Articles : Purification and Characterization of Mouse Liver Rhodanese

Abstract

Rhodanese from mouse liver was purified to near homogeneity by ammonium sulfate precipitation, CM-Sephadex ion exchange, hydroxyapatite and Sephacryl S-200-HR gel filtration chromatographies with a purification of 776 folds. The molecular weight was determined by Sephadex G-150 gel filtration and found to be 34.8 KDa. SDS-PAGE showed molecular weight 34 KDa and two identical subunits splitting by aging for 3 weeks at -70℃ the molecular weight of which was 17 KDa. The optimal pH of enzyme activity was 9.4 and the pI value of the enzyme was 6.6. Rhodanese showed the optimal reaction temperature of 25℃ and near linear increasing pattern until 10 min. incubation. KM values of rhodanese for KCN and Na₂S₂O₃ as substrates were 12.5 mM and 8.3 mM, respectively. Rhodanese activity was inhibited by more than 70% at a concentration of 100 μM of Ni^(2+), Zn^(2+), Cd^(2+), Hg^(2+) and Cu^(2+). Other metal ions, such as Mn^(2+), Mg^(2+), Ca^(2+), and Fe^(2+) showed no effect on rhodanese activity.