Welcome guest, Sign in

Home

X
加载中

The acetylesterases are hydrolytic enzymes which in plants cleave acetyl groups from acetylated cell wall components, primarily polysaccharides. To estimate acetylesterase activity in plant apoplast, two assays can be used. First assay is a direct measurement of the acetylesterase activity in protein extract using synthetic substrate, pNP-acetyl. In this assay, amount of pNP released after hydrolysis of pNP-acetyl is determined by measuring the intensity of developed yellow color using spectrophotometer. The absorbance of reaction mixture is directly proportional to the activity of acetylesterases in the reaction mixture. Second assay is a determination of acetylesterase activity and its specificity towards natural polysaccharides and based on interaction between ferric perchlorate and acetyl residues resulting in ferric acetohydroxamic complex that can be quantified using spectrophotometer. In this assay, commercially available acetylated polysaccharides (xylan from Birchwood for acetylxylan esterase; pectin from citrus fruit for rhamnogalacturonan acetylesterase; or any other available polysaccharide of interest) incubated with apoplastic extract and amount of acetyl residues released from this polysaccharide is estimated using ferric perchlorate reagent (protocol was modified from McComb and McCready, 1957). The absorbance of produced colored complex is directly proportional to the amount of acetyls released from acetylated polysaccharide.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Assays for Determination of Acetylesterase Activity and Specificity Using pNP-acetyl and Acetylated Polysaccharides as Substrates

Plant Science > Plant biochemistry > Protein > Activity
Authors: Gennady Pogorelko
Gennady PogorelkoAffiliation: Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, USA
Bio-protocol author page: a1137
 and Olga A. Zabotina
Olga A. ZabotinaAffiliation: Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, USA
For correspondence: zabotina@iastate.edu
Bio-protocol author page: a1138
Vol 4, Iss 3, 2/5/2014, 2604 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.1037

[Abstract] The acetylesterases are hydrolytic enzymes which in plants cleave acetyl groups from acetylated cell wall components, primarily polysaccharides. To estimate acetylesterase activity in plant apoplast, two assays can be used. First assay is a direct measurement of the acetylesterase activity in protein extract using synthetic substrate, pNP-acetyl. In this assay, amount of pNP released after hydrolysis of pNP-acetyl is determined by measuring the intensity of developed yellow color using spectrophotometer. The absorbance of reaction mixture is directly proportional to the activity of acetylesterases in the reaction mixture. Second assay is a determination of acetylesterase activity and its specificity towards natural polysaccharides and based on interaction between ferric perchlorate and acetyl residues resulting in ferric acetohydroxamic complex that can be quantified using spectrophotometer. In this assay, commercially available acetylated polysaccharides (xylan from Birchwood for acetylxylan esterase; pectin from citrus fruit for rhamnogalacturonan acetylesterase; or any other available polysaccharide of interest) incubated with apoplastic extract and amount of acetyl residues released from this polysaccharide is estimated using ferric perchlorate reagent (protocol was modified from McComb and McCready, 1957). The absorbance of produced colored complex is directly proportional to the amount of acetyls released from acetylated polysaccharide.

Keywords: Cell wall, Acetyl esterase activity, Polysaccharide acetylation, Apoplast

Materials and Reagents

  1. Plant material
  2. 4-Nitrophenyl acetate (Sigma-Aldrich, catalog number: N8130)
  3. 4-Nitrophenyl (Sigma-Aldrich, catalog number: N1048)
  4. Tris-HCl
  5. EDTA
  6. MgCl2
  7. Xylan from Birchwood (Sigma-Aldrich, catalog number: X4252)
  8. Pectin from citrus fruit (Sigma-Aldrich, catalog number: P9436)
  9. Sodium phosphate
  10. Sodium hydroxide
  11. Hydroxylamine hydrochloride (Acros Organics, catalog number: 5470-11-1)
  12. Perchloric acid (Sigma-Aldrich, catalog number: 311421-50ML)
  13. Absolute methanol
  14. Glucose-penta-acetate (Sigma-Aldrich, catalog number: G2354-25G)
  15. Deionized water
  16. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006) (optional)
  17. Extraction/reaction buffer (see Recipes)
  18. Re-suspension buffer (see Recipes)
  19. Acid-alcohol solution (see Recipes)
  20. Ferric Perchlorate reagent (MP Biomedicals, catalog number: 215875) (see Recipes)

Equipment

  1. Sharp razor blade
  2. pH meter
  3. Microtiter plate reader
  4. Microcentrifuge
  5. Centrifuge compatible with 15 ml vials
  6. 96-well microtiter plate
  7. Parafilm
  8. Sharp razor blade
  9. 10 ml syringe
  10. Light water flow laboratory vacuum
  11. SpeedVac dryer
  12. Vortex
  13. NanoDrop spectrophotometer (optional)
  14. Shaker for microtubes

Procedure

  1. Apoplast protein extraction from plant material
    Note: Acetylesterase activity assay can be applied not only for apoplast proteins but also for total proteins extracts and purified enzymes.
    1. Harvest 2-5 g of aerial parts (stems, leaves, siliques, flowers, and buds) of plants and immediately cut into 5 mm segments using a sharp razor blade.
    2. Place plant material into a 10 ml syringe which had its tip sealed with Parafilm (Figure 1A-B).
    3. Add 5 ml of pre-cooled extraction/reaction buffer. 
    4. Place the syringe into a centrifuge tube (tip is showing downwards, Figure 1C) and treat under vacuum in exicator (Figure 1D-E) twice for 15 min with a 5 min break in a cold room at 10 °C.
    5. Open the tip of syringe, carefully drain the buffer by gravity flow and discard it.
    6. Centrifuged at 1,000 x g for 10 min at 10 °C.
    7. Place apoplast extracts accumulates at the bottom of the tube on ice.
    8. Estimate protein concentration by NanoDrop or by Bradford assay described at Bradford Protein Assay (He, 2011).


      Figure 1. Apoplast extraction tools. A. Initial set: 5 ml syringe, 15 ml centrifuge tube, parafilm. B. Syringe tip sealed with parafilm. C. Syringe with sealed tip placed into centrifuge tube. D. Syringe with sealed tip in centrifuge tube placed into vacuum exicator. E. Vacuum exicator connected to the vacuum line.

  2. Total activity of acetylesterases
    1. Dilute apoplast extract with extraction/reaction buffer to obtain 0.128 mg in 100 µl.
      Note: Amount of protein for assay will vary depending on the enzymatic activity and specificity of acetylesterases present in extract. Here we perform example made on Arabidopsis extracts.
    2. Prepare blank negative control by adding of 1 μl of 200 mM pNP-acetyl to the 100 μl of boiled for 10 min apoplast extract containing 0.128 mg of protein.
    3. Add 1 μl of 200 mM pNP-acetyl to 100 µl of diluted apoplast extract.
    4. Incubate at room temperature for 0, 5, 15, 30, 60, 120, 180 min reading blank and samples absorbance at 460 nm in microplate reader at each time point.
    5. Prepare standard curve by measuring absorbance at 460 nm of 100 μl pNP solution diluted in series of concentrations ranging from 0.1 mM to 2 mM. All dilutions prepare in 3 replicates. Calculate the slope of the line which will give STD1 value equal to absorbance of 0.1 μmoles of pNP.
    6. Measure OD460 sample-OD460 blank for each time point and calculate the slope.
    7. Calculate the amount of released pNP using the following formula:
      Slope/STD1*0.1/0.128
      Slope (from step B6)
      0.1 and STD1 (from step B5)
      0.128 (from step B2)
      Obtaining valueу will indicate amount of released pNP µmoles per 1 min per 1 mg of apoplast proteins

  3. Assay for acetylesterase activity using  natural substrates (cell wall derived polysaccharides)
    1. Apply 0.12 mg of apopalst protein in extraction/reaction buffer to 20 mg of natural substrate in total volume 0.6 ml.
    2. Prepare blank negative control by adding 0.12 mg of boiled apoplast protein in extraction/reaction obuffer to 20 mg of natural substrate.
    3. Incubate at room temperature for 0, 30 min, 1 h, 3 h, 6 h shaking at 130 rpm.
    4. At each time point spin down polysaccharide material (1 min at 17,000 x g) and take an aliquot 100 μl of supernatant.
      Note: After taking the aliquot vortex the pellet and continue incubation.
    5. Dry the aliquot using SpeedVac.
    6. Re-suspend dry material in 40 μl of Re-suspension buffer (use vortex).
    7. Add 100 μl of deionized water and mix carefully.
    8. Add 100 μl of acid-alcohol solution and mix carefully.
    9. Add 260 μl of Ferric Perchlorate reagent and mix carefully.
    10. Incubate at room temperature 15-30 min.
    11. Measure the absorbance at 510 nm.
    12. Measure OD510 sample-OD510 blank for each time point and calculate the slope.
    13. Prepare standard curve using glucose-penta-acetate with a series of dilutions from 0.1 mM to 2 mM in 40 μl solution following by steps C7-12 paragraphs from above. Calculate slope which will give STD2 value equal to absorbance of 0.2 nmoles of acetyl residues.
    14. Calculate the amount of released acetyls from natural substrates by following formula:
      Slope*0.2/STD2/0.02
      Slope (from step C12)
      0.2 and STD2 (from step C8)
      0.02 (from a. 0.18 mg of apoplast protein was used in total 6 measurements)
      Determined value will indicate amount of released acetyls (nmoles) per 1 min per 1 mg of apoplast protein

Recipes

  1. Extraction buffer
    25 mM Tris-HCl
    50 mM EDTA
    150 mM MgCl2 (pH 7.4)
  2. Re-suspension buffer
    0.25 M hydroxylamine hydrochloride
    1 M sodium hydroxide solution
  3. Acid-alcohol solution
    5% perchloric acid in absolute methanol
  4. Ferric Perchlorate reagent
    0.3% ferric perchlorate (non-yellow)
    0.5% perchloric acid dissolved in 88% methanol

Acknowledgments

The authors acknowledge Carver Funding for the support of this work.

References

  1. He, F. (2011). Bradford protein assay. Bio-protocol 1(6): e45.
  2. McComb, E. and McCready, R. (1957). Determination of acetyl in pectin and in acetylated carbohydrate polymers. Anal Chem 29(5): 819-821.
  3. Pogorelko, G., Lionetti, V., Fursova, O., Sundaram, R. M., Qi, M., Whitham, S. A., Bogdanove, A. J., Bellincampi, D. and Zabotina, O. A. (2013). Arabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens. Plant Physiol 162(1): 9-23.
  4. Pogorelko, G., Fursova, O., Lin, M., Pyle, E., Jass, J. and Zabotina, O. A. (2011). Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast. Plant Mol Biol 77(4-5): 433-445. 


How to cite this protocol: Pogorelko, G. and Zabotina, O. A. (2014). Assays for Determination of Acetylesterase Activity and Specificity Using pNP-acetyl and Acetylated Polysaccharides as Substrates . Bio-protocol 4(3): e1037. DOI: 10.21769/BioProtoc.1037; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook