I having problem regarding calculation.Please guide me with any 1 example.I have taken reading after every 15 sec till 3 mints.
You should measure the slope of the initial linear part of the reaction progress curve. This slope is directly proportional to the initial rate of product formation. To convert the slope to the rate of product formation, you need to know the constant of proportionality, which requires preparing a standard curve.
For catalase activity as per Anderson et al. (1995) -
Let us presume that you observe decrease in OD (Absorbance) at 240 nm by 0.9 (i.e. A = 0.9) in one minute.
Molar extinction coefficient for H2O2 at 240 nm is 36 M-1cm-1
Path length is 1 cm
Then concentration of H2O2 consumed (in M) = 0.9/36 = 9/360 = 1/40 = 0.025 M
In other words 0.025 M H2O2 was consumed. 0.025 M = 25 mM
25 mM means 25 micromoles/ml
In case if total volume of reaction mixture in cuvette is one ml you can use this value directly (if it is 2 ml then mutiply it by 2 and if it is 2.5 ml multiply 25 micromoles/ml with 2.5).
Let us now suppose that you had taken 100 microliters (i.e. 0.1 ml) enzyme extract (may be crude enzyme extract) and it contains 20 micrograms of protein.
In other words - catalase in 20 micrograms (i.e. 0.02 mg) of protein has potential to catalyze breakdown of 25 micromoles of H2O2 in one minute.
Catalase activity/mg protein/min = 25/0.02 = 2500/2 = 1250
You can calculate peroxidase activity in a similar manner using extinction coefficient for tetraguaicol.
Can catalase measurements without protein measure ? The final formula for calculating mean?
Hello Mahdieh,
Yes, you can calculate on basis of fresh weight or dry weight of whatever you used (living system or its component). You can even caculate per seed or per bacterium etc. In several publications, researchers expressed enzyme activity per gm fresh or dry weight.
I think that the best methods for assessment Catalase activity are:
1. Hadwan MH, kadhum Ali S. New spectrophotometric assay for assessments of catalase activity in biological samples. Analytical biochemistry. 2018 Feb 1;542:29-33.
https://www.ncbi.nlm.nih.gov/pubmed/29175424
2. Hadwan MH. Simple spectrophotometric assay for measuring catalase activity in biological tissues. BMC biochemistry. 2018 Dec;19(1):7.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091033/
Please go through the publications attached. I hope to find useful informations.
Best wishes
I think that the best methods for assessment Catalase activity are:
1. Hadwan MH, kadhum Ali S. New spectrophotometric assay for assessments of catalase activity in biological samples. Analytical biochemistry. 2018 Feb 1;542:29-33.
https://www.ncbi.nlm.nih.gov/pubmed/29175424
2. Hadwan MH. Simple spectrophotometric assay for measuring catalase activity in biological tissues. BMC biochemistry. 2018 Dec;19(1):7.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091033/
Please go through the publications attached. I hope to find useful informations.
Best wishes
@P. Pardha-Saradh sir, do we need another enzyme or chemical as standard or positive control for the assessment of catalase activity?
Dear Rupert Anand,
There is no need to add any chemical or another enzyme for assessing catalase activity. pH 7 is optimal.
If you are using crude enzyme extract, and in case if crude enzyme extract contain high protease activity, you should use protease inhibitor.
Good luck
we need help regarding enzyme activity calculation, everyone have different formula that confusing about actual one, please suggest the most liable formula regarding CAT, POD and other enzyme activity, thnkyou
I need help as regards catalase activity measurement, I using potassium phosphate buffer . How do I prepare 0.05N hydrogen peroxide for the analysis. Finally calculation formula. Thanks
Hydrogen peroxide is usually supplied as a 30% solution, which is 9.8 M (also N, in the case of peroxide ion O22-).
To make a 0.05 N solution, dilute 5.1 microliters of 30% H2O2 to 1 ml.
think that the best methods for assessment Catalase activity are:
1. Hadwan MH, kadhum Ali S. New spectrophotometric assay for assessments of catalase activity in biological samples. Analytical biochemistry. 2018 Feb 1;542:29-33.
https://www.ncbi.nlm.nih.gov/pubmed/29175424
2. Hadwan MH. Simple spectrophotometric assay for measuring catalase activity in biological tissues. BMC biochemistry. 2018 Dec;19(1):7.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091033/
Please go through the publications attached. I hope to find useful informations.
Best wishes
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