How do I have to present in data form? I have the reading absorbance at 470 nm for 3min and every 30 sec interval but now how do I proceed?
There is mainly two method for calculation..
1) U can calculate change in OD/min/ g or
2) change in OD/min/mg of protein (if u estimated protein)
Estimation of peroxidase (POX) activity:
POX catalyses the dehydrogenation of a large number of organic compounds as phenols and aromatic amines. It was determined following the dehydrogenation of guaiacol as a substrate according to Malik and Singh (1980).
Reagents:
1- Phosphate buffer 0.1 M (pH 7.0)
2- Guaiacol solution 20 mM: Dissolve 240 mg guaiacol in water and make up to 100 ml. It can be stored frozen for many months.
3- Hydrogen peroxide solution (0.042% = 12.3 mM): Dilute 0.14 ml of 30% H2O2 to 100 ml with water, prepare freshly.
Enzyme extraction:
Extract a known weight of plant material in 3 ml of 0.1 M phosphate buffer pH 7.0 by grinding in a pre-chilled mortar. The homogenate is then centrifuged at 18000 g for 15 min at 5 °C. Use the supernatant as enzyme source within 2-4 h, store on ice till the assay is carried out.
Pipette out 3 ml of the buffer solution, 0.05 ml guaiacol solution, 0.1 ml enzyme extract and 0.03 ml hydrogen peroxide solution in a cuvette. The mixture was well shaken and placed in the spectrophotometer. The time required for the mixture optical density to be increased by 0.1 (Δt) at 436 nm was recorded and used in calculations.
The enzyme specific activity units g-1 f wt = [500/ Δt] x [1/1000] x [TV / VU] x [1/ f wt];
Δt = time change in minute; TV = total volume of the extract (ml); VU = volume used (ml); f wt = weight of the fresh leaf tissue (g)
Sir,
Can you explain What is "Δt = time change in minute"
And value "500" taken in your enzyme calculation formula.
If you explain this with the example if will be more beneficial to me.
235
Hi Rupert
You read this reference1
https://clu-in.org/download/ert/2035-R00.pdf
2
https://www.indiana.edu/~l113/independent_projects/addlprocedures/AP-VEnzymes.pdf
3
http://www.abcam.com/ps/products/155/ab155895/documents/ab155895%20Peroxidase%20Activity%20Assay%20Kit_protocol%20(website).pdf
4
https://onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.2004.01139.x/full
Aminoantipyrine assay for research peroxidase activity in the stool
For reagents and methods see you: http://www.protocol-online.org/forums/topic/13062-peroxidase-assay/?hl=aminoantipyrine#entry181898
Somebody have experience about the MPO after use of Sigma TMB solution?
I am checking peroxidase activity of my sample but my blank (guaiacol + H2O2 + buffer) itself producing color without peroxidase. If anybody could help?
Dear Navjot Kaur
Let me know specific of the single reagents and reaction procedure used!
Hello,
The final reaction. I am using is as follows
40mM Guaiacol
8mM H2O2
50mM buffer
Then I am incubating it with my crude enzyme (100ul) enzyme extract at 37 degrees until the pink color develops approximately for 30min. The the blank which is without enzyme extract produces pink color also.
Hi!, try this one: dispense in 800 µl of guaiacol buffer (dissolve 40 mM guaiacol in sodium acetate buffer 100 mM at pH 6.0 [dissolve in 80 ml dH2O, 1.28 g di sodium acetate trihydrate, add 50 µl of glacial acetic acid and adjust the pH solution to 6.0 with HCl or NaOH 0.1 M and then fill up to 100 ml]), 100 µl of enzyme extract and incubate for 1-2 min. at 37 °C and then add 100 of 8 mM H2O2 mix and incubated for 30 min at 37 °C in the dark. In addition, be careful if there may be contaminants in the solutions.
Let me know!
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