Purification and characterization of glutathione reductase from rainbow trout ( Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity

Abstract

Glutathione reductase (E C: 1.8.1.7; GR) was purified from rainbow trout ( Oncorhynchus mykiss) liver, and some characteristics of the enzyme were investigated. The purification procedure consisted of four steps: preparation of homogenate, ammonium sulfate fractionation, affinity chromatography on 2′,5′-ADP Sepharose-4B and gel filtration chromatography on Sephadex G-200. The enzyme, with a specific activity of 27.45 U/mg protein, was purified 1,654-fold with a yield of 41%. Optimal pH, stable pH, optimal temperature, optimum ionic strength, molecular mass, K M and V max values for GSSG and NADPH were also determined for the enzyme. In addition, K i values and inhibition types were determined for GSH and NADP +. Additionally, inhibitory effects of metal ions (Cd + 2 , Cu + 2 , Pb + 2 , Hg + 2 , Fe + 3 and Al + 3 ) on glutathione reductase were investigated. K i constants and IC 50 values for metal ions were determined by Lineweaver–Burk graphs and plotting activity % vs. [I], respectively. IC 50 values of Cd + 2 ,Cu + 2 , Pb + 2 , Hg + 2 , Fe + 3 and Al + 3 were 0.0655, 0.082, 0.122, 0.509, 0.797 and 0.804 mM, and the K i constants for Cd + 2 and Cu + 2 were 0.104 ± 0.001, 0.117 ± 0.001, respectively.