(R.G.Q.)的支持。
该循环测定是Bernofsky和Swan(1973)首先描述的方案的修改版本,使用Frezza等人描述的提取缓冲液 (2011),随后是由Gibbon和Larher(1977)描述的减少的MTT沉淀。

参考文献

  1. Bernofsky,C。和Swan,M。(1973)。  用于烟酰胺腺嘌呤二核苷酸的改进的循环试验。 Anal Biochem 53(2):452-458。
  2. Freestyle,C.,Zheng,L.,Tennant,DA,Papkovsky,DB,Hedley,BA,Kalna,G.,Watson,DG和Gottlieb,E。 href ="http://www.ncbi.nlm.nih.gov/pubmed/21912692"target ="_ blank">缺氧细胞的代谢分析揭示了细胞存活所需的分解代谢特征。 6(9):e24411。
  3. Gibon,Y。和Larher,F。(1997)。  烟酰胺腺嘌呤二核苷酸的循环测定:还原四唑的NaCl沉淀和乙醇溶解。 Anal Biochem 251(2):153-157。
  4. Nisselbaum,JS和Green,S.(1969)。  一种用于测定组织中吡啶核苷酸的简单超微方法。 Anal Biochem 27(2):212-217。
  5. Ying,W。(2008)。  NAD + /NADH和NADP + /NADPH在细胞功能和细胞死亡中的表达:调节和生物学结果 Antioxid Redox Signal 10 179-206。
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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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