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

This chapter discusses the preparation of nitrogenase from nodules and separation into components. The chapter reviews Bergerson's 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.