Dear All,
I have checked GPX activity of wheat leaves, the absorbance was not stable. I have got this reading Treatment 1= 1.3, 1.40, 1.50 Control 1.0, 1.11, 110 . GPX activity was determined following the formation of tetraguaiacol by measuring the absorbance at 470 nm and using an extinction coefficient of 26.6 mM−1 cm−1. The reaction mixture contained 2.75 mL of 50 mM phosphate buffer (pH 7.0), 100 μL of 30 mM guaiacol and 100 μL of 40 mM H2O2 along with 50 μL enzyme extract. The reaction was carried out for 3 min. One unit (U) of peroxidase activity was represented by the amount of enzyme catalysing the oxidation of l mol of guaiacol in 1 min and the enzyme activity was expressed as U g−1 FW
May be I am doing a mistake. Do I need to take two readings i.e. at two time intervals? Could anyone explain me.
Thanks
Hi,
So you need to do kinetics (multiple measurements at equal intervals) instead of end point absorbance measurement. GPX is quite fast so 1 - 2 min (measurement every 1-2 s) should be ok. Depending on the samples everything could be over in 30 s. Typically you prepare each reaction mixture and add last the enzyme extract after which you start the measurements immediately. You may have to do the activation with both peroxide and plant extract since the former is quite reactive. Afterwards you can calculate the activity based on the obtained curves (most absorbance readers have analysis options as well, otherwise you use the exponential phase). You also might have to adjust the amount of plant extract and of peroxide depending on the activity.
Hope that helps!
Svetlana
Great Madam. Some researchers make absorbance curves, I could not understand the absorbance curves, Do I need to take the different concentrations of original solvent (I mean not diluted, as it is from company) for example ascorbic acid /Guicol concentrations verses absorbance values? However as you said I should take multiple readings at different times and need to put an equation to get the values, in this case, I donot need to make the absorbance curves? If you explain these two conditions, It would be really helpfull for me. Thanks
Hi,
Normally the spectrophotometer you are using should have kinetics function. It can be set up to measure at certain intervals and usually will generate its own curve. Of course this can be done manually but will take more time and would be more complicated to analyse. Typically you should premix your buffer, guaiacol, and plant extract, prepare to start the reaction immediately and only then add the H2O2. Your absorbance should start increasing until it reaches a plateau and the reaction is saturated. If you think it goes too fast, you should decrease the amount of plant extract (for example by half) and vice versa. If you are recording the measurements manually you will end up with a curve. To analyse the enzyme activity you should use a portion of the curve before the plateau. This might help: Chapter Peroxidase
Madam, I have got readings of the treatment and control as follows: For GPX, Guicol per oxidase activitiy Treatment: 0 sec=.154, 30 sec=.201, 1 minute= .239, 1: 30 minute=271, 2 min=.306, 2:30=.339, 3 min= .370 Control: 0 sec = .125, 30 sec=.176, 1 minute=.221, 1: 30 minute=.262 ,2 min= .300, 2:30 min= .337, 3 min=.377.
In the Treatment OD is high as compared to control but in the control the OD increase at later time Why? This is GPX assay. Here in this spectrophotometer, I don't find the curve, I followed the protocol of Chance and Maehly
(1955).
Thanks
Hi,
I don't know what kind of treatment you have but many stress factors which cause oxidative damage would activate the anti-oxidative enzymes. Higher OD will mean higher activity of the enzyme.
For a proper curve you will need a lot more points (for example every 5 sec). Like this you will know when saturation occurs (not linear). If your spectrophotometer doesn't do curves you can plot your values yourself with time on the X axis and OD on the Y axis.
Thanks. I applied biocontrol agents to wheat seeds(Treatment) and one set of wheat seeds without biocontrol agents(control).
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