Diaphorase activity of ferredoxin: NADP oxidoreductase in the presence of dibromothymoquinone

Effect of cadmium on ferredoxin:NADP+ oxidoreductase activity

“…FNR was isolated from market spinach leaves by procedure of Apley et al [33] with modification of Bojko and Wie z ckowski [18]. The crude extract (obtained after leaf homogenisation and subsequent precipitation by acetone in range 20-80%) was purified successively at three steps: (1) on a DEAE 23 SS cellulose (Serva) column equilibrated with 10 mM Tris-HCl buffer (Tris, Tris(hydroxymethyl)aminomethane hydrochloride, derived from Fluka, A.G.), pH 8.0, and the fraction enriched with FNR was eluted with step gradient of NaCl (0.1; 0.2; 0.3; 0.4 and 0.5 mM) in 10 mM Tris-HCl buffer, pH 8.0, (2) after dialysis against 50 mM buffer TricineNaOH, pH 8.0, collected fraction was loaded on a column of Fraktogel TSK AF Red (Sigma) equilibrated with the same buffer and FNR was eluted with the KCl gradient (0-0.5 mM) in 50 mM Tricine-NaOH buffer, pH 8 (Tricine, N[Tris(hyroxymethyl)methyl]glycine, derived from Sigma), (3) the fraction enriched with FNR obtained in the step 2 was dialysed against 10 mM phosphate buffer (pH 8) overnight and loaded on a Hydroxyapatyt Bio-Gel HTP (Bio-Rad) column equilibrated with 10 mM phosphate buffer, pH 8.…”

“…In addition, FNR is able to catalyse the NADPH-dependent reduction of many non-physiological substrates such as FeCy, methyl viologen, dichlorophenol indophenol [2,15], and polynitroamines [16,17]. The NADPH-FNR system may also be engaged in the reduction of some quinones, as DBMIB, plastoquinone-1, plastoquinone-3 [18,19]. We hypothesised, that three distinct sites at FNR molecule are involved in binding either NADP þ (H), Fd, or quinone, and quinone binding site at the membrane-anchored FNR molecule participates in the cyclic electron flow around photosystem 1 [20].…”

Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization

“…Other procedures and chemicals Ferredoxin:NADP + oxidoreductase from spinach and ferredoxin from spinach were isolated and purified, from leaves obtained at a local market, by the procedure described in Bojko and Więckowski (1995). The protein concentration was determined spectrophotometrically with Bradford assay (Sigma-Aldrich Co) and the calibration curve was made with bovine serum albumin (SigmaAldrich Co).…”

“…Ferredoxin (1 mg), purified from spinach by the method described in Bojko and Więckowski (1995) was immobilized on a 1 ml NHSactivated HiTrap column (Supelco) using the standard protocol supplied by the manufacturer. After the immobilization, the column was equilibrated with 40 mM Tris/HCl pH 7.7.…”

“…Diaphorase activity FNR diaphorase activity was determined by an in vitro assay, as described by Bojko and Więckowski (1995) with small modifications. The reaction mixture (in total volume 2 ml) contained 100 lM NADPH (Sigma-Aldrich Co.), 25 lM DBMIB (Sigma-Aldrich Co.) and enzyme solution, in 40 mM Tris/HCl buffer pH 8.7.…”