Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/30/2004
Publication Date: 7/28/2004
Citation: Banowetz, G.M., Dierksen, K.P., Azevedo, M.D., Stout, R. 2004. Microplate quantification of plant leaf superoxide dismutases. Analytical Biochemistry.332:314-320. Interpretive Summary: During normal respiration, and during times of climatic stress, plants produce reactive oxygen molecules that can oxidize and damage plant proteins, membranes, or DNA. Plants have a system of anti-oxidative enzymes to reduce the amount of reactive oxygen molecules and consequently reduce the risk for irreversible damage to plant growth and crop production. One of these anti-oxidative enzymes is superoxide dismutase (SOD). Scientists that study SOD in order to produce new varieties with enhanced resistance to oxidative stresses need to quantify SOD in large numbers of plants in order to discover individuals that produce increased quantities of this enzyme. Doing so has been a problem because complicated and expensive procedures were required to purify SOD from plant extracts before SOD could be quantified. Our study modified a previously described approach for quantifying SOD that permits measuring SOD activity in crude, unpurified plant extracts. We also found that certain plants contain molecules that inhibit SOD activity. Further study of these inhibitors will help to develop plants with enhanced genetic resistance to climatic stresses.
Technical Abstract: Superoxide dismutases (SODs; EC 18.104.22.168) catalyze the dismutation of superoxide radicals in a broad range of organisms including plants. Quantification of SOD activity in crude plant extracts has been problematic due to the presence of compounds that interfere with the linearity of the assay. Although strategies exist to partially purify SODs from plant extracts, the requirement for purification limits the rapidity and practical number of assays that can be conducted. We describe modification of a procedure using o-dianisidine as substrate that permits relatively rapid quantification of SOD activity in crude leaf extracts in a microplate format. The method employs use of a commercial apparatus that permits lysis of twelve tissue samples at once and use of PIPES buffer to reduce interference from compounds present in crude leaf extracts. The assay provided a linear response from 1- 50 units of SOD. We demonstrate the utility of the procedure using tissue extracts prepared from a group of taxonomically diverse plants and document the occurrence of interfering compounds in certain plant tissues. This approach provides a means to quantify SOD activity in relatively large numbers of plant samples provided that the possibility for the presence of interfering compounds is considered. We document the presence of interfering compounds in certain plant tissues and show that caution is needed in interpreting the effects of plant stresses on SOD.