Harold J. Evans

[41] Preparation of nitrogenase from nodules and separation into components

Publisher Summary This chapter discusses the preparation of nitrogenase from nodules and separation into components. The chapter reviews Bergersons utilization of a specially designed anaerobic press for the preparation of nodule brei, which exhibited nitrogen-fixing capacity. Cell-free extracts of nodule bacteroids could not be prepared until it is realized that the phenolic compounds that are prevalent in legume nodules react with, and denature, the nitrogenase when nodules are macerated in air. In the method, nodules are macerated and extracts of bacteroids are prepared under conditions where contact with oxygen is minimized. Ascorbate is used in the preparative medium to prevent the oxidation of phenols, and phenolic compounds in nodule breis are removed as an insoluble complex with polyvinylpolypyrrolidone (PVP). By use of these procedures, crude extracts of nodule bacteroids with specific activities comparable to those of extracts of free-living nitrogen-fixing bacteria may be prepared consistently. The preparations are purified and fractionated into two components, both of which are essential for nitrogenase activity.

[67] Techniques for measurement of hydrogen evolution by nodules

Publisher Summary This chapter discusses the techniques for the measurement of hydrogen evolution by nodules. The evolution of H2 by nodules has been measured by mass spectrometry, gas chromatography, and amperometry. All of these methods, except the amperometric technique, require sampling of the gas mixture over nodules at intervals and subsequent analyses. The amperometric technique allows continuous measurement of H2 evolution by samples of nodules or bacteroids isolated from nodules. Hoch employed mass spectrometry to measure H2 evolution by nodules. The gas mixture above them was sampled and analyzed for masses 2 and 4. In these experiments, helium was used as an internal standard. A gas chromatograph equipped with a thermal conductivity detector has been used for the quantitation of H2 evolved from legume nodules. In this method, investigators employed a column 6.5 mm diameter by 2 m length of silica gel at room temperature with N2 as the carrier gas. Argon (Ar), whose thermal conductivity is one-tenth that of H2, is the most readily available carrier gas for use in the detection of H2 with a thermal conductivity detector. Nitrogen, whose thermal conductivity is one-seventh that of H2, is less expensive than Ar and may be used as a carrier gas with only a slight loss of sensitivity.

Hydrogen uptake by Robinia pseudoacacia nodules

SummaryHydrogen uptake is thought to increase the efficiency of nitrogen fixation by recycling H2 produced by nitrogenase that would otherwise be lost by diffusion. Here we demonstrate the capacity of eight Rhizobium strains to take up molecular hydrogen. Uptake by nodule homogenates from Robinia pseudoacacia was measured amperometrically under nitrogenase repression. Markedly lower activities were found than in soybean nodules. In addition hydrogenase activity was detected by the ability of bacteroids to reduce methylene blue in the presence of hydrogen. It was demonstrated that hydrogenase structural genes are present in the black locust symbiont, Rhizobium sp. strain R1, using hybridization with a plasmid, which contained hydrogenase genes from R. leguminosarum bv. viceae.