Oxidative Stress Response in Yeast: Purification and Characterization of Glutathione Reductase from Hansenula mrakii

Abstract

Glutathione reductase was purified from a yeast, Hansenula mrakii IFO 0895, to approximately 3500-fold with 59% activity yield. The enzyme was homogenous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis, and 123 kDa by gel filtration using a calibrated Sephadex G-150 column. The K_m values for glutathione disulfide and NADPH were 21.3 μM and 14.3 μM, respectively. The enzyme was most active at pH 7.5, 55°C. The enzyme was stable up to 40°C, and between pHs 4 and 10. The enzyme was inhibited by p-chloromercuribenzoate and metal ions such as Fe³⁺, Cd²⁺, Cu²⁺, and Zn²⁺.