Purification by affinity chromatography of glutathione reductase (EC 1.6.4.2) from Escherichia coli and characterization of such enzyme.
The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N6-2'.5'-ADP-Sepharose affinity column from which it was specifically eluted by a 0-10 mM NADP+ linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C8-ATPR-Sepharose column, from which it was eluted by means of the same NADP+ gradient. Starting from 182 g of E. coli cells, 6.9 mg of pure enzyme was obtained after a 2632-fold purification, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein, with an A272/A450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 A hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH, respectively, as reductants. Apparent K'm values of 16, 377, and 66 microM were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD, NADP+, dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing significantly only after 10 min at 70 degrees C. A marked activity loss was observed however, even at 0 degrees C, in the presence of 20 microM NADPH. The enzyme was inactivated by low concentrations of para-hydroximercuribenzoate; the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.
D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D005980:Glutathione Reductase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013816:Thermodynamics
D046911:Macromolecular Substances; D005980:Glutathione Reductase