Are you planning to detect the superoxide dismutase, catalase and peroxidase activity?

Hi Divya

These enzymes have been extensively studies in diverse organisms....go through some papers in pubmed (some early papers) and you'll be able to get enough information in the way data is anayzed for enzyme assays...essentially you'll use the basic 'enzyme kinetics' for such kind of analysis..

bye

There are different methods available to study antioxidant activities in plants, based on your research work, suggest you to go through research papers related at Google Scholar, PubMed or ScienceDirect. Try to express the units of enzymes in terms of μmol (H2O2 destroyed) g-1(FW) min-1.

I have studied several research papers but unable to get the equation through which i can calculate enzyme units in terms of μmol (H2O2 destroyed) g-1(FW) min-1.

Hi Divya

See the post below from Researchgate only which deals with aspects similar to your query

https://www.researchgate.net/post/I_want_to_measure_antioxidant_enzyme_activities

bye

Hi Divya,

For measuring all those enzymatic activities, you will measuring the appearance or disappearance of something (H2O2, NADH, an so on) in your cuvette or well, in a known time. It could be after a few minutes (10 m, before and after) or you can measure it continuously in plate reader (every 20 seconds). At this stage you have μmol of something min-1. Then, you have to figure out how much protein (Bradford) or tissue (taking into account your dilutions) you had in the well or cuvette. And finally you can express you enzymatic activity gram of tissue-1 min-1. Let me know in case you need a hand with the calculations.

Good luck!

Dear Divya,

you can perform antioxidant assay by following the protocol as follows well as you can perform the native page analysis for knowing the isoenzyme pattern for more clarification you may send me e mail on my id [email protected]

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.

Assay and activity staining of superoxide dismutase (SOD, EC: 1.15.1.1)

The activity of SOD was assayed according to the procedure of Das et al., (2000). The reaction mixture was prepared by mixing 1.110 ml of 50 mM phosphate buffer (pH 7.4), 0.075 ml of 20 mM L-methionine, 0.040 ml of 1% (v/v) Triton X-100, 0.075 ml of 10 mM hydroxylamine hydrochloride and 0.1 ml of 50 µM EDTA. To this mixture 100 µl of enzyme extract (50 µg protein) and 80 µl of riboflavin (50 µM) were added. The cocktail was mixed and then illuminated for 10 min in an aluminium foil coated wooden box containing two 20 W-Philips fluorescent lamps fitted parallel to each other. equal amount of buffer was added to the control tube instead of sample. The sample and its respective control were run together. After 10 min exposure, 1 ml of Greiss reagent (prepared freshly by mixing equal volume of 1% sulphanilamide in 5% phosphoric acid and 0.1% N-1-napthyl ethylene diamine) was added to each tube and the absorbance was measured at 543 nm. The activity was calculated as nKat/ mg of protein

SOD activity staining was done as per the classical method of Beauchamp and Fridovich (1971). The gel was stained in staining buffer containing 50 mM Phosphate buffer, 0.1 ml EDTA, 28 mM TEMED, 0.003 mM riboflavin and 0.25 mM NBT for 30 min in dark condition. After 30 min exposure to darkness the gel was then placed on a glass plate and kept inside the illumination with white light until the bands become visible.

Assay and activity staining of guaiacol peroxidase (GPX, EC: 1.11.1.7)

Peroxidase activity was measured in a reaction mixture (3 ml) containing 100mM Na2HPO4 buffer pH 6.8 (1.5ml), 12mM H2O2 (0.5ml), 3mM guaiacol (0.5ml) and 0.5ml enzyme extract according to the method of Kar and Fierabend (1984).The increase in absorbance due to formation of tetra guaiacol was recorded at 470 nm. Peroxidase activity was calculated by using the extinction co efficient of 26.6M-1cm-1 for H2O2 at 470 nm and was expressed as nKat/ mg of protein.

10 % gel was stained by the procedure of Ulmer et al. (1971). Gels were immersed in 0.018M guaiacol for 30 mins at room temp. Rinsed twice in deionized water and immersed in 0.015% H2O2 in 1% glacial acetic acid till the development of dark brown bands.

hi guys...

I carried out pyrogallol assay to check the activity of SOD1 enzyme.But i got stuck with the calculations.I referrd Marklund's paper in which they have mentioned that amount of enzyme resulting in 50%inhibtion corresponds to 1U.From this value how specific activity is calculated.Actual I am not sure with my calculations.I am unable to relate this value with specific activity.So please can anyone help me out and elaborate the calculations.Though in one of the answers below there is one formula mentione related to my problem

Enzyme unit (U) = (% of inhibition/50) x common dilution factor. (6)

[50% inhibition = 1 U]

But i am not getting what is the common dilution factor here?

Hi, Divya

Hope you are doing well at BHU. First of all why you want to express the enzyme activity in term of Unit/fresh weight. Because, it may lead to give you an incorrect result due to the water content of the cell, the inaccuracy in our measuring system especially the buffer etc. Therefore, it is best to describe the results in unit/mg pr which is usually accepted. For POD, you can contact your Physics Dept. if they have any system to measure the spin of atoms in liquid phase. There are very common methods to measure SOD and CAT activity. You can contact Prof. Trigun of your Department to get the best live answers to your wet lab problems on SOD and CAT. expressing CAT activity depends upon you, there are many ways you can express it as you have proposed your self in your question.

Dear Dr. Alaaeldin A. Hamza

Thank you very much for your scientific support about the SOD and CAT calculations.

I really need to learn: Can I use lyophilized (dried under -55 degree) samples for enzyme analyzes?

I finished some of my analyses with fresh samples then I had to move to abroad. I brought my samples after I dry them under - 55 (with lyophilization method).

So Can I do my other analyses (GR, APX and POX) in abroad with my lyophilized samples? 

And I would be gratefull if there is a specific method for that and if you let me know?

I apreciate everyone who can help me with this?

Thank you

I need best used methodology for Peroxidase activity (POD) and its calculation formulae.