[40] Nitrogenase complex and its components

This chapter discusses the assay method and properties of nitrogenase complex and its components. Three principal methods are available for the measurement of nitrogenase activity: (1) the colorimetric measurement of ammonia formed from N2, (2) the manometric measurement of H2 formation in the absence of reducible substrates, and (3) the measurement of ethylene from acetylene reduction by gas chromatography. The H2 evolution assay is convenient for kinetic studies and is the only known reaction in which all reducing electrons appear in a single product. The acetylene reduction assay is the most sensitive and is preferred when low levels of activity are to be detected. Nitrogenase from Azotobacter vinelandii can be isolated as a purified complex (PNC) providing a more stable and easily stored preparation useful for many enzyme studies. PNC can then be separated into its component proteins for individual study. This procedure reduces the complications accompanying separate isolation procedures for each component. After the initial purification step, the preparations are unstable in air and the entire isolation is conducted under anaerobic conditions.