Purification by affinity chromatography of glutathione reductase (EC 1.6.4.2) from Escherichia coli and characterization of such enzyme.

摘要: 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 adsorbed an N6-2'.5'-ADP-Sepharose column which it specifically eluted 0-10 mM NADP+ linear gradient. finally after second step in C8-ATPR-Sepharose column, means same Starting 182 g E. cells, 6.9 mg pure obtained 2632-fold purification, with total yield 63%. showed specific activity 361 U/mg, its absorption spectrum characteristic flavoprotein, A272/A450 7.84. dimer molecular weight 109 000 40 A hydrodynamic radius. optimum pH were 7.5 4.5 NADPH NADH, respectively, as reductants. Apparent K'm values 16, 377, 66 microM determined at for NADPH, GSSG, respectively. Upon storage stable ranging 9.5, being additionally stabilized FAD, NADP+, dithiothreitol, or glycerol. quite heat stable, denaturing significantly only 10 min 70 degrees C. marked loss observed however, even 0 C, presence 20 NADPH. inactivated low concentrations para-hydroximercuribenzoate; sensitivity towards such mercurial greatly enhanced reduction