I need to measure the MnSOD enzyme activity in yeast mitochondria. I know that that the IMS contains some Cu/Zn isozyme but I am interested in the MnSOD (which is in the mit. matrix). I have not found yet any specific methods to measure the activity specifically in yeast and specifically MnSOD. I would assume measuring the SOD (kit does not distinguish where the H2O2 comes from) of the mitochondrial lysate would be primarily from the Mn isozyme. If there is no such method, then another question would be how would I seperate the IMS proteins from the matrix proteins?
Thank you.
There are three version of SOD. Mn based, Cu/Zn based and extracellular. If you have only mitochondria it is Mn based because mitochondria has Cu/Zn SOD. Cu/Zn SOD is in only cytosol (I think).
Total cytosolic SOD is composed of MnSOD+ Cu/Zn SOD. Add NaCN sodium cyanide to inhibit Cu/Zn SOD and get the MnSOD activity. So you have to estimate total SOD and Mn SOD to calculate Cu/Zn SOD.
You can use either calorimetry (NBT based) or fluorescence (DNA+ Ethidium bromide based). See this paper for NBT http://www.ncbi.nlm.nih.gov/pubmed/2547324 and this paper http://www.ncbi.nlm.nih.gov/pubmed/11230333 for EtBr based method. Fluroescence method is a very sensitive assay and recommended.
There is another way to inhibit MnSOD but its very difficult.
Sorry I dont know about the second question.
You can treat mitochondria with a hypotonic buffer. The outer membrane will rupture due to mitochondrial swelling. As a consequence, most IMS proteins are removed. The resulting mitoplasts can be used as starting material for measuring MnSOD activity.
Another option is to use in gel activity assays. CuZnSOD and MnSOD are separated electrophoretically and can be assayed differentially. The downside of that method is that it is not strictly quantitative.
Regards, Christian
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