Take care that your catalase assay must contain a H2O2 concentration of about 10 mM, in order to take a constant absorbance decrease (vs time) measured at 240 nm, for the first 30-90 sec of the reaction. After this time the rate is usually decreased because of the consumption of the substrate (H2O2) and the inactivation of the enzyme.

Take care that your catalase assay must contain a H2O2 concentration of about 10 mM, in order to take a constant absorbance decrease (vs time) measured at 240 nm, for the first 30-90 sec of the reaction. After this time the rate is usually decreased because of the consumption of the substrate (H2O2) and the inactivation of the enzyme.

may your substrate H2O2 is not in the proper concentration

Also, look at your samples. If it is very cloudy, it can interfere in the stability of your assay.

Good activity of CAT is detected when we added 200 uL of H2O2 at 40 mM in a total volume of 1020 uL (800 uL of buffer and 20 uL of microsomal fraction). I suggest monitoring the absorbance at 240 nm each 30 seconds by 90 seconds. Our research team use the method proposed by Radi et al. 1991.

http://www.ncbi.nlm.nih.gov/pubmed/1657986

I experienced similar problems. I suspect that the formation of oxygen bubbles by the catalytic activity of catalase is responsible for the rapidly fluctuating absorbance values. Using cuvettes with a bigger volume partially solved this problem (although photometer reads are still not completely stable).

Cheers, Christian

thanks all

You have to use special cuvet which is Quartz cuvet and check your assay again

Analyses must be performed using diluted samples (and also using a quartz cuvet)

Dear Ayad,

This is a common problem one experiences, when catalase activity is high. Oxygen bubbles that are released, often get stuck on to the walls of the cuvette (obviously you must be using quartz cuvettes) and interfere with transmission of light. Therefore, we prefer using polarographic assay (using oxygen electrode), instead of spectrophotometric assay. Or else, as suggested by Dr. Jose, please dilute your enzyme samples. 

Just like what others said, the concentration of hydrogen peroxide is the problem. used the correct  diluted concentration of H2O2 to avoid formation of bubbles

It is very important concentration of H2O2 and adequate dilution of samples

Cuvettes might be dirty! Sock them overnight in acid solution!

thanks all 

1. Proper mixing of your homogenate sample with reaction buffer and hydrogen peroxide is very much important.

2. you need to leave your sample and reaction mixture undisturbed for homogeneity for 10-15 seconds. The system will stabilize after that and you will see stable disappearance of absorbency of  hydrogen peroxide at 240 nm.

You may use stopper and invert the cuvette 2-3 times. After mixing, leave it for 10 seconds. After that detect decrease in absorbance at 240 nm.

3. You need to use cuvettes compatible at 240 nm.

I recommend you to use ELISA Kits and applay the protocol on ELISA reader. however, for your problem, I think the main challenge for measuring the aborbance is the purification of the samples so please prepare your samples adequetely. use the resonable Cuvette and advanced spectrophotometers.

Hi,

The sample dilution can be increased; for example 1/500 or 1/1000

I experienced the same problem when my samples where not clear and mixed with pigments. Once I filtered them the problem was solved.

The reliability depends on the nature of the biological sample. What is your biological sample?

Because the reaction carried out catalase causes oxygen gas. The resulting oxygen gas bubbles are sometimes caused to shift absorption but not everytime:)

Omer is right, you may tap cuvette gently to remove bubbles and then take measurements.

thanks all

its for serum sample Mourad Aribi 

H2O2 is easily decomposed so u need to prepare fresh  one each time u assay ur samples 

I have found that this changes in absorbance is due to bubbles formation. Try using lower concentrations of hydrogen peroxide and the kinetic should be less than 5 min. 

All these factors mentioned above could interfere in catalase reaction avoiding linear reaction progress:

- H2O2 concentration around 10 mM is recommended, but you should verify your enzyme Km if specific determinations are required. And then you should work at a H2O2 concentration of about 100 Km. Fast, you should test extremes concentration (lower and higher from the previously used concentration. This may provide you a clue of what is happening with your specific enzyme during your catalase assay (of course, having in mind the possibility of protein denaturation for H2O2). Protein denaturation is less probably if you are trying with exocellular enzymes.

- Temperature and bubbles also interfere. Avoid them, at less those great variations which may affect reproducibility.

- Substrate preparation should be used immediately or in a short period after preparation because of spontaneous hydrolysis. Besides your blank (cuvette references) you must test the spontaneous hydrolysis and subtracting this value from the global enzymatic determination.

- Denaturation is possible through both, UV and visible light exposure. So keep your H2O2 into a dark/opaque container/flask.

- And finally use the data that best fit to linearity (initial minutes where r2 or ∆A between intervals does not varies significantly). Due to instabilities of this reaction, sometimes I prefer to check reaction kinetics with intervals longer than 15 seconds, because the kinetic pursue a progression instead of those regularities and statistical analysis should be better. I’ve read articles in Web of Science journals with r2 less than 0.8 considering it as a good report, which is justifiable from all these inconvenients we have all discussed.

Hope to be useful for you colleagues,

Regards, Frenkel

For temperature avoid using ice cold reagents

Hello. I would like you to give me your opinion on my answer. I am worried because I do not understand why someone selected "downvoted" for my answer. It may have been a confusion by voting criteria based on the principle that someone has acted ethically.

Is there an error in content? Please, could someone review the content and give me feedback about voting? I appreciate if you message me directly correcting my answer if I was wrong.

Greetings, Frenkel

I appreciate if you discuss and correct my answer as well.

Your sample has to be as unclouded as possible and you have to mix the reaction mixture very quickly after adding H2O2 before starting measuring.

Hi Konstadina. I did that, so continued unstable. I used H2O2 30 mM, and I did meansure every 10 seconds in 90 seconds. So I not used pure sample, I determined 100 ug of protein. How you do?

Thanks.

I have the same problem , now I would try to minimize the substrate concentration.

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

H2O2 is easily decomposed so you need to prepare fresh  one each time