The reference that you mention looks very interesting. Older protocols like in gel activity stainings are not quantitative and take rather long. The important advantage of the method in the article you mention is that pH is set to a definitive value and buffered accordingly.

Mechanistically pyrogallol autooxidizes and produces superoxide anions. In subsequent steps purpurogallin is formed which can be measured spectrophotometrically. The presence of antioxidants or antioxidant enzymes alters the reaction kinetics. This allows you to calculate the amount of antioxidant in your sample.

Regards, Christian

Ok..it's means for Sod estimation the most significant reagents are the buffer and pyrogallol..the reaction is what matters..i.e the formations of purpurogallin.any protocol which give you purpurogallin can be used for SOD estimation?becoz in other research papers..riboflavin,methionine and xanthine are used.Which protocol can be more reliable?

In principle the assay is not specific for SOD. It basically can be used to give an estimate on overall antioxidant levels. To get specific SOD levels you would require a specific inhibitor, like cyanide to inhibit CuZnSOD. This is complicated by the fact that you are usually confronted with multiple SOD proteins, like MnSOD or FeSOD. So there is no perfect assay in my opinion. Still I would give the protocol you mention in your question a try if it can help you to answer your research question.

Ok Sir.Will try with this protocol.Thanks for the suggestions.

Regards

Nisha K.

Pyrogallo method is the best.leg. I believe you are referring to Marklund method.

Thanks for the reply Sir Katyare...I am referring to the link I've attached with the question..plz let me know your suggestion..

I am attaching a PDF which will give you all the protocol. You can stay with alkaline pH.

All the best.

Thanks a lot sir for the attachment and clearing my doubt on SOD estimation.Mine is a plant extract.Hope the protocol is applicable to both animal and plant cells.

The principle of SOD activity assay was based on the inhibition of nitroblue tetrazolium (NBT) reduction. Illumination of riboflavin in the presence of O2 and electron donor like methionine generates superoxide anions and this has been used as the basis of assay of SOD. The reduction of NBT by superoxide radicals to blue coloured formazan was followed at 560 nm. “One unit of SOD activity is defined as that amount of enzyme required to inhibit the reduction of NBT by 50% under the specified conditions”. The preparation of hemolysate was done by the method of McCord and Fridovich. In brief, washed erythrocytes treated with ethanol and chloroform, were centrifuged after 15 minutes, at 4°C and supernatant was used for the assay. The procedure adopted was that of Beauchamp and Fridovich. The reaction mixture contained, 1.9ml of phosphate buffer [pH 7.8], 1x 10-2 M methionine, 16.8x10-5 M NBT and 1.17x10-6 M riboflavin, with suitably diluted erythrocyte hemolysate in a total volume of 3ml. Illumination of the solution taken in 10ml beaker was carried out in an aluminium foil lined box, with a 15W fluorescent lamp for 10minutes.Control without the enzyme source was always included. The absorbance was measured at 560 nm. The values were expressed in Units/GmHb.



Mn SOD is mitochondrial2+,, 2+M SOD g is cytosolici.e. microsomal and RBCs have Fe2+ SOD. Purogallol method is applicable for all SODs. Makes no distonction between animal and plant system.It should work as long as there is SOD.

Ok.Thanks Sir Sarhath and Katyare for suggestions.

Regards

Nisha