Large-scale purification of high activity Azotobacter vinelandII nitrogenase.

A large scale, rapid, high-yield purification procedure for Azotobacter vinelandii nitrogenase proteins has been developed. Yields of approx. 600 mg of the FeMo protein (Av1) and approx. 550 mg of the Fe protein Av2 are routinely obtained using a procedure that requires only 28 h. The specific activities of Av1 and Av2, respectively, are 3000 and 2100 nmol H2 evolved/min per mg. These activities are significantly higher higher than those comonly used in reactivity studies. Procedures for the isolation and concentration of large quantities of iron-molybdenum cofactor of nitrogenase are also reported. Techniques for anaerobic protein manipulation, generally applicable to the purification of oxygen sensitive proteins are also described.