SUPEROXIDE DISMUTASE PURIFIED FROM THE ROOTS FROM Stemona tuberosa

• Published in: Preparative biochemistry & biotechnology 2014-10, Vol.44 (7), p.663-679
• Main Authors: Niyomploy, P., Boonsombat, R., Karnchanatat, A., Sangvanich, P.
• Format: Article
• Language: English
• Subjects: Ananas comosus; Biochemistry; Cell Proliferation - drug effects; Cellulose; characterization; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Flowers & plants; Fractions; Hydrogen-Ion Concentration; Metals - pharmacology; Molecular Weight; Nitroblue Tetrazolium - pharmacology; Plant Proteins - isolation & purification; Plant Roots - enzymology; Proteins; purification; Riboflavin - pharmacology; Solanum; Stemona tuberosa; Stemonaceae; Stemonaceae - enzymology; superoxide dismutase; Superoxide Dismutase - chemistry; Superoxide Dismutase - isolation & purification; Superoxide Dismutase - metabolism; Tandem Mass Spectrometry; Temperature
• ISSN: 1532-2297; 1082-6068; 1532-2297
• DOI: 10.1080/10826068.2013.868356

Proteins from the fresh roots of Stemona tuberosa (Stemonaceae) were extracted into 20 m M phosphate buffer, pH 7.2/0.1 M NaCl, precipitated with 90% saturation ammonium sulfate, and enriched by diethylaminoethanol (DEAE) cellulose. The protein eluted as a single main peak from the unbound fractions (ST-1), and appeared as a single band with superoxide dismutase (SOD) activity after native polyacrylamide gel electrophoresis (PAGE) resolution and zymogram development. ST-1 was classified as SOD due to its strong inhibition by HCN and H ₂O ₂. The amino acid sequence of three tryptic peptides of ST-1 matched with the SOD isozymes from Ananas comosus and Solanum lycopersicum . The SOD consisted of at least two heterologous protein subunits with molecular mass of 17.6 and 31.5 kD, respectively, and had an optimal SOD activity at pH 5 and over a temperature range of 0–50°C. MgCl ₂, MnCl ₂, and HgCl ₂ were strongly inhibitory at all concentrations tested. The SOD activity was completely negated in the presence of 0.5 m M SDS or 5 m M HgCl ₂. The relationship between riboflavin and nitroblue tetrazolium (NBT) on SOD activity was linear, giving K ₘ and V ₘₐₓ values of the purified SOD of 62.414 ± 0.015 M and 101.010 ± 0.022 µmol/min/mg protein for NBT and 27.389 ± 0.032 M and 38.167 ± 0.021 µmol/min/mg protein for riboflavin, respectively.