Extraction and characterization of glutathione reductase enzyme from rosemary plant

In this study, glutathione reductase enzyme (Glutathione: NADP+, oxidoreductase, E.C 1.8.1.7: GR) was extracted from the rosemary (Rosmarinus officinalis L.) plant and purified 18 times in 54.80% yield by optimized triple phase purification method. The purified rosemary GR enzyme molecular weight was monitored by SDS-PAGE as a single band of approximately 75 kDa. In the enzyme characterization studies, optimum ionic strength, temperature, pH, and kinetic properties were investigated by using raw extracts. Results are as follows; 400 mM phosphate buffer, 60°C, 7.5, and Km value of NADPH was 0.047 mM; Vmax was found to be 0.0033 EU/mL while Km value of GSSG substrate was 0.029 mM; Vmax was found to be 0.0019 EU/mL. The effects of magnesium, mercury, cobalt, iron, nickel, lead, barium, manganese, aluminum and calcium metals on the enzyme activity were investigated. Among these metals, Mg+2, Mn+2 and Al+3 metals increased the enzyme activity, while the most other metals exhibited inhibitory effect. As a result of storage stability, it was observed that the enzyme was unstable in the long term. In addition, the inhibitory effect of GSH on the GR enzyme from rosemary plant was investigated and the Ki value of GSH was found to be 38.03 mM with the Lineweaver-Bruk graph.