Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas

Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Jap anese founder Paralichthys olivaceus hepato-pancreas, The purification of t he enzyme was carried out by an ethanol/chloroform treatment and acetone pr ecipitation, and then followed by column chromatographies on Q-Sepharose, S -Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a sin gle protein band with molecular mass of 17.5 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular ma ss under non-reducing conditions. Three bands were obtained when the purifi ed enzyme was subjected to native-PAGE, both on protein and activity staini ng, but the electrophoretic mobility of the purified enzyme differed from t hat of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-termin al amino acid sequence of the purified enzyme was determined for 25 amino a cid residues, and the sequence was compared with other Cu, Zn-SODs. The N-t erminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60 degreesC, the thermostability of the enzyme was much lower than th at of bovine Cu, Zn-SOD. (C) 2001 Elsevier Science Inc. AII rights reserved .