How should I calculate SOD activity? How can I calculate the percentage of inhibition? I am using spectrophotometric method. Thanks in advance.
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.
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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
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%.
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