Graduate Thesis Or Dissertation
 

A biochemical investigation of the nitrogenase system from soybean root nodules

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/qj72p994d

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  • The primary purpose of this investigation was to determine the conditions necessary for the preparation of cell-free extracts of nodule bacteroids capable of catalysing the reduction of acetylene to ethylene and nitrogen to ammonia. Initial investigations were conducted to determine whether intact soybean root nodules would reduce acetylene to ethylene and to establish the optimum conditions for the reaction. An extraction procedure for the preparation of cell-free extracts from nodule bacteroids has been developed which involves the use of anaerobic conditions, a buffered ascorbate medium, and insoluble polyvinylpolypyrrolidone. Low-molecular-weight compounds were removed from extracts by use of polyacrylamide gel chromatography. Both acetylene reduction and nitrogen fixation reactions required an ATP-generating system, an electron donor such as Na₂S₂O₄, and cell-free extract containing the nitrogenase system. The optimum conditions are reported for catalysis of nitrogen fixation by these preparations. Reaction mixtures containing an ATP-generating system, Na₂S₂O₄, and nitrogen evolved hydrogen during the course of nitrogen fixation. Under an atmosphere of argon the rate of hydrogen evolution was much greater than under nitrogen. The rate of nitrogen fixation by the bacteroid nitrogenase system was strongly inhibited by hydrogen. The nitrogenase system from nodule bacteroids was fractionated into two components by use of either protamine sulfate or polypropylene glycol precipitation, followed by chromatography on DEAE-cellulose. Iron and molybdenum were concentrated in one fraction and iron in the other. Combination of fractions resulted in a striking stimulation of activity relative to the activity of individual fractions. The effect of different proportions of the two fractions on specific activities was studied. The ratio of the rates of reduction of acetylene and nitrogen by extracts or fractions of different purities was relatively constant.
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file details KochBurtonLee1971.pdf 2017-08-07 Público Baixar