Dear Imteyaz

Catalase unit definition according to Aebi 1974 is: one unit is equivalent to 0.01 decrease in absorbance at  240 nm/mg protein/min.

What you do is to react a known amount of protein extract (say 5 mg protein, which should be calculated based on the protein content of you extract to find out how much volume of sample to use) with a known concentration of peroxide. Once you add all reaction components you read the initial absorbance at time zero. You then wait for a given time (30 s) and then read the absorbance again and record the decrease in absorbance.

To do the calculations:

(Decrease in absorbance x 100/1 ) divided by protein amount in mg divided by time in min=units/mg protein/min. 

I hope this helps.

Dear Imteyaz

Catalase unit definition according to Aebi 1974 is: one unit is equivalent to 0.01 decrease in absorbance at  240 nm/mg protein/min.

What you do is to react a known amount of protein extract (say 5 mg protein, which should be calculated based on the protein content of you extract to find out how much volume of sample to use) with a known concentration of peroxide. Once you add all reaction components you read the initial absorbance at time zero. You then wait for a given time (30 s) and then read the absorbance again and record the decrease in absorbance.

To do the calculations:

(Decrease in absorbance x 100/1 ) divided by protein amount in mg divided by time in min=units/mg protein/min. 

I hope this helps.

Hi there,

              Just to give you one example with details- 

Enzyme protein takes was 50 µg.

CAT activity was determined by measuring the decrease in absorbance at 240 nm as a result of degradation of H2O2 (ε = 39.4 mM-1 cm-1), which was followed in the reaction

mixture containing 50 mM sodium phosphate buffer at pH 7.0 and 10 mM of H2O2 and expressed in nmol of H2O2 utilized mg-1 protein min-1.

Total reaction mixture was 3 ml. This was performed in Onion (A. cepa) root cells.

Thermostatable spectrophotometer is needed or not for catalase estimation??

Hi Ahmad,

Thermostatable spectrophotometer is needed for all enzymatic assays.

 Regards,

 Vesna

Hello,

I´m having the same problem and I would like to be sure about the calculation, so I would appreciate any help. I´m working according to protocol by Chance and Maehly (1955).

My catalase mixture (3 ml) consists of 0,1M phosphate buffer (2,4 ml / pH 7), 10mM H2O2 (0,5 ml) and extract (100 ul). I took reading at OD=240 nm every 10 sec with ε = 39.4 M-1 cm-1. 

Calculation of catalase activity:

ΔA 240 nm (20. - 40. sec x 3 - so it woud be per minute) x ε

But now I´m not sure how to express it either in U/min/mg protein or in umol H2O2/min/mg protein, or even in nmol H2O2/min/mg protein.

(I´ll measure the protein content afterwards). I know there is a factor for conversion the ΔA 240nm into micromoles of utilized H2O2 but I don´t know exactly how to use it. 

Thank you for any help. 

I am also working according to protocol by the method of Aebi (1984). This method based on the hydrogen peroxide decomposition rate. In this connection

I would like to ask you about calculation of catalase activity. Do you calculated catalase activity according only to the formula or also with counting kinetic curves?

Hi Anna, 

I calculate the activity only according to the formula. 

you can calculate enzyme activity of any enzyme using the following formulas

enzyme activity= change in OD/time taken (min) x 1/extinction cofficient of enzyme x total reaction volume/ volume of enzyme extrct taken x total volume of enzyme extract/ Fresh wt of tissue (g) x total protein x 1000 = nano moles of enzyme present per g of sample tissue. for catalase ext coff is 39.4 mM-1cm-1and for peroxidases 26.6 mM-1cm-1. In case of SOD % inhibition = control OD- treatment OD/ control x 100 =X% inhibition. 50% inhibition is equal to 1 unit of enzyme. then X% is equal to 1/50 x X= Y unit. Calculate activity by inserting value of Y in above formula of activity in place of change in OD w.r.t. time. Rest of formula will be same.

Sadiq sir, Can you please tell what is meant by 'total protein' in the above formula?