Abstract: To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the partially purified MoFe protein (Av1) preparation was obtained from Azotobacter vinelandii Lipmann OP by chromatography on DEAE-cellulose (DE52) and Sephacryl S-200 columns and analyzed by PAGE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The Av1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Av1 on the SDS gel. However, on the anoxic native PAGE, in addition to the Av1 band, the preparation was shown to have three other main bands that migrated slower than Av1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Av1, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase (PGI). In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaCl concentration gradient. Thus, Av1 with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.

Key words: Azotobacter vinelandii, chaperonin GroEL, glucose-6-phosphate isomerase, nitrogenase Av1.