Purification of ferredoxin-NADP + oxidoreductase from cyanobacteria by affinity chromatography on 2′,5′-ADP-Sepharose 4B

Abstract

A simple and rapid method for the purification to homogeneity of ferredoxin-NADP + oxidoreductase (EC 1.18.1.2) from the nitrogen-fixing filamentous cyanobacterium Anabaena sp. strain 7119 is described. A crude extract prepared by solubilizing the cells with a detergent was first partially purified on a DEAE-cellulose column and then chromatographed on 2′,5′-ADP-Sepharose 4B. Ligand-bound ferredoxin-NADP + oxidoreductase was eluted by a linear gradient of NaCl. The overall procedure provided an enzyme purified about 400-fold with a yield of 60 to 70%. The final enzyme preparation exhibited a specific activity of 120 units/mg protein and an absorbance ratio A 280 A 458 of 8.26. The enzyme protein migrated as a single band when subjected to polyacrylamide gel electrophoresis and chromatographed as a single isoelectric species under chromatofocusing.