Ann Clin Biochem 1987; 24: 511-512 Radial diffusion as a simple and rapid method for screening superoxide dismutase activity A HUNAITI From the Department of Biological Sciences, Laboratory of Biochemistry and Molecular Biology, Yarmouk University, Irbid, Jordan SUMMARY. Superoxide dismutases are of great interest due to their increasing medical applications in therapy and diagnosis of some diseases. The radial diffusion assay was evaluated for its usefulness as a simple, cheap and accurate assay for screening superoxide dismutase activity. In this assay O 2 radicals were generated from the interaction of reduced riboflavin with molecular oxygen upon exposure of agar gel containing riboflavin and N,N,N,N -tetramethylethylene diamine (TEMEO) to light. If nitrotetrazolium dye is also present, it will be reduced to the blue insoluble formazan, whilst if superoxide dismutase is present it will prevent this blueing. I The developed assay was found to give reproducible estimates of pure samples of superoxide dismutase with a lower limit of measurements of about )() mg. It can be adapted to measure the levels of superoxide dismutase in various crude biological samples. Materials and methods Chemicals. All chemicals were obtained from Sigma Chemical Company (St Louis, USA). Reagents. Buffer A: )() mM potassium phos- phate pH 7·8. Standard. Bovine erythrocyte supcroxide dis- mutase (3()(K) U/mg) was obtained from Sigma product No. S-8254. Stock solution (I mglmL) was prepared by dissolving the lyopholised powder in buffer A and then dialysing over- night against buffer A. Preparation of radial dlffusion plates. Nobel agar 0·4 g was suspended in )()mL buffer A and melted in a boiling watcrbath. The solution was allowed to cool down to 55°C and )() mL of solution containing 2S0 ug 4-nitro blue tetrazo- lium chloride and UK) ug riboflavin dissolved in buffer A was added the mixture was carefully stirred and then quickly poured into a Petri dish held horizontally and left to cool at room temperature for 2 h. Wells of uniform diameter (40101) were punched in the gel using Pharma- cia gel punch (Pbarmacia Fine Chemicals AB, Uppsala, Sweden). The plates were covered with aluminium foil and kept in the dark at 4°C until used. Assay procedure. Samples (15 ilL) were care- fully pipetted into the wells and the plates were incubated overnight at room temperature in a dark moist chamber. With each batch of measurements a standard curve was con- structed using serial dilutions of a standard superoxide dismutase. After the incubation period the plates were removed and soaked in a solution containing 1 g N,N,N,N-tetramethy- lethylene diamine (TEMEO) dissolved in HK) mL buffer A (10 mUplate). The plates were exposed to an appropriate light source (light box) and colourless rings were formed against a blue background. Results and discussion There is a linear relationship between the concentration of superoxide dismutase and the square of the difference between the final radial diffusion diameter (D) and the well diameter (DO) when bovine superoxide dismutase was used as a standard Fig. 1. Similar results were also obtained with human placenta enzyme. The linear relationship was observed between concentration range ng/IS ItL). Lower concentrations gave weak rings which were difficult to measure whilst at higher concentra- tions nonlinear relationship was observed. 511 by guest on July 6, 2016 acb.sagepub.com Downloaded from