Cell extraction

Rat tissues of liver, lung, and kidneys were excised and perfused with ice-cold perfusion solution (0.15 M KCl, 2 mM EDTA, pH-7.4). Tissues were homogenized in Tris-HCl buffer (50 mM, pH 7.4), and the homogenates were centrifuged at 10,000 g and 4°C for 30 min to obtain supernatant. Thereafter, it was separated and then used for enzyme activities measurement and estimation of protein concentration .

Assay of catalase (CAT) activity

Catalase activity in the tissue supernatant was measured following the method of . The reaction contained 5 mM ethylenediaminetetraacetic acid (pH= 7.0), 0.05 M Tris-buffer, and 10 mM hydrogen peroxide (in 0.1 M potassium phosphate buffer, pH= 7.0). The supernatant was added to the above mixture in a final reaction volume of 3 ml. The rate of change in absorbance per minute was measured at 240 nm. Catalase activity in terms of µmoles hydrogen peroxide consumed per min per mg of protein was calculated using the molar extinction coefficient of 43.6 M–1 cm–1 for hydrogen peroxide.

Hello,

yes there are some formula.

Check the following link:

http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-catalase.html

Also check Prof. Laszlo Góth profile in reseachgate. He has an old paper on catalase assay using ammonium molybdate and H2O2 as well as its calculation formula.

Have a fun...

To convert the absorbance change to µM, use Beer's Law. You need to know (1) the difference in extinction coefficient at the measured wavelength between product and substrate, (2) the path length through the sample, and (3) the absorbance change. Then divide by the reaction time and the amount of catalase in mg in the reaction.

you can follow the above said method

or

you can also mention the activity in terms of change in absorbance. say 1 unit enzyme activity is change in absorbance by 0.01...

You may try some kits. Check sigma-aldrich website.

You can calculate using Molar extinction coefficient for H2O2 at 240 nm. Let me help you with a simple example

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

Very very thank you Dr. P. Pardha-Saradhi for your valuable answer. Procedure given by you seems  interesting and simple to calculate catalase activity. I have 30% H2O2 in our lab. Will the procedure mentioned by you be same with 30% H2O2 or any change is needed? what does path length means in your explanation?

Procedure will remain same with any commercially available H2O2 irrespective of its concentration..

Path length is the distance the light travels through the solution. In a standard spectrophotometer cuvette, the path length is 1 cm, but cuvettes with other path lengths are also used. In a clear microplate used in an absorbance-based assay, the path length is the depth of the solution in the well, which depends on the sample volume, well geometry, and meniscus shape.

Dear Adam,

I certainly agree with you and that is the precise reason why I stated path length to be 1 cm (which is the path length in standard spectrophotometric cuvettes). Thanks for providing additional information that is applicable for clear microplate used in absorbance based assays. I hope most of our junior colleagues are aware of the Beer–Lambert law.

You can also use an oxygen electrode. Add hydrogen peroxide and enzymatic levels of catalase and observe the production of molecular oxygen with time. This is quite simple.

I am using ABTS as substrate for Laccase Assay..What will be the standard to be run to measure enzyme activity as in case of using xylan as substrate we use xylanose

Cell extraction

Rat tissues of liver, lung, and kidneys were excised and perfused with ice-cold perfusion solution (0.15 M KCl, 2 mM EDTA, pH-7.4). Tissues were homogenized in Tris-HCl buffer (50 mM, pH 7.4), and the homogenates were centrifuged at 10,000 g and 4°C for 30 min to obtain supernatant. Thereafter, it was separated and then used for enzyme activities measurement and estimation of protein concentration .

Assay of catalase (CAT) activity

Catalase activity in the tissue supernatant was measured following the method of . The reaction contained 5 mM ethylenediaminetetraacetic acid (pH= 7.0), 0.05 M Tris-buffer, and 10 mM hydrogen peroxide (in 0.1 M potassium phosphate buffer, pH= 7.0). The supernatant was added to the above mixture in a final reaction volume of 3 ml. The rate of change in absorbance per minute was measured at 240 nm. Catalase activity in terms of µmoles hydrogen peroxide consumed per min per mg of protein was calculated using the molar extinction coefficient of 43.6 M–1 cm–1 for hydrogen peroxide.

Dear Prem Rajak,

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