| Superoxide dismutate (SOD, EC 1.15.1.1), one kind of metalloenzyme, which has been found ubiquitously among oxygen-metabolizing organisms, is most widely studied and reported in all enzymes we know. According to the type of metal in the active site of the enzymes, SOD has been classified into four types: Cu/Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD. It could catalyse the dismutation of superoxide anion radical to molecular oxygen and hydrogen peroxide. This enzyme has been proposed to play an important role in protecting living cells from the cytotoxic effects of the superoxide anion radical and other reactive oxygen species formed from the superoxide anion radical, such as hydrogen peroxide, the hydroxyl radical and singlet oxygen. Furthermore, SOD was related to caducity, tumour, autoimmunity disease and radioprotection, so it has fine foreground of development and application. This text summarizes the research about SOD, such as type, distribution, chemical structure, chemical character, the measurement of activity and content. Besides, this text emphasize discusses the application of SOD and some problem in application.Cell debris was clarified by centrifugation at 4 000 ｒ/min for 20 min at 0℃ after whetting with quartzite to give rise to the supernatant fraction. Ammonium sulfate powder was added to 35% saturation over night and the precipitated proteins were centrifuged as above. Ammonium sulfate was further added to the supernatant to 55% saturation, which was centrifuged again as above. The precipitate, collected by centrifugation, was suspended(dissolved) in a minimum volume of PBK, and was dialyzed against several (the same buffer for several times) at 4 ℃. The dialysate was clarified by centrifugation and 100 ml of the supernatant was applied to a DEAE-52 ion exchange chromatography column (3.0×35 cm), which had been equilibrated at 4 ℃ with the above buffer. Batches of DEAE-52 cellulose were successively eluted potassium phosphate buffer with 0.1 mol/L,0.2 mol/L,0.3 mol/L and 0.5 mol/L NaCl respectively. The concentrated solution (1 ml) was subjected to gel filtration using a column of Sephadex G-100(1.0×100 cm) previously equilibrated with 25 mM potassium phosphate, pH 7.8. The enzyme was eluted with the equilibrating buffer and the best fractions were recovered by freeze-dry. The resulting faint blue precipitates were suspended in 2 ml 1.5 mol/L (NH4)2SO4. Further purification was carried out by adsorption on a Phenyl SepharoseTM 6 Fast Flow column(1.5×15 cm) equilibrated with 1.5 mol/L (NH4)2SO4. The column was eluted with 0.75 mol/L (NH4)2SO4, 0.1 mol/L (NH4)2SO4 and H2O. The purified SOD had about 13943 units of enzymatic activity and specific activity was 4034.4 units per mg protein. Then the SOD obtained by Phenyl SepharoseTM 6 Fast Flow chromatography was dialyzed, freeze-dry and proceeded isoelectric focusing electrophoresis. The purified SOD showed one protein band by SDS-PAGE and isoelectric focusing electrophoresis. This explained that purified SOD was homogeneous. Finally 27.6 % of the SOD activity was recovered. SOD was 81.8-fold purified.Protein concentration was determined by the Folin method using bovine serum albumin as the standard. SOD activity was assayed by the pyrogallol autoxidation method. One unit of SOD enzymatic activity was defined as the amount of enzyme inhibited the half of the rate of pyragallol autoxidation per min at 325 nm, 25 ℃.Protein was visualized by staining with Coomassie brilliant blue R 250 and whereas SOD activity was negatively stained by the riboflavin mediated photoreduction of Nitroblue Tetrazolium. Cu/Zn-SOD is sensitive to cyanide and hydrogen peroxide, while Fe -SOD is inhibited by hydrogen peroxide but not by cyanide. Mn-SOD, on the other hand, is inhibited by azide but not by either cyanide or hydrogen peroxide. 5 mmol/L KCN restrained 92 % activity of purified SOD from Camellia Pollen, and 5 mmol/L H2O2 restrained completely the activity of purified enzyme. It appeared to be Cu/Zn-SOD according to its specific sensitivity to hydrog...
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