title: A superoxide dismutase purified from the roots from Stemona tuberosa.
abstract: Proteins from the fresh roots of Stemona tuberosa (Stemonaceae) were extracted into 20 mM 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 H2O2. 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. MgCl2, MnCl2, and HgCl2 were strongly inhibitory at all concentrations tested. The SOD activity was completely negated in the presence of 0.5 mM SDS or 5 mM HgCl2. 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.
authors.affiliation: a Program in Biotechnology, Faculty of Science , Chulalongkorn University , Pathumwan , Bangkok , Thailand.
authors.forename: P
authors.initials: P
authors.lastname: Niyomploy
authors.forename: R
authors.initials: R
authors.lastname: Boonsombat
authors.forename: A
authors.initials: A
authors.lastname: Karnchanatat
authors.forename: P
authors.initials: P
authors.lastname: Sangvanich
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009580:Nitroblue Tetrazolium / Q000494:pharmacology; D010940:Plant Proteins / Q000302:isolation & purification; D018517:Plant Roots / Q000201:enzymology; D012256:Riboflavin / Q000494:pharmacology; D041901:Stemonaceae / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D008670:Metals; D010940:Plant Proteins; D009580:Nitroblue Tetrazolium; D013482:Superoxide Dismutase; D012256:Riboflavin
keywords: Stemona tuberosa; characterization; purification; superoxide dismutase