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Ferredoxin–NADP + Reductase from Tsuga canadensis . A Procedure for Isolation and Properties
Author(s) -
RIOV JOSEPH,
BROWN GREGORY
Publication year - 1976
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1976.tb04875.x
Subject(s) - ferredoxin , indophenol , sephadex , ferredoxin—nadp(+) reductase , chemistry , reductase , enzyme , oxidoreductase , electron acceptor , size exclusion chromatography , chromatography , electron donor , column chromatography , enzyme assay , biochemistry , catalysis
Activity of ferredoxin‐NADP + reductase in leaf extracts of eastern hemlock [ Tsuga canadensis (L.) Carr.] was relatively low, but could be markedly increased by use of protective agents. The best method employed polyvinylpolypyrrolidone (PVP) in the extraction medium plus removal of phenolic compounds by filtering the extracts through an insoluble PVP (Polyclar AT) column. Further purification of the enzyme was achieved by means of DEAE cellulose chromatography and DEAE Sephadex chromatography. A 94‐fold purification of the enzyme with a total recovery of 43% was obtained. The eastern hemlock ferredoxin‐NADP + reductase was characterized by its diaphorase activity, i.e. the transfer of electrons from NADPH to an electron acceptor. 2,6‐dichlorophenol indophenol. The pH optimum for the oxidation of NADPH is between 8.5 and 9.0. The enzyme is highly specific for its electron donor. NADPH, but shows low specificity for electron acceptors. The apparent Michaelis constant values of the enzyme for NADPH. NADH, and 2,6‐dichlorophenol indophenol are 2.4 × 10 −5 , 5.4 × 10 −3 , and 4.7 × 10 −5 M respectively. The molecular weight of the enzyme, as estimated by gel filtration, is about 45,000. The enzyme is inhibited by both organic and inorganic mercurials and certain cations. Comparison of properties of eastern hemlock ferredoxin‐NADP + reductase and spinach ferredoxin‐NADP + reductase shows that both enzymes are similar.