Purification and characterization of superoxide dismutase from chicken liver.
Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme has been isolated, purified and partially characterized from chicken liver. The following steps were carried out in order to purify chicken liver SOD. Initially, the liver was homogenized and hemoglobin was removed. Subsequently protein precipitation was effected with (NH(4))(2)SO(4), methanol, (NH(4))(2)SO(4)-methanol and polyethylene glycol methods. The product from polyethylene glycol-3350 precipitation was found to have the highest SOD activity. Polyethylene glycol was removed by chromatography using a PD-10 column. After passing through an ultrafilter, the superoxide dismutase was fractionated by DEAE-ion chromatography and then Sephadex G-75 gel filtration chromatography. During this purification procedure, a specific activity of 4818.2 IU/mg was reached, corresponding to 285.8-fold purification. The purified enzyme, which was characterized as cyanide-sensitive SOD, contained two subunits having Cu and Zn elements with a molecular weight of 16000+/-500 for each. The optimum pH of purified CuZnSOD was determined to be 8.9. The enzyme was found to have good pH stability in the pH range 6.0-7.5 at 25 degrees C over a 2-h incubation period and displayed good thermal stability up to 45 degrees C at pH 7.4 over a 1-h incubation period. The SOD enzyme was not inhibited by DTT and beta-mercaptoethanol, but inhibited by CN(-) and H(2)O(2). In the presence of 2 mM iodoacetamide, the enzyme showed an approximately 40% activity loss. Finally, the inhibitory effect of ionic strength on SOD was also investigated.
Oztürk-Urek
R
R
Department of Chemistry, Faculty of Art and Science, University of Dokuz Eylül, 35150, Izmir, Buca, Turkey
Tarhan
L
L
D000818:Animals; D002645:Chickens; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007477:Ions; D008099:Liver / Q000201:enzymology*; D008623:Mercaptoethanol / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000302:isolation & purification*; D013696:Temperature; D013997:Time Factors
D007477:Ions; D008623:Mercaptoethanol; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D004229:Dithiothreitol; D007460:Iodoacetamide