Molecular mass determination of NifZ. (A) Coomassie blue-stained 10–20% gradient SDS/PAGE of purified, streptavidin-tagged NifZ. Lane 1, 10-μg protein standard; lane 2, 5-μg purified NifZ. The molecular mass of the polypeptide of streptavidin-tagged NifZ is ≈19 kDa, consistent with that determined by mass spectrometry (data not shown). (B) Determination of the native molecular mass of streptavidin-tagged NifZ by Ultrogel AcA 34 gel filtration (Pall Life Science, East Hills, NY). V0, void volume; Ve, elution volume. Protein standards (white circles) are as follows: ribonuclease A (14 kDa), carbonic anhydrase (29 kDa), and ovalbumin (43 kDa). The apparent native molecular mass of streptavidin-tagged NifZ (red circle) is ≈20 kDa, suggesting a monomeric composition of the protein.