[37] Isolation and characterization of nitrate reductase from Escherichia coli

This chapter provides information on the isolation and characterization of nitrate reductase from Escherichia coli. E. coli nitrate reductase (NR) is an inducible membrane bound enzyme that serves as the terminal electron acceptor in cells growing anaerobically. This enzyme can be isolated in large amounts, for it makes up 15–52% of the total protein of the cytoplasmic membrane. It is easily purified and once removed from the membrane, is soluble in aqueous solutions. Owing to its spherical shape and large size it makes an ideal standard for calibrating Sepharose and Bio–Gel columns. NR is assayed colorimetrically by measuring the reduction of nitrate to nitrite using methylviologen as an artificial electron donor. This method is very useful in detecting proteins that might be precursors, mutant proteins, or proteins modified in some other manner. Maximum amounts of NR are produced in cultures growing anaerobically in medium containing nitrate and molybdenum.