ANALYTICAL BIOCHEMISTRY 90, 408-412 (1978)

Affinity Chromatography of Anacystis nidulans Ferredoxin-Nitrate Reductase and

NADP Reductase on Reduced Ferredoxin-Sepharose

CARLOS MANZANO,~EDRO CANDAU, AND MIGUEL G.GUERRERO

Departamento de Bioquimica, Facultad de Ciencias y C.S.I.C.,

Universidad de Sevilla, Sevilla, Spain

Received April 27, 1978

The behavior of two ferredoxin-dependent enzymes-nitrate reductase and NADP reductase-from Anacystis nidulans on a ferredoxin-Sepharose gel was examined. The oxidized gel-bound ferredoxin exhibited very low aftinity for these enzymes but effectively bound both nitrate reductase and NADP reductase when reduced by dithionite. Selective procedures are described for the elution of each of these two enzymes from the reduced ferredoxin-Sepharose gel. These simple methods allow substantial purification of both enzymes.

The first step in the assimilatory reduction of nitrate to ammonia is the two-electron reduction of nitrate to nitrite, catalyzed by molybdoprotein nitrate reductase (1,2). In blue-green algae, reduced ferredoxin is the physiological electron donor for nitrate reduction, a process intimately linked to photosynthesis (2-4), as it is also the case for the reaction catalyzed by ferredoxin-NADP reductase (5).

It has been reported recently that chromatography on immobilized ferre- doxin is an effective purification step for the ferredoxin-dependent en- zymes nitrite reductase and glutamate synthase from higher plants (6,7). In this report we show that reduction of the ferredoxin-Sepharose gel is required in order to obtain effective binding of nitrate reductase and NADP reductase, and that efficient purification of both enzymes can be easily achieved by means of this reduced ferredoxin-affinity gel.

MATERIALS AND METHODS

Anacystis nidulans cells (strain L 1402-l from the University of Gotingen) were cultivated as previously described (8). Extracts containing nitrate reductase and NADP reductase were prepared by overnight extraction of ethanol-dried cells with 50 mM Tris-HCl buffer (pH 7.3, 0.1 M KNOB, 0.1 mM EDTA. After centrifugation at 40,OOOg for 20 min, the supernatant

0003-2697/78/0901-0408$02.00/O Copyright 6 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.

408

REDUCED FERREDOXIN-AFFINITY GEL 409

was desalted by passing it through a Sephadex G-25 column equilibrated with 10 mM Tris-HCl buffer (pH 8.0), and used as enzyme extract. A. nidulans ferredoxin was prepared as described by Smillie and Entsch (9) and further purified by chromatography on DEAE-Sephadex A-25. The A mnm~&mm ratio of the obtained ferredoxin preparation was above 0.5. For the preparation of ferredoxin-Sepharose, 100 mg of A. nidufans ferredoxin was added to a suspension of 2.5 g (10 ml) of Pharmacia CNBr- activated Sepharose 4B in 20 ml of 0.1 M NaZC03/NaHC03 buffer (pH 10.2). The mixture was gently stirred at 4°C for 12 h and the unreacted ferredoxin was removed by washing first with 5 mM potassium phosphate buffer (pH 7.5), 0.3 mM EDTA, and finally with 0.1 M Tris-HCl buffer (pH 7.5). Spectrophotometric analysis at 423 nm of the washings showed that the binding yield was about 80%.

Ferredoxin-NADP reductase activity was assayed by using cytochrome c as electron acceptor (5). Nitrate reductase activity was determined by measuring nitrite production (3). One unit of enzyme is defined as that amount which catalyzes the reduction of 1 pmol of the corresponding substrate per minute at 30°C. Protein was determined according to Kalckar (10) and Lowry et al. (11). When necessary, the dithionite present in the eluates was oxidized by gentle shaking prior to the determination of en- zyme activity and protein.

Ferredoxin-Sepharose gel was packed into columns (1 x 8 cm) which were equilibrated with 10 mM Tris-HCI buffer (pH 8.0). When indicated, I mg of sodium dithionite per milliliter of buffer was added. After applica- tion of the sample in the corresponding equilibration buffer, the columns were washed first with the equilibration buffer and later, before specific elution, as indicated.

RESULTS AND DISCUSSION

Attempts to bind nitrate reductase and NADP reductase of A. nidufans to a ferredoxin-Sepharose column by using the conditions described as optimal for the binding of nitrite reductase and glutamate synthase to similar affinity gels (6,7) were unsuccessful. Changes in ionic strength, type of buffer, and pH did not improve at all the adsorption of these proteins to the column, and both enzymes emerged with the void volume, together with the bulk of the protein. By contrast, in the presence of the reductant dithionite, both nitrate reductase and NADP reductase were ef- fectively retained by the gel material. After washing of the reduced column, the bound enzymes could be specifically eluted with dithionite-free buffer. This treatment resulted in re-oxidation of ferredoxin, which recovered its original brown color, and simultaneous elution of both nitrate reductase and NADP reductase (experiments not shown). An increase in the con- centration of NaCl in the elution buffer resulted in sharper activity peaks.

410 MANZANO, CANDAU. AND GUERRERO

The affinity of reduced ferredoxin-Sepharose for nitrate reductase is higher than for other proteins retained also by this material. NADP re- ductase and some other proteins could be eluted from the reduced gel by concentrations of NaCl(150 mM) which otherwise did not affect the bind- ing of nitrate reductase. Figure 1 depicts the results of an experiment where an enzyme extract in 150 mM NaCl was applied to a ferredoxin- Sepharose column kept under reducing conditions with dithionite. Nitrate reductase activity was bound and could be later eluted by washing with a buffer of the same composition from which dithionite had been excluded. This procedure resulted in a 72-fold purification of the enzyme with a 67% recovery. The resulting nitrate reductase preparation had a specific activ- ity of 10 units/mg of protein and was essentially free of NADP reductase activity.

Pyrophosphate is a competitive inhibitor with respect to reduced ferre- doxin of NADP reductase (12). For the selective purification of NADP reductase from A. nidufans we have developed a procedure consisting of adsorption of the enzyme to a reduced ferredoxin-Sepharose bed, washing of the column, and specific elution with 25 mM sodium pyrophosphate (Fig. 2). By this single step, NADP reductase was purified 140-fold with a yield of 50%. After the pyrophosphate treatment, nitrate reductase re- mained bound to the column and could be eluted by further washing with

0 10 20 30 40

ELUTION VOLUME (ml)

FIG. 1. Elution profile for nitrate reductase from reduced ferredoxin-Sepharose. One

milliliter of enzyme extract was applied to a column of ferredoxin-Sepharose equilibrated with 10 mM Tris-HCI buffer (pH 8.0) 150 mM NaCl plus sodium dithionite, and washed with the same buffer. The arrow shows when dithionite was omitted from the buffer. Total nitrate reductase activity applied: 677 mu.

REDUCED FERREDOXIN-AFFINITY GEL 411

il.6

ELUTION VOLUME (ml)

FIG. 2. Elution profile for NADP reductase from reduced ferredoxin-Sepharose. One milliliter of enzyme extract was applied to a column of ferredoxin-Sepharose equilibrated with 10 mM Tris-HCI buffer (pH 8.0) plus sodium dithionite. The column was washed with 50 mM NaCl in the equilibration buffer. The arrow shows when 25 mM sodium pyrophos- phate in 10 mM Tris-HCI buffer (final pH 8.0) was substituted for the washing buffer. Total NADP reductase activity applied: 560 mu.

150 mM NaCl in dithionite-free buffer. However, this procedure was not adequate for the purification of nitrate reductase because a significant loss of enzyme activity took place. This inactivation of nitrate reductase seems to be due to the combined action of pyrophosphate and dithionite.

The purified nitrate reductase and NADP reductase preparations were not still homogeneous, since a few minor contaminant proteins were de- tected on polyacrylamide gel electrophoresis. The affinity chromatography step efficiently removed phycobiliproteins, which represent a major com- ponent in blue-green algae and are hard to eliminate by the conventional purification procedures.

The obtained results are good examples of how the change in the redox state of the ligand (ferredoxin) modifies its affinity for a given enzyme. The introduction of reducing conditions in affinity chromatography on ferre- doxin-Sepharose gel extends the possibilities of application of this ma- terial for the purification of ferredoxin-dependent enzymes which do not bind under the previously described conditions (6,7). It is conceivable that changes in the redox state of other affinity-chromatography materials (such as FAD-Sepharose (13) or flavodoxin-Sepharose (14)) could also improve their efficiency and increase the number of proteins which can bind to them.

412 MANZANO, CANDAU, AND GUERRERO

ACKNOWLEDGMENTS

We wish to thank Professor M. Losada for his helpful advice and criticism. This work was supported by grants from Philips Research Laboratories (Eindhoven, The Netherlands) and the National Science Foundation (GF-44115).

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4. Candau, P., Manzano, C., and Losada, M. (1976) Nature (London) 262, 715-717. 5. Shin, M. (1971) in Methods in Enzymology (San Pietro, Anthony, ed.), Vol. 23, pp.

440-447. Academic Press, New York. 6. Ida, S., Kobayakawa, K., and Morita, Y. (1976) FEBS Left. 65, 305-308. 7. Wallsgrove, R. M., and Miflin, B. J. (1977) Biochem. Sot. Trans. 5, 269-271. 8. Guerrero, M. G., Manzano, C., and Losada, M. (1974) Plant Sci. Left. 3, 273-278. 9. Smillie, R. M., and Entsch. B. (1971) in Methods in Enzymology (San Pietro, Anthony,

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