Hi, Kenza, can you explain more precise the problem? Are you measure kinetcs of A240? Which tissue you used for protein isolation? Whar was treatment? How long?

Actually you will have higher catalase activity after tretaments with DPI, because tretament with DPI lead to increasing in peroxisomes activity and therefore, you will have higher catalase activity. Do you mean this?

Good luck"!

Yours question is not clear. There should be a decrease in absorbance at 240 nm if catalase is active in your homogenate. You should follow kinetics and calculate rate of H2O2 decomposition. NADPH oxidase is an enzyme responsible for superoxide anion generation which subsequently dismutated to H2O2. I suggest you to measure H2O2 in your sample after DPI treatment. If NADPH oxidase is solely responsible for H2O2 generation in your sample, then there should be a decrease in H2O2 level after DPI treatment.

Hi, Kenza, you should consider that NADPH oxidase produce mainly extracellular superoxide, which nothing to do with peroxisomal H2O2. So, it is not surprisingly that after DPI tretaments you have more peroxisomal H2O2 and more catalase activity.

Adding H2O2 will initially increase your absorbance because it will block more light than your sample without H2O2. Keep an eye on the reading for a minute and it should start coming down. Alternatively you may have too much catalase in your added sample and the bubbles which are produced during the reaction are causing the increase. Give your cuvette a mix to displace the bubbles from around the sides to the top and place back in the spectrophotometer and see if the reaction jumps down and then starts climbing again (too much catalase and too many bubbles being produced).

You need to follow the kinetics of the reaction with absorbance progressively decreasing as H2O2 gets broken down.

thanks for your responses !! i try to quantify catalase activity in a colon homogenate treated with apocynine and when i add the H2O2 the absorbance increase !! and there are not any bubbles !!