The spectrophotometric analysis of antioxidant enzymes viz catalase (Luck et a.l) and Superoxide dismutase (Kakkar et al.)?
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.
If you wish you may visit our lab and learn protocols along with calculations with me, my doctoral and post-doctoral students.
P. Pardha- Saradhi: Dear Sir, Thank you very much for your kind reply. I find the information provided by your kind self to be very much useful. And I would be extremely happy to visit your lab and learn some techniques. Thank you once again sir.
DR Pardha Saradhi has given a very nice reply. I hope you will be able to do your studies
@Dr Premendu Sir: Yes sir. Thank you.... And as u have mentioned, suggestions by Pardha Saradhi sir are really helpful. I am thankful to him for having me enlightened about the enzyme activity calculation.
Dr. Pardha Saradhi,
Nice to see you helping young researchers may your tribe increase so that young researchers, will prosper with knowledge.
Dear Colleagues,
Thanks for your compliments. As a teacher, it is my duty to spread knowledge & help youngsters as much as possible. In fact this has been the goal of ResearchGate.
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