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Figure 7 | BMC Microbiology

Figure 7

From: Zymographic differentiation of [NiFe]-Hydrogenases 1, 2 and 3 of Escherichia coli K-12

Figure 7

The heterodimeric HyaB-His-HyaA complex of Hydrogenase 1 catalyzes the hydrogen-dependent reduction of NBT. Aliquots of crude extracts (25 μg total protein) derived from strains MC4100 and DHP-F2 (ΔhypF) grown anaerobically in TGYEP, pH 6.5 or 8 μg of purified Hyd-1 from strain FTH004 were subjected to 7.5% (w/v polyacrylamide) non-denaturating PAGE and the gels were treated as follows: A. transferred to a nitrocellulose membrane and analyzed with antibodies directed against Hyd-1; B. transferred to a nitrocellulose membrane and analyzed with monoclonal His-tag antibody; C. the gel containing purified Hyd-1 and the molecular mass standard was stained with Coomassie Brilliant Blue. The masses of the standard proteins (Sigma) are given on the right hand of the panel. Alternatively, the extracts and purified enzyme were: D. stained for 10 minutes under a 100% hydrogen atmosphere with PMS and NBT as electron acceptors; or E. stained under a hydrogen atmosphere with BV and TTC as electron acceptors. The bands assigned to Hyd-1 activity or the His tagged version of HyaA-Hyd-1 activity are indicated on the right hand of the gels.

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