Purification and characterization of Cu, Zn-superoxide dismutase from black soybean

Abstract A superoxide dismutase (SOD) was purified from seeds of the Chinese black soybean( Glycine max cv ). The purification procedure comprised ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, affinity chromatography on Affi-gel blue gel and HP LC on DEAE 650-C. The final enzyme was purified 15.40 fold with a specific activity of 4619.3 U/mg protein and a recovery of enzyme was 29.41% from (NH 4 ) 2 SO 4 precipitation. The SDS-PAGE of the purified protein exhibited a molecular mass of 31.0 kDa and could be depolymerized spontaneously to a band at about 14.5 kDa in non-reducing conditions, and a diffusion band at about 15.5 kDa in reducing conditions, indicating a double-strand protein with intra-molecular disulfide bridges. The isoelectric point was determined to be 5.6, indicating it's an acidic protein. The enzyme is a type of copper plus zinc superoxide dismutase (Cu, Zn-SOD) because it was greatly inhibited by hydrogen peroxide, insensitive to chloroform–ethanol, and the maximum ultraviolet absorbance was found to be 266 nm. Thermostability experiments showed that the activity of the purified enzyme was stable after exposure to temperatures below 50 °C, and the activity could be well maintained between pH 6.0 and 8.0. Moreover, the SOD exerted an obvious protective effect against oxidative stress of UVC radiation in normal human liver cell strain L-02 cells.