A Superoxide Dismutase Purified from the Rhizome of <Emphasis Type="Italic">Curcuma aeruginosa</Emphasis> Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line |
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Authors: | Wanwisa Moon-ai Ploypat Niyomploy Ruethairat Boonsombat Polkit Sangvanich Aphichart Karnchanatat |
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Institution: | (1) Program in Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok, 10330, Thailand;(2) Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok, 10330, Thailand;(3) Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok, 10330, Thailand; |
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Abstract: | Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of
the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has
long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested
for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate
precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall
SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa,
as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C.
With nitroblue tetrazolium and riboflavin as substrates, the K
m values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V
max values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min−1 mg protein−1. This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen
peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn2+and Fe2+ ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production
in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC50 = 14.36 ± 0.15 μg protein/ml). |
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