Purification and characterization of cytosolic isoenzyme III of Cu,Zn-superoxide dismutase from tobacco leaves

Abstract Two isoenzymes of cytosolic Cu,Zn-superoxide dismutase (SOD) (I and III) from tobacco ( Nicotiana tobacum ) leaves have been purified to apparent homogeneity by precipitation with ammonium sulfate, ethanol-chloroform and acetone, and column chromatography on DEAE-52, Sephadex G-75. Some properties of Cu,Zn-SODIII have been investigated. The relative molecular mass of the Cu,Zn-SODIII was determined by MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) to be 22,976.6 Da. SDS–PAGE showed that Cu,Zn-SODIII was composed of one subunit of 23,000 (±500) Da. Amino acid composition of Cu,Zn-SODIII was unique, and it contained 187 amino acids. The purified enzyme, which was characterized as a cyanide-sensitive SOD, contained approximately one copper atom and 0.5 zinc atom per subunit, indicating that it belongs to the copper-zinc SOD. Cu,Zn-SODIII was found to have good pH stability in the pH range 5.0–6.0 at 25  °C over a 1-h incubation period, and the activation energy of the thermal denaturation process was 143.5 kJ mol −1 . The fluorescence spectra showed that the enzyme contained Trp, which was in a relative hydrophobic environment. The EPR and absorption spectra of the enzyme were also investigated.