Purification and partial characterization of Cu, Zn superoxide dismutase from haemolymph of Oriental river prawn Macrobrachium nipponense |
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Authors: | Cui-Luan Yao An-Li Wang Zhi-Yong Wang Wei-Na Wang Ru-Yong Sun |
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Institution: | aKey Laboratory of Science and Technology for Aquaculture and Food Safety, Fujian Province University, Fisheries College, Jimei University, Xiamen 361021, China bCollege of Life Science, South China Normal University, Guangzhou 510631, China |
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Abstract: | The copper plus zinc superoxide dismutase (Cu, Zn-SOD) was purified from haemolymph of the Oriental river prawn, Macrobrachium nipponense and partially characterized. Partial protein precipitation in crude extract was affected by using heat treatment and (NH4)2SO4 fractionated precipitation methods. Fractionation of superoxide dismutase was performed by DEAE-cellulose 32 ion-exchange chromatography and followed by CM-cellulose cation-exchange chromatography. The molecular weight of it was about 66.1 kDa, as judged by SDS polyacrylamide gel electrophoresis. The enzyme was sensitive to cyanide and H2O2, and contained 1.08 ± 0.14 atom of copper and 0.98 ± 0.11 zinc per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Cu, Zn superoxide dismutase. The purified enzyme had an absorption peak of 269 nm in ultraviolet region and the enzyme remained stable at 25–45 °C within 60 min. But it was rapidly inactivated at higher temperature (50 °C). The activity of purified shrimp Cu, Zn-SOD was remained stable over the range pH 5.8–8.3. Treated with 10 mM mercaptoethanol, the enzyme activity significantly increased. However, the enzyme activity was obviously inhibited by 10 mM CaCl2, ZnCl2, SDS, EDTA–Na2 and 1 mM and 10 mM K2Cr2O7. The results showed that it might be a kind of EC-SOD. And it was the first report of some characterizations of this EC-SOD in M. nipponense. |
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Keywords: | Superoxide dismutase Macrobrachium nipponense Haemolymph Purification Characterization |
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