Dear Debaprasad,

Using NBT method, one unit of SOD activity would be defined as the amount of enzyme required to cause 50% inhibition of the NBT photoreduction rate and the results expressed as Umg-1 of dry mass.

Classic protocols have been published by Mysra ans Fridovich (1972), and Winterbourn (1975). Other interesting protocols are available in Nature Protocols (Weydert and Cullen, 2010). For protocols and calculations please see the following links.

Finally, please if you consider this answer appropriate, please upvote it using the green up arrow click.

Best regards,

Jose L. Mauirz.

http://www.jbc.org/content/247/10/3170.long

http://www.worthington-biochem.com/sodbe/assay.html

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2830880/pdf/nihms173323.pdf

Dear  Mauriz,

I am facing difficulties with calculation. I set zero with the assay buffer. I have taken reading without homogenate and there was an increase in the absorbance. I have calculated the rate of increase per min. Then, I have taken reading with the homogenate and calculated the rate of increase per min. Now how I will calculate the percentage of inhibition? Δ absobnace/min with sample is lesser than Δ absobnace/min without sample, which indicating an inhibition in the reaction. So, what is the actual formula to calculate it. I am confused.

Dear Debrapasad,

See page 2 of the following link; maybe could be useful for you.

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/6/19160dat.pdf

Thank you very much Mauriz for your help.

What's is the ideal concentration of SOD enzyme (Sigma) for the assay? 

In base to Winterbourn (1975). 

I am trying to detect SOD activity using NBT. But I can not get any visible blue color formation?: Any suggestion?. We are using ribolavine and methionine?

@ José Antonio Cervantes Chávez. If not getting colour, please check whether the samples are kept in light of right intensity and for sufficient time (sometimes it may take 30min.-1hr. for developing colour.

In an aerobic medium, the riboflavin/methionine/NBT mixture

gave a blue coloration, the optical density of which was measured at

580 nm. One unit of SOD corresponds to the amount of protein

necessary to inhibit photoreduction by 50%.

I am getting green colour