I performed catalase assay according to A.K Sinha with modification. I took 1 ml of reaction mixture containing 20uL sample, 780uL dH2O, 0.1M Na-P buffer (final conc. 0.01M) and 100uL H2O2 soln (0.2M). I found OD in the range of 0.9 to 1.5. How can I calculate Catalase activity in this case? I also made standard curve for the above purpose ranging from 25-800mM conc.
What amount of 30% H2O2 (w/w) should be added in ddH2O to make 10ml of 1mM of H2O2 soln. according to me, i should add 1uL 30% H2O2 (w/w) in 10 ml of ddH2O as the strength of H2O2 is 9.76M/L. Is it right? Kindly help me in this regard. Thanking you in advance.
The dilution is correct.
You can use your standard curve to determine the amount of catalase activity in the extract. However, you must be sure that your reaction was running at the initial rate when you made the measurement. Otherwise, you will underestimate the activity.
We use another method of catalase activity measuring. You need 4% ammonium molybdate and 0.03% H2O2. 0.03% H2O2 = 0.88 mM. You shall take 2 ml of 0.03% H2O2, add 0,05-0.1 ml of sample. Then you should wait for 10 min and add 1 ml of 4% ammonium molybdate. It's necessary to make two controls:
1) 2 ml of 0.03% H2O2+0.05-0.1 ml of water+ 1 ml of 4% ammonium molybdate. This is OD of molibdate/H2O2 complex. Catalase decreases the content of H2O2 in the solution, and OD decreases too. You should to subtract your data on sample OD from OD of this control.
2) 2 ml of H2O+0.05-0.1 ml of water+ 1 ml of 4% ammonium molybdate. This is calibration control.
You shall made standard curve. 0.03% H2O2 you can serially diluted and add 4% ammonium molybdate at once. Your sample data you should found on this curve and subtract from the data of control (1). This is the amount of H2O2 destroyed by catalase for 10 min. If you divided to 10 the units of catalase activity will be [ml H2O2/min].
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