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NADPH oxidase is a membrane-bound enzyme that generates (O2-) by transferring electrons from NADPH to molecular oxygen O2. O2-is spontaneously dismasted to the more stable form H2O2. Both O2-and H2O2 are forms ofreactive oxygen species (ROS), which are involved in regulation of many cellular activities such as transcription, intracellular signaling, and host defense. The NADPH oxidase - dependent generation of O2- in total membrane fraction of plant tissue has been determined by the reduction of the tetrazolium salt XTT by O2-. In the presence of O2-, XTT generates a soluble yellow formazan that can be quantified spectrophotometrically.

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Measurement of NADPH Oxidase Activity in Plants

Plant Science > Plant biochemistry > Protein > Activity
Authors: Amita Kaundal
Amita KaundalAffiliation: Plant Biology Department, The Samuel Roberts Noble Foundation, Ardmore, USA
Bio-protocol author page: a132
Clemencia M. Rojas
Clemencia M. RojasAffiliation: Plant Biology Department, The Samuel Roberts Noble Foundation, Ardmore, USA
Bio-protocol author page: a133
 and Kirankumar S. Mysore
Kirankumar S. MysoreAffiliation: Plant Biology Department, The Samuel Roberts Noble Foundation, Ardmore, USA
For correspondence: ksmysore@noble.org
Bio-protocol author page: a134
Vol 2, Iss 20, 10/20/2012, 7015 views, 1 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.278

[Abstract] NADPH oxidase is a membrane-bound enzyme that generates (O2-) by transferring electrons from NADPH to molecular oxygen O2. O2-is spontaneously dismasted to the more stable form H2O2. Both O2-and H2O2 are forms ofreactive oxygen species (ROS), which are involved in regulation of many cellular activities such as transcription, intracellular signaling, and host defense. The NADPH oxidase - dependent generation of O2- in total membrane fraction of plant tissue has been determined by the reduction of the tetrazolium salt XTT by O2-. In the presence of O2-, XTT generates a soluble yellow formazan that can be quantified spectrophotometrically.

Materials and Reagents

  1. Sucrose
  2. HEPES
  3. EDTA
  4. DTT
  5. L-cysteine
  6. MgCl2
  7. PVP
  8. Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (F. Hoffmann-La Roche, catalog number: 04693159001)
  9. BSA
  10. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006)
  11. Tris-HCl
  12. Sodium 3,3'-( -[(phenylamino)carbonyl] -3,4-tetrazolium)-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) (Sigma-Aldrich, catalog number: X4626)
  13. NADPH (Sigma-Aldrich, catalog number: N1630)
  14. Protein extraction working solution (see Recipes)

Equipment

  1. Microtiter plate reader (Infinite M200 Pro, Tecan)
  2. Microcentrifuge (AqquSpin Micro R) (Thermo Fisher Scientific)
  3. Ultracentrifuge (Optima TLX, Beckman)
  4. Microtiter plate (BD Biosciences, catalog number: 353075)

Procedure

  1. Protein extraction and separation of membrane fraction from plant tissues
    1. Harvest tissue in liquid nitrogen. If not used immediately, keep at -80 °C until processing.
    2. Grind tissue in liquid nitrogen and weigh out 0.5 g of the ground tissues in empty Falcon tube that has been pre-chilled in liquid nitrogen and used to tare the scale.
    3. Add 6 ml of ice-cold protein extraction buffer to ground tissues on ice.
    4. Vortexat room temperature to mix thoroughly.
    5. Filter homogenized tissue through four layers of cheese cloth and transfer filtrate (flow-through) to 2-ml microcentrifuge tubes, on ice.
    6. Centrifuge at 10,000 x g for 45 min at 4 °C and transfer supernatant to ultra- centrifuge tube.
    7. Separate total membrane fractions by ultra-centrifugation at 203,000 x g for 60 min at 4 °C.
    8. Discard supernatant and resuspend pellet in 1 ml ice-cold 10 mM Tris-HCl (pH 7.4).

  2. Protein estimation using Bradford microassay (160 μl)
    1. Prepare BSA standards ranging from 5 μg-25 μg/ml as follows:



      These standards will be used to generate a standard curve.
    2. Use 96 well microtiter plate to prepare reaction mix.
    3. Prepare blank byadding 160 μl of water to one well in triplicates.
    4. Prepare test samples by adding 2 μl of supernatant (from section 1) to 158 μl of water.
    5. Add 40 μl of Bradford Assay reagent to BSA standards, blank and test samples.
    6. Mix and incubate at room temperature for 5 min and read absorbance at 595 nm (A595) on plate reader spectrophotometer.
      Note: If spectrophotometer does not include a software to generate standard curve to automatically estimate protein content, generate a BSA standard curve by plotting known protein concentration (X-axis) vs. Absorbance (in Y-axis). Protein concentration for a given unknown sample is estimated by plotting the A595 absorbance of the unknown (in the y-axis) and determining the intersection point with the BSA standard curve and then find the concentration associated with that particular point (in the x-axis). If using excel, after plotting concentration vs A595, obtain the trendline and use the equation for the line and the A595 of the unknown to resolve the unknown concentration.

  3. NADPH oxidase activity assay
    1. Prepare fresh solution of 1 mM XTT and 1 mM NADPH.
    2. Prepare two different assay solutions A and B:


      Note: Because membrane fraction can spontaneously reduce XTT, even in the absence of substrate (NADPH), it is necessary to prepare two blanks, one without NADPH, to correct for this background levels of activity.
    3. Prepare blanks:
      Blank 1: Add 10 μl of water to 240 μl of Assay solution A.
      Blank 2: Add 10 μl of membrane fraction to 240 μl Assay solution B.
    4. Prepare samples by adding 10 μl of membrane fraction (from section 1) to 240 μl of Assay solution A. Read the absorbance at 492 nm (A492) at 0 min and then 20 min intervals for one hour or until saturation point reached.
    5. To get final A492 Blank reading, subtracts A492 Blank1 and A492 Blank 2.
    6. Calculate rate of O2– generation by using an extinction coefficient 2.16 x 104 cm-(Jiang and Zhang 2002).
      A492nm/min testA492nm/min blank) / (2.16X104M-1CM-1) (0.04)
      ΔA492nm/min Test= A492 (sample X) at saturation point - A492 (sample X) at 0 min
      ΔA492nm/min blank = A492nm (blank) at saturation point - A492nm (blank) at 0 min
      0.04 = dilution factor (10 μl/250 μl)
      To calculate specific activity, divide the value obtained in equation by the amount of protein present in the sample (converted to mg/ml).

Recipes

  1. Protein extraction working solution
    0.25 M sucrose
    50 mM HEPES
    3 mM EDTA
    1 mM DTT
    3.6 mM L-cysteine
    0.1 mM MgCl2
    0.6% PVP
    10 Tablets of Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets.
    Prepare the following stock solutions:
    1 M sucrose
    1 M HEPES (pH 7.2)
    0.25 M EDTA
    1 M DTT
    100 mM MgCl2
    In 80 ml of water add the following reagents:



    Add 10 Tablets of Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets.
    Mix well and adjust volume to 100 ml

Acknowledgments

This protocol has been adapted and modified to use in Arabidopsis from Jiang and Zhang (2002). This work was supported by the Samuel Roberts Noble Foundation.

References

  1. Able, A. J., Guest, D. I. and Sutherland, M. W. (1998). Use of a new tetrazolium-based assay to study the production of superoxide radicals by tobacco cell cultures challenged with avirulent zoospores of phytophthora parasitica var Nicotianae. Plant Physiol 117(2): 491-499.
  2. Jiang, M. and Zhang, J. (2002). Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 215(6): 1022-1030.
  3. Rojas, C. M., Senthil-Kumar, M., Wang, K., Ryu, C. M., Kaundal, A. and Mysore, K. S. (2012). Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis. Plant Cell 24(1): 336-352.
  4. Sagi, M. and Fluhr, R. (2001). Superoxide production by plant homologues of the gp91(phox) NADPH oxidase. Modulation of activity by calcium and by Tobacco mosaic virus infection. Plant Physiol 126(3): 1281-1290.


How to cite this protocol: Kaundal, A., Rojas, C. M. and Mysore, K. S. (2012). Measurement of NADPH Oxidase Activity in Plants. Bio-protocol 2(20): e278. DOI: 10.21769/BioProtoc.278; Full Text



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11/8/2012 2:10:55 PM  

a short summery of limitation of mtt and xtt for measuring cell viability
thanks

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