I've been studying plant metabolism and in my lab we ar used to measure superoxid dismutase activity using the methodology of Giannopolitis e Ries, (1977), using the nitrotetrazolium blue reaction.
Sometimes, using the same reagents, the same concentration the reaction does'nt occur, neither the reaction with the enzyme, not the blank. Is there anything wrong in my studies? Is there anything I can change or be cautious in this metodology?
The methods described by Giannopolitis and Ries (1977) relies on the formation of superoxide radical by riboflavine when exposed to light. The superoxide will then react with NBT changing its color from yellow to blue. One thing you can check is your illuminating system.
Alternatively, I would recommend other methods for generating superoxide radicals that do not depend on illuminating system (light source and other materials can greatly interfere in the assay). You can use the xanthine oxidase+hypoxanthine system:
Article McCOrd, J. M., and Fridovich, I. Superoxide dismutase. An en...
or the mercaptoethanol+manganese system:
Article Sensitive spectrophotometric method for the determination of...
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