Purification and some properties of Cu,Zn superoxide dismutase from Radix lethospermi seed, kind of Chinese traditional medicine

Copper-zinc superoxide dismutase (Cu,Zn SOD) has been extracted, purified and characterized from Radix lethospermi seed (RLS), a kind of Chinese traditional medicine. Before extraction, the lipid was removed by super critical fluid extraction (SCF). Partial protein fractionation in the crude extract was affected by using 50-75% (NH4)2SO2. Subsequently, superoxide dismutase was fractionated by column chromatographies on DEAE-52, Sephadex G-200 and DEAE-52 again. Pure Cu,Zn SOD had a specific activity of 4843 U/mg protein and was purified 267.2-fold, with a yield of 23.55%. The purified enzyme has a molecular weight of about 30,500 ± 100 and is composed of two non-covalently joined equal subunits. Purity was confirmed by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), HPLC and mass spectroscopy. Amino acid content has been investigated. The enzyme was found to remain stable in the pH range 6.0-9.0 at 25 °C and up to 45 °C at pH 7.8 for a 30 min incubation period. RLS Cu,Zn SOD appeared to have significant thermal stability lower than other Cu,Zn SODs, as revealed by irreversible heat inactivation at 60 °C. The enzyme was not inhibited by DTT, NaN3 and β-mercaptoethanol, but was inhibited by cyanide and hydrogen peroxide. Finally, in the presence of 2 mM ethylendiamine tetra acetic acid (EDTA) and sodium dodecyl sulphate (SDS), the enzyme showed approximately 18 and 34% activity loss.