Dear Dilki,

the results seem that you have a problem with the reaction as all absorbance values are in negative .. you can send the procedure leaflet to discuss more.

what is the princple that this kit based on? And is your procedure that you used for the extraction of the enzyme from the tissue? What is the control you used?

All these questions may help in solving youer problem.

normally Cu/Zn SOD is in cytoplasm and Mn SOD at mitochondria . did you used the whole tissue extract or centrifuged in different level means the kit may work out or it will not give proper result.

In SOD assays you are looking for an inhibition of an auto-oxidation, which involves the superoxide radical anion (O2.- ) as a mediator of this chain reaction. A come reaction which used in this type of assay is the auto-oxidation of adrenaline (colorless) in an alkaline solution to give adrenochrome (pink). If you get an negative answer for your SOD activity, the sample that you are adding is promoting the auto-oxidation rather that inhibiting it. This could be caused by the presence of certain transition metal ions of possibly an enzyme present in the sample that is promoting this oxidation.

It's either your kit is bad or you didn't get the calculation of your concentration well.

Philip G Penketh please can you provide an article that can give more explanation. Because i got negative answer too and can't fine paper to explain this. thanks

If you look for articles on auto-oxidation reactions, or on the older SOD assay, which was based on alkaline oxidation of adrenaline to pink adrenochrome. I think you will understand. In the adrenochrome based SOD assay, SOD blocked the production of pink adrenochrome and this was the basis for the assay. The rate of auto oxidation of adrenaline to adrenochrome is catalyzed by trace quantities of transition metal ions in the buffer and can be increased by the addition of micromolar iron EDTA. I just made the attached figure which I hope will clarify my potential explanation. You could always perform a few experiments involving spiking assay solutions with very small quantities of iron or copper EDTA (in the presence of absence of SOD) and running your SOD assay to verify the artifactual appearance of a negative SOD activity.

You have to specification of either Cu /Zn SOD or Mn and their presence in cytosol or mitochondria and which method you followed these factors are important normally this experiment will give positive answer follow standard procedure .