Serum samples will add to the absorbance at 240 nm. So the simplest way to do it is to add the serum first, blank on this and then add the peroxide which indeed (at 11 mM) should have an absorbance of about 0.45 at 240 nm. This assumes a recording spectrophotometer and quick readings (every couple of seconds). You mention taking points every 15 seconds. If this is a recording (or computer-linked machine) data points should be obtainable more frequently. You could check your system using a commercial catalase preparation whose 240 nm absorbance would be much less than that of serum.

You may use the simple method for serum catalase determination

Góth L. Clinica Chimica Acta 1991: 96: 143-152.

Good luck

Góth László

Perform Ingel Assya for that activity.i will confirm u.

You may follow the following method

Assay and activity staining of catalase (CAT, EC: 1.11.1.6)

Catalase activity was measured in a reaction mixture (3 ml) containing 100 mM Na2HPO4 buffer pH 6.8 (2 ml), 30 mM H2O2 (0.5 ml) and 0.5 ml enzyme extract according to the protocol of (Aebi, 1984). The decrease in absorbance due to hydrogen peroxide depletion was recorded at 240 nm by Hitachi model 200-20 UV-VIS spectrophotometer. Catalase activity was calculated by using the extinction co- efficient of 40 M-1cm-1 for H2O2 at 240 nm and was expressed as nKat moles of H2O2 decomposes per second/ mg of protein.

The gel was stained by following the method of (Woodbury et al., 1971). The electrophoresed samples in the gel was incubated in 0.01% H2O2 for 10 min and developed in a 2% FeCl3 and 2% K3FeCN6 solution for 10min. The principle involves the reaction of H2O2 with Potassium Ferricyanide (III) by reducing it to Ferrocyanide (II). The peroxide is oxidized to molecular O2. FeCl3 reacts with Ferrocyanide (II) to form stable, insoluble prussiano blue pigment. Catalase signaled its location by scavenging H2O2, causing transparent bands on the gel.

The detailed references are as follows

Aebi H (1984). Catalase, In: Packer L (Eds.). Methods in enzymology. Academic press, Orlando. PP. 121-126.

Woodbury W, Spencer AK, Stahmann MA (1971). An improved procedure using ferricyanide for detecting catalse isozymes. Anal. Biochem.44: 301-305.

@ Peter

I have tried as you suggested i.e adding serum first for blank and then add peroxide. But now I am getting spectro values in negative...

Azeem-

Then you should check the system with a control. A tiny amount of red cell hemolysate should have a lot of catalase activity (it will need diluting 100-fold or so and then adding only a few microlitres).

At 240 nm 11 mM peroxide should always give an increase in absorbance over the starting material provided that the latter is stable.

Hard to say more without knowing what spectrophotometer is involved!

Hii all...I am trying to do a catalase assay,i badly need thefull text Lucks paper,it will help me a lot for my experiment,,thanks in advace.

[email protected]

Subrata -

You don't need Luck (not very accessible at Bergmeyer HU, Methods of enzyme analysis. Academic Press: New York; 1971). You need a decent UV-capable spectrophotometer. Blank on buffer, add peroxide - between 10 and 30 mM for A (at 240nm) between 0.4 and 1.2, add a few microliters of enzyme and track absorbance decline. Commercial catalase at a final concentration of 5 nanomolar heme will give a decomposition half time of about 15 seconds.

how can you have 50ml of enzyme solution in 1 ml of total volume?

Please rewrite the question.

if anybody having copy of paper on catalase activity (Aebi H (1984). Catalase, In: Packer L (Eds.). Methods in enzymology. Academic press, Orlando. PP. 121-126) please send me.

Kamal - The abstract of Aebi's Methods in Enzymology paper is publicly available. It is quite a general survey. Details may not be necessary at this lapse of time (it was 30 years ago) unless for historical purposes. Dr. Aebi is no longer with us but others, myself included, may be able to answer specific related questions.

sir peter,

i am working on CKD patients and want to check serum catalase activity. if sir you could any help me for this purpose so please send me copy of paper. if any other method which currently use for catalase activity investigation send me.

thank with regard.

Dear all

I am trying to assay CAT in wheat grains extract but in 2 ml reaction mix containing Hydrogen peroxide (giving around 0.9 absorbance at zero time) in phosphate buffer and 40 ul extract I observe no decrease in absorbance over time lapse of 5 min. I used pure CAT to ensure that the reagents and spectro are ok and they are. The question is why I do not observe CAT activity in the extract (I do not have much vol of it)?

Thanks

Dear All i myself doing the catalase activity from HIV serum samples. for our lab we have standardise the protcol.

Hello Azeem,

High resting absorbance you mentioned give me the idea that your serum samples are not clear! They might have suspended materials, like lipids (lipemic serums). If yes, I suggest you try the Goth method posted in this topic.

Good luck

Does anyone has the formula for calculating catalase activity in tissue by Aebi method and superoxide dismutase by Madesh and Balasubramanian (1998)? Please provide me if you can.

Dear vinay kant.......

i can provide you with the calculations for catalase activity..

and for SOD i have followed the protocol of marklund and markund..

Respected Shreewardhan Haribhau Rajopadhye, Please send me the formula. I shall be thankful to you 

These very high absorbances probably are a result of unclear plasma samples (centrifuge them at high speed) or plasma that underwent too much hemolysis (very red samples - this cannot be undone), or the use of spectrophotometric cuvettes that are not fit to be used at this low wavelength of 240 nm (use UV cuvettes)

Dear Scientists,

 Try a a simple method for determination of serum catalase activity which appeared in

Clin ChimActa 1991: 96:143-152

with refrerence ranges.

Best regardsűLaszko Goth DSc

Dear colleague,

Please find the following references dealing with assays of CAT activity. 

http://www.indjst.org/index.php/indjst/article/view/80499

http://www.sciencedirect.com/science/article/pii/S2352340915003777

Dear Dr. Mahmoud Hadwan,

Please, could you suggest some reference about this procedure: 

H2O2 (65 mM) in 50 mmol/L sodium, potassium

phosphate buffer: This solution is freshly diluted and

standardized daily using a molar extinction coefficient

of 39.4 M-1 cm-1 at 240 nm.

Thank you

I propose to check the H2O2 molar extinction coefficients you use. When I used 43.6 M-1cm-1 for H2O2 at 240 nm and then performed glutathione peroxidase reaction, I obtained an excellent concondance between decrease of GSH and H2O2 concentration.

The other wavelengths in an ultraviolet range can be used, for example, 250 nm with molar extinction coefficient of 26.4 M-1cm-1 (to find out more, click this link from SpringerNature: https://rdcu.be/b4LjP )