Glycolate Oxidase Activity Assay in Plants

Materials and Reagents

1. Sucrose
   
2. HEPES
   
3. EDTA
   
4. DTT
   
5. L-cysteine
   
6. MgCl₂
   
7. PVP
   
8. BSA
   
9. Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (F. Hoffmann-La Roche, catalog number: 04693159001 ).
   
10. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 )
    
11. Horseradish peroxidase(HRP) (Sigma-Aldrich, catalog number: P8375 )
    
12. O-Dianisidine (Sigma-Aldrich, catalog number: D9143 )
    
13. Sodium glycolate (Thermo Fisher Scientific, Acros Organics,catalog number: 351570250 )
    
14. Potassium phosphate
    
15. Triton X-100
    
16. Protein extraction buffer (see Recipes)
    
17. Glycolate oxidase assay buffer (see Recipes)
    

Equipment

1. Microtiter plate reader (Infinite M200 Pro, Tecan)
   
2. Microcentrifuge (AqquSpin Micro R) (Thermo Fisher Scientific)
   
3. 96-well microtiter plate flat bottom(BD Biosciences, catalog number: 353075 )
   
4. 2 ml-microcentrifuge tubes
   

Procedure

1. Total Protein extraction 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. Vortex at room temperature to mix thoroughly.
      
   5. Filter homogenized tissue through four layers of cheese cloth andtransfer 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 new tubes on ice.
      
   7. Use supernatant to estimate protein concentration and to measure glycolate oxidase activity.
      
   
   
2. Protein estimation using Bradford microassay (160 μl)
   8. Prepare BSA standards ranging from 5 μg-25 μg/ml as follows.
      
      
      
   9. Use 96 well microtiter plate to estimate protein content.
      
   10. Prepare blank by160 μl of water to one well in triplicates.
       
   11. Prepare test samplesby adding 2 μl of supernatant (from section 1) to 158 μl of water.
       
   12. Add 40 μl of Bradford Assay reagent to BSA standards, blank and test samples.
       
   13. 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 vsA595, obtain the trendline and use the equation for the line and the A595 of the unknown to resolve the unknown concentration.
       
   
   
3. Glycolate oxidase activity assay
   14. Prepare blank by adding 10 μl of 0.1 M sodium phosphate buffer (pH 8.3), to 250 μl of glycolate oxidase assay bufferin 96 well microtiter plate.
       
   15. Prepare test samples by adding 10 μl of supernatant (from section 1) to 250 μl of glycolate oxidase assay buffer.
       
   16. Read blank and samples at 440 nm for 0 min and then at 20 min intervals for one hour or until saturation point reached, on plate reader spectrophotometer.
       
   17. Calculate the generation of O-dianisidine radical using the following formula:
       (ΔA_440nm/min _test-ΔA_440nm/min _blank))/ (11.60) (0.04)
       ΔA_440nm/min_test =A_{440 (sample X)} at saturation point - A_{440 (sample X)} at 0 min
       ΔA_440nm/min _blank = A_{440nm (blank)} at saturation point - A_{440nm (blank)} at 0 min
       Where
       11.60 =extinction coefficient for O-dianisidine. (Macheroux et al, 1991)
       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 buffer
   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.
   Protein extraction buffer (100 ml)
   Use following stock solutions to make working solution:
   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.
   
2. Glycolate oxidase assay buffer
   Working solution:
   10 μg ml^-1 HRP
   0.4 mM O-dianisidine
   10 mM sodium glycolate
   in 0.1 M potassium phosphate (pH 8.3)
   Glycolate oxidase assay buffer (1 ml)
   PrepareGlycolate oxidase assay buffer using following stock solutions:
   
   
   

Acknowledgments

This protocol has been adapted and modified to use in Arabidopsis from Macheroux et al. (1991). This work was supported by the Samuel Roberts Noble Foundation.

References

1. Macheroux, P., Massey, V., Thiele, D. J. and Volokita, M. (1991). Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization. Biochemistry 30(18): 4612-4619.
   
2. 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.